580

Participation of Complement in the Phagocytosis of the Conidia of Aspergillus fumigatus by Human Polymorphonuclear Cells Joy Sturtevant and Jean-Paul Latge

Unite de Mycologie, lnstitut Pasteur. Paris, France

Nonspecific immunity plays a major role in the clearance of the opportunistic fungal pathogen, Aspergillus fumigatus. However, the mode of recognition of Aspergillus conidia by the different

Infections due to Aspergillus fumigatus have increased in recent years because of organ transplant surgery, corticosteroid therapy, and the prolongation of life in agranulocytic patients [1-5]. The high incidence ofinvasive aspergillosis in leukopenic and agranulocytic patients clearly indicates an important role for phagocytic cells in the containment of infection in the normal host. The importance of an active phagocyte system has been confirmed by animal studies (reviewed in [6, 7]), Several laboratories have demonstrated that monocytes, lung macrophages, and polymorphonuclear cells (PMNL) can phagocytose conidia of A. fumigatus in vitro [8-15]. However, the initial recognition of the infective conidia by the phagocytic cells is poorly understood. PMNL are thought to be of major importance in opportunistic fungal infections [16-19], and indeed, neutropenia is considered the most predisposing factor in invasive aspergillosis [20]. In addition, PMNL actively infiltrated the lungs in Aspergillus infections in animal models under normal conditions [21-23] but were not detected in immunosuppressed mice that succumbed to infection [22]. PMNL are recruited during lung inflammation and may outnumber macrophages [22, 24] and participate in pulmonary immunity [20]. Because of these facts and the documented clinical importance of neutrophils, we studied the initial interaction between PMNL and infective conidia.

Received 4 September 1991; revised 30 March 1992. Presented in part at Federation of American Societies for Experimental Biology, American Society for Biochemistry and Molecular Biology, and American Association of Immunologists Joint Meeting, June 1990, New Orleans (abstract 3198), and a NATO Workshop, Fungal Cell Wall and Immune Response, Elounda, Greece. September 1990. Grant support: French Foreign Office; Phillipe Foundation. Reprints or correspondence: Dr. Joy Sturtevant, Unite de Mycologic, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. The Journal of Infectious Diseases

1992;166:580-6

© 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0019$01.00

Materials and Methods Fungal cells. A. fumigatus CBS 144.~9 was isolated from a patient with aspergilloma at Institut Pasteur and maintained on malt extract agar slants at 22°C. For experimental use, conidia were transferred to 2% malt extract (Touzart & Matignon, Vitrysur-Seine, France) and cultured at 26°C for 5-6 days. Conidia harvested later than this had slower and lower germination rates. To obtain conidia free of culture nutrients, conidia were harvested by vacuum into sterile distilled water, filtered through gauze to separate conidia from contaminating mycelium, centrifuged, and washed twice with distilled water. These were defined as resting conidia. Resting conidia (2-4 X 106/mL) were incubated in Sabouraud medium with 2% glucose and I%peptone at 37°C for 4-5 h. At the end of this time, conidia were increased in volume 1.5-2.0 times and were defined as swollen conidia [6]. Swollen conidia were washed twice with distilled water before use. Reagents and antibodies. Culture medium 199 (M 199), fluorescein isothiocyanate (FITC), dextran (molecular weight, 124,000), latex beads (3 #Lm), protein A-Sepharose, and antibodies (goat anti-human complement C3, goat anti-human immunoglobulin, goat anti-rat immunoglobulin, fluoresceinconjugated rabbit anti-goat immunoglobulin, and rhodamineconjugated goat anti-rabbit immunoglobulin) were obtained from Sigma (St. Louis). Goat anti-human complement C5 was from Nordic Immunology (Tilburg, Netherlands). Lymphocyte separation medium was from Eurobio (Les VIis, France). Heparin sulfate was from Diagnostics Pasteur (Paris), and fetal calf serum (FCS) from Laboratoire Sorga (La Plaine-Saint-Denis, France). FCS was always heat inactivated at 56°C for 30 min before use. Anti-conidia antibody was produced by injecting rabbits with a homogenate of conidia (I mg) in complete Freund's adjuvant, subcutaneously (I: I VOl/vol). To enrich for conidia surface proteins, rabbits were boosted twice in the ear vein with 2.5%paraformaldehyde-fixed intact conidia (5 X lOS/rabbit) and then boosted twice more with conidia homogenate in incomplete Freund's adjuvant. Conidia homogenate was prepared by disrupting 1.5 X 109 conidia in 10 mL of distilled water for 3

