- Email: [email protected]
GP43 from Paracoccidioides brasiliensis inhibits macrophage functions. An evasion mechanism of the fungus
Cellular Immunology 218 (2002) 87–94 www.academicpress.com
GP43 from Paracoccidioides brasiliensis inhibits macrophage functions. An evasion mechanism of the fungus Ana Flavia Popi, Jose Daniel Lopes, and Mario Mariano* Discipline of Immunology, Department of Microbiology, Immunology, and Parasitology, Federal University of S~ ao Paulo, S~ ao Paulo, Brazil Received 4 July 2002; accepted 11 September 2002
Abstract Macrophages constitute one of the primary cellular mechanisms that impairs parasite invasion of host tissues. The phagocytic and microbicidal properties of these cells can be modulated by specific membrane receptors involved in cell–microorganism interactions. Gp43, the main antigen secreted by Paracoccidiodes brasiliensis (Pb), the causative agent of Paracoccidioidomycosis, is a high mannose glycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known. Here, we describe the influence of this molecule on the interaction between peritoneal murine macrophages and Pb. Phagocytosis of Pb, live or heatkilled, by adherent peritoneal cells from both, B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations of gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed Pb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis of zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It was also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages. Finally, we demonstrated that gp43 inhibits the fungicidal ability of macrophages from both lineages. Based on these data, it is suggested that gp43 can be considered one of the evasion mechanisms for the installation of primary infection in susceptible hosts. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Paracoccidiodes brasiliensis; gp43; Macrophages; Phagocytosis; Microbicidal activity; Escape mechanism
1. Introduction Paracoccidioides brasiliensis (Pb) is the causative agent of human paracoccidioidomycosis, a deep mycosis prevalent in Latin America. Considering the pathogenesis of the disease, it is well established that cellular immune response is the main mechanism of protection against fungal infection [5,6,14]. In this context and considering that Pb is a facultative intracellular microorganism, macrophages may play a pivotal role in the pathogenesis of the disease. Therefore, the investigation of the mechanisms that govern phagocytosis of Pb by macrophages is relevant for the understanding of this peculiar type of host–parasite relationship. It has been demonstrated that Pb is phagocytosed by macrophages in vivo [4] and in vitro, the fungus is in-
*
Corresponding author. Fax: +55-11-5572-3328. E-mail address: [email protected] (M. Mariano).
ternalized and multiply in non-activated human monocytes or macrophages [3]. Conversely, it has been demonstrated that activated macrophages are fungicidal for Pb in vitro and in vivo [3,10]. Evidences that macrophages are relevant in the process of elimination of Pb in vivo have been reported. Kashino et al. [9] demonstrated that blocking macrophage functions by treatment of animals with colloidal carbon turns resistant mice (A/Sn) susceptible to infection by Pb [5]. It has been suggested that the mechanism by which the fungus modulate macrophage microbicidal behavior depends on the specificity of macrophage membrane receptors activated during parasite-cell uptake [8,11]. Therefore, the type of macrophage receptor, and its complementary ligand on the microorganism surface, may have considerable influence on the fate of infection [12,16]. In this direction, tests performed in vitro indicate that fucose and mannose residues on the surface of the
0008-8749/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 8 - 8 7 4 9 ( 0 2 ) 0 0 5 7 6 - 2
88
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
fungus influence phagocytosis of the parasite by macrophages. Evidences that gp43, the main antigenic glycoprotein secreted by the fungus [17] was involved in the process of endocytosis of the fungus was addressed by the observation that sheep red blood cells coated with gp43 were phagocytosed by mouse peritoneal macrophages and, that the addition of fucose or manose to the culture medium partially inhibited the process. These results suggest that gp43 may be involved in the adherence and uptake of the fungus by macrophages [1]. Here we demonstrate that gp43 inhibits phagocytosis of Pb by non-activated mouse peritoneal macrophages in vitro. Further, that this molecule also inhibits activation and the ability of these cells to kill the parasite. Based on these observations it is suggested that gp43 mediates an escape mechanism of the fungus that facilitates the establishment and fate of primary infection in susceptible host.
determined by the Bradford method [2]. All steps of gp43 purification were monitored by SDS–PAGE. 2.4. Peritoneal macrophages Peritoneal cells were collected from the abdominal cavity of mice (B10.A or A/Sn) by repeated washing with 2 ml of RPMI-1640 medium (Sigma). Cell viability was evaluated using the trypan blue dye exclusion. Cells (2 105 cells/ml) were dispensed over round glass coverslips (13 mm) in 24-well flat bottom microtest plates and the cultures incubated at 37 °C in 5% CO2 for 60 min. After incubation, the culture supernatants were aspirated and the non-adherent cells removed. Adherent monolayers were rinsed with RPMI-1640 (Sigma). Subsequently, 1 ml of R10 medium (RPMI-1640 containing 10% of heat inactivated fetal bovine serum) was added to the cultures. The cultures were incubated at 37 °C in 5% CO2 for 3 days. 2.5. Phagocytic test
2. Materials and methods 2.1. Mice Male A/Sn and B10.A mice, 6–12 weeks old, were obtained from the Animal House of the Federal University of S~ ao Paulo. 2.2. Fungi Paracoccidioides brasiliensis isolates, Pb 18 and Pb 339, were maintained in solid YPD medium (yeast extract, casein peptone, D -glucose, agar, and bi-distilled water) at 35 °C in yeast forms or cultivated in liquid TOM medium (yeast extract, glucose, casein peptone, K2 HPO4 , KH2 PO4 , MgSO4 7H2 O, MnSO4 7H2 O, NaCl, and FeSO4 ) at 37 °C on rotating shaker to exoantigen preparation. Yeast cells of Pb grown in liquid TOM medium were used in phagocytic tests. After a resting time, large cells were allowed to sediment and the culture supernatant, containing small cells, was harvested. Pb cells were washed and ressuspended in R10 (8 105 cells/ml). Fungal particles were heat-killed by autoclaving the cultures at 120 °C for 15 min. 2.3. Gp43 purification The 43 kDa glycoprotein (gp43) antigen was purified by affinity chromatography on Affi-Gel 10 column (BioRad) coupled with anti-gp43 mouse monoclonal antibody (mAb 17C) [7] from exoantigen preparation [13]. Gp43 was eluted with citric acid buffer (50 mM, pH 2.8), neutralized with 1 M Tris–HCl, pH 9.0, and concentrated in an Amicon apparatus. Protein content was
After 3 days, the culture supernatants were aspirated and the monolayers of adherent peritoneal cells challenged with 1 ml of the suspension of Pb cells, live or heat-killed, prepared as described above. After incubation of the cultures at 37 °C in 5% CO2 for 1, 2, 4, and 8 h, the cultures were rinsed with PBS for removal of non-internalized Pb cells. The cells were fixed for 1 h in BouinÕs fixative and stained with hematoxylin–eosin. The influence of gp43 on the phagocytic indexes was evaluated by adding different concentrations of the purified protein to the culture medium at time zero of the experiments. The ability of the fungus and or activated macrophages to secrete factors that might inhibit macrophage functions was investigated using transwell microplates (Costar). 2.6. Phagocytosis of sheep red blood cells (SRBC) Macrophages (2 105 cells/ml), isolated and cultured as described above, were challenged with a suspension of SRBC (0.5%) or IgG opsonized SRBC (0.5%) and incubated at 37 °C for 1 h. SRBC were opsonized by incubation with rabbit IgG anti-sheep SRBC (1:100) at 37 °C for 1 h. SRBC were washed in PBS and added to the cultures. The influence of purified gp43 on the phagocytic indexes of SRBC was evaluated by adding different concentrations of purified protein to the SRBC suspensions. After incubation, non-internalized SRBC were lysed by a brief hypotonic shock with haemolytic buffer (NH4 Cl 0.15 M, NaHCO3 0.01 M, and EDTA 0.1 mM). Subsequently, the cultures were rinsed with PBS, fixed for 1 h with a 2.5% glutaraldehyde solution and stained with Giemsa.
