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Intracellular trafficking of opsonized and unopsonized Pseudomonas aeruginosa in human monocyte-derived… Leung, Wendy Wen Sie 1994

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iNTRACELLULAR TRAFFICKING OF OPSONIZED AND UNOPSONIZEDPseudomonas aeruginosa IN HUMAN MONOCYTE-DERIVED MACROPHAGESbyWENDY WEN STE LEUNGB. Sc., The University of Victoria, 1991A THESIS SUBMITFED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Microbiology and Immunology)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust, 1994© Wendy Wen Sie Leung, 1994.In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of MIC€o/3fQLo7 AtThe University of British ColumbiaVancouver, CanadaDate AurusT 2C, irJDE6 (2/88)11ABSTRACTThe intracellular fate of microorganisms ingested by phagocytes may be determinedby the specific receptors mediating their uptake. To test this hypothesis, the subcellularlocation of Pseudomonas aeruginosa in human monocyte-derived macrophages uponphagocytosis via opsonic versus non-opsonic receptors was investigated by indirect doubleimmunofluorescence. Opsonic ingestion of P. aeruginosa is glucose-independent while nonopsonic phagocytosis of this bacterium by macrophages requires the presence of glucose.Therefore, opsonic and non-opsonic phagocytosis of P. aeruginosa were differentiated bypresenting immunoglobulin-coated bacteria to macrophages in glucose-free medium andunopsonized bacteria to the phagocytes in the presence of glucose. Compartments of theopsonic and non-opsonic phagocytic pathways of P. aeruginosa in human monocyte-derivedmacrophages were defined by double-labelling infected macrophages with antibodies specificfor different endocytic compartments (lysosomes, endosomes, etc.) and with polyclonalantibodies to P. aeruginosa. Both opsonized and unopsonized P. aeruginosa colocalized withthe lysosomal-associated membrane glycoprotein, LAMP-i, which is found predominantly inlysosomes. Ingested opsonized P. aeruginosa appeared to colocalize with this LAMP-i +compartment at a faster rate than unopsonized P. aeruginosa. When cells were preloaded withrhodamine-ovalbumin to label secondary lysosomes, a small fraction of ingested bacteria thatwere phagocytosed via the two routes entered these labelled compartments. Brefeldin A, adrug which inhibits transport of newly synthesized membrane proteins and secretory proteins,and monensin, an ionophore which inhibits endosome acidification , did not influence theingestion and intracellular fate of either opsonized or unopsonized P. aeruginosa. Mannose1116-phosphate receptor (MPR), an antigenic marker enriched in the late endosome, showed littlecolocalization with either opsonized or unopsonized P. aeruginosa. These studies suggest thatboth opsonized and unopsonized P. aeruginosa enter functionally similar pathways afterphagocytosis by macrophages and that the phagosomes ultimately fuse with LAMP-1compartments regardless of the receptor mediating the ingestion. Although it is not possibleto determine definitively the stage at which the phagocytosed P. aeruginosa converged withthe endocytic pathway, they appeared to do so at a stage that is distal to the late endosome,probably with prelysosomes.ivTABLE OF CONTENTSAbstract iiTable of Contents ivList of Tables VList of Figures viAcknowledgements viiIntroduction 1Methods 8Results 16Discussion 41Bibliography 57VLIST OF TABLESTable 1. Phagocytosis of opsonized and unopsonized P. aeruginosa byhuman monocyte-macrophages in the absence or presence of glucose. 16Table 2. Kinetics of uptake of unopsonized versus opsonized P. aeruginosa 18Table 3. Quantitative assessment of colocalization of P. aeruginosa withLAMP-i at different timed intervals post-infection. 29Table 4. Effect of monensin on the phagocytosis of unopsonized and opsonizedP. aeruginosa. 38viLIST OF FIGURESFig. 1. Indirect immunofluorescence of lysosomal membrane-associated glycoprotein, LAMP-i in human monocyte-derived macrophages. 23Fig. 2. Colocalization of ingested unopsonized P. aeruginosa with LAMP-i atvarious timed intervals post-infection. 24Fig. 3. Colocalization of ingested opsonized P. aeruginosa with LAMP-i. 25Fig. 4. Colocalization of phagocytosed P. aeruginosa with rhodamineovalbumin-labelled lysosomes. 26Fig. 5. Colocalization studies of P. aeruginosa phagocytosed via the non-opsonicand opsonic pathways with the MPR-enriched late endosome. 31Fig. 6. Effect of brefeldin A on the colocalization of P. aeruginosa withLAMP-i. 36Fig. 7. Effect of monensin on the colocalization of ingested P. aeruginosawith LAMP-i. 39Fig. 8. Proposed model of the phagocytic pathway of P. aeruginosa in relationto the endocytic and biosynthetic pathways in human monocyte-derivedmacrophages. 56viiACKNOWLEDGEMENTSI wish to thank Dr. D. P. Speert for providingopportunity, facilities and guidancewhich allowed me to complete this project; other members of mythesis advisory committee,Drs. B. B. Finlay, L. Matsuuchi, for their constructive input; colleagues in the laboratory forimparting their invaluable knowledge and expertise; Dr. F. Garcia-del Portillo for providingadvice and for sharing of some of his reagents used in the studies. A studentship from theCanadian Cystic Fibrosis Foundation provided financial support for me from 1992-1994. Lastbut not least, I would like to extend my thanks tomy family and friends who have showntremendous support and tolerance through timesof difficulty and frustrations.1INTRODUCTIONThe intracellular fate of ingested microorganisms may be determined by the receptormediating their phagocytosis. In recent years, there has been growing interest inunderstanding the formation and development of phagosomal compartments in relation toendosomal compartments within the host cell. Microorganisms including Trypanosoma cruziJardieux et at., 1992), Leishmania mexicana (Russell et at., 1992), Leishmania amazonensis(Lang et al., 1994), Borrelia burgdoferi (Montgomery et al., 1993), Salmonella lyphimuriumand Yersinia enterocolitica (Garcia del-Portillo et at., 1993) have been demonstrated tocolocalize with compartments containing lysosomal associated membrane glycoproteins (lgp’sor LAMPs for short). Phagosomes containing latex beads which serve as an inert,nondegradable phagocytic stimulus or erythrocytes opsonized with immunoglobulins alsoacquire lysosomal glycoproteins (Rabinowitz et al., 1992; Desjardins et at., 1994).Toxoplasma gondii survives in host cells by inhibition of phagosome-lysosome fusion (Joineret at., 1990). This inhibition is probably due to the mode of entry of the Toxoplasmatachyzoites into the host cell, since only parasitophorous vacuoles containing viable,unopsonized tachyzoites fail to fuse with lysosomal compartments, while dead orimmunoglobulin-coated parasites show fusion with lysosomes. It appears that the intracellularroute of T. gondii is determined by the receptors which mediate the phagocytosis event. It isunclear, however, as to whether this observation can be extended to other microorganisms thatcan enter host cells via distinct receptors.Phagocytes take up extracellular components by two different mechanisms.Extracellular fluid, solutes and receptor-bound ligands are internalized by endocytosis, while2large particles are ingested by phagocytosis. Endocytosis occurs continuously via clathrincoated pits on the cell surface, whereas phagocytosis is a local membrane response to theengagement of the appropriate cell surface receptors by particles such as bacteria.Collective data show that endocytosed material en route to lysosomes appears to passsequentially through several distinct structures beyond the coated vesicle (Kornfeld andMeilman, 1989). These structures include the early endosome, the spherical endosome carriervesicle (or multivesicular bodies), the cation-independent mannose-6-phosphate receptor(MPR)- enriched late endosome, the prelysosomal compartment and the MPR-negativelysosome. During endocytosis, the plasma membrane of a cell invaginates and pinches off,internalizing membrane proteins, lipids and extracellular solutes (Hubbard, 1989).Internalization of membrane components and solutes can occur through a receptor-mediatedprocess involving clathrin-coated pits or by non-selective fluid phase pinocytosis mediated bynon-clathrin-coated pits. Once within the cell, the plasma membrane and the contents aredelivered to early endosomes which are tubulo-reticular in appearance and are found at the cellperiphery. Both membrane and contents can be transported to lysosomes via late endosomes.Until recently, lysosomes were considered to be an end-stage organelle where degradation tookplace whereas endosomes were the sorting compartments. However, recent data suggest thatlysosomes are actually more dynamic structures than previously believed and the distinctionbetween endosomes and lysosomes has become blurred. Particles ingested by phagocytosishave been shown to colocalize with markers of the endocytic pathway (see below). However,the exact mechanism by which the phagocytic and endocytic pathways associate with eachother is not completely understood.3Phagocytosis is a dynamic process which involves the attachment of bacteria to thephagocyte plasma membrane followed by the subsequent uptake of the bacteria. Theattachment step is mediated by the specific receptors on the macrophage surface and is aprocess that depends on the nature of the bacterial surface. (Speert, 1992). Binding of thephagocytic particle to the cell surface receptors stimulates the engulfment by the host cellplasma membrane. The distal regions of the plasma membrane encompassing the phagocyticparticle eventually meet and fuse, resulting in the formation of a phagosome. Followingengulfment, the plasma membrane-derived phagosome is transformed into an acidic andprotease-rich