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elements of an intact phagocyte system remains poorly understood. This study concentrated on the initial interaction between infective conidia and human polymorphonuclear cells (PMNL) using a double-label immunofluorescent method. At 37°C, association was rapid. Although ingestion rates were slower than association, both association and ingestion plateaued at 90 min and were considerably reduced in the presence of fetal calf serum. In the presence of autologous plasma, association increased as the conidia-to-cell ratio increased, but the percentage of associated conidia that were ingested decreased. At 4°C, phagocytosis was negligible. Phagocytosis experiments in the presence of plasma treated in various ways to inhibit the complement pathways demonstrated that optimal association was dependent on an active alternative complement pathway.

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Aspergillis Conidia and PMNL

6.7% treated or nontreated autologous plasma for 30 min at 37°C or 56°C, with antiserum from aspergilloma patients or anti-conidia rabbit antiserum at 10%, or with an enriched human IgG fraction (4 mg/ml.) for 60 min at 4°C. In one experiment, conidia were also opsonized at 37°C with purified C3 (I mg/ml.) donated by T. Kozel (Reno, NV). Conidia were washed three times with PBS before use. The human IgG fraction was prepared by 3.3% caproic acid precipitation, concentrated by 50%ammonium sulfate precipitation [27], and further purified by protein A affinity chromatography. Plasma treatments. Treatment of plasma included chelation of cations by 10 mMEDTA or 10 mMEGTA with 5 mMMg++. Plasma was heated at 56°C for 30 min to inactivate both complement pathways or at 50°C to inactivate the alternative pathway. Plasma was partially depleted ofC3, C5, or IgG by incubating 33%plasma with 10% anti-C3, anti-C5, or anti-human IgG antibodies for 60 min at 4°C. Immunofluorescence assays. Conidia (10 7; 108/mL) were incubated in 6.7% plasma for 30 min at 37°C, then washed three times. Opsonized conidia were then incubated with goat antihuman immunoglobulin or anti-C3 at a dilution of I:20 or in buffer alone for I h at 4°C. Conidia were washed thoroughly and finally probed with fluorescein-conjugated anti-goat immunoglobulin at a dilution of I:200 for I h at 4°C, washed, and observed by fluorescent microscopy. All washes and antibody dilutions were in PBS with 5% FCS. Microscopy. A Zeiss microscope with an epiluminescence device was used for light microscopy with an excitation filter of 510-560 nm for rhodamine and 450-490 nm for fluorescein. Statistics. Means were compared by using the two-sample, two-tailed Student's t test for independent means or two-factor variance analysis [28]. For calculations, Epistat software (T. L. Gustafson, Richardson, TX) was used.

Results Association and ingestion. Figure 1 shows the ability of the double-labeling immunofluorescent method to discriminate between association and ingestion of conidia by PMNL. At 37°C, association was rapid, within 15 min, and plateaued between 30 and 60 min in the presence of autologous plasma (figure 2A, solid lines). Maximal association at a 5: 1 conidia-to-PlvtNl. ratio was 84% after 90 min. The number of conidia per cell usually plateaued at 90 min (figure 2A, dashed lines). The increase in number of conidia per PMNL but not in percentage of association was possibly due to the free conidia always present in suspension. Although absolute numbers were lower, ingestion curves closely followed association (figure 2B). In the presence of autologous plasma, ingestion was slower than association, but maximal ingestion and association were both observed at 90 min. In the presence of FCS only, association and ingestion were considerably less but continued to increase throughout the 3-h incubations. Association increased significantly as the conidia-to-cell ratio increased (figure 3A). In the presence of FCS, associa-