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
2.7. Phagocytosis of zymosan particles Macrophages cultured for 72 h were challenged with zymosan (Sigma) particles (10 mg/ml) and incubated at 37 °C for 1 h. After incubation, cells were rinsed with PBS and fixed for 1 h in a 2.5% glutaraldehyde solution and stained with Giemsa. The influence of purified gp43 on the phagocytic indexes of these particles was also tested. 2.8. Phagocytic indexes (PI) An average of 200 macrophages were counted to determine the phagocytic indexes (PI), calculated as the percent of phagocytic cells multiplied by the mean number of internalized particles. 2.9. NO release in the culture medium Cultures of murine resident peritoneal cells were prepared as described above. After 3 days, the culture supernatants were aspirated and macrophages were challenged with a suspension of Pb cells and/or gp43 (100 lg/ml). After incubation of the cultures for 2, 4, 6, and 8 h, the supernatant was harvested. Four aliquots (50 ll) of the culture medium from each sample were transferred to wells of 96-well flat bottom microtiter plate. A serial dilution of NaNO2 solution (5–60 lmol) was prepared for control. A sample of 50 ll of Greiss reagent solution (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrocloride in 2.5% phosphoric acid) was added to wells containing sample aliquots and NaNO2 standard solutions. Thereafter, the plates were read in a microtiter plate reader (MR 4000, Dynatech Chantilly) with absorbance at 550 nm. The amount of NO2 in the culture samples was extrapolated from the standard curve made with different concentrations of NaNO2 . 2.10. Influence of gp43 on the release of hydrogen peroxide (H2 O2 ) by macrophages Peritoneal cells were collected from the abdominal cavity of the animals (A/Sn and B10.A) by repeated washing with 2 ml of RPMI-1640 medium (Sigma). The cells were adjusted to 2 10 cells/ml in a Phenol Red solution (PBS, 5 mM dextrose, 50 lg of horseradish type II, and 0.28 mM phenol red). A 100 ll of the peritoneal cell suspensions was dispensed in 96-well flat bottom microtiter plate (Costar). Hundred lg/ml of gp43 solution, was added to the wells. The plates were incubated at 37 °C for 1 h and the reaction stopped by the addition of 10 ll of 1 N NaOH solution. A standard curve for the determination of H2 O2 concentration in the experimental tests was constructed using H2 O2 solutions varying from 0.5 to 4 nmol. Ab-
89
sorbance was measured at 620 nm in a microtiter plate reader (MR 4000, Dynatech Chantilly). Results were expressed as nanomoles of H2 O2 per 2 106 cells. 2.11. Colony forming units (CFU) determination The determination of the number of viable fungi after phagocytosis by macrophages was made by CFU counts. Macrophages were challenged with live Pb and incubated for 4 h, as described in phagocytic tests. After this time, cultures were rinsed with PBS for removal of non-internalized Pb cells. Distilled water was added to lyse macrophages. The cellular suspension was harvested, washed in PBS, and the final pellets ressuspended in 1 ml of PBS. Aliquots of 100 ll of each sample were plated in agar plates (4% SFB, 5% BHI solid medium). Colonies per plate were counted after 8–10 days of incubation at 37 °C. 2.12. Statistical analysis Statistical comparisons were made by analysis of variance (ANOVA) and by the Tukey–Kramer test. All values are reported as the mean standard error deviation of the mean, with significance assumed in the range of p < 0:05.
3. Results 3.1. Phagocytosis of Pb by peritoneal macrophages Contrary to what happens with other particles, such as zymosan and SRBC, fungal particles were internalized by macrophages after 4–8 h of culture (data not shown). A possible explanation for the observed phenomenon was the impairment of contact between fungal particles and macrophages membranes. This hypothesis could not be sustained considering that centrifugation of the cultures (1062g for 5 min) in order to allow a closer contact between the particles and the macrophage cell membrane did not speed up endocytosis of Pb (data not shown). Considering that the delay of macrophages to ingest Pb particles was not due to the contact between cells and fungi particles, we investigated whether this phenomenon could be due to the release of inhibitory factors by macrophages induced by the contact between Pb particles and these cells. This hypothesis was tested in a model where the phagocytic indexes of zymosan by macrophages which were previously challenged with dead Pb particles was evaluated. Cultured macrophages were challenged with Pb particles for 4 h. The cultures were washed and added with zymosan (z) particles. Results show that previous ingestion of Pb particles inhibits the phagocytic indexes of zymosan by these cells
90
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
(Fig. 1A). A less significative inhibition was observed when Pb plus zymosan particles were simultaneously added to macrophage cultures. These data suggest that either the fungus down-regulate macrophage functions or induces macrophages to secret a macrophage deactivating factor. In order to investigate the last hypothesis, macrophages were cultured on the bottom of transwell microplates. On half of the plates macrophages were also cultured on the top of the filter. All the plates were added with heat-killed Pb particles. Results show that the contact between macrophages and Pb particles induces a significant decrease in the phagocytic indexes of zymosan when macrophages cultured on the bottom of the wells were tested (Fig. 1B). Considering that heatkilled Pb was used in these experiments, these results suggest that the contact between Pb and macrophage membrane induces these cells to secrete a macrophage deactivating factor/s.
Fig. 1. Interaction Pb–macrophages decreases phagocytic indexes (PI) of zymosan by macrophages. (A) Macrophages were challenged with: zymosan for 1 h (zymosan), Pb and zymosan simultaneously for 1 h (Pb + z) and Pb for 4 h and zymosan for 1 h (Pb 4 h + z). (B) Phagocytosis of zymosan by macrophages cultured on the bottom of transwell microplates. The top of the filters were added with: culture medium (control), macrophage + heat-killed Pb (M/ + Pb), live Pb or heat-killed Pb. Cells were stained with Giemsa and counted to determine PI with the aid of an optic microscope (100). *p < 0:01 is significant when compared with macrophages challenged with zymosan particles.
