Anti-IL-4 antibody therapy causes regression of chronic lesions caused by medium-dose Leishmania major infection in BALB/c mice.

Experimental infection of BALB/c mice with a high number of Leishmania major parasites results in a predominant Th2 response and rapidly progressing, non-healing lesions. Disease can be prevented in such mice by simple therapies, such as administering neutralizing anti-IL-4 or anti-CD4 antibody prior to or around the time of infection, but not once the infection is well established. Established infections can be resolved by combined therapies, such as intralesional administration of massive doses of IL-12 and of anti-leishmanial drugs. We explored the possibility of using simple therapies to cure mice with stable, chronic and large lesions, a state that corresponds more closely to human cutaneous leishmaniasis than does the rapidly progressing model. The anti-parasite immune responses of mice bearing such chronic lesions have a mixed Th1/Th2 phenotype. Administration of either anti-IL-4 or anti-CD4 antibody alone results in the reliable regression of such lesions even when large. Cured mice display a dominant Th1 response with increased L. major-specific IgG2a antibody, increased production of IL-12p40 and of nitric oxide by macrophages, indicating increased parasiticidal activity. Cured mice resist a normally pathogenic L. major challenge. These findings may have implications for treatment of human cutaneous leishmaniasis and other chronic infectious diseases caused by intracellular pathogens.

Immune elimination of Leishmania major in mice: implications for immune memory, vaccination, and reactivation disease.

Infection of susceptible BALB/c mice with a large, moderate, or low number of Leishmania major parasites respectively results in progressive disease, the formation of substantial but stable lesions, denoted as borderline disease, and the absence of a visible lesion. Infection with a low number of parasites results over the long term in either subclinical infections or an asymptomatic state. Subclinical mice produce a predominant Th1 response and are resistant to challenge, in contrast to their asymptomatic counterparts. Statistical and other evidence suggest that the asymptomatic state can arise from a subclinical state following parasite clearance, with consequent loss of resistance. Cell transfer studies demonstrate unequivocally that immune cells from subclinical mice can protect naive mice against a pathogenic challenge and can clear the parasite, leaving the mice susceptible to a rechallenge infection. This susceptibility is associated with the disappearance of both parasite-specific effector and memory T cells from secondary lymphoid organs. These findings have implications for vaccination, maintenance of memory, and prevention of reactivation disease.

Susceptibility and resistance to Trypanosoma congolense infections.

We have put emphasis on recent findings in experimental Trypanosoma congolense infections in highly susceptible BALB/c and relatively resistant C57Bl/6 mice. Based on various analyses, it has been shown that a major difference in resistance to T. congolense infections is expressed early in infection at the macrophage level. A novel plastic-adherent Thy1.2(+) suppressor lymphocyte, which in absolute synergy with a Thy 1.2(-) cell exerts its suppression via interleukin-10 and interferon-gamma opens up an exciting new field of research.

Innate resistance to experimental African trypanosomiasis: differences in cytokine (TNF-alpha, IL-6, IL-10 and IL-12) production by bone marrow-derived macrophages from resistant and susceptible mice.

Resistance to African trypanosomiasis is under multigenic control. BALB/c mice are highly susceptible while C57Bl/6 mice are relatively resistant. Macrophages eliminate opsonized trypanosomes from the bloodstream and are involved in immunosuppression. We therefore investigated the production of a number of cytokines (IL-10, IL-6, TNF-alpha and IL-12) by bone marrow-derived macrophages (BMDM) from C57Bl/6 and BALB/c mice following challenge with either Trypanosoma congolense or Trypanosoma brucei. BMDM from C57Bl/6 mice, upon challenge with whole cell extracts (WCE) of T. congolense or T. brucei, produced significantly more TNF-alpha and IL-12 than those from BALB/c mice. The production of these cytokines was significantly enhanced by pretreatment of the cells with IFN-gamma. BMDM from BALB/c mice, however, produced significantly more IL-6 and IL-10 than those from C57Bl/6 mice. In contrast to LPS stimulation, simultaneous treatment of cells with WCE and IFN-gamma enhanced IL-10 synthesis by BMDM from BALB/c mice. These results indicate that cytokine genes are differentially regulated in macrophages from trypanosome-susceptible and -resistant mice and are consistent with our previous findings wherein retrovirus-immortalized macrophage cell lines from BALB/c and C57Bl/6 mice produce differential amounts of cytokines after phagocytosis of trypanosomes.

Innate resistance to Trypanosoma congolense infections: differential production of nitric oxide by macrophages from susceptible BALB/c and resistant C57Bl/6 mice.