functional phagolysosome by extensive membrane exchange (Pitt et at., 1992).In recent years, there has been an increasing interest in understanding the interactionsof ingested particles with organelles in the endocytic pathway. Rabinowitz et at. (1992)performed studies with phagocytes and ingested latex beads; they demonstrated that thephagocytic and endocytic pathways converge at the level of an extensive tubulo-reticularcompartment, the late endosome! prelysosomal compartment, which is enriched in MPR.Desjardins et al. (1994) showed that newly formed phagosomes containing latex beads areinvolved in rapid multiple contacts with late components of the endocytic pathway. There isalso evidence that endosomes can interact with phagosomes containing other non-pathogenicparticles such as Bacillus subtilis (Lang et aT., 1988) and formaldehyde-fixed Staphylococcusaureus (Mayorga et al.,1991 and Pitt et al., 1992). However, whether the transformation ofthe phagosome into a phagolysosome occurs via a maturation process in which the phagosomematures into a phagolysosome by the gradual accrual of endocytic markers from constitutivesecretory vesicles in the protein biosynthetic route, or via a vesicle-shuttle mechanism4involving the delivery of the plasma membrane-derived phagosome to distinct pre-existingstructures in the endocytic pathway is still not fully understood. As mentioned above, anumber of intracellular pathogens have been demonstrated to colocalize with compartmentscontaining lgp’s, markers of prelysosomal and lysosomal compartments. Yet, the study of theintracellular trafficking of microorganisms which are generally considered to be extracellularis limited.The phagocytic process can be classified into two types: opsonic and non-opsonic.Opsonization involves the binding of opsonins, e.g. immunoglobulin G (IgG) or thebreakdown products of complement component 3 (C3b and iC3b), specifically to the integralsurface membrane molecules on the phagocytes on the one hand and to the bacteria on theother. When IgG binds to the appropriate surface epitope on the bacteria via the (Fab’)2 siteson the Ig, the Fc portion can bind to the Fc receptors that are found on the phagocyte surface,thereby allowing the phagocyte to recognize a large variety of bacterial species with varioussurface characteristics. Analogously, C3b and iC3b can attach to the bacterial surfacescovalently and to the phagocyte surface via complement receptors 1 and 3 respectively.Although opsonization is required for the efficient phagocytosis of microbial pathogens,some particles can be recognized by phagocytes where no exogenous source of complementor Ig is present. This non-opsonic phagocytosis, also known as “non-specific” phagocytosis,actually involves specific receptors, some of which participate in lectin-like interactions (Ofeket al., 1988). Of these, the mannosyl/fucosyl receptor that recognizes mannose residues is oneof the best characterized.5Pseudomonas aeruginosa, a Gram-negative bacterium, is one of the major causes ofnosocomial (hospital acquired) infections in North America. It is an opportunistic pathogenwhich, under normal circumstances, poses no danger to healthy individuals. Individuals whoare at risk from Pseudomonas infections include those who are immunosuppressed, either dueto the nature of their injuries or to chemotherapy such as for the treatment of cancer. Patientswho have low neutrophil counts are particularly susceptible to P. aeruginosa infections, anindication that phagocytes play an important role in the defending the host against challengewith this bacterium. Common sites of infection include: severe bum wounds, the urinary,intestinal or lower respiratory tracts (in the case of cystic fibrosis), the conjunctiva of the eyeor the ear.Although P. aeruginosa is the predominant cause of pulmonary infection in patientswith cystic fibrosis, the means by which this bacterium evades defense mechanisms of the hostis not clearly understood. Macrophages are part of the first line of defense in the protectionof the lung and mucosal surfaces against infection and may need to perform their functions inthe absence of opsonins before the recruitment of neutrophils to the site of infection and theevolution of an inflammatory response. Certain non-mucoid strains of P. aeruginosa frompatients with cystic fibrosis are phagocytosed by human neutrophils and macrophages in theabsence of serum opsonins (Speert et al., 1984). This non-opsonic phagocytosis appears tobe facilitated by bacterial piliation and hindered by the presence of the mucoidexopolysaccharide (Speert et al., 1986; Cabral et al., 1987). Although functionalcharacterization studies have been done, the specific structure of this non-opsonic receptor hasyet to be elucidated. A receptor having some of the characteristics of the mannosyl/ fucosyl6receptor seems to play a role in the phagocytosis of unopsonized P. aeruginosa by humanmonocyte-derived macrophages as mannnan, D-mannose and L-fucose inhibit phagocytosis(Speert et al., 1988).Speert and Gordon (1992) have demonstrated that phagocytosis of unopsonized P.aeruginosa by murine peritoneal and pulmonary alveolar macrophages is absolutely dependentupon the presence of glucose. In the absence of glucose, macrophages efficiently bind P.aeruginosa but do not ingest the bacterium. Glucose-dependent phagocytosis appears to bespecific for uptake of P. aeruginosa by macrophages, as unopsonized zymosan, erythrocytesopsonized with IgG (EIgG), erythrocytes opsonized with complement and 1gM [E(IgM)C] andopsonized P. aeruginosa are ingested by macrophages in the absence as well as in the presenceof glucose. Based on these observations, non-opsonic and opsonic phagocytosis of P.aeruginosa can be differentiated by presenting unopsonized bacteria to macrophages in thepresence of glucose and opsonized bacteria coated with antibodies to the phagocytes in theabsence of glucose, since opsonic phagocytosis of P. aeruginosa mediated by Fe receptors isnot glucose-dependent. This provides a good system for studying two different receptor-mediated phagocytic processes for uptake of the same particle. By differentiating these twomechanisms of uptake, I was able to study the fate and the intracellular traffic of P.aeruginosa phagocytosed via opsonic and non-opsonic receptors and to determine thesubcellular location of the bacteria in human monocyte-derived macrophages at different timespost-ingestion using indirect double immunofluorescence. These studies were undertaken todetermine if the route of ingestion of P. aeruginosa mediated by different phagocytic receptorsdetermines the ultimate fate of this bacterium inside the phagocyte. I investigated if there was7any difference in the intracellular location of opsonized and unopsonized P. aeruginosa afterthey became phagocytosed by human monocyte-derived macrophages. I observed thatopsonized and unopsonized P. aeruginosa colocalized with LAMP-i + compartments uponphagocytosis by the macrophages. The bacteria probably entered similar pathways. Theresults suggest that phagosomes containing P. aeruginosa ultimately fuse with LAMP-i +compartments regardless of the receptor mediating the phagocytosis process. P. aerugiiwsacontaining phagosomes appeared to converge with the endocytic pathway at the prelysosomalstage at a point distal to the late endosome.8METHODSBacterial strain.P. aeruginosa strain P-i is a spontaneous non-mucoid laboratory revertant of a mucoid strainisolated from the sputum of a cystic fibrosis patient (Speert et al., 1984). It has a roughlipopolysaccharide (LPS), is susceptible to the bactericidal effect of human serum and ispiiated (Speert et al., 1986). Bacteria were maintained in frozen aliquots at -70°C and weregrown fresh for each experiment. Bacteria for each experiment were grown overnight understatic conditions at 37°C in L broth (10 g tryptone [Becton Dickinson Microbiology Systems,Cockeysville, MD], 5 g yeast extract [Oxoid, Basingstoke, UK] and 10 g NaC1 [BDH, Inc.,Toronto, Ontario, Canada] per litre distilled water). The bacterial culture was gently vortexedto disrupt the pellicle and then diluted in L broth. The diluted culture was incubated at 37°Cwith shaking until mid-log phase. Immediately before use in phagocytosis experiments,unopsonized bacteria were gently vortexed and diluted to an O.D. reading of approximately0.6 at 600 nm on a Bausch and Lomb Spectronic 2000 spectrophotometer. This 0. D. readingcorresponds to approximately iO bacteria / ml.Hyperinunune anti-Pseudomonas serum.P. aeruginosa strain P-i was grown in 10 ml L broth overnight as described above, washedin PBS twice, incubated in 10% formalin/ normal saline for 15 mm at room temperature,washed twice in PBS and resuspended in one ml PBS. The bacteria were then suspended inan equal volume of complete Freund’s adjuvant and injected in 0.25 ml aliquotsintramuscularly into two adult New Zealand white rabbits. This process was repeated afterfour and six weeks with bacteria suspended in incomplete Freund’s adjuvant. The animals9received a final intravenous injection 12 weeks later, with bacteria in PBS, and wereexsanguinated by cardiac puncture. Anti-Pseudomonos rat serum was also obtained byrepeated immunization of two Wistar female rats with formalin-killed P. aeruginosa. The ratswere injected intramuscularly with formalin-killed strain P-i in Freund’s adjuvant in 0.1 mlaliquots. The procedure was repeated after three and six weeks with bacteria resuspended inPBS and total blood from the rats were taken ten days after the last boost.Opsonization of P. aeruginosa.P. aeruginosa was preopsonized immediately before phagocytosis experiments by tumblingthe bacteria for 15 mm in 2% hyperimmune polyclonal rabbit or rat serum which had beenheat-inactivated at 56°C for 30 mm. After