Leishmania braziliensis amastigotes stimulate production of IL-1ß, IL-6, IL-10 and TGF-ß by peripheral blood mononuclear cells from nonendemic area healthy residents.

Leishmania (Viannia) braziliensis causes cutaneous and mucosal leishmaniasis in several countries in Latin America. In mammals, the parasites live as amastigotes, interacting with host immune cells and stimulating cytokine production that will drive the type of the specific immune responses. Generation of Th17 lymphocytes is associated with tissue destruction and depends on IL-1ß, IL-6, TGF-ß and IL-23 production, whereas IL-10 and TGF-ß are associated with tissue protection. Here, we evaluate whether amastigotes stimulate peripheral blood mononuclear cells (PBMCs) from healthy donors to produce the major cytokines responsible for the generation of Th17. Seven L. (V.) braziliensis isolates from patients with different clinical forms of leishmaniasis were expanded in interferon-γ knockout mice to obtain amastigotes and in culture to get promastigotes. The parasites were used to stimulate PBMCs from healthy donors, and cytokine production was evaluated by ELISA or qPCR. Amastigotes and promastigotes induced IL-10 production in PBMCs; however, only amastigotes induced IL-1ß, IL-6 and TGF-ß. These data demonstrate for the first time that L. (V.) braziliensis amastigotes directly stimulate production of a unique pattern of cytokines that could contribute to the generation of Th17.

Leishmania (Leishmania) amazonensis infection and dissemination in mice inoculated with stationary-phase or with purified metacyclic promastigotes.

Leishmania (Leishmania) amazonensis is a protozoan of the American Continent that causes localized cutaneous leishmaniasis and, rarely, the diffuse cutaneous form of disease in humans. It has become clear in recent years that the course of Leishmania major infection in the mouse model differs when low numbers of purified metacyclic forms are used as inocula in comparison with the traditionally hitherto studied infection models that used large numbers of stationary-phase (SP) promastigotes. The low-number metacyclic inocula are thought to reproduce more closely the natural infection transmitted by the vector. In the present study the course of L. amazonensis infection, its local and distant dissemination patterns, and parasite load were compared in susceptible BALB/c and relatively resistant C57BL/6 mice infected in the footpad with inocula of 107 SP-promastigotes or with 104 purified metacyclic forms. Longer lag-phases were observed for infection with purified metacyclics but the characteristic patterns of disease susceptibility and cytokine production for either mouse strain were similar to those observed for SP-promastigote inocula. An inoculation dose of the order of 104 metacyclics was required to obtain consistent infections; 10- or 100-fold lower doses resulted in variable infection rates. Characteristically, L. amazonensis infection spread to distant organs and persisted there also in the relatively resistant C57BL/6 mice examined after 6 months of infection.

Host and bacterial factors affecting induction of immune responses to flagellin expressed by attenuated Salmonella vaccine strains.

Previous observations demonstrated that the delivery of recombinant Salmonella enterica serovar Dublin strains to mice via mucosal routes did not efficiently activate systemic and secreted antibody responses to either type d flagellin or genetically fused heterologous B-cell epitopes, thus reducing the usefulness of the protein as a carrier of epitopes for vaccine purposes. In this work, we investigated murine systemic and mucosal flagellin immunogenicity after oral immunization with attenuated Salmonella strains. The reduced anti-type d flagellin antibody responses in mice immunized via mucosal routes with three doses of flagellated S. enterica serovar Dublin strains were not caused by oral tolerance and could not be restored by coadministration of a mucosal adjuvant. The induction of antibody responses to Salmonella flagellins was shown to differ according to the genetic background, but not the haplotype, of the mouse lineage. Moreover, BALB/c mice orally immunized with S. enterica serovar Typhimurium strains developed anti-type i flagellin sera and secreted antibody responses, which indicated that the serovar of the Salmonella vaccine strain also affected flagellin immunogenicity. Analyses of cytokine responses of BALB/c mice immunized with three oral doses of flagellated S. enterica serovar Dublin vaccine strains showed that, in spite of the lack of antibody responses, elevated type d flagellin-specific CD4-cell-activation-dependent gamma interferon (IFN-gamma) and interleukin-10 responses were elicited after the administration of the vaccine strains via either parenteral or mucosal routes. Similar cytokine production patterns were detected to a T-cell heterologous epitope, derived from the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC), in mice orally immunized with a Salmonella vaccine strain expressing hybrid flagella. These results indicate that the immunogenicities of Salmonella flagellins can differ significantly, depending on the murine host and on the bacterial vector used, and demonstrate that the induction of CD4-cell-activation-dependent IFN-gamma production represents a major immune response triggered by flagellin and in-frame fused heterologous T-cell epitopes after the oral administration of recombinant S. enterica serovar Dublin vaccine strains.

