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Crystal interactions with isolated human neutrophils : effect of adsorbed proteins Gadkari, Seema Jayant 1991

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CRYSTAL INTERACTIONS WITH ISOLATED HUMAN NEUTROPHILS: EFFECT OF ADSORBED PROTEINS by SEEMA JAYANT GADKARI B. Pharm.; U n i v e r s i t y o f Bombay, 1984 M. Pharm.; U n i v e r s i t y o f Bombay, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE  i n  THE FACULTY OF GRADUATE STUDIES ( F a c u l t y o f Pharmaceutical Sciences) D i v i s i o n o f Pharmaceutics  We accept t h i s t h e s i s as conforming t o t h e r e q u i r e d standard.  THE UNIVERSITY OF BRITISH COLUMBIA August 1991 Seema Jayant Gadkari  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by his or  her  representatives.  It  is understood that copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  F A U L T Y  Department of  rHARftftr\C€uriCM.  The University of British Columbia Vancouver, Canada Date **)CrV$T  DE-6 (2/88)  20,.1991  <i>OEN(j£S  ABSTRACT  C r y s t a l d e p o s i t i o n d i s e a s e s such as gout and pseudogout a r e characterized monohydrate  by (MSUM)  (CPPD) c r y s t a l s arises  from  respectively  and  neutrophils.  various  and c a l c i u m  of  pyrophosphate  o f these  dihydrate Inflammation  crystals  polymorphonuclear  urate  with the  leukocytes  or  We have s t u d i e d t h e n e u t r o p h i l responses t o by MSUM and CPPD c r y s t a l s  protein  coatings  and t h e e f f e c t o f  on t h e c r y s t a l  neutrophil  responses.  The n e u t r o p h i l  were  respiratory  burst  the  monosodium  i n the j o i n t s .  the interaction  synoviocytes  stimulation  the deposition  surface  responses  accompanying  on t h e studied  phagocytosis  l e a d i n g t o p r o d u c t i o n o f superoxide  anion and g e n e r a t i o n o f  chemiluminescence.  o f t h e n e u t r o p h i l was  also and  s t u d i e d by m o n i t o r i n g  MSUM and CPPD  anion rate  induced  t o t h e same degree o f production  reduction  inhibitable  the generation  although  o f superoxide  of ferricytochrome  inhibitable  the  the release of  myeloperoxidase  lysozyme enzymes.  Both  to  Degranulation  f o r MSUM/neutrophil o f superoxide  p r o t e i n onto  CPPD c r y s t a l s  with  anion  induced than  a  faster  CPPD.  c was superoxide  f o r CPPD/neutrophil  inactivation  MSUM  o f superoxide  incubations  The  dismutase but not  i n c u b a t i o n s , probably due dismutase  MSUM c r y s t a l s . Immunoglobulin  by a d s o r p t i o n o f  Precoating  o f MSUM and  G, Bovine serum  albumin  iH  or  plasma  proteins  superoxide The  d i d not i n f l u e n c e  the generation of  significantly.  generation  of  chemiluminescence  luminol  enhanced  by  was  induced  MSUM  neutrophil  of  a  greater  magnitude and t h e maximal response was a t t a i n e d f a s t e r than f o r CPPD c r y s t a l s . immunoglobulin while  plasma  P r e c o a t i n g MSUM o r CPPD c r y s t a l s  G enhanced  t h e chemiluminescence  precoating  inhibited  the  with  response  chemiluminescence  response. R e l e a s e o f myeloperoxidase not  be measured  MSUM.  the  degranulation lysozyme also  crystals  adsorption  of  was monitored  with  proteins  t o adsorb  onto  did  myeloperoxidase.  by measuring  MSUM  e x t e n s i v e l y than myeloperoxidase.  not  Hence  the release of  induced by MSUM and CPPD c r y s t a l s .  found  could  due t o a d s o r p t i o n o f myeloperoxidase by  P r e c o a t i n g MSUM  influence  induced by MSUM c r y s t a l s  Lysozyme was  crystals  although  less  There was no s i g n i f i c a n t  e f f e c t o f p r o t e i n c o a t i n g on MSUM and CPPD induced  lysozyme  release. The  particulate  activation  of  chemiluminescence responses,  and  stimulants neutrophils  MSUM  and  with  CPPD  produced  superoxide  release,  g e n e r a t i o n and d e g r a n u l a t i o n . N e u t r o p h i l in  particular  the  chemiluminescence  responses, t o t h e c r y s t a l s c o u l d be modulated of t h e p r o t e i n adsorbed t o t h e c r y s t a l  by t h e nature  surface.  iv  TABLE OF CONTENTS NO.  TITLE  PAGE  ABSTRACT  ii  LIST OF FIGURES  xi XVi  LIST OF TABLES LIST OF SCHEMES  XVii  LIST OF ABREVIATIONS  xviii XXI  ACKNOWLEDGEMENTS I.  BACKGROUND  1  C r y s t a l induced a r t h r i t i s  1  2  Deposition of c r y s t a l s  1  3  Mechanism o f c r y s t a l induced inflamination  3  3.1. 4.  Inflammatory mediators Crystal-neutrophil  4.1. 4.2.  interactions  Phagocytosis  7 9 9  Components o f the r e s p i r a t o r y b u r s t  12  4.2.1. Chemiluminescence  12  NO.  4.3. 5.  TITLE  PAGE  4.2.2. P r o d u c t i o n o f superoxide anion  16  Degranulation  20  E f f e c t o f c r y s t a l h i s t o r y on inflammatory  21  potential  of c r y s t a l s  5.1.  Crystal  size  21  5.2.  C r y s t a l pretreatment  21  E f f e c t of protein adsorption  22  6.1.  Plasma p r o t e i n s  24  6.2.  Immunoglobulin G (IgG)  23  6.3.  Lipoproteins  24  6.4.  Albumin  24  6.  7.  Hypothesis  and O b j e c t i v e s  II.  EXPERIMENTAL  25  Materials  26  Reagents and S o l v e n t s  27  Buffer Solutions  28  vi  NO.  1  TITLE  PAGE  Instruments  28  Labware  29  Methods  30  P r e p a r a t i o n of c r y s t a l s  30  1.1.  Monosodium Urate Monohydrate  30  1.2.  Calcium  31  Pyrophosphate Dihydrate  1.2.1. S y n t h e s i s of c a l c i u m dihydrogen  31  pyrophosphate 1.2.2. S y n t h e s i s of c a l c i u m pyrophosphate  32  dihydrate 2.  C h a r a c t e r i z a t i o n of c r y s t a l s  33  2.1  X-ray d i f f r a c t i o n a n a l y s i s  33  2.2  D i f f e r e n t i a l scanning  33  2.3  Particle size analysis  34  2.4  Scanning e l e c t r o n microscopy  34  P r e p a r a t i o n of n e u t r o p h i l suspension  35  3.1  E s t i m a t i o n of n e u t r o p h i l count  36  3.2  E s t i m a t i o n of n e u t r o p h i l v i a b i l i t y  36  3.  calorimetry  vii  NO.  TITLE  PAGE  3.2.1. CL d e t e r m i n a t i o n  36  3.2.2. S t a i n i n g w i t h t r y p a n b l u e  38  4.  Protein coating  5.  Measurement o f n e u t r o p h i l r e s p o n s e  39  Superoxide anion release  39  5.1  of crystals  5.1.1. S u p e r o x i d e r e l e a s e stimulation  from n e u t r o p h i l s on  39  by u n c o a t e d MSUM  5.1.2. S u p e r o x i d e r e l e a s e  from n e u t r o p h i l s  s t i m u l a t i o n by u n c o a t e d 5.1.3. E f f e c t  38  on  40  CPPD  o f SOD o n s u p e r o x i d e r e l e a s e  41  5.1.4. A d s o r p t i o n o f SOD by MSUM c r y s t a l s  41  5.1.5. S u p e r o x i d e r e l e a s e  42  from n e u t r o p h i l s on  s t i m u l a t i o n by p r o t e i n c o a t e d MSUM and 5.2  CPPD  Chemiluminescence  5.2.1. C h e m i l u m i n e s c e n t  43 response o f n e u t r o p h i l s  on s t i m u l a t i o n by u n c o a t e d MSUM a n d CPPD  crystals  43  viii  NO.  TITLE  PAGE  5.2.2. Chemiluminescent response o f n e u t r o p h i l s  43  on s t i m u l a t i o n by p r o t e i n coated MSUM and 5.3  CPPD c r y s t a l s  Degranulation  i n d i c a t o r : r e l e a s e o f MP0  44  and LYZ 5.3.1. MP0 and LYZ r e l e a s e from n e u t r o p h i l s  44  on s t i m u l a t i o n by uncoated MSUM and CPPD c r y s t a l s 5.3.2. Measurement o f LYZ  45  5.3.3. Measurement o f MPO  46  5.3.4. MPO and LYZ r e l e a s e from n e u t r o p h i l s on  46  s t i m u l a t i o n by p r o t e i n coated MSUM and CPPD c r y s t a l s 6.  Statistical tests  III.  46  RESULTS AND DISCUSSION  1.  Preparation  of c r y s t a l s  48  1.1  Monosodium u r a t e monohydrate (MSUM)  48  1.2  Calcium pyrophosphate d i h y d r a t e  50  (CPPD)  IX  NO.  TITLE  PAGE  1.2.1. S y n t h e s i s  o f c a l c i u m dihyrogen  pyrophosphate 1.2.2. S y n t h e s i s  (CDPP)  o f c a l c i u m pyrophosphate  dihydrate 2.  50  54  (triclinic)  Characterization of c r y s t a l s  54  2.1  X-ray d i f f r a c t i o n  54  2.2  D i f f e r e n t i a l scanning c a l o r i m e t r y  2.3  Scanning e l e c t r o n microscopy (SEM)  62  E s t i m a t i o n o f n e u t r o p h i l count and  69  3.  (DSC)  62  viability 4. 4.1  Measurement o f n e u t r o p h i l responses  70  C r y s t a l s t i m u l a t e d superoxide r e l e a s e  70  from n e u t r o p h i l s 4.1.1. E f f e c t o f SOD on superoxide r e l e a s e  76  4.1.2. E f f e c t o f p r o t e i n s on superoxide r e l e a s e  81  4.2  83  C r y s t a l s t i m u l a t e d chemiluminescent response o f n e u t r o p h i l s  4.2.1. E f f e c t o f temperature on the CL response  93  X  NO.  TITLE  PAGE  4.2.2. E f f e c t o f p r o t e i n s on the chemiluminescent 4.3  93  response  Crystal stimulated degranulation  99  response o f n e u t r o p h i l s 4.3.1. C r y s t a l s t i m u l a t e d MPO r e l e a s e  100  from n e u t r o p h i l s 4.3.2. E f f e c t o f p r o t e i n s on MPO r e l e a s e  105  4.3.3. C r y s t a l s t i m u l a t e d LYZ r e l e a s e  109  from n e u t r o p h i l s 4.3.4. E f f e c t o f p r o t e i n s on LYZ r e l e a s e  115  5.  Future work  115  IV.  SUMMARY AND CONCLUSIONS  119  V.  REFERENCES  122  xi  LIST OF FIGURES NO.  TITLE  PAGE  1. a) S t r u c t u r e o f s y n o v i a l j o i n t  2  b) Release o f c r y s t a l s i n t o t h e j o i n t  2  2. C r y s t a l n e u t r o p h i l i n t e r a c t i o n  4  3. Schematic r e p r e s e n t a t i o n o f p h a g o c y t o s i s  10  4. P a r t i c l e s i z e o f MSUM c r y s t a l s  49  (Method o f B u r t and Jackson, 1983) 5. P a r t i c l e s i z e o f MSUM c r y s t a l s  51  ( M o d i f i e d method) 6. X-ray d i f f r a c t i o n p a t t e r n o f c a l c i u m  52  dihydrogen pyrophosphate 7. P a r t i c l e s i z e d i s t r i b u t i o n o f CPPD c r y s t a l s  55  8. X-ray d i f f r a c t i o n p a t t e r n o f monosodium  56  u r a t e monohydrate (Burt e t a l . ,  1983)  9. X-ray d i f f r a c t i o n p a t t e r n o f monosodium  58  u r a t e monohydrate ( M o d i f i e d method) 10. X-ray d i f f r a c t i o n p a t t e r n o f c a l c i u m pyrophosphate  60  dihydrate  11. DSC thermogram o f CPPD c r y s t a l s  63  TITLE  NO.  12. DSC thermogram o f MSUM (Burt  et al.,  crystals  1983)  13. DSC thermogram o f MSUM (Modified  crystals  method)  14. S c a n n i n g e l e c t r o n m i c r o g r a p h o f CPPD  crystals  15. S c a n n i n g e l e c t r o n m i c r o g r a p h o f MSUM c r y s t a l s  (Burt e t a l . ,  1983)  16. S c a n n i n g e l e c t r o n m i c r o g r a p h o f MSUM c r y s t a l s  (Modified  17. S u p e r o x i d e g e n e r a t i o n stimulated  method)  by n e u t r o p h i l s  by u n c o a t e d MSUM a n d CPPD  crystals 18. S u p e r o x i d e g e n e r a t i o n  i n d u c e d by MSUM  crystals 19.  Superoxide generation  i n d u c e d by CPPD  crystals 20. E f f e c t  o f c y t o c h a l a s i n B o n MSUM i n d u c e d  superoxide 21.  Effect release  generation  o f SOD on CPPD i n d u c e d s u p e r o x i d e from  neutrophils  xiiI  NO.  TITLE  22. Superoxide p r o d u c t i o n  PAGE by MSUM s t i m u l a t e d  79  n e u t r o p h i l s i n t h e presence o f superoxide dismutase 23. E f f e c t o f MSUM t r e a t e d SOD on CPPD induced  80  superoxide r e l e a s e from n e u t r o p h i l s 24. E f f e c t o f p r o t e i n c o a t i n g on t h e g e n e r a t i o n  82  o f superoxide induced by MSUM 25. E f f e c t o f p r o t e i n c o a t i n g on t h e superoxide generation  84  induced by CPPD c r y s t a l s  26. Chemiluminescence  generated by n e u t r o p h i l s  85  s t i m u l a t e d by 5 mg uncoated MSUM 27. E f f e c t o f MSUM c o n c e n t r a t i o n  on  87  generated by n e u t r o p h i l s  89  chemiluminescence 28. Chemiluminescence  s t i m u l a t e d by 50 mg uncoated CPPD 29. E f f e c t o f CPPD c o n c e n t r a t i o n  on n e u t r o p h i l  90  chemiluminescence 30. E f f e c t o f p r o t e i n c o a t i n g on  95  chemiluminescence response t o MSUM 31. E f f e c t o f p r o t e i n c o a t i n g on t h e chemiluminescent response t o CPPD  96  TITLE  NO.  32. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f s u p e r n a t a n t o n t h e MPO a s s a y  ( a t t = 60 s )  33. M y e l o p e r o x i d a s e r e l e a s e f r o m c y t o c h a l a s i n B pretreated uncoated  n e u t r o p h i l s on s t i m u l a t i o n  CPPD  crystals  34. M y e l o p e r o x i d a s e r e l e a s e pretreated  with  from c y t o c h a l a s i n B  n e u t r o p h i l s on s t i m u l a t i o n  w i t h u n c o a t e d MSUM c r y s t a l s 35. E f f e c t o f a d d i t i o n o f i n c r e a s i n g o f MSUM o n m y e l o p e r o x i d a s e 36. M y e l o p e r o x i d a s e r e l e a s e pretreated  amounts  activity  from c y t o c h a l a s i n B  n e u t r o p h i l s on s t i m u l a t i o n  with  p r o t e i n c o a t e d CPPD ( n = 6) 37. M y e l o p e r o x i d a s e r e l e a s e pretreated  neutrophils  from c y t o c h a l a s i n B  on s t i m u l a t i o n  p r o t e i n c o a t e d MSUM 38. S t a n d a r d c u r v e f o r l y s o z y m e a s s a y b y micrococcus  method  39. L y s o z y m e r e l e a s e  i n d u c e d b y MSUM  40. L y s o z y m e r e l e a s e  i n d u c e d b y CPPD  with  XV  NO.  TITLE  41. Study o f lysozyme a d s o r p t i o n onto MSUM  PAGE 114  and t h e e f f e c t on t h e lysozyme assay 42. Lysozyme r e l e a s e induced by p r o t e i n  116  c o a t e d MSUM 43. Lysozyme r e l e a s e induced by p r o t e i n c o a t e d CPPD  117  L I S T OF TABLES NO.  PAGE  TITLE  1.  X-ray p a t t e r n o f CDPP  53  2.  X-ray p a t t e r n o f MSUM (Burt and Jackson,  57  1983) 3.  X-ray p a t t e r n o f MSUM ( M o d i f i e d method)  59  4.  X-ray p a t t e r n o f CPPD  61  5.  E f f e c t o f MSUM c o n c e n t r a t i o n on  88  chemiluminescence 6.  E f f e c t o f CPPD c o n c e n t r a t i o n on  91  chemiluminescence 7.  E f f e c t o f sample temperature on  94  chemiluminescence 8.  E f f e c t o f p r o t e i n c o a t i n g on MSUM and CPPD induced  chemiluminescence  98  xv ii  L I S T OF SCHEMES NO.  TITLE induced neutrophil  PAGE  1.  Crystal  stimulation  2.  E f f e c t o f complement a c t i v a t i o n b y MSUM  8  3.  Activation  13  4.  Generation o f chemiluminescence  15  5.  Generation of superoxide  17  of luminol  5  xviii  L I S T OF  A  ABREVIATIONS  Change i n absorbance  A450  Absorbance a t 450  A550  Absorbane a t 550  ANOVA  Analysis of Variance  AUC  Area under t h e curve  BSA  Bovine serum albumin  CDPP  Calcium dihydrogen pyrophosphate  CL  nanometers nanometers  Chemiluminescence  CPPD  Calcium pyrophosphate d i h y d r a t e  DSC  D i f f e r e n t i a l scanning c a l o r i m e t r y  jum  micrometers  g  Gravity  HDL  High d e n s i t y l i p o p r o t e i n s  HP  Hewlett Packard  Ig G  Immunoglobulin  KV  Kilovolts  G  LDH  Lactate  LDL  Low  LYZ  Lysozyme  dehydrogenase  density  lipoproteins  mA  milliamperes  MPO  Myeloperoxidase  MSUM  Monosodium u r a t e monohydrate  mV  millivolts  NL  N a t i v e luminescence  nm  Nanometer  nmoles  Nanomoles  02~  Superoxide anion  PC  P e r s o n a l computer  PMNL  Polymorphonuclear l e u k o c y t e s  rpm  R e v o l u t i o n s per minute  SEM  Scanning e l e c t r o n microscopy  SOD  Superoxide dismutase  U/mL  U n i t s per m i l l i l i t e r  UBC  U n i v e r s i t y of B r i t i s h  Columbia  Ultraviolet Volts.second  ACKNOWLEGEMENTS I  am g r a t e f u l  t o Dr. Helen  Burt  f o r her s u p e r v i s i o n and  encouragement d u r i n g t h e course o f t h i s work.  I would  also  l i k e t o thank Dr. Frank Abbott, Dr. Mike B r i d g e s , Dr. David Godin and Dr. A l a n M i t c h e l l  f o r t h e i r e n t h u s i a s t i c support  and guidance. I  would  like  assistance. Michael  t o thank  Mr.  The a s s i s t a n c e  Gentleman  appreciated.  and Ms.  John  Jackson  f o r technical  o f Ms. Barbara McErlane, Mr. Sukhbinder  Panesar  i s greatly  Thanks a r e a l s o due t o Mr. Ron Aoyama and Ms.  P a s c a l Schmidt f o r t h e i r h e l p d u r i n g t h i s p r o j e c t . The guidance and f r i e n d s h i p o f Dr. Wayne Riggs, Mr. Matthew Wright and Dr. A l b e r t Chow i s g r e a t l y a p p r e c i a t e d . I  am  grateful  friendship  t o Mr.  