opsonization, the bacteria were washed once andresuspended in phosphate-buffered saline (PBS) pH 7.4 which was reconstituted from tablets(Oxoid Ltd., Basingstoke, UK).Macrophage cultivation.Macrophages were derived from human peripheral blood monocytes by culturing in suspensionin Teflon beakers as described previously (Speert and Silverstein, 1985). Thirty ml ofheparinized peripheral venous blood and 20 ml of clotted blood were drawn from a healthyvolunteer for each experiment. The heparinized blood was diluted 1:1 with saline (BaxterCorp., Toronto, Ontario, Canada) and then gently layered onto a Ficoll-Paque (PharmaciaLKB, Piscataway, NJ) gradient and centrifuged at 400xg for 30 mm at room temperature.The “buffy coat” found at the interface between the ficoll and the serum was retrieved anddiluted with cold RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented withpenicillin and streptomycin (Terry Fox Laboratory, B.C. Cancer Research Centre, Vancouver,10B.C., Canada) followed by several washes. The mononuclear cells were cultured in acid-washed teflon beakers with 15% autologous fresh human serum in RPMI supplemented withpenicillin and streptomycin at 37°C in 5% CO2. The cells received fresh medium containingautologous serum on day 3 and were washed free of serum on the day of the experiment andplated on glass coverslips. These cells were viable as determined by trypan blue dyeexclusion.Assessment of phagocytosis.In all experiments, macrophages were used between 4-7 days of in vitro cultivation, at whichtime they had all the characteristics of mature macrophages, including the maturation ofphagocytic receptors, but had not begun to fuse with each other. Cells harvested from teflonbeakers were washed free of serum and resuspended in RPMI containing 0.2% bovine serumalbumin (Boehringher Mannheim, Mannheim, Germany). Approximately 4-8 X iO cells wereplated onto acid-washed 11-mm diameter round glass coverslips in 24-well plastic tissueculture plates (Becton Dickinson Labware, Lincoln Park, NJ). The coverslips were incubatedat 37°C in 5 % CO2 for 2 hours to allow the macrophages to adhere to the coverslips. Thecoverslips were then dipped several times in PBS to remove nonadherent cells and put intofresh plates containing 400 l glucose-free phagocytosis medium (138 mM NaC1, 8. 1 mMNa2HPO4,1.5 mM KH2PO4,2.7 mM KC1, 0.6 mM CaC12.2H0, 1 mM MgCl2.6H0)perwell. The plates were equilibrated at 37°C in ambient CO2 for 30 mm to allow the cells todeplete their glucose stores. 50 l of opsonized or unopsonized bacteria (prepared asdescribed above) were added to each of the wells, giving a bacteria: macrophage ratio ofapproximately 1000:1. Uptake of the opsonized or unopsonized P. aeruginosa was11synchronized so that the time at which the bacteria were internalized by the macrophages couldbe determined and compared. To synchronize the ingestion of unopsonized P. aeruginosa,bacteria were first allowed to bind to macrophages in the absence of glucose for 30 mm afterwhich the addition of glucose (final concentration = 10mM) to each well instigated uptake ofthe bound unopsonized bacteria. For bacteria phagocytosed via the opsonic route, theingestion phase of phagocytosis was synchronized by allowing the preopsonized P. aeruginosato adhere to phagocytes at 4°C for 15 mm and then shifting the cells to 37°C to permit particleingestion. Ten mm after the shift, the coverslips were washed to remove extracellularbacteria. Phagocytosis of opsonized P. aeruginosa was carried out in glucose-freephagocytosis medium. Cells infected with either unopsonized or opsonized bacteria wereincubated at 37°C in ambient CO2 for the specified times. Any uningested bacteria were lysedas follows: 500 .d of ice-cold lysozyme (5 mg/mI; Sigma Chemical Co., St. Louis, MO) in0.25 M Tris buffer, pH 8.0, was added to each well. The plates were incubated at roomtemperature for 5 mm and then the wells were washed with PBS. Bacterial spheroplasts werelysed by the addition of 500 d 50% PBS for 2 mm 15 sec and the coverslips were washedwith PBS and fixed with methanol. The coverslips were air-dried, mounted on glassmicroscope slides, and stained with Giemsa stain (BDFI Inc., Toronto, Ontario, Canada).Phagocytosis was assessed by bright-field microscopy. All experiments were performed induplicates and were repeated twice on separate days with macrophages from different donors.Sixty macrophages were examined on each coverslip and the number of P. aeruginosaphagocytosed by macrophages were counted. Ingested bacteria were those associated withmacrophages after the lysozyme treatment.12Antibodies.Crude and affinity-purified rabbit anti-bovine cation-independent mannose-6-phosphatereceptor (MPR) serum were kindly provided by Dr. S. Komfeld, Washington UniversitySchool of Medicine. The monoclonal antibody H4A3 that is specific for the human lysosomalmembrane glycoprotein, LAMP-i, developed by Drs. J.T. August and J.E.K. Hildreth wasobtained from the Developmental Studies Hybridoma Bank (DSHB) maintained by theDepartment of Pharmacology and Molecular Sciences, The Johns Hopkins University Schoolof Medicine, Baltimore, MD 21205, and the Department of Biology, University of Iowa, IowaCity, IA 52242, under contract NOi-HD-2-3144 from the NICHD. LM2/1.6.11 andM1/ which are specific for CD11b (CR3), developed by Dr. T. A. Springer,were also obtained from DSHB. OKM1, a monoclonal antibody recognizing CR3 wasobtained from American Type Culture Collection (ATCC). The monoclonal antibody H68.4which is specific for the cytoplasmic tail of the trarisferrin receptor was a gift from Dr. I.S.Trowbridge, the Salk Institute for Biological Studies, San Diego. Goat anti-clathrindelipidized whole antiserum and goat anti-human transferrin fractionated antiserum werepurchased from Sigma ImmunoChemicals.Fluorescein isothiocyanate (FITC)- and Texas Red-conjugated secondary antibodieswere purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Allsecondary agents used were antibodies that had been isolated from antisera by immunoaffinitychromatography using antigens coupled to agarose gels and were F(ab’)2 fragments tominimize steric hindrance of secondary antibody binding to Fc receptors on macrophages.13Adsorption of rabbit and rat antisera.The rabbit and rat antisera specific for P. aeruginosa that were used in the doubleimmunofluorescence studies were adsorbed against whole blood and monocyte-derivedmacrophages. Ten ml of heparinized human blood was centrifuged and the cells resuspendedin PBS containing 0.02% Na azide (Sigma). The antisera were diluted 1/10 in PBS in thepresence of phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor to prevent thedegradation of the immunoglobulins. The diluted antisera were added to the blood cells andwere allowed to sit at room temperature overnight. The mixture was centrifuged and thesupernatant was kept. The above adsorption process was repeated with human monocytederived macrophages.Immunofluorescence.Infected macrophage monolayers were fixed in 2% paraformaldehyde (Sigma)/PBS for 15 mmat room temperature. After washing the coverslips with PBS, primary antibodies diluted inPBS/3 % BSA/ 0.2% saponin (Sigma) were added to each coverslip and allowed to incubatefor 1 hr at room temperature. After washing with 0.2% saponin/PBS three times, antibodieswere visualized with the appropriate fluorochrome-conjugated, species-specific secondaryantibodies diluted in PBS! 3% BSA/ 0.2% saponin. Coverslips were washed with PBS andmounted on a drop of mounting media (Sigma). Samples were examined on a Zeiss Axioskopmicroscope or on an Olympus BFIS-313 research microscope.The labelling experiments were conducted in parallel with controls omitting theprimary antibodies. The controls were consistently negative at the concentrations of14fluorochrome-conjugated secondary antibodies used in the studies. Non-specific labelling ofby the primary antibodies were also not likely because of the following observations:(1). The monoclonal antibody H4A3 (IgGi isotype) specific for LAMP-i which was usedto label the macrophages showed a distinct pattern of LAMP-i + structures while the isotypematched monoclonal antibody H68.4 specific for the human transferrin receptor did not givea detectable signal.(2). Since the rabbit serum specific for P. aeruginosa did not label the uninfectedmacrophages, the labelling of the macrophages with the rabbit anti-MPR serum was not dueto non-specific labelling.Fluorescent labelling of lysosomes in macrophages with fluid tracers.Human monocyte-derived macrophages adhered onto coverslips were incubated with RPMI/0.2% BSA containing rhodamine-ovalbumin (Molecular Probes, Eugene, OR) at aconcentration of 500 g/ml and incubated overnight at 37°C in 5% CO2 followed by a 60 mmchase in medium without the tracer to label lysosomal compartments within the cells.Leupeptin (Sigma), a protease inhibitor, at a concentration of 10 g/ml was also present in themedium throughout the experiment to prevent the degradation of the tracer by lysosomalenzymes. Colocalization experiments with opsonized and unopsonized P. aeruginosa werethen performed as described above.Inhibitors of the endocytic pathway.Brefeldin A and monensin (Sodium salt) were purchased from Sigma. BFA is a yeastmetabolite that inhibits transport of newly synthesized membrane proteins and secretoryproteins and impairs endocytic transport to lysosomes. Monensin is an inhibitor of endosome15acidification. In some experiments, BFA at a concentration of 5 g/ml or monensin at a finalconcentration of 20 mM was present in the phagocytosis medium during the equilibration stepprior to the addition of bacteria to the wells. The inhibitor was present throughout thephagocytosis experiments.Labelling of Golgi apparatus in macrophages.Human monocyte-derived macrophages