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Originally published online as doi:10.1189/jlb.1005603 on May 2, 2006

Published online before print May 2, 2006
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(Journal of Leukocyte Biology. 2006;80:36-44.)
© 2006 by Society for Leukocyte Biology

Nitric oxide-dependent killing of Cryptococcus neoformans by B-1-derived mononuclear phagocyte

Eliver Eid Bou Ghosn*, Montchilo Russo# and Sandro Rogerio Almeida*,1

* Departamento de Analises Clinicas e Toxicologicas, Faculdade de Ciências Farmacêuticas,
# Departamento de Imunologia, Universidade de São Paulo, Brazil

1 Correspondence: Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, Avenida Prof. Lineu Prestes, 580, Bloco 17, São Paulo, Brazil, CEP: 05508-900. E-mail: sandroal{at}usp.br

ABSTRACT

The role of B lymphocytes in protecting the host against pulmonary Cryptococcus neoformans infection is until now, uncertain. A recent study using B lymphocyte-deficient mice suggests that B lymphocytes play a protective role in cryptococcal infection. It has been well established that at least three B cell subsets, B-1a, B-1b, and B-2, are present in the mouse periphery. B-1 cells constitute a minor fraction of the B cell population in the spleen and are not detected in lymph nodes of mice. We demonstrated that B-1 cells migrate to a nonspecific, inflammatory focus and differentiate into macrophage-like cells. However, the role these cells might play on the kinetics and evolution of the inflammatory response and on fungal infection has not yet been established. Based on these data, we decided to investigate the interaction of B-1-derived mononuclear phagocytes (BDMP) with C. neoformans to elucidate the possible influence of this cell in the progression of the disease. In this study, we demonstrated that the BDMP cell internalized C. neoformans and that this process was mediated by complement receptor 3. Thus, our results showed that the BDMP cell was more fungicidal than a macrophage and up-regulated major histocompatibility complex type II and the CD86 costimulatory molecule with the production of proinflammatory cytokines. The phagocytosis of C. neoformans results in the nitric oxide (NO)-mediated death of the fungus, as demonstrated by experiments using NO synthase 2 knockout and aminoguanidine-treated, wild-type mice.

Key Words: inflammation • fungus • lymphocyte

INTRODUCTION

The Cryptococcus neoformans yeast is an encapsulated fungus that can cause infection in immunocompetent and immunocompromised individuals. The AIDS epidemic has dramatically increased its incidence during the last two decades, although in general, impaired, cell-mediated immunity represents the main predisposing factor for the development of cryptococcosis [1 ]. Meningoencephalitis is the most frequent clinical presentation, but any organ can be affected [2 ].

The lung is the entry gateway of C. neoformans in humans. In immunocompetent hosts, infection is presumably contained in the lung, and the majority of human infections are asymptomatic [3 ]. The effective tissue response for controlling C. neoformans infection is the granulomatous inflammation [4 ]. The inflammatory infiltrate seen in the lung following C. neoformans infection includes macrophages, lymphocytes, neutrophils, and eosinophils. B lymphocytes have been reported to be the predominant type of cells in the inflammatory response to C. neoformans [5 ].

It is well established that at least three B cell subsets, B-1a, B-1b, and B-2, are present in the mouse periphery. The B-2 cells seem to be responsible for T cell-dependent responses to exogenous antigens and for generating memory B cells, which display a characteristic pattern of cell surface markers such as immunogloblin Mlo (IgMlo), IgDhi, B220hi, CD23+, membrane-activated complex-1– (Mac-1–), and CD5– [6 ]. B-1 cells constitute a minor fraction of the B cell population in the spleen and are not detected in lymph nodes of mice. Nevertheless, they represent the main B cell population in the peritoneal and pleural cavities of these animals. They express high levels of IgM and low levels of B220 and IgD, but not CD23. B-1 cells also express low levels of Mac-1 and a subset, B-1a, which has intermediate levels of CD5 on its surface [7 8 9 ]. However, IgM and CD5 antigens are lost when B-1 cells migrate from the peritoneal cavity [10 ].

