Surfactant Protein-A Reduces Binding and ... - ATS Journals

Surfactant Protein-A Reduces Binding and Phagocytosis of Pneumocystis carinii by Human Alveolar Macrophages In Vitro Henry Koziel, David S. Phelps, Jay A. Fishman, Martine Y. K. Armstrong, Frank F. Richards, and Richard M. Rose Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston; Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, Pennsylvania; and MacArthur Center for Molecular Parasitology, Yale University School of Medicine, New Haven, Connecticut

Surfactant protein-A (SP-A) levels are increased in Pneumocystis carinii pneumonia, but the role of SP-A in the pathogenesis of P. carinii pneumonia is not completely understood. This study investigated the effect of SP-A on the in vitro binding and phagocytosis of P. carinii by normal human alveolar macrophages (AM). Determination of binding and phagocytosis was done with a fluorescence-based assay, utilizing fluorescein isothiocyanate (FITC)-labeled P. carinii. Binding and phagocytosis of P. carinii to AM correlated inversely with the levels of SP-A present on the surface of the organisms (r 5 20.6323, P 5 0.0086; and r 5 20.9827, P , 0.0001, respectively). The addition of exogenous SP-A to organisms with low surface-associated SP-A reduced P. carinii binding by 30% (P , 0.05) and reduced phagocytosis by 20% (P , 0.05), whereas this effect was reversed with ethylenediamine tetraacetic acid (EDTA) or anti-SP-A antibody. Furthermore, binding and phagocytosis were enhanced after enzymatic removal of P. carinii surface-associated SP-A, and this effect was reversed with the addition of exogenous SP-A. The observed inhibitory effect of SP-A on P. carinii binding and phagocytosis reflected binding of SP-A to the organisms rather than a direct effect of SP-A on the macrophages. These data suggest that increased levels of SP-A may contribute to the pathogenesis of P. carinii pneumonia through binding to the surface of the organism and interfering with AM recognition of this opportunistic pulmonary pathogen. Koziel, H., D. S. Phelps, J. A. Fishman, M. Y. K. Armstrong, F. F. Richards, and R. M. Rose. 1998. Surfactant protein-A reduces binding and phagocytosis of Pneumocystis carinii by human alveolar macrophages in vitro. Am. J. Respir. Cell Mol. Biol. 18:834–843.

Pneumocystis carinii pneumonia remains a frequent and serious pulmonary complication in the immunocompromised host (1). P. carinii is an obligate extracellular parasite, and with rare exception infection by this organism is limited to the lungs (2). Although the conditions predisposing hosts to this infection have been well described (3, 4), the factors involved in the pathogenesis of clinical P. carinii disease and responsible for the predisposition to infection in the lungs have not been completely identified. Alveolar macrophages (AM), as the principal resident phagocytic cells of the lungs, are capable of binding and (Received in original form June 17, 1997 and in revised form October 22, 1997) Address correspondence to: Henry Koziel, M.D., Division of Pulmonary and Critical Care Medicine, Palmer Building, Room 108, Beth Israel Deaconess Medical Center, West, One Deaconess Road, Boston, MA 02215. E-mail: [email protected] Abbreviations: alveolar macrophages, AM; Dulbecco’s modified Eagle’s medium, DMEM; enzyme-linked immunosorbent assay, ELISA; fluorescein isothiocyanate, FITC; surfactant protein-A, SP-A. Am. J. Respir. Cell Mol. Biol. Vol. 18, pp. 834–843, 1998

phagocytosing P. carinii (5–9), and phagocytosis is associated with rapid degradation of the organisms (5). Binding may be partly mediated by immunoglobulin (5) and fibronectin (7), and phagocytosis may be facilitated by immunoglobulin (5). The macrophage mannose receptor mediates binding and phagocytosis of nonopsonized P. carinii (8), presumably through interaction with the mannose-rich glycoprotein surface of the organism (9). Selective depletion of AM renders rodents susceptible to P. carinii pneumonia (10). Thus, AM represent an important component of host-cell defense against this opportunistic pathogen. Interaction of P. carinii organisms with host-defense cells occurs within the alveolar lining material (ALM) (11), which consists of the pulmonary surfactant monolayer at the air–liquid interface and the alveolar hypophase, a thin layer of fluid between the alveolar epithelium and the surfactant monolayer. The hypophase is complex, and contains surfactant apoproteins, immunoglobulins, extracellular matrix (ECM) proteins, and pulmonary immune cells (12). Early clearance of pathogens by AM in the lungs may also involve humoral components of innate im-