plated on coverslips were fixed in 0.5%glutaraldehyde in PBS for 30 mm at room temperature. Cells were incubated in phagocytosismedium in the absence or in the presence of BFA (5 g/ml) for 30 mm at room temperaturein ambient CO2. The cells were washed with PBS twice and incubated on ice for 30 mm. 2501 of 7-nitrobenz-2-oxa-l,3diazole (NI3D)-ceramide was added to each well and left on ice for30 mm to label the Golgi apparatus in the macrophages. Coverslips were washed with PBStwice. Cells were then washed 4 times with 10 % fetal bovine serum (30 mm per wash) atroom temperature. Coverslips were mounted on a drop of serum and the cells were visualizedimmediately on a fluorescence microscope using a fluorescein filter.16RESULTSDifferentiation of opsonic versus non-opsonic pbagocytosis of P. aeruginosa bymacrophages.Initial experiments were done using human monocyte-derived macrophages to optimizethe conditions for phagocytosis of P. aeruginosa P-i mediated by opsonic and non-opsonicreceptors and to ascertain that these two routes of ingestion can be clearly distinguished.Macrophages plated on coverslips were infected with either unopsonized or opsonized bacteriathat had been preincubated in 2% heat-inactivated polyclonal rabbit antiserum in the absenceor presence of glucose for 1 hour and washed. Any uningested extracellular bacteria werelysed osmotically by a lysozyme-50% PBS treatment. Table 1 shows that unopsonized P.aeruginosa were ingested only when glucose was present in the medium while antibody-coatedbacteria could be phagocytosed regardless of the presence of glucose. It was demonstrated thatthe two different routes of phagocytosis of P. aeruginosa by human monocyte-derivedmacrophages could be distinguished clearly.Table 1. Pbagocytosis of opsonized and unopsonized P. aeruginosa by human monocytederived macrophages in the absence or presence of glucose. Cells were infected withunopsonized or antibody-coated bacteria for 1 hour in phagocytosis medium +1- glucose.P. aeruginosa Number of ingested bacteria /macrophage(average of 2 experiments)no glucose +10 mM glucoseunopsonized 0.5 (0.8) 15.2 (8.8)opsonized >30 >30( ) = standard error17Kinetics of phagocytosis of unopsonized and opsonized P. aeruginosa by macrophages.To investigate the kinetics of uptake of unopsonized and opsonized P. aeruginosa bymacrophages at various timed intervals post-infection, the ingestion of the bacteria taken upvia the two different routes were synchronized. For non-opsonic phagocytosis, glucose wasinitially withheld to allow adherence of the bacteria to the phagocytic receptor onmacrophages. Glucose was subsequently added to induce the ingestion process to occur. Inthe case of P. aeruginosa preopsonized with heat-inactivated antiserum, phagocytosis wassynchronized by first allowing the binding of the bacteria to macrophages to occur at 4°C, atemperature at which phagocytosis cannot occur, and then shifting the cells to 37°C at whichphagocytes can ingest bacteria. After 10 mm to allow for temperature equilibration, theextracellular opsonized bacteria were washed away with PBS. The kinetics of phagocytosisof opsonized P. aerugiiusa was observed to be faster than that of the unopsonized bacteria(Table 2). Although this observation was clear only for the 10 mm point from the datapresented, experiments where uptake of opsonized P. aeruginosa was not synchronized by thetemperature shifts showed that ingestion occurred almost immediately upon the addition of thebacteria to the phagocytes at 37°C when the binding step of the antibody-coated bacteria tomacrophages at 4°C was omitted (data not shown). In contrast, unopsonized P. aeruginosawas not ingested efficiently until 10-20 mm after the addition of glucose to the phagocytosismedium.18Table 2. Kinetics of uptake of unopsonized versus opsonized P. aeruginosa. Quantitationof P. aeruginosa phagocytosed by human monocyte-derived macrophages via the opsonic andnon-opsonic routes was performed at various timed intervals post-infection. Afterphagocytosis, extracellular bacteria were lysed by treatment with lysoszyme (5 mg/mi)followed by the addition of 50% PBS to the coverslips. In the experiments that determine theeffects of brefeldin A on phagocytosis of P. aeruginosa, the drug was present in theequilibration step as well as throughout the experiments.Time (mm) Number of bacteria / macrophage (average of 3 experiments)aUnopsonized P. aeruginosa bOpsonized P. aeruginosano BFA +BFA no BFA +BFA(5g/m1) (5ig/ml)0 0.3(0.8) 0.4(1.1) NA NA10 2.6 (3.4) 5.2 (5.4) 6.3 (4.9) 9.3 (6.8)20 11.4 (8.6) 15.1 (10.0) 12.9 (7.6) 15.7 (8.9)30 >20 >20 17.4(9.3) 18.6(11.1)60 >20 >20 >20 >20aTime for non-opsonic phagocytosis of P. aeruginosa was the time after the addition of glucoseto the phagocytosis medium. (See Methods)“Time for opsonic phagocytosis of P. aeruginosa was the time after the infected cells had beenshifted from 4°C to 37°C. (See Methods)( ) = standard errorNA = not applicable. Since there was a lag period when the temperature needed to rise from4°C to 37°C in order to allow the macrophages to regain their phagocytic ability, the timepoint at which the cells had just been shifted to 37°C was not evaluated.19limnunofluorescence studies with various structures in the endocytic pathway.Immunofluorescence studies were done to determine if there was differentialassociation of P. aeruginosa ingested via the opsonic or the non-opsonic pathway with variousstructures involved in the endocytic process. Polyclonal antiserum specific for clathrin wasemployed to label the coated pits involved in receptor-mediated endocytosis. However, thecommercially available product showed non-specific labelling and cross-reacted with the P.aeruginosa used in the studies. Therefore, colocalization studies with this reagent were notperformed.Antibodies that recognize the transferrin receptor were also tried in an attempt to labelcompartments involved in endocytosis, exclusive of lysosomes. This receptor is not normallyrouted to the lysosome for degradation but is recycled back to the plasma membrane instead(Hanover and Dickson, 1985). Therefore, this receptor should be enriched in early endosomalcompartments. The monoclonal antibody, H68.4, which is specific for the cytoplasmic tailof the transferrin receptor also non-specifically labelled the bacteria, making the colocalizationstudies technically not feasible. Different monoclonal antibodies recognizing the humancomplement receptor 3 (CR3), another opsonic phagocytic receptor, were also tried. OKM1,LM2/1.6.11 and M1/ which recognize CD11b of the CR3 did not give strong,detectable fluorescent signals. Thus, colocalization studies using these aforementionedmarkers were not pursued. Several antibodies that recognized proteins enriched in otherendocytic compartments (lysosomes, late endosomes etc.) did not cross-react with bacteria andwere used to define the bacteria-containing compartments (see below).20Colocalization of phagocytosed P. aeruginosa with lysosomes.To define the compartments of opsonic and non-opsonic phagocytic pathways of theP. aeruginosa in human monocyte-derived macrophages, double immunofluorescence studiesusing antibodies specific for various endocytic compartments such as lysosomes andendosomes were used to label macrophages in conjunction with antibodies specific for P.aeruginosa. Non-opsonic phagocytosis of P. aeruginosa was synchronized as describedabove. Infected macrophages were fixed and double stained with antibodies against P.aeruginosa and the lysosomal membrane-associated glycoprotein, LAMP-i, a marker foundpredominantly in lysosomes (Mane et al., 1989) followed by the appropriate fluorochromeconjugated secondary antibodies. In uninfected cells, lysosomes stained heavily in theperinuclear region of the phagocyte (Fig. 1). Some elongated tubular extensions whichseemed to be interconnected as a network were also LAMP- 1. In cells which were infectedwith opsonized or unopsonized P. aeruginosa (Figs. 2 and 3), the tubular LAMP-i + networkstarted to retract after phagocytosis of the bacteria, and the shape of the LAMP-1 structuresappeared to conform to the ingested bacteria since indirectly-labelled bacteria showed a similarstaining pattern to that of the LAMP-i + compartments. In cells where the number of ingestedbacteria was low, some of these tubular lysosomal structures were still detected. However,the tubules almost completely disappeared in cells that had ingested a larger number ofbacteria. The membrane of all phagosomes was strongly and uniformly labelled.Colocalization of ingested unopsonized P. aeruginosa with lysosomes was studied attimed intervals after the addition of glucose to the phagocytosis mixture to synchronize uptakeof the bacteria. At the earlier time points, i.e. less than 20 mm post-ingestion, no21colocalization of the phagosome with the lysosome was detected (Fig. 2a and b). This lackof colocalization was not due to the bacteria not being inside the macrophages since uningestedP. aeruginosa in the background and intracellular bacteria were on a slightly different planeof focus when examined under the fluorescence microscope. At about 20 mm after the additionof glucose, some colocalization with LAMP-i was observed (Fig. 2c and d). By 60 mm,phagosomes containing unopsonized P. aeruginosa colocalized completely with the LAMP-i +compartments as observed by the similarities in the staining patterns of both the FITC-stainedingested bacteria and the Texas Red-stained LAMP-i + compartments in the doubleimmunofluorescence studies (Fig. 2e and f).When phagocytosis of opsonized P. aeruginosa was synchronized, doubleimmunofluorescence studies showed that the phagosomes colocalized with LAMP-i +compartments as early as 10 mm after the removal of unbound bacteria (Fig. 3a and b).The colocalization of