Recent reports have shown evidences that the lymphoid and myeloid lineages share an unexpectedly close relationship. This is based on studies demonstrating that a number of mice [11 , 12 ] and human [13 , 14 ] B-1 malignancies can generate phagocytic descendants exhibiting macrophage-like characteristics. The same phenomenon has been described for normal mouse B-1a cells (CD5+) when they were cocultured with fibroblasts. The authors called these cells bi/phenotypic macrophages [15 ]. The same group demonstrated that B/macrophage cells express cyclooxygenase 1 (COX-1) and up-regulate COX-2 expression as well as prostaglandin E2 production in response to proinflammatory signals. They have also confirmed the existence of B-1 cells in the normal peritoneal cavity of mice [16 ]. However, despite the large volume of information concerning the origin [17 18 19 20 ], proprieties [11 , 18 , 21 , 22 ], and participation of these cells in normal [17 , 23 , 24 ] or pathologic conditions [14 , 25 26 27 28 29 30 31 ], the demonstration of whether the generation of mononuclear phagocytes from B-1 cells is one of the evolution consequences of these cells in vivo remains to be established.

We have recently demonstrated that B-1 cells proliferate spontaneously in stationary cultures of normal, adherent mouse peritoneal cells. Furthermore, we have also demonstrated that B-1 cells migrate to a nonspecific, inflammatory focus and differentiate into macrophage-like cells [32 ]. Nevertheless, the role these cells might play on the kinetics and progression of the inflammatory response and on fungal infection has not yet been established.

The role of B lymphocytes in protecting the host against pulmonary C. neoformans infection is until now, uncertain. A recent study using B lymphocyte-deficient mice suggests that B lymphocytes play a protective role in cryptococcal infection [33 ]. Other significant data include the abundance of B lymphocytes in the lung infiltrate in cryptococcal infection, suggesting the importance of evaluating the contribution of this cell to pathogenesis [5 ].

Based on these data, we decided to investigate the interaction of B-1-derived mononuclear phagocytes (BDMP) with C. neoformans to elucidate the possible influence that this cell has on the progression of infection.

MATERIALS AND METHODS

Mice
Female BALB/c, C57BL/6 nitric oxide synthase 2 (NOS2)-deficient and NOS2-sufficient mice, ranging from 8- to 12-weeks old, were obtained at the Isogenic Breeding Unit of the Department of Immunology, Institute of Biomedical Sciences, University of São Paulo (Brazil).

Strain of C. neoformans
In this study, we used the Centraalbureau voor Schimmelcultures (CBS; The Netherlands) 132 strain of C. neoformans var neoformans, obtained from the CBS collection.

Culture of B-1 cell
B-1 cells were produced according to the method proposed by Almeida et al. [32 ]. Briefly, peritoneal cells of normal mice were collected after peritoneal washing with phosphate-buffered saline (PBS) and isolated by selective adherence on glass Petri dishes for 2 h at 37°C. Adherent cells were detached by scraping using a silicone rubber and were then washed and suspended in RPMI-1640 complete medium (Sigma-Aldrich, St. Louis, MO) containing 10% heat-inactivated fetal calf serum (FCS; Gibco-BRL, Grand Island, NY). The cells were plated in 24-well plates at a concentration of 2 x 106 cells per well and incubated at 37°C in an atmosphere of 5% CO2 for 7 days. During this period, the culture medium was not changed. Analysis of nonadherent cell phenotypes showed that more than 86% of cells expressed Mac-1 and IgM markers.

Obtaining BDMP
BDMP cells were produced according to the method proposed by Almeida et al. [32 ]. Briefly, after 7 days of culture, the nonadherent cells were harvested, washed, and recultured in fresh medium (RPMI 1640 plus 10% FCS) in 24-well tissue-culture plates containing a round glass coverslip in the base of each well. After 24 h of culture, these cells became larger and projected pseudopods from two poles of the cell. These pseudopods became more prominent, and typical bipolar cells appeared. A high proportion of these populations (more than 80%) was composed of IgM+ and Mac-1+.