Koziel, Phelps, Fishman, et al.: SP-A Impairs Phagocytosis of Pneumocystis carinii

munity (13). Prior investigators have described enhanced in vitro recognition and ingestion of pathogens by AM in the presence of lung lavage fluid (14–17). Alterations in the composition of the ALM in disease states may therefore disrupt normal local host-defense function, and may contribute to the pathogenesis of certain infections. In P. carinii pneumonia, it is unclear whether disease represents a primary defect of macrophage function, or reflects the effect of local factors in the ALM that inhibit an effective macrophage response against this opportunistic pathogen, or both. Surfactant protein-A (SP-A) is a well-characterized major glycoprotein component of pulmonary surfactant (12). The monomeric form has a mass of 28 to 35 kD, whereas the native form exists as a complex structure of six trimers (18). Through carbohydrate-recognition domains, binding of SP-A to carbohydrates is calcium-dependent, a characteristic of C-type lectins such as complement component C1q, conglutinin, and mannose-binding protein (19, 20). In vitro, SP-A may enhance the phagocytosis of organisms by macrophages (21–24). However, others have described either no effect on certain pathogens (24) or SP-A-mediated impairment of phagocytosis (23, 25). Thus, in addition to having surface-tension-reducing properties, SP-A may influence host-cell interaction with various pathogens (26). The effect of increased SP-A levels on the pulmonary effector-cell response to P. carinii has not been completely characterized. In studies with the rodent model of P. carinii pneumonia, SP-A is present on the surface of organisms obtained from rats with active P. carinii pneumonia (27). SP-A levels are increased in the lungs of uninfected, glucocorticoid-immunosuppressed rats, and are further increased during P. carinii pneumonia (28), and SP-A may enhance attachment of P. carinii to rat AM (29). Human studies show that bronchoalveolar lavage fluids (BALFs) from individuals with acquired immune deficiency syndrome (AIDS)-related P. carinii pneumonia have a 5-fold increase in SP-A levels in the absence of corticosteroids (30). Recent studies have reported increased SP-A levels in the BALF of asymptomatic human immunodeficiency virus (HIV)-seropositive individuals (31). However, the effect of SP-A on P. carinii interaction with human AM has not been previously investigated. In human P. carinii pneumonia it remains unclear whether increased levels of SP-A are beneficial to the host or impair host recognition of this opportunistic pathogen. The goal of this study was to examine the effect of SP-A on the in vitro binding and phagocytosis of P. carinii by human AM.

Materials and Methods AM Bronchoalveolar cells were obtained from normal, healthy, nonsmoking, HIV-seronegative persons with normal spirometric findings, through bronchoalveolar lavage (BAL) done with standard technique (32). All BAL procedures were performed on consenting adults under the auspices of the Institutional Review Board of the Beth Israel Deaconess Medical Center, West Campus. Briefly, after topical 1% lidocaine anesthesia, a fiberoptic bronchoscope was wedged in a subsegment of the right middle lobe. BAL was per-

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formed by instilling six 50-ml aliquots of warm normal saline (0.9%), followed by gentle suction after the instillation of each aliquot. The cell pellet was isolated from the pooled BALF by centrifugation at 100 3 g for 10 min and was washed in RPMI 1640 medium (Cellgro; Mediatech, Washington, DC) supplemented with penicillin 100 U/ml and streptomycin 100 mg/ml (Sigma Chemical Co., St. Louis, MO), and the cells were counted with a hemacytometer. Cell morphologic determination and differential determination were done with light microscopy following cytocentrifugation (Shandon, Pittsburgh, PA) and modified Giemsa staining (Diff-Quik; Sigma). AM (7.5 3 105) were isolated by adherence in 24-well plastic tissue-culture plates for 2 h at 378C in 5% CO2, and were washed and kept overnight in complete RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS; JRH Biosciences, Lanexa, KS), penicillin 100 U/ml, streptomycin 100 mg/ml, and L-glutamine 2 mM (Sigma) at 378C in 5% CO2. By this method, the remaining adherent cells were . 98% viable as determined by trypan blue dye exclusion, were . 95% positive with nonspecific esterase staining, and were . 98% actively phagocytic of 1.1-mm latex beads. SP-A Preparation Human SP-A was obtained from the BALF of a patient with alveolar proteinosis. SP-A was isolated from acidic fractions following preparative isoelectric focusing in a Rotophor preparative isoelectric focusing cell (Bio-Rad, Richmond, CA), as previously described (33). The fractions containing SP-A were then extensively dialyzed in distilled H2O. The SP-A was lyophilized and resuspended in a small aliquot of distilled H2O, and was maintained at 2708C prior to use. With this method, no detectable levels of contaminating immunoglobulin (IgG) were present as determined by silver staining following two-dimensional gel electrophoresis. Pneumocystis carinii Organisms Organisms were isolated from the lungs of the immunosuppressed rat model of P. carinii pneumonia. For the purposes of these studies, both cultured and freshly obtained organisms were used. Cultured P. carinii. Barrier-raised, male Sprague–Dawley rats (Hilltop Laboratory Animals, Scotsdale, PA) were immunosuppressed by adding dexamethasone (1 mg/liter) to their drinking water, and were maintained on low-protein (8%) normocaloric diets (34–36). After 9 to 10 wk, when the animals developed moderate to severe P. carinii pneumonia, they were killed and the lungs aseptically removed, homogenized, and filtered through sterile wire mesh in phosphate-buffered saline (PBS). P. carinii were isolated from the supernatant by differential centrifugation, washed in PBS, and maintained in short-term culture with a feeder-cell layer of mink-lung cell line MV 1 Lu (ATCC CCL64) (35) to disperse the native P. carinii clusters. After 8 days, culture supernatants containing the organisms were centrifuged and washed in PBS. Fresh P. carinii. Barrier raised, male Sprague–Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were immunosuppressed by adding dexamethasone (1 mg/liter)

