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Crystal interactions with isolated human neutrophils : effect of adsorbed proteins 1991

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CRYSTAL INTERACTIONS WITH ISOLATED HUMAN NEUTROPHILS: EFFECT OF ADSORBED PROTEINS by SEEMA JAYANT GADKARI B. Pharm.; University of Bombay, 1984 M. Pharm.; University of Bombay, 1986 A THESIS SUBMITTED IN REQUIREMENTS MASTER PARTIAL FULFILLMENT OF THE OF THE DEGREE OF OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) D i v i s i o n of Pharmaceutics We accept t h i s thesis as conforming to the required 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. <i>OEN(j£S The University of British Columbia Vancouver, Canada Date **)CrV$T 20,.1991 DE-6 (2/88) A B S T R A C T C r y s t a l deposition diseases such as gout and pseudogout are characterized by the deposition of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) c r y s t a l s respectively i n the j o i n t s . Inflammation a r i s e s from the int e r a c t i o n of these c r y s t a l s with the synoviocytes and polymorphonuclear leukocytes or neutrophils. We have studied the neutrophil responses to stimulation by MSUM and CPPD c r y s t a l s and the e f f e c t of various protein coatings on the c r y s t a l surface on the neutrophil responses. The neutrophil responses studied were the respiratory burst accompanying phagocytosis leading to production of superoxide anion and generation of chemiluminescence. Degranulation of the neutrophil was also studied by monitoring the release of myeloperoxidase and lysozyme enzymes. Both MSUM and CPPD induced the generation of superoxide anion to the same degree although MSUM induced a fast e r rate of production of superoxide anion than CPPD. The reduction of ferricytochrome c was superoxide dismutase i n h i b i t a b l e f or CPPD/neutrophil incubations but not in h i b i t a b l e f or MSUM/neutrophil incubations, probably due to i n a c t i v a t i o n of superoxide dismutase by adsorption of the protein onto MSUM c r y s t a l s . Precoating of MSUM and CPPD c r y s t a l s with Immunoglobulin G, Bovine serum albumin i H or plasma proteins did not influence the generation of superoxide s i g n i f i c a n t l y . The generation of luminol enhanced neutrophil chemiluminescence induced by MSUM was of a greater magnitude and the maximal response was attained f a s t e r than for CPPD c r y s t a l s . Precoating MSUM or CPPD c r y s t a l s with immunoglobulin G enhanced the chemiluminescence response while plasma precoating i n h i b i t e d the chemiluminescence response. Release of myeloperoxidase induced by MSUM c r y s t a l s could not be measured due to adsorption of myeloperoxidase by MSUM. Precoating MSUM c r y s t a l s with proteins d i d not influence the adsorption of myeloperoxidase. Hence degranulation was monitored by measuring the release of lysozyme induced by MSUM and CPPD c r y s t a l s . Lysozyme was also found to adsorb onto MSUM c r y s t a l s although less extensively than myeloperoxidase. There was no s i g n i f i c a n t e f f e c t of protein coating on MSUM and CPPD induced lysozyme release. The p a r t i c u l a t e stimulants MSUM and CPPD produced a c t i v a t i o n of neutrophils with superoxide release, chemiluminescence generation and degranulation. Neutrophil responses, and i n p a r t i c u l a r the chemiluminescence responses, to the c r y s t a l s could be modulated by the nature of the protein adsorbed to the c r y s t a l surface. i v TABLE OF CONTENTS NO. TITLE PAGE ABSTRACT i i LIST OF FIGURES xi LIST OF TABLES XVi LIST OF SCHEMES XVii LIST OF ABREVIATIONS x v i i i ACKNOWLEDGEMENTS XXI I. BACKGROUND 1 Cryst 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 of c r y s t a l induced inflamination 3 3.1. Inflammatory mediators 7 4. Crystal-neutrophil interactions 9 4.1. Phagocytosis 9 4.2. Components of the respiratory burst 12 4.2.1. Chemiluminescence 12 NO. TITLE PAGE 4.2.2. Production of superoxide anion 16 4.3. Degranulation 20 5. E f f e c t of c r y s t a l history on inflammatory 21 po t e n t i a l of c r y s t a l s 5.1. C r y s t a l s i z e 21 5.2. Cryst a l pretreatment 21 6. E f f e c t of protein adsorption 22 6.1. Plasma proteins 24 6.2. Immunoglobulin G (IgG) 23 6.3. Lipoproteins 24 6.4. Albumin 24 7. Hypothesis and Objectives 25 I I . EXPERIMENTAL Materials 26 Reagents and Solvents 27 Buffer Solutions 28 vi NO. TITLE PAGE Instruments 28 Labware 29 Methods 30 1 Preparation of c r y s t a l s 30 1.1. Monosodium Urate Monohydrate 30 1.2. Calcium Pyrophosphate Dihydrate 31 1.2.1. Synthesis of calcium dihydrogen 31 pyrophosphate 1.2.2. Synthesis of calcium pyrophosphate 32 dihydrate 2. Characterization 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 analysis 33 2.2 D i f f e r e n t i a l scanning calorimetry 33 2.3 P a r t i c l e s i z e analysis 34 2.4 Scanning electron microscopy 34 3. Preparation of neutrophil suspension 35 3.1 Estimation of neutrophil count 36 3.2 Estimation of neutrophil v i a b i l i t y 36 v i i 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 blue 38 4. P r o t e i n c o a t i n g of c r y s t a l s 38 5. Measurement of n e u t r o p h i l response 39 5.1 Superoxide anion r e l e a s e 39 5.1.1. Superoxide r e l e a s e from n e u t r o p h i l s on 39 s t i m u l a t i o n by uncoated MSUM 5.1.2. Superoxide r e l e a s e from n e u t r o p h i l s on 40 s t i m u l a t i o n by uncoated CPPD 5.1.3. E f f e c t o f SOD on superoxide r e l e a s e 41 5.1.4. A d s o r p t i o n of SOD by MSUM c r y s t a l s 41 5.1.5. Superoxide r e l e a s e from n e u t r o p h i l s on 42 s t i m u l a t i o n by p r o t e i n coated MSUM and CPPD 5.2 Chemiluminescence 43 5.2.1. Chemiluminescent response of 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 uncoated MSUM and CPPD c r y s t a l s v i i i NO. TITLE PAGE 5.2.2. Chemiluminescent response of neutrophils 43 on stimulation by protein coated MSUM and CPPD c r y s t a l s 5.3 Degranulation indicator: release of MP0 44 and LYZ 5.3.1. MP0 and LYZ release from neutrophils 44 on stimulation by uncoated MSUM and CPPD c r y s t a l s 5.3.2. Measurement of LYZ 45 5.3.3. Measurement of MPO 46 5.3.4. MPO and LYZ release from neutrophils on 46 stimulation by protein coated MSUM and CPPD c r y s t a l s 6. S t a t i s t i c a l t e sts 46 I I I . RESULTS AND DISCUSSION 1. Preparation of c r y s t a l s 48 1.1 Monosodium urate monohydrate (MSUM) 48 1.2 Calcium pyrophosphate dihydrate (CPPD) 50 IX NO. TITLE PAGE 1.2.1. Synthesis of calcium dihyrogen 50 pyrophosphate (CDPP) 1.2.2. Synthesis of calcium pyrophosphate 54 dihydrate ( t r i c l i n i c ) 2. 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 calorimetry (DSC) 62 2.3 Scanning electron microscopy (SEM) 62 3. Estimation of neutrophil count and 69 v i a b i l i t y 4. Measurement of neutrophil responses 70 4.1 Cr y s t a l stimulated superoxide release 70 from neutrophils 4.1.1. E f f e c t of SOD on superoxide release 76 4.1.2. E f f e c t of proteins on superoxide release 81 4.2 Cr y s t a l stimulated chemiluminescent 83 response of neutrophils 4.2.1. E f f e c t of temperature on the CL response 93 X NO. TITLE PAGE 4.2.2. E f f e c t of proteins on the 93 chemiluminescent response 4.3 Cryst a l stimulated degranulation 99 response of neutrophils 4.3.1. Cryst a l stimulated MPO release 100 from neutrophils 4.3.2. E f f e c t of proteins on MPO release 105 4.3.3. Cryst a l stimulated LYZ release 109 from neutrophils 4.3.4. E f f e c t of proteins on LYZ release 115 5. Future work 115 IV. SUMMARY AND CONCLUSIONS 119 V. REFERENCES 122 x i LIST OF FIGURES NO. TITLE PAGE 1. a) Structure of synovial j o i n t 2 b) Release of c r y s t a l s into the j o i n t 2 2. C r y s t a l neutrophil i n t e r a c t i o n 4 3. Schematic representation of phagocytosis 10 4. P a r t i c l e s i z e of MSUM c r y s t a l s 49 (Method of Burt and Jackson, 1983) 5. P a r t i c l e s i z e of MSUM c r y s t a l s 51 (Modified method) 6. X-ray d i f f r a c t i o n pattern of calcium 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 of CPPD c r y s t a l s 55 8. X-ray d i f f r a c t i o n pattern of monosodium 56 urate monohydrate (Burt et a l . , 1983) 9. X-ray d i f f r a c t i o n pattern of monosodium 58 urate monohydrate (Modified method) 10. X-ray d i f f r a c t i o n pattern of calcium 60 pyrophosphate dihydrate 11. DSC thermogram of CPPD c r y s t a l s 63 NO. TITLE 12. DSC thermogram of MSUM c r y s t a l s (Burt e t a l . , 1983) 13. DSC thermogram o f MSUM c r y s t a l s ( M o d i f i e d method) 14. Scanning e l e c t r o n micrograph of CPPD c r y s t a l s 15. Scanning e l e c t r o n micrograph of MSUM c r y s t a l s (Burt e t a l . , 1983) 16. Scanning e l e c t r o n micrograph of MSUM c r y s t a l s ( M o d i f i e d method) 17. Superoxide 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 by uncoated MSUM and CPPD c r y s t a l s 18. Superoxide g e n e r a t i o n induced by MSUM c r y s t a l s 19. Superoxide g e n e r a t i o n induced by CPPD c r y s t a l s 20. E f f e c t o f c y t o c h a l a s i n B on MSUM induced superoxide g e n e r a t i o n 21. E f f e c t o f SOD on CPPD induced superoxide r e l e a s e from n e u t r o p h i l s x i i I NO. TITLE PAGE 22. Superoxide production by MSUM stimulated 79 neutrophils i n the presence of superoxide dismutase 23. E f f e c t of MSUM treated SOD on CPPD induced 80 superoxide release from neutrophils 24. E f f e c t of protein coating on the generation 82 of superoxide induced by MSUM 25. E f f e c t of protein coating on the superoxide 84 generation induced by CPPD c r y s t a l s 26. Chemiluminescence generated by neutrophils 85 stimulated by 5 mg uncoated MSUM 27. E f f e c t of MSUM concentration on 87 chemiluminescence 28. Chemiluminescence generated by neutrophils 89 stimulated by 50 mg uncoated CPPD 29. E f f e c t of CPPD concentration on neutrophil 90 chemiluminescence 30. E f f e c t of protein coating on 95 chemiluminescence response to MSUM 31. E f f e c t of protein coating on the 96 chemiluminescent response to CPPD NO. TITLE 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 on 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 om 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 on s t i m u l a t i o n w i t h u n c o a t e d CPPD c r y s t a l s 34. M y e l o p e r o x i d a s e r e l e a s e 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 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 amounts o f MSUM on m y e l o p e r o x i d a s e a c t i v i t y 36. M y e l o p e r o x i d a s e r e l e a s e 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 on s t i m u l a t i o n w i t h 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 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 on s t i m u l a t i o n w i t h 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 lysozyme a s s a y by m i c r o c o c c u s method 39. Lysozyme r e l e a s e i n d u c e d by MSUM 40. Lysozyme r e l e a s e i n d u c e d by CPPD XV NO. TITLE PAGE 41. Study of lysozyme adsorption onto MSUM 114 and the e f f e c t on the lysozyme assay 42. Lysozyme release induced by protein 116 coated MSUM 43. Lysozyme release induced by protein 117 coated CPPD LIST OF TABLES NO. TITLE PAGE 1. X-ray pattern of CDPP 53 2. X-ray pattern of MSUM (Burt and Jackson, 1983) 57 3. X-ray pattern of MSUM (Modified method) 59 4. X-ray pattern of CPPD 61 5. Ef f e c t of MSUM concentration on chemiluminescence 88 6. Ef f e c t of CPPD concentration on chemiluminescence 91 7. E f f e c t of sample temperature on chemiluminescence 94 8. Ef f e c t of protein coating on MSUM and CPPD induced chemiluminescence 98 xv i i LIST OF SCHEMES NO. TITLE PAGE 1. C r y s t a l induced n e u t r o p h i l s t i m u l a t i o n 5 2. E f f e c t of complement a c t i v a t i o n by MSUM 8 3. A c t i v a t i o n of lu m i n o l 13 4. Gen e r a t i o n of chemiluminescence 15 5. G e n e r a t i o n o f superoxide 17 x v i i i LIST OF ABREVIATIONS A Change i n absorbance A 4 5 0 Absorbance at 450 nanometers A 5 5 0 Absorbane at 550 nanometers ANOVA Analysis of Variance AUC Area under the curve BSA Bovine serum albumin CDPP Calcium dihydrogen pyrophosphate CL Chemiluminescence CPPD Calcium pyrophosphate dihydrate DSC D i f f e r e n t i a l scanning calorimetry jum micrometers g Gravity HDL High density lipoproteins HP Hewlett Packard Ig G Immunoglobulin G KV K i l o v o l t s LDH Lactate dehydrogenase LDL Low density lipoproteins LYZ Lysozyme mA milliamperes MPO Myeloperoxidase MSUM Monosodium urate monohydrate mV m i l l i v o l t s NL Native luminescence nm Nanometer nmoles Nanomoles 02~ Superoxide anion PC Personal computer PMNL Polymorphonuclear leukocytes rpm Revolutions per minute SEM Scanning electron microscopy SOD Superoxide dismutase U/mL Units per m i l l i l i t e r UBC University of B r i t i s h Columbia U l t r a v i o l e t Volts.second ACKNOWLEGEMENTS I am gr a t e f u l to Dr. Helen Burt for her supervision and encouragement during the course of t h i s work. I would also l i k e to thank Dr. Frank Abbott, Dr. Mike Bridges, Dr. David Godin and Dr. Alan M i t c h e l l for t h e i r enthusiastic support and guidance. I would l i k e to thank Mr. John Jackson f o r technical assistance. The assistance of Ms. Barbara McErlane, Mr. Michael Gentleman and Ms. Sukhbinder Panesar i s greatly appreciated. Thanks are also due to Mr. Ron Aoyama and Ms. Pascal Schmidt for t h e i r help during t h i s project. The guidance and friendship of Dr. Wayne Riggs, Mr. Matthew Wright and Dr. Albert Chow i s greatly appreciated. I am gr a t e f u l to Mr. Krishnaswamy Yeleswaram f o r his friendship and encouragement during the course of t h i s work. I would also l i k e to thank my family for t h e i r support during t h i s time., I would l i k e to thank Red Cross, Vancouver, f o r t h e i r generosity i n providing blood for the experiments. F i n a n c i a l support from the University of B r i t i s h Columbia i s g r a t e f u l l y acknowledged. 1 BACKGROUND 1 . CRYSTAL-INDUCED ARTHRITIS Cr y s t a l deposition disease has been defined as a pathological condition associated with the presence of c r y s t a l s which then contribute to the ti s s u e damage (Dieppe and Calvert, 1983). These c r y s t a l s have been i d e n t i f i e d as being those of monosodium urate monohydrate (MSUM), calcium pyrophosphate dihydrate (CPPD) and i n some cases, basic calcium phosphates (McCarty, 1989). C r y s t a l deposition diseases can be c l a s s i f i e d based on the type of c r y s t a l deposited. The condition known as gout involves deposition of MSUM c r y s t a l s and pseudogout or CPPD deposition disease i s characterized by the presence of CPPD, t r i c l i n i c and monoclinic c r y s t a l s (Ryan and McCarty, 1989). 