Similar experiments were performed using live Pb particles. As shown in Fig. 1B, when live Pb particles were added to the top of the wells, a significant decrease in the phagocytic index of zymosan ingestion by macrophages cultured in the bottom of the wells was observed. These data allow us to conclude that live Pb secrets a factor/s which deactivates macrophage functions. 3.2. Influence of gp43 on the phagocytic activity of peritoneal macrophages Based on results above described we hypothesized that gp43 might have a down-regulatory effect on macrophages. The addition of purified gp43 to the cultures, simultaneously to the challenge with heat-killed fungus, led to a significant inhibition of phagocytosis by macrophages from the A/Sn or B10A strains. Different concentrations of gp43 added to the culture medium determined a decrease in the phagocytic index in a dose-dependent pattern (Fig. 2). Similar results were obtained when macrophages were challenged with live Pb (data not shown). Considering that zymosan is endocytosed by macrophages via mannose receptors [16], we decided to investigate whether gp43 would influence the uptake of this particle. As shown in Fig. 3A, the addition of gp43 to the cultures decreased the phagocytic indexes when the cells were challenged for 1 h with zymosan particles (10 mg/ml).
Fig. 2. Influence of gp43 on the PI of heat-killed Pb by peritoneal macrophages from A/Sn and B10.A mice. Gp43 inhibited phagocytosis when added to the culture medium, simultaneously with addition of the fungal particles at the concentrations of 25, 75, 100, and 150 lg/ml. Cells were stained with HE and counted to determine the PI with the aid of an optic microscope (100). Significance (*p < 0:05 and **p < 0:001) was obtained when treated cells were compared with those from control group.
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
91
gate the influence of Pb and/or gp43 on the liberation of NO and H2 O2 by macrophages in vitro. As shown in Fig. 4, gp43 inhibits H2 O2 liberation by macrophages from the A/Sn or B10.A mice stimulated or not by zymosan particles (Fig. 4). The amount of NO spontaneously liberated by peritoneal macrophages from both strains of mice increases with time (Fig. 5). Addition of Pb, gp43 or Pb + gp43 did not induce increase in NO liberation. Otherwise, these stimuli caused inhibition of NO production when compared with results obtained with non-stimulated macrophages. Gp43 also inhibited NO liberation by macrophage stimulated with zymosan. The addition of different concentration of zymosan stimulated NO production by macrophage at a dose-dependent manner. As shown in Fig. 6A, gp43 is able to inhibit NO liberation by macrophages. It is important to note that macrophage from B10.A mice did not induce a significant increase in NO liberation as observed with macrophages obtained from mice from the A/Sn strain (Fig. 6B). 3.4. Influence of gp43 on the viability of internalized fungus
Fig. 3. Influence of gp43 on the phagocytosis of zymosan and opsonized SRBC. (A) Purified gp43 (100 lg/ml) inhibited phagocytosis of zymosan particles (10 mg/ml) by peritoneal macrophages from A/Sn and B10.A mice. *p < 0:01 is significant when compared with control group not added with gp43. (B) Phagocytic indexes of IgG opsonized sheep red blood cells (SRBC) by macrophages from both A/Sn and B10.A mice. Non-opsonized sheep red blood cells (SRBC) were used as control (data not shown). The addition of gp43 did not influence phagocytosis P ¼ 0.044. Cells were stained with Giemsa and counted to determine the phagocytic indexes with the aid of an optic microscope (100).
Results above described did not bring evidences that the killing capacity of macrophages from resistant (A/Sn) or susceptible (B10.A) mice is different. Results obtained with the colony forming units method clearly demonstrate that macrophages from the A/Sn strain have a higher ability to kill the fungus when compared with macrophages from the B10.A lineage. However, it is also clearly demonstrated that the addition of gp43 to the cultures, significantly inhibits the killing capacity of
The inhibitory effect of gp43 on the endocytosis of zymosan particles could be due to a general metabolic down-regulation of the phagocytes. This hypothesis was discharged considering that gp43 did not influence the phagocytic indexes of opsonized sheep red blood cells by macrophages, as shown in Fig. 3B. 3.3. Influence of gp43 on the liberation of NO and H 2 O2 by peritoneal macrophages It has been reported that NO, H2 O2 , and other free metabolites are involved with the killing capacity of macrophages [11,12]. Therefore, we decided to investi-
Fig. 4. Influence of gp43 on the release of hydrogen peroxide (H2 O2 ) by murine macrophage (A/Sn and B10.A). Peritoneal cells were stimulated with gp43, zymosan, zymosan + gp43 or not stimulated. After 1 h, the culture supernatants were harvested and absorbance (620 nm) was measured to determine the amount of H2 O2 released. p < 0:01 when the values were compared between them.
92
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
Fig. 5. Influence of gp43 on NO release by peritoneal macrophages from A/Sn and B10.A mice challenged with Pb. Macrophages, after 3 days in culture, were simultaneously challenged with Pb cells, gp43 or both. Macrophages were not stimulated in control group. The culture supernatants were harvested and the amount of nitrite analyzed in a microtiter plate reader with absorbance at 550 nm. p < 0:05 when values were compared with control group.
macrophages obtained from both resistant or susceptible mice (Fig. 7).
4. Discussion It is well established that P. brasiliensis (Pb) is a facultative intracellular parasite [14]. It is also established that the interaction between molecules on the surface of the parasite and homologous receptors on the cell membrane of macrophages modulates phagocytosis [8,11]. Molecular complexes containing proteins and polysaccharides with a high proportion of mannose have been detected on the surface of the fungus by Almeida et al. [1]. These authors demonstrated that phagocytosis of Pb by murine peritoneal macrophages in vitro was
Fig. 6. (A) and (B)—Influence of gp43 on NO liberation by peritoneal macrophages challenged with zymosan particles. The culture supernatants were harvested and the amount of nitrite analyzed in a microtiter plate reader with absorbance at 550 nm. *p < 0:01 when the values obtained with macrophages from B10.A mice were compared, **p < 0:001 when the values obtained with macrophages from A/Sn mice were compared, # p < 0:001 when the values were compared between them.
inhibited when D -mannose and D -fucose was added to the culture medium. These data indicate that endocytosis of the fungus by macrophages is mediated by the mannose receptor. The authors have also demonstrated that policlonal antibodies directed to epitopes of gp43 also induce a marked decrease on the phagocytic indexes of macrophages challenged with Pb. However, a monoclonal antibody which recognizes a peptidic epitope of gp43 did not interfere with the interaction macrophage– Pb. Finally, the authors have also demonstrated that gp43 coupled to the surface of sheep red blood cells was endocytosed by macrophages and this interaction was inhibited by the addition of D -mannose and D -fucose to the culture medium. These data attest the participation of gp43, the main antigenic molecule secreted by the fungus, on the uptake of the fungus by macrophages.
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
Fig. 7. (A) and (B)—Viability of Pb (CFU) after phagocytosis of fungi by macrophages in the presence or absence of gp43. Results show that the addition of gp43 to the cultures inhibited the fungal killing by peritoneal macrophages. *p < 0:01 when compared with control group (A). Differences are illustrated in (B).