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 18 1998

and tetracycline (500 mg/liter) to their drinking water, and were maintained on low-protein (8%) normocaloric diets (ICN Biomedicals, Costa Mesa, CA). After 7 d, the rats received intratracheal inoculation with 106 to 107 freshly isolated P. carinii organisms by methods previously described (34). After 6 to 12 wk the animals were killed and the lungs surgically removed and homogenized in a stomacher apparatus (Tekmar Co., Cincinnati, OH). The homogenate was centrifuged and the supernatant diluted in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (100 U/ml), streptomycin (100 mg/ ml), and amphotericin-B (25 mg/ml) (Sigma). Contaminating red cells were lysed with ammonium chloride buffer (1 g KHCO3/liter, 8.21 g NH4Cl, pH 5 7.4). For the purpose of removing contaminating host cells and disrupting the native clusters of organisms, the P. carinii suspension was sequentially filtered through Nucleopore filters (Poratics, Livermore, CA) with pore sizes of 10 and 8 mm, and twice through 5-mm filters. The P. carinii organisms were collected by centrifugation, washed, and resuspended in PBS. Morphologically, P. carinii were verified by staining slide preparations with a modified Giemsa method, which identified the trophozoite and cyst nuclei, and by toluidine blue O staining, which stained the cyst form. Quantitation was done by counting P. carinii nuclei with light microscopy. Both preparations yielded approximately 90% trophozoite forms and 10% cyst forms of P. carinii, and were . 98% free of contaminating host cells. Viability was measured as . 85% for all preparations as determined by flow cytometry (37). Importantly, the organisms were dispersed as single trophozoite and cyst forms, and were therefore well-suited for quantitative phagocytosis studies. All P. carinii preparations were cultured for contaminating bacteria or fungi, and contaminated preparations were excluded. Preparation of P. carinii for binding and phagocytosis studies. For the current studies, macrophage binding and phagocytosis were determined for three preparations of P. carinii organisms: (1) Cultured organisms, maintained in short-term culture as described previously, without further modification (This preparation is routinely used in our laboratory); (2) Fresh organisms, freshly isolated and purified as described previously, without modification; (3) Enzyme-treated fresh organisms, freshly isolated and purified as described previously, with subsequent enzymatic digestion with Zymolyase (38) (Zymolyase 100T; ICN Biomedicals). Zymolyase is a crude enzyme preparation containing b(1,3)-glucan laminaripentaohydrolase, an enzyme which cleaves b(1,3)-glucosidic linkages. This treatment has been used to remove the outer cell surface of P. carinii (38). Fresh P. carinii preparations were divided equally, and one portion was incubated in the presence of Zymolyase (0.33 mg/ml in PBS) at 218C for 30 min, followed by centrifugation and washing to remove excess enzyme. The other portion was not treated with Zymolyase. Zymolyase treatment did not affect the viability of the organisms, and did not affect the morphology of the P. carinii as determined by light microscopy. Quantitation of P. carinii Surface-associated SP-A Estimates of the SP-A present on the surface of P. carinii were obtained through an indirect enzyme-linked immu-

nosorbent assay (ELISA) (39). P. carinii were isolated by centrifugation, and the pellet was extracted with 3 M urea in 1% 2-mercaptoethanol to remove organism-bound SP-A. Carbonate/bicarbonate coating buffer was added to the SP-A-containing extracts, and the mixture was used to coat the wells of an Immulon II ELISA plate (Dynatech, Chantilly, VA) in serial dilutions. The sample was then sequentially incubated with rabbit antihuman SP-A IgG, and with horseradish peroxidase-coupled goat antirabbit IgG (Bio-Rad). Following color development with o-phenylenediamine as a substrate, optical density at 490 nm was recorded, and calculations of SP-A concentration were based on comparison with a standard curve generated with SP-A in the same buffers. With this method, SP-A quantitation by ELISA correlated well with the results of Western blot analysis. FITC Labeling of P. carinii For the purpose of facilitating identification of P. carinii, organisms from the three different preparations were labeled with fluorescein isothiocyanate (FITC) (8). Briefly, 10-ml suspensions of P. carinii (1 3 107/ml) were incubated with FITC 0.1 mg/ml in PBS at pH 5 7.4 for 30 min at 378C. Following centrifugation at 1,300 3 g for 20 min, the pellet was washed four times with PBS to remove all detectable free FITC as determined by fluorescence measurement of the supernatant compared with PBS alone. Enumeration of organisms was accomplished by counting with a hemacytometer, using epifluorescence microscopy. This method labeled trophozoite, cyst, and intermediate forms of P. carinii uniformly. FITC is tightly bound to the 120-kD major surface glycoprotein of P. carinii, GP-A (37). The organisms remained as individual trophozoite and cyst forms following FITC labeling, and viability was not affected. Candida albicans A clinical laboratory isolate of Candida albicans (kindly provided by Dr. P. DeGerolomi, Clinical Microbiology Facility, Department of Pathology, Beth Israel Deaconess Medical Center) was cultured on agar plates at 378C. Isolated colonies were heat-fixed at 1008C for 10 min, washed in PBS, and pelleted by centrifugation at 1,200 3 g for 30 min. FITC labeling was accomplished by incubating the suspension with FITC 0.1 mg/ml in PBS at pH 7.4 for 30 min at 378C, followed by centrifugation and repeated washing with PBS to remove free FITC label. The organisms were counted with a hemacytometer, using epifluorescence microscopy, resuspended to a final concentration of 1 3 107 FITC-labeled C. albicans/ml in PBS, and stored at 2708C in 1-ml aliquots. Binding and Phagocytosis Assay Binding and internalization of P. carinii or C. albicans were estimated with a plate fluorescence measuring system (37) (Cytofluor 2300 Measurement System; Millipore, Bedford, MA). Briefly, AM monolayers were incubated with FITClabeled organisms at a ratio of 10:1 (P. carinii to AM) for 30 to 180 min at 378C in 5% CO2 in balanced salt solution containing calcium and magnesium (BSS1). This multiplicity of infection was previously determined to be optimal


