Recombinant soluble interleukin-4 (IL-4) receptor acts as an antagonist of IL-4 in murine cutaneous Leishmaniasis.

This study was performed to evaluate the soluble interleukin-4 receptor (sIL-4R) as a potential antagonist of interleukin-4 (IL-4) in an infectious disease. It is shown that antigen-triggered proliferation and cytokine secretion of Leishmania major-specific, cloned Th2 cells in vitro can be inhibited dose dependently by recombinant murine, but not control human, sIL-4R. In vivo, we found that endogenous synthesis of IL-4 mRNA is upregulated during the first week of infection, while an increase of IL-4R mRNA occurred later after infection of BALB/c mice with L. major. To interfere successfully with the IL-4 ligand-receptor interaction, we therefore chose to treat infected BALB/c mice with recombinant sIL-4R during the onset (e.g., days 0 to 7) of the immune response. Treatment with murine, but not with human, sIL-4R during the first week of infection rendered BALB/c mice clinically resistant to L. major, led to a 7- to 12-fold reduction of the parasite load in spleen and lymph nodes at 7 weeks of infection, shifted the pattern of cytokines towards a Th1 type, and provided durable resistance against reinfection. Thus, it could be demonstrated that the balance among sIL-4R, membrane-bound IL-4R, and their ligand IL-4 can be modulated in vivo, thereby modifying the antiparasitic immune response. These results suggest a therapeutic value of sIL-4R in diseases in which neutralization of IL-4 is desirable.     

Interleukin-4 but not gamma interferon production correlates with the severity of murine cutaneous leishmaniasis.

For murine cutaneous leishmaniasis, data to date suggest a correlation between the presence of gamma interferon (IFN-gamma) and resistance in C57BL/6 mice and the presence of interleukin-4 (IL-4) and disease in BALB/c mice. In this study, 13 inbred strains of mice covering the range of susceptibility to disease were infected with Leishmania major to determine whether the subsequent expression of IFN-gamma or IL-4 is a reliable indicator of cure or progressive disease. The presence of IL-4 and IFN-gamma mRNAs in the draining lymph nodes was examined 9 weeks after infection, when differences in disease severity became obvious. There were large differences in the levels of IL-4 mRNA among the different strains, whereas IFN-gamma mRNA was detected at similar levels in all strains. The levels of IL-4 mRNA correlated with lesion score, with susceptible and intermediate strains containing up to 100-fold more than any of the resistant strains. Differences in the levels of IFN-gamma mRNA were within only a fourfold range, with significant overlap among susceptible, intermediate, and resistant strains. Similarly, the levels of IFN-gamma secreted in vitro by lymph node cells from infected mice in response to L. major antigens were within a 10-fold range for most strains, and there was no correlation with lesion score. Analysis of Leishmania-specific antibody levels revealed a correlation between immunoglobulin G1 (IgG1) titers and lesion score, consistent with the role of IL-4 as a switch factor for IgG1. In contrast, there was no correlation between IgG2a titers and lesion score, supporting the notion that IFN-gamma synthesis (which promotes IgG2a production) is not correlated with disease state. These data suggest that along the spectrum of murine cutaneous leishmaniasis, IL-4 is a reliable indicator of disease, but IFN-gamma is not prognostic for resistance.     

Control of New World cutaneous leishmaniasis is IL-12 independent but STAT4 dependent

Leishmania mexicana, a New World protozoan parasite, induces small, chronic, but non-progressive lesions in C57BL/6 (B6) mice. In this study we investigated the role of IL-12, and subsequent Th1 factors, in controlling cutaneous L. mexicana infection. IL-12 treatment failed to promote disease resolution, suggesting that the inability of mice to heal is not related to a deficiency of endogenous IL-12 production. Surprisingly, L. mexicana-induced cutaneous lesions in wild-type and IL-12p40-deficient mice were indistinguishable, with similar parasite burdens, immune responses, and lesion histopathology. In contrast, iNOS, IFN-gamma, and STAT4-deficient mice developed progressive disease and uncontrolled parasite growth. These results differ dramatically from L. major infection, in which IL-12p40-deficient mice are highly susceptible, with very rapid lesion growth, very large parasite burdens, and the development of a strong Th2 response. These data uncover the existence of an alternate IFN-gamma and iNOS pathway for control of Leishmania lesions, which is IL-12 independent, but which unexpectedly requires STAT4.     

STAT-4 mediated IL-12 signaling pathway is critical for the development of protective immunity in cutaneous leishmaniasis.