Bone marrow-derived macrophages (BMDM)
BMDM were generated as described [34 ] from 6- to 8-week-old BALB/c mice. Cultures were kept at 37°C in a 5% CO2 in RPMI supplemented with 10% fetal bovine serum and 5% of conditioned medium derived from cultures of L929 cells, known to be high producers of macrophage-colony stimulating factor.

Phagocytosis of C. neoformans
BDMP cells or macrophages were obtained as described above, and 1 x 106 cells were placed in 24-well plates containing glass coverslips with 1 ml complete RPMI-1640 medium, with or without complement in the presence of 5 x 106 C. neoformans yeast cells. As source of complement, we used 5% of normal mouse sera. BDMP cells plus C. neoformans were incubated for 4 h at 37°C in a 5% CO2 atmosphere. The glass coverslips were then washed with PBS and stained with Giemsa. An average of 100 cells was counted on several microscopic fields to determine the percentage of BDMP or macrophages that internalized at least one conidia (P) and the average number of fungal cells in these cells (F). The phagocytic index (PI) was calculated by multiplying P x F [35 ]. For inhibition studies, prior to addition of the yeast cells, the macrophages and BDMP were incubated for 1 h at 4°C with 10 µg/ml antibodies against CD11b (M1/70) and CD18 (M18/2; BD PharMingen, San Diego, CA).

Fungicidal activity of BDMP and macrophages
BDMP and macrophages were obtained and cultured with C. neoformans, as described above, for 24, 48, and 72 h in 24-well plates. Nonphagocytosed fungi were removed, and fungicidal activity was measured by the disruption of these cells using 200 µl distilled and sterilized water for 2 min, followed by the addition of 800 µl PBS for completing the volume. The cellular suspensions were plated on Sabouraud agar supplemented with antibiotics. The plates were incubated at 37°C, and colony-forming units (CFU) were counted from the fifth day until no increase in counts was observed (30th day).

Cell viability of C. neoformans-infected macrophages and BDMP were determined by counting the number of cells that remained adherent to the substratum. Briefly, cell suspensions containing 1 x 106 macrophages or BDMP were layered onto 24-well tissue-culture microplates (Corning Costar Co., Cambridge, MA) for 1 h to allow for adherence. The attached cells were rinsed in PBS and incubated with 1 mL complete RPMI-1640 medium containing yeast at a 1:5 ratio in 100 µL in RPMI complete medium for 24, 48, and 72 h at 37°C in a 5% CO2 atmosphere. The remaining cells were washed with PBS and stained and fixed with 100 µL 0.05% crystal violet dissolved in 3% acetic acid (final solution) for 10 min. Afterwards, the wells were washed with distilled water and left to dry at room temperature. The numbers of stained cells were determined by the mean of the number of stained macrophages or BDMP present in three random fields at high-power field magnification (400x).