2. DEPOSITION OP CRYSTALS The synovial j o i n t s of the extremities (hands, feet, elbows, knees) are p a r t i c u l a r l y prone to c r y s t a l deposition diseases. The structure of a synovial j o i n t i s shown i n Figure l a . The common s i t e s for deposition of c r y s t a l s are i n the a r t i c u l a r c a r t i l a g e , synovium or d i r e c t l y i n the synovial f l u i d of the j o i n t . Crystals may be released into the j o i n t f l u i d by the rupture of preformed synovial Bony a) STRUCTURE OF SYNOVIAL JOINT b) RELEASE OF CRYSTALS INTO THE JOINT FIGURE 1 3 deposits, by a " c r y s t a l shedding" mechanism from the c a r t i l a g e as seen i n Figure lb, or they may p r e c i p i t a t e i n the synovial f l u i d i t s e l f due to transient hyperuricemia i n the synovium or a transient f a l l i n temperature (McCarty, 1989). 3. MECHANISM OF CRYSTAL-INDUCED INFLAMMATION Crystal-induced inflammation involves several stages. The i n i t i a l response i s through in t e r a c t i o n of the c r y s t a l s with the synoviocytes or phagocytic c e l l s l i n i n g the synovium (Wallingford and McCarty, 1971) rather than with polymorphonuclear leukocytes (PMNL). The binding of the c r y s t a l s to the synoviocytes may be enhanced by adsorbed proteins such as immunoglobulin G (Ig G) (Kozin and McCarty, 1976). Crystal-induced l y s i s of the phagocytic synoviocytes leads to release of a crystal-induced chemotactic factor which promotes the migration of polymorphonuclear leukocytes (PMNL/neutrophils) into the area (McCarty, 1989). This chemotactic factor has been i d e n t i f i e d as a low molecular weight peptide with a molecular weight of 8,500 daltons (Phelps et a l . , 1981) but other reports indicate a molecular weight of 11,500 daltons (Spilberg and Mandell, 1983). Following increased migration of PMNL into the j o i n t , the i n t e r a c t i o n of the c r y s t a l s with neutrophils plays a ce n t r a l r o l e i n the crystal-induced inflammatory response and i s shown i n Figure 2 and Scheme 1 and summarized below. 4 A C U T E C R Y S T A L L I Z A T I O N C R Y S T A L SHSDOINO. C O M P L E M E N T CONTACT SYSTEM SYNOVIAL FIBROBLAST MONONUCLEAR PHAGOCYTE (synovial OnUtg. [pint fluid) 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 OXYGEN OXIDATIVE ^ n A k n n m „ „ DEGRANULATION PHAGOCYTOSIS UPTAKE METABOLISM SUPEROXIDE ANION HYDROXYL RADICAL HYDROGEN PEROXIDE CHEMILUMINESCENCE LYSOSOMAL ENZYME RELEASE MYELOPEROXIDASE MANNOSIDASE LYSOZYME C E L L U L A R A U T O L Y S I S L A C T A T E D E H Y D R O G E N A S E R E L E A S E SCHEME 1 6 The naked c r y s t a l upon release into the j o i n t f l u i d adsorbs several proteins such as immunoglobulin G (Ig G) , albumin, complement fragments, c l o t t i n g factors and lipo p r o t e i n s from the synovial f l u i d (Kozin and McCarty, 1976; Hasselbacher and Schumacher, 1978; Hasselbacher, 1979a, 1979b; Terkeltaub et a l . , 1983). The protein coated c r y s t a l may in t e r a c t with receptors on the macrophage/neutrophil plasma membrane through the mediation of the adsorbed proteins e.g. Ig G (Kozin and McCarty, 1976) and t h i s surface stimulation of neutrophils 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 increase i n oxidative metabolism within the macrophage/neutrophil (Simchowitz et a l . , 1982). The c r y s t a l i s phagocytosed by the PMNL and l i e s within the cytoplasm i n a sac known as a phagosome (McCarty, 1962; Schumacher and Phelps, 1971). This i s followed by fusion of the lysosomes of the PMNL with the phagosome producing a phagolysosome. Degranulation then occurs and the contents of the neutr o p h i l i c granules of the lysosomes are emptied into the phagolysosome (Shirahama and Cohen, 1974). I t has been proposed that the release of the lysosomal contents within t h i s phagolysosome r e s u l t s i n digestion of the proteins adsorbed onto the c r y s t a l , leaving the membrane of the phagolysosome exposed to the "naked" c r y s t a l thereby allowing a crystal-membrane interaction to take place (McCarty, 1989; Gordon et a l . , 1988). The crystal-membrane i n t e r a c t i o n i s thought to occur in two consecutive steps, c r y s t a l membrane binding followed by membrane rupture or 7 membranolysis (Burt and Jackson, 1988). Rupture of the phagolysosomal membrane and r e l e a s e of the lysosomal enzymes i n t o the cytoplasm f o l l o w s , l e a d i n g t o c e l l u l a r a u t o l y s i s and r e l e a s e of the c r y s t a l i n t o the s y n o v i a l f l u i d . The r e l e a s e of the c y t o s o l i c contents of the PMNL a c t s as a powerful s t i m u l u s f o r the m i g r a t i o n of more n e u t r o p h i l s i n t o the j o i n t l e a d i n g t o f u r t h e r i n t e r a c t i o n w i t h the c r y s t a l s and p o t e n t i a t i o n of the inflammatory response (Gordon e t a l . , 1988). T h i s mechanism of c r y s t a l - i n d u c e d c y t o l y s i s i s known as the " p e r f o r a t i o n from w i t h i n " h y p o t h e s i s and was f i r s t d e s c r i b e d by A l l i s o n i n h i s work on the t o x i c i t y of s i l i c a p a r t i c l e s ( A l l i s o n e t a l . , 1966). W a l l i n g f o r d and McCarty (1971) l a t e r extended t h i s h y p o t h e s i s t o the mechanism of c r y s t a l - i n d u c e d inflammation. 3.1. I N F L A M M A T O R Y M E D I A T O R S a) MSUM a c t i v a t e s t h e complement system by the c l a s s i c a l and a l t e r n a t e pathways in vitro (Naff and Byers, 1973; Byers e t a l . , 1973; Hasselbacher, 1979a, 1979b; Tenner and Cooper, 1982) . In v i v o , these a c t i v a t e d complement f a c t o r s may t r i g g e r a cascade of events e v e n t u a l l y p r o d u c i n g f a c t o r s t h a t enhance the chemotactic m i g r a t i o n of n e u t r o p h i l s , i n c r e a s e p h a g o c y t o s i s and cause c e l l l y s i s (Scheme 2). b) A c t i v a t i o n of t h e Hageman f a c t o r , which i s f a c t o r XII of the c l o t t i n g cascade, by c r y s t a l s has been r e p o r t e d (activation of C3) +- + C5/6/7 Chemotactic factor enhanced phagocytosis + C8/9 * -CeU lysis M S U M INDUCED COMPLEMENT ACTIVATION SCHEME 2 9 (Kellermeyer, 1967). The a c t i v a t e d f a c t o r has been shown t o a c t i v a t e the c l o t t i n g cascade and the k i n i n system i n v i t r o , the l a t t e r r e s u l t i n g i n the form a t i o n of potent v a s o d i l a t o r s which may enhance the inflammatory response (Ginsberg e t a l . , 1980) but the s i g n i f i c a n c e of t h i s f i n d i n g i n v i v o has not been e s t a b l i s h e d ( S p i l b e r g , 1974; Green e t a l . , 1982). c) MSUM i s i n i t i a l l y phagocytosed by the s y n o v i o c y t e s and t h e r e i s an a s s o c i a t e d s y n t h e s i s and r e l e a s e of p r o s t a g l a n d i n s and i n t e r l e u k i n s which cause v a s c u l a r changes and which are mediators of the p a i n s t i m u l u s (Alwan e t a l . , 1989; Guerne e t a l . , 1989; Woolf and Dieppe, 1987) . The r e l e a s e of superoxide, lysosomal enzymes and other products of the polymorphonuclear c e l l i n j u r y i n t o the s y n o v i a l f l u i d may a l s o i n f l u e n c e the inflammatory process (Rosen e t a l . , 1986). 4. CRYSTAL-NEUTROPHIL INTERACTIONS 4.1. PHAGOCYTOSIS F i g u r e 3 i s a schematic r e p r e s e n t a t i o n of p h a g o c y t o s i s of p a r t i c l e s by n e u t r o p h i l s (Trush et a l . , 1978). The PMNL i s s t i m u l a t e d on c o n t a c t with the opsonized c r y s t a l through the proc e s s of p a r t i c l e r e c o g n i t i o n . The p r o t e i n s adsorbed onto the c r y s t a l s u r f a c e are b e l i e v e d t o p l a y an important r o l e , i n p a r t i c u l a r adsorbed Ig G on the c r y s t a l s u r f a c e opsonizes the c r y s t a l thereby enhancing the n e u t r o p h i l response (Roos • O P S O N I Z E O P H » c o c r r o s A ( K . e P A R T I C L E 1. P A R T I C L E R E C O G N I T I O N A N O B I N O I N C 2. M E M B R A N E I N V A G I N A T I O N A N O P H A G O C Y T O S I S J . F O R M A T I O N O F P H A C O S O M E 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. F I G U R E 3 11 e t a l . , 1981). The s t i m u l u s p r o v i d e d by the opsonized c r y s t a l causes i n v a g i n a t i o n of the plasma membrane of the n e u t r o p h i l . The lysosomes a l s o migrate towards the i n v a g i n a t e d areas of the plasma membrane and some of the lysosomes fuse with the plasma membrane b e f o r e the p a r t i c l e i s c ompletely phagocytosed and d i s c h a r g e t h e i r c o ntents i n t o the e x t r a c e l l u l a r medium (Weissmann e t a l . , 1972; Hawkins, 1972; B a g g i o l i n i and Dewald, 1984). Polymorphonuclear l e u k o c y t e s are capable o f responding t o s t i m u l a t i o n by e i t h e r p a r t i c u l a t e or s o l u b l e s t i m u l i which produce d i s t i n c t m e t a b o l i c changes i n the c e l l s . PMNL norma l l y e x h i b i t a n aerobic metabolism. However, on exposure t o p a r t i c u l a t e or s o l u b l e s t i m u l i the m e t a b o l i c p a t t e r n changes and PMNL e x h i b i t i n c r e a s e d o x i d a t i v e metabolism (Babior e t a l . , 1973). T h i s i s manifested i n terms of i n c r e a s e d oxygen uptake and the g e n e r a t i o n of o x i d a t i o n by- products and h i g h l y r e a c t i v e s p e c i e s such as the superoxide anion, the h y d r o x y l r a d i c a l and s t r o n g o x i d i z i n g agents such as hydrogen p e r o x i d e (Simchowitz e t a l . , 1982). P h a g o c y t o s i s of p a r t i c u l a t e s t i m u l i by the PMNL i s accompanied by t h i s i n c r e a s e i n o x i d a t i v e metabolism which i s termed the x r e s p i r a t o r y b u r s t ' . 12 4.2. COMPONENTS OF THE RESPIRATORY BURST 4.2.1. CHEMILUMINESCENCE Chemiluminescence (CL) i s the p r o d u c t i o n of l i g h t as a r e s u l t of a chemical r e a c t i o n and PMNL e x h i b i t CL on s t i m u l a t i o n by p a r t i c u l a t e and n o n - p a r t i c u l a t e s t i m u l i ( A l l e n e t a l . , 1972; Cheson e t a l . , 1976; W e s t r i c k e t a l . , 1980; Roschger e t a l . , 1988; Harber and Topley, 1986). T h i s luminescence can be measured through the use of l i g h t a m p l i f y i n g compounds such as l u m i n o l and l u c i g e n i n . CL r e f l e c t s the e x c i t a t i o n s t a t e of the n e u t r o p h i l and thus s e r v e s as an 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 (Trush e t a l . , 1978) . Luminol, (5-amino-2,3-dihydro-l,4-phthalazinedione), has been r o u t i n e l y used t o a m p l i f y the C L produced by v a r i o u s chemiluminescent r e a c t i o n s (Trush et a l . , 1978). The nature of l u m i n o l a c t i v a t i o n i s shown i n Scheme 3 ( A l l e n , 1982). Luminol e x h i b i t s a high C L quantum y i e l d i . e . t h e r e i s an i n c r e a s e i n the number of photons r e l e a s e d per e l e c t r o n i c a l l y e x c i t e d s p e c i e s generated, thereby i n c r e a s i n g the s e n s i t i v i t y of the C L measurement. The CL of l u m i n o l depends on d i o x y g e n a t i o n of luminol and hence i t can be used i n the study of b i o l o g i c a l oxygenation r e a c t i o n s such as phagocyte o x i d a t i v e metabolism. Luminol has been shown not t o a l t e r the r e s p i r a t o r y b u r s t i n n e u t r o p h i l s ( A l l e n , 1982) . RADICAL D i O X Y G E N A T I O N Proposed biochemical mechanisnvfor cyclic hydrazide chemiluminescence at neuiral to acid pH. ACTIVATION OF LUMINOL SCHEME 3 14 Scheme 4 d e p i c t s the r e a c t i o n s o c c u r r i n g i n the c e l l t h a t may p o s s i b l y r e s u l t i n CL (adapted from Wilson e t a l . , 1978) . The o x i d a t i v e products produced d u r i n g the r e s p i r a t o r y b u r s t may r e a c t with components of the n e u t r o p h i l t o y i e l d luminescent products. The g e n e r a t i o n of l u m i n o l - a m p l i f i e d CL was shown t o f o l l o w two pathways (Dahlgren, 1988), one dependent on MPO and hydrogen p e r o x i d e and the other independent of hydrogen p e r o x i d e , p o s s i b l y dependent on superoxide s i n c e l u m i n o l may be oxygenated by r e a c t i o n with superoxide or hydrogen p e r o x i d e . The c o n t r i b u t i o n of superoxide t o CL has been s t u d i e d u s i n g superoxide dismutase (SOD) (Webb e t a l . , 1974; A l l e n and Loose, 1976), an enzyme normally p r e s e n t i n PMNL which c a t a l y s e s the d i s m u t a t i o n of superoxide t o hydrogen p e r o x i d e and oxygen as shown i n equation 1 below (McCord e t a l . , 1971). ° 2 ~ + °2~ + 2 H + > H 2 ° 2 + °2 Eqn. 1 The r o l e of MPO i n the g e n e r a t i o n of CL has been s t u d i e d u s i n g sodium a z i d e as an i n h i b i t o r of MPO (Nurcombe and Edwards, 1989). Luminol-dependent CL was found t o be a f f e c t e d by f a c t o r s such as o p s o n i z a t i o n of the p a r t i c u l a t e s t i m u l i , i n c u b a t i o n temperature and the presence of c a l c i u m and albumin i n the b u f f e r (Harber and Topley, 1986; Roschger 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 CELLULAR (PMN) ACTIVATION PARTICLE ENGULFMENT ANO/OR OEGRANULATION MPO STIMULATION CF OXIDATIVE METABOLISM (HMP SHUNT) JNADPH/NAOP* \ H ' ° PYRIOINE NUCLEOTIDE DEPENDENT ENZYMIC FORMATION OF 0»" MPO + H,0, + Cr 27,000 a porllculali (oronulO fraction from ocli»ol«d cill Oj* + Ot* + 2H* — H t 0 » + 0j °l + H,0, —0H*+ 0H« + 0 2' Nontniymie Rtoclisni b«lw«in Oiyojn Spiclll H,0+CI- + Oj' OXIDIZABLE SUBSTRATES ON PHAGOCYTOSABLE OR -» PROSTAGLANDIN SYNTHESIS ANO/OR LIPID PEROXIDATION PHAGOCYTtZEO PARTICLE I "^•GENERATION OF ELECTRONIC , *VEXCITATION STATE(S) - * (Slnoltt Oiyotn anil a' ' corbonyl groups) CELL * TREE ENZYME SYSTEMS I. Xonlftint ci it fon, pyrifti l y p i r o i i d t q i M r o h ' f l f Z.M;llopifoii40U, M , 0 . . holidt r«oclipn 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. SCHEME 4 H 16 The concentrations of luminol and PMNL as w e l l as s t i m u l i a l l a f f e c t the observed response (Wilson et a l . , 1978). Other f a c t o r s such as d i e t may al s o a f f e c t the CL response of PMNL (Magaro et a l . , 1988). 4 . 2 . 2 . PRODUCTION OF SUPEROXIDE ANION Superoxide anion i s the term used t o des c r i b e the e l e c t r o n i c a l l y e x c i t e d species a r i s i n g from s i n g l e t molecular 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 the generation of superoxide and other e l e c t r o n i c a l l y e x c i t e d s t a t e s of oxygen (Green and H i l l , 1984) . Molecular oxygen (O2) contains two unpaired e l e c t r o n s i n the outermost o r b i t a l and hence can e x i s t i n e i t h e r of two s p i n s t a t e s , t r i p l e t and s i n g l e t s t a t e s . In the t r i p l e t s t a t e the unpaired e l e c t r o n s possess p a r a l l e l s p i n w h i l e i n the s i n g l e t s t a t e one of the e l e c t r o n s i s f l i p p e d so t h a t the s p i n i s a n t i - p a r a l l e l . Energy must be s u p p l i e d t o cause the e l e c t r o n t o f l i p ; thus the s i n g l e t s t a t e of oxygen i s an e l e c t r o n i c a l l y e x c i t e d s t a t e . This can undergo f u r t h e r r e d u c t i o n , i . e . a d d i t i o n of an e l e c t r o n , l e a d i n g t o formation of superoxide anion, 0 2~. Further a d d i t i o n of e l e c t r o n s leads t o the formation of other o x i d a t i v e products such as hydrogen peroxide and hydroxy1 r a d i c a l . N e u t r o p h i l s generate the e l e c t r o n i c a l l y e x c i t e d species of oxygen during the r e s p i r a t o r y burst ( A l l e n et a l . , 1972). 