Based on these information we decided to broaden these investigations in order to clarify the role of gp43 on the interaction macrophage–fungus. Our results show that the phagocytic indexes of macrophages challenged with live or heat-killed fungi are similar and are inhibited when gp43 is added to the culture medium. These observations suggest that heatkilled fungi retain on their surface molecules that interact with macrophage cell membrane and that this interaction is modified by the presence of gp43. Also, that macrophages from both lineages, A/Sn and B10.A, have similar phagocytic indexes either when live or heatkilled cells were tested. Gp43 induces a dose-dependent decrease in the phagocytic indexes of macrophages stimulates with Pb. These data suggest a possible interaction of Pb–macrophage and gp43–macrophage with the same receptor, probably through the mannose receptor. This hypothe-
93
sis was reinforced by the observation that gp43 also blocked endocytosis of zymosan particles which are phagocytosed by macrophages via the mannose receptor [15]. Furthermore, gp43 did not alter the phagocytic indexes of IgG opsonized sheep red blood cells indicating a selective effect of the molecule on the endocytic mechanisms of macrophages. The long period needed for macrophages to ingest heat-killed fungal particles (4–8 h) is an intriguing phenomenon considering that these cells phagocytose other particles in a shorter period of time. In order to understand this peculiarity we hypothesized that the contact macrophage–Pb induces deactivation of these cells by release of autocrine factors by macrophages. The decrease in phagocytic indexes of zymosan by macrophages previously challenged with dead Pb particles suggest that either the fungus down-regulate macrophage functions or induces macrophages to secret a macrophage deactivating factor. Our data strengthen the hypothesis that gp43 impairs the ingestion of Pb, and the interaction macrophage– fungus induces deactivation of these cells. This hypothesis is reinforced by the fact that gp43 inhibits the liberation of intermediary reactives of oxygen and nitrogen, mediators involved in the microbicidal activity of macrophages. The liberation of H2 O2 by macrophages from the A/Sn and B10.A mice was inhibited by gp43. However, this molecule did not influence the liberation of this metabolite when phagocytes were stimulated with PMA. It is possible that the interaction of gp43 and PMA with macrophage cell membrane activates different pathways of signal transduction in these cells (data not shown). The spontaneous production of NO by macrophages was inhibited when gp43 and/or Pb were added to the cultures. The addition of Pb to these cultures did not induce a significant increase in NO liberation as occurs with macrophages challenged with zymosan. However, gp43 also showed inhibitory effect on NO liberation in these circumstances. These data reinforced the hypotheses that the fungus induce macrophages to secret autocrine deactivating factor/s. Summing up, we may conclude that gp43 has an inhibitory effect on the liberation of NO and H2 O2 by murine peritoneal macrophages stimulated by the contact with Pb. Further, that the inhibitory effect of gp43 on the microbicidal activity of macrophages was clearly demonstrated, considering that treatment of macrophages with gp43 impairs the fungicidal capacity of these cells as demonstrated by the CFU tests. Based on results here described and considering that macrophages are pivotal in the control of fungal infection, it is licit to put forward the hypothesis that gp43 is involved in an evasion mechanism of the fungus that facilitates its homing in the host tissues in primary infections.
94
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
Acknowledgments We are indebted to Prof. Michel Rabinowich for his skilful suggestions during the development of this work and to Luiz Cl audio Godoy and Sandro Rogerio de Almeida for their guidance during the execution of many experiments. Ana Flavia Popi was the recipient of a grant as a graduating student of Biomedicine from Fundacß~ao de Amparo a Pesquisa do Estado de S~ ao Paulo (FAPESP) (Process: 98/11790-0).
[8]
[9]
[10]
[11]
References [12] [1] S.R. Almeida, C.S. Unterkircher, Z.P. Camargo, Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages, Med. Mycol. 36 (1998) 405–411. [2] M.M Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [3] E. Brummer, L.H. Hanson, A. Restrepo, D.A. Stevens, In vivo and in vitro activation of pulmonary macrophages by IFN-c for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis, J. Immunol. 140 (1988) 2786–2789. [4] E. Brummer, L.H. Hanson, A. Restrepo, D.A. Stevens, Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages, Infect. Immun. 57 (1989) 2289–2294. [5] V.L. Calich, L.M. Singer-Vermes, A.M. Siqueira, E. Burger, Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis, Br. J. Exp. Pathol. 66 (1985) 585–594. [6] V.L. Calich, C.A. Vaz, E. Burger, Immunity to Paracoccidioides brasiliensis infection, Res. Immunol. 149 (1998) 407–417, discussion 499–500. [7] J.L. Gesztesi, R. Puccia, L.R. Travassos, A.P. Vicentini, J.Z. de Moraes, M.F. Franco, J.D. Lopes, Monoclonal antibodies against
[13]
[14]
[15]
[16]
[17]
the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis, Hybridoma 15 (1996) 415–422. S. Gordon, V.H. Perry, S. Rabinowitz, L.P. Chung, H. Rosen, Plasma membrane receptors of the mononuclear phagocyte system, J. Cell Sci. Suppl. 9 (1988) 1–26. S.S. Kashino, L.M. Singer-Vermes, V.L. Calich, E. Burger, Alterations in the pathogenicity of one Paracoccidioides brasiliensis isolate do not correlative with its in vitro growth, Mycopathologia 111 (1990) 173–180. M. Moscardi-Bacchi, E. Brummer, D.A. Stevens, Support of Paracoccidioides brasiliensis multiplication by human monocytes or macrophages: inhibition by activated phagocytes, J. Med. Microbiol. 40 (1994) 159–164. L. Ohman, G. Maluszynska, K.E. Magnusson, O. Stendahl, Surface interaction between bacteria and phagocytic cells, Prog. Drug Res. 32 (1988) 131–147. D.M Paulnock, The molecular biology of macrophage activation, Immunol. Ser. 60 (1994) 47–62. R. Puccia, L.R. Travassos, The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis, Arch. Biochem. Biophys. 289 (1991) 298–302. L.M. Singer-Vermes, E. Burger, V.L. Calich, L.H. ModestoXavier, T.N. Sakamoto, M.F. Sugizaki, D.A. Meira, R.P. Mendes, Pathogenicity and immunogenicity of Paracoccidioides brasiliensis isolates in the human disease and in an experimental murine model, Clin. Exp. Immunol. 97 (1994) 113–119. D.P. Speert, S.C. Silverstein, Phagocytosis of unopsonized zymosan by human monocyte-derived macrophages: maturation and inhibition by mannan, J. Leukoc. Biol. 38 (1985) 655– 658. P.D. Stahl, R.A. Ezekowitz, The mannose receptor is a pattern recognition receptor involved in host defense, Curr. Opin. Immunol. 10 (1998) 50–55. L.R. Travassos, R. Puccia, P. Cisalpino, C. Taborda, E.G. Rodrigues, M. Rodrigues, J.F. Silveira, I.C. Almeida, Biochemistry and molecular biology of the main diagnostic antigen of Paracoccidioides brasiliensis, Arch. Med. Res. 26 (1995) 297– 304.