Cytokines and inducible NOS (iNOS) mRNA analysis
The mRNA expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Total cellular RNA from BDMP cell was extracted by TRIzol (Invitrogen, Life Technologies, Carlsbad, CA), according to the protocol of the manufacturer. The first-strand cDNA was synthesized from 1 µg total RNA by Superscript II RNAase H-RT, following the instructions of the manufacturer using 0.5 µg oligo(dT)12–18 (Invitrogen, Life Technologies). One-tenth of synthesized cDNA was amplified by PCR using 0.33 µM sense and 0.33 µM antisense primers (Invitrogen Brasil, Life Technologies, SP) with Platinum mix (Invitrogen, Life Technologies) in a 1.5-U concentration of Taq polymerase and 2.5 mM Mg++ in a total volume of 30 µl. PCR cycles were performed for 1 min at 94°C for denaturation, 1.5 min at 55°C (iNOS), and at 62°C (others) for annealing, 1 min at 72°C for extension, and at the first cycle, denaturation was run for 3 min at 94°C. The sequence of the sense and antisense primers, the size of products, and PCR cycles are as follows: hypoxanthine-guanine phosphoribosyltransferase (HPGRT), 5'-GTT GGA TAC AGG CCA GAC TTT GTT G-3' and 5'-GAG GGT AGG CTG GCC TAT GGC T-3', 352 base pairs (bp), 35 cycles; iNOS, 5'-(F): CAT GGC TTG CCC CTG GAA GTT TCT CTT CAA AG-3' and 5'-GCA GCA TCC CCT CTG ATG GTG CCA TCG-3', 828 bp, 35 cycles; tumor necrosis factor {alpha} (TNF-{alpha}), 5'-GGC AGG TCT ACT TTG GAG TCA TTG C-3' and 5'-ACA TTC GAG GCT CCA GTG AAT TCG G-3', 309 bp, 35 cycles; interferon-{gamma} (IFN-{gamma}), 5'-AGC GGC TGA CTG AAC TCA GAT TGT AG-3' and 5'-GTC ACA GTT TTC AGC TGT ATA GGG-3', 244 bp, 40 cycles; interleukin (IL)-10, 5'-CCA GTT TTA CCT GGT AGA AGT GAT G-3' and 5'-TGT CTA GGT CCT GGA GTC CAG CAG ACT CAA-3', 324 bp, 40 cycles; IL-4, 5'-CAT CGG CAT TTT GAA CGA GGT CA-3' and 5'-CTT ATC GAT GAA TCC AGG CAT CG-3', 240 bp, 40 cycles. PCR products (10 µl) were submitted to electrophoresis using a 1.4% agarose gel. After ethidium bromide staining (Sigma-Aldrich), PCR products were visualized by ultraviolet illumination. The intensity of each band formed was estimated using the Kodak 1D 3.5 software (Eastman Kodak Co., Rochester, NY). Expression of HPGRT was evaluated in parallel and used for proper normalization of each sample load in analytic runs.

Determination of cytokines
The levels of cytokines in supernatants of BDMP cell cultures were determined using a sandwich enzyme-linked immunosorbent assay (ELISA) kit, according to the suggestions of the manufacturer. BDMP cells were incubated with C. neoformans as described above, and after 4 h or 72 h, the supernatants were harvested and stored at –70°C for cytokine quantification. IL-10, IL-4, TNF-{alpha}, and IFN-{gamma} were quantified in the supernatants by ELISA (R&D Systems, Minneapolis, MN), according to the instructions of the manufacturer. Cytokine activity was determined applying curves with serial dilutions of mouse recombinant cytokines.

BDMP phenotyping
The effects of C. neoformans on surface molecule expression by BDMP were investigated by using cells incubated with yeast (6:1) for 24 h. Phenotype cells were analyzed by flow cytometry with a FACScan (Becton Dickinson, San Jose, CA). To determine the class II major histocompatibility complex (MHC) and costimulatory molecules, phycoerythrin (PE)-labeled monoclonal antibodies (mAb) were used against mouse anti-MHC-II (M5/114.15.2), CD80 (16-10A1), Mac-1 (M1/70), and CD86 (GL1) or isotype controls. Results are expressed as the mean of fluorescence intensity obtained with specific antibodies.

NO production
NO production was quantified by the accumulation of nitrite (as a stable end-product) in the supernatants of BDMP cell cultures in the presence of C. neoformans by the standard Griess reaction as described previously. Conversion of absorbency to NO micromolar concentrations was deduced from a standard curve using a known concentration of NaNO2 diluted in RPMI medium.

In vitro NOS2 inhibition by aminoguanidine (AG) hemisulfate treatment
To inhibit NO production in vitro, BDMP were obtained and cultured with C. neoformans, as described above, for 24 h and 72 h in 24-well plates, with or without 1 mM AG (Sigma-Aldrich). Nonphagocytosed fungi were removed, and fungicidal activity was measured by the disruption of these cells using 200 µl distilled and sterilized water for 2 min, followed by the addition of 800 µl PBS for completing the volume. The cellular suspensions were plated on Sabouraud agar plus antibiotics. The plates were incubated at 37°C, and colonies were counted from the fifth day until no increase in counts was observed (30th day).