17 3 0 2 - J 0 2 c O7 H O i c pK>l< pK-l\,6 01- HO7 H t 0 2 c 2H+ (OH - H 2 0 p A ' = l i . 9 O - O H - 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 n e u t r o p h i l s i n v o l v e s a membrane bound NADPH-dependent oxidase (Figure 3) b e l i e v e d t o be l o c a t e d on the plasma membrane of the PMNL (Babior and Pe t e r s , 1981; G o l d s t e i n et a l . , 1977; Badwey and Karnovsky, 1979; Dewald et a l . , 1979). The superoxide anion i s res p o n s i b l e f o r the b a c t e r i c i d a l a c t i o n of phagocytes (Babior et a l . , 1973) and may a l s o be r e s p o n s i b l e f o r damage t o the host t i s s u e (Hsie et a l . , 1986). MSUM and CPPD c r y s t a l - i n d u c e d superoxide r e l e a s e has been s t u d i e d and MSUM was found to be a stronger stimulus than CPPD (Nagase et a l . , 1989; Naccache et a l . , 1991). Methods have been developed f o r the measurement of superoxide generation by n e u t r o p h i l s such as the n i t r o b l u e t e t r a z o l i u m r e d u c t i o n assay (NBT) and r e d u c t i o n of ferr i c y t o c h r o m e c. Measurement of oxygen uptake and consumption by manometric methods have a l s o been reported (Babior and Cohen, 1981). Manometry r e q u i r e s high c o n c e n t r a t i o n s of c e l l s and i s not a very s e n s i t i v e method wh i l e potentiometry i s more s e n s i t i v e but u n s u i t a b l e f o r long-term in c u b a t i o n s . The method most commonly used i n the study of n e u t r o p h i l r e s p i r a t o r y burst i s based on the re d u c t i o n of ferricytochrome c to ferrocytochrome c by superoxide as described i n equation 2 (McCord and F r i d o v i c h , 1969). 19 > FeJ--Lcyt c + 0 2 Eqn. 2 This reduction i s accompanied by a s h i f t i n UV absorption maximum from 520 nm to 550 nm and hence can be r e a d i l y monitored. The reduction of ferricytochrome c can be made s p e c i f i c f or superoxide through the use of superoxide dismutase (SOD), a Cu-Zn metalloenzyme normally present within the PMNL as part of the protective mechanisms against superoxide-inflicted damage. SOD catalyses the dismutation of superoxide as described previously i n equation 1 (McCord et a l . , 1971). °2~ + °2~ + 2 H + > H2°2 + °2 Eqn. 1 The reduction of ferricytochrome c by superoxide i s in h i b i t e d by the presence of SOD i n the reaction mixture and t h i s can be used to confer s p e c i f i c i t y on the assay (Babior and Cohen, 1981). Ferricytochrome c being a high molecular weight compound i s excluded from the c e l l and hence t h i s assay can only measure changes i n the l e v e l of e x t r a c e l l u l a r superoxide. However, i t has been found that a large proportion of superoxide i s released e x t r a c e l l u l a r l y by PMNL (Roos and Weening, 1979). Fe i : i : icyt c O- 20 4 . 3 . D E G R A N U L A T I O N Degranulation of the neutrophil occurs into the e x t r a c e l l u l a r medium during phagocytosis and into the phagosome af t e r phagocytosis (Baggiolini and Dewald, 1984; Weissmann et a l . , 1972; Hawkins, 1972). Degranulation can occur even i n the absence of phagocytosis (Weissmann et a l . , 1972; Hawkins, 1972; Blackburn et a l . , 1987). The granules release t h e i r contents, including several enzymes such as lysozyme, myeloperoxidase and alpha mannosidase (Jones and Cross, 1988), into the phagosome containing the opsonized c r y s t a l . I t has been hypothesized that proteases such as lysozyme s t r i p the proteins adsorbed on the c r y s t a l surface to expose the "naked" c r y s t a l to the phagolysosomal membrane. Myeloperoxidase (MPO) catalyzes a hydrogen peroxide dependent oxidation reaction forming part of the resp i r a t o r y burst. The group of compounds known as the cytochalasins are useful i n the study of degranulation since such agents i n h i b i t phagocytosis by i n t e r f e r i n g with the polymerization of the actin-myosin network (Davis et a l . , 1971; Malawista et a l . , 1971). This r e s u l t s i n degranulation and release of granule contents into the e x t r a c e l l u l a r f l u i d , thereby f a c i l i t a t i n g measurement of the contents. Degranulation can be monitored by measuring the release of any of the enzymes released during t h i s process. The 21 presence of MPO i n the e x t r a c e l l u l a r f l u i d i s u s e f u l as an i n d i c a t o r of n e u t r o p h i l degranulation s i n c e i t i s present e x c l u s i v e l y i n the a z u r o p h i l i c granules of the n e u t r o p h i l (Andrews and Kr i n s k y , 1985) . MPO can be measured by the hydrogen peroxide dependent MPO-catalyzed o x i d a t i o n of o- d i a n i s i d i n e . Lysozyme (LYZ) rel e a s e d from the lysosomes of the PMNL can be assayed by monitoring the decrease i n t u r b i d i t y of a suspension of Micrococcus lysodeikticus on a d d i t i o n of LYZ (Babior and Cohen, 1981) . LYZ hydrolyzes the muramic a c i d - c o n t a i n i n g mucopeptide i n the poly s a c c h a r i d e of the c e l l w a l l s of the Micrococcus thus c o n v e r t i n g the 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 i n t u r b i d i t y . 5. EFFECT OF CRYSTAL HISTORY ON INFLAMMATORY POTENTIAL OF CRYSTALS 5.1. CRYSTAL SIZE The s i z e of i n d i v i d u a l c r y s t a l s i s known t o be important i n acute inflammation. Amorphous deposits and l a r g e c r y s t a l s of monosodium urate were found to be r e l a t i v e l y i n e f f e c t i v e , whereas c r y s t a l s of about 5 jum i n length were the most r e a c t i v e (Schumacher et a l . , 1975; Burt et a l . , 1989). 5.2. CRYSTAL PRETREATMENT G r i n d i n g of c r y s t a l s t o reduce p a r t i c l e s i z e , a p r a c t i c e reported by e a r l i e r workers, has now been demonstrated t o 22 e f f e c t a change i n the c r y s t a l s t r u c t u r e and hence a l t e r the inflammatory p o t e n t i a l of the c r y s t a l s (Burt et a l . , 1986). S i m i l a r l y , depyrogenation of the c r y s t a l s by heating at 200 - 250 °C f o r 2 hr was found to produce a change i n the c r y s t a l s t r u c t u r e l e a d i n g t o a l t e r a t i o n of i t s inflammatory p o t e n t i a l (Hasselbacher, 1979; Mandel, 1980; Cheng and P r i t z k e r , 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 surface t h a t can adsorb many d i f f e r e n t p r o t e i n s and other molecules from the s y n o v i a l f l u i d . I t i s now w e l l recognized t h a t p r o t e i n s bound t o the inflammatory c r y s t a l s play an important r o l e i n determining 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 to adsorb s e v e r a l p r o t e i n s 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 are pa r t of the c l o t t i n g cascade, p r o t e i n s of the complement system, immunoglobulins and l i p o p r o t e i n s ( W a l l i n g f o r d and McCarty, 1974; Terkeltaub et a l . , 1983). I t has been suggested' t h a t the spontaneous remission which i s c h a r a c t e r i s t i c of gout and pseudogout may be due t o the p r o t e i n s adsorbed on the c r y s t a l surface (Kozin and McCarty, 1976). As the inflammatory episode proceeds, the accompanying increase i n va s c u l a r p e r m e a b i l i t y a l t e r s the composition of the s y n o v i a l f l u i d such t h a t i t c l o s e l y 23 resembles t h a t of plasma (Swan, 1978). The l i p o p r o t e i n s and other p r o t e i n s present i n s y n o v i a l f l u i d are adsorbed onto the c r y s t a l surface and may i n t e r f e r e w i t h 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 r e m i s s i o n of the inflammatory episode (Terkeltaub et a l . , 1984). Recently ^ - a c i d g l y c o p r o t e i n has been shown t o i n h i b i t n e u t r o p h i l responses such as chemotaxis, aggregation and superoxide anion generation (Laine et a l . , 1990). Serum 2 ~ HS g l y c o p r o t e i n had e a r l i e r been shown to be a potent and s p e c i f i c i n h i b i t o r of n e u t r o p h i l s t i m u l a t i o n by hydroxyapatite c r y s t a l s (Terkeltaub et a l . , 1988). 6.2. IMMUNOGLOBULIN G ( I g G) Ig G i s s t r o n g l y adsorbed t o MSUM but l e s s so t o CPPD c r y s t a l s (Hasselbacher and Schumacher, 1978). The bi n d i n g of Ig G t o MSUM has been shown t o be a f u n c t i o n of the charge d e n s i t y of the Ig G molecule (Hasselbacher, 1979) and the 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 nature. The F a b p o r t i o n of the Ig G molecule i s more p o s i t i v e than the F c strands and hence i t i s the F ak part t h a t binds t o the c r y s t a l l e a v i n g the F c fragments exposed. Ig G-coated MSUM has been shown t o enhance n e u t r o p h i l responses t o the c r y s t a l s w i t h increases i n the extent of phagocytosis of the c r y s t a l s , e levated l e v e l s of superoxide and enhanced lysosomal enzyme r e l e a s e (Kozin et a l . , 1979; Hasselbacher et a l . , 1978; Abramson et a l . , 1982; Rosen et a l . , 1986). The enhanced response of the n e u t r o p h i l to the c r y s t a l s i s 24 apparently through i n t e r a c t i o n of the exposed F c fragments w i t h the c e l l u l a r receptors present on the PMNL (Hasselbacher, 1979). 6.3. L I P O P R O T E I N S MSUM and CPPD c r y s t a l s bind low de n s i t y l i p o p r o t e i n (LDL) and high d e n s i t y l i p o p r o t e i n (HDL) (Terkeltaub et a l . , 1986; Burt et a l . , 1989). The p r o t e i n moiety of LDL i s mostly a p o l i p o p r o t e i n B and th a t of HDL i s a p o l i p o p r o t e i n A l and A2. Terkeltaub et a l . (1983, 1984, 1986) monitored the extent of phagocytosis, CL and degranulation produced by n e u t r o p h i l s on exposure t o HDL and LDL coated MSUM c r y s t a l s . LDL coated MSUM c r y s t a l s i n h i b i t e d a l l MSUM induced n e u t r o p h i l responses wh i l e HDL coated MSUM c r y s t a l s apparently d i d not have any s i g n i f i c a n t i n h i b i t o r y e f f e c t . Burt et a l . , (1989) s t u d i e d the e f f e c t s of LDL and HDL bound t o MSUM and CPPD on c r y s t a l - i n d u c e d n e u t r o p h i l c y t o l y s i s measured by l a c t a t e dehydrogenase re l e a s e (LDH). Both HDL and LDL s t r o n g l y i n h i b i t e d CPPD and MSUM induced n e u t r o p h i l c y t o l y s i s (Burt et a l . , 1989). 6 . 4 . ALBUMIN The s y n o v i a l f l u i d i s an u l t r a f i l t r a t e of plasma (Swan, 1978). Albumin, a major c o n s t i t u e n t of plasma i s among the p r o t e i n s present i n the s y n o v i a l f l u i d . MSUM and CPPD c r y s t a l s have been reported t o bind albumin (Kozin and McCarty, 1976). The e f f e c t of adsorbed albumin on the 25 p a r t i c l e s t i m u l a t e d l u m i n o l dependent luminescence and n a t i v e luminescence (NL) has been r e p o r t e d by Roschger e t a l . (1988). T h e i r r e s u l t s i n d i c a t e t h a t w hile l u m i n o l dependent luminescence i s i n h i b i t e d , g e n e r a t i o n o f NL i s enhanced by albumin. In t h e i r s t u d i e s of the i n h i b i t o r y e f f e c t s of l i p o p r o t e i n s on MSUM induced n e u t r o p h i l s t i m u l a t i o n , T e r k e l t a u b e t a l . (1983, 1984, 1986) i n c l u d e d albumin i n the b u f f e r s used f o r n e u t r o p h i l / c r y s t a l i n c u b a t i o n s . They s t a t e d t h a t the purpose of the albumin was t o "to e l i m i n a t e non s p e c i f i c i n h i b i t i o n " . However i n s i m i l a r s t u d i e s by Burt e t a l . (1989) of the i n h i b i t o r y e f f e c t of l i p o p r o t e i n s on n e u t r o p h i l c y t o l y s i s , albumin was not added t o the b u f f e r s . 7. HYPOTHESIS AND OBJECTIVE I t i s our hyp o t h e s i s t h a t the presence of p r o t e i n s on the c r y s t a l s u r f a c e can i n f l u e n c e 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 . The o b j e c t i v e of our work i s to study the n e u t r o p h i l responses (CL, superoxide i o n p r o d u c t i o n and deg r a n u l a t i o n ) induced by uncoated and p r o t e i n coated MSUM and CPPD c r y s t a l s as a f u n c t i o n of i n c u b a t i o n time. P r o t e i n s used i n these s t u d i e s were bovine serum albumin, immunoglobulin g and plasma p r o t e i n s . 26 EXPERIMENTAL MATERIALS Albumin, F r a c t i o n V, f a t t y a c i d f r e e , from bovine serum, Boehringer Mannheim GmbH, W. Germany. Calcium t e t r a h y d r o g e n di-orthophosphate, CaH 4 ( P 0 4 ) 2 • H 2 O , BDH Chemicals L t d . , Poole, England. C a t a l a s e , from bovine l i v e r , Sigma Chemical Co., S t . L o u i s , MO. U.S.A. C y t o c h a l a s i n B, from Helminthosporium dematioideum, Sigma Chemical Co., St. L o u i s , MO. U.S.A. Cytochrome c, Type I I I , from horse h e a r t , Sigma Chemical Co., St. L o u i s , MO. U.S.A. Dextran T 7 0 , Pharmacia LKB, Biotechnology AB, Uppsala, Sweden. o - D i a n i s i d i n e d i h y d r o c h l o r i d e , Sigma Chemical Co., St. L o u i s , MO. U.S.A. F i c o l l - P a q u e ( R ) , Pharmacia LKB, Biotechnology AB, Uppsala, Sweden. Hydrogen Peroxide, 3 0 % , Sigma Chemical Co., St. L o u i s , MO. U.S.A. 27 Immunoglobulin G, Human, Sigma Chemical Co., St. Lo u i s , MO. U.S.A. Lac t a t e Dehydrogenase Assay K i t , Sigma Chemical Co., St. Lou i s , MO. U.S.A. Luminol, (5-amino-2,3-dihydro-l,4-phthalazinedione) Sigma Chemical Co., St. Louis, MO. U.S.A. Lysozyme, Grade I , from chicken egg white, Sigma Chemical Co., St. Louis, MO. U.S.A. Micrococcus lysodeikticus, Sigma Chemical Co., St. Lo u i s , MO. U.S.A. Superoxide dismutase, from bovine e r y t h r o c y t e s , Sigma Chemical Co., St. Louis, MO. U.S.A. T r i t o n X-100, (OctylPhenoxy Polyethoxyethanol), Sigma Chemical Co., St. Louis, MO. U.S.A. U r i c a c i d , ( 2 , 6 , 8 - t r i o x y p u r i n e ) , Sigma Chemical Co., St. Lou i s , MO. U.S.A. REAGENTS AND SOLVENTS Acetone, BDH. Ethanol Calcium acetate, Sigma. H y d r o c h l o r i c a c i d , BDH. 28 Orthophosphorous a c i d , 80% J.T. Baker. Sodium c h l o r i d e , BDH. Sodium hydroxide, F i s h e r . BUFFER SOLUTIONS Hanks b u f f e r (NaCl, 137 mM; KCl, 5.4 mM; Na 2HP0 4. 