for this assay (37). Total remaining macrophage monolayer-associated fluorescence was measured after vigorously washing non-adherent organisms from each well. This fluorescence measurement represented both attached and internalized organisms. An estimate of internalized organisms was then accomplished by quenching extracellular fluorescence with the addition of trypan blue dye (1 mg/ ml) to each well, followed by immediate repeat fluorescence determination. Trypan blue is excluded from intact macrophages, and FITC-labeled P. carinii phagocytosed by the macrophages are therefore protected from quenching by trypan blue dye. Measurements were made at an excitation wavelength of 485 nm and emission wavelength of 520 nm. For all experimental conditions, three macrophageassociated fluorescence (F) determinations were made for each well: (1) initial fluorescence measurement (F Initial), representing the initial inoculum of FITC-labeled organisms, (2) total cell-associated fluorescence measurement, made after incubation for the desired period and removal of nonadherent organisms (FTotal), and representing bound and internalized FITC-labeled P. carinii; and (3) internalized fluorescence measurement, made after quenching of extracellular fluorescence (FInternal), and representing P. carinii phagocytosed by macrophages. Values for macrophage monolayer autofluorescence are subtracted from all fluorescence measurements used in the calculation of data. The lower limit of detectability was approximately 20,000 P. carinii per well in a 24-well culture plate. Data were calculated and expressed as percentages of the initial inoculum of FITC-labeled P. carinii organisms as follows: (1) total cell-associated P. carinii, PCTotal 5 [(FTotal /FInitial) 3 100], which represents both bound and internalized organisms; (2) internalized P. carinii, PCInternal 5 [(FInternal/FInitial) 3 100]; and (3) P. carinii bound to the surface, PCBound 5 [(FTotal 2 FInternal)/FInitial 3 100]. The comparison of fluorescence measurements following washing and quenching with the initial fluorescence measurement for each P. carinii preparation normalized data for differences in FITC labeling of different P. carinii preparations. In select experiments as noted, data were expressed as a percent change from values under control conditions. Determination of Time Dependence for P. carinii Binding and Phagocytosis by AM Experiments were first conducted to determine the time dependence of P. carinii binding and phagocytosis by AM. For these experiments, a single preparation of cultured P. carinii was used to interact with AM from different individuals. FITC-labeled P. carinii were incubated with AM at a ratio of 10:1 (organisms to macrophages) at 378C in 5% CO2 for 30, 60, 90, 120, and 180 min. At the different time points, fluorometric measurements were made on different sample wells, as previously outlined in detail. The Effect of Native SP-A on Binding and Phagocytosis of P. carinii We investigated the effect of native SP-A on P. carinii binding and phagocytosis by AM. This was done by measuring the surface-associated levels of SP-A in the different preparations of organisms, and then examining the binding and

