Effect of cytokines on the in vitro fungicidal activity of monocytes from paracoccidioidomycosis patients

Peripheral blood monocytes obtained from paracoccidioidomycosis patients and healthy individuals were preactivated with recombinant gamma interferon (IFN-gamma) in different concentrations (250, 500 and 1000 U/ml) and evaluated for fungicidal activity against Paracoccidiodes brasiliensis strain 18 (Pb 18, high-virulence strain) and strain 265 (Pb 265, low-virulence strain) by plating of cocultures and counting of colony-forming units, after 10 d. Monocytes from healthy individuals failed to present fungicidal activity against P. brasiliensis even after IFN-gamma activation at the three concentrations. However, patient monocytes activated with IFN-gamma (1000 U/ml) showed a significant fungicidal activity when compared to that obtained with non-activated or activated cells with other IFN-gamma concentrations (250 and 500 U/ml). Moreover, patient monocytes presented higher fungicidal activity than the control, even before the activation process. These results may be explained by the activation state of patients' cells as a function of the in vivo contact with the fungus, which was confirmed by their higher capacity to release H(2)O(2) in vitro. Unlike the results obtained with Pb 18, patient and control cells presented a significant fungicidal activity against Pb 265, after priming with IFN- gamma. These results are explained by the higher levels of TNF-alpha in supernatants of cultures challenged with Pb 265. Moreover, higher levels of the cytokine were obtained in patient cell supernatants. Taken together, our results suggest that for effective killing of P. brasiliensis by monocytes, an initial activation signal induced by IFN-gamma is necessary to stimulate the cells to produce TNF-alpha. This cytokine may be involved, through an autocrine pathway, in the final phase activation process. The effectiveness of this process seems to depend on the virulence of the fungal strain and the activation state of the challenged cells.     

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for 18 hours increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism.     

Effect of interleukin-10 on the Paracoccidioides brasiliensis killing by gamma-interferon activated human neutrophils

Paracoccidioidomycosis, a deep mycosis endemic in Latin America, is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Phagocytic cells play a critical role against this fungus, and several studies have shown the effects of activator and suppressive cytokines on macrophage and monocyte functions. However, studies on polymorphonuclear neutrophils (PMNs), that are the first cells recruited to the infection sites, are scarcer. Thus, the objective of this paper was to assess whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to block the activity of IFN-gamma-activated human PMNs upon P. brasiliensis intracellular killing, in vitro. The results showed that IFN-gamma-activated PMNs have an effective fungicidal activity against the fungus. This activity was associated with the release of high levels of H(2)O(2), the metabolite involved in phagocytic cells antifungal activities. However, the concomitant incubation of these cells with IFN-gamma and IL-10 significantly blocked IFN-gamma activation. As a consequence, PMNs killing activity and H(2)O(2) release were inhibited. Together, our results show the importance of PMNs exposure to activator or suppressor cytokines in the early stages of paracoccidioidomycosis infection.     

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O??), hydrogen peroxide (H?O?), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O?? and H?O? release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection.     

Killing of <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> yeast cells by IFN-γ and TNF-α activated murine peritoneal macrophages: evidence of H<Subscript>2</Subscript>O<Subscript>2</Subscript> and NO effector mechanisms

Paracoccidioidomycosis is a deep mycosis, endemic in Latin America, caused by Paracoccidioides brasiliensis. Macrophage activation by cytokines is the major effector mechanism against this fungus. This work aimed at a better understanding of the interaction between yeast cells-murine peritoneal macrophages and the cytokine signals required for the effective killing of high virulence yeast-form of P. brasiliensis. In addition, the killing effector mechanisms dependent on the generation of reactive oxygen or nitrogen intermediates were investigated. Cell preincubation with IFN-gamma or TNF-alpha, at adequate doses, resulted in effective yeast killing as demonstrated in short-term (4-h) assays. Both, IFN-gamma and TNF-alpha activation were associated with higher levels of H(2)O(2) and NO when compared to nonactivation. Treatment with catalase (CAT), a H(2)O(2 )scavenger, and N(G)-monomethyl-L: -arginine (L: -NMMA), a nitric oxide synthase inhibitor, reverted the killing effect of activated cells. Taken together, these results suggest that both oxygen and L: -arginine-nitric oxide pathways play a role in the killing of highly virulent P. brasiliensis.     

