Cure of progressive murine leishmaniasis: interleukin 4 dominance is abolished by transient CD4(+) T cell depletion and T helper cell type 1-selective cytokine therapy.

Progressive infection with Leishmania major in susceptible BALB/c mice is mediated by interleukin (IL)-4-producing T helper cell type 2 (Th2) CD4(+) T cells that, once established, become resistant to Th1-deviating therapies with recombinant (r)IL-12 and/or neutralizing anti-IL-4 antibodies. We sought to restore protective immunity in advanced leishmaniasis by depletion of Th2-biased CD4(+) populations and by cytokine-directed reconstitution of Th1 cellular responses during lymphocyte recovery. Treatment with cytolytic GK1.5 anti-CD4 mAb alone did not reverse disease in 3 wk-infected BALB/c mice, but GK1.5 combined with anti-IL-4 antibody and intralesional rIL-12 cured cutaneous lesions in 80% of mice and established a Th1-polarized cytokine response to L. major antigen protective against reinfection. The curative effects of GK1.5 were not replaced by cytotoxic anti-CD8 monoclonal antibody 2.43 or nondepleting anti-CD4 mAb YTS177, confirming that depletion of CD4(+) cells was specific and essential for therapeutic effect. Finally, combined CD4(+) depletion and IL-4 neutralization were curative, indicating that neither increased parasite burden nor altered accessory cell function independently biased towards Th2 reconstitution in advanced leishmaniasis. Advanced leishmaniasis can be cured by T cell depletion and cytokine-directed recovery of Th1 cellular responses, suggesting novel interventions for other immune-mediated diseases and identifying distinct roles for CD4(+) T cell and non-T cell in the maintenance of Th2 and Th1 phenotypes.

Increased capacity for interleukin-2 synthesis parallels disease progression in mice infected with Leishmania major.

Lymph node cells of BALB/c mice with progressive leishmaniasis produced sixfold more interleukin-2 (IL-2) in culture than those of healing C57BL/6 mice. IL-2 synthesis also increased in C57BL/6 mice made susceptible by IL-12 or gamma interferon deficiency. However, IL-2 mRNA levels in vivo did not reflect IL-2 production in vitro. Because IL-2 contributes to the pathogenesis of progressive leishmaniasis, the functional significance of these findings should be further explored.

Underproduction of interleukin-12 in susceptible mice during progressive leishmaniasis is due to decreased CD40 activity.

Interleukin-12 promotes Th1 lymphocyte responses necessary for the cure of murine Leishmania major infection. We found that IL-12 p40 mRNA expression peaked at 4 weeks of infection in resistant C57BL/6 mice at levels threefold greater than in BALB/c mice. Peak IL-12 p40 expression in both strains was reduced threefold following treatment with neutralizing anti-CD40 ligand antibody and disease worsened in C57BL/6 mice. Direct activation of cultured lymph node cells by anti-CD40 MAb or soluble CD40 ligand failed to restore deficient IL-12 production by infected BALB/c mice unless recombinant IFN-beta was added to culture. Infected BALB/c lymph nodes also contained two- to threefold fewer low-density CD40+ accessory cells compared to that in C57BL/6 mice. We conclude that CD40-dependent responses are continually required for healing of leishmaniasis and that progressive disease is associated with decreased CD40-stimulated IL-12 synthesis as a consequence of either altered cytokine environment or inadequate accessory cell number.

In vivo production and function of IL-12 p40 homodimers.

The bioactivity of IL-12 is mediated by heterodimers of disulfide-linked p35 and p40 protein subunits. Homodimeric p40 competes with heterodimer for binding to the high affinity IL-12R and inhibits IL-12 bioactivity in vitro. However, the production and significance of p40 homodimer as a cytokine antagonist in vivo have not been determined. In these studies, we observed increased amounts of both IL-12 p40 monomer and homodimer in the serum of C57BL/6 mice following injection of 300 microg of Salmonella enteritidis LPS. Homodimer constituted between 20 and 40% of the total circulating p40 in endotoxemic sera, as confirmed by both Sephacryl S-100 gel filtration and p40-specific immunoprecipitation analyses. Similar relative amounts of homodimer and monomer were observed in endotoxemic BALB/c, C57BL/6, IFN-gamma-deficient C57BL/6 mice and C57BL/6 mice previously infected with bacille Calmette-Guérin. To determine whether IL-12 p40 homodimer was capable of antagonizing IL-12-dependent IFN-gamma responses in vivo, we pretreated C57BL/6 mice with purified rIL-12 p40 homodimer before i.p. challenge with endotoxin. Mice treated with 40 to 80 microg of p40 homodimer generated 80 to 82% less circulating IFN-gamma during acute endotoxemia than saline controls (p < 0.01). We conclude that p40 homodimer is produced in vivo, functions as a cytokine antagonist in the context of the mouse model of acute endotoxemia, and may represent a novel form of self-regulating cytokine response.

IFN-gamma-independent production of IL-12 during murine endotoxemia.