2H 20, 0.33 mM; KH 2P0 4, 0.44 mM; MgS0 4.7H20, 0.41 mM; CaCl 2.2H 20, 1.3 mM; Glucose, 5.6 mM; MgCl 2.6H 20, 0.5 mM; NaHC0 3, 4.2 mM), pH 7.4 C i t r a t e b u f f e r , pH 5.5, 0.1M Potassium phosphate b u f f e r pH 6.2, 0.065M INSTRUMENTS B i o - o r b i t 12 50 Luminometer i n t e r f a c e d w i t h PC; data c o l l e c t i o n software program obtained from manufacturer, F i s h e r S c i e n t i f i c Co. and equipped w i t h F i s h e r S c i e n t i f i c Isotemp Dry Bath 147. Dupont D i f f e r e n t i a l Scanning C a l o r i m e t e r S e r i e s 910 wit h S e r i e s 99 Thermal a n a l y z e r . Eppendorf c e n t r i f u g e 5412, Brinkmann Instruments. F i s h e r Dyna-Mix F i s h e r S c i e n t i f i c Accumet pH meter with O r i o n pH probe Model 91-02. 29 Haake c i r c u l a t i n g water bath Hewlett Packard Vectra spectrophotometer i n t e r f a c e d w i t h PC and equipped w i t h data c o l l e c t i o n software. H i t a c h i S-57 scanning e l e c t r o n microscope and Hummer s p u t t e r g o l d coater. Magnetic s t i r r e r and h o t p l a t e . M e t t l e r balances models AJ100 and AE163. Nikon d i f f e r e n t i a l i n t e r f e r e n c e microscope Model R w i t h Nikon o b j e c t i v e and o c u l a r micrometers. Oven, Johns S c i e n t i f i c P e r k i n Elmer D i f f e r e n t i a l Scanning Calorimeter, Model IB. Rigaku G i e g e r f l e x X-Ray Di f f r a c t o m e t e r System. VWR Vortexer 2, S c i e n t i f i c I n d u s t r i e s , N.Y. LABWARE Polypropylene tubes, 50 mL, 10 mL, Nalgene Polypropylene Eppendorf tubes, 1.5 mL, 0.5 mL, Brinkmann Instruments P o l y s t y r e n e cuvettes, 1 mL, F i s h e r S c i e n t i f i c 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 of the method of Burt et a l . (1983) was used t o prepare MSUM samples. To 800 mL of d i s t i l l e d water was added 4.0 g of u r i c a c i d with s t i r r i n g and the suspension heated t o 55°C. Approximately 2 5 mL of 1M sodium hydroxide was then added s l o w l y and the u r i c a c i d d i s s o l v e d t o giv e a c l e a r s o l u t i o n . The s t i r r i n g was continued f o r 45 minutes a t 55°C a f t e r which the pH was checked. The pH was adjusted t o 7.5 wi t h 1M h y d r o c h l o r i c a c i d . The s o l u t i o n was f i l t e r e d hot through 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 allowed t o stand undisturbed f o r 24 hours at room temperature. The r e s u l t i n g c r y s t a l s were harvested by f i l t r a t i o n through Whatman # 1 f i l t e r paper under vacuum. 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 of d i s t i l l e d water saturated w i t h MSUM at room temperature and were then allowed to dry overnight at 60 °C. The c r y s t a l s were stored i n t i g h t l y capped amber g l a s s b o t t l e s . The c r y s t a l s were c h a r a c t e r i z e d by X-ray d i f f r a c t i o n a n a l y s i s and d i f f e r e n t i a l scanning c a l o r i m e t r y . P a r t i c l e 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 microscopy. 31 1.2. CALCIUM PYROPHOSPHATE DIHYDRATE (CPPD) The p r e p a r a t i o n of CPPD was a two step process, the f i r s t step being the prep a r a t i o n of calcium a c i d pyrophosphate/calcium dihydrogen pyrophosphate. 1.2.1. Synthesis of calcium dihydrogen pyrophosphate (CDPP) In a 600 mL g l a s s beaker, 250 mL of 80% orthophosphoric a c i d was added and heated w i t h s t i r r i n g to 215°C. A d d i t i o n of calcium tetrahydrogen diorthophosphate was begun at an i n i t i a l r a t e of 1 g/min. This was continued u n t i l about 40 g of calcium tetrahydrogen diorthophosphate had been added a f t e r which the r a t e of a d d i t i o n was about 0.5 g/min. This was continued t i l l a f u r t h e r 35-40 g had been added. At t h i s stage the mixture formed a white s l u r r y and s t i r r i n g became d i f f i c u l t . This s l u r r y was then f i l t e r e d through a s i n t e r e d g l a s s f i l t e r heated t o approximately 170°C by passing orthophosphoric a c i d at t h a t temperature through the f i l t e r . The c r y s t a l s were allowed t o c o o l t o room temperature i n the f i l t e r . They were then washed w i t h three 100 mL a l i q u o t s of acetone to 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 c r y s t a l s were c h a r a c t e r i z e d by X-ray d i f f r a c t i o n t o confirm t h a t they were CDPP p r i o r to use i n the next step. 32 1.2.2. Synthesis of calcium pyrophosphate dihydrate ( t r i c l i n i c ) : 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 heated i n a water bath to 60 + 2°C and s t i r r e d c o n s t a n t l y w i t h a T e f l o n coated s t i r bar. The s t i r r i n g was slowed and 0.71 mL of concentrated h y d r o c h l o r i c a c i d and 0.32 mL of g l a c i a l a c e t i c a c i d were added, followed by 0.6 g of calcium a c e t a t e . A 150 mL beaker c o n t a i n i n g 20 mL of d i s t i l l e d water was heated t o 60 °C i n a water bath and 0.6 g of calcium acetate added. The r a t e of s t i r was increased i n the 250 mL beaker, and 2 g of CDPP added r a p i d l y . When the CDPP was ne a r l y a l l d i s s o l v e d , the r a t e of s t i r r i n g was reduced f o r 5 minutes, then over a p e r i o d of 15 seconds, the contents of the small beaker were poured i n t o the la r g e beaker w i t h vigorous s t i r r i n g . This l e d t o the formation of a white g e l . S t i r r i n g was disc o n t i n u e d and the g e l allowed t o stand undisturbed overnight. The g e l c o l l a p s e d w i t h the formation of CPPD c r y s t a l s . The c r y s t a l s were washed i n d i s t i l l e d water three times, washed i n ethanol, then acetone and allowed t o a i r dry. The c r y s t a l s were c h a r a c t e r i z e d by X-ray d i f f r a c t i o n a n a l y s i s and d i f f e r e n t i a l scanning c a l o r i m e t r y . 33 2. CHARACTERISATION OF CRYSTALS 2.1. X-RAY DIFFRACTION ANALYSIS: The X-ray d i f f r a c t i o n analyses were done using a Rigaku G i e g e r f l e x X-Ray Di f f r a c t o m e t e r System w i t h a b i p l a n a r goniometer, a D/max-B c o n t r o l l e r i n t e r f a c e between the goniometer and an IBM compatible 28 6 PC and equipped w i t h a s c i n t i l l a t i o n counter. The X r a d i a t i o n used was copper K wavelength = 1.56 Angstrom u n i t s , which was obtained by combining a copper source with a n i c k e l f i l t e r . Data analyses were done wi t h a software program provided by the manufacturer which allowed i n t e n s i t y and r e l a t i v e i n t e n s i t y determinations. The X-ray tube was operated at a p o t e n t i a l of 4 0 KV and at a current of 20 mA. The sample was scanned over a range of 5 t o 55 degrees 2 at a r a t e of 5 degrees/minute. The instrument c o l l e c t e d data every 0.05 degrees 2 /sample. The unground samples were packed i n an aluminum sample holder f o r MSUM and s p r i n k l e d onto a double s i d e d Scotch tape attached t o the holder f o r CPPD samples. 2.2. DIFFERENTIAL SCANNING CALORIMETRY Weighed samples of MSUM prepared by both the method of Burt et al. (1983) and the modified method and CPPD were analyzed by DSC. The MSUM data 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 at 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 temperature of 350°C, w i t h a heating r a t e of 10°C/min i n open crimped aluminum pans. The data f o r 34 CPPD c r y s t a l s were a l s o c o l l e c t e d on the same instrument i n open crimped aluminum pans i n a n i t r o g e n atmosphere and heated a t 10°C/min from 150° to 350°C. CPPD samples were h e l d i s o t h e r m a l l y a t 350°C f o r 30 minutes. V a p o r i z a t i o n of water of h y d r a t i o n from the pans c o n t a i n i n g MSUM and CPPD samples was estimated q u a n t i t a t i v e l y by weighing the pans a f t e r the appearance of the endothermic peak. Percent water l o s s e s were c a l c u l a t e d f o r MSUM and CPPD samples. 2 . 3 . PARTICLE SIZE ANALYSIS 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 of the c r y s t a l s was determined by o p t i c a l microscopy u s i n g a 0.01 mm Nikon o b j e c t i v e micrometer and eyepiece micrometer w i t h a Nikon d i f f e r e n t i a l i n t e r f e r e n c e microscope. The samples were suspended i n l i q u i d p a r a f f i n and 100 c r y s t a l s were measured f o r each of CPPD, MSUM (method of Burt e t al. , 1983) and MSUM (modi f i e d method) samples. 2 . 4 . SCANNING ELECTRON MICROSCOPY Samples of MSUM and CPPD c r y s t a l s were a t t a c h e d t o the s u r f a c e of a metal stub with g r a p h i t e p a i n t and the samples were g o l d coated on a Hummer s p u t t e r g o l d c o a t e r i n an Argon atmosphere. The samples were then examined on a H i t a c h i S- 57 scanning e l e c t r o n microscope. 35 3 . PREPARATION OF NEUTROPHIL SUSPENSION: N e u t r o p h i l s were separated from f r e s h l y c o l l e c t e d human whole blood obtained from the Red Cross, Vancouver, B.C. or from h e a l t h y v o l u n t e e r s . Red Cross blood was from a s i n g l e donor and not pooled. To 450 mL of c i t r a t e t r e a t e d whole human blood i n polypropylene stopper b o t t l e s were added 100 mL of 3% dextran s o l u t i o n i n Hanks b u f f e r and the blood/dextran suspension allowed t o stand undisturbed. The e r y t h r o c y t e s sedimented i n the presence of dextran and the n e u t r o p h i l r i c h supernatant was removed as soon as i t appeared, t o reduce l o s s due t o s e t t l i n g of the n e u t r o p h i l s . A l i q u o t s of the n e u t r o p h i l r i c h supernatant (5 mL) were layered over 4 mL of F i c o l l - P a q u e i n polypropylene tubes. The tubes were c e n t r i f u g e d at 400 x g at 20 °C f o r 15 minutes. The n e u t r o p h i l p e l l e t thus obtained was suspended i n 1.5 mL of d i s t i l l e d water at 4°C with gentle v o r t e x i n g t o l y s e contaminating e r y t h r o c y t e s . T o n i c i t y was r e s t o r e d a f t e r 10 seconds by the a d d i t i o n of 0.5 mL of 0.6 M sodium c h l o r i d e . The tubes were c e n t r i f u g e d at 200 x g at 4°C f o r 5 minutes. The supernatant was discarded and the l y s i s treatment was repeated. The p e l l e t obtained a f t e r the second l y s i s step was resuspended i n 6 mL Hanks b u f f e r and kept on i c e u n t i l used. 36 3.1. ESTIMATION OF NEUTROPHIL COUNT: Samples of n e u t r o p h i l suspensions obtained by the method desc r i b e d above were sent p e r i o d i c a l l y t o the Dept. of Laboratory Medicine, U n i v e r s i t y H o s p i t a l (UBC S i t e ) f o r d i f f e r e n t i a l c e l l counts. On a r o u t i n e b a s i s , the t o t a l c e l l count (and t h e r e f o r e an estimate of the n e u t r o p h i l count) i n the c e l l suspension was determined from the l a c t a t e dehydrogenase (LDH) content i n an a l i q u o t of l y s e d c e l l s using the Sigma LDH assay k i t . A 10% s o l u t i o n of T r i t o n X-100 was added t o 0.4 mL of the c e l l suspension, vortexed f o r 5 minutes and c e n t r i f u g e d at 200 x g f o r 5 minutes. To 2.2 mL of l a c t a t e reagent at 30°C was added 110 J I L of supernatant i n a U V / v i s i b l e cuvette and the absorbance measured at 340 nm using the HP Vect r a spectrophotometer i n the k i n e t i c s mode f o r a p e r i o d of 240 seconds. The absorbance was read every 3 0 seconds and the d i f f e r e n c e between the absorbances at 180 and 60 seconds used t o c a l c u l a t e the change i n absorbance per minute. The c e l l count was then estimated from a standard curve. 3.2. ESTIMATION OF NEUTROPHIL VIABILITY: 3.2.1. CL determination The time elapsed between blood c o l l e c t i o n and p r e p a r a t i o n of the n e u t r o p h i l suspension was between 24-48 hours f o r Red Cross blood and 4-6 hours f o r blood from UBC v o l u n t e e r s . As 37 a r a p i d t e s t t o determine whether Red Cross blood n e u t r o p h i l s were s t i l l v i a b l e f o l l o w i n g completion of the sep a r a t i o n procedure, the MSUM stim u l a t e d chemiluminescent response of Red Cross blood n e u t r o p h i l s was compared t o th a t of UBC volunt e e r blood n e u t r o p h i l s . To a 1.5 mL polypropylene Eppendorf tube was added 5 mg uncoated MSUM, 10 ill of 1 x 10E-3 M luminol s o l u t i o n i n DMSO ( f i n a l 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 of the c e l l suspension t o giv e a f i n a l n e u t r o p h i l c o n c e n t r a t i o n of 2 x 10E6 cells/mL. The CL produced by the n e u t r o p h i l s was monitored on a B i o - O r b i t 1250 Luminometer. The instrument was i n t e r f a c e d w i t h an IBM compatible PC and the data c o l l e c t i o n was done through a software package f o r the same obtained from the manufacturer. The tube was placed i n the chemiluminometer and maintained at 37 °C by means of a jacket e d tube holder connected to a c i r c u l a t i n g water bath. Every 3 minutes the tube was removed from the tube holder and the contents g e n t l y a g i t a t e d . The a g i t a t i o n was re q u i r e d t o overcome the problem of s e t t l i n g of the c r y s t a l s w i t h i n the tube l e a d i n g to a reduced i n t e r a c t i o n of c e l l s w i t h the c r y s t a l s . The response was stu d i e d f o r 3 0 minutes w i t h 1000 sampling p o i n t s i n t h i s i n t e r v a l i . e . readings were taken every 1.8 seconds. 38 3 . 2 . 2 . S t a i n i n g with Trypan b l u e An a l i q u o t of the n e u t r o p h i l suspension was mixed with an equal volume of 0.5% tryp a n blue i n 0.9% s a l i n e , incubated a t room temperature f o r 5 minutes and the n e u t r o p h i l s examined under the microscope f o r dye uptake. 4 . PROTEIN COATING OF CRYSTALS Samples of 5 mg MSUM or 50 mg CPPD were weighed i n t o 1.5 mL po l y p r o p y l e n e Eppendorf tubes. P r o t e i n s o l u t i o n s were prepared 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 or Hanks b u f f e r p r e s a t u r a t e d with MSUM at 37° (Hanks s a t u r a t e d with MSUM) f o r c o a t i n g MSUM. One mL of the f o l l o w i n g p r o t e i n s o l u t i o n s was added t o the c r y s t a l s and the tubes tumbled a t 37°C f o r 30 minutes: 6 mg/mL Ig G (3 mg/mL f o r CPPD), 16.67 mg/mL BSA and plasma d i l u t e d 50:50 wi t h Hanks / Hanks s a t u r a t e d with MSUM. The tubes were c e n t r i f u g e d i n an Eppendorf c e n t r i f u g e a t 16,000 x g f o r 5 minutes. The c r y s t a l p e l l e t was washed by resuspending i n 1 mL of Hanks b u f f e r or Hanks s a t u r a t e d with MSUM, c e n t r i f u g a t i o n a t 16,000 x g and d i s c a r d i n g the supernatant. 