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min in a glass bead homogenizer (MSK; Braun, Melsungen, Germany). Antiserum from patients with aspergilloma were donated by V. Hearn (Public Health Laboratory Service, Mycological Reference Laboratory, London). PMNL. PMNL were isolated by differential centrifugation [25]. Blood donated by healthy volunteers in heparin (10 units/ mL) was diluted I: I in M 199. Granulocytes were separated from mononuclear cells by lymphocyte separation medium [25]. The top layer was kept as a source of autologous plasma and considered to be 33%plasma. PMNL were further purified by dextran sedimentation and hypotonic lysis with distilled water [26]. Phagocytosis assay. To optimize visualization of conidia and discriminate from cellular vacuoles and contaminating platelets, conidia were labeled with FITC basically as described for yeast [26]. Freshly harvested conidia (~5 X 108/mL) in 0.1 M carbonate buffer, pH 9.0, were incubated with FITC at a final concentration of 0.1 mg/ml. overnight with rotating at 4°C. FITC-Iabeled conidia were incubated with PMNL (10 6/mL in M 199,0.5% FCS) in suspension with angular rotation (20 revolutions/min) at different conidia-to-cell ratios in the presence of 10% heat-inactivated FCS or 6.7% autologous plasma at 37°C for I h unless otherwise noted. Ratios > 10:I were not used because of high levels of aggregation of conidia and subsequent inability to determine accurately the number of conidia per PMNL. Results were expressed as percentage of association: the number of PMNL out of 100 that had one or more associated conidia. The average number of conidia per PMNL was calculated by counting the total number of conidia associated with 100 PMNL and dividing by 100. To obtain an index of association, percentage of association was multiplied by the average number of conidia per PMNL. Ingestion. To quantitate ingestion, it was necessary to adapt a method that allowed discrimination between attached and ingested conidia. This was accomplished by labeling free conidia with an anti-conidia antibody. After the desired incubation period, the PMNL-FITC-conidia mixture was washed and either fixed or not in 2.5%paraformaldehyde for I h at 22°C for counting before antibody treatments. To avoid nonspecific binding of antibodies by PMNL, mixtures were preincubated with goat anti-rat IgG (I :50) for 30 min at 4°C and then washed. For specific labeling of exposed conidia, mixtures were probed with rabbit anti-conidia antibody (I :500), washed, and finally incubated in rhodamine-conjugated anti-rabbit antibody (1:50). Consequently, free and attached conidia were labeled with both fluorescein and rhodamine, while the ingested conidia, which were not exposed to the antibody treatments, were labeled only with fluorescein. The percentage of ingestion was the number of PMNL out of 100 that had ingested ~ I conidia. The average number of ingested conidia was estimated after counting 100 PMNL and dividing by 100. Preliminary studies demonstrated that under otherwise similar conditions (M 199, 6.7% autologous plasma, I h at 37°C), association was significantly more efficient when PMNL were in suspension (63%) than if adhered to coverslips (26%). Consequently, all further studies were done in sterile Eppendorftubes with rotation. Opsonization. Conidia at 5 X 107/ml, were incubated with

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tion was the highest at conidia-to-cell ratios of 5: I and 10:I, but the average number of conidia per 100 PMNL remained low, between I and 2 (figure 3B). In the presence of plasma, the average number of conidia per PMNL was as high as 7.6 at a ratio of 10:I. Although the absolute number of conidia ingested in the presence of plasma increased as the conidiato-cell ratio increased, the percentage of PMNL-associated conidia that were ingested decreased (60%vs. 39%at ratios of