RESULTS

Phagocytosis of C. neoformans by BDMP cells
We recently demonstrated that B-1 cells proliferate spontaneously in stationary cultures of normal, adherent mouse peritoneal cells. B-1 cells were characterized by morphology, immunohistochemistry, and flow cytometry; the major cell population analyzed expresses the B-1b phenotype. When these cells were transferred to a new culture, a large proportion of them adhered to the plastic surface and spread as bipolar cells endowed with the capacity to phagocytose via Fc and mannose receptors. Flow cytometry analysis of these adherent cells (BDMP) demonstrated that the great majority of them (more than 80%) shares IgM and Mac-1 antigens (Fig. 1B ). In this work, we first investigated whether BDMP cells would internalize C. neoformans yeast cells. For this purpose, we determined the PI of BDMP cells cocultured with C. neoformans during 4 h. We found that the majority of BDMP cells ingested yeast in the presence of complement (Fig. 1C) . The phagocytosis of C. neoformans by BDMP in the presence of complement was higher than by macrophages. In the absence of complement, a low PI was observed. A blocking experiment revealed that complement-mediated phagocytosis occurred through CR3 in both cells (Fig. 1D) .


Figure 1
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  		Figure 1. In vitro phagocytosis of C. neoformans by macrophages and BDMP, and BDMP cells were obtained as described in Materials and Methods, removed from the plastic surface with the aid of a rubber policeman, and analyzed by flow cytometry. Cells were double-stained for IgM and Mac-1 (B) and isotype control (A). A high proportion of these cells were IgM and Mac-1+ cells. FITC, Fluorescein isothiocyanate. (C) A total of 106 cells were cocultivated with 5 x 106 C. neoformans yeast cells (1:5 ratio) for 4 h at 37°C 5% CO2 in RPMI medium, with or without 5% serum (supplement of complement proteins). (D) Inhibition of phagocytosis by antibodies against complement receptor 3 (CR3). C. neoformans cells were added to the macrophages and BDMP at a ratio of 1:5 in the presence of complement. Prior to the addition of yeast, the cells were incubated for 1 h at 4°C with 10 µg/ml antibodies against CD11b and CD18. The values were expressed in PI, as described, and represent the mean as well as the SD obtained in three different experiments. *, P < 0.05, when compared with PI of macrophage phagocytosis (with complement); #, P < 0.05, when compared with PI of macrophages and BDMP without treatment with CD11b and CD18.

 
C. neoformans viability test
We were interested in determining the evolution of C. neoformans after its uptake by the BDMP cell. For this, we analyzed the fungicidal capacity of the BDMP cell and compared it with that of macrophages. A significant decrease in the number of viable yeast cells after 72 h of incubation was observed, showing that phagocyted yeasts were destroyed by the BDMP cell (Fig. 2A ). Conversely, macrophages were not able to destroy the yeast cells (Fig. 2C) . To determine BDMP and macrophage viability after phagocytosis, these cells were infected with yeast, and after different intervals of time, the culture was washed, fixed, and stained. After this, the cell cultures were examined under light microscopy. We observed that the number of BDMP cells was the same at different times, showing that these cells remained viable (Fig. 2B) . In contrast, when macrophages were counted in high-power magnification, an increased number of macrophages were found to have died (Fig. 2D) .