BALB/c and C57B1/6 mice differ in resistance to Trypanosoma congolense infections. Evidence suggests that macrophages play a central role in the resistance to trypanosomiasis. Nitric oxide (NO) produced by macrophages in response to various stimuli or pathogens is one of the important arms of nonspecific immunity. We investigated the production of NO by the peritoneal macrophages and bone marrow-derived macrophages (BMDM) from trypanosome-resistant C57B1/6 and -susceptible BALB/c mice following stimulation with T. congolense whole cell extract (WCE) or following phagocytosis of T. congolense mediated by anti-variant surface glycoprotein (VSG) antibodies of IgM or IgG2a isotype. C57B1/6 peritoneal macrophages as well as BMDM produced significantly more NO than similar BALB/c macrophages in response to T. congolense lysate and IFN-gamma. In both BALB/c and C57B1/6 BMDM cultures, phagocytosis of T. congolense mediated by anti-VSG antibodies of IgG2a isotype in the presence of IFNgamma induced two- to ninefold more NO than phagocytosis mediated by IgM antibodies and C57B1/6 BMDM produced significantly higher amounts of NO than BALB/c BMDM under these conditions. NO produced by BMDM was found to exert trypanostatic effect on T. congolense in vitro, but was not found to influence the in vivo infectivity of these treated parasites under the conditions used in this study.

Cytokines and antibody responses during Trypanosoma congolense infections in two inbred mouse strains that differ in resistance.

We studied IL-4, IL-10 and IFN-gamma secretion by splenocytes and the plasma levels of different isotypes of antibodies against various antigens of Trypanosoma congolense in highly susceptible BALB/c and relatively resistant C57BL/6 mice during the early course of infection with T. congolense. The patterns of appearance of cytokine spotforming cells in the spleens were essentially similar in the two mouse strains although higher numbers were detected in the spleens of BALB/c than C57BL/6 mice on some days post-infection. However, the amount of IL-4, IL-10 and IFN-gamma secreted into the culture fluids was dramatically different. From day 4 forward, splenocytes from BALB/c mice secreted very high levels of these cytokines. In contrast, splenocytes from infected C57BL/6 mice did not secrete detectable levels of IL-4 throughout the period tested. The secretion of IL-10 and IFN-gamma by C57BL/6 splenocytes only became appreciable on day 6 and was down-regulated by day 8, when the first wave of parasitaemia was being controlled. At days 6-8, splenocytes from infected C57BL/6 mice secreted two-fold higher amounts of IL-12 p40 than those from BALB/c mice. Infected BALB/c mice mounted an earlier IgM antibody response to variant surface glycoprotein (VSG), formalin-fixed T. congolense and whole T. congolense lysates than did infected C57BL/6 mice. However, they failed to make any detectable IgG3 and IgG2a antibody responses to these antigens whereas infected C57BL/6 mice made strong IgG3 and IgG2a responses. We speculate that enhanced resistance against T. congolense infections in mice may be mediated by IL-12 dependent synthesis of IgG2 antibodies to VSG and possibly also common trypanosomal antigens.

Innate resistance to experimental Trypanosoma congolense infection: differences in IL-10 synthesis by macrophage cell lines from resistant and susceptible inbred mice.

BALB/c and C57Bl/6 mice differ in resistance to T. congolense infections. We investigated the production of various cytokines (IL-10, IL-6, TNF-alpha and TGF-beta) by macrophages from these mice. Macrophage cell lines (BALB.BM cells) of BALB/c mice but not (ANA-I cells)of C57BL/6 mice constitutively produced IL-10. Challenge of these cells with trypanosomes induced the production of 50-100 times more IL-10 in BALB.BM cells than in ANA-1 cells. Pre-incubation of the cell lines with IFN-gamma. prior to the trypanosome challenge, further upregulated this IL-10 production in BALB.BM but not in ANA-1 cells. Primary cultures of bone marrow-derived macrophages (BMDM) from BALB/c mice also produced more IL-10 following challenge with IFN-gamma and opsonized trypanosomes than did the C57Bl/6 BMDM. Similarly after challenge with trypanosomes, BALB.BM and BALB/c BMDM produced significantly more IL-6 than did the analogous cells from the C57Bl/6 mice following such challenges. Higher steady state levels of TNF-alpha mRNA accumulated in ANA-1 cells than in BALB.BM cells following challenge with IFN-gamma and opsonized trypanosomes. Findings of this study for the first time indicate a differential regulation of cytokines (IL-10, IL-6 and TNF-alpha) in macrophages of mice that significantly differ in their susceptibility to infections with T. congolense.