Krishnaswamy  and encouragement  Yeleswaram  during  for  t h e course  of  his this  work. I  would  also  like  t o thank  my  family  for their  support  d u r i n g t h i s time., I  would  like  generosity Financial  t o thank  in  Red Cross,  providing  blood  Vancouver, for  support from t h e U n i v e r s i t y  is gratefully  acknowledged.  the  for their  experiments.  of B r i t i s h  Columbia  1  BACKGROUND 1 . CRYSTAL-INDUCED ARTHRITIS Crystal  deposition  pathological  disease  condition  has  been  associated  with  defined the  as  a  presence  of  c r y s t a l s which then c o n t r i b u t e t o t h e t i s s u e damage (Dieppe and C a l v e r t , being  1983).  These c r y s t a l s have been i d e n t i f i e d as  t h o s e o f monosodium u r a t e monohydrate (MSUM),  pyrophosphate  dihydrate  (CPPD) and  calcium  phosphates (McCarty, 1989).  Crystal  deposition  type  of crystal  cases,  can be c l a s s i f i e d  deposited.  involves deposition deposition disease  diseases  i n some  The c o n d i t i o n  calcium basic  based on t h e  known  as gout  o f MSUM c r y s t a l s and pseudogout o r CPPD i s c h a r a c t e r i z e d by t h e presence o f CPPD,  t r i c l i n i c and m o n o c l i n i c  c r y s t a l s (Ryan and McCarty, 1989).  2. DEPOSITION OP CRYSTALS The s y n o v i a l j o i n t s o f t h e e x t r e m i t i e s knees)  are  diseases. Figure in  la.  particularly  The s t r u c t u r e  the a r t i c u l a r  joint  to  crystal  of a synovial  joint  deposition i s shown i n  The common s i t e s f o r d e p o s i t i o n o f c r y s t a l s a r e  synovial f l u i d the  prone  (hands, f e e t , elbows,  c a r t i l a g e , synovium  of the j o i n t .  fluid  by  the  or d i r e c t l y  i n the  C r y s t a l s may be r e l e a s e d  rupture  of  preformed  into  synovial  Bony  a) STRUCTURE OF SYNOVIAL JOINT  b) RELEASE OF CRYSTALS INTO THE JOINT  FIGURE 1  3  deposits, cartilage  by  a  "crystal  mechanism  from  the  as seen i n F i g u r e l b , o r they may p r e c i p i t a t e i n  the s y n o v i a l f l u i d the  shedding"  synovium  itself  due t o t r a n s i e n t h y p e r u r i c e m i a i n  or a t r a n s i e n t  fall  i n temperature  (McCarty,  1989). 3. MECHANISM OF CRYSTAL-INDUCED INFLAMMATION Crystal-induced  inflammation  i n i t i a l response  i s through  the  synoviocytes  (Wallingford  crystals  The  i n t e r a c t i o n of the c r y s t a l s with  o r phagocytic  and  polymorphonuclear  involves s e v e r a l stages.  McCarty, leukocytes  t o the synoviocytes  cells 1971)  (PMNL). may  lining  t h e synovium  rather  than  The b i n d i n g  be enhanced  by  with of the  adsorbed  p r o t e i n s such as immunoglobulin G (Ig G) (Kozin and McCarty, 1976). leads  Crystal-induced l y s i s of the phagocytic to release  synoviocytes  o f a c r y s t a l - i n d u c e d chemotactic  factor  which promotes t h e m i g r a t i o n o f polymorphonuclear l e u k o c y t e s (PMNL/neutrophils)  into  chemotactic  has been  weight  factor  peptide  (Phelps e t a l . ,  with  the area  (McCarty,  identified  a molecular  1989).  as a low m o l e c u l a r  weight  o f 8,500  daltons  1981) but other r e p o r t s i n d i c a t e a m o l e c u l a r  weight o f 11,500 d a l t o n s ( S p i l b e r g and Mandell, Following  This  increased migration  o f PMNL i n t o  1983).  the j o i n t , the  i n t e r a c t i o n o f the c r y s t a l s w i t h n e u t r o p h i l s p l a y s a c e n t r a l role  i n t h e c r y s t a l - i n d u c e d inflammatory  response  shown i n F i g u r e 2 and Scheme 1 and summarized below.  and i s  4  ACUTE  CRYSTALLIZATION  CRYSTAL  COMPLEMENT  CONTACT SYSTEM  SYNOVIAL FIBROBLAST  SHSDOINO.  MONONUCLEAR PHAGOCYTE ( s y n o v i a l OnUtg. [pint  1 PAIN, VASODILATATION, OEDEMA, FEVER, PMN INFILTRATION  CHANGES IN CRYSTALUNE-PROTEIN-COATING CRYSTAL DISSOLUTION IMHI81TION OF PHAGOCYTOSIS ACTIVATION OF ANTI-INFLAMMATORY PATHWAYS UNKNOWN FACTO«(S)  C R Y S T A L NEUTROPHIL INTERACTION  FIGURE 2  fluid)  OXYGEN UPTAKE  OXIDATIVE METABOLISM  SUPEROXIDE ANION HYDROXYL RADICAL HYDROGEN PEROXIDE  CHEMILUMINESCENCE  ^ „ „ DEGRANULATION n  A  k  n  n  m  LYSOSOMAL ENZYME RELEASE  MYELOPEROXIDASE MANNOSIDASE LYSOZYME  SCHEME 1  PHAGOCYTOSIS  C E L L U L A R AUTOLYSIS  LACTATE DEHYDROGENASE R E L E A S E  6  The naked c r y s t a l upon r e l e a s e i n t o t h e j o i n t f l u i d several proteins  such as immunoglobulin  adsorbs  G ( I g G) ,  albumin,  complement fragments, c l o t t i n g f a c t o r s and l i p o p r o t e i n s from the  synovial  fluid  (Kozin and McCarty,  1976; Hasselbacher  and Schumacher, 1978; Hasselbacher, 1979a, 1979b; T e r k e l t a u b et  al.,  1983).  receptors through (Kozin  on  The p r o t e i n coated c r y s t a l may i n t e r a c t w i t h t h e macrophage/neutrophil  t h e mediation and McCarty,  o f t h e adsorbed  1976) and t h i s  plasma  proteins  surface  membrane e.g. I g G  stimulation of  n e u t r o p h i l s by MSUM/CPPD c r y s t a l s r e s u l t s i n enhanced oxygen uptake  and an i n c r e a s e  macrophage/neutrophil crystal  i n oxidative (Simchowitz  i s phagocytosed  cytoplasm  i n a s a c known  lysosomes  of  within  this  T h i s i s f o l l o w e d by f u s i o n o f t h e phagosome p r o d u c i n g a  that  (Shirahama  the release  phagolysosome  phagolysosome  exposed  o f the lysosomal  results  a  (McCarty,  1989; Gordon e t a l . ,  interaction crystal  crystal-membrane  i s thought  membrane  binding  contents of the  l e a v i n g t h e membrane o f  interaction 1988).  t o occur  I t has  i n digestion  t o t h e "naked"  allowing  a r e emptied  and Cohen, 1974).  p r o t e i n s adsorbed onto t h e c r y s t a l , the  within the  (McCarty, 1962;  g r a n u l e s o f t h e lysosomes  i n t o t h e phagolysosome proposed  and l i e s  The  D e g r a n u l a t i o n then occurs and t h e c o n t e n t s  the neutrophilic  been  within the  a l . , 1982).  as a phagosome  o f t h e PMNL w i t h  phagolysosome.  et  by t h e PMNL  Schumacher and Phelps, 1971). the  metabolism  crystal to  take  thereby place  The crystal-membrane  i n two c o n s e c u t i v e s t e p s ,  f o l l o w e d by membrane  rupture or  7  membranolysis  (Burt  and  Jackson,  p h a g o l y s o s o m a l membrane and into and  the  cytoplasm  release  release  of  of  powerful  stimulus  joint  and  potentiation  leading  1988). as  first  described  the  by  factors  b)  1982)  may  producing  in et  his  the  enzymes  autolysis  fluid.  PMNL  The  acts  as  o f more n e u t r o p h i l s the  response  work  on  this  into  (Gordon  et  cytolysis is  the  a l . , 1966).  a  crystals  hypothesis  and  was  toxicity  Wallingford  hypothesis  of and  to  the  inflammation.  complement s y s t e m by in  .  factors of  cause c e l l  lysis  A c t i v a t i o n of  the  clotting  vitro  In v i v o , a  neutrophils, (Scheme  and  1979a,  of  by  and  Byers,  1973;  1979b;  Tenner  complement  events  eventually  enhance  the  increase  phagocytosis  chemotactic and  2).  Hageman f a c t o r , w h i c h  cascade,  classical  these a c t i v a t e d  cascade  that  the  (Naff  Hasselbacher,  trigger  migration  the  the  of  MEDIATORS  a l . , 1973;  Cooper,  of  within"  extended  pathways  et  synovial  crystal-induced  from  (Allison  cellular  interaction with  of  crystal-induced  alternate  to  inflammatory  Allison  MSUM a c t i v a t e s t h e  and  further  later  lysosomal  the  migration  the  of the  contents  "perforation  INFLAMMATORY  Byers  into  mechanism  (1971)  mechanism o f  a)  of  particles  McCarty  3.1.  to  This  known  silica  f o r the  Rupture  leading  crystal  cytosolic  the  al.,  follows,  the  the  release  1988).  crystals  i s f a c t o r XII  has  been  of  reported  (activation of C3)  +- + C5/6/7  + C8/9  Chemotactic factor enhanced phagocytosis *-CeU lysis  M S U M INDUCED C O M P L E M E N T ACTIVATION  SCHEME 2  9  (Kellermeyer, shown  to  system of  1967).  activate  i n vitro,  potent  established  of  is  changes (Alwan  an  and et  which  enzymes  and  formation  enhance  vivo  are mediators  The  other  inflammatory  by  has  the  but the  not  been  Guerne  and  which  of  products  of  and  release  of  cause  vascular  of the pain  stimulus  e t a l . , 1989;  release  process  the synoviocytes  synthesis  i n j u r y into the synovial  3  particles  is a  Woolf  superoxide, the  fluid  and  lysosomal  polymorphonuclear may  also  influence  (Rosen e t a l . , 1 9 8 6 ) .  INTERACTIONS  process  schematic  by n e u t r o p h i l s  stimulated  the  in  kinin  PHAGOCYTOSIS  Figure  in  may  been  the  e t a l . , 1980)  interleukins  a l . , 1989;  4. CRYSTAL-NEUTROPHIL  the  which  phagocytosed  and  1987) .  the  and  has  r e s u l t i n g i n the  finding  associated  Dieppe,  cell  cascade  (Ginsberg  this  factor  ( S p i l b e r g , 1974; G r e e n e t a l . , 1 9 8 2 ) .  prostaglandins  4.1.  the l a t t e r  is initially  there  the c l o t t i n g  response  significance  MSUM  activated  vasodilators  inflammatory  c)  The  on c o n t a c t  of p a r t i c l e  crystal  surface  thereby  (Trush  of phagocytosis  e t a l . , 1978).  the opsonized  The p r o t e i n s  are believed  to play  I g G on t h e c r y s t a l  enhancing  through the  adsorbed  an i m p o r t a n t surface  the neutrophil  of  The PMNL i s  crystal  recognition.  p a r t i c u l a r adsorbed crystal  with  representation  onto role,  opsonizes  response  (Roos  •  OPSONIZEO  1. P A R T I C L E 2. M E M B R A N E J.  FORMATION  PH»cocrrosA(K.e RECOGNITION INVAGINATION  ANO ANO  PARTICLE  BINOINC PHAGOCYTOSIS  O F PHACOSOME  Schematic representation of the endocytic events and accompanying metabolic processes that occur during phagocytosis by polymorphonuclear leukocytes (PMNs). For more detailed discussions of these processes see: (a) B. D. Cheson, J. T. Curnutte, and B. M. Babior, in "Progress in Clinical Immunology" (B. S. Schwartz, ed.). Vol. 3. p. I. Game & Stratton, New York. 1977. PMN biochemistry, (b) S. J. Klebanoff, Semin. Hematol. 12, 117 (1975). PMN antimicrobial mechanisms, (c) T. P. Stossel.A/. Engl. J. Med. 290,717.774,833 (1974). General review on phagocytosis.  FIGURE 3  11  et  a l . , 1981).  crystal  The  causes  invagination  neutrophil. invaginated lysosomes is the  The areas  fuse  extracellular Baggiolini  stimulation produce  lysosomes  also  o f t h e plasma  medium  the opsonized  membrane  migrate  membrane  membrane  distinct  towards  a n d some  particulate  (Babior  e t a l . , 1973).  increased products anion,  PMNL  oxygen  and h i g h l y  reactive  accompanied termed  of  oxidative  such  (Simchowitz stimuli  PMNL  pattern  metabolism of  o f o x i d a t i o n byas t h e superoxide  et by  i n oxidative  burst'.  which  i n terms  and s t r o n g o x i d i z i n g  increase  the respiratory x  the metabolic  i s manifested  species  particulate  by t h i s  stimuli  However, o n e x p o s u r e  and t h e g e n e r a t i o n  peroxide  Phagocytosis  of responding t o  i n the cells.  increased This  the hydroxyl radical  hydrogen  stimuli  exhibit  uptake  contents into  or soluble  changes  soluble  and  of the  e t a l . , 1972; Hawkins,  a n a e r o b i c metabolism.  changes  the  1984).  metabolic  or  of the  before the p a r t i c l e  leukocytes are capable  by e i t h e r  particulate  (Weissmann  a n d Dewald,  normally exhibit  is  by  o f t h e plasma  w i t h t h e plasma  Polymorphonuclear  as  provided  c o m p l e t e l y p h a g o c y t o s e d and d i s c h a r g e t h e i r  1972;  to  stimulus  agents  a l . , the  such  1982).  PMNL  metabolism  is  which  12  4.2.  COMPONENTS OF  THE  RESPIRATORY BURST  4.2.1. CHEMILUMINESCENCE (CL)  Chemiluminescence result  of  a  stimulation (Allen 1980;  et  is  chemical by  the  reaction  particulate  a l . , 1972;  Cheson  R o s c h g e r e t a l . , 1988;  luminescence amplifying reflects serves  can  as  be  an  et  a l . , 1976;  excitation  state  CL  of  and  the  on  et a l . ,  1986).  use  This  of  light  lucigenin.  neutrophil  i n d i c a t o r of n e u t r o p h i l f u n c t i o n  a  stimuli  Westrick  the  luminol  as  exhibit  Topley,  through  as  light  non-particulate  H a r b e r and  such  of  PMNL  and  and  measured  compounds  the  production  CL  and  thus  (Trush e t a l . ,  1978) . Luminol, been  (5-amino-2,3-dihydro-l,4-phthalazinedione),  r o u t i n e l y used  to  amplify  chemiluminescent r e a c t i o n s of  luminol  Luminol  activation  exhibits a  increase  in  the  electronically the  in  the  high  shown  the  in  of  of  the  Scheme  of  of  biological  and  by The  nature  (Allen,  1982).  Luminol  is  released  an per  thereby  increasing  The  of  CL  hence i t can  oxygenation  various  i . e . there  photons  luminol  respiratory burst  3  yield  C L measurement.  phagocyte o x i d a t i v e metabolism. to alter  e t a l . , 1978).  C L quantum  number  dioxygenation  study  (Trush  CL produced  e x c i t e d species generated,  sensitivity  d e p e n d s on  is  the  has  reactions has  in neutrophils  luminol be  such  b e e n shown (Allen,  used as not  1982) .  RADICAL  DiOXYGENATION  Proposed biochemical mechanisnvfor cyclic hydrazide chemiluminescence at neuiral to acid pH.  ACTIVATION OF LUMINOL SCHEME 3  14  Scheme may  4  depicts the reactions occurring i n the c e l l  possibly  1978) .  result  The  respiratory neutrophil  i n CL  oxidative  burst  The  generation  two  pathways  from  products  may  to yield  (adapted  react  luminescent  produced  with  1988),  p o s s i b l y d e p e n d e n t on s u p e r o x i d e  peroxide.  The  other  reaction  of  superoxide  Allen  and Loose,  normally  which  catalyses the dismutation  al.,  °2~  +  role  using  +  2  H  o f MPO  sodium  Edwards,  of  hydrogen  or  hydrogen  t o CL  has  been  (Webb e t a l . , 1974; present  superoxide  a s shown i n e q u a t i o n  >  +  H  2°2  to  1 below  i n PMNL hydrogen  (McCord e t  azide  as  an  o f CL  inhibitor  of  has been  MPO  1  studied  (Nurcombe and  1989).  CL  was  found  t o be  as o p s o n i z a t i o n o f the p a r t i c u l a t e  temperature buffer  Eqn.  °2  +  i n the generation  Luminol-dependent such  an enzyme  and  1971).  °2~  The  and o x y g e n  the  follow  MPO  of  superoxide  (SOD)  peroxide  of  s i n c e l u m i n o l may  s t u d i e d u s i n g superoxide dismutase 1976),  the  on  independent  with  contribution  during  shown t o  dependent  peroxide,  by  the  one  peroxide  oxygenated  and  al. ,  products.  o f l u m i n o l - a m p l i f i e d CL was (Dahlgren,  et  components  hydrogen  be  Wilson  that  and t h e p r e s e n c e  (Harber  and  Topley,  of calcium 1986;  affected stimuli,  by  incubation  and a l b u m i n  Roschger  factors  i n the  e t a l . , 1988).  STIMULANTS 1 OOVC^'MO P o M i t l l l  (bacttria.ijmoion] 2 So'uDU Aqinll pAo'bol m j n l l G U e««tOI« coicigm ioftophOM A23I87 3 14 G Coolid Filttri  PARTICLE ENGULFMENT ANO/OR OEGRANULATION MPO  CELLULAR (PMN) ACTIVATION  STIMULATION CF OXIDATIVE METABOLISM (HMP  MPO + H,0, + Cr  SHUNT)  JNADPH/NAOP*  \  H  ' °  PYRIOINE NUCLEOTIDE DEPENDENT ENZYMIC FORMATION OF 0»"  H,0+CI- + Oj' 27,000 a porllculali (oronulO fraction from ocli»ol«d cill  Oj* + Ot* + 2H* — H 0 » + 0 j  OXIDIZABLE SUBSTRATES ON PHAGOCYTOSABLE OR -» PHAGOCYTtZEO PARTICLE  t  °l + H,0, —0H*+ 0H« + 0 ' 2  I  Nontniymie Rtoclisni b«lw«in Oiyojn Spiclll  "^•GENERATION OF ELECTRONIC , *VEXCITATION STATE(S) PROSTAGLANDIN  SYNTHESIS  '  CELL * TREE ENZYME SYSTEMS I. Xonlftint ciitfon, pyrifti lypiroiidt qiMroh'flf Z.M;llopifoii40U, M , 0 . . holidt r«oclipn  * (Slnoltt Oiyotn anil a' corbonyl groups)  ANO/OR LIPID PEROXIDATION EMISSION OF LIGHT-*— UPON RELAXATION TO GROUND STATE  F I G . 