Figure 2
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  		Figure 2. C. neoformans killing by BDMP. The numbers represent the recovery of C. neoformans CFU after 24, 48, and 72 h of conducting the phagocytosis assay (1:5 ratio) using BDMP cells (A) and macrophages (C). Fungal cytotoxicity was determined by cell count by colorimetric assay. The number of stained BDMP cells (B) and macrophages (D) was determined by counting the number of stained cells with crystal violet present in three random fields at a high-power magnification field (400x). The values represent the mean obtained from three different experiments. *, P < 0.05, when compared with a 24-h CFU count.

 
mRNA iNOS and cytokine expression by BDMP cells in the presence of C. neoformans
It has been shown that iNOS expression is essential to control murine pulmonary cryptococcosis; therefore, mRNA expression of C. neoformans-infected BDMP cells was analyzed. An increase of mRNA iNOS by BDMP cells after C. neoformans phagocytosis was observed. The most interesting fact was that incubation of BDMP cells in the absence of fungus resulted in the expression of iNOS mRNA. Indeed, the mRNA expression of cytokine was also evaluated. The results showed an increase of TNF-{alpha}, IFN-{gamma}, and IL-4 expression after C. neoformans phagocytosis by the BDMP cell. In contrast, the IL-10 expression was decreased in the presence of fungus. As in the iNOS expression experiment, the nonstimulated BDMP cell expressed IL-10 and TNF-{alpha}, which was measured by determining their mRNA (Fig. 3 ). The possibility that this basal expression of cells was induced by lipopolysaccharide (LPS) contamination can be excluded, as the cultures were monitored using the Limulus amebocyte lysate (LAL) test (Endosafe LAL, Charles River, Charleston, SC), and the maximal concentration of LPS detected in the cultures was always <0.1 ng LPS/ml.


Figure 3
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  		Figure 3. Kinetics of iNOS and cytokine expression in BDMP cells. BDMP RNA samples were collected after 4 h and 72 h of conducting the phagocytosis assay (1:5 ratio) performed with C. neoformans yeast cells. The expression of iNOS, TNF-{alpha}, IL-10, IFN-{gamma}, and IL-4 in BDMP cells was assessed. The expression of HPGRT was evaluated as a control. 4h//BDMP, Basal expression of BDMP cultivated for 4 h without stimulus. In panels, bands represent the level of mRNA in each sample.

 
Cytokine and NO production
The next step was to determine cytokine and NO production by C. neoformans-infected BDMP cells. We found that infected BDMP cells produced significant levels of TNF-{alpha} and decreased IL-10 when compared with noninfected BDMP cells. However, we could not detect IL-4 and IFN-{gamma} in the supernatants of infected or noninfected BDMP cells (Fig. 4 ).


Figure 4
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  		Figure 4. Cytokines released by BDMP. A total of 106 cells was cocultivated with 5 x 106 C. neoformans yeast cells (1:5 ratio) during 4 h and 72 h at 37°C, 5% CO2, in RPMI medium in a final volume of 1 mL. BDMP cells were cultivated without any stimulus as control. The supernatant was collected after 4 h and 72 h, and the presence of IL-10 (A) and TNF-{alpha} (B) was measured by ELISA. The values represent the mean and the SD obtained in three different experiments.

 
Conversely, we evaluated the production of NO by BDMP cells in the presence of fungus. It is interesting that we observed a basal liberation of NO by BDMP cells without stimuli, and this production was increased after 72 h of incubation. A difference in NO production when the fungus was added to the culture was not observed (Fig. 5 ).


Figure 5
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  		Figure 5. Kinetic of NO release. The phagocytosis assay (1:5 ratio) was performed in a final volume of 0.5 mL, and the supernatant was collected after 4 h, 24 h, and 72 h of culture essay. The amount of nitrites was measured using the Griess reagent. The values represent the mean and the SD obtained from three different experiments. *, P < 0.05, when compared with the amount of nitrites after 4 h and 24 h.

 
Expression of MHC-II and costimulatory molecules by C. neoformans-infected BDMP
We examined whether C. neoformans infection could influence the expression of MHC-II, CD80, CD86, and Mac-1 by BDMP. We found that surface expression of MHC-II and CD86 antigens increased significantly after infection with C. neoformans (Fig. 6 ), whereas the level of Mac-1 and CD80 expression was not modified significantly. Thus, phagocytosis of C. neoformans up-modulates the expression of MHC-II and CD86 costimulatory molecule.