39 5. MEASUREMENT OF NEUTROPHIL RESPONSE: 5.1. SUPEROXIDE ANION RELEASE 5.1.1. Superoxide 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 uncoated MSUM To a 1.5 mL Eppendorf tube was added 0.2 mL of a 25 mg/mL MSUM suspension i n Hanks saturated w i t h MSUM, 0.1 mL of a 10 mg/mL ferricytochrome c s o l u t i o n i n Hanks saturated w i t h MSUM, 0.2 mL of Hanks saturated w i t h MSUM and 0.5 mL of a 1 x 10E7 cells/mL n e u t r o p h i l suspension. The tubes contained 5 mg MSUM and 5 x 10E6 n e u t r o p h i l s . The tubes were r o t a t e d end-over-end at 15 rpm at 37°C f o r 180 minutes. At i n t e r v a l s , tubes were removed and c e n t r i f u g e d i n an Eppendorf c e n t r i f u g e at 16,000 x g f o r 30 seconds and sto r e d on i c e . A 0.75 mL a l i q u o t of the supernatant was withdrawn and the absorbance of the sample at 550 nm measured on a diode array spectrophotometer i n a polystyrene 1 mL cuvette 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 to the samples w i t h the s u b s t i t u t i o n of 0.2 mL of Hanks saturated w i t h MSUM f o r the 0.2 mL of c r y s t a l suspension. Blanks were 0.1 mL of ferricytochrome 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 the samples. 40 5.1.2. Superoxide 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 uncoated CPPD: Tubes c o n t a i n i n g 50 mg CPPD and 5 x 10E6 n e u t r o p h i l s i n Hanks b u f f e r were prepared as described above and r o t a t e d end-over-end at 15 rpm and 37 ° C f o r 180 minutes. At i n t e r v a l s tubes were withdrawn and c e n t r i f u g e d i n an Eppendorf c e n t r i f u g e at 16,000 x g f o r 30 seconds and stored on i c e . A 0.75 mL a l i q u o t of the supernatant was withdrawn and the absorbance of the sample at 550 nm measured on the HP V e c t r a spectrophotometer i n a polystyrene 1 mL cuvette. 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 to the samples w i t h the s u b s t i t u t i o n of 0.2 mL of Hanks b u f f e r f o r the 0.2 mL of c r y s t a l suspension. Blanks were 0.1 mL of ferricytochrome 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 to 1 mL and t r e a t e d s i m i l a r l y t o the samples. The r a t e of superoxide production was determined from the l i n e a r change i n absorbance at 550 nm t h a t occurs a f t e r an i n i t i a l phase of no absorbance change followed by an i n c r e a s i n g r a t e of change. Rates were reported i n nmoles C>2~/min/106 PMNL. The r a t e was c a l c u l a t e d u s i n g the formula shown below: nmoles O 2~/min/10 6 PMNL = A 5 5 0 ( 0 . 0 2 1 1 ) ( c e l l cone)(10~ 6) where 0.0211 i s the e x t i n c t i o n c o e f f i c i e n t of f e r r i c y t o c h r o m e c. 41 5.1 . 3 . E f f e c t of SOD on superoxide release To show that ferricytochrome c was reduced by superoxide anion and not other species, the level s of reduced ferricytochrome c were monitored in the presence of SOD, an enzyme which dismutates superoxide. These experiments were performed as described above with the following modifications: 0.1 mL of 3000 U/mL SOD solution i n Hanks buffer (3 00 U/tube) was added to the tubes instead of 0.1 mL of buffer and tubes were withdrawn only at 10 minutes i n the case of MSUM and at 60 minutes in the case of CPPD. Control tubes were as described previously. 5.1.4. Adsorption of SOD by MSUM cr y s t a l s In the following studies MSUM cr y s t a l s were incubated with solutions containing SOD, centrifuged and the supernatant added to tubes containing CPPD and neutrophils. The rat i o n a l e for t h i s approach was that i f MSUM c r y s t a l s adsorb s i g n i f i c a n t amounts of SOD from solution, then the reduced concentration of SOD i n the supernatant added to the CPPD/neutrophil suspensions should r e s u l t i n a decreased i n h i b i t i o n of CPPD induced superoxide release. To each of three 1.5 mL Eppendorf tubes was added 0.2 mL of a 20 mg/mL ferricytochrome c solution (4 mg/tube), 0.2 mL of Hanks saturated with MSUM, 0.1 mL of a 3000 U/mL SOD solu t i o n (300 U/tube) and 10 mg MSUM c r y s t a l s . Another set of three tubes without the cr y s t a l s was run as controls. 42 The tubes were tumbled end-over-end at 15 rpm at 37°C f o r 30 minutes. The tubes were then c e n t r i f u g e d i n an Eppendorf c e n t r i f u g e at 16,000 x g f o r 30 seconds and 0.5 mL of the supernatant added t o another set of 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 suspension (50 mg/tube). F i v e hundred m i c r o l i t e r s of a 1 x 10E7 cells/mL c e l l suspension were added t o the tubes which were then incubated at 37°C f o r 60 minutes. Another set of tubes w i t h CPPD c r y s t a l s but not c o n t a i n i n g any SOD was a l s o run as a p o s i t i v e c o n t r o l . The absorbance was measured at 550 nm as described e a r l i e r . 5.1.5. Superoxide release from neutrophils on stimulation by protein coated MSUM and CPPD To tubes c o n t a i n i n g 5 mg p r o t e i n coated MSUM or 50 mg p r o t e i n coated CPPD were added 0.1 mL of a 10 mg/mL ferricyt o c h r o m e c s o l u t i o n (1 mg/tube), 0.3 mL of Hanks sat u r a t e d w i t h MSUM or Hanks b u f f e r and 0.5 mL of a 1 x 10E7 cells/mL n e u t r o p h i l suspension. Tubes c o n t a i n i n g uncoated c r y s t a l s , ferricytochrome c and n e u t r o p h i l s were a l s o prepared as described above. Co n t r o l tubes ( c r y s t a l s absent) were prepared as above. The tubes were tumbled end over end at 15 rpm at 37°C f o r 10 minutes f o r MSUM co n t a i n i n g tubes and 60 minutes f o r CPPD c o n t a i n i n g tubes. A l l tubes were c e n t r i f u g e d at 16,000 x g f o r 30 seconds, 0.75 mL of the supernatant withdrawn and the absorbance at 550 nm measured. 43 5.2. CHEMILUMINESCENCE 5.2.1 Chemiluminescent response of n e u t r o p h i l s 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 of uncoated MSUM or 10, 20, 30, 40, 50 mg of uncoated CPPD c r y s t a l s , 10 / i l of a 1 x 10E-3 M luminol s o l u t i o n i n DMSO ( f i n a l c o n c e n t r a t i o n 10E-5 M) and 0.5 mL of a 1 x 10E7 cells/mL n e u t r o p h i l suspension. The tubes were maintained at 37°C i n the F i s h e r Isotemp Dry Bath and read i n the tube holder of the chemiluminometer at the same temperature. Every 1.5-3 minutes the tube was removed from the tube holder and the contents g e n t l y a g i t a t e d . The response was st u d i e d f o r 60 minutes with 2000 sampling p o i n t s i n t h i s i n t e r v a l i . e . readings were taken every 1.8 seconds or t i l l the response abated. The response was recorded as a graph of mV versus time. 5.2.2. Chemiluminescent response of 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 coated MSUM and CPPD c r y s t a l s Tubes c o n t a i n i n g 5 mg of uncoated or p r o t e i n coated MSUM and 3 0 mg of uncoated or p r o t e i n coated CPPD were prepared. To each tube was added 0.5 mL of a 1 x 10E7 cells/mL n e u t r o p h i l suspension and the tubes maintained at 37°C i n the F i s h e r Isotemp Dry Bath. The response was recorded f o r 15 seconds f o r each tube i n succession, i . e . uncoated c r y s t a l tube f o l l o w e d by tubes c o n t a i n i n g BSA-coated, Ig G-coated and 44 plasma p r o t e i n coated c r y s t a l s i n tha t order f o r a p e r i o d of 30 minutes. The tubes were mixed p r i o r to measurement. The r a t i o n a l e f o r t h i s p a r t i c u l a r sequence of measurements was because i t was necessary to minimize any d i f f e r e n c e s i n response due to changing c e l l v i a b i l i t y . 5 . 3 . DEGRANULATION INDICATOR: RELEASE OF MPO AND LYZ Degranulation and r e l e a s e of MPO and LYZ were s t u d i e d i n n e u t r o p h i l s p r e t r e a t e d w i t h c y t o c h a l a s i n B t o prevent phagocytosis of c r y s t a l s and subsequent n e u t r o p h i l c y t o l y s i s . 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 n e u t r o p h i l suspension t o give a f i n a l c o n c e n t r a t i o n of 10 jug/mL c y t o c h a l a s i n B and the suspension incubated at 37°C f o r 30 minutes. 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 uncoated 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 of 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 suspension at 2.5 x 10E7 cells/mL was added 0.1 mL of e i t h e r a 25 mg/mL MSUM suspension i n Hanks sat u r a t e d w i t h MSUM or a 250 mg/mL CPPD suspension i n Hanks b u f f e r . C o n t r o l tubes ( c r y s t a l s absent) were a l s o prepared as described above. The tubes were tumbled end over end at 15 rpm at 37°C and withdrawn at 5, 10,20, 30, 45, 60, 90, 120, 150 and 180 minutes. The tubes were 45 c e n t r i f u g e d i n a Beckmann Cent r i f u g e at 200 x g f o r 2 minutes at 0°C and the supernatant removed f o r a n a l y s i s . 5 . 3 . 2 . Measurement of LYZ LYZ was determined using the method of Ginsberg et al. (1977). A standard curve f o r the LYZ assay was prepared over a c o n c e n t r a t i o n range of 0-200 units/mL. LYZ from chicken egg white, obtained from Sigma Chemical Co. was used as the standard. A s e r i e s of s o l u t i o n s c o n t a i n i n g 25, 50, 75, 100, 125, 150, 175 and 200 units/mL were prepared and the change i n absorbance at 4 50 nm per minute, produced when 100 m i c r o l i t e r s of each LYZ s o l u t i o n were added to a suspension of Micrococcus lysodeikticus at 25°C was monitored. The change i n absorbance per minute was p l o t t e d a g a i n s t the c o n c e n t r a t i o n of LYZ i n units/mL. To a quartz cuvette c o n t a i n i n g 2.5 mL of Micrococcus l y s o d e i k t i c u s suspension (A450nm = 0.6-0.7) was added 100 m i c r o l i t e r s of supernatant obtained 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 / c r y s t a l incubations and the decrease i n t u r b i d i t y monitored over a p e r i o d of 12 0 seconds. The change i n absorbance per minute was c a l c u l a t e d and the c o n c e n t r a t i o n of LYZ c a l c u l a t e d using the equation f o r the standard curve. 46 5.3.3. Measurement of MPO A 0.05 mL a l i q u o t of the supernatant obtained 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 / c r y s t a l incubations was added t o a 1 mL cuvette c o n t a i n i n g 0.89 mL 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 of 1% T r i t o n X-100, and 0.01 mL of 10 mM H 20 2 and the change i n absorbance at 450 nm measured f o r 120 seconds at 25°C. The r a t e of o x i d a t i o n of d i a n i s i d i n e which i s d i r e c t l y p r o p o r t i o n a l t o MPO concent r a t i o n was c a l c u l a t e d from the f o l l o w i n g equation: Rate (nmol/min) = 50 X A (units) where A i s the change i n absorbance 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 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 Tubes c o n t a i n i n g 2.5 mg p r o t e i n coated MSUM or 25 mg p r o t e i n coated CPPD were prepared and incubated as described above except t h a t i n c u b a t i o n times were 4 5 minutes f o r MSUM co n t a i n i n g tubes and 60 minutes f o r CPPD c o n t a i n i n g tubes. 6. STATISTICAL TESTS S t a t i s t i c a l e v a l u a t i o n s were performed using e i t h e r a two sample t t e s t or one way ANOVA to compare mean values between groups. A s i g n i f i c a n c e l e v e l of p < 0.05 two t a i l e d was used. A l l t e s t s were done using the Statview program on 47 a Macintosh computer. The values shown i n the graphs represent the mean + standard e r r o r of the 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 prepared i n t h i s l a b o r a t o r y by the method described by Burt et al. ( 1 9 8 3 ) . The previous procedure i n v o l v e d d i s s o l u t i o n of u r i c a c i d i n d i s t i l l e d water t o which sodium hydroxide had been added, fo l l o w e d by a d d i t i o n of sodium hydroxide t o adjust the pH of the r e a c t i o n mixture to 8.9. 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 of the r e s u l t i n g c r y s t a l s showed a high p r o p o r t i o n w i t h l a r g e p a r t i c l e s i z e s (over 50% of c r y s t a l s were 75 ̂ m or greater) Figure 4. This n e c e s s i t a t e d an a d d i t i o n a l step of s i z i n g of the 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 s i z e c r y s t a l s f o r use i n experiments (Burt and Jackson, 1989). T h e o r e t i c a l l y , an increase i n the degree of s u p e r s a t u r a t i o n of the sodium urate s o l u t i o n would increase the d r i v i n g f o r c e f o r c r y s t a l l i z a t i o n l e a d i n g to the production of sma l l e r s i z e c r y s t a l s . The degree of su p e r s a t u r a t i o n of monosodium urate i n the s o l u t i o n can be increased by e i t h e r adding more s o l u t e or by a l t e r i n g the c o n d i t i o n s such t h a t the s o l u b i l i t y i s reduced. The s o l u b i l i t y of sodium urate at pH 8.9 i s gre a t e r than at pH 7.5. Hence, i t was f e l t t h a t a decrease i n the pH of the PARTICLE SIZE OF M S U M CRYSTALS (Method of Burt and Jackson, 1983) 0 25 50 75 100 125 150 175 200 225 L E N G T H (MICRONS) FIGURE 4 50 supersaturated s o l u t i o n from 8.9 to 7.5 would cause a s i g n i f i c a n t increase i n the degree of s u p e r s a t u r a t i o n and r e s u l t i n the production of smaller c r y s t a l s of 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 of MSUM c r y s t a l s obtained by the modified method i s shown i n Figure 5. The m o d i f i c a t i o n , which i n v o l v e d a l t e r a t i o n of pH of the s o l u t i o n p r i o r t o c r y s t a l l i z a t i o n , r e s u l t e d i n smaller p a r t i c l e s i z e s . The range of p a r t i c l e s i z e was between 0.1-50 /xm wi t h a mean p a r t i c l e s i z e of 21 /xm and a smaller standard d e v i a t i o n about the mean. 1.2. CALCIUM PYROPHOSPHATE DIHYDRATE (CPPD) 1.2.1. Synthesis of calcium dihydrogen pyrophosphate (CDPP) The s y n t h e s i s of CDPP was c a r r i e d out using the p r e v i o u s l y reported method (Burt and Jackson, 1987). The CDPP c r y s t a l s obtained were c h a r a c t e r i z e d by X-ray powder d i f f r a c t i o n p r i o r t o use i n the syn t h e s i s of CPPD (Figure 6, Table 1) . In Tables 1-4, the term x d space' r e f e r s t o the di s t a n c e between the c r y s t a l planes w h i l e N I ( r e l ) ' r e f e r s t o the r e l a t i v e i n t e n s i t y of the peak compared to the i n t e n s i t y of the strongest peak. CDPP p r e v i o u s l y synthesized i n the l a b was used f o r comparison purposes. The d i f f r a c t i o n peaks at 2 values of 23.8, 25.25, 26.7, 28.0, 33.25 and 40.55 were used f o r i d e n t i f i c a t i o n of CDPP. PARTICLE SIZE OF M S U M CRYSTALS (Modified method) F IGURE 5 H XRAY DIFFRACTION PATTERN OF C A L C I U M DIHYDROGEN PYROPHOSPHATE 3 0 0 0 r 2 4 0 0 1 8 0 0 o CO to c p o o £ 1 2 0 0 10 c 6 0 0 0 10 15 2 0 25 2 Theta 30 35 40 FIGURE 6 53 TABLE 1 X-RAY PEAKS OBTAINED FOR CDPP Sample I d e n t i f i c a t i o n : Calcium dihydrogen pyrophosphate (CDPP) Peak 2-Theta d space I ( r e l ) 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. S y n t h e s i s o f c a l c i u m p yrophosphate d i h y d r a t e ( t r i c l i n i c ) T r i c l i n i c c r y s t a l s o f CPPD were o b t a i n e d w i t h i n a p e r i o d o f 24 h r t o 4 days f o l l o w i n g s y n t h e s i s . 