2. Representation of the enzymic and nonenzymic mechanisms involved in the generation of chemiluminescence (CL) by polymorphonuclear leukocytes (PMNs) following cellular activation by both particulate and soluble stimulants. Dashed line represents two processes, prostaglandin synthesis and lipid peroxidation, known to generate CL; however, the contribution, if any, of these processes to the CL response of PMNs has yet to be defined. H  SCHEME 4  16  The  concentrations  all  affect  of luminol  t h e observed  and PMNL as w e l l as s t i m u l i  response  (Wilson  e t a l . , 1978).  Other f a c t o r s such as d i e t may a l s o a f f e c t t h e CL r e s p o n s e o f PMNL (Magaro e t a l . , 1988). 4.2.2.  PRODUCTION OF SUPEROXIDE  Superoxide  anion  electronically  i s the  excited  ANION  term  used  species  to  arising  describe  the  from  singlet  m o l e c u l a r oxygen f o l l o w i n g a one e l e c t r o n r e d u c t i o n  process.  Scheme 5 i l l u s t r a t e s t h e g e n e r a t i o n electronically  excited  states  o f s u p e r o x i d e and o t h e r  o f oxygen  (Green  and H i l l ,  1984) . M o l e c u l a r oxygen (O2) c o n t a i n s  two u n p a i r e d e l e c t r o n s i n t h e  o u t e r m o s t o r b i t a l and hence can e x i s t i n e i t h e r o f two s p i n states, the  triplet  unpaired  singlet  and s i n g l e t s t a t e s .  electrons  state  electron  to flip;  electronically reduction,  possess p a r a l l e l  one o f t h e e l e c t r o n s  spin i sa n t i - p a r a l l e l .  In the t r i p l e t spin while  i n the  i s f l i p p e d so t h a t t h e  Energy must be s u p p l i e d t o cause t h e  thus the s i n g l e t s t a t e  excited  state  state.  i . e . addition  of  formation  of superoxide  anion,  electrons  leads t o t h e formation  This an 0 ~. 2  o f oxygen i s an  can undergo electron, Further  further  leading  to  addition of  of other o x i d a t i v e products  such a s hydrogen p e r o x i d e and hydroxy1 r a d i c a l . Neutrophils  generate the e l e c t r o n i c a l l y e x c i t e d  oxygen d u r i n g  the r e s p i r a t o r y burst  species of  ( A l l e n e t a l . , 1972).  17  3  0  - 0 J  2  2  c  O7  HOi  c  01-  pK>l<  HO7  pK-l\,6  H 0 t  2  c 2H+  (OH  -H 0 2  pA'=li.9  O-  OH-  e  2H  O  j  +  -H,0  The products derived from the successive one-electron reductions of dioxygen.  GENERATION OF SUPEROXIDE SCHEME 5  18  The  production  of superoxide  i n neutrophils  membrane bound NADPH-dependent o x i d a s e  (Figure  involves  a  3) b e l i e v e d  t o be l o c a t e d on t h e plasma membrane o f t h e PMNL ( B a b i o r and P e t e r s , 1981; 1979; The  G o l d s t e i n e t a l . , 1977;  Dewald e t a l . , superoxide  1979).  anion  i s responsible  a c t i o n o f phagocytes responsible 1986).  Badwey and Karnovsky,  f o r the bactericidal  ( B a b i o r e t a l . , 1973) and may a l s o be  f o r damage t o t h e h o s t  tissue  (Hsie  MSUM and CPPD c r y s t a l - i n d u c e d s u p e r o x i d e  been s t u d i e d  Methods  have  superoxide  been  1991).  f o r t h e measurement  c.  assay  (NBT)  Measurement  of  and  reduction  oxygen  uptake  consumption by manometric methods have a l s o been (Babior  and  concentrations while  Cohen,  1981).  i s more  long-term incubations. study  of  of neutrophil  sensitive  of and  reported  requires  high  s e n s i t i v e method  but u n s u i t a b l e f o r  The method most commonly used i n t h e respiratory  reduction  of ferricytochrome  superoxide  as d e s c r i b e d  1969).  Manometry  o f c e l l s and i s n o t a v e r y  potentiometry  stimulus  by n e u t r o p h i l s such as t h e n i t r o b l u e  reduction  ferricytochrome  Naccache e t a l . ,  developed  generation  tetrazolium  r e l e a s e has  and MSUM was found t o be a s t r o n g e r  t h a n CPPD (Nagase e t a l . , 1989;  et a l . ,  c  i n equation  burst  i s based  t o ferrocytochrome  on t h e c by  2 (McCord and F r i d o v i c h ,  19  Fe  This  i : i : i  cyt c  reduction  maximum  from  >  O-  i s accompanied  520 nm  Fe -- cyt c J  L  by a s h i f t  t o 550 nm  +  0  Eqn. 2  2  i n UV a b s o r p t i o n  and hence  can be  readily  monitored. The  r e d u c t i o n of f e r r i c y t o c h r o m e c can be made s p e c i f i c f o r  superoxide  through  t h e use of superoxide  dismutase  (SOD), a  Cu-Zn metalloenzyme normally present w i t h i n t h e PMNL as p a r t of  t h e p r o t e c t i v e mechanisms  damage.  SOD c a t a l y s e s  against  superoxide-inflicted  the dismutation  o f superoxide  d e s c r i b e d p r e v i o u s l y i n equation 1 (McCord e t a l . , °2~  The  +  °2~  +  2  H  reduction  +  >  of  H  2°2  +  ferricytochrome  i n h i b i t e d by t h e presence  1971).  Eqn.  °2  c  by  as  1  superoxide  is  of SOD i n t h e r e a c t i o n mixture and  t h i s can be used t o c o n f e r s p e c i f i c i t y on t h e assay  (Babior  and Cohen, 1981). Ferricytochrome excluded  c being a high molecular  from t h e c e l l and hence t h i s assay can o n l y measure  changes i n t h e l e v e l of e x t r a c e l l u l a r it  weight compound i s  has been found  superoxide.  However,  t h a t a l a r g e p r o p o r t i o n o f superoxide i s  r e l e a s e d e x t r a c e l l u l a r l y by PMNL (Roos and Weening, 1979).  20  4.3.  DEGRANULATION  Degranulation  of  extracellular  the  medium  phagosome a f t e r  neutrophil  during  phagocytosis  Weissmann e t a l . , 1972;  occurs  phagocytosis (Baggiolini  Hawkins,  Hawkins, 1972;  release  their  lysozyme, Cross,  Blackburn  contents,  myeloperoxidase  1988),  crystal.  into  I t has  the  and  alpha  expose  the  membrane. peroxide  "naked"  crystal  Myeloperoxidase dependent  The  mannosidase  can  granules such  as and  opsonized  that proteases  such  as  on the c r y s t a l s u r f a c e to  (MPO)  1984;  (Jones  phagosome c o n t a i n i n g the  been hypothesized  the  (Weissmann e t a l . ,  s e v e r a l enzymes  lysozyme s t r i p the p r o t e i n s adsorbed to  into  Dewald,  e t a l . , 1987).  including  the  Degranulation  occur even i n the absence of phagocytosis 1972;  and  and  1972).  into  the  phagolysosomal  catalyzes  oxidation reaction  forming  a  hydrogen  part  of  the  respiratory burst. The group of compounds known as the c y t o c h a l a s i n s are u s e f u l in  the  study  phagocytosis  of by  degranulation interfering  a c t i n - m y o s i n network 1971). contents  since  with  such  agents  inhibit  the p o l y m e r i z a t i o n of  (Davis e t a l . , 1971;  Malawista  the  et a l . ,  T h i s r e s u l t s i n d e g r a n u l a t i o n and r e l e a s e of granule i n t o the e x t r a c e l l u l a r  fluid,  thereby  facilitating  measurement of the c o n t e n t s . Degranulation any  of  the  can  be  monitored  enzymes  released  by measuring the r e l e a s e of during  this  process.  The  21  p r e s e n c e o f MPO i n t h e e x t r a c e l l u l a r indicator  of neutrophil  exclusively  hydrogen  PMNL  turbidity addition the  1985) .  MPO can be measured by t h e oxidation  o f o-  Lysozyme (LYZ) r e l e a s e d from t h e lysosomes o f  c a n be assayed  by m o n i t o r i n g t h e d e c r e a s e i n  o f a s u s p e n s i o n o f Micrococcus  lysodeikticus  o f LYZ ( B a b i o r and Cohen, 1981) .  muramic  polysaccharide  i t i s present  granules of the neutrophil  p e r o x i d e dependent MPO-catalyzed  dianisidine. the  i s u s e f u l as an  degranulation since  i n the azurophilic  (Andrews and K r i n s k y ,  fluid  acid-containing of the c e l l  LYZ h y d r o l y z e s  mucopeptide  walls  on  in  o f t h e Micrococcus  the thus  c o n v e r t i n g t h e spores t o p r o t o p l a s t s r e s u l t i n g i n a decrease in turbidity. 5. EFFECT  OF CRYSTAL HISTORY  ON  INFLAMMATORY  POTENTIAL OF  CRYSTALS 5.1.  CRYSTAL SIZE  The s i z e o f i n d i v i d u a l c r y s t a l s acute inflammation.  i s known t o be i m p o r t a n t i n  Amorphous d e p o s i t s and l a r g e  crystals  of monosodium u r a t e were found t o be r e l a t i v e l y i n e f f e c t i v e , whereas  crystals  o f about  5 jum i n l e n g t h  were  t h e most  r e a c t i v e (Schumacher e t a l . , 1975; B u r t e t a l . , 1989). 5.2. CRYSTAL PRETREATMENT  Grinding  of c r y s t a l s  t o reduce  reported  by e a r l i e r w o r k e r s ,  particle  size,  a  practice  has now been demonstrated t o  22  e f f e c t a change i n t h e c r y s t a l s t r u c t u r e and hence a l t e r t h e i n f l a m m a t o r y p o t e n t i a l o f t h e c r y s t a l s (Burt e t a l . , 1986). S i m i l a r l y , d e p y r o g e n a t i o n o f t h e c r y s t a l s by h e a t i n g a t 200 -  250 °C  f o r 2 h r was found  t o produce  a change  in  the  c r y s t a l s t r u c t u r e leading t o a l t e r a t i o n of i t s inflammatory potential  (Hasselbacher,  Pritzker,  1979; Mandel,  1980; Cheng and  1981).  6. EFFECT OF PROTEIN ADSORPTION MSUM and CPPD c r y s t a l s have a h i g h l y r e a c t i v e s u r f a c e  that  can a d s o r b many d i f f e r e n t p r o t e i n s and o t h e r m o l e c u l e s from the s y n o v i a l f l u i d .  I t i s now w e l l r e c o g n i z e d  that proteins  bound t o t h e i n f l a m m a t o r y c r y s t a l s p l a y an i m p o r t a n t determining  rolei n  the b i o l o g i c a l a c t i v i t y of the c r y s t a l s .  6.1. PLASMA PROTEINS MSUM c r y s t a l s have been  shown t o adsorb  several  proteins  such as f i b r o n e c t i n , f i b r i n o g e n , Hageman f a c t o r , which a r e part  of the c l o t t i n g  system,  cascade,  immunoglobulins  McCarty,  that  characteristic  the  o f gout  As  accompanying composition  the increase  ( W a l l i n g f o r d and  e t a l . , 1983).  spontaneous  I t has been  remission  which  and pseudogout may be due t o  p r o t e i n s adsorbed on t h e c r y s t a l s u r f a c e 1976).  o f t h e complement  and l i p o p r o t e i n s  1974; T e r k e l t a u b  suggested'  proteins  inflammatory i n vascular  of the synovial  fluid  is the  ( K o z i n and McCarty,  episode  proceeds,  permeability such  that  the  alters the i tclosely  23  r e s e m b l e s t h a t o f plasma  (Swan, 1978).  other proteins present i n synovial the  crystal  surface  The l i p o p r o t e i n s and  fluid  and may i n t e r f e r e  a r e adsorbed with  the c r y s t a l -  n e u t r o p h i l i n t e r a c t i o n t o b r i n g about spontaneous of  t h e inflammatory  Recently  ^-acid  neutrophil  responses  episode  (Terkeltaub  glycoprotein such  has been  shown  as chemotaxis,  g l y c o p r o t e i n had e a r l i e r  specific  inhibitor  6.2.  Ig  a g g r e g a t i o n and 1990).  Serum  2  ~  neutrophil  stimulation  (Terkeltaub e t a l . ,  by  1988).  IMMUNOGLOBULIN G ( I g G)  G  i s strongly  crystals of  to inhibit  been shown t o be a p o t e n t and  of  hydroxyapatite c r y s t a l s  remission  e t a l . , 1984).  superoxide anion g e n e r a t i o n (Laine e t a l . , HS  onto  adsorbed  t o MSUM b u t l e s s  ( H a s s e l b a c h e r and Schumacher, 1978).  I g G t o MSUM has been  so t o CPPD The b i n d i n g  shown t o be a f u n c t i o n  of the  charge d e n s i t y o f t h e I g G m o l e c u l e ( H a s s e l b a c h e r , 1979) and t h e b i n d i n g may, t h e r e f o r e be e l e c t r o s t a t i c i n n a t u r e . F  ab  F  c  p o r t i o n o f t h e I g G m o l e c u l e i s more p o s i t i v e t h a n t h e s t r a n d s and hence i t i s t h e F k p a r t t h a t b i n d s t o t h e a  crystal leaving the F has  The  been  shown  c  fragments exposed.  t o enhance  neutrophil  I g G-coated MSUM responses  t o the  c r y s t a l s w i t h increases i n the extent of phagocytosis o f t h e crystals,  elevated  levels  l y s o s o m a l enzyme r e l e a s e et a l . , The  1978;  enhanced  of  superoxide  (Kozin e t a l . ,  Abramson e t a l . ,  1982;  1979;  and  enhanced  Hasselbacher  Rosen e t a l . ,  1986).  response of t h e n e u t r o p h i l t o t h e c r y s t a l s i s  24  apparently with  through  the  interaction  cellular  o f t h e exposed F  receptors  present  fragments  c  on  the  PMNL  ( H a s s e l b a c h e r , 1979). 6.3.  LIPOPROTEINS  MSUM and CPPD c r y s t a l s  bind  and h i g h d e n s i t y l i p o p r o t e i n Burt  e t a l . , 1989).  apolipoprotein A2. extent  l i p o p r o t e i n (LDL)  (HDL) ( T e r k e l t a u b  e t a l . , 1986;  The p r o t e i n moiety o f LDL i s m o s t l y  B and t h a t  Terkeltaub  low d e n s i t y  et a l .  o f phagocytosis,  o f HDL i s a p o l i p o p r o t e i n A l and (1983,  1984, 1986) m o n i t o r e d t h e  CL and d e g r a n u l a t i o n  produced by  n e u t r o p h i l s on exposure t o HDL and LDL c o a t e d MSUM c r y s t a l s . LDL  coated  MSUM  neutrophil apparently  crystals  responses  while  inhibited HDL  a l l MSUM  coated  d i d n o t have any s i g n i f i c a n t  induced  MSUM  crystals  inhibitory  effect.  B u r t e t a l . , (1989) s t u d i e d t h e e f f e c t s o f LDL and HDL bound to  MSUM and CPPD  on c r y s t a l - i n d u c e d  measured by l a c t a t e and  neutrophil (LDH).  Both HDL  LDL s t r o n g l y i n h i b i t e d CPPD and MSUM i n d u c e d  neutrophil  cytolysis  ( B u r t e t a l . , 1989).  6.4.  ALBUMIN  The  synovial  1978).  dehydrogenase r e l e a s e  cytolysis  fluid  i s an u l t r a f i l t r a t e  o f plasma  (Swan,  A l b u m i n , a major c o n s t i t u e n t o f plasma i s among t h e  proteins  present  crystals  have  McCarty,  1976).  i n the synovial  been  reported  The e f f e c t  fluid.  t o bind  MSUM  albumin  o f adsorbed  and CPPD  ( K o z i n and  albumin  on t h e  25  particle native al.  stimulated  luminol  luminescence  (1988).  dependent  (NL) h a s been  Their  results  luminescence  enhanced  by a l b u m i n .  effects  of  stimulation, albumin  incubations. was  similar effect not  7. It  MSUM  et al.  (1983,  buffers  They  stated  used that  et al.  while  o f NL i s  induced  neutrophil  1984, 1986) i n c l u d e d  for  neutrophil/crystal o f t h e albumin  inhibition". (1989)  o f l i p o p r o t e i n s on n e u t r o p h i l  luminol  of the inhibitory  t h e purpose  non s p e c i f i c  by B u r t  that  studies  and  by R o s c h g e r e t  generation  on  the  studies  However i n  of the inhibitory  cytolysis,  a l b u m i n was  added t o t h e b u f f e r s .  HYPOTHESIS AND OBJECTIVE i s our hypothesis  crystal  surface  interaction. neutrophil  that  can  t h e presence  influence  The o b j e c t i v e responses  degranulation) and  lipoproteins  t o "to eliminate  indicate  In t h e i r  luminescence  reported  i s inhibited,  Terkeltaub  in  dependent  induced  used  i n these  the  (CL, s u p e r o x i d e by u n c o a t e d  studies  immunoglobulin g and plasma  i s t o study t h e  i o n production  and p r o t e i n  of incubation were  proteins.  on t h e  crystal-neutrophil  o f o u r work  CPPD c r y s t a l s a s a f u n c t i o n  Proteins  of proteins  bovine  coated  and MSUM  time. serum  albumin,  26  EXPERIMENTAL MATERIALS Albumin,  Fraction  V,  fatty  acid  B o e h r i n g e r Mannheim GmbH, W. Calcium  from  bovine  serum,  Germany.  tetrahydrogen di-orthophosphate, CaH (P0 ) •H2O,  Catalase, MO.  L t d . , Poole,  from  bovine  2  BDH  England.  liver,  Sigma  Chemical  Co., S t . L o u i s ,  U.S.A.  Cytochalasin  B,  Chemical Cytochrome Co.,  4  4  Chemicals  Dextran  free,  from  Helminthosporium  Co., S t . L o u i s ,  c,  Type  I I I , from  S t . L o u i s , MO. T70,  MO.  Pharmacia  dematioideum,  Sigma  U.S.A.  horse  heart,  Sigma  Chemical  AB,  Uppsala,  U.S.A. LKB,  Biotechnology  Sweden. o-Dianisidine Louis, Ficoll-Paque  dihydrochloride,  MO.  Sigma  Chemical  Co.,  St.  U.S.A.  ( R ) , Pharmacia  LKB, B i o t e c h n o l o g y AB,  Uppsala,  Sweden. Hydrogen  Peroxide,  U.S.A.  3 0 % , Sigma  Chemical  Co., S t . L o u i s ,  MO.  27  Immunoglobulin G, Human, Sigma Chemical Co., S t . L o u i s , MO. U.S.A. Lactate  Dehydrogenase Assay  K i t , Sigma  Chemical  Co.,  St.  