Gene expression analysis in a murine model of allergic asthma reveals overlapping disease and therapy dependent pathways in the lung.

Accumulating evidence in animal models and human asthma support a central role for IL-13 signaling in disease pathogenesis. In order to identify asthma and therapy associated genes, global transcriptional changes were monitored in mouse lung following antigen challenge (ovalbumin (OVA)), either alone or in the presence of a soluble IL-13 antagonist. Changes in whole lung gene expression after instillation of mIL-13 were also measured both in wild type and STAT6 deficient mice. A striking overlap in the gene expression profiles induced by either OVA challenge or mIL-13 was observed, further strengthening the relationship of IL-13 signaling to asthma. Consistent with results from functional studies, a subset of the OVA-induced gene expression was significantly inhibited by a soluble IL-13 antagonist while IL-13-modulated gene expression was completely attenuated in the absence of STAT6-mediated signaling. Results from these experiments greatly expand our understanding of asthma and provide novel molecular targets for therapy and potential biomarkers of IL-13 antagonism.

Evaluation of the safety of recombinant P-selectin glycoprotein ligand-immunoglobulin G fusion protein in experimental models of localized and systemic infection.

P-selectin is a major component in the early interaction between platelets, endothelial cells, and inflammatory cells in the initial phases of the innate immune response. The major ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1) and this ligand is expressed on the surface of monocyte, lymphocyte, and neutrophil membranes. A truncated form of recombinant human P-selectin glycoprotein ligand-1 has been covalently linked to immunoglobulin G (rPSGL-Ig) and this fusion peptide functions as a competitive inhibitor of PSGL-1. As an inhibitor of neutrophil-endothelial cell adherence, rPSGL-Ig is in early clinical development for the treatment of ischemia reperfusion injury. To determine the potential for deleterious effects from inhibition in P-selectin-mediated neutrophil attachment in the presence of bacterial infection, the effects of therapeutic doses of rPSGL-Ig were tested in three standard laboratory sepsis models. The experimental models included: the murine systemic Listeria monocytogenes infection model, the Pseudomonas aeruginosa bacteremia model in neutropenic rats, and the cecal ligation and puncture (CLP)-induced peritonitis model in rats. Recombinant human PSGL-Ig had no adverse effects on mortality or immune clearance in systemic bacterial infection in any of the three infection models. The PSGL-1 inhibitor did significantly decrease local neutrophil infiltration and bacterial clearance in the peritoneum following CLP, but this did not increase the systemic levels of proinflammatory cytokines, the quantitative levels of bacteremia, or the overall mortality rate following CLP. The results indicate that rPSGL-Ig did not exacerbate infection in these experimental sepsis models.

Interleukin-12 regulates the response to chemotherapy in experimental visceral Leishmaniasis.

In experimental visceral leishmaniasis, interleukin (IL)-12 initiates control over Leishmania donovani via Th1 cell activation, interferon (IFN)-gamma secretion, and granuloma formation. Because the leishmanicidal effect of conventional therapy, pentavalent antimony (Sb), also requires T cells and endogenous IFN-gamma, we tested IL-12 as a determinant of host responsiveness to chemotherapy. L. donovani-challenged IL-12p35 gene knockout (KO) mice permitted uncontrolled hepatic infection and failed to respond to Sb. In contrast, 96% of liver parasites in KO mice were killed by amphotericin B, which acts independently of immune responses. Exogenous IL-12 combined with Sb was tested in normal mice: low-dose Sb was converted from weakly to strongly leishmanicidal, and a no-effect Sb dose was converted to approximately 100% leishmanistatic. IL-12 plus Sb synergism in normal mice was IFN-gamma dependent; however, IL-12 also increased responsiveness to Sb in IFN-gamma KO mice. Thus, IL-12 regulates host IFN-gamma-dependent and -independent responses that permit and/or enhance the leishmanicidal activity of Sb.

Gastrointestinal nematode expulsion in IL-4 knockout mice is IL-13 dependent.

Female IL-4 knockout (KO) mice on a C57BL/6 background (F4KOC57) are susceptible to infection with the cecal-dwelling nematode Trichuris muris whereas wild-type C57BL/6 mice are resistant and expel the parasite. In this study we show that in sharp contrast, female IL-4 KO mice on a BALB/c background (F4KOB/c) are resistant to infection as are wild-type BALB/c mice. Although susceptible F4KOC57 make negligible levels of all type 2 cytokines, resistant F4KOB/c were capable of producing significant levels of antigen-specific IL-13 (a cytokine shown to be critical in resistance to T. muris). To examine if the IL-13 in F4KOB/c mice was of functional importance, it was neutralized in vivo using a fusion protein, A25 (sIL-13 R.Fc). The results presented here clearly demonstrate that neutralization of IL-13 in vivo did indeed prevent T. muris expulsion in normally resistant F4KOB/c mice. In addition, administration of recombinant mouse IL-13 to normally susceptible male IL-4KO BALB/c mice (M4KOB/c) caused an 87.85 % reduction in worm burden. Collectively, these data show that IL-13 is important in the poorly understood effector mechanisms resulting in the expulsion of T. muris from the gut. Moreover, the present data highlight the functional importance of gender and background strain in interpretation of studies using gene-targeted animals.