P. aeruginosa-containing phagosomes with lysosomes was alsoassessed quantitatively (Table 3). Ingested opsonized P. aeruginosa appeared to colocalizewith the lysosomes at a faster rate than unopsonized bacteria. By 10 mm after the extracellularbacteria were removed by washing, over half of the phagocytosed opsonized P. aeruginosahad colocalized with LAMP-i while it took the unopsonized P. aeruginosa longer to enter theLAMP-i + compartment. Almost all of the ingested bacteria, opsonized and unopsonized,colocalized with LAMP-i after 1 hour.Although LAMP-i is found predominantly in lysosomes and was thus used as a markerfor lysosomes in my studies, trace amounts of this membrane glycoprotein may also be foundin late endosomes (Rabinowitz et al., 1992). Therefore, to determine if the phagocytosed22opsonized and unopsonized bacteria were actually colocalizing with true, functional lysosomesas opposed to LAMP-i + non-lysosomal constitutive secretory vesicles or late endosomes, afluid phase tracer, rhodamine-ovalbumin, was employed with a pulse and chase to probe forsecondary lysosomes within the macrophages (Swanson, 1989). Prior to infection withbacteria, cells were preloaded with the probe overnight to label all endocytic compartmentsfollowed by a i hour chase in fresh medium containing no rhodamine-ovalbumin so as to clearorganelles located in the early stages of the endocytic route (i.e. early endosomes) and to chasethe pinocytosed tracer into the lysosomal compartment. Of the 60 macrophages examined percoverslip, it appeared that only a small fraction of ingested bacteria colocalized with therhodamine-ovalbumin labelled lysosomes (Fig. 4a-d). These results suggested that thephagosomes containing opsonized or unopsonized P. aeruginosa did not fuse with functionallysosomes. Quantitation of the colocalization event was not done because the fluorescenceemitted by the rhodamine-ovalbumin labelled lysosomes was too strong , thus rendering thecounting of the number of phagosomes difficult to perform.23Fig. 1. Indirect immunofluorescence of lysosomal membrane-associated glycoprotein,LAMP-i, in human monocyte-derived macrophages. Uninfected macrophages werelabelled with mouse monoclonal antibodies specific for human LAMP-i followed by TexasRed conjugated donkey anti-mouse IgG. LAMP-i stained heavily in the perinuclear regionof the cells. Tubular extensions were also observed. Bar, 5gm.24Fig. 2. Colocalization of ingested unopsonized P. aeruginosa with the lysosomal marker,LAMP-i, at various timed intervals post-infection. Double indirect immunofluorescencewas done by labelling unopsonized P. aeruginosa with adsorbed rabbit antiserum and LAMP-iwith mouse monoclonal antibody against this marker followed by staining with FITCconjugated goat anti-rabbit IgG and Texas Red-conjugated donkey anti-mouse IgG. Panels(a,b) 10 mm after the addition of glucose. Panels (c,d) 20 mm after glucose addition. Arrowsindicate partial colocalization of the bacteria with LAMP-i. Panels (e,f) 60 mm after glucoseaddition. Panels (a,c,e) unopsonized bacteria. Panels (b,d,f) LAMP-i. Bar, 5gm.aCe25Fig. 3. Colocalization of opsonized P. aeruginosa with the lysosomal compartment.Phagocytosis was synchronized by incubating macrophages and preopsonized P. aeruginosaat 4°C for 15 mm and then shifting to 37°C for 10 mm before the extracellular bacteria wereremoved by washing. 10 mm after the removal of the uningested bacteria, cells were fixedand labelled with LAMP-i followed staining with FITC-conjugated goat anti-rabbit IgG andTexas Red-conjugated donkey anti-mouse IgG. Panel (a) Opsonized P. aeruginosa and (b)LAMP-i showed similar staining patterns. Bar, 5pm.ab26Fig. 4. Colocalization of phagocytosed P. aeruginosa with rhodamine-ovalbumin-labelledlysosomes. Lysosomes were stained by preloading the cells overnight with rhodamineovalbumin followed by a 1 hr chase. Upon ingestion of (a) unopsonized and (c) opsonized P.aeruginosa, some of the phagosomes colocalized with rhodamine-ovalbumin-labelledlysosomes (b and d), see arrows. Bar, 5gm.LiI8129Table 3. Quantitative assessment of colocalization of P. aeruginosa with LAMP-i atdifferent timed intervals post infection.Time % colocalization of phagosome with LAMP-i(mm)Unopsonized P. aeruginosa Opsonized P. aeruginosano BFA +BFA no BFA +BFA(5g/m1) (5g/m1)10 21.2 (11.8) 5.5(4.9) 55.6 (17.2) 4.6 (11.7)20 69.3 (14.0) 64.5 (15.4) 81.3 (5.9) 76.5(16.7)30 95.2 (2.9) 86.9 (9.8) 93.3 (6.6) 71.4 (7.4)60 95.3 (4.4) 94.0 (6.4) 90.8 (10.3) 100 (0)( ) = standard errorColocalization of phagocytosed P. aeruginosa with late endosomes.Antibodies against the mannose-6-phosphate receptor (MPR), an antigenic markerenriched in the late endosome (Griffiths et al., 1988), were used to stain human monocytederived macrophages. This compartment contains only trace amounts of LAMP-i (Rabinowitzet al., 1992). A punctate pattern was observed in uninfected cells when stained with the antiMPR antibodies (data not shown). In most cases, when the phagocytes were infected witheither opsonized or unopsonized P. aeruginosa, neither a redistribution of the antigenic markersimilar to that of the LAMP-i nor a colocalization of the majority of bacteria with the MPR30compartments was observed. On careful examination, the phagosomes containing unopsonizedP. aeruginosa appeared to occupy a hollow area devoid of MPR in the macrophages (Fig. 5aand b). However, in some cases, a small amount of MPR was found around the phagosome,forming a very faint halo around the phagocytosed P. aeruginosa (Fig. 5c and d). Yet, thiswas a rare event. Similar results were observed when macrophages phagocytosed opsonizedP. aeruginosa (data not shown).31Fig. 5. Colocalization of P. aeruginosa phagocytosed via the non-opsonic pathway withthe MPR-enriched late endosome. (a,c) Ingested unopsonized bacteria were recognized byrat antiserum specific for P. aeruginosa and (b , d) infected cells were labelled with rabbitantiserum specific for the cation-independent bovine mannose-6-phosphate receptor (MPR).(a, b) Ingested P. aeruginosa and MPR did not colocalize in most cases. Occasionally, asmall amount of MPR was found around the phagosome, forming a halo around thephagocytosed P. aeruginosa (c,d), see arrows. Similar results were observed with opsonizedP. aeruginosa (data not shown). Bar, 5gm.ab33Effect of inhibitors of the endocytic pathway on the phagocytosis of opsonized andunopsonized P. aeruginosa in macrophages.LAMP-i is enriched in the lysosomal compartment; however, small amounts of thismarker is found in the prelysosomal compartment and constitutive secretory vesicles buddingout of the trans-Golgi network in the biosynthetic route. In order to determine whether thecolocalization of the bacteria-containing phagosome was with pre-existing lysosomes or withLAMP-1 secretory vesicles, I tested the effect of inhibitors which block traffic in thebiosynthetic as well as endocytic pathways on the colocalization of the phagosomes withlysosomes. Two inhibitors, namely brefeldin A (BFA) and monensin, were tested. BFA, ayeast metabolite that has been reported to inhibit transport of newly synthesized membraneproteins and secretory proteins (Lippincott-Schwartz et al., 1989, 1990) as well as impairendocytic transport to lysosomes (Lippincott-Schwartz et at., 1991), was used. BFA blockstraffic from the Golgi apparatus onward, by causing the redistribution of markers from theGolgi to the endoplasmic reticulum (Lippincott-Schwartz et at., 1989,1990). Thisredistribution ought to block the transport of new secretory vesicles carrying LAMP-i fromthe biosynthetic pathway. To ascertain that BFA, at the concentration used in the experiments,was actually inhibiting endocytic transport in the macrophages, positive controls wereperformed. Since the drug causes Golgi stacks to disassemble and the Golgi proteins to betransported back into the endoplasmic reticulum (ER) (Lippincott-Schwartz et al., 1989,1990), cells were stained with NBD-ceramide to label the Golgi stacks. In the presence ofBFA at a concentration of 5 jLg/ml, the Golgi apparatus was observed to have disassembled(data not shown). This inhibitor, at the concentration used, did not impair the ability of the34macrophages to phagocytose opsonized and unopsonized P. aeruginosa (Table 2). Also, thisinhibitor did not exert any effect on the colocalization of the opsonized and unopsonized P.aeruginosa with the LAMP-i + compartments since in the presence of BFA, both opsonizedand unopsonized bacteria showed colocalization with LAMP compartments with patterns thatwere similar to that observed when the experiments were done in the absence of BFA (Fig.6a-d).Monensin, an ionophore which inhibits vacuolar acidification was also tested.Monensin exerts its most profound effects on the trans cisternae of the Golgi apparatus stacksin those regions of the apparatus primarily associated with the final stages of secretory vesiclematuration and in post-Golgi structure primarily associated with endocytosis and membrane!product sorting. (Mollenhauer, H. H. et al., 1990). This drug did not affect the ingestionof opsonized and unopsonized P. aeruginosa since the number of bacteria phagocytosed permacrophage was similar in the presence and in the absence of monensin (Table 4).Colocalization of the phagosomes containing unopsonized or opsonized P. aeruginosa withLAMP-i + compartments was also observed when monensin was present (Fig. 7a -d).Inhibitors of vacuolar acidification have been reported to perturb the distribution of lysosomes(Tardieux et al., 1992). Alkalinization of the cytosol with such inhibitors can induce asignificant depletion of peripheral lysosomes by withdrawing the lysosomes from the cellperiphery to the cell centre (Tardieux et al., 1992). In my immunofluorescence studies,uninfected celis treated with