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.     

Synergistic activation of human monocytes by granulocyte-macrophage colony-stimulating factor and IFN-gamma. Increased TNF-alpha but not IL-1 activity

TNF-alpha and IL-1 activities and PGE2 levels were investigated in the supernatants of highly purified human monocytes cultured for 18 h with recombinant human granulocyte-macrophage CSF (GM-CSF). GM-CSF alone did not stimulate IL-1 or TNF-alpha activities or the production of PGE2. GM-CSF with IFN-gamma, but not with LPS, consistently activated the monocytes for TNF-alpha activity. In contrast, for increased IL-1 activity, GM-CSF synergized weakly and irregularly with LPS, but not at all with IFN-gamma. For the third monocyte product investigated, GM-CSF was a weak and inconsistent inducer of PGE2 and only in the co-presence of IFN-gamma. Thus, GM-CSF can elicit different responses in human monocytes depending both on the co-stimulus as well as the monocyte product being investigated.     

High expression of human monocyte iNOS mRNA induced by Paracoccidioides brasiliensis is not associated with increase in NO production

In this study we investigated the role of nitric oxide (NO) in monocyte fungicidal activity against Paracoccidioides brasiliensis. We found that cells primed with IFN-γ, TNF-α or GM-CSF and challenged with a high-(Pb18) or low-virulence (Pb265) strain of the fungus increase their fungicidal activity. Expression of iNOS mRNA was increased after priming cells with each cytokine, and tended to be inhibited by Pb18. Despite up-regulation of iNOS mRNA expression by Pb265, an equivalent increase in NO production was not detected, as metabolite levels were similar in all cultures. The results indicated that high expression of human monocyte iNOS mRNA induced by P. brasiliensis is not correlated with NO concentrations produced.     

Interleukin-10 but not Transforming Growth Factor beta inhibits murine activated macrophages Paracoccidioides brasiliensis killing: Effect on H2O2 and NO production

Paracoccidioidomycosis is caused by the thermally dimorphic fungus Paracoccidioides brasiliensis (P. brasiliensis). Most often, this mycosis runs as a chronic progressive course affecting preferentially the lungs. In vitro fungicidal activity against a high virulent strain of P. brasiliensis by murine peritoneal macrophages preactivated with IFN-γ or TNF-α is high and correlates with increased NO and H₂O₂ production. Within this context, the purpose of this work was to study the role of suppressor cytokines, such as IL-10 and TGF-β, in this process. Incubation of either IFN-γ or TNF-α with IL-10 inhibits fungicidal activity of these cells. However, TGF-β had no effect on fungicidal activity of IFN-γ or TNF-α-activated macrophages. The suppression of fungicidal activity by IL-10 correlated with the inhibition of NO and H₂O₂ production supporting the involvement of these metabolites in P. brasiliensis killing. These results suggest that IL-10 production in vivo could represent an evasion mechanism of the fungus to avoid host immune response.     

Granulocyte macrophage colony-stimulating factor enhances the modulatory effect of cytokines on monocyte-derived multinucleated giant cell formation and fungicidal activity against Paracoccidioides brasiliensis

Multinucleated giant cells (MGC) are cells present in characteristic granulomatous inflammation induced by intracellular infectious agents or foreign materials. The present study evaluated the modulatory effect of granulocyte macrophage colony-stimulating factor (GM-CSF) in association with other cytokines such as interferon-gamma (IFN-γ), tumour necrosis factor-alpha, interleukin (IL)-10 or transforming growth factor beta (TGF-β1) on the formation of MGC from human peripheral blood monocytes stimulated with Paracoccidioides brasiliensis antigen (PbAg). The generation of MGC was determined by fusion index (FI) and the fungicidal activity of these cells was evaluated after 4 h of MGC co-cultured with viable yeast cells of P. brasiliensis strain 18 (Pb18). The results showed that monocytes incubated with PbAg and GM-CSF plus IFN-γ had a significantly higher FI than in all the other cultures, while the addition of IL-10 or TGF-β1 had a suppressive effect on MGC generation. Monocytes incubated with both pro and anti-inflammatory cytokines had a higher induction of foreign body-type MGC rather than Langhans-type MGC. MGC stimulated with PbAg and GM-CSF in association with the other cytokines had increased fungicidal activity and the presence of GM-CSF also partially inhibited the suppressive effects of IL-10 and TGF-β1. Together, these results suggest that GM-CSF is a positive modulator of PbAg-stimulated MGC generation and on the fungicidal activity against Pb18.     

Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

Involvement of prostaglandins in the immunosuppression occurring during experimental infection by Paracoccidioides brasiliensis

We investigated whether PGE2 mediates the immunosuppression observed during Paracoccidioides brasilensis infection. Con-A-stimulated splenocytes, isolated from mice on days 15 and 60 of infection, release high amounts of PGE2, this release was inhibited by the treatment of animals with indomethacin, sodium salicylate or meloxicam. The treatment of the animals with salicylate or meloxicam, but not indomethacin, enhanced the release of IL-2 by splenocytes from animals on day 15, but not on day 60 of infection. Furthermore, we demonstrated that the productions of TNF-alpha, IFN-gamma, IL-4, and IL-10 by Con-A-stimulated splenocytes from mice at 15 days of infection were inhibited by treatment with salicylate or meloxicam. Indomethacin inhibited only TNF-alpha and IFN-gamma production. The three treatments caused reduction of granuloma areas in the liver and lungs of infected mice. In conclusion, results suggest that the PGE2 released by COX-2 mediates the immunosuppression early on (day 15), but not during the later phase (60 days) of P. brasiliensis infection by a mechanism dependent upon IL-4 and IL-10.     

Role of TNF-alpha in the induction of fungicidal activity of mouse peritoneal exudate cells against Cryptococcus neoformans by IL-12 and IL-18

We have recently demonstrated that two IFN-gamma-inducing cytokines, interleukin (IL)-12 and IL-18, synergistically induced the fungicidal activity of mouse peritoneal exudate cells (PEC) against Cryptococcus neoformans through NK cell production of interferon (IFN)-gamma and nitric oxide (NO) synthesis. In the present study, we further dissected these effects by examining the involvement of tumor necrosis factor (TNF)-alpha in the induction of IL-12/IL-18-stimulated PEC fungicidal activity. The addition of neutralizing anti-TNF-alpha mAb significantly suppressed IL-12/IL-18-stimulated PEC anticryptococcal activity. This effect was ascribed to the inhibition of macrophage NO synthesis, but not of IFN-gamma production by NK cells, because the same treatment inhibited the former response, but not the latter one. On the other hand, combined treatment with IL-12 and IL-18 synergistically induced the production of TNF-alpha by PEC and this effect was almost completely abrogated by neutralizing anti-IFN-gamma mAb. The cell type producing TNF-alpha among PEC was mostly macrophage. TNF-alpha significantly promoted macrophage NO production and anticryptococcal activity induced by IFN-gamma, and furthermore anti-TNF-alpha mAb partially inhibited these responses. Considered together, our results indicated that TNF-alpha contributed to the potentiation of IL-12/IL-18-induced PEC fungicidal activity against C. neoformans through enhancement of IFN-gamma-induced production of NO by macrophages, but not through increased production of IFN-gamma by NK cells.     

Selective modulation of BV-2 microglial activation by prostaglandin E(2). Differential effects on endotoxin-stimulated cytokine induction.