GP43 from Paracoccidioides brasiliensis inhibits macrophage functions. An evasion mechanism of the fungus Ana Flavia Popi, Jose Daniel Lopes, and Mario Mariano* Discipline of Immunology, Department of Microbiology, Immunology, and Parasitology, Federal University of S~ ao Paulo, S~ ao Paulo, Brazil Received 4 July 2002; accepted 11 September 2002
Abstract Macrophages constitute one of the primary cellular mechanisms that impairs parasite invasion of host tissues. The phagocytic and microbicidal properties of these cells can be modulated by specific membrane receptors involved in cell–microorganism interactions. Gp43, the main antigen secreted by Paracoccidiodes brasiliensis (Pb), the causative agent of Paracoccidioidomycosis, is a high mannose glycoprotein. The role played by gp43 in the pathogenesis of the disease is not completely known. Here, we describe the influence of this molecule on the interaction between peritoneal murine macrophages and Pb. Phagocytosis of Pb, live or heatkilled, by adherent peritoneal cells from both, B10.A (susceptible) and A/Sn (resistant) mice, was evaluated. Addition of different concentrations of gp43 to the culture medium inhibited, in a dose-dependent pattern, phagocytosis of live or heat-killed Pb by peritoneal macrophages from both B10.A and A/Sn mice. Gp43 also inhibits phagocytosis of zymosan particles but did not interfere with the uptake of opsonized sheep red blood cells. It was also shown that both gp43 and heat-killed Pb have an inhibitory effect on the release of NO by zymosan stimulated macrophages. Finally, we demonstrated that gp43 inhibits the fungicidal ability of macrophages from both lineages. Based on these data, it is suggested that gp43 can be considered one of the evasion mechanisms for the installation of primary infection in susceptible hosts. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Paracoccidiodes brasiliensis; gp43; Macrophages; Phagocytosis; Microbicidal activity; Escape mechanism
1. Introduction Paracoccidioides brasiliensis (Pb) is the causative agent of human paracoccidioidomycosis, a deep mycosis prevalent in Latin America. Considering the pathogenesis of the disease, it is well established that cellular immune response is the main mechanism of protection against fungal infection [5,6,14]. In this context and considering that Pb is a facultative intracellular microorganism, macrophages may play a pivotal role in the pathogenesis of the disease. Therefore, the investigation of the mechanisms that govern phagocytosis of Pb by macrophages is relevant for the understanding of this peculiar type of host–parasite relationship. It has been demonstrated that Pb is phagocytosed by macrophages in vivo [4] and in vitro, the fungus is in-
*
Corresponding author. Fax: +55-11-5572-3328. E-mail address: [email protected] (M. Mariano).
ternalized and multiply in non-activated human monocytes or macrophages [3]. Conversely, it has been demonstrated that activated macrophages are fungicidal for Pb in vitro and in vivo [3,10]. Evidences that macrophages are relevant in the process of elimination of Pb in vivo have been reported. Kashino et al. [9] demonstrated that blocking macrophage functions by treatment of animals with colloidal carbon turns resistant mice (A/Sn) susceptible to infection by Pb [5]. It has been suggested that the mechanism by which the fungus modulate macrophage microbicidal behavior depends on the specificity of macrophage membrane receptors activated during parasite-cell uptake [8,11]. Therefore, the type of macrophage receptor, and its complementary ligand on the microorganism surface, may have considerable influence on the fate of infection [12,16]. In this direction, tests performed in vitro indicate that fucose and mannose residues on the surface of the
0008-8749/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 8 - 8 7 4 9 ( 0 2 ) 0 0 5 7 6 - 2
88
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
fungus influence phagocytosis of the parasite by macrophages. Evidences that gp43, the main antigenic glycoprotein secreted by the fungus [17] was involved in the process of endocytosis of the fungus was addressed by the observation that sheep red blood cells coated with gp43 were phagocytosed by mouse peritoneal macrophages and, that the addition of fucose or manose to the culture medium partially inhibited the process. These results suggest that gp43 may be involved in the adherence and uptake of the fungus by macrophages [1]. Here we demonstrate that gp43 inhibits phagocytosis of Pb by non-activated mouse peritoneal macrophages in vitro. Further, that this molecule also inhibits activation and the ability of these cells to kill the parasite. Based on these observations it is suggested that gp43 mediates an escape mechanism of the fungus that facilitates the establishment and fate of primary infection in susceptible host.
determined by the Bradford method [2]. All steps of gp43 purification were monitored by SDS–PAGE. 2.4. Peritoneal macrophages Peritoneal cells were collected from the abdominal cavity of mice (B10.A or A/Sn) by repeated washing with 2 ml of RPMI-1640 medium (Sigma). Cell viability was evaluated using the trypan blue dye exclusion. Cells (2 105 cells/ml) were dispensed over round glass coverslips (13 mm) in 24-well flat bottom microtest plates and the cultures incubated at 37 °C in 5% CO2 for 60 min. After incubation, the culture supernatants were aspirated and the non-adherent cells removed. Adherent monolayers were rinsed with RPMI-1640 (Sigma). Subsequently, 1 ml of R10 medium (RPMI-1640 containing 10% of heat inactivated fetal bovine serum) was added to the cultures. The cultures were incubated at 37 °C in 5% CO2 for 3 days. 2.5. Phagocytic test
2. Materials and methods 2.1. Mice Male A/Sn and B10.A mice, 6–12 weeks old, were obtained from the Animal House of the Federal University of S~ ao Paulo. 2.2. Fungi Paracoccidioides brasiliensis isolates, Pb 18 and Pb 339, were maintained in solid YPD medium (yeast extract, casein peptone, D -glucose, agar, and bi-distilled water) at 35 °C in yeast forms or cultivated in liquid TOM medium (yeast extract, glucose, casein peptone, K2 HPO4 , KH2 PO4 , MgSO4 7H2 O, MnSO4 7H2 O, NaCl, and FeSO4 ) at 37 °C on rotating shaker to exoantigen preparation. Yeast cells of Pb grown in liquid TOM medium were used in phagocytic tests. After a resting time, large cells were allowed to sediment and the culture supernatant, containing small cells, was harvested. Pb cells were washed and ressuspended in R10 (8 105 cells/ml). Fungal particles were heat-killed by autoclaving the cultures at 120 °C for 15 min. 2.3. Gp43 purification The 43 kDa glycoprotein (gp43) antigen was purified by affinity chromatography on Affi-Gel 10 column (BioRad) coupled with anti-gp43 mouse monoclonal antibody (mAb 17C) [7] from exoantigen preparation [13]. Gp43 was eluted with citric acid buffer (50 mM, pH 2.8), neutralized with 1 M Tris–HCl, pH 9.0, and concentrated in an Amicon apparatus. Protein content was
After 3 days, the culture supernatants were aspirated and the monolayers of adherent peritoneal cells challenged with 1 ml of the suspension of Pb cells, live or heat-killed, prepared as described above. After incubation of the cultures at 37 °C in 5% CO2 for 1, 2, 4, and 8 h, the cultures were rinsed with PBS for removal of non-internalized Pb cells. The cells were fixed for 1 h in BouinÕs fixative and stained with hematoxylin–eosin. The influence of gp43 on the phagocytic indexes was evaluated by adding different concentrations of the purified protein to the culture medium at time zero of the experiments. The ability of the fungus and or activated macrophages to secrete factors that might inhibit macrophage functions was investigated using transwell microplates (Costar). 2.6. Phagocytosis of sheep red blood cells (SRBC) Macrophages (2 105 cells/ml), isolated and cultured as described above, were challenged with a suspension of SRBC (0.5%) or IgG opsonized SRBC (0.5%) and incubated at 37 °C for 1 h. SRBC were opsonized by incubation with rabbit IgG anti-sheep SRBC (1:100) at 37 °C for 1 h. SRBC were washed in PBS and added to the cultures. The influence of purified gp43 on the phagocytic indexes of SRBC was evaluated by adding different concentrations of purified protein to the SRBC suspensions. After incubation, non-internalized SRBC were lysed by a brief hypotonic shock with haemolytic buffer (NH4 Cl 0.15 M, NaHCO3 0.01 M, and EDTA 0.1 mM). Subsequently, the cultures were rinsed with PBS, fixed for 1 h with a 2.5% glutaraldehyde solution and stained with Giemsa.