monensin were observed to have a high concentration of LAMP-iin the perinuclear region of the macrophages, rendering the area around the cell nucleusexcessively bright (data not shown). Based on this observation, it was believed that monensin35at 2OM did indeed block acidification. The kinetics of the colocalization event was notanalyzed for these monensin studies because a lot of the phagosomes were found in perinuclearareas of the macrophages that were heavily stained with LAMP-i. Therefore, it was difficultto enumerate the bacterial particles that had colocalized with LAMP-i.The results from these two studies with BFA and monen sin suggested that phagosomescontaining both opsonized and unopsonized P. aeruginosa acquired LAMP-i by fusion withpre-existing lysosomal or prelysosomal structures but not with constitutive secretory vesicles.36Fig. 6. Effect of BFA on the colocalization of ingested P. aeruginosa with LAMP-i.Cells were preincubated in phagocytosis medium containing brefeidin A (5 g/m1) for 30 mmprior to phagocytosis. The inhibitor was present throughout the experiment. (a) Unopsonizedand (c) opsonized P. aeruginosa showed colocalization with LAMP-i (b,d). Bar, 5pm.abp0L38Table 4. Effect of monensin on the phagocytosis of unopsonized and opsonized P.aeruginosa. Quantitation of P. aeruginosa phagocytoseci by human monocyte-derivedmacrophages via the opsonic and non-opsonic routes were performed at various timed intervalspost-infection.Time Number of bacteria / macrophage (average of 3 experiments)(mm)aUnopsonized P. aeruginosa bOpsonized P. aeruginosano +20MM no +20uMmonensin monensin monensin monensin0 0.2 (0.7) 0.6 (0.9) NA NA10 6.2 (5.2) 7.1 (5.2) 4.2 (3.1) 3.5 (2.5)20 14.3 (9.3) 12.2 (8.7) 7.4 (4.6) 4.4 (3.0)30 15.4 (9.8) 13.8 (9.2) 9.3 (5.4) 8.6 (6.4)60 16.7 (11.7) 15.7 (12.4) 11.3 (7.0) 10.9 (7.8)9’ime for non-opsonic phagocytosis of P. aeruginosa was the time after the addition of glucoseto the phagocytosis medium. (See Methods)bTime for opsonic phagocytosis of P. aeruginosa was the time after the infected cells havebeen shifted from 4°C to 37°C. (See Methods)( ) = standard errorNA = not applicable. Since there was a lag period when the temperature needed to rise from4°C to 37°C in order to allow the macrophages to regain their phagocytic ability, the timepoint at which the cells had just been shifted to 37°C was not evaluated.39Fig. 7. Effect of monensin on the colocalization of ingested P. aeruginosa with LAMP-i.Macrophages were preincubated in phagocytosis medium containing monensin (20 mM) for30 mm prior to phagocytosis. The inhibitor was present throughout the experiment. (a)Unopsonized and (c) opsonized P. aeruginosa showed colocalization with LAMP-i (b,d).Bar, 5gm.abp30,41DISCUSSIONAlthough the endosomal system of eukaryotic cells has been extensively studied(Griffiths et at., 1988; Komfeld and Meilman, 1989), the formation and development ofphagosomal compartments have become a subject of growing interest in recent years. Someof the studies done were based on observations with phagosomes formed by inert particles e.g.latex beads. However, such particles do not provide sufficient information to assess to whatextent pathogens modify the host cell phagosome. Most pathogens examined previously arethose considered to be intracellular pathogens which have the capability of establishingthemselves in intracellular habitats within the host cell. Of those studied, Toxoplasma gondii(Joiner et al., 1990) survives intracellularly by inhibition of phagosome-lysosome fusion. Thisinhibition is likely due to the mode of entry of the parasite into the host cell as non-viable orIgG-coated parasites have been found in organelles that contain lysosomal glycoproteins. Onthe other hand, Leishmania rnexican&s survival in macrophage depends on its ability tosurvive within an acidic intracellular compartment having the characteristics of a lysosome(Russell et al., 1992). However, most of the studies have been restricted to thedemonstration of the subcellular fate of intracellular pathogens inside host cells while the studyof the intracellular trafficking of microorganisms which are generally considered to beextracellular bacteria is very limited. In this series of immunofluorescence studies of a typicalphagocyte, namely the macrophage, I have used various antibodies specific for the differentendocytic markers and rhodamine-ovalbumin, a fluid phase endocytic tracer, to define theendosomal/ lysosomal compartments and to determine the subcellular location of P.42aeruginosa phagocytosed via the opsonic or non-opsonic route in relation to these endocyticcompartments.Immunofluorescence microscopic analysis provided evidence that the phagosomalmembrane surrounding phagocytosed unopsonized P. aeruginosa and antibody-coated P.aeruginosa possessed human LAMP-i (Figs. 2 and 3). The targeting of intracellularpathogens to lysosomal glycoprotein-containing vacuoles has also been described for otherparasites including dead or IgG-coated Toxoplasma gondii (Joiner et al., 1990), Trypanosomacruzi (Tardieux et al., 1992), Leishmania mexicana (Russell et al., 1992), Leishmaniaamazonensis (Lang et al., 1994), Borrelia burgdoferi (Montgomery et al., 1993), Salmonellatyphimurium and Yersinia enterocolitica (Garcia del-Portillo et al., 1993). Latex beads thatserve as a nondegradable phagocytic stimulus also show a similar colocalization with LAMPs(Rabinowitz et al., 1992; Desjardins et al., 1994). The colocalization of P. aeruginosaphagocytosed via the opsonic and non-opsonic receptors with LAMP-i provided evidence offlow of LAMP-i + vesicles to phagosomes and suggested that either (a) phagosome fusion witha LAMP-i + compartment had occurred or (b) the phagosome had gradually accumulated smallamounts of LAMP-i present in surrounding small vesicles to eventually mature into afunctional lysosome.One of the problems encountered in my studies with human monocyte-derivedmacrophages was the susceptibility of the phagocytes to osmotic lysis induced by lysozyme.In phagocytosis systems involving murine peritoneal macrophages, lysozyme was found todisrupt the extracellular bacteria while leaving the murine macrophages intact, thus facilitatingthe quantitation of P. aeruginosa phagocytosed by macrophages (Speert and Gordon, 1992).43However, the same lysozyme-water treatment caused the lysis of the human macrophages aswell. Therefore, I used 50% PBS instead of water to bring about the bacterial lysis inducedby lysozyme since this treatment was not as harsh on the cells. Moreover, in those cells thatsurvived the lysozyme treatment, the lysosomal compartment underwent tremendous osmoticexpansion, thus changing the normal morphology of lysosomes. As a result of thisobservation, the lysozyme-50% PBS treatment was omitted in the immunofluorescence studiesto ascertain that the LAMP-i + compartments and the rhodamine-ovalbumin labelled structuresseen reflect the true morphology of these structures under normal conditions.Although LAMP-l was used as a marker of lysosomes in this series ofimmunofluorescence studies, its colocalization with both opsonized and unopsonized P.aeruginosa did not necessarily indicate that this endocytic structure, with which the ingestedbacteria colocalized, was truly a functional lysosomal compartment; trace amounts of thisglycoprotein have also been found in a prelysosomal compartment of mammalian cells(Rabinowitz et a!., 1992). Rhodamine-ovaibumin was therefore used as a probe for functionallysosomes (Swanson, 1989) to determine if these structures were truly mature lysosomes andnot just simply a compartment containing LAMP-i. It was shown that only a small fractionof the ingested bacteria, both opsonized and unopsonized, colocalized with the rhodamineovalbumin-containing lysosomal compartment. This observation suggested that by one hourafter ingestion, most of the phagosomes that possessed LAMP-i were not in true, maturelysosomes, but were in a prelysosomal compartment proximal to the compartment possessingall the necessary hydroiases which are characteristic of mature, functional lysosomes. To testthis possibility, further studies could be performed using lysosomai enzymes, such as44cathepsins, as markers of true lysosomes. The results could be compared to those obtainedwith the LAMP-i studies to determine if the colocalization of phagosomes containingunopsonized or antibody-coated P. aeruginosa with these lysosomal enzymes coincided withthe colocalization of the bacteria with LAMP-i.Since the direct comparison of the two systems used to synchronize the ingestion of P.aeruginosa via the opsonic and non-opsonic routes was not ideal, the results observed in thesestudies have to be interpreted with caution. The synchronization of phagocytosis ofunopsonized P. aeruginosa was accomplished by withholding glucose to allow only bindingof the bacteria to the phagocyte membrane surface to occur since the adherence step is glucose-independent (Speert and Gordon, 1992). Glucose was subsequently added to the phagocytosismedium, a requirement for ingestion of the bacteria to occur. On the other hand, uptake ofopsonized P. aeruginosa was synchronized by incubating the cells at 4°C, a temperature atwhich adherence of the bacteria to the macrophage surface can occur while phagocytosiscannot (Silverstein et al., 1989). The cells with the bound bacteria were then shifted from 4°Cto 37°C to induce ingestion of the bacteria by the macrophages. At low temperatures, bacteriawere very loosely bound to the macrophage surface and the monocyte-derived macrophageswere easily detached from the glass coverslips by washing. To prevent subsequent washesfrom dislodging the loosely bound bacteria from the phagocyte surface as well as to avoidremoving the loosely attached macrophages from the coverslips, the macrophages