Immature macrophages derived from mouse bone marrow produce large amounts of IL-12p40 after LPS stimulation.

Production of IL-12 is an important indicator of the macrophage's ability to regulate immune responses. In this study, we investigated the IL-12 production by macrophages in different developmental stages. To this end, macrophages were generated in vitro from precursors stimulated with M-CSF, GM-CSF or IL-3. Density separation yielded populations enriched in different maturation stages. Invariably, only cells banding at the 40-50% Percoll interface produced large amounts of IL-12p40 when stimulated with LPS, whereas only low levels of IL-12p70 were produced. These cells represented immature macrophages, as indicated by the absence of precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 and ER-MP58, and by the low level of expression of mature-cell markers like ER-HR3, scavenger receptor and CD11b/Mac-1. Upon further maturation, the macrophages' ability to produce IL-12p40 decreased, coinciding with increased nitric oxide production upon LPS stimulation. These results show that immature macrophages produce high levels of IL-12p40 and thus may either contribute to IL-12p70 production or regulate it.

Modulation of delayed-type hypersensitivity during the time course of immune response to a protein antigen.

Delayed-type hypersensitivity reactions elicited in the footpad of ovalbumin-sensitized mice after challenge with aggregated ovalbumin on day 4 or 8 of immunization are distinct. The former was characterized by a dense mononuclear infiltrate and, macroscopically, the reaction peaked at 48 hr after antigen challenge; the latter was preceded by immediate-type reactions, reached the maximum at 24 hr and faded drastically later. Histologically, oedema and a mixed granulocytic-lymphocytic infiltrate was found at this time-point. Immunoglobulin G1 (IgG1), IgG2a and IgE antibodies were detected only in plasma obtained after 8 days of immunization. Regarding the cytokines produced by draining lymph node cells after in vitro restimulation, interleukin-4 (IL-4) and IL-10 were predominant after 4 days and interferon-gamma and IL-2 after 8 days of immunization. These two types of delayed-type hypersensitivity (DTH) were used to study the influence of antibody-mediated responses on the inductive and effector phases of cell-mediated immunity. The effector phase of DTH was not affected by immediate-type reactions, as abrogation of these reactions by mediators' antagonists on day 8 or induction of passive reactions by transfer of immune serum on day 4 did not change the extent or kinetics of either type of DTH. Only transfer, before immunization, of whole or T-cell-enriched spleen cells, but not sera, from hyperimmunized donors (high antibody producers) abolished the induction of pure DTH in 4-day immunized recipient mice and changed their cytokine profile to a T helper 2 type. These results indicate that in a non-polarized immune response to a protein antigen there is initially a bias towards cell-mediated immunity, which is gradually dampened by the development of antibody-mediated immunity.

Most parasite-specific CD8+ cells in Trypanosoma cruzi-infected chronic mice are down-regulated for T-cell receptor-alphabeta and CD8 molecules.

