In vitro granuloma formation, NO production and cytokines profile from human mononuclear cells induced by fractionated antigens of Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity represents the main mode of protection against this fungal infection. We investigated in vitro the response of peripheral blood mononuclear cells (PBMC) from paracoccidioidomycosis (PCM) patients presenting different clinical forms to antigenic fractions from P. brasiliensis yeast cell lysate (PbAg). These fractions designated F0 to FV were obtained using anion-exchange chromatography on a FPLC system. Our studies showed variation in the cellular responses induced by different antigenic fractions. The fraction F0 caused significant decrease in cellular proliferation, granuloma formation, accompanied by significant elevation in the production of IL-10. The fractions FII and FIII increased in vitro granuloma formation associated with high production of TNF-alpha. Besides that, FII and FIII evoked decrease in NO production but not F0 that induced very high levels, among patients with PCM from acute form. The findings suggest that P. brasiliensis antigenic components participate in the modulation or activation of PBMC response in PCM, and IL-10 and NO could be important in the regulation of in vitro granuloma formation.

Nitric oxide interaction with IL-10, MIP-1alpha, MCP-1 and RANTES over the in vitro granuloma formation against different Schistosoma mansoni antigenic preparations on human schistosomiasis.

Nitric oxide (NO) produced by cytokine-activated macrophages is reported to be cytotoxic against the helminth Schistosoma mansoni, although this is a controversial issue. Previous work in our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to soluble egg antigen (SEA) or to SWAP. Here we report that, in comparison to other S. mansoni antigenic preparations (SEA and SWAP), supernatants of PBMC cultivated with PIII possess higher concentrations of interleukin-10 (IL-10) and macrophage inflammatory protein (MIP-1alpha), concomitantly with lower concentrations of monocyte chemoattractant protein (MCP-1) and regulated on activation, normal T expressed and secreted (RANTES). In the particular case of NO inhibition, supernatants of PBMC cultivated with PIII present decreased IL-10 levels. Altogether, these results indicate that IL-10, MIP-1alpha, MCP-1 and RANTES are distinctively important elements in the PIII modulating role, while NO seems to be pivotal in the regulation of granulomatous responses.

Antigenic stimulation is more efficient than LPS in inducing nitric oxide production by human mononuclear cells on the in vitro granuloma reaction in schistosomiasis.

Nitric oxide (NO) is an extremely important and versatile messenger in biological systems. It has been identified as a cytotoxic factor in the immune system, presenting anti- or pro-inflammatory properties under different circumstances. In murine monocytes and macrophages, stimuli by cytokines or lipopolysaccharide (LPS) are necessary for inducing the immunologic isoform of the enzyme responsible for the high-output production of NO, nitric oxide synthase (iNOS). With respect to human cells, however, LPS seems not to stimulate NO production in the same way. Addressing this issue, we demonstrate here that peripheral blood mononuclear cells (PBMC) obtained from schistosomiasis-infected patients and cultivated with parasite antigens in the in vitro granuloma (IVG) reaction produced more nitrite in the absence of LPS. Thus, LPS-induced nitrite levels are easily detectable, although lower than those detected only with antigenic stimulation. Concomitant addition of LPS and L-N-arginine methyl ester (L-NAME) restored the ability to produce detectable levels of nitrite, which had been lost with L-NAME treatment. In addition, LPS caused a mild decrease of the IVG reaction and its association with L-NAME was responsible for reversal of the L-NAME-exacerbating effect on the IVG reaction. These results show that LPS alone is not as good an NO inducer in human cells as it is in rodent cells or cell lines. Moreover, they provide evidence for interactions between LPS and NO inhibitors that require further investigation.

Antigenic stimulation is more efficient than LPS in inducing nitric oxide production by human mononuclear cells on the in vitro granuloma reaction in schistosomiasis

Nitric oxide (NO) is an extremely important and versatile messenger in biological systems. It has been identified as a cytotoxic factor in the immune system, presenting anti- or pro-inflammatory properties under different circumstances. In murine monocytes and macrophages, stimuli by cytokines or lipopolysaccharide (LPS) are necessary for inducing the immunologic isoform of the enzyme responsible for the high-output production of NO, nitric oxide synthase (iNOS). With respect to human cells, however, LPS seems not to stimulate NO production in the same way. Addressing this issue, we demonstrate here that peripheral blood mononuclear cells (PBMC) obtained from schistosomiasis-infected patients and cultivated with parasite antigens in the in vitro granuloma (IVG) reaction produced more nitrite in the absence of LPS. Thus, LPS-induced nitrite levels are easily detectable, although lower than those detected only with antigenic stimulation. Concomitant addition of LPS and L-N-arginine methyl ester (L-NAME) restored the ability to produce detectable levels of nitrite, which had been lost with L-NAME treatment. In addition, LPS caused a mild decrease of the IVG reaction and its association with L-NAME was responsible for reversal of the L-NAME-exacerbating effect on the IVG reaction. These results show that LPS alone is not as good an NO inducer in human cells as it is in rodent cells or cell lines. Moreover, they provide evidence for interactions between LPS and NO inhibitors that require further investigation.