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phagocytosis of these different P. carinii preparations with AM at a single time point. The Effect of Exogenous SP-A on Binding and Phagocytosis of P. carinii We used three approaches to investigate the effect of exogenous SP-A on P. carinii binding and phagocytosis by AM macrophages: (1) preincubating organisms with or without exogenous SP-A; (2) preincubating the alveolar monolayers with exogenous SP-A prior to the addition of P. carinii; and (3) simultaneously adding exogenous SP-A and P. carinii to the macrophage monolayers. For these experiments, we used cultured P. carinii. Preincubation with exogenous SP-A was done at a range of concentrations from 0.01 to 10.0 mg/ml (corresponding to levels measured in human BAL specimens) in PBS at pH 7.4 for 20 min at 378C. Following washing, the FITClabeled P. carinii were incubated with AM at 378C in 5% CO2. These conditions were compared with the effect of mannan (1 mg/ml) and monoclonal anti-P. carinii antibody (the generous gift of Dr. Joseph Kovacs, NIH, NHLBI, Bethesda, MD) on P. carinii binding and phagocytosis, and with the effect of SP-A on C. albicans binding and phagocytosis by AM. Characterization of SP-A Effect on the Binding and Phagocytosis of P. carinii by AM To determine the role of divalent cations on the SP-A effect on binding and phagocytosis of P. carinii, experiments were performed with the chelating agent ethylenediamine tetraacetic acid (EDTA) (Sigma). For these experiments, preincubation of P. carinii with SP-A was done in the presence or absence of 0.6 mM EDTA for 20 min. These organisms were used as described earlier. The role of SP-A was further investigated with specific antibody directed against SP-A. A rabbit polyclonal antiserum directed against human SP-A was prepared as previously described (39). This antiserum cross-reacts with rat SP-A as determined by Western blot analysis and ELISA. An IgG fraction was purified from the antiserum with protein-A Sepharose chromatography (Pharmacia, Piscataway, NJ). For the phagocytosis assay, P. carinii were preincubated with SP-A in the absence or presence of anti-SP-A IgG (15 ng/ml) for 20 min at 378C, and were washed before their addition to macrophage monolayers. To control for nonspecific effects of SP-A or IgG on the binding and phagocytosis of P. carinii, experiments were done with bovine serum albumin (BSA; Sigma) or nonimmune rabbit IgG (Chemicon, Temecula, CA). In these experiments, P. carinii were preincubated in the presence or absence of BSA (10 mg/ml) or rabbit antihuman IgG (1:200) prior to use under the experimental conditions. Results with and without SP-A pretreatment were compared. Statistical Analysis All experiments were performed in duplicate on at least three different occasions, using AM from three different individuals except where noted. The results of each experiment are presented as mean 6 SEM or as percent change compared with control conditions. The binding and phagocytosis data for each experimental condition were evalu-

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Figure 1. Representative photomicrograph of human AM following incubation with FITC-labeled, cultured P. carinii. By brightfield illumination (a), P. carinii organisms are difficult to distinguish from other macrophage inclusions. The same microscopic field viewed with epifluorescence microscopy (b) readily identifies the FITC-conjugated P. carinii bound to or phagocytosed by the macrophages. Fluorescence determination by plate fluorimetry measured the total macrophage-associated fluorescence per well (see MATERIALS AND METHODS). Note that the macrophages exhibit limited autofluorescence. This figure illustrates that the P. carinii cyst (solid arrow) and trophozoite (hollow arrows) forms were individual and readily identifiable, and were not clumped in the presence or absence of SP-A. (Most trophozoite forms are out of the plane of focus.)

ated with Student’s t test for paired observations. Correlations between P. carinii binding and phagocytosis and surface-associated SP-A measurements were calculated with Pearson’s test. Statistical data were calculated with the INSTAT statistical package (Graphpad Software, San

Diego, CA) on an IBM PS/2 386 computer. Statistical significance was accepted for P < 0.05.

Results Time-dependent Interaction of P. carinii with AM Figure 1 illustrates a representative microscopic field of human AM following incubation with FITC-labeled P. carinii. The fluorescence detection system used in this study measured the fluorescence associated with the adherent monolayer of macrophages per well for each experimental condition. The interaction of P. carinii with AM was time-dependent (Figure 2). The number of organisms bound to or phagocytosed by AM reached a maximum by 60 min at 378C, without showing a significant increase before 180 min. On the basis of this observation, all subsequent experiments were conducted for 60 min.

Figure 2. Time-dependent interaction of P. carinii with adherent normal human AM. Cultured, FITC-labeled P. carinii (PC) were incubated with AM at 378C in 5% CO2. P. carinii association with macrophages (including both bound and phagocytosed organisms) was maximal by 60 min (n 5 3).

Determination of AM Binding and Phagocytosis Comparing Different Preparations of P. carinii Following incubation at 378C for 60 min at a P. cariniito-macrophage ratio of 10:1, 17.4 6 4.8% (range: 8.1 to 29.2%) of the initial P. carinii inoculum was bound to the surface, and 5.34 6 2.01% (range: 0.8 to 9.0%) was phagocytosed. As previously reported (37), the relationship be-


tween measured fluorescence signal and number of organisms was linear over the range used under experimental conditions. In the absence of macrophages, there was no detectable binding of FITC-labeled P. carinii to the plastic wells, and the addition of trypan blue to a full inoculum of P. carinii completely quenched the fluorescence signal, to background levels (data not shown). Macrophage viability was not affected by incubation with P. carinii up to 180 min, and the FITC signal was not quenched during the course of incubation (data not shown). Quantitation of P. carinii Surface-associated SP-A In general, freshly isolated P. carinii showed the lowest binding and phagocytosis, whereas cultured P. carinii showed the highest binding and phagocytosis. To investigate the possibility that the observed variation in binding and phagocytosis was attributable to the presence of variable amounts of SP-A on the surface of P. carinii, we quantitated SP-A present on the surface of the different preparations of the P. carinii organisms. Table 1 shows that SP-A was present in highest amounts on the surface of P. carinii freshly extracted from rats with active P. carinii pneumonia. These levels were reduced by Zymolyase enzymatic treatment (P , 0.05). Levels of SP-A were lower on the surface of cultured organisms (P , 0.001) than on the surfaces of freshly harvested organisms with or without Zymolyase pretreatment. The Effect of Native SP-A on Binding and Phagocytosis of P. carinii A correlation plot was generated, comparing the amount of P. carinii surface-associated SP-A with the macrophage binding and phagocytosis of P. carinii for each P. carinii preparation. The ability of AM to bind and phagocytose P. carinii correlated inversely with the amount of SP-A present on the surface of different preparations of P. carinii (Figure 3). The correlation coeficient (r) for binding was 20.6323 (P 5 0.0086). The correlation coefficient (r) for phagocytosis was 20.9827 (P , 0.0001).