Recent studies have demonstrated that two IL-12 signaling pathways, a STAT 4 - dependent and STAT4 - independent, are involved in the development of a Th1-like response. To determine their roles in the development of protective immunity against Leishmania major, we monitored progression of cutaneous Leishmania major infection in STAT4-deficient mice (STAT4-/-) compared to similarly infected wild-type (STAT4+/+) mice. Although the onset of lesion growth was delayed in STAT4-/- mice during the early phase of infection, these mice eventually developed large, non-healing lesions, whereas STAT4+/+ mice resolved their lesions. As infection progressed, both STAT4+/+ and STAT4-/- mice infected with L. major displayed similar titers of Leishmania-specific IgG1 and IgE but later produced lower IgG2a. On days 20 and 40 post-infection, Leishmania antigen-stimulated lymphnode cells from STAT4-/- mice produced significantly lower amounts of IFN-gamma than those from STAT4+/+ mice as measured by enzyme-linked immunosorbent assay. There was no significant difference, however, in IL-4 and IL-12 production between the two groups. These results indicate that STAT4-mediated IL-12 signaling is critical for the development of protective Th1 response following L. major infection in genetically resistant mice. Additionally, they demonstrate that, although genetically resistant mice lacking STAT4 signaling pathway develop large, non-healing lesions, they do not default towards a Th2-like response.     

Ability of macrophage subsets to transfer resistance to murine leishmaniasis is dependent on IL-12 production

Leishmania major, which causes cutaneous leishmaniasis in humans, can also infect mice, in which can cause either a local cutaneous lesion or a fatal disseminated infection. We have previously shown that BALB/c mice injected with antigen-pulsed macrophages derived from bone marrow with granulocyte-macrophage colony-stimulating factor (GMMphi), but not other types of macrophages, were protected from an otherwise lethal infection with L. major The protection induced by GMMphi was shown to be antigen specific and correlated with the induction of a Th1-like response characterized by production of high levels of IFN-gamma, delayed-type hypersensitivity reactivity and long-lived resistance to reinfection. In this report, we show that the protection induced in this disease model correlates with the sustained production of IL-12 synthesis by GMMphi in vitro and in vivo. Moreover, GMMphi deficient in the production of IL-12 were unable to induce protection in this model. Our data, therefore, suggest that a defect in macrophage activation or differentiation may play a significant role in the inability of BALB/c mice to respond effectively to L. major, and this mechanism may also be important in determining resistance to other intracellular parasites.     

Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro

The recombinant form of the extracellular domain of the IL-4 receptor (sIL-4R) is a potential candidate to neutralize IL-4; however, murine sIL-4R displayed both antagonistic and agonistic activity in vivo. Here we show that human recombinant sIL-4R induced the formation of complexed IL-4 in supernatants of activated T cells in a dose-dependent manner as measured by newly developed enzyme-linked immunosorbent assays. These IL-4/sIL-4R complexes liberated free IL-4 even after prolonged culturing. In contrast, in the absence of exogenously added sIL-4R, free IL-4 was rapidly consumed or proteolytically degraded in cultures of activated T cells. Thus, no IL-4 bioactivity could be determined in supernatants of T cells activated in the presence of IL-4 for 6 days. In contrast, the same cultures carried out in the presence of sIL-4R showed marked IL-4 bioactivity. While low concentrations of sIL-4R enhanced IL-4-driven inhibiton of IFN-gamma production by activated T cells, higher concentrations neutralized IL-4. Together, human sIL-4R, besides its activity as an antagonist to IL-4, also possesses protective and agonistic functions for IL-4, which may be relevant for clinical studies aiming to neutralize IL-4 in vivo.     

The Role of IL-4 and IL-12 in the Regulation of Collagen Synthesis by Fibroblasts

Chronic sclerodermifomic graft versus host disease is a rare but important complication of allogeneic hematopoietic stem cell transplantation that especially occurs in patients who are treated with donor lymphocyte infusions for relapse of a malignant disease. Today most knowledge about the pathogenesis of chronic Graft-versus-Host Disease is based on mice models. In this report we describe the development of an allogeneic in vitro model that allows studying the pathogenesis of chronic sclerodermifomic Graft-versus-Host Disease in the human setting. We report that priming of mononuclear cells in the presence of allogeneic fibroblasts and Interleukin (IL)-4 induces fibroblast collagen synthesis, whereas priming in the presence of IL-12 suppresses collagen synthesis during subsequent coculture of primed mononuclear cells with allogeneic fibroblasts. Since IL-12 is also known to mediate anti-tumor effects by stimulation of Natural Killer cell and Lymphokine Activated Killer cell activity, these findings indicate that treatment of patients with IL-12 or pretreatment of donor lymphocytes with IL-12 might strengthen a graft versus leukemia effect and at the same time decrease the risk of chronic sclerodermifomic Graft-versus-Host Disease development.     