Experimental murine Trypanosoma congolense infections. II. Role of splenic adherent CD3+Thy1.2+ TCR-alpha beta- gamma delta- CD4+8- and CD3+Thy1.2+ TCR-alpha beta- gamma delta- CD4-8- cells in the production of IL-4, IL-10, and IFN-gamma and in trypanosome-elicited immunosuppression.

Trypanosome-induced suppression of T and B cell responses to parasite-related and -unrelated Ags is considered a major mechanism of evasion of the host's immune defenses by the parasite. Reduced T and B cell responses have been attributed to suppressor T cells, suppressor macrophages, or both. We have previously shown that endogenously produced IL-10 and IFN-gamma mediate the suppression of T cell responses in Trypanosoma congolense-infected mice. Here, we show for the first time that splenic CD3+ Thy1.2+ alphabeta- gammadelta- CD4+ 8- and CD3+ Thy1.2+ alphabeta- gammadetla- CD4- 8- cells that copurify with plastic-, nylon wool-, or Sephadex G-10-adherent cell populations, in synergy with adherent Thy1.2- cells, are the major producers of IL-4, IL-10, and IFN-gamma in T. congolense-infected mice. We further demonstrate the involvement of these cells in the suppression of T cell proliferative responses to mitogen and in B cell responses to a parasite-unrelated Ag.

Experimental murine Trypanosoma congolense infections. I. Administration of anti-IFN-gamma antibodies alters trypanosome-susceptible mice to a resistant-like phenotype.

The mechanisms regulating resistance or susceptibility to African trypanosomes have been enigmatic. In this study, we assessed the production of several cytokines (IL-4, IFN-gamma, and TNF-alpha) in vivo and in vitro using genetically susceptible (BALB/c) or resistant (C57BL/6) mice infected with cloned Trypanosoma congolense and the role of these cytokines in pathogenesis of this infection. Plasma of infected BALB/c mice contained higher levels of IL-4 and IFN-gamma than the plasma of infected C57BL/6 mice. Conversely, plasma TNF-alpha levels were elevated significantly in the resistant mice relative to the susceptible ones. Splenic IFN-gamma mRNA appeared earlier and were maintained at higher levels in infected BALB/c than in C57BL/6 mice. Both spontaneous and Con A-induced secretions of IL-4 and IFN-gamma by splenocytes from infected BALB/c mice were significantly higher than those from their C57BL/6 counterparts. Con A-induced proliferation of splenocytes from infected BALB/c mice was progressively suppressed. Nitric oxide was not involved in this suppression, but the suppression was positively correlated with IFN-gamma secretion. Addition of neutralizing Abs to IFN-gamma to cultures of Con A-stimulated spleen cells from infected BALB/c mice effectively reversed this suppression. Furthermore, administration of anti-IFN-gamma Abs to BALB/c mice early during infection dramatically shifted the phenotype of these susceptible mice to a more resistant-like phenotype, as expressed by a low and undulating parasitemia and a >300% increase in survival period. These results strongly suggest that the enhanced induction and secretion of IFN-gamma during T. congolense infections contribute to the relative susceptibility of BALB/c mice to the disease.

Immunoregulation in experimental murine Trypanosoma congolense infection: anti-IL-10 antibodies reverse trypanosome-mediated suppression of lymphocyte proliferation in vitro and moderately prolong the lifespan of genetically susceptible BALB/c mice.

We infected highly susceptible BALB/c and relatively resistant C57BL/6 mice with cloned Trypanosoma congolense and followed the effects of these infections on the circulating parasite numbers, mouse mortality and cytokine expression. C57BL/6 mice controlled their parasitaemia and survived for up to 163 +/- 12 days, while BALB/c mice could not control their parasitaemia and succumbed to the infection within 8.4 +/- 0.5 days. Susceptible BALB/c mice had dramatically higher plasma levels of IL-10 than the resistant C57BL/6 mice from day 7 forward. This was preceded by an earlier and higher level induction of splenic IL-10 messenger RNA (mRNA) expression in the infected BALB/c mice. There was a strong negative correlation between the splenocyte proliferative responses to Concanavalin-A (Con-A) and their production of IL-10 in these infected BALB/c mice. Co-treatment of the Con-A-stimulated spleen cell cultures with monoclonal anti-IL-10 antibodies, but not isotype-matched control antibodies, could completely reverse this suppression of the splenocyte proliferative response. Finally, in three experiments, anti-IL-10 antibody treatment in vivo reduced the peak circulating parasitaemia of infected BALB/c mice by 43% and increased their median survival periods by 38% relative to isotype-matched control antibody-treated mice.