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AM after 60 min incubation at 378C in 5% CO2. Under these conditions, approximately 64% of the cell-associated organisms were bound to the surface, and 36% were phagocytosed by the AM. The FITC signal was not quenched by exogenous SP-A (data not shown). Preincubation of the cultured P. carinii organisms with exogenous SP-A reduced binding of the organisms to AM monolayers by up to 30% (P , 0.05) over a range of concentrations of SP-A of 0.01 to 10 mg/ml (Figure 4A), in a concentration-dependent manner. Phagocytosis of cultured P. carinii by AM was reduced by up to 20% (P , 0.05) in the presence of exogenous SP-A (Figure 4B). The simultaneous addition of exogenous SP-A and cultured P. carinii to the macrophage monolayers (i.e., omitting preincubation), or preincubation of AM with exogenous SP-A failed to demonstrate any significant effect on P. carinii binding or phagocytosis as compared with control conditions (data not shown). As control conditions for the phagocytosis assay, binding and phagocytosis of P. carinii were increased by 47% in the presence of specific monoclonal anti-P. carinii antibodies, and were reduced by 81% in the presence of mannan (data not shown). Characterization of SP-A Effect on Binding and Phagocytosis of P. carinii by AM With cultured P. carinii, the reduction in binding and phagocytosis after the addition of exogenous SP-A was reversed in the presence of EDTA (0.6 mM) or in the presence of

Effect of Exogenous SP-A on Binding and Phagocytosis of P. carinii by AM To examine the effect of exogenous SP-A on P. carinii binding and phagocytosis, we used organisms with the lowest quantity of surface-associated SP-A, cultured P. carinii. In the absence of added opsonins, 22.7% of the initial inoculum of cultured P. carinii remained associated with the

TABLE 1

Quantitation of surface-associated SP-A for different Pneumocystis carinii preparations Preparation

PC (fresh) PC-Z (fresh, Zymolyase treated) PC (cultured) Values represent means 6 SEM. * P , 0.05 compared with PC (fresh). † P , 0.001 compared with PC (fresh). § P , 0.001 compared with PC-Z.

SP-A (pg/PC)

4.28 6 2.5 1.92 6 1.9* 0.03 6 0.01†§

Figure 3. Correlation plot comparing P. carinii surface-associated SP-A levels with AM-mediated binding and phagocytosis of P. carinii. SP-A was eluted from different preparations of P. carinii, including freshly isolated (n 5 3), freshly isolated and Zymolyase treated (n 5 3), and cultured organisms (n 5 2), and was quantitated with ELISA as detailed in MATERIALS AND METHODS. Each point represents measurements from a single preparation of P. carinii. (For each curve, the three points on the extreme right represent freshly isolated organisms, and the two points on the extreme left represent cultured organisms.) Pearson’s correlation coefficient (r) for binding 5 20.6323 (P 5 0.0086) and for internalization 5 20.9827 (P , 0.0001).

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Figure 5. Characterization of the effect of exogenous SP-A on P. carinii binding and phagocytosis by macrophages. Cultured P. carinii were incubated in the presence of SP-A. The reduction in binding due to SP-A (0.1 mg/ml) was abrogated in the presence of EDTA (0.6 mM) or specific antibody to SP-A (15 ng/ml). Substitution with an irrelevant antibody or nonspecific protein (BSA 10 mg/ml) produced no significant effect on binding or phagocytosis. Data are expressed as percentages of control values (n 5 3).

Figure 4. Exogenous SP-A reduced P. carinii binding and phagocytosis by AM. Cultured, FITC-labeled P. carinii were preincubated in the presence or absence of SP-A (0.01 to 10.0 mg/ml), and were then incubated with AM for 60 min at 378C in 5% CO2. Binding and phagocytosis of P. carinii are each expressed as a percent change from control values without SP-A (n 5 4).