Regulation of cell-mediated immunity in cutaneous leishmaniasis

There is now good evidence that cell-mediated immunity (CMI) rather than humoral antibody plays a causal role in acquired immunity to leishmaniasis. In genetically susceptible strains of mice, the failure to control the disease progression is associated with a population of Lyt-2-T cells which can prevent the induction or expression of curative CMI and hence exacerbate disease development. Susceptible BALB/c mice can be rendered resistant to L. major infection by prior sublethal dose gamma-irradiation, anti-mu antibody treatment from birth, anti-L3T4 antibody treatment or intravenous (i.v.) or intraperitoneal (i.p.) route of immunisation with killed L. major promastigotes or isolated leishmanial antigens. The route of immunisation, however, appears crucial in the induction of prophylactic immunity. Subcutaneous (s.c.) and intramuscular routes of immunisation with killed promastigotes are not only ineffective, they induce a population of Lyt-2- L3T4+ T cells which inhibit the prophylactic effect of i.v. immunisation. Although both the disease-promoting T cells and the host-protective T cells express the same phenotypic cell surface markers, they differ functionally. Protective T cells produce interferon-gamma (IFN-gamma) and macrophage-activating factor (MAF) when cultured in vitro with leishmanial antigens, whereas the disease-promoting T cells do not. In addition, these latter cells are able to produce substances in their antigen-specific culture supernatant which inhibits the MAF activity of the host protective T cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of LPS induced IL-12 production by IFN-gamma and IL-4 through intracellular glutathione status in human alveolar macrophages.

Interleukin-12 (IL-12) is secreted from monocytes and macrophages; it exerts pleiotropic effects on T cells and natural killer (NK) cells, and stimulates interferon-gamma (IFN-gamma) secretion. Glutathione tripeptide regulates the intracellular redox status and other aspects of cell physiology. We examined whether IFN-gamma and IL-4 affect the balance between intracellular reduced glutathione (GSH) and oxidized (GSSG) glutathione, as this may affect IL-12 production in human alveolar macrophages (AM). We used both AM from healthy non-smokers obtained by bronchoalveolar lavage and the monocytic THP-1 cell line in this study. Incubation of AM for 2 h with the GSH precursor N-acetylcysteine (NAC) increased the intracellular GSH/GSSG ratio, and enhanced lipopolysaccharide (LPS)-induced IL-12 secretion by AM. In THP-1 cells, NAC increased the GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA, whereas L-buthionine-[S,R]-sulphoximine (BSO) decreased these. NAC and BSO offset their own effects on the intracellular GSH/GSSG ratio and the expression of LPS-induced IL-12 mRNA. Furthermore, exposure of AM to the helper T cell type 1 (Th1) cytokine IFN-gamma or the helper T cell type 2 (Th2) cytokine IL-4 for 72 h increased and decreased the GSH/GSSG ratio, respectively. Lipopolysaccharide (LPS)-induced secretion of IL-12 in AM was enhanced by IFN-gamma but inhibited by IL-4. These results suggest that IFN-gamma and IL-4 oppositely affect the GSH/GSSG balance, which may regulate IL-12 secretion from AM in response to LPS.     

CD4(+) cells are indispensable for ulcer development in murine cutaneous leishmaniasis

One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection. Leishmania amazonensis-infected mice show similar ulcerative lesions. Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from either Leishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4(+), CD8(+), or B220(+) cell populations and the remaining cells were injected into Leishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4(+) cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4(+) cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.     

Regulation of murine macrophage function by IL-4: IL-4 and IFN-gamma differentially regulate macrophage tumoricidal activation.

To understand the differential role of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in the process of macrophage tumoricidal activation, we investigated the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide in activated murine macrophages and the effects of those lymphokines on prostaglandin E2 (PGE2)-mediated down-regulation. IFN-gamma and IL-4 increased lipopolysaccharide (LPS)-induced TNF-alpha production by different mechanisms because IL-4, unlike IFN-gamma, failed to overcome the LPS-hyporesponsiveness in C3H/HeJ mice. Moreover, only IFN-gamma synergized with LPS to induce nitric oxide production and blocked eicosanoid-mediated down-regulation. These differential effects of IFN-gamma and IL-4 on the select efferent cytolytic activities may be the result of an altered or different signal transduction pathway. Because potentiation of protein kinase C (PKC) activity by IFN-gamma has been previously documented, we next studied the role of IFN-gamma and IL-4 in alteration of enzymatic activity of PKC. Two lymphokines caused translocation of PKC from cytosol to membrane with different levels, providing a biochemical basis for explaining how two lymphokines lead to different phenotypic responses. Although treatment of macrophages with IFN-gamma and IL-4 gave rise to a similar enhancing effect on macrophage TNF-alpha production, these two lymphokines appeared to differentially regulate the overall functional state of macrophages for tumour cell killing capability. Additionally, this differential regulation seems to be accomplished in part by different biochemical events.     

Retrovirally induced switch from production of IL-12 to IL-4 in dendritic cells.