L o u i s , MO. U.S.A. Luminol,  (5-amino-2,3-dihydro-l,4-phthalazinedione)  Sigma  C h e m i c a l Co., S t . L o u i s , MO. U.S.A. Lysozyme, Grade I , from  c h i c k e n egg w h i t e , Sigma  Chemical  Co., S t . L o u i s , MO. U.S.A. Micrococcus  lysodeikticus,  Sigma  Chemical  Co., S t . L o u i s ,  MO. U.S.A. Superoxide  dismutase,  from  bovine  erythrocytes,  Sigma  C h e m i c a l Co., S t . L o u i s , MO. U.S.A. Triton  X-100,  (OctylPhenoxy  Polyethoxyethanol),  Sigma  C h e m i c a l Co., S t . L o u i s , MO. U.S.A. Uric  acid,  (2,6,8-trioxypurine),  L o u i s , MO. U.S.A. REAGENTS AND SOLVENTS  A c e t o n e , BDH. Ethanol Calcium acetate, Hydrochloric  Sigma.  a c i d , BDH.  Sigma  Chemical  Co.,  St.  28  Orthophosphorous a c i d , Sodium c h l o r i d e ,  80% J . T . B a k e r .  BDH.  Sodium h y d r o x i d e ,  Fisher.  BUFFER SOLUTIONS Hanks b u f f e r  (NaCl,  mM;  KH P0 ,  0.44  mM;  Glucose,  5.6 mM;  2  4  137 mM;  mM;  K C l , 5.4 mM;  MgS0 .7H20, 4  MgCl .6H 0, 2  2  0.41  Na HP0 . 2H 0, 2  mM;  0.5 mM;  4  2  0.33  CaCl .2H 0, 2  1.3  2  NaHC0 , 4.2 mM), 3  pH  7.4  Citrate  buffer,  Potassium  pH 5.5, 0.1M  phosphate b u f f e r  pH 6.2, 0.065M  INSTRUMENTS Bio-orbit  12 50  collection Fisher  software  Scientific  Scientific Dupont  Luminometer  Eppendorf  with  program o b t a i n e d from Co.  and  equipped  PC;  data  manufacturer, with  Fisher  Isotemp D r y B a t h 147.  Differential  Series  interfaced  Scanning  Calorimeter  Series  910  with  99 T h e r m a l a n a l y z e r .  centrifuge  5412, B r i n k m a n n  Instruments.  Fisher  Dyna-Mix  Fisher  S c i e n t i f i c Accumet pH m e t e r w i t h O r i o n pH p r o b e M o d e l  91-02.  29  Haake c i r c u l a t i n g  water bath  Hewlett Packard V e c t r a spectrophotometer and  equipped w i t h d a t a c o l l e c t i o n  H i t a c h i S-57  i n t e r f a c e d w i t h PC  software.  s c a n n i n g e l e c t r o n microscope and Hummer s p u t t e r  gold coater. M a g n e t i c s t i r r e r and h o t p l a t e . M e t t l e r b a l a n c e s models AJ100 and AE163. Nikon  differential  interference  microscope  Model  R  with  N i k o n o b j e c t i v e and o c u l a r m i c r o m e t e r s . Oven, Johns  Scientific  P e r k i n Elmer D i f f e r e n t i a l  Scanning C a l o r i m e t e r , Model IB.  R i g a k u G i e g e r f l e x X-Ray D i f f r a c t o m e t e r System. VWR  V o r t e x e r 2, S c i e n t i f i c I n d u s t r i e s ,  N.Y.  LABWARE  P o l y p r o p y l e n e t u b e s , 50 mL, Polypropylene  Eppendorf  10 mL,  tubes,  1.5  Nalgene mL,  0.5  Instruments P o l y s t y r e n e c u v e t t e s , 1 mL,  Fisher  Scientific  mL,  Brinkmann  30  METHODS 1. PREPARATION OF CRYSTALS 1.1. MONOSODIUM URATE MONOHYDRATE  (MSUM)  A m o d i f i c a t i o n o f t h e method o f B u r t e t a l . (1983) was used t o p r e p a r e MSUM samples. added  4.0 g o f u r i c  h e a t e d t o 55°C.  To 800 mL o f d i s t i l l e d water was  acid with s t i r r i n g  and t h e s u s p e n s i o n  A p p r o x i m a t e l y 2 5 mL o f 1M sodium h y d r o x i d e  was t h e n added s l o w l y and t h e u r i c a c i d d i s s o l v e d t o g i v e a clear solution.  The s t i r r i n g was c o n t i n u e d f o r 45 minutes  a t 55°C a f t e r which t h e pH was checked. t o 7.5 w i t h 1M h y d r o c h l o r i c a c i d .  The pH was a d j u s t e d  The s o l u t i o n was f i l t e r e d  h o t t h r o u g h Whatman # 1 f i l t e r paper under vacuum t o remove any suspended p a r t i c l e s .  The f i l t r a t e was a l l o w e d t o s t a n d  u n d i s t u r b e d f o r 24 hours a t room t e m p e r a t u r e . crystals  were h a r v e s t e d by f i l t r a t i o n  f i l t e r paper under vacuum.  The r e s u l t i n g  t h r o u g h Whatman # 1  The c r y s t a l s were washed w i t h 4  x 250 mL p o r t i o n s o f d i s t i l l e d water s a t u r a t e d w i t h MSUM a t room t e m p e r a t u r e and were then a l l o w e d t o d r y o v e r n i g h t a t 60 °C.  The c r y s t a l s  were  stored  i n tightly  capped  amber  glass bottles. The  crystals  analysis  were  characterized  and d i f f e r e n t i a l  by  X-ray  diffraction  scanning calorimetry.  s i z e a n a l y s i s was a l s o done by o p t i c a l m i c r o s c o p y .  Particle  31  1.2. CALCIUM PYROPHOSPHATE DIHYDRATE (CPPD) The p r e p a r a t i o n o f CPPD was a two s t e p p r o c e s s , t h e step  being  the  preparation  pyrophosphate/calcium  dihydrogen  of  first  calcium  acid  pyrophosphate.  1.2.1. S y n t h e s i s of c a l c i u m dihydrogen  pyrophosphate (CDPP)  I n a 600 mL g l a s s beaker, 250 mL o f 80% o r t h o p h o s p h o r i c a c i d was  added and heated  calcium  with s t i r r i n g  tetrahydrogen  t o 215°C.  diorthophosphate  i n i t i a l r a t e o f 1 g/min.  was  A d d i t i o n of begun  at  an  T h i s was c o n t i n u e d u n t i l about 40  g o f c a l c i u m t e t r a h y d r o g e n d i o r t h o p h o s p h a t e had been added a f t e r which t h e r a t e o f a d d i t i o n was about 0.5 g/min. was this  continued t i l l  a further  stage the mixture  became d i f f i c u l t . sintered  glass  35-40 g had been added.  formed a w h i t e s l u r r y  and  T h i s s l u r r y was then f i l t e r e d filter  heated  This  to  approximately  At  stirring through a 170°C  by  p a s s i n g o r t h o p h o s p h o r i c a c i d a t t h a t temperature t h r o u g h t h e filter. temperature  The  crystals  i n the f i l t e r .  100 mL a l i q u o t s o f acetone  were  allowed  to  cool  to  room  They were then washed w i t h t h r e e t o remove any adsorbed  a c i d and  allowed t o a i r dry i n the f i l t e r . The  crystals  were  characterized  by  X-ray  diffraction  c o n f i r m t h a t t h e y were CDPP p r i o r t o use i n t h e next s t e p .  to  32  1.2.2.  Synthesis  of  calcium  pyrophosphate  dihydrate  (triclinic):  A 250 mL beaker c o n t a i n i n g 103 mL d i s t i l l e d water was h e a t e d in  a w a t e r bath  T e f l o n coated of  t o 60 + 2°C and s t i r r e d  s t i r bar.  concentrated  acetic  acid  acetate. water  added,  acetate  t o 60 °C  added.  and 0.32 mL o f g l a c i a l  followed  A 150 mL beaker  was h e a t e d  calcium  The s t i r r i n g was slowed and 0.71 mL  hydrochloric acid  were  constantly with a  by  containing i n a water  0.6  g  of  20 mL o f d i s t i l l e d bath  The r a t e o f s t i r  and 0.6 g o f  was i n c r e a s e d i n  t h e 250 mL beaker, and 2 g o f CDPP added r a p i d l y . CDPP was n e a r l y  calcium  a l l dissolved, the rate  When t h e  o f s t i r r i n g was  r e d u c e d f o r 5 m i n u t e s , t h e n over a p e r i o d o f 15 seconds, t h e contents  of the small  beaker  beaker w i t h v i g o r o u s s t i r r i n g . a white g e l . to  poured  into  the large  This l e d t o the formation of  S t i r r i n g was d i s c o n t i n u e d and t h e g e l a l l o w e d  stand undisturbed  formation  were  overnight.  o f CPPD c r y s t a l s .  The g e l c o l l a p s e d w i t h t h e The c r y s t a l s  were washed i n  d i s t i l l e d water t h r e e t i m e s , washed i n e t h a n o l , t h e n acetone and a l l o w e d t o a i r d r y . The  crystals  were  characterized  a n a l y s i s and d i f f e r e n t i a l s c a n n i n g  by  X-ray  calorimetry.  diffraction  33  2. CHARACTERISATION OF CRYSTALS 2.1.  X-RAY DIFFRACTION ANALYSIS:  The  X-ray  diffraction  Giegerflex  X-Ray  goniometer,  a  a n a l y s e s were  Diffractometer  D/max-B  done  System  controller  using  with  a Rigaku  a  interface  biplanar  between t h e  goniometer and an IBM c o m p a t i b l e 28 6 PC and equipped w i t h a scintillation wavelength combining  counter.  The X r a d i a t i o n  = 1.56 Angstrom a  copper  source  units, with  a  used was copper K  which  was o b t a i n e d by  nickel  a n a l y s e s were done w i t h a s o f t w a r e program  filter.  p r o v i d e d by t h e  m a n u f a c t u r e r which a l l o w e d i n t e n s i t y and r e l a t i v e determinations. of  a  range  degrees/minute.  intensity  The X-ray tube was o p e r a t e d a t a p o t e n t i a l  4 0 KV and a t a c u r r e n t o f 20 mA.  over  Data  The sample was scanned  o f 5 t o 55 degrees  2  at a rate  The i n s t r u m e n t c o l l e c t e d  degrees 2 /sample.  The unground  of 5  d a t a e v e r y 0.05  samples were packed  i n an  aluminum sample h o l d e r f o r MSUM and s p r i n k l e d onto a double s i d e d S c o t c h t a p e a t t a c h e d t o t h e h o l d e r f o r CPPD samples. 2.2.  DIFFERENTIAL SCANNING  CALORIMETRY  Weighed samples o f MSUM p r e p a r e d by b o t h t h e method o f B u r t e t al. (1983) and t h e m o d i f i e d method and CPPD were a n a l y z e d by DSC.  The MSUM d a t a were c o l l e c t e d on a Dupont DSC under  a n i t r o g e n atmosphere  a t 20 p s i , from an i n i t i a l  temperature  of  25°C t o a f i n a l t e m p e r a t u r e o f 350°C, w i t h a h e a t i n g r a t e  of  10°C/min i n open crimped  aluminum pans.  The d a t a f o r  34  CPPD c r y s t a l s  were a l s o  open  aluminum  crimped  heated held  a t 10°C/min  isothermally  Vaporization MSUM  and  weighing peak.  pans  from  in a  nitrogen  150° t o 350°C.  of hydration  samples  t h e pans Percent  on t h e same i n s t r u m e n t i n atmosphere  CPPD  samples  and were  a t 350°C f o r 30 m i n u t e s .  o f water  CPPD  collected  was  after  water  from t h e pans  estimated  t h e appearance  losses  were  containing  quantitatively  by  o f t h e endothermic  calculated  f o r MSUM a n d  CPPD s a m p l e s . 2.3.  PARTICLE S I Z E ANALYSIS  The  particle  determined objective  by  for  each  distribution  optical  micrometer  differential suspended  size  microscopy  and e y e p i e c e  interference  i n liquid o f CPPD,  (method  ( m o d i f i e d method) s a m p l e s .  2.4.  SCANNING ELECTRON MICROSCOPY  surface  o f MSUM  a n d CPPD  of a metal stub with  were g o l d  coated  atmosphere.  using  a  crystals 0.01  micrometer The  a n d 100 c r y s t a l s  MSUM  Samples  the  microscope.  paraffin MSUM  of  of Burt  crystals graphite  57 s c a n n i n g e l e c t r o n m i c r o s c o p e .  Nikon  samples  were  were  measured 1983) a n d  to the  and t h e samples  coater  T h e s a m p l e s were t h e n examined  Nikon  a  attached  paint  on a Hummer s p u t t e r g o l d  with  e t al. ,  were  mm  was  i n an Argon  o n a H i t a c h i S-  35  3 . PREPARATION OF NEUTROPHIL SUSPENSION: Neutrophils  were  separated  from  freshly  whole b l o o d o b t a i n e d from t h e Red from h e a l t h y v o l u n t e e r s .  Red  collected  human  C r o s s , Vancouver, B.C.  Cross b l o o d was  or  from a s i n g l e  donor and not p o o l e d . To  450  mL  of  polypropylene dextran  citrate  stopper  solution  treated  bottles  in  Hanks  whole  were  buffer  added and  allowed to stand undisturbed.  sedimented  i n the  rich  supernatant  reduce  was  of  removed  as  as  of  3%  erythrocytes  the n e u t r o p h i l i t appeared,  to  l o s s due t o s e t t l i n g of t h e n e u t r o p h i l s . A l i q u o t s o f  the n e u t r o p h i l r i c h supernatant mL  soon  and  mL  in  blood/dextran  The  dextran  blood  100  the  suspension  presence  human  (5 mL)  were l a y e r e d over  of F i c o l l - P a q u e i n polypropylene tubes.  centrifuged  at  400  x  g  at  20 °C  for  n e u t r o p h i l p e l l e t t h u s o b t a i n e d was distilled  water  at  4°C  with  contaminating erythrocytes.  15  gentle  mL  vortexing  of 0.6  mL  to  of lyse 10  M sodium c h l o r i d e .  The  supernatant  the  The  restored after  t u b e s were c e n t r i f u g e d a t 200 x g a t 4°C d i s c a r d e d and  were  minutes.  The  was  tubes  suspended i n 1.5  T o n i c i t y was  seconds by t h e a d d i t i o n of 0.5  The  4  lysis  for 5  minutes.  treatment  was  repeated. The  pellet  resuspended  obtained  after  the  second  lysis  step  i n 6 mL Hanks b u f f e r and k e p t on i c e u n t i l  was used.  36  3.1.  ESTIMATION OF NEUTROPHIL COUNT:  Samples  o f n e u t r o p h i l suspensions  described  above  Laboratory  were  Medicine,  differential cell  sent  obtained  periodically  University  by t h e method  t o t h e Dept. o f  Hospital  (UBC S i t e ) f o r  counts.  On a r o u t i n e b a s i s , t h e t o t a l c e l l  count  (and t h e r e f o r e an  e s t i m a t e o f t h e n e u t r o p h i l count) i n t h e c e l l s u s p e n s i o n was determined  from t h e l a c t a t e dehydrogenase  (LDH) c o n t e n t i n  an a l i q u o t o f l y s e d c e l l s u s i n g t h e Sigma LDH assay k i t .  A  10% s o l u t i o n o f T r i t o n X-100 was added t o 0.4 mL o f t h e c e l l suspension,  v o r t e x e d f o r 5 minutes and c e n t r i f u g e d a t 200 x  g f o r 5 minutes.  To 2.2 mL o f l a c t a t e r e a g e n t  added 110 J I L o f s u p e r n a t a n t absorbance  measured  spectrophotometer seconds. difference  at  a t 30°C was  i n a U V / v i s i b l e c u v e t t e and t h e 340  nm  using  the  HP  Vectra  i n t h e k i n e t i c s mode f o r a p e r i o d o f 240  The absorbance was read every between  t h e absorbances  3 0 seconds and t h e  a t 180 and 60 seconds  used t o c a l c u l a t e t h e change i n absorbance p e r minute. c e l l count was t h e n e s t i m a t e d from a s t a n d a r d 3.2.  The  curve.  ESTIMATION OF NEUTROPHIL VIABILITY:  3.2.1. CL d e t e r m i n a t i o n The t i m e e l a p s e d between b l o o d c o l l e c t i o n and p r e p a r a t i o n o f the  n e u t r o p h i l suspension  was between 24-48 hours f o r Red  C r o s s b l o o d and 4-6 hours f o r b l o o d from UBC v o l u n t e e r s .  As  37  a  rapid  test  to  determine  n e u t r o p h i l s were s t i l l separation  procedure,  viable  whether following  Red  Cross  blood  completion of the  t h e MSUM s t i m u l a t e d  chemiluminescent  r e s p o n s e o f Red C r o s s b l o o d n e u t r o p h i l s was compared t o t h a t of UBC v o l u n t e e r b l o o d n e u t r o p h i l s . To  a 1.5 mL p o l y p r o p y l e n e Eppendorf  uncoated MSUM, 10 ill o f 1 x 10E-3 (final  tube  was added  5 mg  M l u m i n o l s o l u t i o n i n DMSO  c o n c e n t r a t i o n 10E-5 M) and s u f f i c i e n t  amount o f t h e  c e l l suspension t o give a f i n a l neutrophil concentration of 2 x 10E6 c e l l s / m L . monitored was  The CL produced  on a B i o - O r b i t  interfaced  with  by t h e n e u t r o p h i l s was  1250 Luminometer.  an IBM c o m p a t i b l e  The i n s t r u m e n t  PC and t h e d a t a  c o l l e c t i o n was done t h r o u g h a s o f t w a r e package f o r t h e same o b t a i n e d from t h e m a n u f a c t u r e r . chemiluminometer  by means  of a  j a c k e t e d tube h o l d e r connected t o a c i r c u l a t i n g water  bath.  Every and  3 minutes  and m a i n t a i n e d  The tube was p l a c e d i n t h e a t 37 °C  t h e tube was removed from t h e tube h o l d e r  the contents  gently  agitated.  The a g i t a t i o n  was  r e q u i r e d t o overcome t h e problem o f s e t t l i n g o f t h e c r y s t a l s within  t h e tube  leading  with the crystals. with  1000 s a m p l i n g  t o a reduced  interaction of cells  The response was s t u d i e d f o r 3 0 points i n this  were t a k e n e v e r y 1.8 seconds.  interval  minutes  i . e . readings  38  3.2.2. An  aliquot  equal at  Staining with  Trypan  blue  of the n e u t r o p h i l suspension  v o l u m e o f 0.5% t r y p a n  room  temperature  blue  for 5  4.  i n 0.9% s a l i n e ,  minutes  examined under t h e microscope  was m i x e d  w i t h an incubated  and t h e n e u t r o p h i l s  f o r dye uptake.  PROTEIN COATING OF CRYSTALS  Samples  o f 5 mg MSUM o r 50 mg CPPD were w e i g h e d  polypropylene prepared buffer MSUM)  Eppendorf  tubes.  Protein  f o rcoating  with  MSUM a t 37° (Hanks  MSUM.  One mL  3 7 ° C f o r 30 m i n u t e s : BSA  with  Eppendorf  centrifuge  crystal  pellet  buffer  or  MSUM.  diluted  and t h e t u b e s  50:50  The tubes a t 16,000  was washed  Hanks  saturated  with  protein  tumbled a t  6 mg/mL I g G (3 mg/mL f o r CPPD), 16.67  and plasma  saturated  were  o r Hanks  of the following  s o l u t i o n s was added t o t h e c r y s t a l s  1.5 mL  solutions  i n Hanks b u f f e r f o r c o a t i n g CPPD c r y s t a l s  presaturated  mg/mL  into  x  with  were g  16,000 x g a n d d i s c a r d i n g t h e  with  /  centrifuged  for 5  by r e s u s p e n d i n g  saturated  Hanks  MSUM,  supernatant.  