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 CPPD c r y s t a l s i s shown i n F i g u r e 7. The range o f p a r t i c l e s i z e was between 0.1-95 jum w i t h a mean p a r t i c l e s i z e o f 29 /xm. 2. CHARACTERIZATION OF CRYSTALS 2.1. X-RAY DIFFRACTION MSUM and CPPD c r y s t a l s were i d e n t i f i e d by t h e X-ray d i f f r a c t i o n p a t t e r n s o b t a i n e d on a 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 . The 2 v a l u e s , d s p a c i n g s and r e l a t i v e i n t e n s i t i e s o f t h e d i f f r a c t i o n peaks of MSUM p r e p a r e d by t h e p r e v i o u s method o f B u r t e t a l . (1983) and t h e m o d i f i e d method a r e g i v e n i n T a b l e s 2 and 3 r e s p e c t i v e l y w h i l e t h e d i f f r a c t i o n p a t t e r n s a r e shown i n F i g u r e s 8 and 9 r e s p e c t i v e l y . The d i f f r a c t i o n peaks a t 2 v a l u e s o f 3.16, 3.48, 4.69, 4.92, 7.58 and 9.40 were compared t o c o n f i r m t h a t t h e c r y s t a l s p r e p a r e d by the m o d i f i e d method were t h o s e o f monosodium u r a t e monohydrate. The X - ray d i f f r a c t i o n p a t t e r n o f CPPD c r y s t a l s i s shown i n F i g u r e 10 and t h e d a t a g i v e n i n T a b l e 4. The d i f f r a c t i o n peaks a t 2 v a l u e s o f 3.12, 3.24, 7.07 and 8.1 were compared t o t h e s t a n d a r d p a t t e r n f o r CPPD ( J o i n t Committee on Powder PARTICLE SIZE DISTRIBUTION OF CPPD CRYSTALS (n = 100) 3 0 T L E N G T H (MICRONS) F I G U R E 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 20.0 22.5 25.0 27.5 30.0 2Theta FIGURE 8 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 X R A Y D I F F R A C T I O N P A T T E R N O F M O N O S O D I U M U R A T E M O N O H Y D R A T E ( M O D I F I E D M E T H O D ) 1300 r 1170 - 1040 - o <u to \ 910 - to r 780 - (c ou i 650 - >̂ 'to 520 • I 390 - 2 Theta F I G U R E 9 TABLE 3 X-RAY PEAKS OBTAINED FOR MSUM (modified method) Peak 2-Theta d space I ( r e l ) 1 8.300 10.6442 22 . 65 2 9.400 9.4009 31.86 3 11.650 7.5899 37.45 4 16.650 5.3202 23 .82 5 17.950 4.9377 37.01 6 18.900 4.6916 60.54 7 19.550 4.5371 17.57 8 25.150 3.5381 16.26 9 25.550 3.4836 31.14 10 28.200 3.1620 100.00 11 29.300 3.0457 27 . 09 12 33.650 2.6613 39.96 13 36.400 2.4663 26. 56 X R A Y 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 P Y R O P H O S P H A T E D I H Y D R A T E 1200 r o > C o o o c 0) 1080 960 840 720 600 480 360 240 120 0 8 11 14 17 20 23 2 Theta FIGURE 10 26 29 32 35 TABLE 4 X-RAY PEAKS OBTAINED FOR CPPD Peak 2-Theta d space I ( r e l ) 1 11.000 11.9443 32.84 2 12.650 10.3915 100 3 16.950 7.7679 6.79 4 19.750 6. 6753 7 . 60 5 22.100 5.9730 13.49 6 23.050 5.7299 7.99 7 25.750 5.1377 10.72 8 27.550 4.8079 43 . 03 9 28.600 4.6349 23 . 66 10 29.050 4.5646 19.57 11 30.000 4.4232 13 .13 12 32.200 4.1282 8.75 13 33.500 3.9723 12 . 03 62 D i f f r a c t i o n Standards, JCPDS pattern) (Burt and Jackson, 1987) t o confirm t h a t the prepared c r y s t a l s were those of calcium pyrophosphate dihydrate ( t r i c l i n i c ) . 2.2. DIFFERENTIAL SCANNING CALORIMETRY (DSC) The DSC curve obtained f o r calcium pyrophosphate d i h y d r a t e samples i s shown i n Figure 11. The presence of two endothermic peaks, the f i r s t between 190 t o 285°C and the second between 285 to 300°C corresponded t o the l o s s of two moles of water from the c r y s t a l s (average weight l o s s 12.3% at 300°C; molecular weight of CPPD 290) confirming the presence of the calcium pyrophosphate as a dih y d r a t e . C r y s t a l s of monosodium urate prepared by the Burt et a l . (1983) method as w e l l as the modified method were analyzed by DSC and the curves obtained are shown i n Figures 12 and 13 r e s p e c t i v e l y . The broad endothermic peak between 230 t o 300°C corresponded t o the l o s s of one mole of water of hy d r a t i o n (average weight l o s s 10.2%; molecular weight of MSUM 209) confirming the monohydrate form of these c r y s t a l s . 2.3. SCANNING ELECTRON MICROSCOPY (SEM) Figures 14-16 show scanning e l e c t r o n micrographs of CPPD c r y s t a l s and MSUM c r y s t a l s prepared by the method of Burt et a l . (1983) and the modified method. The micrometer length p r i n t e d i n the lower r i g h t hand corner of the micrographs r e f e r s t o the length of the l a r g e s t c r y s t a l i n the 1 3 U 300 c DSC THERMOGRAM OF CPPD CRYSTALS FIGURE 11 3 0 0 ° DSC THERMOGRAM OF MSUM CRYSTALS (BURT et al., 1983) FIGURE 12 DSC THERMOGRAM 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 MSUM CRYSTALS (Burt etal., 1983) FIGURE 15 68 SCANNING E L E C T R O N MICROGRAPH OF MSUM CRYSTALS (Modified method) FIGURE 16 69 micrograph . The MSUM c r y s t a l s prepared by e i t h e r method were found t o have the t y p i c a l long needle shaped c r y s t a l h a b i t and the CPPD c r y s t a l s the t y p i c a l elongated p r i s m a t i c h a b i t . 3. ESTIMATION OF NEUTROPHIL COUNT AND VIABILITY D i f f e r e n t i a l counts of the prepared n e u t r o p h i l suspension were used t o determine the % p u r i t y of the p r e p a r a t i o n as w e l l as t o determine the c e l l count. The n e u t r o p h i l count thus o b t a i n e d was s i m i l a r t o the count e s t i m a t e d by the r e l e a s e of LDH from n e u t r o p h i l s l y s e d w i t h T r i t o n X-100. The prepared n e u t r o p h i l suspensions were found t o be more than 95% pure i . e . >95% of the c e l l s were polymorphonuclear l e u k o c y t e s . The a b i l i t y of the v i a b l e n e u t r o p h i l t o exclude the dye t r y p a n b l u e from the c e l l has p r e v i o u s l y been used as a measure of the v i a b i l i t y of i s o l a t e d human n e u t r o p h i l s (Babior and Cohen, 1981). The prepared n e u t r o p h i l suspension when examined by t h i s method had >95% c e l l v i a b i l i t y . Thus the method of i s o l a t i o n of n e u t r o p h i l s was found t o y i e l d c e l l p r e p a r a t i o n s of >95% p u r i t y and >95% v i a b i l i t y . The a b i l i t y of n e u t r o p h i l s t o produce CL depends on the v i a b i l i t y of the c e l l s . Thus the chemiluminescent response of 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 a r t i c u l a t e or s o l u b l e s t i m u l i c o u l d serve as an i n d i c a t o r of the v i a b i l i t y of the 70 c e l l p r e p a r a t i o n . Hence, the chemiluminescent response of ne u t r o p h i l s separated from Red Cross blood (processing time 24-48 hr) and st i m u l a t e d with uncoated MSUM c r y s t a l s i n the presence of luminol was used as a r a p i d t e s t of the v i a b i l i t y of the c e l l p r e p a r a t i o n . A l l observed responses were compared t o the response obtained when the n e u t r o p h i l c e l l p r e p a r a t i o n i s o l a t e d from blood c o l l e c t e d from human vo l u n t e e r s (processing time, 4-5 hours) was st i m u l a t e d w i t h MSUM i n the presence of lum 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 considered acceptable i f the CL response was grea t e r than or equal to 150 mV with a time t o maximum response of between 5-10 minutes. 4. MEASUREMENT OF NEUTROPHIL RESPONSES The weights chosen represented the weights corresponding t o the maximal response. In our s t u d i e s , surface areas of 5 mg MSUM and 50 mg CPPD were s i m i l a r (Burt et a l . , 1 9 8 9 ) . 4.1. CRYSTAL STIMULATED SUPEROXIDE RELEASE FROM NEUTROPHILS Figur e 17 shows the t y p i c a l time course of superoxide anion generation by n e u t r o p h i l s s t i m u l a t e d with uncoated MSUM and CPPD c r y s t a l s . There was a r a p i d increase i n superoxide l e v e l s up to about 20 minutes f o r MSUM to reach a peak value of about 15 nmoles 02~ generated per 10E6 neutrophils/mL f o r MSUM. The response of the n e u t r o p h i l s t o s t i m u l a t i o n w i t h CPPD c r y s t a l s was slower than the response t o s t i m u l a t i o n w i t h MSUM c r y s t a l s though the magnitude of the responses S U P E R O X I D E GENERATION BY N E U T R O P H I L S STIMULATED B Y UNCOATED M S U M AND C P P D C R Y S T A L S 20 T TIME ( m m ) FIGURE 17 72 were not d i f f e r e n t . The maximum superoxide anion l e v e l s were a t t a i n e d i n about 60 minutes f o r CPPD s t i m u l a t e d n e u t r o p h i l s and were about 14 nmoles per 10E6 neutrophils/mL. Controls were about 50% of the maximum values f o r superoxide r e l e a s e . Figures 18 and 19 show the graphs obtained f o r superoxide r e l e a s e when data from 4 experiments each f o r MSUM and CPPD were pooled. Naccache et a l . , (1991) a l s o found t h a t MSUM c r y s t a l s (3 mg/mL) produced a more r a p i d generation of superoxide than CPPD c r y s t a l s (3 mg/mL). The amounts of superoxide produced were maximal w i t h i n 10 min f o r both c r y s t a l s at about 9 nmoles and 5 nmoles superoxide per 10E6 n e u t r o p h i l s f o r MSUM and CPPD c r y s t a l s , r e s p e c t i v e l y . Terkeltaub et a l . (1984, 1988) expressed 0 2~ generation i n terms of nmoles ferricytochrome c reduced. At c r y s t a l concentrations of 5 mg/mL f o r both MSUM and CPPD, they reported values of between 3.2-5.4 nmoles ferricytochrome c reduced per 10E6 n e u t r o p h i l s f o r MSUM and 2.4 nmoles f erricytochrome c reduced per 10E6 n e u t r o p h i l s f o r CPPD. Other s t u d i e s of c r y s t a l - i n d u c e d superoxide r e l e a s e have included c y t o c h a l a s i n B i n the in c u b a t i o n medium t o prevent phagocytosis and i n t e r n a l i z a t i o n of the membrane bound NADPH dependent oxidase i n v o l v e d i n superoxide generation (Nagase et a l . , 1987; Rosen et a l . , 1986; Abramson et a l . , 1982; Higson et a l . , 1984). We have stud i e d the e f f e c t of adding 10 /xg/mL c y t o c h a l a s i n B t o the incubation medium on superoxide r e l e a s e and the r e s u l t s are shown i n Figure 20. 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 20 25 30 TIME ( m i n ) FIGURE 18 SUPEROXIDE GENERATION INDUCED BY CPPD CRYSTALS (n = 4) 30 x FIGURE 19 E F F E C T OF CYTOCHALASIN B ON M S U M INDUCED S U P E R O X I D E GENERATION FIGURE 20 76 s i g n i f i c a n t r e d u c t i o n i n the superoxide generation induced by MSUM c r y s t a l s (p < 0.05). C y t o c h a l a s i n B i s known t o i n h i b i t phagocytosis which i s accompanied by the r e s p i r a t o r y burst i n v o l v i n g superoxide r e l e a s e and CL. Hence i n h i b i t i o n of phagocytosis may r e s u l t i n i n h i b i t i o n of the r e s p i r a t o r y b u r s t as w e l l . Simchowitz et a l . (1982) reported the i n h i b i t i o n of MSUM induced oxygen uptake by c y t o c h a l a s i n and suggested t h a t the s t i m u l a t i o n of the r e s p i r a t o r y burst could be dependent on phagocytosis. 4 . 1 . 1 . EFFECT OF SOD ON SUPEROXIDE RELEASE The use of f erricytochrome c t o monitor the r e l e a s e of superoxide i s based on the re d u c t i o n of ferricytochrome c t o ferrocytochrome c. This r e d u c t i o n can be e f f e c t e d by s e v e r a l agents but only the red u c t i o n of f e r r i t o f e r r o by superoxide can be i n h i b i t e d by the enzyme SOD. To confirm t h a t the r e d u c t i o n being measured i n the assay was indeed due t o the superoxide being generated, the re d u c t i o n of ferricytochrome c was monitored i n the presence of the enzyme SOD. Re s u l t s are expressed i n terms of nmoles superoxide generated which represents the nmoles of ferricytochrome c reduced. The r e s u l t s obtained when CPPD was incubated w i t h n e u t r o p h i l s i n the presence of SOD are shown i n Figure 21. The re d u c t i o n of ferricytochrome c by superoxide generated by CPPD s t i m u l a t e d n e u t r o p h i l s was suppressed i n the presence of SOD i n d i c a t i n g t h a t the re d u c t i o n of ferricytochrome c measured when CPPD c r y s t a l s E F F E C T OF SOD ON CPPD INDUCED SUPEROXIDE R E L E A S E FROM N E U T R O P H I L S 2 0 T 16-- Q <D 00 12--<D O CD 8--a CO CO 6 4 - G 0 0 6 0 1 2 0 TIME IN MINUTES 1 8 0 O CONTROLS • 50 mg CPPD A CPPD + SOD 2 4 0 FIGURE 21 «0 78 were incubated w i t h n e u t r o p h i l s was due to the presence of superoxide. The r e s u l t s obtained when MSUM was incubated with n e u t r o p h i l s i n the presence of SOD are shown i n Figure 22. The r e d u c t i o n of ferricytochrome c by superoxide 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 not s i g n i f i c a n t l y suppressed i n the presence of SOD. This could be caused e i t h e r by the re d u c t i o n of ferricytochrome c being d r i v e n by another reducing species other than superoxide, or by the i n a c t i v a t i o n of SOD f o r example by adsorption of t h i s p r o t e i n onto the MSUM c r y s t a l s . To determine whether SOD was being adsorbed t o the MSUM c r y s t a l surface and thereby being i n a c t i v a t e d , the supernatant from the in c u b a t i o n of SOD w i t h MSUM c r y s t a l s was added t o CPPD/neutrophil i n c u b a t i o n s . The r e s u l t s of these incubations are shown i n Figure 23. The l e v e l s of reduced ferricytochrome c f o r CPPD/neutrophil incubations to which the MSUM/SOD supernatants had been added were not decreased and i n f a c t were g r e a t e r than f o r CPPD/neutrophil i n c u b a t i o n s . I t was l i k e l y the SOD had been i n a c t i v a t e d , probably by adsorption onto the surface of 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 of ferricytochrome c assay has been widely used t o monitor the generation of superoxide anion by n e u t r o p h i l s incubated w i t h MSUM c r y s t a l s (Terkeltaub et a l . , 1984; Abramson et a l . , 1982; Rosen et a l . , 1986; Naccache et a l . , 1991). However the i n a b i l i t y of 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 THE P R E S E N C E OF SUPEROXIDE DISMUTASE F I G U R E 22 E F F E C T OF M S U M TREATED SOD ON CPPD INDUCED S U P E R O X I D E R E L E A S E F R O M NEUTROPHILS 5 T CU +-> co cu c cu ciO V, 'x o u cu a 00 CO QJ o £ 4 3 ~ 2 ~ 0 0 4 5 1: CPPD 50 mg/mL ; 2: CPPD 50 mg/mL + SOD 300 U/mL ; 3: CPPD 50 mg/mL +MSUM TREAT FIGURE 23 81 SOD t o i n h i b i t the redu c t i o n of ferricytochrome c by superoxide generated from MSUM stim u l a t e d n e u t r o p h i l s has not been p r e v i o u s l y reported. The presence of BSA i n the b u f f e r s used i n previous s t u d i e s may have prevented any i n a c t i v a t i o n of SOD due to p r e f e r e n t i a l adsorption of BSA onto the MSUM c r y s t a l surface thus i n h i b i t i n g any subsequent ad s o r p t i o n of SOD onto the MSUM c r y s t a l s . 