Dendritic cells (DC) in HIV-1 infection show a reduced capacity to stimulate primary T cell proliferation. Exposure of bone marrow-derived DC to Rauscher leukemia virus (RLV) provides a mouse model for studying retrovirally induced reduction in stimulatory capacity for T cells. Treatment with IL-12, a cytokine that promotes the development of Th1 cells, has been postulated as a treatment for AIDS and is effective at restoring cell-mediated immunity in mice infected with mouse AIDS virus or with RLV (see Knight, S. C. and Patterson, S., Annu. Rev. Immunol. 1994. 15: 593-615 for references). Here we studied the direct effect of RLV and of IL-12 on bone marrow-derived DC. Normal DC produced IL-12 and IL-10 and stimulated primary allogeneic T cell proliferation. Exposure of DC to RLV caused reduced production of IL-12, production of IL-4 was seen in DC for the first time and T cell stimulation was inhibited. Addition of IL-12 reinstated and enhanced IL-12 synthesis in RLV-treated DC, abrogated production of IL-10 and IL-4 and restored stimulatory activity. Manipulation of cytokine production in DC could be a stratagem that has evolved in the retrovirus to avoid stimulation of cellular responses.     

The role of the macrophage in cutaneous leishmaniasis.

The investigation of the role of the macrophage in cutaneous leishmaniasis has been prompted by observations of the clinical behaviour of the infection. In contrast to the self-healing oriental sore, chronic leishmaniasis is characterized by persistent lesions and leishmania recidiva by lesions that flare up locally long after clinical healing. In both clinical types, the parasite is thought to be maintained inside the macrophages. It will be shown that the normal macrophages of mice and guinea-pigs are parasitized by L. tropica; the parasite is not killed by the macrophages but it multiplies within these cells. Incubation of the macrophages with rabbit or human anti-Leishmania sera on the other hand, leads to the attachment of specific immunoglobulins to the macrophage cell surface and consequently to the prevention of parasitization by L. tropica under the experimental conditions. The parasite appears to be immobilized at the macrophage cell surface. Normal rabbit or human sera did not interfere with parasitization. It is postulated that the parasite specifically immobilized at the cell surface might possibly be better exposed to and affected by the immune response than intracellular parasites. Furthermore, infected parasitized macrophages contribute to the immune response by processing soluble antigens from the intracellular parasites and presenting them on their surfaces, as seen by the greater affinity (higher dilution) of anti-Leishmania antibody for the cell membrane of infected macrophages than for normal macrophages.     

Suppression of IL-12 production by phosphodiesterase inhibition in murine endotoxemia is IL-10 independent

Phosphodiesterase (PDE) inhibitors are potent regulators of various immune processes. Immune cells contain type IV and type III PDE. Here we studied in mice the effects of rolipram, a selective PDE IV inhibitor, and amrinone, a selective PDE III blocker, on plasma levels of IL-12 (p70), IFN-γ, IL-1, TNF-α, and nitric oxide (NO) induced by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (80 mg/kg). Pretreatment of BALB/c mice with both rolipram (1 – 25 mg/kg) and amrinone (10 – 100 mg/kg) decreased plasma IL-12 levels in a dose-dependent manner. Similarly, LPS-elicited plasma IFN-γ concentrations were suppressed by both rolipram and amrinone. However, LPS-induced plasma IL-1α levels were not affected by either of these compounds. In addition, rolipram inhibited IL-12, IFN-γ, TNF-α and nitrite/nitrate (breakdown products of NO) production in C57BL/6 IL-10^+/+ mice as well as in their IL-10-deficient counterparts (C57BL/6 IL-10^−/−). Our results suggest that rolipram and amrinone decrease the immune activation in endotoxemia through inhibition of the production of pro-inflammatory mediators IL-12, IFN-γ, TNF-α and NO. These effects are not the consequences of the increase in IL-10 production by PDE inhibition.     

TNF-alpha reverses the disease-exacerbating effect of subcutaneous immunization against murine cutaneous leishmaniasis.

Earlier studies have demonstrated that mice injected subcutaneously or intramuscularly with leishmanial antigens develop significantly exacerbated disease compared with unimmunized controls when challenged with the cutaneous protozoan parasites Leishmania major. We report here that this disease enhancement can be prevented, and protective immunity induced, by the incorporation of recombinant tumour necrosis factor (TNF-alpha) in the immunizing inoculum. This effect of TNF-alpha is dose-dependent and is not evident when TNF-alpha and the antigens are injected into separate sites. Furthermore, TNF-alpha injected together with p183, a peptide known to preferentially stimulate Th2 cells and disease exacerbation in H-2d mice, activates spleen and lymph node cells secreting more interferon-gamma (IFN-gamma) and less interleukin-4 (IL-4) and induces a modest but significant degree of resistance against L. major infection in highly susceptible BALB/c mice.