Hanks i n an  minutes.  The  i n 1 mL o f Hanks centrifugation  at  39  5. MEASUREMENT OF NEUTROPHIL RESPONSE: 5.1. SUPEROXIDE ANION RELEASE 5.1.1. S u p e r o x i d e r e l e a s e from n e u t r o p h i l s on s t i m u l a t i o n by u n c o a t e d MSUM To a 1.5 mL  Eppendorf  tube was added 0.2 mL o f a 25 mg/mL  MSUM s u s p e n s i o n i n Hanks s a t u r a t e d w i t h MSUM, 0.1 mL o f a 10 mg/mL f e r r i c y t o c h r o m e  c solution  i n Hanks  saturated  with  MSUM, 0.2 mL o f Hanks s a t u r a t e d w i t h MSUM and 0.5 mL o f a 1 x 10E7 c e l l s / m L n e u t r o p h i l s u s p e n s i o n . 5 mg MSUM and 5 x 10E6 n e u t r o p h i l s . end-over-end intervals,  a t 15 rpm a t 37°C tubes  were  removed  The t u b e s  contained  The t u b e s were r o t a t e d f o r 180 m i n u t e s .  and  centrifuged  At  i n an  Eppendorf c e n t r i f u g e a t 16,000 x g f o r 30 seconds and s t o r e d on i c e . A 0.75 mL  a l i q u o t o f t h e s u p e r n a t a n t was withdrawn  and  o f t h e sample a t 550 nm measured on a  t h e absorbance  diode array spectrophotometer i n a polystyrene  1 mL c u v e t t e  C o n t r o l s were t r e a t e d s i m i l a r l y t o t h e samples w i t h t h e s u b s t i t u t i o n o f 0.2 mL o f Hanks s a t u r a t e d w i t h MSUM f o r t h e 0.2  mL  of crystal  ferricytochrome  suspension.  Blanks  were  0.1 mL o f  c s o l u t i o n d i l u t e d w i t h b u f f e r t o 1 mL and  t r e a t e d s i m i l a r l y t o t h e samples.  40  5.1.2. S u p e r o x i d e r e l e a s e from n e u t r o p h i l s on s t i m u l a t i o n by u n c o a t e d CPPD: Tubes  containing  50  mg  Hanks b u f f e r  were p r e p a r e d  end-over-end  at  intervals  15  tubes  rpm  were  5 x  CPPD and as  described 37 °C  and  10E6  above and  for  withdrawn  neutrophils  180  and  rotated  minutes.  centrifuged  and HP  A 0.75  the  mL  a l i q u o t of the s u p e r n a t a n t was  absorbance of t h e  Vectra  Controls  sample a t 550  were of  treated 0.2  mL  c r y s t a l suspension.  similarly of  to  the  Hanks b u f f e r  B l a n k s were 0.1  mL  s o l u t i o n d i l u t e d w i t h b u f f e r t o 1 mL  The  withdrawn  1 mL  samples f o r the  with 0.2  treated  the  cuvette.  mL  of f e r r i c y t o c h r o m e and  an  stored  measured on  spectrophotometer i n a polystyrene  substitution  t o the  nm  At  in  Eppendorf c e n t r i f u g e a t 16,000 x g f o r 30 seconds and on i c e .  in  the of c  similarly  samples.  rate  of  superoxide production  was  determined  l i n e a r change i n absorbance a t 550  nm  initial  change  phase  increasing  rate  of  no  of  C>2~/min/10 PMNL.  change. The  6  absorbance  that occurs a f t e r followed  Rates were r e p o r t e d  r a t e was  from  by  the an an  i n nmoles  c a l c u l a t e d using the  formula  shown below: nmoles O ~/min/10  6  2  PMNL =  A  5  5  0  (0.0211)(cell cone)(10~ ) 6  where  0.0211  ferricytochrome  is c.  the  extinction  coefficient  of  41  5.1.3. To  E f f e c t o f SOD on superoxide r e l e a s e  show  anion  that  and  f e r r i c y t o c h r o m e c was reduced  not other  species,  f e r r i c y t o c h r o m e c were monitored enzyme which performed  as  described  levels  of  reduced  i n t h e presence o f SOD, an  dismutates superoxide.  modifications: buffer  the  by superoxide  These  above  experiments  with  the  were  following  0.1 mL o f 3000 U/mL SOD s o l u t i o n  i n Hanks  (3 00 U/tube) was added t o the tubes i n s t e a d o f 0.1 mL  of b u f f e r and tubes were withdrawn o n l y a t 10 minutes case o f MSUM and a t 60 minutes  i n the case o f CPPD.  i n the Control  tubes were as d e s c r i b e d p r e v i o u s l y . 5.1.4. A d s o r p t i o n o f SOD by MSUM c r y s t a l s In t h e f o l l o w i n g solutions added  s t u d i e s MSUM c r y s t a l s  containing  t o tubes  SOD, c e n t r i f u g e d  containing  CPPD  were i n c u b a t e d w i t h and t h e supernatant  and n e u t r o p h i l s .  The  r a t i o n a l e f o r t h i s approach was t h a t i f MSUM c r y s t a l s adsorb significant  amounts o f SOD from  concentration  of  CPPD/neutrophil  SOD  solution,  then t h e reduced  i n t h e supernatant  suspensions  should  result  added in a  to the decreased  i n h i b i t i o n o f CPPD induced superoxide r e l e a s e . To each o f t h r e e 1.5 mL Eppendorf  tubes was added 0.2 mL o f  a 20 mg/mL f e r r i c y t o c h r o m e c s o l u t i o n  (4 mg/tube), 0.2 mL o f  Hanks  of a  saturated  solution of  three  with  MSUM,  0.1 mL  3000  (300 U/tube) and 10 mg MSUM c r y s t a l s . tubes  without  the c r y s t a l s  U/mL SOD  Another s e t  was r u n as c o n t r o l s .  42  a t 1 5 rpm a t 37°C f o r 30  The t u b e s were tumbled end-over-end minutes.  The t u b e s were then c e n t r i f u g e d  c e n t r i f u g e a t 16,000 x g f o r 3 0 seconds  i n an Eppendorf  and 0.5 mL o f t h e  s u p e r n a t a n t added t o a n o t h e r s e t o f tubes c o n t a i n i n g 0.2 mL of a 250 mg/mL CPPD c r y s t a l  s u s p e n s i o n (50 mg/tube).  hundred m i c r o l i t e r s o f a 1 x 10E7 c e l l s / m L c e l l  Five  suspension  were added t o t h e t u b e s which were then i n c u b a t e d a t 37°C f o r 60 m i n u t e s . not c o n t a i n i n g  Another s e t o f tubes w i t h CPPD c r y s t a l s b u t any SOD was a l s o r u n as a p o s i t i v e c o n t r o l .  The absorbance was measured a t 550 nm as d e s c r i b e d 5.1.5. Superoxide r e l e a s e protein  To  from n e u t r o p h i l s  earlier.  on s t i m u l a t i o n by  coated MSUM and CPPD  tubes  protein  containing coated  ferricytochrome  5 mg p r o t e i n  CPPD  were  c solution  added  coated 0.1 mL  (1 mg/tube),  MSUM o r 50 mg of a  10 mg/mL  0.3 mL o f Hanks  s a t u r a t e d w i t h MSUM o r Hanks b u f f e r and 0.5 mL o f a 1 x 10E7 cells/mL neutrophil crystals, prepared  suspension.  ferricytochrome as d e s c r i b e d  c  above.  Tubes c o n t a i n i n g  and n e u t r o p h i l s Control  tubes  uncoated  were  also  (crystals  absent) were p r e p a r e d as above.  The tubes were tumbled end  over  f o r 10 minutes  end a t 15  containing All  rpm a t 37°C  f o r MSUM  t u b e s and 60 minutes f o r CPPD c o n t a i n i n g  t u b e s were c e n t r i f u g e d  a t 16,000 x g f o r 30 seconds,  0.75 mL o f t h e s u p e r n a t a n t withdrawn 550 nm measured.  tubes.  and t h e absorbance a t  43  5.2. CHEMILUMINESCENCE 5.2.1  Chemiluminescent  response  of  neutrophils  on  s t i m u l a t i o n by uncoated MSUM and CPPD c r y s t a l s To a 1.5 mL p l a s t i c Eppendorf tube was added, 2, 5, 10 mg o f uncoated  MSUM o r 10, 20, 30, 40, 50 mg o f u n c o a t e d  crystals, (final  10 / i l  o f a 1 x 10E-3 M l u m i n o l s o l u t i o n  concentration  cells/mL neutrophil  CPPD  i n DMSO  10E-5 M) and 0.5 mL o f a 1 x 10E7 suspension.  The t u b e s were m a i n t a i n e d  a t 37°C i n t h e F i s h e r Isotemp Dry Bath and r e a d i n t h e tube holder Every holder  o f t h e chemiluminometer 1.5-3 minutes  t h e tube  and t h e c o n t e n t s g e n t l y  studied  f o r 60 minutes  with  a t t h e same was removed agitated.  temperature.  from  t h e tube  The r e s p o n s e was  2000 s a m p l i n g p o i n t s  i n this  i n t e r v a l i . e . r e a d i n g s were t a k e n every 1.8 seconds o r t i l l t h e r e s p o n s e abated.  The response was r e c o r d e d as a graph  o f mV v e r s u s t i m e . 5.2.2.  Chemiluminescent  response  of  neutrophils  on  s t i m u l a t i o n by p r o t e i n c o a t e d MSUM and CPPD c r y s t a l s Tubes c o n t a i n i n g 5 mg o f uncoated o r p r o t e i n c o a t e d MSUM and 3 0 mg o f uncoated o r p r o t e i n c o a t e d CPPD were p r e p a r e d .  To  each tube was added 0.5 mL o f a 1 x 10E7 c e l l s / m L n e u t r o p h i l s u s p e n s i o n and t h e t u b e s m a i n t a i n e d a t 37°C i n t h e F i s h e r Isotemp Dry B a t h . for  each  followed  tube  The response was r e c o r d e d f o r 15 seconds  i n succession,  by t u b e s  containing  i . e . uncoated BSA-coated,  crystal  tube  I g G-coated and  44  plasma p r o t e i n c o a t e d c r y s t a l s i n t h a t o r d e r 30 m i n u t e s . The  f o r a period of  The t u b e s were mixed p r i o r t o measurement.  rationale for this  particular  sequence o f measurements  was because i t was n e c e s s a r y t o m i n i m i z e any d i f f e r e n c e s i n r e s p o n s e due t o c h a n g i n g c e l l 5.3.  viability.  DEGRANULATION INDICATOR: RELEASE OF MPO AND LYZ  Degranulation neutrophils  and r e l e a s e pretreated  phagocytosis  of  o f MPO and LYZ were s t u d i e d i n  with  crystals  cytochalasin and  B  to  subsequent  prevent  neutrophil  cytolysis. A 1 mg/mL c y t o c h a l a s i n B s o l u t i o n was added t o a 2.5 x 10E7 cells/mL of  n e u t r o p h i l suspension t o give a f i n a l  concentration  10 jug/mL c y t o c h a l a s i n B and t h e s u s p e n s i o n i n c u b a t e d a t  37°C f o r 30 m i n u t e s . 5 . 3 . 1 . MPO and LYZ r e l e a s e  from n e u t r o p h i l s  on s t i m u l a t i o n  by u n c o a t e d MSUM and CPPD c r y s t a l s To a 0.5 mL Eppendorf tube c o n t a i n i n g 0.4 mL o f c y t o c h a l a s i n B  pretreated  was added  neutrophil  s u s p e n s i o n a t 2.5 x 10E7 c e l l s / m L  0.1 mL o f e i t h e r a 25 mg/mL MSUM s u s p e n s i o n i n  Hanks s a t u r a t e d w i t h MSUM o r a 250 mg/mL CPPD s u s p e n s i o n i n Hanks b u f f e r . prepared  Control  as d e s c r i b e d  tubes above.  (crystals  absent)  were  also  The tubes were tumbled end  o v e r end a t 15 rpm a t 37°C and withdrawn a t 5, 10,20, 30, 45,  60, 90, 120, 150 and 180 minutes.  The t u b e s  were  45  centrifuged  in a  Beckmann  Centrifuge at  200  x  g  for  2  minutes a t 0°C and t h e s u p e r n a t a n t removed f o r a n a l y s i s . Measurement of LYZ  5.3.2.  LYZ  was  determined  (1977). over  A  a  using  the  standard curve  method  of  f o r t h e LYZ  c o n c e n t r a t i o n range  assay  was  et  al.  prepared LYZ  from  c h i c k e n egg w h i t e , o b t a i n e d from Sigma Chemical Co. was  used  as t h e s t a n d a r d . 75,  100,  125,  of  Ginsberg  0-200 units/mL.  A s e r i e s o f s o l u t i o n s c o n t a i n i n g 25,  150,  175  and  200  units/mL  were p r e p a r e d  50, and  t h e change i n absorbance a t 4 50 nm per minute, produced when 100  microliters  of  each  LYZ  Micrococcus  solution  suspension  of  monitored.  The change i n absorbance  were  lysodeikticus  added  at  to  25°C  per minute was  a was  plotted  a g a i n s t t h e c o n c e n t r a t i o n of LYZ i n units/mL. To  a  quartz  lysodeikticus microliters pretreated in  in  containing  suspension of  (A45 nm 0  supernatant  monitored  absorbance  c o n c e n t r a t i o n o f LYZ standard curve.  2.5 =  of  from  minute  Micrococcus  was  added  cytochalasin  i n c u b a t i o n s and  over a p e r i o d per  mL  0.6-0.7) was  obtained  neutrophil/crystal  turbidity  change  cuvette  the  B  decrease  of 12 0 seconds. calculated  100  and  The the  c a l c u l a t e d u s i n g the equation f o r the  46  5.3.3. Measurement o f  A  0.05  mL  MPO  aliquot  of  cytochalasin B pretreated added  to  a  1  mL  the  supernatant  obtained  from  n e u t r o p h i l / c r y s t a l incubations  cuvette  containing  0.89  mL  was  3.2  mM  d i a n i s i d i n e s o l u t i o n i n pH 5.5 c i t r a t e b u f f e r , 0.05 mL o f 1% T r i t o n X-100, and 0.01 mL o f 10 mM H 0 2  2  and t h e change i n  absorbance a t 450 nm measured f o r 120 seconds a t 25°C. The  rate  of oxidation  proportional following  t o MPO  concentration  5.3.4.  which  i s directly  was c a l c u l a t e d  from t h e  equation:  Rate (nmol/min) = 50 X where  of d i a n i s i d i n e  A (units)  A i s t h e change i n absorbance and LYZ r e l e a s e  MPO  from n e u t r o p h i l s  on s t i m u l a t i o n  by p r o t e i n c o a t e d MSUM and CPPD c r y s t a l s Tubes c o n t a i n i n g  2.5 mg p r o t e i n c o a t e d MSUM o r 25 mg p r o t e i n  c o a t e d CPPD were p r e p a r e d and i n c u b a t e d as d e s c r i b e d except  that  incubation  times  were  45  minutes  f o r MSUM  c o n t a i n i n g t u b e s and 60 minutes f o r CPPD c o n t a i n i n g 6. S T A T I S T I C A L  Statistical sample  t  between groups. was used.  tubes.  TESTS  evaluations  test  above  were performed  o r one way  ANOVA  using  either  a two  mean  values  t o compare  A s i g n i f i c a n c e l e v e l o f p < 0.05 two t a i l e d  A l l t e s t s were done u s i n g t h e S t a t v i e w  program on  47  a  Macintosh  computer.  The  values  shown  i n t h e graphs  r e p r e s e n t t h e mean + s t a n d a r d e r r o r o f t h e mean.  48  RESULTS AND DISCUSSION 1. PREPARATION OF CRYSTALS 1.1.  MONOSODIUM URATE MONOHYDRATE (MSUM)  MSUM had e a r l i e r  been p r e p a r e d  method  by B u r t  described  procedure  involved  i n this  e t al.  dissolution  l a b o r a t o r y by t h e The  (1983).  of u r i c  acid  previous  i n distilled  water t o w h i c h sodium h y d r o x i d e  had been added, f o l l o w e d by  addition  t o adjust  o f sodium  r e a c t i o n mixture the r e s u l t i n g  hydroxide  t o 8.9.  t h e pH  of the  The p a r t i c l e s i z e d i s t r i b u t i o n o f  c r y s t a l s showed a h i g h p r o p o r t i o n w i t h  large  p a r t i c l e s i z e s (over 50% o f c r y s t a l s were 75 ^m o r g r e a t e r ) F i g u r e 4.  T h i s n e c e s s i t a t e d an a d d i t i o n a l s t e p o f s i z i n g o f  t h e c r y s t a l s i n which c e n t r i f u g a t i o n was used t o p h y s i c a l l y separate  the smaller  ( B u r t and J a c k s o n ,  size  crystals  f o r use i n e x p e r i m e n t s  1989).  T h e o r e t i c a l l y , an i n c r e a s e i n t h e degree o f s u p e r s a t u r a t i o n of  t h e sodium  force  s o l u t i o n would  for crystallization  smaller size The  urate  degree  leading  increase  the driving  t o the production  of  crystals. of supersaturation  o f monosodium u r a t e  i n the  s o l u t i o n c a n be i n c r e a s e d by e i t h e r a d d i n g more s o l u t e o r by a l t e r i n g t h e c o n d i t i o n s such t h a t t h e s o l u b i l i t y i s reduced. The  s o l u b i l i t y o f sodium u r a t e a t pH 8.9 i s g r e a t e r t h a n a t  pH 7.5. Hence, i t was f e l t t h a t a d e c r e a s e i n t h e pH o f t h e  P A R T I C L E SIZE OF M S U M C R Y S T A L S (Method of Burt and Jackson, 1983)  0  25  50  75  100  125  150  L E N G T H (MICRONS)  FIGURE 4  175  200  225  50  supersaturated significant  solution  increase  from  8.9 t o 7.5 would  cause  a  i n t h e degree o f s u p e r s a t u r a t i o n and  r e s u l t i n t h e p r o d u c t i o n o f s m a l l e r c r y s t a l s o f MSUM. The  p a r t i c l e s i z e d i s t r i b u t i o n o f MSUM c r y s t a l s o b t a i n e d by  t h e m o d i f i e d method i s shown i n F i g u r e 5. The m o d i f i c a t i o n , which  involved  alteration  crystallization,  resulted  range o f p a r t i c l e particle  size  size  o f pH o f t h e s o l u t i o n i n smaller  particle  prior to  sizes.  was between 0.1-50 /xm w i t h  o f 21 /xm and a s m a l l e r  standard  The  a mean  deviation  about t h e mean. 1.2.  CALCIUM PYROPHOSPHATE DIHYDRATE (CPPD)  1.2.1. S y n t h e s i s o f c a l c i u m d i h y d r o g e n pyrophosphate (CDPP) The  s y n t h e s i s o f CDPP was c a r r i e d  out using t h e p r e v i o u s l y  r e p o r t e d method ( B u r t and J a c k s o n , 1 9 8 7 ) . obtained  were  characterized  by X-ray  The CDPP c r y s t a l s powder  diffraction  p r i o r t o u s e i n t h e s y n t h e s i s o f CPPD ( F i g u r e 6, T a b l e 1) . In Tables  1-4, t h e term  between t h e c r y s t a l  x  d space' r e f e r s  planes while  N  t o the distance  I (rel)'  refers  to the  r e l a t i v e i n t e n s i t y o f t h e peak compared t o t h e i n t e n s i t y o f t h e s t r o n g e s t peak. was 2  CDPP  p r e v i o u s l y synthesized i n the  used f o r comparison purposes. v a l u e s o f 23.8, 25.25,  used f o r i d e n t i f i c a t i o n  lab  The d i f f r a c t i o n peaks a t  26.7, 28.0, 33.25 and 40.55 were  o f CDPP.  