Nitric oxide and IL-10 production induced by PIII--a fraction of Schistosoma mansoni adult worm antigenic preparation--associated with downregulation of in vitro granuloma formation.

Identification and characterization of Schistosoma mansoni antigens that can provide protective immunity, as well as an investigation of their role in host-parasite interaction, is crucial for understanding the complex immunoregulatory events that modulate granuloma formation in schistosomiasis. Previous work by our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, obtained by anionic chromatography on fast protein liquid chromatography (FPLC). This fraction was able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to SWAP or to soluble egg antigens (SEA). In the present work, we investigate some biological activities of PIII, such as the stimulation of nitric oxide (NO) and cytokine production. Our data demonstrated that SEA, SWAP and specially PIII were able to induce a time-dependent increased NO production during in vitro granuloma reaction. Besides that, PIII evoked increased IL-10 production, but not IL-2 or IFNgamma. Collectively, our results indicate the possibility that the modulation role of PIII on in vitro granuloma might be mediated in part by its ability to induce the higher production, initially of IL-10, and lately of NO.

Evidence for nitric oxide action on in vitro granuloma formation through pivotal changes in MIP-1alpha and IL-10 release in human schistosomiasis.

Nitric oxide (NO) has been implicated, both and paradoxically, as a pro- and anti-inflammatory agent in a wide range of circumstances. It is of common concern that NO can be either up- or downregulated by different inflammatory cytokines. Attempting to assess the contribution of NO to the granulomatous response, we used the in vitro granuloma (IVG) model which consists on a reaction of mononuclear cells around polyacrylamide beads conjugated to antigens. Our assays employed Schistosoma mansoni antigens and human peripheral blood mononuclear cells (PBMC) from schistosomiasis patients. Recently, we have described evidence for a regulatory role of NO, with the aid of an inhibitor of NO synthesis, L-NAME. The addition of L-NAME to IVG cultures elicited an increase on the granuloma formation index. Based on these data we decided to investigate the mechanisms involved in the effects of L-NAME-enhanced granuloma formation. Cytokines and chemokines are involved in inflammatory responses by, particularly the latter, inducing migration and adhesion of leukocytes, which led us on this search for their interactions with NO on granulomatous reaction. We evaluated the cytokine/chemokine-secreting profile of PBMC (treated and not treated with L-NAME) on the IVG reaction in order to investigate how NO could interfere on the release of these soluble mediators. Comparison of cell culture releasing amounts of IL-2, IL-10, TNFalpha, IFNgamma, MIP-1alpha, MCP-1, and RANTES demonstrated that MIP-1alpha had increased levels when NO production was blocked with L-NAME, whereas IL-10 secretion decreased in presence of L-NAME. The other tested cytokines (IL-2, TNFalpha, and IFNgamma) and chemokines (MCP-1 and RANTES) showed no significant differences between the presence or absence of L-NAME. Results obtained in this work suggest that inhibition of NO production could upregulate the IVG reaction on human schistosomiasis through changes in the cytokine/chemokine profile released by PBMC. The mechanisms involved may lead to a MIP-1alpha-increased and IL-10-decreased secretion under our experimental conditions, which could partly account for the previously ascribed IVG-exacerbating action of NO inhibition.

Phenotype and cytokine profile of Schistosoma mansoni specific T cell lines and clones derived from schistosomiasis patients with distinct clinical forms.

It is essential to distinguish the role of T lymphocytes on the physiopathology associated to more severe forms of schistosomiasis and on the immunomodulation that evolves in the majority of infected people. In this study, we generated Schistosoma mansoni-specific T cell lines and clones from patients with the acute and chronic (intestinal and hepatosplenic forms) phases of disease, from former ones, and from uninfected individuals sensitized to parasite soluble antigens. T cell lines derived from nontreated acute infected donors were capable of producing IL-4 and IL-5, while cells from treated patients secreted IFN-gamma. Lines from intestinal chronic and antigen-sensitized donors preferentially produced IFN-gamma, while those from hepatosplenic patients secreted all three cytokines. The cytokine analysis of CD4+ T cell clones revealed a Th2/Th0 pattern (clones producing IL-4 and IL-5 and clones producing all three cytokines) for those derived from infected patients, while cells from antigen-sensitized donors exhibited an opposite Th1/Th0 pattern (clones producing IFN-gamma and clones producing all three cytokines). The possible role of these T cell populations on human schistosomiasis mansoni is discussed.