anti SP-A IgG (15 ng/ml) (Figure 5). Furthermore, preincubation of P. carinii with an irrelevant antibody or a nonspecific protein, BSA, failed to demonstrate any significant effect on P. carinii binding or phagocytosis. The Effect of SP-A on C. albicans Binding and Phagocytosis In contrast to P. carinii, C. albicans, in identical experiments, showed enhanced binding and phagocytosis in the presence of exogenous SP-A. Following preincubation of C. albicans with SP-A over an SP-A concentration of 0.1 to 10 mg/ml, binding and phagocytosis were increased by 19.0 6 7.2% (P , 0.05; data not shown). No significant effect was observed on the binding or phagocytosis of C. albicans in the presence of BSA. Effect of Enzymatic Digestion of P. carinii Surface Molecules on Binding and Phagocytosis by Macrophages To determine the role of P. carinii surface-associated molecules, including native SP-A, on the surface of P. carinii obtained from animals with active pneumonia, we per-

formed experiments in which we compared the binding and phagocytosis of freshly isolated P. carinii organisms before and after enzymatic pretreatment with Zymolyase. Preincubation of freshly isolated P. carinii with exogenous SP-A did not significantly affect the binding or phagocytosis of these organisms by AM as compared with conditions lacking SP-A pretreatment (Figure 6). However, enzymatic digestion of the outer surface of the organisms with Zymolyase increased the binding and phagocytosis of P. carinii (P , 0.05). Furthermore, preincubation of enzymatically pretreated P. carinii with exogenous SP-A reduced binding and phagocytosis to levels observed before enzymatic digestion (P , 0.05).

Discussion This study demonstrated that SP-A reduced in vitro binding and phagocytosis of P. carinii by normal human AM. Binding and phagocytosis of rat-derived P. carinii correlated inversely with the amount of surface-associated SP-A on the organisms. The reduced binding and phagocytosis were observed after the addition of purified exogenous SP-A to cultured P. carinii organisms with low levels of native surface-associated SP-A, or to freshly harvested P. carinii that were enzymatically pretreated to remove surface-associated SP-A. This effect was concentration dependent and calcium dependent, and was abrogated in the presence of specific anti-SP-A antibody. Although Zymolyase may remove other surface-associated surfactant glycoproteins and ECM proteins, or reveal important antigenic determinants, the complete reversal of enhanced binding of Zymolyase-treated P. carinii with the addition of exogenous SP-A suggests that SP-A is sufficient to account for the observed effect. The precise mechanism for the observed reduction in P. carinii binding and phagocytosis was not established in


Figure 6. Enzymatic removal of surface-associated molecules of freshly isolated P. carinii enhanced binding and internalization by AM. Preincubation of freshly isolated organisms with high levels of native SP-A (PCfresh) with exogenous SP-A produced no significant effect on binding or phagocytosis of P. carinii. However, Zymolyase pretreatment of these same freshly isolated organisms (PC-Zfresh) produced increased binding and phagocytosis as compared with untreated organisms (PCfresh). Addition of exogenous SP-A to enzymatically pretreated organisms (PC-Zfresh) reduced binding and phagocytosis to levels comparable to those for freshly isolated, untreated material (PCfresh) (n 5 3).

the current study. The observation that SP-A enhanced binding and phagocytosis of C. albicans, as noted in the current study and by other investigators (22, 23, 25, 40), suggests different mechanisms of macrophage recognition for these two microorganisms. The reduced P. carinii binding and phagocytosis represented an interaction of SP-A with the organism, rather than an interaction with the macrophage. The elimination of the effect with EDTA suggests the involvment of carbohydrate binding by SP-A, a characterstic of C-type lectins. SP-A binds to a variety of monosaccharides, including D-mannose, L-fucose, D-glucose, and D-galactose (19), and investigators have shown SP-A binding to the mannose-rich surface of P. carinii (27). In this regard, binding of SP-A to the surface of P. carinii may interfere with the recognition of the organism by the AM mannose receptor (8, 9) or another important receptor. This mechanism of utilizing host-cell molecules to evade host recognition is a strategy used by other parasites, and may in part serve as a means by which P. carinii eludes host-defense-cell recognition in the lungs. This possibility is particularly intriguing because SP-A levels are increased in the lungs of glucocorticoid-immunosuppressed rats prior to infection with P. carinii (28). Similarly, SP-A levels are increased in the lungs of asymptomatic HIV-infected individuals (31), including persons at late clinical stages (pe-