P A R T I C L E SIZE OF M S U M C R Y S T A L S (Modified method)  FIGURE 5  H  XRAY DIFFRACTION P A T T E R N OF C A L C I U M DIHYDROGEN P Y R O P H O S P H A T E 3000  r  2400 o CO  to  c 1800 p o o  £  1200  10  c 600  0 10  15  20  2 Theta  25  FIGURE 6  30  35  40  53 TABLE 1 X-RAY PEAKS OBTAINED FOR CDPP Sample  Identification:  Calcium dihydrogen  pyrophosphate  (CDPP) Peak  2-Theta  d space  I  (rel)  1  17.700  5.0069  7.94  2  20.050  4.4250  8.59  3  23.800  3.7356  20.24  4  26.650  3.3422  39.92  5  28.000  3.1841  100.00  6  33.200  2.6963  11.23  7  40.200  2.2415  11.43  8  40.550  2.2229  8.41  54  1.2.2.  Synthesis  of  calcium  pyrophosphate  dihydrate  (triclinic) Triclinic  crystals  o f CPPD w e r e o b t a i n e d  within a period of  24 h r t o 4 d a y s f o l l o w i n g s y n t h e s i s . The  particle  F i g u r e 7.  size  distribution  o f CPPD c r y s t a l s  The r a n g e o f p a r t i c l e  w i t h a mean p a r t i c l e  size  size  i s shown i n  was b e t w e e n 0 . 1 - 9 5 jum  o f 29 /xm.  2. CHARACTERIZATION OF CRYSTALS 2.1.  X-RAY DIFFRACTION  MSUM  and  diffraction  crystals  CPPD  patterns  diffractometer. intensities previous method  The 2  method  d  are  o f monosodium u r a t e X-ray  Figure  diffraction  values  t h e standard  and  relative  p e a k s o f MSUM p r e p a r e d (1983)  X-ray  by t h e  and t h e m o d i f i e d  shown  in  Figures  peaks a t 2  8  values  compared  and  9  o f 3.16,  t o confirm  by t h e m o d i f i e d method were  those  monohydrate. p a t t e r n o f CPPD c r y s t a l s  10 a n d t h e d a t a  peaks a t 2  t h e X-ray  Giegerflex  spacings  4 . 6 9 , 4 . 9 2 , 7.58 a n d 9.40 w e r e prepared  by  2 and 3 r e s p e c t i v e l y w h i l e t h e  The d i f f r a c t i o n  that the c r y s t a l s  to  on a R i g a k u  et a l .  i n Tables  patterns  identified  values,  of Burt  a r e given  respectively.  The  obtained  o f the d i f f r a c t i o n  diffraction  3.48,  were  given  o f 3.12,  i n Table  3.24,  p a t t e r n f o r CPPD  4.  i s shown i n  The d i f f r a c t i o n  7.07 a n d 8.1 w e r e c o m p a r e d (Joint  C o m m i t t e e o n Powder  PARTICLE SIZE DISTRIBUTION OF CPPD CRYSTALS (n = 30  100)  T  LENGTH  (MICRONS)  FIGURE  7  XRAY DIFFRACTION PATTERN OF MONOSODIUM URATE MONOHYDRATE (Burt et al., 1983) 1300  r  1170 1040 h u CO GO  \ CO  _J  c O  o oo  c  CD  5.0  7.5  10.0  12.5  15.0  17.5  2Theta  20.0  FIGURE 8  22.5  25.0  27.5  30.0  TABLE 2 X-RAY PEAKS OBTAINED FOR MSUM  (Burt e t a l . ,  1983)  Peak  2-Theta  d space  I( r e l )  1  11.650  7.5899  32.31  2  16.700  5.3044  100.00  3  17.950  4.9377  41.13  4  18.900  4.6916  45.14  5  19.550  4.5371  14.38  6  25.150  3.5381  40.43  7  25.600  3.4769  44 . 66  8  27.100  3.2877  12.64  9  27.900  3.1953  22 . 07  10  28.200  3.1620  53.19  11  29.300  3.0457  26.59  12  33.700  2.6574  44.99  XRAY DIFFRACTION PATTERN MONOSODIUM URATE MONOHYDRATE  1300  r  1170  -  1040  -  o <u  910  -  r  780  -  650  -  520  •  (coui  to \ to  >^  'to  I  390 -  2 Theta  FIGURE 9  OF  (MODIFIED  METHOD)  TABLE 3 X-RAY PEAKS OBTAINED FOR MSUM ( m o d i f i e d method) Peak  2-Theta  d space  I (rel)  1 2 3  8.300 9.400 11.650  4  16.650  10.6442 9.4009 7.5899 5.3202  22 . 65 31.86 37.45 23 .82  5 6  17.950 18.900  7 8 9 10 11 12  19.550 25.150 25.550 28.200 29.300 33.650  4.9377 4.6916 4.5371  37.01 60.54 17.57  3.5381 3.4836 3.1620 3.0457 2.6613  16.26 31.14 100.00 27 . 09 39.96  13  36.400  2.4663  26. 56  XRAY DIFFRACTION P A T T E R N OF CALCIUM PYROPHOSPHATE  1200  DIHYDRATE  r  1080 960  o  > C o  o o  840 720 600 480  c 0)  360 240 120  0 8  11  14  17  20  2 Theta  23  FIGURE 10  26  29  32  35  TABLE 4 X-RAY PEAKS OBTAINED FOR CPPD Peak  2-Theta  d space  I(rel)  1 2  11.000 12.650  11.9443 10.3915  32.84 100  3 4  16.950 19.750  7.7679 6. 6753  6.79 7 . 60  5 6 7  22.100  5.9730  8  23.050 25.750 27.550  9 10 11 12 13  28.600 29.050 30.000 32.200 33.500  5.7299 5.1377 4.8079 4.6349 4.5646 4.4232 4.1282 3.9723  13.49 7.99 10.72 43 . 03 23 . 66 19.57 13 .13 8.75 12 . 03  62  Diffraction  Standards,  1987) t o c o n f i r m  JCPDS  that  pattern)  (Burt  and J a c k s o n ,  t h e p r e p a r e d c r y s t a l s were t h o s e o f  c a l c i u m pyrophosphate d i h y d r a t e  (triclinic).  2.2. DIFFERENTIAL SCANNING CALORIMETRY (DSC) The DSC c u r v e o b t a i n e d f o r c a l c i u m pyrophosphate samples  i s shown  endothermic  i n Figure  peaks,  the f i r s t  11.  dihydrate  The p r e s e n c e  o f two  between 190 t o 285°C and t h e  second between 285 t o 300°C corresponded t o t h e l o s s o f two moles o f water from t h e c r y s t a l s at  300°C;  molecular weight  (average w e i g h t l o s s 12.3%  o f CPPD  290) c o n f i r m i n g t h e  p r e s e n c e o f t h e c a l c i u m pyrophosphate as a d i h y d r a t e . Crystals  o f monosodium  (1983) method as w e l l  u r a t e p r e p a r e d by t h e B u r t e t a l . as t h e m o d i f i e d method were a n a l y z e d  by DSC and t h e c u r v e s o b t a i n e d a r e shown i n F i g u r e s 12 and 13 r e s p e c t i v e l y . 300°C  The broad endothermic peak between 230 t o  corresponded  hydration  t o the loss  (average w e i g h t  loss  o f one mole  o f water o f  10.2%; m o l e c u l a r w e i g h t o f  MSUM 209) c o n f i r m i n g t h e monohydrate form o f t h e s e c r y s t a l s . 2.3. SCANNING ELECTRON MICROSCOPY (SEM) Figures  14-16 show  scanning electron  micrographs  o f CPPD  c r y s t a l s and MSUM c r y s t a l s p r e p a r e d by t h e method o f B u r t e t al.  (1983) and t h e m o d i f i e d method.  The micrometer  length  printed  i n t h e lower r i g h t hand c o r n e r o f t h e m i c r o g r a p h s  refers  t o the length  of the largest  crystal  i n the  1 3 U  300  DSC T H E R M O G R A M OF CPPD CRYSTALS  FIGURE 11  c  300°  DSC T H E R M O G R A M OF MSUM CRYSTALS (BURT et al., 1983)  FIGURE 12  DSC T H E R M O G R A M OF MSUM CRYSTALS (Modified method)  FIGURE 13  SCANNING E L E C T R O N MICROGRAPH OF CPPD CRYSTALS  FIGURE 14  SCANNING E L E C T R O N MICROGRAPH OF M S U M CRYSTALS (Burt etal.,  1983)  FIGURE 15  68  SCANNING  E L E C T R O N MICROGRAPH  (Modified method)  FIGURE 16  OF  MSUM  CRYSTALS  69  micrograph  .  were  t o have  found  habit  The  MSUM  crystals  the t y p i c a l  and t h e CPPD c r y s t a l s  prepared long  by  needle  the t y p i c a l  either shaped  elongated  method crystal  prismatic  habit. 3.  ESTIMATION OF NEUTROPHIL COUNT AND  Differential  counts  of  were  used  well  as t o determine  thus  obtained  release The  t o determine  of  was  LDH  prepared  than  the  purity  the c e l l  count.  to  the  neutrophil  suspension  o f t h e p r e p a r a t i o n as The  count  neutrophils lysed  neutrophil  95% pure  prepared  the %  similar  from  VIABILITY  suspensions  neutrophil estimated  with  were  i . e . >95% o f t h e c e l l s  were  by  Triton  found  count the  X-100.  t o be  more  polymorphonuclear  leukocytes. The  ability  trypan  of  blue  measure  from  of  (Babior  the  the  and  viable  the  cell  viability  Cohen,  when  viability.  Thus t h e method  to  yield  examined  cell  has of  1981).  suspension  found  neutrophil  by  to  previously isolated The this  been  the  used  dye as  a  human  neutrophils  prepared  neutrophil  method  of i s o l a t i o n  preparations of  exclude  had  >95%  cell  o f n e u t r o p h i l s was  >95%  purity  and  >95%  viability. The  ability  viability of  of  neutrophils to  of the c e l l s .  n e u t r o p h i l s on  stimuli  produce  CL  depends  Thus t h e c h e m i l u m i n e s c e n t  stimulation  with  c o u l d s e r v e a s an i n d i c a t o r  particulate  or  of the v i a b i l i t y  on  the  response soluble of the  70  cell  preparation.  Hence, t h e c h e m i l u m i n e s c e n t  response o f  n e u t r o p h i l s s e p a r a t e d from Red C r o s s b l o o d ( p r o c e s s i n g t i m e 24-48  h r ) and s t i m u l a t e d w i t h uncoated  presence  of luminol  was used  viability  of the c e l l  preparation.  MSUM  crystals i n the  as a r a p i d  test  A l l observed  of  the  responses  were compared t o t h e response o b t a i n e d when t h e n e u t r o p h i l cell  preparation  volunteers  isolated  from  blood c o l l e c t e d  from  human  ( p r o c e s s i n g t i m e , 4-5 hours) was s t i m u l a t e d w i t h  MSUM i n t h e p r e s e n c e o f l u m i n o l .  The v i a b i l i t y  of a c e l l  p r e p a r a t i o n was c o n s i d e r e d a c c e p t a b l e i f t h e CL response was greater  than  o r equal  t o 1 5 0 mV w i t h  a time  t o maximum  r e s p o n s e o f between 5-10 minutes. 4. MEASUREMENT OF NEUTROPHIL RESPONSES The w e i g h t s chosen r e p r e s e n t e d t h e w e i g h t s c o r r e s p o n d i n g t o the  maximal r e s p o n s e .  MSUM and  50  I n o u r s t u d i e s , s u r f a c e a r e a s o f 5 mg  mg CPPD were s i m i l a r  (Burt e t a l . ,  1989).  4.1. CRYSTAL STIMULATED SUPEROXIDE RELEASE FROM NEUTROPHILS F i g u r e 17 shows t h e t y p i c a l t i m e c o u r s e o f s u p e r o x i d e a n i o n g e n e r a t i o n by n e u t r o p h i l s s t i m u l a t e d w i t h uncoated MSUM and CPPD c r y s t a l s .  There was a r a p i d  l e v e l s up t o about of  20  minutes f o r  increase  MSUM  i n superoxide  t o r e a c h a peak v a l u e  about 15 nmoles 0 2 ~ g e n e r a t e d p e r 1 0 E 6 n e u t r o p h i l s / m L f o r  MSUM.  The response o f t h e n e u t r o p h i l s t o s t i m u l a t i o n  CPPD c r y s t a l s with  was s l o w e r t h a n t h e response  MSUM c r y s t a l s  though  t h e magnitude  with  t o stimulation  o f t h e responses  S U P E R O X I D E G E N E R A T I O N B Y N E U T R O P H I L S STIMULATED B Y U N C O A T E D M S U M AND C P P D C R Y S T A L S 20  T  TIME  (mm)  FIGURE 17  72  were  not d i f f e r e n t .  were  attained  neutrophils  The maximum  i n about and  neutrophils/mL.  superoxide  60 minutes  were  about  levels  f o r CPPD s t i m u l a t e d  14  C o n t r o l s were  anion  nmoles  about  per  10E6  50% o f t h e maximum  values f o r superoxide release.  F i g u r e s 18 and 19 show t h e  graphs  r e l e a s e when  obtained  f o r superoxide  data  e x p e r i m e n t s each f o r MSUM and CPPD were p o o l e d . al.,  (1991) a l s o found t h a t MSUM c r y s t a l s  from  4  Naccache e t  (3 mg/mL) produced  a more r a p i d g e n e r a t i o n o f s u p e r o x i d e than CPPD c r y s t a l s (3 mg/mL).  The amounts o f s u p e r o x i d e  within  10 min f o r b o t h  nmoles  superoxide  crystals,  crystals  produced  a t about  were  maximal  9 nmoles and 5  p e r 10E6 n e u t r o p h i l s f o r MSUM and CPPD  respectively.  Terkeltaub  e t a l . (1984, 1988)  e x p r e s s e d 0 ~ g e n e r a t i o n i n terms o f nmoles f e r r i c y t o c h r o m e 2  c reduced. MSUM  and CPPD,  nmoles MSUM  At crystal  and 2.4 nmoles  superoxide incubation  values  release medium  oxidase  of  Other  have to  Rosen e t a l . , 1984).  cytochalasin  c  reduced  for  p e r 10E6  cytochalasin  prevent  t h e membrane  B  bound  NADPH  generation  1986; Abramson e t a l . ,  t o the incubation  in  phagocytosis  medium  the and  dependent  (Nagase e t a l . , 1982; Higson e t  We have s t u d i e d t h e e f f e c t o f a d d i n g B  3.2-5.4  studies of crystal-induced  included  i n v o l v e d i n superoxide  o f between  p e r 10E6 n e u t r o p h i l s  ferricytochrome  f o r CPPD.  internalization  al.,  reported  f e r r i c y t o c h r o m e c reduced  neutrophils  1987;  they  c o n c e n t r a t i o n s o f 5 mg/mL f o r b o t h  on  r e l e a s e and t h e r e s u l t s a r e shown i n F i g u r e 20.  10 /xg/mL superoxide  There was a  S U P E R O X I D E  G E N E R A T I O N  I N D U C E D  B Y  M S U M  C R Y S T A L S  (n = 4) 20  T  0  5  10  15  TIME  (min)  FIGURE 18  20  25  30  SUPEROXIDE GENERATION INDUCED BY CPPD CRYSTALS (n =  4)  30 x  FIGURE 19  E F F E C T OF C Y T O C H A L A S I N B ON M S U M INDUCED S U P E R O X I D E G E N E R A T I O N  FIGURE 20  76  significant by  reduction  MSUM c r y s t a l s  i n t h e superoxide  (p < 0.05).  i n h i b i t phagocytosis  burst  r e l e a s e and CL.  Simchowitz  i n h i b i t i o n o f MSUM induced that  B i s known t o  Hence i n h i b i t i o n  may r e s u l t i n i n h i b i t i o n o f t h e r e s p i r a t o r y  as w e l l .  suggested  Cytochalasin  induced  which i s accompanied by t h e r e s p i r a t o r y  burst i n v o l v i n g superoxide of p h a g o c y t o s i s  generation  e t a l . (1982)  reported the  oxygen uptake by c y t o c h a l a s i n and  the stimulation of the r e s p i r a t o r y  burst  c o u l d be dependent on p h a g o c y t o s i s . 4 . 1 . 1 . EFFECT OF SOD ON SUPEROXIDE RELEASE The  use o f ferricytochrome  superoxide  c t o monitor  the release of  i s based on t h e r e d u c t i o n o f f e r r i c y t o c h r o m e c t o  ferrocytochrome  c.  This  reduction  can be e f f e c t e d by  s e v e r a l agents b u t o n l y t h e r e d u c t i o n o f f e r r i superoxide  c a n be i n h i b i t e d by t h e enzyme SOD.  t h a t t h e reduction being due  t o t h e superoxide  ferricytochrome enzyme  SOD.  superoxide  t o f e r r o by  generated  was i n c u b a t e d  being  which  the reduction of  i n t h e presence  a r e expressed  c reduced. with  i n t h e a s s a y was indeed  generated,  c was m o n i t o r e d Results  ferricytochrome  measured  To c o n f i r m  i n terms  represents  the  of the  o f nmoles nmoles  The r e s u l t s o b t a i n e d  of  when CPPD  n e u t r o p h i l s i n t h e p r e s e n c e o f SOD a r e  shown i n F i g u r e 21.  The r e d u c t i o n o f f e r r i c y t o c h r o m e  superoxide  generated  by CPPD  suppressed  i n t h e presence  reduction of ferricytochrome  stimulated o f SOD  c by  n e u t r o p h i l s was  indicating  c measured when CPPD  that the crystals  E F F E C T OF SOD ON C P P D I N D U C E D S U P E R O X I D E R E L E A S E F R O M  20  N E U T R O P H I L S  T  16-Q <D 00  <D  12--  O CONTROLS • 50 mg CPPD A CPPD + SOD  O CD  a  8--  CO CO  6  4-  G  0  0  60  120  180  240  TIME IN MINUTES  FIGURE 21  «0  78  were i n c u b a t e d  w i t h n e u t r o p h i l s was due t o t h e p r e s e n c e o f  superoxide. The  results  obtained  when  MSUM  was  incubated  with  n e u t r o p h i l s i n t h e presence o f SOD a r e shown i n F i g u r e 2 2 . The  r e d u c t i o n o f f e r r i c y t o c h r o m e c by s u p e r o x i d e  produced by  MSUM s t i m u l a t e d n e u t r o p h i l s was n o t s i g n i f i c a n t l y s u p p r e s s e d i n t h e p r e s e n c e o f SOD.  T h i s c o u l d be caused e i t h e r by t h e  reduction  of ferricytochrome  reducing  species  inactivation protein was  SOD  o f SOD  than  driven  superoxide,  f o r example  by  another  or  by t h e  by a d s o r p t i o n  of t h i s  To d e t e r m i n e whether SOD  adsorbed t o t h e MSUM c r y s t a l s u r f a c e and t h e r e b y  inactivated, with  MSUM  incubations. Figure  being  onto t h e MSUM c r y s t a l s .  being  being  other  c  23.  crystals  was  The l e v e l s  from  added  The r e s u l t s o f t h e s e  CPPD/neutrophil supernatants  t h e supernatant  to  CPPD/neutrophil  i n c u b a t i o n s a r e shown i n  o f reduced  incubations  the incubation of  ferricytochrome  to  which  the  c  for  MSUM/SOD  had been added were n o t d e c r e a s e d and i n f a c t  were g r e a t e r t h a n f o r C P P D / n e u t r o p h i l l i k e l y t h e SOD had been i n a c t i v a t e d ,  incubations. probably  I t was  by a d s o r p t i o n  o n t o t h e s u r f a c e o f uncoated MSUM c r y s t a l s . The  SOD i n h i b i t a b l e r e d u c t i o n o f f e r r i c y t o c h r o m e c a s s a y has  been w i d e l y  used  anion  neutrophils  by  (Terkeltaub al.,  1986;  t o monitor  the generation  incubated  e t a l . , 1984; Naccache e t a l . ,  with  of  superoxide  MSUM  crystals  Abramson e t a l . , 1 9 8 2 ; 1991).  