Role of nitric oxide on human schistosomiasis mansoni: upregulation of in vitro granuloma formation by N omega-nitro-L-arginine methyl ester.

Over the past decade, nitric oxide has been intensely studied due to its relevance as a widespread intra- and intercellular messenger and as a cytotoxin released during several physiopathological events, including immunological reactions and inflammation. In the present paper, we investigate the effect of inhibition of NO synthesis, using an analogue of L-arginine, N omega-nitro-L-arginine methyl ester (L-NAME), on in vitro granulomatous formation of human peripheral blood mononuclear cells (PBMC) from Schistosoma mansoni-infected individuals. The results demonstrated that human PBMC are capable of in vitro NO production and that inhibition of its production through the addition of L-NAME is responsible for exacerbating granulomatous reaction. This L-NAME-induced granuloma enhancement (ranging from 30 to 65%) was measured using the granuloma index. Furthermore, we observed a general time-dependent increase in NO production during the period of cell culture (21 days) and an inverse relationship between nitrite detection and granuloma reactivity. Collectively, our results point to a possible regulatory role of NO on the development of granulomatous inflammation.

Cytokine profile associated to effector functions of human T cell clones specific for Schistosoma mansoni antigens.

T cell lines and clones specific for Schistosoma mansoni antigens were established to study cellular immunity in human schistosomiasis. Flow cytometric analysis of the clones demonstrated that all of them were of the helper inducer T cell subset (CD3+, CD4+, CD8-), and expressed the alphabeta T cell receptor, besides the IL-2 low affinity receptor CD25. Lymphokine analysis revealed that clones presented Th1, Th2, or either Th0 patterns of secretion. More interestingly, the capability of clones to induce in vitro granuloma reactions seems to be related to the presence of TNF-alpha and the absence of IL-10. In counterpart, IL-10 producer clones did not help in vitro granuloma formation, even in the presence of TNF-alpha.

Immunization with PIII, a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, affects nitric oxide production by murine spleen cells.

Nitric oxide (NO) is an important effector molecule involved in immune regulation and defense. NO produced by cytokine-activated macrophages was reported to be cytotoxic against the helminth Schistosoma mansoni. Identification and characterization of S. mansoni antigens that can provide protective immunity is crucial for understanding the complex immunoregulatory events that modulate the immune response in schistosomiasis. It is, then, essential to have available defined, purified parasite antigens. Previous work by our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to SEA (soluble egg antigen) or to SWAP. In the present work we report the effect of different in vivo trials with mice on their spleen cells ability to produce NO. We demonstrate that PIII-immunization is able to significantly increase NO production by spleen cells after in vitro stimulation with LPS. These data suggest a possible role for NO on the protective immunity induced by PIII.

IL-10 plays a role in the modulation of human granulomatous hypersensitivity against Schistosoma mansoni eggs induced by immune complexes.

It has been demonstrated that the chronic intestinal form of schistosomiasis is associated with the establishment and maintenance of a variety of immunoregulatory mechanisms that lead to a diminished granulomatous reaction to parasite eggs. Using an in vitro model of granuloma reaction we showed that immune complexes (IC) isolated from the sera of chronic intestinal schistosomiasis patients were able to reduce the granulomatous hypersensitivity (developed by peripheral blood mononuclear cells (PBMC) from schistosomiasis patients) to soluble egg antigen (SEA)-conjugated polyacrylamide beads (PB-SEA). This inhibitory activity, mediated by IC, was also observed in the proliferative response of PBMC stimulated with SEA and soluble worm antigen preparation (SWAP). Furthermore, we observed a decrease in TNF-alpha and an increase in IL-10 production by PBMC treated with IC in an in vitro granuloma reaction. This phenomenon was also seen in a cell proliferation assay when PBMC were treated with IC and stimulated with S. mansoni antigens. These results demonstrate that circulating IC may down-regulate PBMC reactivity to S. mansoni antigens by changing the cytokine pattern produced by these cells.

Human schistosomiasis: modulation of in vitro granulomatous hypersensitivity and lymphocyte proliferative response by macrophages undergoing differentiation.

The mechanisms associated with the modulation of immune response in the chronic phase of human schistosomiasis mansoni infection are complex and involve many cell types. In the present paper the authors demonstrate that antigenic stimulation of peripheral blood mononuclear cells (PBMC) from chronic-intestinal schistosomiasis mansoni patients with polyacrylamide beads (PB) conjugated to Schistosoma mansoni soluble egg antigens (PB-SEA) or adult worm antigen preparation (PB-SWAP) were able to induce a statistically significant increase on the in vitro multinucleated giant cell (MGC) formation after the 15th day in culture. A correlation between an increase in the number of MGC and a decrease in in vitro granuloma formation index to PB-SEA and PB-SWAP was observed. Moreover, the authors demonstrated a down-regulation of lymphocyte proliferative responses to S. mansoni antigens, during the differentiation pathway of monocytes towards MGC formation, due to a decrease in the antigen-presenting capacity of these cells. These phenomena also correlate with a concomitant decrease in the expression of HLA-DR and CD54 adhesion molecules on the surface of MGC. The results suggest that differentiation of monocytes to MGC may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.