4.1.2. EFFECT OF PROTEINS ON SUPEROXIDE RELEASE The e f f e c t of p r o t e i n c o a t i n g on the MSUM c r y s t a l - i n d u c e d superoxide generation by n e u t r o p h i l s i s shown i n Figure 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) between the superoxide r e l e a s e d by uncoated MSUM compared t o Ig G, BSA or plasma p r o t e i n coated MSUM. Other s t u d i e s have shown t h a t p r e c o a t i n g of MSUM c r y s t a l s w i t h Ig G s i g n i f i c a n t l y enhanced MSUM induced superoxide generation (Abramson et a l . , 1982; Rosen et a l . , 1986; Nagase et a l . , 1989). However the superoxide r e l e a s e from n e u t r o p h i l s induced by uncoated MSUM i n our work was approximately 3 f o l d g r e a t e r than i n the previous s t u d i e s . I t i s p o s s i b l e t h a t under the assay c o n d i t i o n s used i n our s t u d i e s , the n e u t r o p h i l s were maximally s t i m u l a t e d by the uncoated MSUM c r y s t a l s such t h a t the c o a t i n g of the c r y s t a l s with Ig G could not enhance the generation of superoxide from the n e u t r o p h i l s . Abramson et a l . (1982) showed th a t precoating MSUM wi t h plasma d i d not a f f e c t superoxide generation from E F F E C T OF PROTEIN COATING ON THE GENERATION OF S U P E R O X I D E INDUCED B Y M S U M FIGURE 24 83 n e u t r o p h i l s , whereas Terkeltaub et a l . (1984) found t h a t p r e c o a t i n g MSUM with plasma s i g n i f i c a n t l y i n h i b i t e d MSUM induced superoxide generation. Figure 25 shows the e f f e c t of p r o t e i n c o a t i n g on the generation of superoxide induced by in c u b a t i o n w i t h CPPD c r y s t a l s . 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) i n the superoxide r e l e a s e induced by uncoated CPPD compared t o Ig G or plasma p r o t e i n coated CPPD. Again, i t i s p o s s i b l e t h a t n e u t r o p h i l s were maximally s t i m u l a t e d by both uncoated CPPD and Ig G-coated CPPD. Nagase et a l . (1989) found no s i g n i f i c a n t enhancement of superoxide generation when CPPD c r y s t a l s were coated w i t h Ig G. To our knowledge, there are no other r e p o r t s on the e f f e c t of p r o t e i n coatings on CPPD c r y s t a l - i n d u c e d superoxide generation. Although no d i f f e r e n c e s i n superoxide r e l e a s e were observed f o r p r o t e i n coated MSUM and CPPD versus uncoated c r y s t a l s , a l l the s t u d i e s were c a r r i e d out at an inc u b a t i o n time of 30 min f o r MSUM and 60 min f o r CPPD. I t i s p o s s i b l e t h a t there may be d i f f e r e n c e s i n the r a t e s of superoxide generation f o r n e u t r o p h i l s s t i m u l a t e d by p r o t e i n coated versus uncoated c r y s t a l s . 4.2. CRYSTAL STIMULATED CHEMILUMINESCENT RESPONSE OF NEUTROPHILS The chemiluminescent response of n e u t r o p h i l s to.. s t i m u l a t i o n by 5 mg of uncoated MSUM c r y s t a l s i s shown i n Figure 26. E F F E C T OF PROTEIN COATING ON SUPEROXIDE GENERATION INDUCED BY C P P D CRYSTALS 40 T CON CPPD BSA IGG PLASMA COATED COATED COATED F I G U R E 2 5 co CHEMILUMINESCENCE GENERATED BY NEUTROPHILS STIMULATED BY 5 mg UNCOATED MSUM 2100 > o z o in 1400 700 + 0 0 o o 0 o o 500 \ o \ O O 5 mg MSUM *0- ° - o - - o ~ o r 0 , n _ n _ 0 _ 0 . 1000 1500 2000 TIME IN SECONDS FIGURE 26 2 5 0 0 03 86 The b e l l shaped response curve was a f f e c t e d by changes i n c r y s t a l c o n c e n t r a t i o n as seen i n Figure 27. Table 5 shows the data expressed i n terms of the area under the curve (AUC) of the p l o t s of CL response i n mV versus time at each c r y s t a l c o n c e n t r a t i o n . An increase i n the c r y s t a l c o n c e n t r a t i o n from 2 mg to 5 mg/mL r e s u l t e d i n a higher maximum response and a decrease i n the time r e q u i r e d t o e l i c i t the maximal response. However f u r t h e r increases i n MSUM c r y s t a l c o n c e n t r a t i o n to 10 mg/mL d i d not s i g n i f i c a n t l y enhance the response observed compared t o 5 mg/mL MSUM. This could be due t o i n t e r f e r e n c e i n measurement due t o l i g h t s c a t t e r i n g by the c r y s t a l s at the higher c r y s t a l c o n c e n t r a t i o n s or due t o the l i m i t e d metabolic c a p a c i t y of the i s o l a t e d n e u t r o p h i l . The CL response of the n e u t r o p h i l s incubated w i t h 5 mg/mL MSUM may be near the maximum p o s s i b l e thus a l l o w i n g only a small increase when the c r y s t a l c o n c e n t r a t i o n i s doubled to 10 mg/mL. The chemiluminescent response of n e u t r o p h i l s t o 50 mg/mL CPPD c r y s t a l s i s shown i n Figure 28. The e f f e c t of CPPD co n c e n t r a t i o n on n e u t r o p h i l CL was st u d i e d over the con c e n t r a t i o n range of 10-50 mg/mL and the r e s u l t s obtained are shown i n Figure 29 and Table 6. The CL generated was in f l u e n c e d by the CPPD c r y s t a l c o ncentration. The CL produced increased as the CPPD concent r a t i o n was increased from 10 mg/mL t o 30 mg/mL but f u r t h e r increases i n the CPPD co n c e n t r a t i o n t o 50 mg/mL r e s u l t e d i n a re d u c t i o n of the EFFECT OF MSUM CONCENTRATION ON CHEMILUMINESCENCE 2700 T TIME IN SECONDS FIGURE 27 T A B L E 5 E F F E C T OF MSUM C R Y S T A L CONCENTRATION ON C H E M I L U M I N E S C E N C E C R Y S T A L C O N C . A U C 1 (mg/ltlL) (V-s) 2 656 (629 - 682) 5 1192 (1127 - 1256) 10 1140 (1139 - 1268) Note : 1 - AUC i s the area under the curve f o r p l o t s of CL response i n mV versus time. Values represent the mean of two determinations w i t h the range i n parentheses. 8 9 PQ W < EH GO GO S OH O E-< P ^ Q ^IP PQ < Q ° fe ^ < P P^ Q fe fe O fe W fe o p ; fe O CO fe P P fe p tlfl o LO Q a . O 0/) o • • • • • • I • • • • • • / • I • / • • • • • d o o cv o o Cv2 oa P o 4- 9co co P i—i E-1 O o CO o o o o o CO o o o o o CD CO w p a I—I 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 o o co P PI o 300 + 200 + 100 0 / \ \ T<$ A • • O • A • x • a 10 me CPPD 20 mg CPPD 30 mg CPPD 40 mg CPPD 50 mg CPPD 1600 TIME IN SECONDS 2400 3 2 0 0 FIGURE 29 VD O 91 TABLE 6 EFFECT OF CPPD CRYSTAL CONCENTRATION ON CHEMILUMINESCENCE CRYSTAL CONC. (mg/mL) 10 62 (50 - 73) 20 57 (56 - 58) 30 79 (74 - 82) 40 63 (55 - 70) 50 55 (47 - 62) Note : 1 - AUC i s the area under the curve f o r p l o t s of CL response i n mV versus time. Values r e p r e s e n t the mean of two de t e r m i n a t i o n s with the range i n parentheses. AUC 1 (V.s) 92 t o t a l amount of CL generated. A s i m i l a r trend of increased CL w i t h i n c r e a s i n g CPPD concentrations upto 30 mg/mL was observed at 25°C. Though the number of 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 s would be expected to increase w i t h c r y s t a l c o n c e n t r a t i o n , the accompanying increase i n s c a t t e r could reduce the amount of l i g h t reaching the p h o t o m u l t i p l i e r tube thus decreasing the CL detected. Another f a c t o r which may a f f e c t the CL response of n e u t r o p h i l s t o CPPD c r y s t a l s i s the tendency of the c r y s t a l s to s e t t l e r a p i d l y even duri n g the time of measurement (15 sec) at high c r y s t a l c o n c e n t r a t i o n s thus decreasing the number of p o s s i b l e 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 s . The n e u t r o p h i l chemiluminescent response appears to be maximal at a CPPD co n c e n t r a t i o n of 30 mg/mL. This CPPD con 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 experiments with CPPD. To our knowledge there are no other r e p o r t s of the e f f e c t of MSUM and CPPD c r y s t a l concentrations on the CL generated by n e u t r o p h i l s . We have s t u d i e d the e f f e c t of adding 10 jug/mL c y t o c h a l a s i n B to the in c u b a t i o n medium on the CL produced. There was a s i g n i f i c a n t (p < 0.05) red u c t i o n i n the CL produced i n the presence of c y t o c h a l a s i n B. The AUC f o r MSUM induced CL i n the absence of c y t o c h a l a s i n B was 588 + 108 V.s (mean of three determinations) w h i l e the AUC f o r MSUM induced CL i n the presence of c y t o c h a l a s i n B was 5.46 + 3 V.s (mean of three d e t e r m i n a t i o n s ) . Hence c y t o c h a l a s i n B was found t o 93 i n h i b i t the g e n e r a t i o n of superoxide and CL p r o d u c t i o n by n e u t r o p h i l s . T h i s may be due to the i n h i b i t i o n of pha g o c y t o s i s by c y t o c h a l a s i n B which may r e s u l t i n the i n h i b i t i o n of the accompanying r e s p i r a t o r y b u r s t (Simchowitz e t a l . , 1982) . 4.2.1. EFFECT OF TEMPERATURE ON THE CL RESPONSE The CL generated by n e u t r o p h i l s i n response t o c r y s t a l - induced s t i m u l a t i o n was dependent on the temperature of the suspending medium f o r n e u t r o p h i l s . Table 7 g i v e s the AUC f o r the CL curves o b t a i n e d when n e u t r o p h i l s were incubated w i t h MSUM c r y s t a l s a t 25°C and 37 °C. The i n c r e a s e i n AUC r e v e a l s t h a t the CL response was s i g n i f i c a n t l y enhanced when the n e u t r o p h i l s were maintained a t 37°C presumably due t o an enhanced o x i d a t i v e m e t a b o l i c a c t i v i t y of the n e u t r o p h i l s a t 37° compared t o t h a t a t 25°. Hence the n e u t r o p h i l s were maintained a t 37 °C f o r a l l f u r t h e r experiments. These r e s u l t s are i n good agreement with those r e p o r t e d by Harber and Topley (1986). 4.2.2. EFFECT OF PROTEINS ON THE CHEMILUMINESCENT RESPONSE F i g u r e s 3 0 and 31 show the t y p i c a l CL curves o b t a i n e d when n e u t r o p h i l s were exposed t o p r o t e i n coated MSUM and CPPD r e s p e c t i v e l y . I t was not p o s s i b l e t o measure the CL generated by a c o n t r o l s e t of tubes s i n c e the CL va l u e s were extremely low and below the d e t e c t i o n l i m i t s of the chemiluminometer. The AUCs of the p l o t s of CL response i n 94 TABLE 7 EFFECT OF SAMPLE TEMPERATURE ON MSUM INDUCED CHEMILUMINESCENCE AUC 1 MSUM (mg/mL) SAMPLE TEMPERATURE 25°C 37°C 2 443 656 (414 - 470) (629 - 682) 5 787 1191 (686 - 886) (1127-1257) 10 967 1198 (877 - 1056) (1139-1268) Note : 1 - AUC i s the area under the curve f o r p l o t s of CL response i n mV versus time. Values represent the mean of two determinations w i t h the range i n parentheses. E F F E C T OF PROTEIN 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 T O M S U M 2000 T 1500 + > Q P O P Q O <X p S 1000 p P CJ GO p p p P I P P CJ 500 + O UNCOATED MSUM • BSA COATED MSUM A Ig G COATED MSUM • A PLASMA COATED MSUM 1000 1500 TIME (sec) FIGURE 30 VO E F F E C T OF PROTEIN COATING ON THE 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 C P P D 350 T TIME (sec) FIGURE 31 VO 97 mV versus time f o r p r o t e i n coated and uncoated MSUM are given i n Table 8. There was s i g n i f i c a n t enhancement of the CL response when the c r y s t a l s were coated w i t h immunoglobulin g. This may be due to the opsonizing e f f e c t of I g G which f a c i l i t a t e s an i n t e r a c t i o n between the F c p o r t i o n of Ig G on the c r y s t a l surface and the F c receptors present on the n e u t r o p h i l membrane. S t a t i s t i c a l a n a l y s i s of the data revealed t h a t there was a s i g n i f i c a n t suppression of the n e u t r o p h i l CL response when the MSUM c r y s t a l s were precoated w i t h plasma p r o t e i n s . Terkeltaub et a l . (1984) reported t h a t plasma pr e c o a t i n g of MSUM c r y s t a l s i n h i b i t e d the CL generated by >50%. They suggested t h a t the low de n s i t y l i p o p r o t e i n f r a c t i o n bound to MSUM from plasma was re s p o n s i b l e f o r a major p o r t i o n of the plasma i n h i b i t i o n of n e u t r o p h i l CL responses t o MSUM. This i s an i n t e r e s t i n g o b s e r v ation because during a gout attack, the synovium of the j o i n t becomes more permeable and s y n o v i a l f l u i d l e v e l s of l a r g e molecules such as l i p o p r o t e i n s increases (Terkeltaub et a l . , 1986). The e f f e c t of 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 versus time p l o t s f o r p r o t e i n coated and uncoated CPPD are shown i n Table 8. Precoating of CPPD c r y s t a l s w i t h plasma was found t o suppress the CL response though t h i s suppression was not found to 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 the CL generated when TABLE 8 EFF E C T OF PROTEIN COATED AND UNCOATED MSUM AND CPPD ON CHEMILUMINESCENCE A U C 1 (V.S) MSUM 5 mg/mL CPPD 3 0 mg/mL Uncoated c r y s t a l s 306 + 85 IgG coated 570 + 97 38 + 8 103 + 22 (*) BSA coated 97 + 34 32 + 9 Plasma coated 20 + 10 (*•) 17 + 6 Note : 1 - AUC i s the area under the curve for plots of CL response i n mV versus time. Values represent the mean of 9 determinations for MSUM + standard error and 7 determinations for CPPD + standard error. * s i g n i f i c a n t at p < 0.05 99 c r y s t a l s were precoated with Ig G was 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). Again, i t i s l i k e l y that adsorbed Ig G enhanced the F c receptor mediated crystal-neutrophil a c t i v a t i o n and CL generation. The s l i g h t reduction in CL observed when c r y s t a l s were coated with BSA was not 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) for 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 ref e r s to the release of the contents of the primary and azurophilic granules of the neutrophil either into the phagosome formed a f t e r phagocytosis or d i r e c t l y into the e x t r a c e l l u l a r medium during invagination of the plasma membrane before phagocytosis i s complete. The primary and azurophilic granules of the neutrophil contain several enzymes, among them, MPO, LYZ, alpha mannosidase, collagenase, gelatinase and ft glucuronidase (Baggiolini and Dewald, 1984). Some of these enzymes are involved i n the generation of tox i c species such as the hydroxyl r a d i c a l , hypochlorous acid and chloramines while others are p r o t e o l y t i c in action. The release of these l y t i c contents into the e x t r a c e l l u l a r f l u i d either as a consequence of c e l l death or during the process of phagocytosis can have deleterious e f f e c t s on the surrounding ti s s u e s . In order to study the k i n e t i c s of degranulation following c r y s t a l 100 s t i m u l a t i o n i t i s necessary to a l t e r the n e u t r o p h i l such t h a t phagocytosis or i n t e r n a l i z a t i o n of the c r y s t a l i s i n h i b i t e d and complete degranulation occurs i n t o the e x t r a c e l l u l a r medium. The c y t o c h a l a s i n s are a group of compounds t h a t i n h i b i t phagocytosis but do not i n h i b i t d e g r a n u l a t i o n (Weissmann et a l . , 1972). Hence n e u t r o p h i l s were t r e a t e d w i t h c y t o c h a l a s i n B p r i o r t o s t i m u l a t i o n w i t h the p a r t i c u l a t e s t i m u l i t o study degranulation. C y t o c h a l a s i n B has been p r e v i o u s l y used i n s t u d i e s with human n e u t r o p h i l s (Nagase et a l . , 1987; Rosen et a l . , 1986; Abramson et a l . , 1982; Higson et a l . , 1984). However, i t i s important t o note t h a t t h i s a l t e r a t i o n of the a b i l i t y of the n e u t r o p h i l t o phagocytose could r e s u l t i n a l t e r a t i o n of other f u n c t i o n s of the c e l l as w e l l . Furthermore some s t u d i e s have revealed t h a t c y t o c h a l a s i n B has a tendency to s t i m u l a t e degranulation (Aaku et a l . , 1990; H o f f s t e i n et a l . , 1980). 