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
2.7. Phagocytosis of zymosan particles Macrophages cultured for 72 h were challenged with zymosan (Sigma) particles (10 mg/ml) and incubated at 37 °C for 1 h. After incubation, cells were rinsed with PBS and fixed for 1 h in a 2.5% glutaraldehyde solution and stained with Giemsa. The influence of purified gp43 on the phagocytic indexes of these particles was also tested. 2.8. Phagocytic indexes (PI) An average of 200 macrophages were counted to determine the phagocytic indexes (PI), calculated as the percent of phagocytic cells multiplied by the mean number of internalized particles. 2.9. NO release in the culture medium Cultures of murine resident peritoneal cells were prepared as described above. After 3 days, the culture supernatants were aspirated and macrophages were challenged with a suspension of Pb cells and/or gp43 (100 lg/ml). After incubation of the cultures for 2, 4, 6, and 8 h, the supernatant was harvested. Four aliquots (50 ll) of the culture medium from each sample were transferred to wells of 96-well flat bottom microtiter plate. A serial dilution of NaNO2 solution (5–60 lmol) was prepared for control. A sample of 50 ll of Greiss reagent solution (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrocloride in 2.5% phosphoric acid) was added to wells containing sample aliquots and NaNO2 standard solutions. Thereafter, the plates were read in a microtiter plate reader (MR 4000, Dynatech Chantilly) with absorbance at 550 nm. The amount of NO2 in the culture samples was extrapolated from the standard curve made with different concentrations of NaNO2 . 2.10. Influence of gp43 on the release of hydrogen peroxide (H2 O2 ) by macrophages Peritoneal cells were collected from the abdominal cavity of the animals (A/Sn and B10.A) by repeated washing with 2 ml of RPMI-1640 medium (Sigma). The cells were adjusted to 2 10 cells/ml in a Phenol Red solution (PBS, 5 mM dextrose, 50 lg of horseradish type II, and 0.28 mM phenol red). A 100 ll of the peritoneal cell suspensions was dispensed in 96-well flat bottom microtiter plate (Costar). Hundred lg/ml of gp43 solution, was added to the wells. The plates were incubated at 37 °C for 1 h and the reaction stopped by the addition of 10 ll of 1 N NaOH solution. A standard curve for the determination of H2 O2 concentration in the experimental tests was constructed using H2 O2 solutions varying from 0.5 to 4 nmol. Ab-
89
sorbance was measured at 620 nm in a microtiter plate reader (MR 4000, Dynatech Chantilly). Results were expressed as nanomoles of H2 O2 per 2 106 cells. 2.11. Colony forming units (CFU) determination The determination of the number of viable fungi after phagocytosis by macrophages was made by CFU counts. Macrophages were challenged with live Pb and incubated for 4 h, as described in phagocytic tests. After this time, cultures were rinsed with PBS for removal of non-internalized Pb cells. Distilled water was added to lyse macrophages. The cellular suspension was harvested, washed in PBS, and the final pellets ressuspended in 1 ml of PBS. Aliquots of 100 ll of each sample were plated in agar plates (4% SFB, 5% BHI solid medium). Colonies per plate were counted after 8–10 days of incubation at 37 °C. 2.12. Statistical analysis Statistical comparisons were made by analysis of variance (ANOVA) and by the Tukey–Kramer test. All values are reported as the mean standard error deviation of the mean, with significance assumed in the range of p < 0:05.
3. Results 3.1. Phagocytosis of Pb by peritoneal macrophages Contrary to what happens with other particles, such as zymosan and SRBC, fungal particles were internalized by macrophages after 4–8 h of culture (data not shown). A possible explanation for the observed phenomenon was the impairment of contact between fungal particles and macrophages membranes. This hypothesis could not be sustained considering that centrifugation of the cultures (1062g for 5 min) in order to allow a closer contact between the particles and the macrophage cell membrane did not speed up endocytosis of Pb (data not shown). Considering that the delay of macrophages to ingest Pb particles was not due to the contact between cells and fungi particles, we investigated whether this phenomenon could be due to the release of inhibitory factors by macrophages induced by the contact between Pb particles and these cells. This hypothesis was tested in a model where the phagocytic indexes of zymosan by macrophages which were previously challenged with dead Pb particles was evaluated. Cultured macrophages were challenged with Pb particles for 4 h. The cultures were washed and added with zymosan (z) particles. Results show that previous ingestion of Pb particles inhibits the phagocytic indexes of zymosan by these cells
90
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
(Fig. 1A). A less significative inhibition was observed when Pb plus zymosan particles were simultaneously added to macrophage cultures. These data suggest that either the fungus down-regulate macrophage functions or induces macrophages to secret a macrophage deactivating factor. In order to investigate the last hypothesis, macrophages were cultured on the bottom of transwell microplates. On half of the plates macrophages were also cultured on the top of the filter. All the plates were added with heat-killed Pb particles. Results show that the contact between macrophages and Pb particles induces a significant decrease in the phagocytic indexes of zymosan when macrophages cultured on the bottom of the wells were tested (Fig. 1B). Considering that heatkilled Pb was used in these experiments, these results suggest that the contact between Pb and macrophage membrane induces these cells to secrete a macrophage deactivating factor/s.
Fig. 1. Interaction Pb–macrophages decreases phagocytic indexes (PI) of zymosan by macrophages. (A) Macrophages were challenged with: zymosan for 1 h (zymosan), Pb and zymosan simultaneously for 1 h (Pb + z) and Pb for 4 h and zymosan for 1 h (Pb 4 h + z). (B) Phagocytosis of zymosan by macrophages cultured on the bottom of transwell microplates. The top of the filters were added with: culture medium (control), macrophage + heat-killed Pb (M/ + Pb), live Pb or heat-killed Pb. Cells were stained with Giemsa and counted to determine PI with the aid of an optic microscope (100). *p < 0:01 is significant when compared with macrophages challenged with zymosan particles.