5: I and 10: I), suggesting a concentration threshhold for ingestion (figure 3B). Self-aggregation of conidia in the presence of plasma at a 10: I conidia-to-PMNL ratio also may have contributed to reduced ingestion. At 4°C, phagocytosis was negligible at low conidia-to-cell ratios. In the presence of high numbers of conidia, percentage of association was as high as 30% but never exceeded an average of I conidia or conidia aggregate/PMNL (data not shown), whereas> 7 conidia/PMNL were associated at 37°C. At conidia-to-PMNL ratios >5: I, after prolonged incubation (from 4-6 h to 24 h), association in the presence ofFCS reached levels not significantly different from the ones observed in autologous plasma after l-h incubations. Indeed, when conidia were incubated with PMNL in the presence of autologous plasma for 4-6 h, association levels decreased significantly (figure 4). This was concomitant with a significant decrease (P = .01, df= 4) in the starting PMNL population when conidia were incubated with PMNL in the presence of autologous plasma (52%); the latter decrease was not observed in the presence of FCS (20%) or latex beads and plasma (20%). Role of plasma. Figures 2-4 demonstrated the importance of plasma in the initial interaction between PMNL and conidia. In experiments where autologous plasma and FCS were mixed (I: I vol/vol), results were similar to those seen with plasma alone. Therefore, FCS was not suppressive but the plasma was stimulative. To determine the components in plasma important for initial recognition, conidia were preincubated in different serum sources before incubation with PMNL. When conidia were opsonized in 6.7% autologous plasma for 30 min at 37°C, the association index did not significantly differ from control levels in the PMNL reaction assay (figure 5). Association was low and similar to levels seen with FCS if conidia were opsonized with heat-inactivated plasma (56°C) or an IgG-enriched fraction from normal human plasma despite the deposition of IgG on the conidia surface as demonstrated by immunofluorescence (figure 6). Also, conidia preincubated in heat-inactivated serum from patients or hyperimmune rabbit anti-conidia antibody did not significantly augment phagocytosis (figure 5). Therefore, IgG did not playa significant role in association. In one experiment, conidia were opsonized in purified C3. Association of C3-opsonized conidia (42%, 0.78 conidium/ PMNL) was not significantly different from that of conidia opsonized in plasma (56%, 0.92 conidium/PMNL). However, SDS-PAGE and immunoblotting showed that the C3 preparation used in these experiments contained a low percentage of C3b, iC3b, and C3d. Association of control conidia opsonized in FCS alone was only 12%, 0.1 conidium/ PMNL. Plasma was then treated in various ways to inhibit the classical or alternative complement pathways (figure 7). Blocking of both pathways by heating at 56°C, chelation with 10

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Figure 1. Phagocytosis of conidia of A. fumigatus by human polymorphonuclear cells (PMNL). PMNL and fluorescein-conjugated conidia were incubated at 5: I for 45 min at 37°C. Attached and ingested conidia were discriminated by postlabeling with rabbit anti-spore antibody and rhodamine anti-rabbit antibody. A, phase contrast; B, fluorescein, and C, rhodamine conditions. Arrowheads indicate ingested spores. Bar = 10 JLm.

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Aspergillis Conidia and PMNL

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" conidia/PMN

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Figure 2. Kinetics of association and ingestion of conidia by polymorphonuclear cells (PMNL). Conidia and PMNL (5: 1) were incubated in presence of 10% heat-inactivated fetal calf serum (FCS) or 6.7%autologous plasma (AP) at 37°C for 5-180 min. A, Percentage ofPMNL with associated conidia. (solid lines, scale at left) and average number ofconidia associated per PMNL (dashed lines, scale at right). B, Percentage with ingested conidia and average number ingested. Results are average of three experiments.

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Discussion In agreement with other studies [8-11], association between the conidia of A. fumigatus and human PMNL was rapid, and results with both swollen and resting conidia were comparable [9]. Although Levitz and Farrell [10] observed only 10% association between conidia and PMNL at a ratio of 2: 1, they stopped the reaction after 10 min. In our system, this was still a suboptimal time of phagocytosis. In addition, they observed higher rates of association with swollen co-

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tion of C5 did not significantly inhibit association. Since C5 is a late-acting component, it is not surprising that it did not play a significant role in recognition or association. After conidia were incubated in plasma, C5could not be detected by indirect immunofluorescence (data not shown).