Rosen e t  However t h e i n a b i l i t y o f  S U P E R O X I D E PRODUCTION B Y M S U M STIMULATED N E U T R O P H I L S IN T H E P R E S E N C E OF S U P E R O X I D E D I S M U T A S E  FIGURE  22  E F F E C T OF M S U M T R E A T E D SOD ON C P P D I N D U C E D S U P E R O X I D E RELEASE FROM NEUTROPHILS 5  CU +->  T  4  co cu  c  cu  ciO  3~  V, 'x o u cu  a  2~  00 CO QJ  o  £  0  4  0  5  1: CPPD 50 m g / m L ; 2: CPPD 50 m g / m L + SOD 300 U / m L ; 3: CPPD 50 m g / m L +MSUM TREAT  FIGURE 23  81  SOD  to inhibit  superoxide not  generated  been p r e v i o u s l y  buffers  used  inactivation  of ferricytochrome  the reduction from  MSUM s t i m u l a t e d  reported.  i n previous  c by  n e u t r o p h i l s has  The presence o f BSA i n t h e  studies  may have  o f SOD due t o p r e f e r e n t i a l  p r e v e n t e d any  adsorption  o f BSA  onto t h e MSUM c r y s t a l s u r f a c e t h u s i n h i b i t i n g any subsequent adsorption  o f SOD onto t h e MSUM c r y s t a l s .  4.1.2. EFFECT OF PROTEINS ON SUPEROXIDE RELEASE  The  effect  of protein  superoxide generation  coating  on t h e MSUM c r y s t a l - i n d u c e d  by n e u t r o p h i l s i s shown i n F i g u r e 24.  There were no s i g n i f i c a n t d i f f e r e n c e s (p < 0.05) superoxide released or  plasma  that  precoating  enhanced al.,  protein  MSUM  1982;  by uncoated MSUM compared t o I g G, BSA coated  MSUM.  Other  o f MSUM c r y s t a l s induced  Rosen  between t h e  superoxide  with  studies Ig G  generation  e t a l . , 1986; Nagase  However t h e s u p e r o x i d e r e l e a s e  have  significantly (Abramson e t  e t a l . , 1989).  from n e u t r o p h i l s  i n d u c e d by  u n c o a t e d MSUM i n o u r work was a p p r o x i m a t e l y 3 f o l d than i n the previous assay c o n d i t i o n s  studies.  shown  greater  I t i s p o s s i b l e t h a t under t h e  used i n o u r s t u d i e s , t h e n e u t r o p h i l s  were  m a x i m a l l y s t i m u l a t e d by t h e uncoated MSUM c r y s t a l s such t h a t t h e c o a t i n g o f t h e c r y s t a l s w i t h I g G c o u l d n o t enhance t h e generation Abramson plasma  o f s u p e r o x i d e from the n e u t r o p h i l s . et a l .  did  (1982)  not  showed  affect  that  precoating  superoxide  MSUM  generation  with from  E F F E C T OF P R O T E I N COATING ON THE G E N E R A T I O N OF INDUCED B Y M S U M  FIGURE 24  SUPEROXIDE  83  neutrophils, precoating  whereas MSUM w i t h  induced superoxide Figure  25  Terkeltaub  shows  plasma  et a l .  found  that  inhibited  MSUM  (1984)  significantly  generation. the effect  of protein  o f superoxide  crystals.  There were no s i g n i f i c a n t d i f f e r e n c e s (p < 0.05)  to  by i n c u b a t i o n  on t h e  generation  i n t h e superoxide  induced  coating  with  CPPD  r e l e a s e induced by uncoated CPPD compared  I g G o r plasma  protein  coated  CPPD.  Again,  i t  is  p o s s i b l e t h a t n e u t r o p h i l s were m a x i m a l l y s t i m u l a t e d by both u n c o a t e d CPPD and I g G-coated CPPD. found  no s i g n i f i c a n t  enhancement  Nagase e t a l . (1989)  of superoxide  when CPPD c r y s t a l s were c o a t e d w i t h I g G.  generation  To o u r knowledge,  t h e r e a r e no o t h e r r e p o r t s on t h e e f f e c t o f p r o t e i n c o a t i n g s on CPPD c r y s t a l - i n d u c e d s u p e r o x i d e  generation.  A l t h o u g h no d i f f e r e n c e s i n s u p e r o x i d e f o r p r o t e i n coated  r e l e a s e were observed  MSUM and CPPD v e r s u s  uncoated  crystals,  a l l t h e s t u d i e s were c a r r i e d o u t a t an i n c u b a t i o n t i m e o f 30 min  f o r MSUM and 60 min f o r CPPD.  I ti s possible that  may be d i f f e r e n c e s i n t h e r a t e s o f s u p e r o x i d e neutrophils  stimulated  by p r o t e i n  coated  there  generation f o r versus  uncoated  crystals. 4.2.  CRYSTAL  STIMULATED  CHEMILUMINESCENT  RESPONSE  OF  NEUTROPHILS  The by  c h e m i l u m i n e s c e n t response o f n e u t r o p h i l s to.. s t i m u l a t i o n 5 mg o f uncoated MSUM c r y s t a l s  i s shown i n F i g u r e 26.  E F F E C T OF P R O T E I N COATING ON S U P E R O X I D E G E N E R A T I O N INDUCED BY CPPD CRYSTALS  40  T  CON  CPPD  BSA COATED  FIGURE  IGG COATED  25  PLASMA COATED  co  CHEMILUMINESCENCE GENERATED BY NEUTROPHILS STIMULATED BY 5 mg UNCOATED MSUM 2100 o  o  > o  o z o  o  1400  in O  O 5 m g MSUM  700 + \  o\ *0-  0  0  °-o--o~o , _ _ _ . r 0  0  500  1000 1500 TIME IN SECONDS F I G U R E 26  2000  n  n  0  0  2500 03  86  The b e l l crystal the  shaped  response  c u r v e was a f f e c t e d by changes i n  c o n c e n t r a t i o n as seen  data  expressed  i n terms  i n F i g u r e 27.  T a b l e 5 shows  o f t h e area under  t h e curve  (AUC) o f t h e p l o t s o f CL response i n mV v e r s u s t i m e a t each crystal  concentration.  concentration maximum  from  response  elicit  An  increase  i n the  crystal  2 mg t o 5 mg/mL r e s u l t e d i n a  and a d e c r e a s e  t h e maximal r e s p o n s e .  i n t h e time  higher  required t o  However f u r t h e r i n c r e a s e s i n  MSUM c r y s t a l c o n c e n t r a t i o n t o 10 mg/mL d i d n o t s i g n i f i c a n t l y enhance This  t h e response  could  light  observed  compared t o 5 mg/mL MSUM.  be due t o i n t e r f e r e n c e i n measurement due t o  scattering  concentrations  by t h e c r y s t a l s  a t the higher  o r due t o t h e l i m i t e d m e t a b o l i c  the i s o l a t e d n e u t r o p h i l .  crystal  capacity of  The CL response o f t h e n e u t r o p h i l s  i n c u b a t e d w i t h 5 mg/mL MSUM may be near t h e maximum p o s s i b l e thus  allowing  only  a  small  increase  when  the crystal  of neutrophils  t o 50 mg/mL  c o n c e n t r a t i o n i s doubled t o 10 mg/mL. The  chemiluminescent  response  CPPD c r y s t a l s  i s shown i n F i g u r e  concentration  on  neutrophil  CL  28.  The e f f e c t  was  studied  over t h e  c o n c e n t r a t i o n range o f 10-50 mg/mL and t h e r e s u l t s are  shown i n F i g u r e  influenced produced  29 and T a b l e 6.  by t h e CPPD  increased  crystal  o f CPPD  obtained  The CL g e n e r a t e d was  concentration.  The CL  as t h e CPPD c o n c e n t r a t i o n was i n c r e a s e d  from 10 mg/mL t o 30 mg/mL b u t f u r t h e r i n c r e a s e s i n t h e CPPD concentration  t o 50 mg/mL r e s u l t e d i n a r e d u c t i o n  of the  EFFECT OF MSUM CONCENTRATION ON CHEMILUMINESCENCE  2700  T  TIME IN SECONDS  FIGURE 27  TABLE  EFFECT  CRYSTAL  5  O F MSUM C R Y S T A L C O N C E N T R A T I O N  CONC.  (mg/ltlL)  ON  AUC  CHEMILUMINESCENCE  1  (V-s)  2  656 (629 - 682)  5  1192 (1127 - 1256)  10  1140 (1139 - 1268)  Note : 1 - AUC i s t h e a r e a under t h e c u r v e f o r p l o t s o f CL r e s p o n s e i n mV v e r s u s t i m e .  Values represent the  mean o f two d e t e r m i n a t i o n s w i t h t h e range i n parentheses.  89  PQ Q  W  a.  <  O 0/)  •  o  •  EH  • •  GO  •  GO  • I  OH Q O fe  P ^  fe  O  W  fe o  P o  ° ^  •  •  •  • /  4-  9co co  Pi—i  •  tlfl  E-  •  o  O  o  LO  CO  fe  O CO fe  P  fe  p  d o o  o o  CO  o o  o o  w p  a  I—I  p;  P  CD CO  I  < P  fe  oa  •  ^IP PQ < P^  Cv2  • /  Q  Q fe  o o  • • • • •  S E-<  o o cv  o  o  1  E F F E C T OF C P P D CONCENTRATION ON N E U T R O P H I L C H E M I L U M I N E S C E N C E  400  T  300 +  o o co  A •  200 +  0  / \  \  T<$  •  O 10 20 • A 30 • x 40 • a50  me mg mg mg mg  CPPD CPPD CPPD CPPD CPPD  P 100 PI  o 1600 TIME IN SECONDS  FIGURE 29  2400  3200  VD O  91  TABLE 6 E F F E C T OF CPPD CRYSTAL CONCENTRATION ON CHEMILUMINESCENCE  CRYSTAL  AUC  CONC.  1  (V.s)  (mg/mL)  10  62 (50 - 73)  20  57 (56 - 58)  30  79 (74  40  - 82) 63  (55 - 70) 50  55 (47 - 62)  Note  : 1 - AUC i s t h e a r e a u n d e r t h e c u r v e response  i n mV v e r s u s t i m e .  f o rplots  Values  o f CL  represent the  mean o f two d e t e r m i n a t i o n s w i t h t h e r a n g e i n parentheses.  92  t o t a l amount o f CL g e n e r a t e d . CL w i t h  A s i m i l a r trend of increased  i n c r e a s i n g CPPD c o n c e n t r a t i o n s  upto  30 mg/mL was  o b s e r v e d a t 25°C.  Though t h e number o f c r y s t a l - n e u t r o p h i l  interactions  be e x p e c t e d  would  concentration,  t h e accompanying  to increase increase  with  crystal  i n scatter  could  r e d u c e t h e amount o f l i g h t r e a c h i n g t h e p h o t o m u l t i p l i e r tube thus decreasing affect  t h e CL d e t e c t e d .  Another f a c t o r w h i c h may  t h e CL r e s p o n s e o f n e u t r o p h i l s t o CPPD c r y s t a l s i s  t h e tendency o f t h e c r y s t a l s t o s e t t l e r a p i d l y even the  time  of  concentrations  measurement thus  crystal-neutrophil  (15  decreasing  sec) a t t h e number  interactions.  The  during  high  crystal  of  possible  neutrophil  c h e m i l u m i n e s c e n t r e s p o n s e appears t o be maximal a t a CPPD c o n c e n t r a t i o n o f 30 mg/mL.  T h i s CPPD c o n c e n t r a t i o n was used  f o r a l l f u r t h e r CL e x p e r i m e n t s w i t h CPPD. To o u r knowledge t h e r e a r e no o t h e r r e p o r t s o f t h e e f f e c t o f MSUM and CPPD c r y s t a l c o n c e n t r a t i o n s on t h e CL g e n e r a t e d by neutrophils. We have s t u d i e d t h e e f f e c t o f adding 10 jug/mL c y t o c h a l a s i n B t o t h e i n c u b a t i o n medium on t h e CL produced. significant  (p < 0.05) r e d u c t i o n i n t h e CL produced i n t h e  p r e s e n c e o f c y t o c h a l a s i n B. the  The AUC f o r MSUM induced  CL i n  absence o f c y t o c h a l a s i n B was 588 + 108 V.s (mean o f  three determinations) the  There was a  w h i l e t h e AUC f o r MSUM induced  CL i n  p r e s e n c e o f c y t o c h a l a s i n B was 5.46 + 3 V.s (mean o f  three  determinations).  Hence c y t o c h a l a s i n B was found t o  93  inhibit  the generation  neutrophils.  This  phagocytosis inhibition et  by  of superoxide may  be  cytochalasin  due B  and CL p r o d u c t i o n  to  which  the  inhibition  may  result  o f t h e accompanying r e s p i r a t o r y b u r s t  by of  i n the  (Simchowitz  a l . , 1982) .  4.2.1. EFFECT OF TEMPERATURE ON THE CL RESPONSE The  CL  induced  generated  with  neutrophils  i n response  s t i m u l a t i o n was d e p e n d e n t  suspending for  by  medium  MSUM  obtained  crystals  when  a t 25°C  Table  7 gives  neutrophils  a n d 37 °C.  neutrophils  enhanced 37°  oxidative  compared  maintained results and  were m a i n t a i n e d metabolic  t o that  at  37 °C  Topley  incubated  The i n c r e a s e  i n AUC  e n h a n c e d when  a t 37°C p r e s u m a b l y due t o a n  activity  a t 25°.  Hence  f o r a l l further  a r e i n good  of the t h e AUC  were  r e v e a l s t h a t t h e CL r e s p o n s e was s i g n i f i c a n t l y the  crystal-  on t h e t e m p e r a t u r e  f o rneutrophils.  t h e CL c u r v e s  to  agreement w i t h  of the neutrophils a t the neutrophils experiments.  those reported  were These  by H a r b e r  (1986).  4.2.2. EFFECT OF PROTEINS ON THE CHEMILUMINESCENT RESPONSE Figures  3 0 a n d 31 show t h e t y p i c a l  neutrophils  were  respectively.  exposed I t was  CL c u r v e s  obtained  to protein  coated  MSUM  not possible  to  measure  a n d CPPD t h e CL  g e n e r a t e d by a c o n t r o l s e t o f t u b e s s i n c e t h e CL v a l u e s extremely  low  and  chemiluminometer.  below  the  detection  The AUCs o f t h e p l o t s  limits  when  were  of the  o f CL r e s p o n s e i n  94  TABLE 7 EFFECT OF SAMPLE TEMPERATURE ON MSUM INDUCED CHEMILUMINESCENCE AUC  1  SAMPLE TEMPERATURE 25°C  37°C  MSUM (mg/mL) 2  443 (414 - 470)  5  787 (686 - 886)  10  967 (877 - 1056)  656 (629 - 682) 1191 (1127-1257) 1198 (1139-1268)  Note : 1 - AUC i s t h e a r e a under t h e c u r v e f o r p l o t s o f CL r e s p o n s e i n mV v e r s u s t i m e .  Values represent the  mean o f two d e t e r m i n a t i o n s w i t h t h e range i n parentheses.  E F F E C T OF P R O T E I N COATING ON C H E M I L U M I N E S C E N C E R E S P O N S E  2000  TO MSUM  T  >  Q PO PQ O P  1500 + O UNCOATED MSUM • BSA COATED MSUM A Ig G COATED MSUM • A PLASMA COATED MSUM  <X  p  S p  1000  CJ GO  p p  p  PP  500 +  PI  CJ  1000  1500  TIME (sec)  FIGURE 30  VO  E F F E C T OF P R O T E I N COATING ON T H E C H E M I L U M I N E S C E N C E R E S P O N S E TO  350  C P P D  T  TIME  (sec)  FIGURE 31  VO  97  mV v e r s u s  time  f o r protein  g i v e n i n T a b l e 8. CL  response  I g G which  and uncoated  when  the  crystals  coated  with  T h i s may be due t o t h e o p s o n i z i n g  effect  facilitates  were  an i n t e r a c t i o n  between  p o r t i o n o f I g G on t h e c r y s t a l s u r f a c e and t h e F p r e s e n t on t h e n e u t r o p h i l membrane. the data  MSUM a r e  There was s i g n i f i c a n t enhancement o f t h e  immunoglobulin g. of  coated  revealed  c  the F  receptors  S t a t i s t i c a l analysis of  t h a t t h e r e was a s i g n i f i c a n t  suppression  of t h e n e u t r o p h i l CL response when t h e MSUM c r y s t a l s precoated reported the  with  plasma p r o t e i n s .  t h a t plasma p r e c o a t i n g  CL g e n e r a t e d  density  by >50%.  lipoprotein  Terkeltaub  suggested  were  e t a l . (1984)  o f MSUM c r y s t a l s  They  c  inhibited  that  the  low  f r a c t i o n bound t o MSUM from plasma was  r e s p o n s i b l e f o r a major p o r t i o n o f t h e plasma i n h i b i t i o n o f neutrophil observation the of  CL r e s p o n s e s t o MSUM. because d u r i n g  This  i s an i n t e r e s t i n g  a gout a t t a c k ,  t h e synovium o f  j o i n t becomes more permeable and s y n o v i a l f l u i d large  molecules  such  as  lipoproteins  levels  increases  ( T e r k e l t a u b e t a l . , 1986). The  e f f e c t o f p r o t e i n c o a t i n g on CL induced  by CPPD c r y s t a l s  f o l l o w e d a s i m i l a r p a t t e r n as f o r MSUM c r y s t a l s .  The AUCs  of CL v e r s u s t i m e p l o t s f o r p r o t e i n coated and uncoated CPPD are  shown i n T a b l e  plasma was found  8.  Precoating  t o suppress  o f CPPD c r y s t a l s  with  t h e CL response though  this  s u p p r e s s i o n was n o t found t o be s t a t i s t i c a l l y s i g n i f i c a n t (p < 0.05) f o r CPPD.  The enhancement i n t h e CL g e n e r a t e d when  TABLE 8  EFFECT  OF P R O T E I N  C O A T E D AND UNCOATED  MSUM AND C P P D ON  CHEMILUMINESCENCE  AUC  1  (V.S)  MSUM  CPPD  5 mg/mL  3 0 mg/mL  Uncoated c r y s t a l s  306 + 85  38  IgG  coated  570  103  BSA  coated  97  + 34  20  + 10  Plasma coated  + 97  (*•)  + 8  + 22 (*)  32  + 9  17  + 6  Note : 1 - A U C i s t h e area under t h e curve f o r p l o t s o f C L response i n mV versus time. mean error  of 9  determinations  and 7 d e t e r m i n a t i o n s  error. * s i g n i f i c a n t a t p < 0.05  Values r e p r e s e n t t h e f o r MSUM f o r CPPD  +  standard  +  standard  99  crystals  were  significant Ig  precoated  (p < 0.05).  G enhanced  the F  with  Ig  Again,  G  was  i t i s likely  r e c e p t o r mediated  c  statistically t h a t adsorbed  crystal-neutrophil  a c t i v a t i o n and CL g e n e r a t i o n . The  slight  reduction  i n CL  observed  coated w i t h BSA was not s t a t i s t i c a l l y for  when  crystals  significant  were  (p < 0.05)  e i t h e r MSUM or CPPD c r y s t a l s .  4.3.  CRYSTAL  STIMULATED  DEGRANULATION  RESPONSE  OF  NEUTROPHILS Degranulation primary  refers  t o the r e l e a s e of the contents  and a z u r o p h i l i c  granules  into  t h e phagosome formed  into  the e x t r a c e l l u l a r  plasma  membrane  primary  and a z u r o p h i l i c  several  enzymes,  collagenase, Dewald,  during  phagocytosis granules them,  acid  species and  i n action.  or  directly  invagination of the i s complete.  