The influence of prostaglandins on glial functions and, more specifically, on glial activation is not well understood. We report here that prostaglandin E(2) (PGE(2)), one of the major prostaglandins produced in the brain, acts as a potent and selective inhibitor of tumor necrosis factor alpha (TNF-alpha) production in lipopolysaccharide-stimulated primary microglia and the microglial cell line BV-2. The IC(50) for this effect is 1 nM, and 100 nM PGE(2) suppresses TNF-alpha production by >95%. More detailed studies of BV-2 cells show that PGE(2) also prevents the secretion of interleukin (IL)-6 but does not significantly modify lipopolysaccharide-stimulated expression of cyclooxygenase-2, pro-IL-1beta, or inducible nitric oxide synthase. PGE(2) appears to act primarily at the level of translation or protein stability, because TNF-alpha and IL-6 mRNA levels were only modestly decreased at high PGE(2) concentrations; concomitantly with this inhibition, PGE(2) up-regulated the levels of IL-1beta mRNA. The effects of PGE(2) could be largely mimicked by 8-bromo-cAMP, suggesting that, as in other cell types, PGE(2) action is mediated at least in part by a rise in intracellular cyclic AMP. However, the protein kinase A inhibitor H89 only partially reversed the inhibition of TNF-alpha production by PGE(2), implying that the PGE(2) effect in BV-2 cells is mediated through both protein kinase A-dependent and -independent pathways.     

Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56(+)granulysin(+) cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.     

Human alveolar macrophages and granulocyte-macrophage colony-stimulating factor-induced monocyte-derived macrophages are resistant to H2O2 via their high basal and inducible levels of catalase activity

Human alveolar macrophages (A-MPhi) and macrophages (MPhi) generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factors (GM-MPhi) express high levels of catalase activity and are highly resistant to H(2)O(2). In contrast, MPhi generated from monocytes by macrophage colony-stimulating factors (M-MPhi) express low catalase activity and are about 50-fold more sensitive to H(2)O(2) than GM-MPhi or A-MPhi. Both A-MPhi and GM-MPhi but not M-MPhi can induce catalase expression in both protein and mRNA levels when stimulated with H(2)O(2) or zymosan. M-MPhi but not GM-MPhi produce a large amount of H(2)O(2) in response to zymosan or heat-killed Staphylococcus aureus. These findings indicate that GM-MPhi and A-MPhi but not M-MPhi are strong scavengers of H(2)O(2) via the high basal level of catalase activity and a marked ability of catalase induction and that catalase activity of MPhi is regulated by colony-stimulating factors during differentiation.     

Effects of TGF-beta, TNF-alpha, IL-beta and IL-6 alone or in combination, and tyrosine kinase inhibitor on cyclooxygenase expression, prostaglandin E2 production and bone resorption in mouse calvarial bone cells

Cyclooxygenase-2 (COX-2) and tyrosine kinase, which are involved in the biosynthesis of prostaglandin E(2) (PGE(2)) in mouse calvarial osteoblasts, are stimulated by cytokine interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-6 (IL-6). IL-1beta and IL-6 and, to a lesser extent, TNF-alpha, enhances COX-2 mRNA levels in calvarial osteoblasts. Simultaneous treatment with IL-6 and IL-1beta and TNF-alpha resulted in enhanced COX-2 mRNA levels accompanied by the cooperative stimulation of PGE(2) biosynthesis compared to cells treated with IL-1beta or TNF-alpha or IL-6 alone. In contrast, the presence of TGF-beta reduced COX-2 mRNA level, PGE(2) biosynthesis and bone resorption induced by IL-1beta, TNF-alpha, IL-6 or a combination thereof. However, neither IL-1beta, TNF-alpha, IL-6 nor a combination of IL-1beta, TNF-alpha, IL-6 enhanced COX-1 mRNA levels in calvarial osteoblasts. A novel Src tyrosine kinase inhibitor, Herbimycin A (HERB), reduced COX-2 mRNA levels as well as PGE(2) production induced by IL-1beta, TNF-alpha and IL-6 or a combination of IL-1beta, TNF-alpha, IL-6, whereas COX-1 mRNA levels remained unaffected. Finally, HERB was found to inhibit in vitro bone resorption. These results indicate that the cooperative effects of IL-beta, TNF-alpha, IL-6 on PGE(2) production are due to the enhanced expression of the COX-2 gene and that tyrosine kinase(s) are involved in COX-2 signal transduction in mouse calvarial osteoblasts. Thus, the Src family of kinase inhibitors may be useful in treating diseases associated with elevated bone loss.