The present study shows that CD8+ T lymphocytes expressing low levels of T-cell receptor (TCR)alphabeta, CD8 and CD3 accumulate in the spleen, blood, peritoneum and liver, but not in the lymph nodes of mice chronically infected with Trypanosoma cruzi. Analysis of spleen lymphocytes reveals that most CD8LOW TCRLOW T cells have an experienced phenotype (CD44HIGH CD62LLOW and CD45RA,B,CLOW). These cells have small size, lack activation markers such as CD69, CD25 and CD11b (Mac-1), and do not spontaneously secrete cytokines, suggesting they are at the resting state. When stimulated in vitro with T. cruzi-infected macrophages, TCRLOW CD8LOW T cells behave as parasite-specific memory cells, readily responding with interferon-gamma (IFN-gamma) production. Indeed, among parasite-activated CD8+ lymphocytes, IFN-gamma production was mostly due to TCRLOW CD8LOW cells. Upon in vitro stimulation with anti-CD3/CD28 monoclonal antibodies, down-regulated cells produce IFN-gamma and tumour necrosis factor-alpha, but not interleukin IL-10 or IL-4. Our results indicate that despite parasite persistence, most T. cruzi-specific experienced CD8+ cells are resting. Nevertheless, when encountering infected macrophages these cells differentiate to Tc1 effectors.

Effects of interleukin-4 deprivation and treatment on resistance to Trypanosoma cruzi.

Trypanosoma cruzi (Y strain)-infected interleukin-4(-/-) (IL-4(-/-)) mice of strains 129/J, BALB/c, and C57BL/6 showed no significant difference in parasitemia levels or end point mortality rates compared to wild-type (WT) mice. Higher production of gamma interferon (IFN-gamma) by parasite antigen (Ag)-stimulated splenocytes was observed only for C57BL/6 IL-4(-/-) mice. Treatment of 129/J WT mice with recombinant IL-4 (rIL-4), rIL-10, anti-IL-4, and/or anti-IL-10 monoclonal antibodies (MAbs) did not modify parasitism. However, WT mice treated with rIL-4 and rIL-10 had markedly increased parasitism and suppressed IFN-gamma synthesis by spleen cells stimulated with parasite Ag, concanavalin A, or anti-CD3. Addition of anti-IL-4 MAbs to splenocyte cultures from infected WT 129/J, BALB/c, or C57BL/6 mice failed to modify IFN-gamma synthesis levels; in contrast, IL-10 neutralization increased IFN-gamma production and addition of rIL-4 and/or rIL-10 diminished IFN-gamma synthesis. We conclude that endogenous IL-4 is not a major determinant of susceptibility to Y strain T. cruzi infection but that IL-4 can, in association with IL-10, modulate IFN-gamma production and resistance.

Cutting edge: mouse IgG1 antibodies comprise two functionally distinct types that are differentially regulated by IL-4 and IL-12.

IL-4-dependent and -independent IgG1 Abs differ in their ability to induce mast cell degranulation as measured by passive cutaneous anaphylaxis (PCA). Mice immunized with OVA or PIII (fraction of Ascaris suum) produced high titers of IgG1 as shown by ELISA and PCA. In contrast, another A. suum fraction, PI, elicited IgG1 Abs with no PCA activity. IgG1 with anaphylactic activity required IL-4, as IgG1 responses to OVA and PIII in IL-4-/- mice gave no PCA. PI-specific IgG1 was IL-4-independent, because no difference was found between the responses of IL-4-/- and IL-4+/+ mice. Significant PCA reactions were elicited, however, with PI-specific IgG1 from IL-12-/- or anti-IFN-gamma Ab-treated mice, although less Ab was measured by ELISA. These results indicate that one type of IgG1 has anaphylactic activity and its synthesis is IL-4-dependent, being inhibited by IL-12 or IFN-gamma; the other lacks this activity and its synthesis is stimulated by IL-12 or IFN-gamma.

Interference of natural mouse hepatitis virus infection with cytokine production and susceptibility to Trypanosoma cruzi.