The  of the n e u t r o p h i l c o n t a i n  MPO,  Some of these  of t o x i c  hypochlorous proteolytic  among  phagocytosis  LYZ, alpha  g e l a t i n a s e and ft g l u c u r o n i d a s e  1984).  generation  before  of the n e u t r o p h i l e i t h e r  after  medium  o f the  mannosidase,  ( B a g g i o l i n i and  enzymes a r e i n v o l v e d i n the  such  as the h y d r o x y l  chloramines  while  The r e l e a s e of these  radical,  others lytic  are  contents  i n t o t h e e x t r a c e l l u l a r f l u i d e i t h e r as a consequence o f c e l l death  or  during  the  process  of  phagocytosis  d e l e t e r i o u s e f f e c t s on the surrounding t i s s u e s . study  the  kinetics  of  degranulation  can  have  In order t o  following  crystal  100  stimulation that  i t i s necessary  phagocytosis  inhibited  and  the  neutrophil  internalization  of  the  crystal  is  occurs  into  the  medium.  that  degranulation  degranulation The  inhibit  cytochalasins  phagocytosis  but  (Weissmann e t a l . , 1972).  were t r e a t e d w i t h the  alter  complete  extracellular compounds  or  to  B  a  do  not  Hence  group  of  inhibit  neutrophils  cytochalasin B p r i o r to stimulation  particulate  Cytochalasin  are  such  stimuli  has  human n e u t r o p h i l s  been  to  study  previously  Abramson e t a l . , 1982;  degranulation.  used  (Nagase e t a l . , 1987;  with  in  studies  with  Rosen e t a l . , 1986;  Higson e t a l . , 1984).  However, i t i s  i m p o r t a n t t o note t h a t t h i s a l t e r a t i o n of t h e a b i l i t y of neutrophil other  to  phagocytose  functions  of  the  s t u d i e s have r e v e a l e d stimulate al.,  cell  as  result well.  in  Furthermore  t h a t c y t o c h a l a s i n B has  degranulation  (Aaku  et  a l t e r a t i o n of  a l . , 1990;  some  a tendency t o Hoffstein  et  1980).  4.3.1.  The  could  the  CRYSTAL STIMULATED MPO  determination  of MPO  RELEASE FROM NEUTROPHILS  r e l e a s e d from n e u t r o p h i l s was  on t h e MPO  c a t a l y z e d o x i d a t i o n of o - d i a n i s i d i n e by  peroxide.  Increasing  Triton  X-100  lysed  amounts of  neutrophil  based  hydrogen  supernatant obtained  from  s u s p e n s i o n s were added t o  a  dianisidine  r e a c t i o n mixture t o determine the r e l a t i o n s h i p  between t h e  volume of  absorbance produced. The  r e l a t i o n s h i p was  s u p e r n a t a n t added and The  r e s u l t s are  found t o be  the  change i n  shown i n F i g u r e  32.  l i n e a r w i t h i n the range of  E F F E C T OF VARIOUS CONCENTRATIONS OF S U P E R N A T A N T ON T H E M P O A S S A Y (AT T = 60 s e c )  1.200  Y = 1.0616E-2 X + 8.6888E-2  T  r = 0.9722 1.000 + 0.800 + 0.600 + 0.400 0.200 0.000 0  10  '  20  30  40  50  60  V O L U M E OF S U P E R N A T A N T ADDED (uL)  FIGURE 32  70  102  30  t o 70 JXL, o f s u p e r n a t a n t  added.  Hence  50 fih o f t h e  s u p e r n a t a n t were used i n a l l f u r t h e r d e t e r m i n a t i o n s . method  of analysis  gives  the rate  of oxidation  This of o -  d i a n i s i d i n e and t h i s r a t e has been shown t o be p r o p o r t i o n a l to  the concentration  1981).  o f t h e enzyme MPO ( B a b i o r  and Cohen,  The r a t e o f appearance o f MPO i n t h e s u p e r n a t a n t was  calculated  from t h e d i f f e r e n c e i n t h e r a t e o f o x i d a t i o n a t  various time i n t e r v a l s . The  time  course  cytochalasin  f o r the release  B pretreated  o f MPO  neutrophils  were  u n c o a t e d CPPD c r y s t a l s i s shown i n F i g u r e  obtained  when  incubated  with  33.  The maximum  amount o f MPO p r e s e n t i n t h e s u p e r n a t a n t was between 10-45 min  and t h e MPO l e v e l s observed on i n c u b a t i o n w i t h CPPD were  s i g n i f i c a n t l y higher for  the release  neutrophils shown  were  i n Figure  than c o n t r o l v a l u e s .  o f MPO  when  incubated  with  34.  The l e v e l  The time  cytochalasin uncoated  o f MPO i n t h e s u p e r n a t a n t s  from  t h e MSUM/neutrophil  than that  f o r t h e c o n t r o l s e t of experiments.  considered  a likely  been p r e v i o u s l y (Terkeltaub al.,  supernatant  possibility  reported  In from  order  was lower This  since  raised  removed from t h e  onto t h e MSUM c r y s t a l s .  T h i s was  MSUM c r y s t a l s have  t o adsorb many d i f f e r e n t p r o t e i n s  e t a l . , 1983; K o z i n  1979).  incubations  t h a t t h e enzyme was b e i n g  s u p e r n a t a n t by a d s o r p t i o n  pretreated  MSUM c r y s t a l s i s  resulting  the p o s s i b i l i t y  B  course  to  neutrophils  and McCarty, 1976; K o z i n e t test  lysed  this with  hypothesis Triton  the  X-100 10%  M Y E L O P E R O X I D A S E R E L E A S E F R O M CYTOCHALASIN B P R E T R E A T E D ON STIMULATION WITH UNCOATED C P P D C R Y S T A L S  FIGURE  33  NEUTRO  MYELOPEROXIDASE RELEASE FROM CYTOCHALASIN B PRETREATED ON STIMULATION WITH UNCOATED M S U M C R Y S T A L S  2.500  T  TIME ( m i n )  FIGURE 3 4  NEUTROPHILS  105  solution  was  incubated  concentrations increasing  with  MSUM  from 0.1 mg/mL t o 10 mg/mL.  t h e amount o f  MPO a c t i v i t y  increasing  The e f f e c t o f  added on t h e % r e d u c t i o n i n  MSUM  can be seen i n F i g u r e  of MSUM r e s u l t e d  crystal  35.  Increasing  i n s i g n i f i c a n t reduction  amounts  o f MPO a c t i v i t y .  Hence i t i s l i k e l y t h a t MPO was b e i n g adsorbed onto t h e MSUM crystal  surface  from  appeared t o t a k e proteins Terkeltaub  t h e supernatant.  place  released  by  et a l . ,  Adsorption  o f MPO  even i n t h e p r e s e n c e o f many degranulation  (1991)  studied  and  cell  other death.  the degranulation  of  n e u t r o p h i l s i n d u c e d by MSUM b u t t h e y s t u d i e d t h e r e l e a s e o f a l p h a mannosidase. 4.3.2.  The  EFFECT  OF PROTEINS ON MPO  RELEASE  e f f e c t o f p r o t e i n c o a t i n g o f t h e c r y s t a l s on t h e r e l e a s e  o f MPO from c y t o c h a l a s i n B p r e t r e a t e d n e u t r o p h i l s s t i m u l a t e d with CPPD  CPPD c r y s t a l s crystals  significantly the  i s shown i n F i g u r e  with  immunoglobulin  enhance t h e r e l e a s e  interaction  with  neutrophil surface.  the F  c  36. g  Precoating of  was  found  to  o f MPO, p o s s i b l y t h r o u g h  receptors  present  on t h e  P r e c o a t i n g w i t h plasma and BSA d i d n o t  have a s i g n i f i c a n t e f f e c t on t h e r e l e a s e o f MPO induced by CPPD c r y s t a l s . As  seen i n F i g u r e  MSUM  was l e s s  crystals  with  3 7 , t h e MPO r e l e a s e  than  control  I g G,  BSA  samples o r plasma  induced by uncoated and p r e c o a t i n g proteins  MSUM  produced  E F F E C T OF ADDITION OF INCREASING AMOUNTS OF M S U M ON M Y E L O P E R O X I D A S E ACTIVITY  M Y E L O P E R O X I D A S E R E L E A S E F R O M C Y T O C H A L A S I N B P R E T R E A T E D NEUTROPHILS ON STIMULATION WITH PROTEIN COATED C P P D (n = 6)  FIGURE 36  o  MYELOPEROXIDASE RELEASE FROM CYTOCHALASIN B PRETREATED ON STIMULATION WITH P R O T E I N COATED M S U M  FIGURE  37  NEUTROPHILS  §  109  significantly  lower  0.05).  Hence,  protein  coat  adsorption  MPO  i t seems on  o f MPO  the  possible  of  o f MPO  were  was  t o study  during  degranulation,  4.3.3.  CRYSTAL STIMULATED  Figure  38 shows t h e s t a n d a r d in  suspension was  added  The  concentration  time  shown  in  between This by  Figure  represented  values  of  when  was  with  MSUM  further  surface  i t was  a s an i n d i c a t o r B  pretreated  crystals.  Hence i t  of another  enzyme  released  NEUTROPHILS  curve obtained a  standard  found  by m e a s u r i n g t h e  Micrococcus  to  f o r the release  neutrophils  with  o f MPO  LYZ RELEASE FROM  lysodeikticus  egg w h i t e from be  500  LYZ  solution  t o 3500  linear  39.  The  with  maximal  U/mL.  over  the  a b o u t 40% o f t o t a l  cytochalasin  uncoated  response  i n t h e range  T r i t o n X-100, w h i c h  reported  o f LYZ from  stimulated  30-60 min and was  lysis  of a  r a n g e 0.1 t o 200 L Y Z u n i t s / m L .  course  pretreated  prevent  cytochalasin  t o i t i n amounts r a n g i n g  relationship  The  nm  not  <  LYZ.  absorbance a t 450  the presence  onto t h e c r y s t a l  the release  (p  surface.  when  stimulated  for controls  even  did  the release  occurring  neutrophils  change  that  crystal  t o monitor  degranulation  decided  likely  than  onto t h e c r y s t a l  Due t o t h e a d s o r p t i o n not  release  was  o f 990  +  enzyme a c t i v i t y i s i n good  by S i m c h o w i t z e t a l .  (1982).  MSUM  B is  observed 290  U/mL.  released  agreement  with  STANDARD C U R V E F O R L Y S O Z Y M E A S S A Y B Y M I C R O C O C C U S METHOD  0.149 Y= 0.0006 X + 0.0046 r = 0.994  C=3 O  0 . 0 9 9 --  z  < ffl  « O  CO  <  5  0.049 -  u <  -6.000E  50  75  100  125  150  200  L Y S O Z Y M E ADDED ( u n i t s ;  FIGURE 38  o  LYSOZYME RELEASE INDUCED- BY MSUM  C/3  0  -I 0  1  1  1  1  20  40  60  80  TIME ( m i n u t e s )  FIGURE 3 9  1  • 100  ; 120  112  The  time  course  crystals  f o r the release  i s shown  approximately  i n Figure  o f LYZ i n d u c e d  by CPPD  The r e s p o n s e  reached  40.  1500 + 240 U/mL i n 45-60 min and r e a c h e d a  maximum o f 2200 + 120 a t about 150 min.  Figures  39 and 40  show t h a t t h e c o n t r o l v a l u e s remained i n t h e same range over the  time  course  f o r MSUM and CPPD c r y s t a l s .  LYZ r e l e a s e  from n e u t r o p h i l s s t i m u l a t e d by MSUM c r y s t a l s was l o w e r and subject from  t o greater  CPPD  variation  stimulated  possibility  that  compared  neutrophils.  analogous  Figure  41  solutions There  shows  was a  incubated  t h e change  o f egg w h i t e  with  slight  MSUM c r y s t a l s .  including  raised  LYZ was a l s o  the being  surface. i n absorbance  LYZ i n c u b a t e d  reduction  studied the adsorption  This  t o MPO,  adsorbed onto t h e MSUM c r y s t a l  t o t h e LYZ r e l e a s e  with  MSUM  i n activity Kozin  produced  by  crystals.  o f LYZ when  and McCarty  (1967)  o f s e v e r a l p r o t e i n s by MSUM c r y s t a l s  LYZ, albumin  and I g G.  They  found  that the  a d s o r p t i o n o f LYZ t o MSUM was g r e a t e r than t h e a d s o r p t i o n o f BSA  t o MSUM.  Kozin  e t a l . (1979) a l s o observed o n l y  small  amounts o f LYZ r e l e a s e d from n e u t r o p h i l s s t i m u l a t e d by MSUM which was p r o b a b l y  due t o a d s o r p t i o n  o f LYZ by MSUM.  Hence  i t i s p r o b a b l e t h a t t h e LYZ l e v e l s measured when n e u t r o p h i l s were  incubated  with  uncoated  MSUM  u n d e r e s t i m a t e d due t o t h e a d s o r p t i o n crystals.  crystals  were  being  o f LYZ onto t h e MSUM  L Y S O Z Y M E R E L E A S E INDUCED B Y C P P D 2500  2000 +  1500 +  O — O CONTROL • — •  CPPD  1000 +  500 +  -O  0 0  20  40  60  80 100 120 TIME ( m i n u t e s )  F I G U R E 40  140  160  180  200  S T U D Y OF L Y S O Z Y M E ADSORPTION ONTO M S U M AND THE E F F E C T ON T H E L Y S O Z Y M E A S S A Y  200  e  o o  o  o  6  o o  175 +  o  O  o  <  150 0  5  10 M S U M ADDED  o  o  o o  15 (mg/mL)  FIGURE 41  20  25  115  4 . 3 . 4 . EFFECT OF PROTEINS ON LYZ RELEASE The  effect  of protein  degranulation monitored 43  from  coating  cytochalasin  by t h e r e l e a s e  respectively.  pretreated  o f MSUM a n d CPPD B  pretreated  induced  from  by CPPD  crystals  by t h e nature o f t h e p r o t e i n c o a t i n g  as  i n Figure  proteins induced  h a d no  43.  Precoating  significant  on  was  and B not  on t h e c r y s t a l  o f MSUM  effect  42  cytochalasin  affected seen  neutrophils  o f LYZ i s shown i n F i g u r e s  T h e LYZ r e l e a s e  neutrophils  c r y s t a l s on  crystals  t h e LYZ  with  release  b y MSUM.  5. FUTURE WORK We h a v e  studied  t h e modulation  MSUM a n d CPPD c r y s t a l s such  at  interesting responses  response effect  such  as  production the  specific  to  since  density  could  treatment  albumin  and plasma  t h e time  course  of the neutrophil  coated  proteins  MSUM  such  crystal  CPPD  different  The e f f e c t deposition  crystals,  proteins.  The  lipoproteins  on t h e n e u t r o p h i l and  be  i n the rate of  a s low d e n s i t y  generation  be s t u d i e d .  and  may e x i s t  with  lipoproteins  of  serum  proteins,  I t would  coated  superoxide  o f adsorbed  intervals.  differences  to crystals  responses t o  time  protein  o f other  high  G, b o v i n e  t o study  particularly  and  by t h e p r e s e n c e  a s immunoglobulin  proteins,  of neutrophil  responses  chemiluminescence o f drugs diseases  used i n on t h e  L Y S O Z Y M E R E L E A S E INDUCED B Y P R O T E I N COATED  F I G U R E 42  MSUM  L Y S O Z Y M E R E L E A S E INDUCED B Y P R O T E I N COATED C P P D  CONTROL  UNCOATED CPPD  IgG COATED CPPD  FIGURE  BSA COATED CPPD  43  PLASMA COATED CPPD  118  n e u t r o p h i l r e s p o n s e s t o t h e uncoated and p r o t e i n c o a t e d MSUM and CPPD c r y s t a l s c o u l d a l s o be  studied.  119  SUMMARY AND CONCLUSIONS  1.  The method  modified  of preparation  to yield  crystals  o f MSUM  was  of a smaller  successfully  and more  uniform  s i z e range. 2. MSUM and CPPD c r y s t a l s a t c o n c e n t r a t i o n s 50 mg/mL r e s p e c t i v e l y anion  by n e u t r o p h i l s .  induced but  induced  the generation  The r a t e  amounts  was no s i g n i f i c a n t  o f superoxide  anion  of superoxide  of superoxide  by CPPD c r y s t a l s was s l o w e r  there  o f 5 mg/mL and  production  t h a n f o r MSUM c r y s t a l s  difference generated  i n t h e maximum from  neutrophils  s t i m u l a t e d by e i t h e r MSUM o r CPPD. 3. The r e d u c t i o n for  of ferricytochrome  CPPD/neutrophil  reduction  incubations  of ferricytochrome  superoxide  anions.  f erricytochrome  c  MSUM/neutrophil inactivation  c was SOD  c  indicating was d r i v e n  However, was  not  incubations.  o f SOD p r o b a b l y  inhibitable  the SOD  This  that primarily  reduction  inhibitable was  attributed  due t o a d s o r p t i o n  the by of for to  onto t h e  s u r f a c e o f MSUM c r y s t a l s . 4. The p r e c o a t i n g  o f CPPD and MSUM c r y s t a l s  plasma had no e f f e c t on s u p e r o x i d e of  an enhanced  opsonized  with  responsiveness  with  generation.  of neutrophils  I g G may be because  under  I g G and  The absence to crystals  the incubation  c o n d i t i o n s employed, t h e n e u t r o p h i l s s t i m u l a t e d by uncoated crystals  were a l r e a d y  producing  maximum p o s s i b l e l e v e l s o f  120  superoxide.  There  precoating  o f MSUM  whereas BSA-coated superoxide 5.  The  was on  no  significant  MSUM  induced  CPPD p r o d u c e d  effect  o f BSA  superoxide  a significant  release,  increase i n  generated. CL  stimulation  generated  by  neutrophils  in  response  b y MSUM was more r a p i d a n d e x t e n s i v e  than  to that  g e n e r a t e d o n s t i m u l a t i o n b y CPPD. 6.  In general,  crystals  t h e nature  strongly  influenced  enhancing  and plasma  Adsorbed  Ig  G  proteins  probably  crystal-neutrophil  component  lipoprotein  fraction  probably  which  pretreated MSUM  induced  binds  may  adsorption  o f MPO  stimulated  degranulation  mediated  be  The m a j o r  t h e low  density  s t r o n g l y t o MSUM a n d CPPD with  the i n i t i a l  degranulation  as monitored  release  t h e CL g e n e r a t e d . receptor  c  Ig G  crystal-  process.  stimulate  MPO  F  on t h e  with  a n d CL g e n e r a t i o n .  interfering  neutrophils  coating  response  inhibiting  o f plasma  n e u t r o p h i l membrane b i n d i n g 7. CPPD c r y s t a l s  t h e CL  enhanced  activation  inhibitory  crystals  of the protein  could  by MSUM.  of cytochalasin B  by t h e r e l e a s e  o f MPO.  n o t be m o n i t o r e d  due t o  Both  MSUM  as monitored  a n d CPPD  crystals  by LYZ r e l e a s e b u t  MSUM a l s o a d s o r b e d L Y Z . 8.  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