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time (minutes)

time (minutes)

mM EDT A, or depletion of complement component C3 by anti-Cs inhibited percentage of association by 73%, 60%,and 75%, respectively. When plasma was heat treated at 50°C, which inactivates factor B, association was equivalent to that of FeS controls. After conidia were incubated in plasma, deposition of C3 could be detected by indirect immunofluorescence (figure 6). Depletion of IgG did not significantly inhibit association. Moreover, addition of enriched human IgG fraction to FCS did not significantly augment phagocytosis (figure 7). These results confirmed opsonization data and indicated that IgG alone did not playa major role in association. Although IgG alone did not aid in phagocytosis, it was possible that it played a secondary role by activation of the classical complement pathway. Chelation with EGT A, which inhibits the classical but not the alternative pathway, did not significantly inhibit phagocytosis of conidia. Deple-

Figure 3. Phagocytosis of conidia by human polymorphonuclear cells (PMNL) as function of conidia-toPMNL ratio after incubation for 60 min. A, Percentage of association in presence of heat-inactivated fetal calf serum (FCS) or autologous plasma (AP). B, Average number of conidia associated (solid lines) or ingested (dashed lines) per PMNL in presence of FCS or AP. Results are average of four experiments. Percentage ofassociation and average number associated or ingested were significantly higher with AP than with FCS at all conidiato-PMNL ratios (P < .01, df= 21, twoway analysis of variance; all values were significantly different (P = .01, df = 3) except for conidia-to-PMNL ratios of 2: I and 5: I in the presence of FCS (not significant).

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lysis of the PMNL (data not shown). In experiments with opsonized latex beads (31lm), in which there was significant association (86%), degranulation but not lysis ofPMNL was observed. This suggests that physicochemical properties of the conidia may induce lysis of PMNL. Resting conidia do not significantly induce degranulation ofPMNL primary and secondary granules [10] nor initiate a respiratory burst [9, 29]; they are relatively resistant to isolated granular proteins and enzyme systems from PMNL [8-10, 30). In contrast, swollen conidia did initiate degranulation [I OJ and consequently may be more susceptible to killing by PMNL [9, 10, 30). These studies also demonstrated the importance of plasma in the initial recognition of conidia. Immunoglobulin plays a secondary role, if any, in this initial interaction between A. fumigatus and PMNL. This agrees with clinical and in vitro animal studies that indicate that humoral immunity is not important [6, 20]. As suggested by other phagocytic studies using heat-inactivated plasma [8, 13] and demonstrated by

I

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100,.---------------------,

Figure 4. Decrease ofassociation ofconidia with polymorphonuclear cells (PMNL) over time. Conidia were incubated with PMNL at different ratios in presence of heat-inactivated fetal calf serum (FCS) or autologous plasma (AP). Data are representative of three different assays. Bars with same letter are not significantly different (P < .01, paired-sample t test).

nidia than with resting conidia. We also had slightly more rapid association rates (data not shown), but levels equalized after 30 min (42% vs. 44% at a I: I conidium-to-PMNL ratio and 82% vs. 72% at a 10: I conidia-to-PMNL ratio for resting and swollen conidia, respectively). With the double fluorescent labeling system we were able to demonstrate that ingestion quickly followed association and that at lower conidia-to-cell ratios, a high percentage of associated conidia were ingested. As observed in earlier studies [8, II], the number of conidia ingested by PMNL was significantly lower when PMNL were challenged with high numbers of resting conidia (10: I). Reduced ingestion also occurred with swollen conidia. The average number of ingested swollen conidia per PMNL was never> I and the percentage of associated conidia that were ingested was 25.83% ± 5.6% for all conidia-to-PMNL ratios (data not shown). The similarity between these two results may be due to the increase in fungal volume or the quantity of metabolic products excreted by the fungus. Ten resting conidia would occupya volume of -." 120 ILm 3 , which would be equivalent to 1-2 (95-113 1lm3) swollen conidia. Alternatively, swollen conidia may excrete different products into the immediate environment. Metabolic excretions of A. fumigatus that inhibit phagocytosis have been reported [13, 29]. Incubation of high numbers of conidia seemed to lead to

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opsonization Figure 5. Effect of opsonization of conidia on phagocytosis. Conidia were opsonized in treated plasma (AP) either autologous (37) or heat-inactivated (56) and incubated in antibody sources: heat-inactivated pool of serum from aspergilloma patients (patAb) or rabbit anti-conidia antibody (RbAb). After opsonization, conidia were incubated with polymorphonuclear cells in presence of 10% heat-inactivated fetal calf serum (FCS). Controls were nonopsonized conidia incubated with PMNL in presence of FCS or AP. Results are average of four experiments and are expressed as percentage ofcontrol incubations done in presence of6. 7% autologous plasma based on phagocytic indices. Bars with same letter are not significantly different (P = .01, paired-sample t test). Control AP and 37 were not significantly different.