wereallowed to ingest the opsonized P. aeruginosa for 10 mm before any uningested extracellularbacteria were removed by washing. The cells challenged with opsonized P. aeruginosa werethen examined at timed intervals after the removal of extracellular bacteria by washing. As45a result, there was a lag period during which the temperature had to increase from 4°C to37°C; however, the exact time at which the macrophages regained the ability to phagocytosebacteria could not be precisely determined. Therefore, the time points referred to in thestudies involving opsonic phagocytosis of P. aeruginosa may not accurately reflect the precisetime at which bacteria entered the cells via the opsonic receptors. An attempt was made toincorporate this same treatment to synchronize the phagocytosis of unopsonized P.aerugiwisa by the phagocyte so as to allow this system to become directly comparable to theopsonic system, but ingestion of the bacteria via the non-opsonic route was quite poor. Nonopsonic phagocytosis of prebound P. aeruginosa has been demonstrated to be time dependent(Barghouthi et al., submitted). The slower rate of non-opsonic phagocytosis of P. aeruginosacompared to that via the Fc receptor-mediated route may explain the poor ingestion observed;thus, 10 mm may not be sufficient for macrophages to take up the unopsonized bacteria viathe non-opsonic pathway. Any bacteria that were bound to the macrophage surface would beremoved by the subsequent washes after the 10 mm lag period before they had adequate timeto be phagocytosed by the macrophages. Since motility has been demonstrated to be arequirement for unopsonized P. aeruginosa to be phagocytosed by murine peritonealmacrophages (Mahenthiralingam et al., 1994), the immobility of the bacteria at the lowtemperature may provide another explanation for the poor ingestion observed when theunopsonized bacteria were preincubated at 4°C prior to incubating at 37°C.From the results reported here, there seemed to be no difference in the intracellular fateof P. aeruginosa ingested via the opsonic receptors or the non-opsonic receptor(s) sincephagosomes containing antibody-coated bacteria or unopsonized bacteria both possessed46LAMP-i. Furthermore, neither BFA nor monensin had any effect on the colocalization of thephagosome with the lysosomal marker. The only observable difference seemed to be in therate of ingestion of P. aeruginosa by the macrophages. Opsonic receptor-mediatedphagocytosis of P. aeruginosa occurred at a faster rate than phagocytosis of the bacteria viathe non-opsonic route even though the systems used to synchronize phagocytosis of P.aeruginosa via these two different routes were not directly comparable. At temperaturespermissive for phagocytosis, the ingestion of opsonized bacteria by macrophages was almostimmediate while little ingestion of unopsonized P. aeruginosa was not observed until 10-20mm after the addition of glucose to the phagocytosis medium (data not shown). Also,opsonized P. aeruginosa entered LAMP- 1 compartments faster than the unopsonizedbacteria. However, there is the possibility that the colocalization event was not faster, butrather that the opsonized P. aeruginosa were internalized faster and thus were available to fusewith LAMP-i + compartments at an earlier time point. Generally, more opsonized thanunopsonized P. aeruginosa particles were ingested by the macrophages. However, at the 10mm time point in Table 4, there were 6.2 ingested unopsonized bacteria per macrophageversus 4.2 ingested opsonized P. aeruginosa per macrophage. The results in this table do nottruly reflect the actual situation, and the inconsistencies seen were probably due to the lagperiod during which the phagocytes had to equilibrate to 37°C as discussed above. Therefore,these results do not provide definitive answers as to whether there was a difference in the rateof the actual fusion event or in the rate of ingestion of bacteria phagocytosed via the twodifferent routes.47Although lysosomes are generally described as small, discrete spherical organelles(Bainton, 1981), they were also observed in these studies to contain elongated, tubularextentions which appeared to be interconnected as a network in uninfected human monocytederived macrophages. This observation of tubular lysosomal structures in human macrophagesis consistent with other published observations on murine macrophages (Swanson et al., 1987;Knapp and Swanson., 1990; Swanson et al., 1992). The morphology of these tubularlysosomes is believed to be maintained by cytoplasmic microtubules (Swanson et al., 1987).The retraction and disappearance of these tubular lysosomal structures upon phagocytosis ofP. aeruginosa is consistent with the observation that tubular lysosomes wrap aroundphagosomes containing latex beads or opsonized erythrocytes (Knapp and Swanson, 1990).BFA is a fungal metabolite that has multiple, species-specific effects on vesiculartransport (for review, see Pelham, 1991). It blocks protein transport into the Golgi apparatus,resulting in the rapid disassembly of the Golgi stack and the transport of resident Golgiproteins back into the ER (Lippincott-Schwartz et al., 1989, 1990). Addition of BFA to cellsalso causes tubulation of the endosomal system, the trans-Golgi network and lysosomes(Lippincott-Schwartz et al., 1991). Traffic between endosomes and lysosomes has beenreported to be impaired in the presence of BFA (Lippincott-Schwartz et al., 1991). Thecolocalization of both opsonized and unopsonized P. aeruginosa with LAMP-i in the presenceor absence of BFA suggested that fusion of the phagosomes with pre-existing LAMP- 1compartments rather than the slow maturation into a functional phagolysosome by the gradualacquisition and accumulation of lysosomal membrane components occurred.48Monensin, an ionophore which inhibits endosome acidification by inducing vacuolaralkalinization, was used to determine if vacuolar acidification is necessary for thecolocalization of phagosomes containing P. aeruginosa with LAMP-i + compartments.Acidification of the endosome can affect eukaryotic receptor recycling to the cell surface (Basuet al., 1981; Lippincott-Schwartz et a!., 1984), potentially affecting bacterial entry. It hasbeen shown to reduce both intracellular dissociation of receptor-ligand complexes and therecycling of certain receptors to the plasma membrane and to inhibit the delivery of thecomplexes to lysosomes (Meliman et al., 1986). An important similarity among most of theorganelles of the vacuolar system, i.e. elements of the exo- and endocytic pathways, is thepresence of H-ATPases responsible for generating an acidic internal environment (Mellmanet a!., 1986). In the endocytic pathway, incoming material encounters progressivelydecreasing pH as it moves through the various endocytic compartment. A similar trend ofprogressively increasing acidity is observed in the exocytic pathway followed by many newlysynthesized secretory products (i.e. from the endoplasmic reticulum to secretory vesicles).It seems that low intravesicular pH plays important roles in the exo- and endocytic pathways.Monensin can bring about alterations in the processing and transport of membrane andsecretory proteins on the exocytic pathway. Disruption of Golgi apparatus pH could lead toinhibition of transport of secretory products in the biosynthetic pathway (Meilman et al.,1986). This study showed that vacuolar acidification was not required for fusion ofphagosomes containing P. aeruginosa with LAMP-1 compartments since the colocalizationoccurred both in the absence and presence of monensin. Also, since disruption of normalGolgi function by monensin did not affect the colocalization of phagosomes containing49opsonized or unopsonized P. aeruginosa with LAMP-i, it suggests that this colocalization wasnot due to fusion of constitutive secretory vesicles containing newly synthesized LAMP- 1.However, it is unclear whether inhibition of vacuolar acidification had any effect on thekinetics of the colocalization of the ingested bacteria with LAMP-i since the heavily stainedLAMP- 1 compartments in the perinuclear area of the phagocytes made the quantitativeassessment of P. aeruginosa colocalization with this marker difficult.The results for the colocalization studies of ingested P. aeruginosa with the MPR werequite difficult to interpret since in most cases, the phagosomes did not appear to possess MPR,but occasionally in some cells the phagosome seemed to be labelled with a small amount ofthe marker. This probably indicated that P. aeruginosa ingested via the opsonic and nonopsonic routes did not generally colocalize with the MPR-enriched late endosome; however,some of the phagosomes acquired trace amounts of MPR and may possibly have entered adifferent pathway where colocalization of the phagosome with the late endosome occurred.One might speculate that the lack of colocalization of the phagosome with MPR was due tothe bacteria not being ingested by the phagocyte since extracellular bacteria were not lysed bylysozyme treatment in the immunofluorescence studies for reasons already mentioned above.This is unlikely because of the observation that in cells challenged with P. aeruginosa, thestained MPR surrounded a hollow MPR-negative area where the bacteria was found. Inaddition, quantitative phagocytosis experiments were done in parallel with theseimmunofluorescence studies to ascertain that there was good ingestion of the bacteria by themacrophages.50Based on the observations obtained from this series of studies and what is currentlyknown about the endocytic and exocytic pathway, I propose a model of the phagocyticpathway of opsonized and unopsonized P. aeruginosa (Fig. 8). Early phagosomes which arebelieved to be functionally distinct from early endosomes, were not examined in this study.Unlike early and late endosomes, newly formed phagosomes have been demonstrated tobecome transiently alkalinized before acidification (Geisow et al., 1981). Results from thepresent studies appear to be in agreement with the model proposed by Rabinowitz et al.