Mouse hepatitis virus (MHV) infection can have a pronounced impact on several investigation areas. Reports on natural MHV outbreaks are rare and most studies have been conducted by deliberately infecting mice with MHV laboratory strains that cause moderate to severe disturbances to the immune system. We have investigated the effects of a natural acute outbreak of MHV in our otherwise specific-pathogen-free (SPF) inbred mouse colonies, and of enzootic chronic MHV infection on cytokine production and resistance to the intracellular pathogen Trypanosoma cruzi. We found that BALB/c and/or C57BL/6 SPF mice that had been injected with T. cruzi blood trypomastigotes from recently MHV-contaminated (MHV+) mice developed significantly higher parasite blood counts, accelerated death, and showed higher IL-10 production by spleen cells than their counterparts whose T. cruzi inoculum was derived from MHV-negative (MHV-) donors. Interferon-gamma (IFN-gamma) production by MHV+ and MHV- mice was not significantly different. In contrast, T. cruzi infection of chronically MHV-infected mice did not result in major changes in the course of infection when compared with that observed in mice from MHV- colonies, although a trend to higher parasitaemia levels was observed in BALB/c MHV+ mice. Nevertheless, both BALB/c and C57BL/6 T. cruzi-infected MHV+ mice had diminished IFN-gamma production to parasite-antigen stimulation in comparison with similarly infected MHV- mice. Interleukin-10 (IL-10) production levels by spleen cells did not differ between chronic MHV+ and MHV- mice, but IFN-gamma neutralization by monoclonal antibody treatment of anti-CD3-stimulated spleen cell cultures showed higher levels of IL-10 synthesis in MHV+ BALB/c mice.

Prostaglandins mediate suppression of lymphocyte proliferation and cytokine synthesis in acute Trypanosoma cruzi infection.

Suppression of host lymphoproliferative responses to mitogens and Ag is characteristically seen during acute infection with the protozoan parasite Trypanosoma cruzi. We investigated the reciprocal regulation of prostaglandins (PG), TNF-alpha, and nitric oxide (NO) production and their effects on cytokine production and lymphoproliferative responses to parasite Ag and to Con A by spleen cells (SC) from T.-cruzi-infected mice. Large amounts of PGE2, TNF-alpha, and NO were produced during infection. TNF-alpha stimulated PG and NO synthesis, while both mediators inhibited TNF-alpha synthesis. Blocking PG also reduced NO synthesis indicating that PG stimulate NO production. Treatment with indomethacin or NMLA stimulated lymphoproliferation on days 6 and 22 of infection; on day 14, when suppression of proliferation and NO production was maximal, combined inhibition of NO and PG production restored parasite Ag specific and Con A proliferative responses. Blocking PG or NO production increased IL-2, IFN-gamma, and TNF-alpha, but not IL-12 production by SC; IL-10 levels were not reduced. Indomethacin-treated infected mice had higher mortality compared to untreated infected animals. The data indicate that PG, together with NO and TNF-alpha, participate in a complex circuit that controls lymphoproliferative and cytokine responses in T. cruzi infection.

The role of polymorphonuclear leukocytes in the resistance to cutaneous Leishmaniasis.

The massive infiltration by polymorphonuclear leukocytes (PMN) soon after skin infection with Leishmania major suggests that PMN could participate in reducing parasite load and controlling the spreading of leishmanial infection. Yet, direct evidence for the participation of PMN in host defense against L. major was lacking. We investigated L. major infection in susceptible and resistant mice treated with the monoclonal (mAb) antibody RB6-8C5 that depletes the population of mature neutrophils and eosinophils. Both BALB/c and C57BL/6 mice depleted of PMN show accelerated parasite spreading and more severe footpad swelling than similarly infected untreated mice. In addition, significant higher parasite numbers were found in the lesion draining lymph nodes from PMN-depleted C57BL/6 mice. Histopathological analysis of the paw confirmed neutrophils containing ingested parasites as the dominant cell type in the infiltrate of the first days after infection and the nearly absolute neutrophil depletion in mAb-treated mice. Our data show the importance of PMN in early control of parasite load and parasitism spreading in cutaneous leishmaniasis.

Activation of CD4+ and CD8+ parasite -specific T-cells by macrophages infected with live T. cruzi amastigotes.