Figure 6
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  		Figure 6. Flow cytometry of BDMP expression of MHC-II, CD80, CD86, and CD11b in the presence or absence of C. neoformans. The filled histograms represent fluorescence-activated cell sorter profiles after staining with corresponding isotype-matched control mAb, and the bold line represents specific fluorescence intensity. Results of one representative experiment out of four are shown. Numbers indicate the median fluorescence intensity values.

 
Viability test of C. neoformans using BDMP cells from NOS2 knockout (KO) mice
To verify the importance of NO in inducing the death of the fungus, we analyzed the fungus’ viability after phagocytosis using BDMP cells from NOS2 KO mice. We observed that cells obtained from NOS2 KO mice did not kill the yeast after 72 h of incubation. In contrast, cells from wild-type (WT) mice destroyed the yeast (Fig. 7A ). To confirm this result, we added AG, a more selective NOS2 substrate inhibitor, to C. neoformans-infected BDMP cultures from WT mice. As shown, the addition of AG blocked NO production (Fig. 7B) and concomitantly increased the number of CFU substantially (Fig. 7C) . These results indicate that NO could be the main fungicidal component of the BDMP cell. Phagocytosis, surface marker expression, and cytokine expression in NOS2 KO mice were made, and no significant difference was observed compared with WT mice (data not shown).


Figure 7
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  		Figure 7. C. neoformans killing by BDMP of NOS2 KO mice. A phagocytosis assay (1:5 ratio) was performed with BDMP cells of NOS2 KO or WT C57BL/6 mice. The numbers obtained represent the recovery of C. neoformans CFU at 24 h and 72 h of the phagocytosis assay (A). The values represent the mean obtained from three different experiments. *, P < 0.05, when compared with WT CFU after 24 h. In vitro, NOS2 inhibition by AG treatment. BDMP cells were obtained as described above and cultured for 72 h, with or without 1 mM AG in the presence of C. neoformans at a ratio of 1:5 yeast cells/BDMP. NO production represents the mean obtained from three different experiments (B). The numbers obtained represent the recovery of C. neoformans CFU at 24 h and 72 h of the phagocytosis assay (C).

 
DISCUSSION

In this study, we demonstrated that BDMP cells internalized C. neoformans, and this process results in the NO-mediated death of the yeast with production of proinflammatory cytokines, suggesting that this cell could be important in protection against this fungus.

First, we investigated whether the BDMP cell phagocyted the yeast. It was demonstrated that the BDMP cell internalized C. neoformans, and this process was complement-dependent. The C. neoformans-encapsulated pathogenic fungus provides an excellent system for studying phagocytosis, as there is practically no ingestion of yeast cells in the absence of opsonins [36 ]. As demonstrated for macrophages, the phagocytosis of C. neoformans is complement-dependent. These results suggest that the polysaccharide capsule is also antiphagocytic to the BDMP cell. Complement-mediated phagocytosis of C. neoformans by BDMP and macrophages after serum opsonization required CR3, as evidenced by the reduction of phagocytosis in the presence of antibodies against CD11b and CD18.

After that, we analyzed the fungicide capacity of these cells. It was shown that the BDMP cell killed the yeast 72 h after internalization. To prove that this result was not an artifact of the methodology, after phagocytosis, the monolayer was washed, and the number of cells was counted. Our results showed that the number of cells adhered to the substratum was the same at all periods of the experiment, confirming that the low number of CFU observed was not derived from lysed BDMP. This was quite startling, as numerous studies have presented C. neoformans as a fungal pathogen surviving phagocytosis by macrophages and proliferating in them [37 ]. This result was also confirmed in our experiments with macrophages. Thus, our results showed that BDMP cells were more fungicidal than macrophages.

Therefore, we now wonder how the BDMP cell was, unlike macrophages, able to kill C. neoformans.