4 . 3 . 1 . CRYSTAL STIMULATED MPO RELEASE FROM NEUTROPHILS The determination of MPO r e l e a s e d from n e u t r o p h i l s was based on the 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 hydrogen peroxide. I n c r e a s i n g amounts of supernatant obtained from T r i t o n X-100 l y s e d n e u t r o p h i l suspensions were added to a d i a n i s i d i n e r e a c t i o n mixture to determine the r e l a t i o n s h i p between the volume of supernatant added and the change i n absorbance produced. The r e s u l t s are shown i n Figure 32. The r e l a t i o n s h i p was found to be 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 THE MPO ASSAY (AT T = 60 sec) 1.200 T 1.000 + 0.800 + 0.600 + 0.400 0.200 0.000 0 Y = 1 . 0 6 1 6 E - 2 X + 8 . 6 8 8 8 E - 2 r = 0.9722 10 ' 20 30 40 50 60 70 VOLUME OF S U P E R N A T A N T ADDED (uL) FIGURE 32 102 30 t o 70 JXL, of supernatant added. Hence 50 fih of the supernatant were used i n a l l f u r t h e r determinations. This method of a n a l y s i s gives the r a t e of o x i d a t i o n 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 t o the co n c e n t r a t i o n of the enzyme MPO (Babior and Cohen, 1981). The r a t e of appearance of MPO i n the supernatant was c a l c u l a t e d from the d i f f e r e n c e i n the r a t e of o x i d a t i o n at va r i o u s time i n t e r v a l s . The time course f o r the re l e a s e of MPO obtained when 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 were incubated w i t h uncoated CPPD c r y s t a l s i s shown i n Figure 33. The maximum amount of MPO present i n the supernatant was between 10-45 min and the MPO l e v e l s observed on incu 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 than c o n t r o l values. The time course f o r the r e l e a s e of MPO when 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 were incubated w i t h uncoated MSUM c r y s t a l s i s shown i n Figure 34. The l e v e l of MPO i n the supernatants r e s u l t i n g from the MSUM/neutrophil incubations was lower than t h a t f o r the c o n t r o l set of experiments. This r a i s e d the p o s s i b i l i t y t h a t the enzyme was being removed from the supernatant by adsorption onto the MSUM c r y s t a l s . This was considered a l i k e l y p o s s i b i l i t y s i n c e MSUM c r y s t a l s have been p r e v i o u s l y reported t o adsorb many d i f f e r e n t p r o t e i n s (Terkeltaub et a l . , 1983; Kozin and McCarty, 1976; Kozin et a l . , 1979). In order to t e s t t h i s hypothesis the supernatant from n e u t r o p h i l s lysed with T r i t o n 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 FROM CYTOCHALASIN B P R E T R E A T E D NEUTRO ON STIMULATION WITH UNCOATED C P P D CRYSTALS F IGURE 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 CYTOCHALASIN B P R E T R E A T E D NEUTROPHILS 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 4 105 s o l u t i o n was incubated with i n c r e a s i n g MSUM c r y s t a l c o n c e n t r a t i o n s from 0.1 mg/mL to 10 mg/mL. The e f f e c t of i n c r e a s i n g the amount of MSUM added on the % red u c t i o n i n MPO a c t i v i t y can be seen i n Figure 35. I n c r e a s i n g amounts of MSUM r e s u l t e d i n s i g n i f i c a n t r e d u c t i o n of 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 being adsorbed onto the MSUM c r y s t a l surface from the supernatant. Adsorption of MPO appeared t o take place even i n the presence of many other p r o t e i n s r e l e a s e d by degranulation and c e l l death. Terkeltaub et a l . , (1991) stud i e d the degranulation of n e u t r o p h i l s induced by MSUM but they s t u d i e d the r e l e a s e of alpha mannosidase. 4 . 3 . 2 . EFFECT OF PROTEINS ON MPO RELEASE The e f f e c t of p r o t e i n c o a t i n g of the c r y s t a l s on the r e l e a s e of MPO from c y t o c h a l a s i n B pr 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 w i t h CPPD c r y s t a l s i s shown i n Figure 36. Precoating of CPPD c r y s t a l s with immunoglobulin g was found to s i g n i f i c a n t l y enhance the r e l e a s e of MPO, p o s s i b l y through the i n t e r a c t i o n with the F c receptors present on the n e u t r o p h i l s u r f a c e . Precoating with plasma and BSA d i d not have a s i g n i f i c a n t e f f e c t on the re l e a s e of MPO induced by CPPD c r y s t a l s . As seen i n Figure 37, the MPO re l e a s e induced by uncoated MSUM was l e s s than c o n t r o l samples and precoating MSUM c r y s t a l s with Ig G, BSA or plasma p r o t e i n s produced E F F E C T OF ADDITION OF INCREASING AMOUNTS OF M S U M ON MYELOPEROXIDASE 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 CYTOCHALASIN 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 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 NEUTROPHILS ON STIMULATION WITH PROTEIN COATED M S U M F I G U R E 3 7 § 109 s i g n i f i c a n t l y lower MPO r e l e a s e than f o r c o n t r o l s (p < 0.05). Hence, i t seems l i k e l y t h a t even the presence of a p r o t e i n coat on the c r y s t a l d i d not prevent f u r t h e r a d s o r p t i o n of MPO onto the c r y s t a l s u r f a c e . Due t o the a d s o r p t i o n of MPO onto the c r y s t a l s u r f a c e i t was not p o s s i b l e t o monitor the r e l e a s e of MPO as an i n d i c a t o r of d e g r a n u l a t i o n o c c u r r i n g when 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 were s t i m u l a t e d with MSUM c r y s t a l s . Hence i t was de c i d e d t o study the r e l e a s e of another enzyme r e l e a s e d d u r i n g d e g r a n u l a t i o n , LYZ. 4 . 3 . 3 . CRYSTAL STIMULATED LYZ RELEASE FROM NEUTROPHILS F i g u r e 38 shows the standard curve obtained by measuring the change i n absorbance of a Micrococcus lysodeikticus suspension a t 450 nm when standard egg white LYZ s o l u t i o n was added t o i t i n amounts ran g i n g from 500 t o 3500 U/mL. The r e l a t i o n s h i p was found t o be l i n e a r over the c o n c e n t r a t i o n range 0.1 to 200 LYZ units/mL. The time course f o r the r e l e a s e of LYZ 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 uncoated MSUM i s shown i n F i g u r e 39. The maximal response was observed between 30-60 min and was i n the range of 990 + 290 U/mL. T h i s r e p r e s e n t e d about 40% of t o t a l enzyme a c t i v i t y r e l e a s e d by l y s i s with T r i t o n X-100, which i s i n good agreement with v a l u e s r e p o r t e d by Simchowitz e t a l . (1982). STANDARD C U R V E FOR L Y S O Z Y M E A S S A Y B Y MICROCOCCUS METHOD 0.149 C=3 O z < ffl « O CO < u < Y = 0.0006 X + 0.0046 r = 0.994 0.099 -- 5 0.049 - - 6 . 0 0 0 E 50 75 100 125 150 L Y S O Z Y M E ADDED (un i t s ; 200 FIGURE 38 o LYSOZYME RELEASE INDUCED- BY MSUM C/3 0 -I 1 1 1 1 1 ; 0 20 40 60 80 • 100 1 2 0 TIME ( m i n u t e s ) FIGURE 3 9 112 The time course f o r the re l e a s e of LYZ induced by CPPD c r y s t a l s i s shown i n Figure 40. The response reached approximately 1500 + 240 U/mL i n 45-60 min and reached a maximum of 2200 + 120 at about 150 min. Figures 39 and 40 show t h a t the c o n t r o l values remained i n the 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 lower and subj e c t t o gre a t e r v a r i a t i o n compared to the LYZ r e l e a s e from CPPD s t i m u l a t e d n e u t r o p h i l s . This r a i s e d the p o s s i b i l i t y t h a t analogous t o MPO, LYZ was a l s o being adsorbed onto the MSUM c r y s t a l surface. Figure 41 shows the change i n absorbance produced by s o l u t i o n s of egg white LYZ incubated with MSUM c r y s t a l s . There was a s l i g h t r e d u c t i o n i n a c t i v i t y of LYZ when incubated w i t h MSUM c r y s t a l s . Kozin and McCarty (1967) s t u d i e d the adsorption of 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 i n c l u d i n g LYZ, albumin and Ig G. They found t h a t the adsor p t i o n of LYZ to MSUM was greater than the adso r p t i o n of BSA t o MSUM. Kozin et a l . (1979) a l s o observed only small amounts of LYZ rel 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 probably due to adsorption of LYZ by MSUM. Hence i t i s probable t h a t the 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 c r y s t a l s were being underestimated due to the adsorption of LYZ onto the MSUM c r y s t a l s . L Y S O Z Y M E R E L E A S E INDUCED B Y CPPD 2500 2000 + 1500 + 1000 + 500 + 0 O — O CONTROL • — • CPPD -O 0 20 40 60 80 100 120 140 160 180 2 0 0 TIME ( m i n u t e s ) FIGURE 40 STUDY OF L Y S O Z Y M E ADSORPTION ONTO M S U M AND THE E F F E C T ON THE L Y S O Z Y M E ASSAY 200 e o o 6 O < 175 + o o o o o o o o o o 150 0 5 10 15 M S U M ADDED ( m g / m L ) 20 25 FIGURE 41 115 4 . 3.4. EFFECT OF PROTEINS ON LYZ RELEASE The e f f e c t of p r o t e i n c o a t i n g of MSUM and CPPD c r y s t a l s on d e g r a n u l a t i o n 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 monitored by the r e l e a s e of LYZ i s shown i n F i g u r e s 42 and 43 r e s p e c t i v e l y . The LYZ r e l e a s e 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 induced by CPPD c r y s t a l s was not a f f e c t e d by the nature of the p r o t e i n c o a t i n g on the c r y s t a l as seen i n F i g u r e 43. P r e c o a t i n g of MSUM c r y s t a l s w i t h p r o t e i n s had no s i g n i f i c a n t e f f e c t on the LYZ r e l e a s e induced by MSUM. 5. FUTURE WORK We have s t u d i e d the modulation of n e u t r o p h i l responses t o MSUM and CPPD c r y s t a l s by the presence of adsorbed p r o t e i n s , such as immunoglobulin G, bovine serum albumin and plasma p r o t e i n s , a t s p e c i f i c time i n t e r v a l s . I t would be i n t e r e s t i n g t o study the time course of the n e u t r o p h i l responses t o p r o t e i n coated MSUM and CPPD c r y s t a l s , p a r t i c u l a r l y s i n c e d i f f e r e n c e s may e x i s t i n the r a t e of response t o c r y s t a l s coated with d i f f e r e n t p r o t e i n s . The e f f e c t of othe r p r o t e i n s such as low d e n s i t y l i p o p r o t e i n s and h i g h d e n s i t y l i p o p r o t e i n s on the n e u t r o p h i l responses such as superoxide g e n e r a t i o n and chemiluminescence p r o d u c t i o n c o u l d be s t u d i e d . The e f f e c t of drugs used i n the treatment of 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 on the L Y S O Z Y M E R E L E A S E INDUCED B Y PROTEIN COATED M S U M FIGURE 42 L Y S O Z Y M E R E L E A S E INDUCED B Y PROTEIN COATED C P P D CONTROL UNCOATED IgG COATED BSA COATED PLASMA CPPD CPPD CPPD COATED CPPD FIGURE 43 118 n e u t r o p h i l responses t o the uncoated and p r o t e i n coated MSUM and CPPD c r y s t a l s could a l s o be st u d i e d . 119 SUMMARY AND CONCLUSIONS 1. The method of prepar a t i o n of MSUM was s u c c e s s f u l l y m o d ified t o y i e l d c r y s t a l s of a smaller and more uniform s i z e range. 2. MSUM and CPPD c r y s t a l s at concentrations of 5 mg/mL and 50 mg/mL r e s p e c t i v e l y induced the generation of superoxide anion by n e u t r o p h i l s . The r a t e of superoxide production induced by CPPD c r y s t a l s was slower than f o r MSUM c r y s t a l s but there was no s i g n i f i c a n t d i f f e r e n c e i n the maximum amounts of superoxide anion generated from n e u t r o p h i l s s t i m u l a t e d by e i t h e r MSUM or CPPD. 3. The r e d u c t i o n of ferricytochrome c was SOD i n h i b i t a b l e f o r CPPD/neutrophil incubations i n d i c a t i n g t h a t the re d u c t i o n of ferricytochrome c was d r i v e n p r i m a r i l y by superoxide anions. However, the r e d u c t i o n of f erricytochrome c was not SOD i n h i b i t a b l e f o r MSUM/neutrophil incubations. This was a t t r i b u t e d t o i n a c t i v a t i o n of SOD probably due to adsorption onto the surf a c e of MSUM c r y s t a l s . 4. The pre c o a t i n g of CPPD and MSUM c r y s t a l s w i t h Ig G and plasma had no e f f e c t on superoxide generation. The absence of an enhanced responsiveness of n e u t r o p h i l s t o c r y s t a l s opsonized w i t h Ig G may be because under the in c u b a t i o n c o n d i t i o n s employed, the n e u t r o p h i l s s t i m u l a t e d by uncoated c r y s t a l s were already producing maximum p o s s i b l e l e v e l s of 120 s u p e r o x i d e . There was no s i g n i f i c a n t e f f e c t o f BSA p r e c o a t i n g o f MSUM on MSUM ind u c e d s u p e r o x i d e r e l e a s e , whereas BSA-coated CPPD produced a s i g n i f i c a n t i n c r e a s e i n s u p e r o x i d e g e n e r a t e d . 5. The CL g e n e r a t e d by n e u t r o p h i l s i n r e s p o n s e t o s t i m u l a t i o n by MSUM was more r a p i d and e x t e n s i v e t h a n t h a t g e n e r a t e d on s t i m u l a t i o n by CPPD. 6. I n g e n e r a l , t h e n a t u r e o f t h e p r o t e i n c o a t i n g on t h e c r y s t a l s s t r o n g l y i n f l u e n c e d t h e CL response w i t h I g G en h a n c i n g and plasma p r o t e i n s i n h i b i t i n g t h e CL g e n e r a t e d . A dsorbed I g G p r o b a b l y enhanced F c r e c e p t o r m e d i a t e d c r y s t a l - n e u t r o p h i l a c t i v a t i o n and CL g e n e r a t i o n . The major i n h i b i t o r y component o f plasma may be t h e low d e n s i t y l i p o p r o t e i n f r a c t i o n w hich b i n d s s t r o n g l y t o MSUM and CPPD c r y s t a l s p r o b a b l y i n t e r f e r i n g w i t h t h e i n i t i a l c r y s t a l - n e u t r o p h i l membrane b i n d i n g p r o c e s s . 7. CPPD c r y s t a l s s t i m u l a t e d e g r a n u l a t i o n o f 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 as m o n i t o r e d by t h e r e l e a s e o f MPO. MSUM i n d u c e d MPO r e l e a s e c o u l d not be m o n i t o r e d due t o a d s o r p t i o n o f MPO by MSUM. Both MSUM and CPPD c r y s t a l s s t i m u l a t e d d e g r a n u l a t i o n as m o n i t o r e d by LYZ r e l e a s e but MSUM a l s o adsorbed LYZ. 8. 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 CPPD i n d u c e d LYZ r e l e a s e but p r e c o a t i n g w i t h I g G s i g n i f i c a n t l y enhanced CPPD in d u c e d r e l e a s e of MPO. P r o t e i n 121 p r e c o a t i n g of MSUM c r y s t a l s d i d not have any e f f e c t on the MSUM induced LYZ r e l e a s e or MSUM induced MPO r e l e a s e . 122 REFERENCES Aaku, E, Sorsa, T. and Wikstrom, M., Human immunoglobulin G p o t e n t i a t e s superoxide p r o d u c t i o n induced by chemotactic p e p t i d e s and causes d e g r a n u l a t i o n i n i s o l a t e d human n e u t r o p h i l s , Biochim. Biophys. Acta, 1052 : 243-247, 1990. 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