Similar experiments were performed using live Pb particles. As shown in Fig. 1B, when live Pb particles were added to the top of the wells, a significant decrease in the phagocytic index of zymosan ingestion by macrophages cultured in the bottom of the wells was observed. These data allow us to conclude that live Pb secrets a factor/s which deactivates macrophage functions. 3.2. Influence of gp43 on the phagocytic activity of peritoneal macrophages Based on results above described we hypothesized that gp43 might have a down-regulatory effect on macrophages. The addition of purified gp43 to the cultures, simultaneously to the challenge with heat-killed fungus, led to a significant inhibition of phagocytosis by macrophages from the A/Sn or B10A strains. Different concentrations of gp43 added to the culture medium determined a decrease in the phagocytic index in a dose-dependent pattern (Fig. 2). Similar results were obtained when macrophages were challenged with live Pb (data not shown). Considering that zymosan is endocytosed by macrophages via mannose receptors [16], we decided to investigate whether gp43 would influence the uptake of this particle. As shown in Fig. 3A, the addition of gp43 to the cultures decreased the phagocytic indexes when the cells were challenged for 1 h with zymosan particles (10 mg/ml).
Fig. 2. Influence of gp43 on the PI of heat-killed Pb by peritoneal macrophages from A/Sn and B10.A mice. Gp43 inhibited phagocytosis when added to the culture medium, simultaneously with addition of the fungal particles at the concentrations of 25, 75, 100, and 150 lg/ml. Cells were stained with HE and counted to determine the PI with the aid of an optic microscope (100). Significance (*p < 0:05 and **p < 0:001) was obtained when treated cells were compared with those from control group.
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
91
gate the influence of Pb and/or gp43 on the liberation of NO and H2 O2 by macrophages in vitro. As shown in Fig. 4, gp43 inhibits H2 O2 liberation by macrophages from the A/Sn or B10.A mice stimulated or not by zymosan particles (Fig. 4). The amount of NO spontaneously liberated by peritoneal macrophages from both strains of mice increases with time (Fig. 5). Addition of Pb, gp43 or Pb + gp43 did not induce increase in NO liberation. Otherwise, these stimuli caused inhibition of NO production when compared with results obtained with non-stimulated macrophages. Gp43 also inhibited NO liberation by macrophage stimulated with zymosan. The addition of different concentration of zymosan stimulated NO production by macrophage at a dose-dependent manner. As shown in Fig. 6A, gp43 is able to inhibit NO liberation by macrophages. It is important to note that macrophage from B10.A mice did not induce a significant increase in NO liberation as observed with macrophages obtained from mice from the A/Sn strain (Fig. 6B). 3.4. Influence of gp43 on the viability of internalized fungus
Fig. 3. Influence of gp43 on the phagocytosis of zymosan and opsonized SRBC. (A) Purified gp43 (100 lg/ml) inhibited phagocytosis of zymosan particles (10 mg/ml) by peritoneal macrophages from A/Sn and B10.A mice. *p < 0:01 is significant when compared with control group not added with gp43. (B) Phagocytic indexes of IgG opsonized sheep red blood cells (SRBC) by macrophages from both A/Sn and B10.A mice. Non-opsonized sheep red blood cells (SRBC) were used as control (data not shown). The addition of gp43 did not influence phagocytosis P ¼ 0.044. Cells were stained with Giemsa and counted to determine the phagocytic indexes with the aid of an optic microscope (100).
Results above described did not bring evidences that the killing capacity of macrophages from resistant (A/Sn) or susceptible (B10.A) mice is different. Results obtained with the colony forming units method clearly demonstrate that macrophages from the A/Sn strain have a higher ability to kill the fungus when compared with macrophages from the B10.A lineage. However, it is also clearly demonstrated that the addition of gp43 to the cultures, significantly inhibits the killing capacity of
The inhibitory effect of gp43 on the endocytosis of zymosan particles could be due to a general metabolic down-regulation of the phagocytes. This hypothesis was discharged considering that gp43 did not influence the phagocytic indexes of opsonized sheep red blood cells by macrophages, as shown in Fig. 3B. 3.3. Influence of gp43 on the liberation of NO and H 2 O2 by peritoneal macrophages It has been reported that NO, H2 O2 , and other free metabolites are involved with the killing capacity of macrophages [11,12]. Therefore, we decided to investi-
Fig. 4. Influence of gp43 on the release of hydrogen peroxide (H2 O2 ) by murine macrophage (A/Sn and B10.A). Peritoneal cells were stimulated with gp43, zymosan, zymosan + gp43 or not stimulated. After 1 h, the culture supernatants were harvested and absorbance (620 nm) was measured to determine the amount of H2 O2 released. p < 0:01 when the values were compared between them.
92
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
Fig. 5. Influence of gp43 on NO release by peritoneal macrophages from A/Sn and B10.A mice challenged with Pb. Macrophages, after 3 days in culture, were simultaneously challenged with Pb cells, gp43 or both. Macrophages were not stimulated in control group. The culture supernatants were harvested and the amount of nitrite analyzed in a microtiter plate reader with absorbance at 550 nm. p < 0:05 when values were compared with control group.
macrophages obtained from both resistant or susceptible mice (Fig. 7).
4. Discussion It is well established that P. brasiliensis (Pb) is a facultative intracellular parasite [14]. It is also established that the interaction between molecules on the surface of the parasite and homologous receptors on the cell membrane of macrophages modulates phagocytosis [8,11]. Molecular complexes containing proteins and polysaccharides with a high proportion of mannose have been detected on the surface of the fungus by Almeida et al. [1]. These authors demonstrated that phagocytosis of Pb by murine peritoneal macrophages in vitro was
Fig. 6. (A) and (B)—Influence of gp43 on NO liberation by peritoneal macrophages challenged with zymosan particles. The culture supernatants were harvested and the amount of nitrite analyzed in a microtiter plate reader with absorbance at 550 nm. *p < 0:01 when the values obtained with macrophages from B10.A mice were compared, **p < 0:001 when the values obtained with macrophages from A/Sn mice were compared, # p < 0:001 when the values were compared between them.
inhibited when D -mannose and D -fucose was added to the culture medium. These data indicate that endocytosis of the fungus by macrophages is mediated by the mannose receptor. The authors have also demonstrated that policlonal antibodies directed to epitopes of gp43 also induce a marked decrease on the phagocytic indexes of macrophages challenged with Pb. However, a monoclonal antibody which recognizes a peptidic epitope of gp43 did not interfere with the interaction macrophage– Pb. Finally, the authors have also demonstrated that gp43 coupled to the surface of sheep red blood cells was endocytosed by macrophages and this interaction was inhibited by the addition of D -mannose and D -fucose to the culture medium. These data attest the participation of gp43, the main antigenic molecule secreted by the fungus, on the uptake of the fungus by macrophages.
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
Fig. 7. (A) and (B)—Viability of Pb (CFU) after phagocytosis of fungi by macrophages in the presence or absence of gp43. Results show that the addition of gp43 to the cultures inhibited the fungal killing by peritoneal macrophages. *p < 0:01 when compared with control group (A). Differences are illustrated in (B).