Several inflammatory mediators, including IL-12, TNF-{alpha}, IFN-{gamma}, and NO, are important for the effective immune response against C. neoformans [38 ]. Aiming to obtain a better understanding of the high microbicidal capacity of BDMP cells, we determined the expression of cytokine and iNOS by BDMP cells in the presence of C. neoformans. We observed that in presence of yeast, the expression of TNF-{alpha}, IFN-{gamma}, and IL-4 by the BDMP cell is increased after 72 h. In contrast, the expression of IL-10 decreased. An early expression of these cytokines (4 h after incubation with C. neoformans) could also be seen. This result shows that the BDMP cell expressed a mixed pattern of T helper cell type 1 (Th1)/Th2 cytokines in presence of the fungus. It is interesting that our results showed the expression of TNF-{alpha} and IL-10 by BDMP cells in the absence of the fungus. When the amount of cytokine produced was determined, we observed that IL-10 was produced in high levels by the BDMP cells without stimulus. However, the BDMP cell decreased the IL-10 production in the presence of C. neoformans, in addition to the high production of TNF-{alpha}. IL-12, IFN-{gamma}, and IL-4 were not detected in the cultures. One possibility for explaining this finding may be the fact that small amounts of cytokine might not be detected by the immunoenzymatic assay used. This approach suggests that the decrease in IL-10 and the increase in TNF-{alpha} production by the BDMP cell could contribute, as an important mechanism, in eliminating C. neoformans from our organism.

We found here that C. neoformans phagocytosis by BDMP induced an up-modulation in the expression of MHC-II and CD86, showing that this cell could function as antigen-specific, professional antigen-presenting cells (APCs).

Borrello and Phipps [39 ] obtained similar results. The authors have shown that B/macrophage cells constitutively express multiple cell-surface molecules necessary for efficient T cell stimulation. These include B7-2, CD40, intercellular adhesion molecule 1, and MHC-II. With antigen-specific Igs on their surface, coupled with their phagocytic ability, B/macrophage cells could function as antigen-specific, professional APCs [40 ].

A particularly interesting aspect of BDMP cells is their possible participation in Th responses, where their singular expertise in specific binding of antigen, as well as phagocytosis and presentation of epitopes to T cells, could be useful in an environment where conventional macrophages function poorly.

Conversely, the generation of NO by iNOS (NOS2) has been involved in the antimicrobial activity of activated macrophages against a variety of intracellular pathogens, including Mycobacterium tuberculosis [41 ], Leishmania major [42 ], and Listeria monocytogenes [43 ]. In our results, we observed a basal expression of mRNA iNOS by BDMP cells in the absence of fungus, and this expression was not modified in the presence of fungus. These results, in addition to the production of cytokines, highly suggest the importance of the BDMP cell in the early immune response against C. neoformans, where this cell produces cytokines and NO independently of inflammatory stimulus.

Depending on its concentration, chemically generated NO is fungistatic or fungicidal for C. neoformans [44 ]. Furthermore, NO derived from cultured human astrocytes is fungistatic against C. neoformans [45 ]. Hence, there is considerable evidence that NO is important for host protection against C. neoformans.

To test the antimicrobial activity of NO produced by the BDMP cell, we used BDMP cells from NOS2 KO mice. Our results showed that BDMP cells from these KO mice were not able to kill phagocyted yeast cells. To confirm this observation, we treated BDMP cells obtained from WT mice with AG, and the same result was observed.

This study provided additional support for highlighting the importance of NO in the protection against C. neoformans and demonstrated, for the first time, the dependence of BDMP cells in this process.

The role that B-1 cells play in infections is still not known. Therefore, some reports about the new attributes of this cell include the participation in the modulation of this infectious disease. Marquis et al. [46 ] showed that CBA/N XID mice, which lack B-1 cells, were highly susceptible to C. neoformans infection. In short, we observed that BDMP cells could participate in the control of cryptococcosis by inhibiting pathogen growth and by inducing the production of cytokines to conduct the adaptive immune response to Th1.

ACKNOWLEDGEMENTS

This work was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP Process: 03/13876-0).

Received October 24, 2005; revised February 23, 2006; accepted February 27, 2006.

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