T. cruzi-infected macrophages are potential candidates for the presentation of parasite antigens to T. cruzi-specific T lymphocytes. To assess this question, we examine the ability of peritoneal exudate macrophages to process exogenous live or dead parasites and to activate defined populations of T. cruzi-specific immune T-cells. Macrophages infected with live amastigotes activated both lymph node CD4+ and spleen CD8 + T-primed cells that proliferated and secreted cytokines. Lymph node CD4+ T-cells produced IFN-gamma and IL-10 while CD8 + T-cells produced IFN-gamma. In contrast, macrophages pulsed with dead parasites activated only lymph node CD4+ T-cells, which proliferated and secreted IFN-gamma. Interestingly, the immunization with heat-killed parasites primed mice for CD8+ T-cells which were expanded in vitro by recognition of infected macrophages. Taken together, these results demonstrated that amastigote infected macrophages present parasite peptides associated with MHC I and II molecules, activating both CD4 + and CD8+ T-cells. Furthermore, the development of T. cruzi-specific CD8+ T-cells in vivo using the immunization protocol with non-living parasites as described in this report could be explored for further studies on the role of CTL in the outcome of infection.

Cytokines in innate and acquired immunity to Trypanosoma cruzi infection.

Resistance to Trypanosoma cruzi infections is critically dependent on cytokine-mediated activation of cell-mediated immune effector mechanisms. This review focuses on the role of IL-10, TNF-alpha, IFN-gamma and IL-12 in controlling T. cruzi replication by the innate and specific immune systems of the vertebrate host. A study performed on mice with disrupted recombinase-activating genes (RAG/KO), which lack T and B lymphocytes, revealed the importance of IL-12, IFN-gamma and TNF-alpha in the resistance against T. cruzi mediated by the innate immune system. In addition, data from experiments using IL-10 KO, RAG/KO and double RAG/IL-10 KO mice indicating an in vivo regulatory role of IL-10 in innate and T. cruzi-specific immunity are discussed.

Susceptibility and resistance to Leishmania amazonensis in H-2q syngeneic high and low antibody responder mice (Biozzi mice).

H-2 syngeneic H and L (Biozzi) mice provide a model to study Leishmania infections in which polar resistant and susceptible phenotypes are independent from H-2 differences. High-Ab-responder (H) and low-Ab-responder (L) mice syngeneic at the H-2 locus (H-2q) were, respectively, susceptible and highly resistant to Leishmania amazonensis infection. L-mice resistance was associated with high IFN-gamma and transient IL-4 production by lymph node (LN) cells, in contrast with sustained IL-4 and decreasing IFN-gamma production by susceptible H mice. IL-12 production could be detected only in LN from resistant mice. The cytokine production pattern was consistent with preferential progression to a Th1-type response in resistant L-mice, and to a Th2-type response in susceptible H-mice. We also investigated whether this shift towards Th1- or Th2-type cytokine responses was dependent upon H or L antigen presenting cells' (APC) intrinsic ability to preferentially stimulate either T-cell subset. To this end, LN-derived T-cell lines were grown from 12-day infected mice, when both strains produced IFN-gamma and IL-4. L-derived T-cell lines developed a Th2 cytokine pattern whereas H-derived T-cell lines produced IFN-gamma, IL-4 and IL-10 whatever the APC origin (H or L) used for their derivation. This work constitutes the first characterization of cellular immune responses to the intracellular parasite, L. amazonensis in H-2 syngeneic mice, an infection model in which polar resistant and susceptible phenotypes are determined by non-MHC genes.

Immunomodulation induced by Ascaris suum extract in mice: effect of anti-interleukin-4 and anti-interleukin-10 antibodies.