Based on these information we decided to broaden these investigations in order to clarify the role of gp43 on the interaction macrophage–fungus. Our results show that the phagocytic indexes of macrophages challenged with live or heat-killed fungi are similar and are inhibited when gp43 is added to the culture medium. These observations suggest that heatkilled fungi retain on their surface molecules that interact with macrophage cell membrane and that this interaction is modified by the presence of gp43. Also, that macrophages from both lineages, A/Sn and B10.A, have similar phagocytic indexes either when live or heatkilled cells were tested. Gp43 induces a dose-dependent decrease in the phagocytic indexes of macrophages stimulates with Pb. These data suggest a possible interaction of Pb–macrophage and gp43–macrophage with the same receptor, probably through the mannose receptor. This hypothe-
93
sis was reinforced by the observation that gp43 also blocked endocytosis of zymosan particles which are phagocytosed by macrophages via the mannose receptor [15]. Furthermore, gp43 did not alter the phagocytic indexes of IgG opsonized sheep red blood cells indicating a selective effect of the molecule on the endocytic mechanisms of macrophages. The long period needed for macrophages to ingest heat-killed fungal particles (4–8 h) is an intriguing phenomenon considering that these cells phagocytose other particles in a shorter period of time. In order to understand this peculiarity we hypothesized that the contact macrophage–Pb induces deactivation of these cells by release of autocrine factors by macrophages. The decrease in phagocytic indexes of zymosan by macrophages previously challenged with dead Pb particles suggest that either the fungus down-regulate macrophage functions or induces macrophages to secret a macrophage deactivating factor. Our data strengthen the hypothesis that gp43 impairs the ingestion of Pb, and the interaction macrophage– fungus induces deactivation of these cells. This hypothesis is reinforced by the fact that gp43 inhibits the liberation of intermediary reactives of oxygen and nitrogen, mediators involved in the microbicidal activity of macrophages. The liberation of H2 O2 by macrophages from the A/Sn and B10.A mice was inhibited by gp43. However, this molecule did not influence the liberation of this metabolite when phagocytes were stimulated with PMA. It is possible that the interaction of gp43 and PMA with macrophage cell membrane activates different pathways of signal transduction in these cells (data not shown). The spontaneous production of NO by macrophages was inhibited when gp43 and/or Pb were added to the cultures. The addition of Pb to these cultures did not induce a significant increase in NO liberation as occurs with macrophages challenged with zymosan. However, gp43 also showed inhibitory effect on NO liberation in these circumstances. These data reinforced the hypotheses that the fungus induce macrophages to secret autocrine deactivating factor/s. Summing up, we may conclude that gp43 has an inhibitory effect on the liberation of NO and H2 O2 by murine peritoneal macrophages stimulated by the contact with Pb. Further, that the inhibitory effect of gp43 on the microbicidal activity of macrophages was clearly demonstrated, considering that treatment of macrophages with gp43 impairs the fungicidal capacity of these cells as demonstrated by the CFU tests. Based on results here described and considering that macrophages are pivotal in the control of fungal infection, it is licit to put forward the hypothesis that gp43 is involved in an evasion mechanism of the fungus that facilitates its homing in the host tissues in primary infections.
94
A. Flavia Popi et al. / Cellular Immunology 218 (2002) 87–94
Acknowledgments We are indebted to Prof. Michel Rabinowich for his skilful suggestions during the development of this work and to Luiz Cl audio Godoy and Sandro Rogerio de Almeida for their guidance during the execution of many experiments. Ana Flavia Popi was the recipient of a grant as a graduating student of Biomedicine from Fundacß~ao de Amparo a Pesquisa do Estado de S~ ao Paulo (FAPESP) (Process: 98/11790-0).
[8]
[9]
[10]
[11]
References [12] [1] S.R. Almeida, C.S. Unterkircher, Z.P. Camargo, Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages, Med. Mycol. 36 (1998) 405–411. [2] M.M Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [3] E. Brummer, L.H. Hanson, A. Restrepo, D.A. Stevens, In vivo and in vitro activation of pulmonary macrophages by IFN-c for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis, J. Immunol. 140 (1988) 2786–2789. [4] E. Brummer, L.H. Hanson, A. Restrepo, D.A. Stevens, Intracellular multiplication of Paracoccidioides brasiliensis in macrophages: killing and restriction of multiplication by activated macrophages, Infect. Immun. 57 (1989) 2289–2294. [5] V.L. Calich, L.M. Singer-Vermes, A.M. Siqueira, E. Burger, Susceptibility and resistance of inbred mice to Paracoccidioides brasiliensis, Br. J. Exp. Pathol. 66 (1985) 585–594. [6] V.L. Calich, C.A. Vaz, E. Burger, Immunity to Paracoccidioides brasiliensis infection, Res. Immunol. 149 (1998) 407–417, discussion 499–500. [7] J.L. Gesztesi, R. Puccia, L.R. Travassos, A.P. Vicentini, J.Z. de Moraes, M.F. Franco, J.D. Lopes, Monoclonal antibodies against
[13]
[14]
[15]
[16]
[17]
the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis, Hybridoma 15 (1996) 415–422. S. Gordon, V.H. Perry, S. Rabinowitz, L.P. Chung, H. Rosen, Plasma membrane receptors of the mononuclear phagocyte system, J. Cell Sci. Suppl. 9 (1988) 1–26. S.S. Kashino, L.M. Singer-Vermes, V.L. Calich, E. Burger, Alterations in the pathogenicity of one Paracoccidioides brasiliensis isolate do not correlative with its in vitro growth, Mycopathologia 111 (1990) 173–180. M. Moscardi-Bacchi, E. Brummer, D.A. Stevens, Support of Paracoccidioides brasiliensis multiplication by human monocytes or macrophages: inhibition by activated phagocytes, J. Med. Microbiol. 40 (1994) 159–164. L. Ohman, G. Maluszynska, K.E. Magnusson, O. Stendahl, Surface interaction between bacteria and phagocytic cells, Prog. Drug Res. 32 (1988) 131–147. D.M Paulnock, The molecular biology of macrophage activation, Immunol. Ser. 60 (1994) 47–62. R. Puccia, L.R. Travassos, The 43-kDa glycoprotein from the human pathogen Paracoccidioides brasiliensis and its deglycosylated form: excretion and susceptibility to proteolysis, Arch. Biochem. Biophys. 289 (1991) 298–302. L.M. Singer-Vermes, E. Burger, V.L. Calich, L.H. ModestoXavier, T.N. Sakamoto, M.F. Sugizaki, D.A. Meira, R.P. Mendes, Pathogenicity and immunogenicity of Paracoccidioides brasiliensis isolates in the human disease and in an experimental murine model, Clin. Exp. Immunol. 97 (1994) 113–119. D.P. Speert, S.C. Silverstein, Phagocytosis of unopsonized zymosan by human monocyte-derived macrophages: maturation and inhibition by mannan, J. Leukoc. Biol. 38 (1985) 655– 658. P.D. Stahl, R.A. Ezekowitz, The mannose receptor is a pattern recognition receptor involved in host defense, Curr. Opin. Immunol. 10 (1998) 50–55. L.R. Travassos, R. Puccia, P. Cisalpino, C. Taborda, E.G. Rodrigues, M. Rodrigues, J.F. Silveira, I.C. Almeida, Biochemistry and molecular biology of the main diagnostic antigen of Paracoccidioides brasiliensis, Arch. Med. Res. 26 (1995) 297– 304.