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ripheral CD41 T-lymphocyte count , 200 cells/mm3) or even at earlier clinical stages (peripheral CD41 T-lymphocyte count 200 to 500 cells/mm3) of HIV-1 infection (31), in the absence of P. carinii pneumonia. These observations suggest that increased SP-A levels may predispose the host to P. carinii pneumonia. The finding that SP-A only modestly reduced binding and phagocytosis suggests that increased levels of SP-A may only partly contribute to host susceptibility, and that SP-A-coated P. carinii may interact with macrophages through different pathways. Whereas unopsonized P. carinii may interact predominantly with the mannose receptor (8), surface-associated SP-A on P. carinii could interact with macrophage receptor for SP-A (41). SP-A may also influence C1q and Fc-receptor-mediated phagocytosis (21). The potential contribution of IgG, which may contaminate SP-A preparations obtained from patients with alveolar proteinosis (42), is unlikely, since contaminating IgG was not detected in the SP-A preparation used in the current study. The potential contributions of other important molecules that influence P. carinii interaction with macrophages, including fibronectin (7), vitronectin (43), and SP-D (44), were not specifically investigated in this study. Increased levels of fibronectin and vitronectin have been measured in the BALF of asymptomatic HIV-infected persons (43), and increased levels of SP-D are observed in the lungs of rats with P. carinii pneumonia (44), creating the opportunity for these molecules to influence P. carinii interaction with AM in vivo. Although specific data are not available, the different P. carinii preparations examined in the present study may also have different surface levels of rat-derived IgG, fibronectin, vitronectin, SP-D, and other molecules, each of which may also contribute to the observed P. carinii binding and phagocytosis. Results from the current study contrast with those in a recent investigation that demonstrated enhanced P. carinii attachment to rat AM in the presence of exogenous SP-A (29) at 48C. In addition to reflecting differences in the species of origin for macrophages, the species of origin for the P. carinii and surface glycosylation patterns, and the methodology for measuring binding and phagocytosis in the current study and in this other study, the results observed in the current study may reflect differences in the kinetics or mechanism(s) of SP-A binding when performed at the physiologic temperature of 378C. Importantly, prior reports have also described diverse effects of SP-A on the interaction of microorganisms with host cells. SP-A was reported to promote attachment of Mycobacterium tuberculosis to human AM (31), to increase the cell-association of serum-opsonized Staphylococcus aureus with rat AM (22), and to enhance the phagocytosis (15, 24, 45) and intracellular killing (15, 46) of bacteria by macrophages. However, other investigators reported either no effect of SP-A on binding or phagocytosis, or SP-A mediated impairment of phagocytic function (23–25, 40). Similarly, the explanations for these differences in the reported effects of SP-A on macrophage function are probably multifactorial, and illustrate the complexity of pathogen–host cell interaction. The reported effects of SP-A depend on the species (23, 25) and specific strain (40) of

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microbial agent studied, the growth phase of the microbes (23), the species origin of the phagocytic cell type investigated (25, 29, 31), specific experimental culture systems, and the specific methods used to measure binding and phagocytosis. Furthermore, differences in the origin, preparation, activity, and concentrations of SP-A probably contribute to the observed differences in phagocyte function. In the current study, attempts to minimize the influence of these variables included the use of a single preparation of human SP-A, the use of a physiologically relevant range of SP-A concentrations, the use of freshly derived primary human AM, and investigation at physiologic temperature. Certain limitations should be considered, however. The current method did not allow for determination of whether the observed reduced binding and phagocytosis represented a reduced proportion of macrophages engaging organisms, or a reduction in the number of organisms bound to or phagocytosed by individual macrophages. The potential influence of exogenous host proteins or soluble factors related to the preparation of P. carinii was also not investigated. Although SP-A derived from persons with alveolar proteinosis is commonly utilized in studies, its function may be different from that of SP-A from healthy individuals or persons with HIV infection and P. carinii pneumonia, and extrapolation of these results may therefore be limited. Futhermore, the observed in vitro effects of SP-A may not accurately represent effects that occur in vivo. Ideally, use of human-derived P. carinii in the current investigation would have provided additional insight into the mechanisms of interaction that were studied. However, the use of rat-derived P. carinii was necessary in the absence of a consistent source of purified human-derived P. carinii (47). Rat-derived P. carinii share many major antigenic determinants with human-derived organisms (48), and histopathologic comparison of P. carinii pneumonia in rats reveals it to have similar characteristics to human infection (49). Examination of the effect of SP-A on the growth phase of this organism as this relates to binding and phagocytosis was not possible, nor did the study allow for determining differences in macrophage interaction with isolated trophozoite forms from cyst forms of P. carinii. In summary, the study demonstrated that SP-A decreased the binding and phagocytosis of P. carinii by AM in vitro, and that binding and phagocytosis inversely correlated with the levels of surface-associated SP-A on P. carinii. We postulate that in the setting of HIV infection or glucocorticoid immunosuppression, increased SP-A levels interfere with host-defense-cell recognition of P. carinii, and perhaps in combination with other factors, may impair macrophage clearance of this opportunistic pathogen and in part contribute to the pathogenesis of P. carinii pneumonia in the susceptible host. Furthermore, in view of the unique anatomic localization of SP-A in the alveolar air space, these observations may in part represent one of the factors related to the predisposition of P. carinii for establishing disease in the lungs. The role of SP-A in the pathogenesis of P. carinii pneumonia could be further defined by examining the susceptibility of SP-A-deficient mice to P. carinii challenge (50). Acknowledgments: The authors express special thanks to Julie Samia, Cheryl Arena, Theresa Kelley, Robert Garland, and Todd Umstead for their dedicated

and excellent technical assistance. The authors also thank Dr. Paula Pinkston for her careful review and helpful suggestions. This work was supported in part by Public Health Service Grants HL43510 (R.M.R., H.K.), HL48006 (D.S.P.), and AI24194 (F.F.R.), Massachusetts Thoracic Society/American Lung Association Research Grant (H.K.), and the Parker B. Francis Foundation (H.K.).

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