Simultaneous immunization of mice with an Ascaris suum extract (Asc) and ovalbumin (OA) markedly affects the immune response to OA. The role of interleukin (IL)-4 and IL-10 induced by Asc immunization on the modulation of antigen-specific and mitogen-induced responses was investigated following single or combined cytokine-specific monoclonal antibody (MoAb) treatment of mice before immunization with OA + Asc. Immediate hypersensitivity reactions to aggregated OA and OA-specific immunoglobulin (Ig)G2a antibody production were completely restored only when both IL-4 and IL-10 were neutralized. These findings were associated with enhanced interferon (IFN)-gamma secretion by OA-stimulated lymph node (LN) cells. In addition, the Asc-specific cytokine response in anti-IL-4 plus anti-IL-10 MoAb treated mice was shifted towards a Th1 phenotype, with an increase in IFN-gamma and IL-2 levels and a decrease in IL-4, but not in IL-10, levels. Consequently, Asc-specific IgG2a antibody production increased, whereas IgE titres diminished in these animals. These results indicate that IL-4 and IL-10 act together in the Asc-induced mechanism of antigen-specific pansuppression. In contrast, modulation of Concanavalin A (Con A)-induced cytokine responses in Asc-immunized mice appears to be essentially mediated by an IL-4-dependent mechanism, since the neutralization of just IL-4 (and not of IL-10), either in vivo or in vitro, changed the cytokine profile from a Th2 towards a Th1 type. However, OA and Asc-specific cell responses were not modified by either anti-IL-4 or by anti-IL-4 + anti-IL-10 MoAbs in vitro treatments, suggesting that the induction of a Th2 response to Asc components concomitant to OA immunization has a strong suppressive effect on the priming stage of OA-specific Th1 type response.

Cytokines in innate and acquired immunity to Trypanosoma cruzi infection

Reistance to Trypanosoma cruzi infections is critically dependent on cytokine-mediated activation of cell-mediated immune effector mechanisms. This review focuses on the role of IL-10, TNF-alpha, IFN-gamma and IL-12 in controlling T. cruzi replication by the innate and specific immune systems of the vertebrate host. A study performed on mice with disrupted recombinase-activating genes (RAG/KO), which lack T and B lymphocytes, revealed the importance of IL-12, IFN-gamma and TNF-alpha in the resistance against T. cruzi mediated by the innate immune system. In addition, data from experiments using IL-10 KO, RAG/KO and double RAG/IL-10 KO mice indicating an in vivo regulatory role of IL-10 in innate and T. cruzi-specific immunity are discussed.

IL-12 enhances proliferation of peripheral blood mononuclear cells from Chagas' disease patients to Trypanosoma cruzi antigen.

Chagas' disease is caused by infection with Trypanosoma cruzi. Patients in the chronic phase of infection were grouped as belonging to the asymptomatic (or indeterminate), cardiac and cardiac plus digestive forms. Previous studies have described abnormal immune responsiveness by peripheral blood mononuclear cells (PBMC) from chronic chagasic patients. We report significant parasite antigen (T-Ag)-stimulated PBMC proliferative responses to be present in all three groups of patients. Treatment of T-Ag-stimulated cultures with rIL-12 significantly amplifies proliferative responses in all patients' groups, with similar rates of increment. IL-12 enhances T-Ag-specific lymphoproliferation without increasing proliferation of unstimulated PBMC from normal individuals or from patients. Comparatively, treatment with rIL-2 enhances both T-Ag-specific and unstimulated proliferation by PBMC from patients and normals. Thus, IL-12 acts on pre-activated cells while IL-2 also stimulates resting cells. No synergism was obtained by the combined use of IL-12 and IL-2. Therefore IL-12 can act as a more selective amplifier of T. cruzi reactive cells than IL-2. IL-12, by enhancing parasite-antigen specific immunity, could be of potential therapeutic use to control reactivated T. cruzi infections concomitant to AIDS or other situations of immunosuppression.

Trypanosoma cruzi: IL-10, TNF, IFN-gamma, and IL-12 regulate innate and acquired immunity to infection.