Effect in vitro of gamma interferon and interleukin-10 on generation of oxidizing species by human granulocytes.

We examined the effect of interleukin-10 (IL-10), gamma interferon (IFN-gamma), phorbol ester (PDB), opsonized zymosan (OZ) and aminophylline (a cAMP phosphodiesterase inhibitor) on the reducing power and oxidizing species generation by human neutrophils, using MTT dye reduction and luminol-dependent chemiluminescence assays, respectively. Gamma interferon (IFN-gamma), phorbol ester (PDB) and opsonized zymosan (OZ) were activators while interleukin-10 (IL-10) and aminophylline were inhibitors. A strong parallelism was observed between oxidizing species generation and cellular reducing power in both activation and inhibition experiments. Our results also demonstrate for the first time the effect of IL-10 on free radical generation by neutrophils. The consequence of these activating and inhibiting effects on the inflammatory process are discussed.

Human giant cell formation induced in vitro by Schistosoma mansoni antigens.

Although multinucleated giant cells have been described for many years in association with different chronic inflammatory responses, their participation in immunoregulatory mechanisms within the schistosome egg granulomas remains to be clarified. In this study we determined if soluble egg antigen (SEA) or adult worm antigen preparations (SWAP) from S. mansoni induce giant cell formation in vitro and their relationship with the intensity of granulomatous reactivity. Antigenic stimulation of peripheral blood mononuclear cells (PBMC) from patients (N = 9) with active schistosomiasis infection increased giant cell formation per field after the 12th day in culture when treated with S. mansoni SEA conjugated to polyacrylamide beads (PB-SEA) (17 +/- 1.2) and SWAP (PB-SWAP) (18.5 +/- 1.5). The increase in the number of giant cells was statistically significant when compared to the control polyacrylamide beads (PB) (9 +/- 1.1) and purified protein derivative conjugated to beads (PB-PPD) (11.6 +/- 1.7). We also observed a correlation between an increase in the number of giant cells and a decrease in in vitro granuloma index (GI) to PB-SEA (GI decreased from 4.3 +/- 0.2 on the 6th day to 3.2 +/- 0.2 on the 12th day) and PB-SWAP (GI decreased from 4.8 +/- 0.3 on the 6th day to 3.5 +/- 0.05 on the 12th day). These data suggest that giant cell formation may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs.

Human giant cell formation induced in vitro by Schistosoma mansoni antigens

Although multinucleated giant cells have been described for many years in association with different chronic inflammatory responses, their participation in immunoregulatory mechanisms within the schistosome egg granulomas remains to be clarified. In this study we determined if soluble egg antigen (SEA) or adult worm antigen preparations (SWAP) from S. mansoni induce giant cell formation in vitro and their relationship with the intensity of granulomatous reactivity. Antigenic stimulation of peripheral blood mononuclear cells (PBMC) from patients (N = 9) with active schistosomiasis infection increased giant cell formation per field after the 12th day in culture when treated with S. mansoni SEA conjugated to polyacrylamide beads (PB-SEA) (17 +/- 1.2) and SWAP (PB-SWAP) (18.5 +/- 1.5). The increase in the number of giant cells was statistically significant when compared to the control polyacrylamide beads (PB) (9 +/- 1.1) and purified protein derivative conjugated to beads (PB-PPD) (11.6 +/- 1.7). We also observed a correlation between an increase in the number of giant cells and a decrease in in vitro granuloma index (GI) to PB-SEA (GI decreased from 4.3 +/- 0.2 on the 6th day to 3.2 +/- 0.2 on the 12th day) and PB-SWAP (GI decreased from 4.8 +/- 0.3 on the 6th day to 3.5 +/- 0.05 on the 12th day). These data suggest that giant cell formation may be one of the immunoregulatory mechanisms involved in the down-regulation of the granuloma reaction against S. mansoni eggs

Modulation of sheep erythrocyte receptor expression by anti-CD4, anti-CD8, and anti-HLA antibodies.

The modulation of E receptors on T cells by anti-CD4 and/or anti-CD8 is reported. The percentage of E rosette-forming cells (RFC) was decreased when sheep erythrocyte and mononuclear cells were incubated in the presence of monoclonal antibodies anti-CD4 and/or anti-CD8. This inhibition was not due to shedding of the E receptor and it was reversed by 8-Br.-3',5' cyclic guanosine monophosphate (cGMP). In contrast, anti-HLA antibodies induced an enhancement of sheep erythrocyte receptor expression evaluated by RFC.