(1992). Their data suggest that phagosomes must either directly or indirectly fuse with atubulo-reticular compartment (TC) whose complex morphology is consistent with themorphological description of tubular lysosomes by Knapp and Swanson (1990). A model inwhich the pathway from early phagosome to the lysosomal compartment which bypasses earlyendosomes has been postulated. Two distinct pathways, one from early endosomes and onefrom early phagosomes, seem to exist based on the following published observations: dextransulphate, a polyanionic compound, blocks phagosome-lysosome fusion but fails to influenceendocytosis (Kielian et al., 1982) while lectins (e.g. lectin Concanavalin A) selectively blocksthe pathway from endosomes to the TC (Rabinowitz et al., 1992; Kielian and Cohn, 1981)without any effects on the route from phagosome to lysosome (Goldman et al., 1976; Kielianand Cohn, 1981). Pathways of lysosome biogenesis have been extensively reviewed byKomfeld and Meilman (1989). Early endosomes in the peripheral cytoplasm are involved inthe constitutive pathway of endocytosis and recycling with the cell surface. As such theywould be expected to contain any MPR and lgp’s internalized from the cell surface. The earlyendosomes then translocate towards the perinuclear cytoplasm via microtubules. The51generation of biochemically distinct late endosomes and preiysosomal compartments ensues.The TC mentioned in the study by Rabinowitz et al. (1992) corresponds to the late endosome/prelysosomal compartment. The late endosomes would now begin to receive new input fromGolgi-derived vesicles (presumably containing MPR, newly synthesized lysosomal membraneglycoproteins and lysosomal enzymes). As a result of the constant protein traffickingthroughtout the cell from the inside to the plasma membrane as well as from the plasmamembrane to the endocytic compartments, there are three potential sources of LAMPs fromwhich the phagosomes containing opsonized or unopsonized P. aeruginosa can acquire them.These include the lysosome, prelysosomal compartment, and constitutive secretory vesiclescontaining LAMP-i newly synthesized in the endoplasmic reticulum and transported throughthe Golgi and the trans-Golgi network (TGN). The acquisition of LAMP-i by the phagosomeis probably not from the LAMP-i + secretory vesicles in the biosynthetic pathway or endocyticvesicles since BFA and monensin, inhibitors of the exo- and endocytic pathways, did not affectthe colocalization of the ingested P. aeruginosa with LAMP-i. Because colocalization ofingested P. aerugilwsa with LAMP-i was more extensive than with the rhodamine-ovalbuminlabelled iysosomes, this observation suggested that the phagosomes fused with a prelysosomalcompartment which has acquired the LAMP-i but may not have possessed some of thelysosomal enzymes found in mature, functional lysosome. To test this hypothesis, furtherstudies using lysosomal enzymes as markers of functional lysosomes are essential. The datafrom the colocalization studies with MPR also pointed to the prelysosomal compartment as theorganelle with which the phagosome fused since the majority of the phagocytosed P.aerugiiwsa did not colocalize with MPR with a few exceptions where a few bacteria acquired52very small amounts of the marker as seen in the faint halo of MPR surrounding the phagosome(Fig. 5c and d). The prelysosomal compartment possibly contains trace amounts of MPR.In contrast, a postulation that phagosomes can fuse with both late and early endosomalcompartments has been put forward (Mayorga et al., 1991; Pitt et al., 1992). However, theirobservations of this fusion event occurred under nonphysiological conditions which involvedan in vitro reconstitution of fusion between phagosomes and endocytic vesicles, and so it isunclear how this may apply to the results obtained with P. aeruginosa.How does the P. aeruginosa-containing phagosome fuse selectively with onecompartment and not with another? “Donor” compartments are precisely targeted to themembrane of “acceptor” compartments with which they fuse. This fusion event results in thedelivery of compartment contents as well as membrane lipids and membrane proteins to thenext organelle in the pathway (Rothman and Orci, 1990). Clearly, fusion of organelles mustbe specific and carefully regulated; otherwise, compartment contents may be delivered to anincorrect acceptor compartment. Such an error would disrupt the ordered, sequential,vectorial processing and trafficking of intracellular materials. An attractive model for themolecular mechanism of selective vesicle fusion among cell organelles has recently beenproposed by Rothman et al. (see reviews by Wilson et al., 1991; Waters et al., 1991, andRothman, 1992). Following targeting of coated vesicles to the correct destination by an as yetundetermined process, possibly involving recognition proteins on the organelles’ membranesuncoating occurs to allow the donor lipid bilayer to become closely associated to that of theacceptor compartment. This juxtaposition could be a signal for the recruitment of Nethylmaleimide (NEM)-sensitive fusion proteins (NSFs) and soluble NSF attachment proteins53(SNAPs) from a common cytoplasmic pool and the assembly of NSF and SNAPs into amultisubunit complex anchored to the membrane by the integral NSF/SNAP receptor. TheNSF/SNAP complex promotes fusion by interacting with fatty acyl coenzyme A, GTP-bindingproteins and additional soluble or membrane-bound recognition proteins. There is evidencethat NSF is also needed for vesicle fusion in the endocytic pathway in addition to its role inthe biosynthetic transport. This model may very well apply to phagocytic systems as well.However, the question of the selectivity of fusion of the phagosome with specific organelleswould have to be addressed. One may speculate that the bacteria may be modifying thephagosome membrane in such a way that the membrane would be recognized by certainacceptor compartments and not with others in the pathway. In order for the ingestedmicroorganism to be capable of modifying the phagosome membrane, the bacteria should beviable upon ingestion. However, attempts at assessing the viability of the phagocytosed P.aeruginosa upon ingestion by human monocyte-derived marophages has never been consistentand reproducible in our laboratory. Another method to test if this speculation can be true isto perform the same kinds of experiments as reported here but with killed P. aeruginosa.Alternatively, the phagosomes may have acquired the proteins that are recognized by acceptorcompartments from the plasma membrane and that these proteins are internalized along withthe phagocytic receptors upon phagocytosis. It is also possible that phagosomes accrue theserecognition proteins in secretory vesicles involved in the biosynthetic route.In order to obtain more definitive results to arrive at a model for the intracellularpathway of P. aeruginosa ingested via the opsonic and non-opsonic receptors, electronmicroscopic analysis using the approach used in the reported immunofluorescence studies54should be done. Experiments employing immunogold to label various intracellular organellescan be performed. Also, ovalbumin or BSA conjugated to gold particles can be used with apulse and chase to probe for the various organelles involved in the endocytic process todetermine the relationship between phagosomes and endosomes.In conclusion, P. aeruginosa phagocytosed via the opsonic and non-opsonic receptorsby human monocyte-derived macrophages appeared to enter pathways that are functionallysimilar and that the phagosomes ultimately fuse with compartments that contain lysosomalmembrane components, a likely candidate being the prelysosomal compartment.55Fig. 8. Proposed model of the phagocytic pathway of P. aeruginosa in relation to theendocytic and biosynthetic pathways in human monocyte-derived macrophages.Endocytosis occurs via clathrin-coated pits on the cell surface. Distal to the clathrin-coatedvesicle step, endocytosed materials pass sequentially through the early endosome (EE), theendosome carrier vesicle (ECV), the MPR-enriched late endosome (LE) and the prelysosomalcompartment (PL) before reaching the lysosome (L). Proteins are also constantly beingtrafficked throughout the cell via the biosynthetic pathway where proteins newly synthesizedfrom the endoplasmic reticulum (ER) are transported to the Golgi apparatus and trans-Golginetwork (TGN) for processing. Secretory proteins are exocytosed via constitutive secretoryvesicles. The acquisition of LAMP-i by P. aeruginosa-containing phagosomes (P) is probablyby fusion with one or more of the three potential sources of LAMP-i, namely theprelysosomal compartment (1), the lysosome (2) or the constitutive secretory vesicles (3).Results reported in this thesis seemed to favour the prelysosomal compartment as the primarysource of LAMP-i acquired by phagosomes containing P. aeruginosa.560APLTGNPhagocytosis Endocytosis1P(3) (1)4(2)recyclingE EQ ECVLAMLQLE/LAMP+LAMP0cons ti tu tiv esecretoryvesicles0GolgiER57BIBLIOGRAPHYBainton, D. F.. 1981. The discovery of lysosomes. J. Cell Biol. 91:66s-76s.Barghouthi, S., K. D. E. Everett, and D. P. Speert. Nonopsonic phagocytosis ofPseudomonas aeruginosa requires facilitative transport of D-glucose by, S. S., J. L. Goldstein, G. W. Anderson and M. S. Brown. 1981. Monensin interruptsthe recycling of low density lipoprotein receptors in human fibroblasts. Cell. 24:493-502.Cabral, D. A., B. A. Loh and D. P. Speert. 1987. 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