We examined whether IFN-gamma deficiency alters the in vivo IL-12 response occurring in the mouse model of acute endotoxemia. C57BL/6 IFN-gamma knockout mice (IFN-gamma 0/0) produced as much circulating IL-12 p40 and IL-12 p70 as did IFN-gamma +/+ mice following injection with S. enteritidis LPS, despite sustaining 11-fold reductions in circulating TNF-alpha. Pretreatment of IFN-gamma 0/0 mice with recombinant mouse IFN-gamma (rIFN-gamma) enhanced circulating TNF-alpha by as much as sixfold, but serum IL-12 p40 and IL-12 p70 responses increased by only twofold or less. Compared with IFN-gamma +/+ mice, the spleens of endotoxemic IFN-gamma 0/0 mice generated two- to threefold fewer IL-12 p40-secreting cells following in vitro or in vivo exposure to endotoxin. The addition of rIFN-gamma to IFN-gamma 0/0 splenocyte culture restored normal levels of LPS-stimulated IL-12 p40 production. Removal of Mac-1+ or F4/80+ cells from endotoxin-stimulated spleen reduced both TNF-alpha and IL-12 p40 production, but 33D1+ dendritic cell removal only affected IL-12 synthesis. These data suggest that the aggregate IL-12 p40 and p70 response to endotoxemia in vivo is IFN-gamma-independent and distinct from IFN-gamma-dependent serum TNF-alpha and splenic IL-12 responses. The cellular and/or cytokine basis for the unexpected preservation of IL-12 production in IFN-gamma-deficient mice may be relevant to normal and pathologic immune responses.

Immunoregulation of murine leishmaniasis by interleukin-12.

Distinct phenotypic outcomes following infection of mice with Leishmania major are closely linked to the emergence of functionally dissimilar Th1 or Th2 CD4+ T-cell responses early in the course of disease. This model of T-cell-dependent microbial pathology has proven useful for the study of cytokine regulatory and effector functions in vivo. To this end, the causal relationships linking synthesis of IFN gamma to cure and of IL4 to disease exacerbation have already been well characterized. IL12 also has a defined role in shaping the immune response against L. major. Early treatment with recombinant IL12, or vaccination using IL12 as an adjuvant, protects genetically susceptible hosts from progressive infection. Protective mechanisms include both suppression of deleterious Th2 cell responses and amplification of beneficial Th1 cell activities. Although Leishmania are poor stimuli for macrophage-derived IL12 when compared to bacteria and other protozoa, in vivo production during infection can be indirectly demonstrated by the worsening of leishmaniasis that follows anti-IL12 injection in normally resistant mice. Whether IL12 production during infection represents constitutive or regulated synthesis by infected macrophages is unresolved and deserves further exploration.

Endogenous IL-12 is required for control of Th2 cytokine responses capable of exacerbating leishmaniasis in normally resistant mice.

We investigated the mechanisms by which treatment with anti-IL-12 Ab prevents cure of infection with Leishmania major in resistant C57BL/6 mice. Consistent with delayed production of IL-12, anti-IL-12 Abs could be administered as late as 2 wk after infection to exacerbate disease. Starting at 2 wk of infection, the cultured lymph node cells from mice treated with either polyclonal or monoclonal anti-IL-12 Abs persistently generated 3- to 10-fold more IL-4 and IL-10 in response to L. major Ag compared with cells from mice receiving preimmune goat IgG. Reciprocal decreases in Ag-specific IFN-gamma production were observed in mice receiving anti-IL-12 Abs. A similar reversal of IFN-gamma and IL-4 production accompanied progressive disease induced by pretreatment with a single dose of anti-IFN-gamma mAb. Although IFN-gamma production was suppressed for up to 4 wk in mice treated with monoclonal anti-IL-12 or anti-IFN-gamma, coadministration of neutralizing anti-IL-4 IgG reversed progressive illness. These findings demonstrate that IL-12 produced in vivo is necessary for both the emergence of IFN-gamma producing cells and the down-regulation of Th2 cell responses during murine leishmaniasis. Furthermore, the uninhibited production of IL-4 was required to sustain progressive infection initiated by the decreased IFN-gamma synthesis observed in anti-IL-12 and anti-IFN-gamma-treated mice.

Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon.

Gamma interferon (IFN-gamma) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock. To address the cellular source of IFN-gamma production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-gamma RNA was abundant in splenic CD4+ and CD8+ T cells and in a T- and B-cell-depleted population of splenocytes containing 34% NK1.1+ natural killer (NK) cells. Because interleukin 12 (IL-12) is a known inducer of IFN-gamma synthesis by cultured T cells and NK cells, we examined whether IL-12 might be involved in IFN-gamma release during endotoxemia. mRNA encoding the p40 subunit of IL-12 increased markedly in the spleens of C57BL/6 mice at 2 h after LPS injection, whereas p35 IL-12 mRNA was constitutively expressed at all times. Bioactive IL-12 (p70 heterodimer) was detected in mouse serum at 2 to 4 h after LPS injection. Similar results were obtained using a p40 subunit-specific enzyme-linked immunosorbent assay. Endotoxin-insensitive C3H/HeJ mice generated threefold less IL-12 p70 and IFN-gamma at these times than endotoxin-sensitive C3H/HeOuJ mice. Pretreatment of mice with polyclonal anti-mouse IL-12 antibody reduced IFN-gamma levels present at 6 h post-LPS nearly sixfold in three separate experiments. These studies support a role for IL-12 as a proximal stimulator of IFN-gamma release during endotoxemia.