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5:1

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pathway, and the fungus adheres to PMNL via C3 fragments. Although this had not been previously demonstrated' for Aspergillus species, the role of C3 as an opsonin and subsequent enhancement of phagocytosis has been documented for Coccidoidies immitis, Cryptococcus neoformans, and Candida albicans (reviewed in [34]). The possibility that other plasma proteins, such as lectins, C-reactive protein, mannanbinding protein, or fibrinogen, playa role in recognition of Aspergillus conidia cannot be excluded [35-40]. This is suggested by the high levels of association we observed after prolonged incubation (6-24 h) of conidia and PMNL in the presence ofFCS only. Kan and Bennett have demonstrated both in an animal study [35J and with human monocytes [36] that significant association occurs in the absence of plasma at 4°C and suggested that this association is via lectin recognition. In conclusion, the process ofassociation and ingestion be-

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our results, complement plays a major role in this interaction. Concomitant with the binding of plasma components by conidia, phagocytosis is enhanced, suggesting that C3 or C3 fragments act as an opsonin and that complement is important for the initial containment of conidia by PMNL. However, the role of C3 in phagocytosis is not fully elucidated, since we have observed recently that conidia can bind and cleave C3 in the absence of other plasma proteins [7, 31]. Other studies have indicated a role for complement in phagocytosis of A. fum igatus. Waldorf and Diamond [23J observed that PMNL were recruited to lungs' of mice after inhalation of conidia and suggested that conidia activated complement and the resulting C5a fragment was responsible for this recruitment. Kozel et al. [32] reported binding of radiolabeled C3 to conidia via the activation of the alternative pathways. Finally, Washburn and colleagues [13, 33] have isolated a polar lipid that inhibits both activation of the alternative pathway and phagocytosis of A. fumigatus and Cryptococcus neoformans by monocytes. However, this component was isolated from mycelial culture filtrate, and it is not known if conidia produce this same component. A hypothetical model by Fukazawa and Kagaya [18] suggests that fungi activate complement through the alternative

_

non treated

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20

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FCS

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plasma treatment Figure 7. Effect ofdepletion of complement pathways on phagocytosis. Plasma was depleted of both classical (CP) and alternate complement pathways (ACP) or of each pathway alone, or not treated. Conidia and PMNL were incubated in presence of 6.7% autologous plasma (AP), 10% heat-inactivated fetal calf serum (FCS), 10% heat inactivated FCS containing 4 mg/mL purified human IgG (FIgG), heat-inactivated AP at 56°C (56) or 50°C (50), AP chelated with 10 mM EDTA or EGTA, or AP depleted of component C3 (C3), orIgG (IgG), or ofcomponent C5 (C5) by incubation with anti-C3, anti-IgG, or anti-C5 antibodies, respectively. Results are average of three experiments and expressed as percentage of control incubations in presence of 6.7%autologous plasma based on phagocytic indices. Bars with same letter are not significantly different (P = .01, paired-sample t test). Results with EGTA, IgG, and C5 treatments were not significantly different from AP control.

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Figure 6. Deposition of plasma components on conidia surfaces. Conidia were incubated with normal human plasma and probed by indirect immunofluorescence for deposition of immunoglobulin (C) or complement component C3 (B). A, Conidia incubated with fluorescent second antibody only. Bar = 10 #Lm.

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tween PMNL and conidia is rapid and dependent on an intact alternative complement pathway. It is now of interest to further explore what role C3 plays in this interaction and whether the method of recognition affects the ensuing intracellular reactions. This may explain the differential responses of human phagocytic cells to the different morphologic forms of Aspergillus organisms.

20.

21.

22. References

23.

24.

25. 26. 27.

28. 29. 30.

31.

32.

33.

34.

35.

36.

37.

38.

39. 40.

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JID 1992; 166 (September)

Participation of complement in the phagocytosis of the conidia of Aspergillus fumigatus by human polymorphonuclear cells.

Nonspecific immunity plays a major role in the clearance of the opportunistic fungal pathogen, Aspergillus fumigatus. However, the mode of recognition...
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