Control of the acute phase of Trypanosoma cruzi infections is critically dependent on cytokine-mediated macrophage activation to intracellular killing. We investigated the roles of IL-10, TNF, IFN-gamma, and IL-12 in the control of parasitism by innate and specific immunity. Mice with disrupted IL-10 genes (IL-10 KO) infected with Y strain T. cruzi have lower parasite numbers in the blood and tissues and higher IFN-gamma and nitric oxide (NO) production by spleen cells than wild type (WT) mice. Treatment of IL-10 KO and WT mice with recombinant IL-10 resulted in increased parasitemia. Mice with disrupted recombinase-activating genes (RAG/KO) that lack B and T cells provided a model for determining the importance of innate immunity to resistance. RAG/KO and WT mice had similar parasitemia levels until Day 13 of infection, suggestive of effective control of parasitism by the innate immune system during the early phase of infection; from them on parasitemia was higher in RAG/KO. Double RAG/IL-10 KO mice and RAG/KO mice had superimposable parasitemia curves, indicating that in the absence of T and B cells, endogenous IL-10 does not limit the efficacy of the innate immune system. Treatment of infected RAG/KO, IL-10/KO, and WT mice with anti-IFN-gamma, anti-TNF, or anti-IL-12 neutralizing mAbs increased parasitemia levels showing the importance of endogenous production of these cytokines in the control of parasitism by innate and specific immune responses. Spleen cells from anti-IL 12-treated WT mice had diminished production of IFN-gamma and NO, suggesting that early IFN-gamma synthesis is most dependent on IL-12 stimulation.

Saponin adjuvant primes for a dominant interleukin-10 production to ovalbumin and to Trypanosoma cruzi antigen.

The adjuvant activity of saponin for T-cell responses was evaluated and compared with that of complete Freund's adjuvant (CFA) in two antigen systems: a lysate of the protozoa Trypanosoma cruzi and ovalbumin (OA). Strong delayed-type hypersensitivity and T-cell proliferate responses, comparable with those stimulated by CFA, were observed for both antigens following immunization with saponin as adjuvant. Upon in vitro secondary antigen stimulation, high interleukin-10 (IL-10) and low interferon-gamma (IFN-gamma) levels were observed in lymph node (LN) cell cultures from saponin-immunized mice in contrast with the high IFN-gamma and decreased IL-10 production by LN cells from CFA-immunized mice. Production of IL-10 and IFN-gamma in these conditions was CD4-activation dependent. Concanavalin A (Con A)-stimulated interleukin-4 (IL-4) production was higher in saponin-immunized mice than in CFA-immunized mice. IL-10 produced by LN cells from saponin-immunized mice suppressed IFN-gamma production and Con A-induced proliferation. Taken together, these data are consistent with in vivo stimulation of both T-helper (Th)1 and Th2-type cells by immunization with saponin; in vitro a Th2-type cytokine response with high IL-10 production predominates, indicating preferential priming towards a Th2-type response. Immunization with CFA induced a Th1-type cytokine response. To our knowledge this is the first report in which an adjuvant is shown to prime for a dominant IL-10 production.

Cytokine and nitric oxide regulation of the immunosuppression in Trypanosoma cruzi infection.

An intense suppression of splenic T cell proliferation to mitogens and to Ags from the parasite is characteristic of the acute phase of Trypanosoma cruzi infection in mice. The impairment of proliferation is coincident with high levels of IFN-gamma and nitrite and decreased production of IL-2 in the supernatants of spleen cell cultures from infected mice. Previous work demonstrated that suppression of proliferation is largely mediated by the population of adherent cells in the infected spleen. In this study we confirmed the active suppression exerted by these cells on Con A, anti-CD3, and parasite Ag-stimulated proliferation of CD4+ splenic T cells. Inasmuch as the high production of IFN-gamma and of nitrite were compatible with intense macrophage activation and nitric oxide (NO) production, we determined the effects of cytokines that regulate macrophage activation and of NO on the proliferation of spleen cells from infected mice. We show that spleen cell proliferation to Ag and to T cell polyclonal stimuli is increased by neutralizing mAbs to IFN-gamma, TNF-alpha and -beta, or by the inhibitor of NO synthase, NG-monomethyl-L-arginine, added to the cultures. The addition of rIL-2 or rIL-4 also contributed to suppression reversal, and the combined addition of rIL-2 and anti-IFN-gamma mAb further increased lymphocyte proliferation. Anti-IL-4, anti-IL-10, or anti-TGF-beta neutralizing mAbs did not modify suppressed proliferative responses, and the addition of rIL-10 or of rTGF-beta also did not recover cell proliferation. Thus, the suppression of proliferative responses in T. cruzi-infected mice resulted largely from increased NO production by macrophages activated by IFN-gamma and TNF allied to insufficient IL-2 to fully support in vitro growth of T lymphocytes.