Recombinant interleukin 12 cures mice infected with Leishmania major.

Resistant C57BL/6 mice infected with Leishmania major are self-healing, whereas susceptible BALB/c mice fail to contain cutaneous infection and subsequently undergo fatal visceral dissemination. These disparate outcomes are mediated by dissimilar expansions of T helper type 1 (Th1) and Th2 CD4+ T lymphocyte subsets in vivo during cure and progression of disease. Because interleukin 12 (IL-12) has potent T cell growth and interferon gamma (IFN-gamma) stimulatory effects, we studied its effect on CD4+ T cell differentiation during murine leishmaniasis. Treatment with recombinant murine (rMu)IL-12 during the first week of infection cured 89% of normally susceptible BALB/c mice, as defined by decreased size of infected footpads and 1,000-10,000-fold reduced parasite burdens, and provided durable resistance against reinfection. Cure was associated with markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, but preserved or increased production of IFN-gamma relative to untreated mice. IL-4 and IFN-gamma mRNA associated with CD4+ T lymphocytes isolated from infected lymph nodes showed similar reciprocal changes in response to rMuIL-12 therapy. A single injection of anti-IFN-gamma monoclonal antibody abrogated the protective effect of rMuIL-12 therapy and restored Th2 cytokine responses. We conclude that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma-dependent fashion during murine leishmaniasis. Since BALB/c leishmaniasis cannot be cured with rMuIFN-gamma alone, additional direct effects of IL-12 during T cell subset selection are suggested. Because rMuIL-12 is uniquely protective in this well-characterized model of chronic parasitism, differences in IL-12 production may underlie heterogenous host responses to L. major and other intracellular pathogens.

IL-2 is necessary for the progression of leishmaniasis in susceptible murine hosts.

BALB/c mice are highly susceptible to disseminated infection with the intracellular protozoa Leishmania major. Progression of disease requires in vivo expansion of Th2 CD4+ lymphocytes and is reversed by treatment with anti-IL-4 monoclonal antibody. Inasmuch as IL-2 may be necessary for both the production of IL-4 and differentiation of Th2 cells, the possible contribution of IL-2 to progressive infection was examined. Four weekly injections of anti-IL-2 mAb (S4B6) cured more than 80% of BALB/c mice infected with L. major, as determined by diminished footpad swelling and decreased numbers of parasites in infected tissues. Multiple doses of S4B6 were necessary for benefit; a single dose given at the time of infection was ineffective. The anti-IL-2R mAb PC61 demonstrated a similar protective effect when administered twice weekly for 4 wk. Anti-IL-2-mediated cure of cutaneous leishmaniasis was associated with increased IFN-gamma and decreased IL-4 production by regional lymph node cells compared to untreated BALB/c mice with progressive illness. Both CD4+ and CD8+ T lymphocytes contributed to the increased expression of IFN-gamma mRNA in cured mice. These data suggest that levels of IL-2 suboptimal for Th2 expansion in vivo do not inhibit Th1 CD4+ and CD8+ T cell activation and IFN-gamma synthesis. Other cytokines or activation pathways that are either IL-2-independent or synergistic with low levels of IL-2 may account for the appearance of curative T cell responses during treatment with anti-IL-2 antibodies.

Photofrin II photosensitization is mutagenic at the tk locus in mouse L5178Y cells.

Photosensitization mediated by Photofrin II (PFII) was found to be mutagenic at the heterozygous thymidine kinase (tk) locus in mouse L5178Y lymphoma strains LY-S1 and LY-R16 but not in strain LY-R83 which is hemizygous at the tk locus. After treatments yielding 37% survival, the mutagenicity of photosensitization with PFII in strain LY-S1 was similar to that of other mutagenic agents including x-radiation, ethyl methanesulfonate, and photosensitization with chloroaluminum phthalocyanine (AlPcCl). Although both strain LY-S1 and strain LY-R16 were mutagenized by photosensitization with PFII, only strain LY-S1 was mutagenized by photosensitization with AlPcCl. The non-mutability of strain LY-R83 following photodynamic treatment with either sensitizer may be because of the poor recovery of mutants with intergenic mutations in this TK+/0 hemizygous strain, whereas the non-mutability of strain LY-R16 subjected to photodynamic treatment with AlPcCl may be because LY-R16 cells sustaining mutagenic damage do not survive for reasons other than the loss of an essential gene.