Autocrine regulation of IL-12 receptor expression is independent of secondary IFN-gamma secretion and not restricted to T and NK cells.

The biological response to IL-12 is mediated through specific binding to a high affinity receptor complex composed of at least two subunits (designated IL-12Rbeta1 and IL-12Rbeta2) that are expressed on NK cells and activated T cells. The selective loss of IL-12Rbeta2 expression during Th2 T cell differentiation suggests that regulation of this receptor component may govern IL-12 responsiveness. In murine assays, down-regulation of IL-12Rbeta2 expression can be prevented by treatment with IFN-gamma, indicating that receptor expression and hence IL-12 responsiveness may be regulated, at least in part, by the local cytokine milieu. In this study, we report that cellular expression of both IL-12Rbeta1 and beta2 mRNA is increased in the lymph nodes of naive mice following systemic administration of murine rIL-12 (rmIL-12). Changes in IL-12R mRNA were associated with increased IFN-gamma secretion following ex vivo activation of lymph node cells with rmIL-12, indicating the presence of a functional receptor complex. Expression of IL-12R mRNA was not restricted to lymph node T cells, and its autocrine regulation was independent of secondary IFN-gamma secretion. Data from fractionated lymph node cells as well as rmIL-12-treated B cell-deficient mice suggest that IL-12-responsive B cells may represent an alternative cellular source for IFN-gamma production. However, the strength of the biological response to rmIL-12 is not governed solely by receptor expression, as rmIL-12-induced IFN-gamma secretion from cultured lymph node cells is accessory cell dependent and can be partially blocked by inhibition of B7 costimulation.

Expression of Th2 cytokines and the stable Th2 marker ST2L in the absence of IL-4 during Leishmania major infection.

In this study we characterized Th2 responses in the absence of IL-4. We show that ST2L, a stable Th2 marker, is expressed at similar levels in Leishmania major-infected IL-4-deficient (IL-4(-/-)) and wild-type BALB/c (IL-4(+/+)) mice. Th2 cytokines are secreted by in vivo differentiated lymphocytes in response to specific activation in the absence of IL-4. Although IL-13 is produced, its neutralization did not alter the outcome of infection. Thus, we demonstrate that Th2 differentiation as assessed by the expression of ST2L and the production of Th2 cytokines can occur in vivo in the absence of IL-4.

Protective immunity using recombinant human IL-12 and alum as adjuvants in a primate model of cutaneous leishmaniasis.

Protection from cutaneous leishmaniasis, a chronic ulcerating skin lesion affecting millions, has been achieved historically using live virulent preparations of the parasite. Killed or recombinant Ags that could be safer as vaccines generally require an adjuvant for induction of a strong Th1 response in murine models. Murine rIL-12 as an adjuvant with soluble Leishmania Ag has been shown to protect susceptible mice. We used 48 rhesus macaques to assess the safety, immunogenicity, and efficacy of a vaccine combining heat-killed Leishmania amazonensis with human rIL-12 (rhIL-12) and alum (aluminum hydroxide gel) as adjuvants. The single s.c. vaccination was found to be safe and immunogenic, although a small transient s.c. nodule developed at the site. Groups receiving rhIL-12 had an augmented in vitro Ag-specific IFN-gamma response after vaccination, as well as increased production of IgG. No increase in IL-4 or IL-10 was found in cell culture supernatants from either control or experimental groups. Delayed hypersensitivity reactions were not predictive of protection. Intradermal forehead challenge infection with 107 metacyclic L. amazonensis promastigotes at 4 wk demonstrated protective immunity in all 12 monkeys receiving 2 microgram rhIL-12 with alum and Ag. Partial efficacy was seen with lower doses of rhIL-12 and in groups lacking either adjuvant. Thus, a single dose vaccine with killed Ag using rhIL-12 and alum as adjuvants was safe and fully effective in this primate model of cutaneous leishmaniasis. This study extends the murine data to primates, and provides a basis for further human trials.

Interleukin-12 is capable of generating an antigen-specific Th1-type response in the presence of an ongoing infection-driven Th2-type response.

Previously we demonstrated that recombinant murine interleukin-12 (rmIL-12) administration can promote a primary Th1 response while suppressing the Th2 response in mice primed with 2,4, 6-trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH). The present studies examined the capacity of rmIL-12 to drive a Th1 response to TNP-KLH in the presence of an ongoing Th2-mediated disease. To establish a distinct Th2 response, we used a murine model of leishmaniasis. Susceptible BALB/c mice produce a strong Th2 response when infected with Leishmania major and develop progressive visceral disease. On day 26 postinfection, when leishmaniasis was well established, groups of mice were immunized with TNP-KLH in the presence or absence of exogenous rmIL-12. Even in the presence of overt infection, TNP-KLH-plus-rmIL-12-immunized mice were still capable of generating KLH-specific gamma interferon (IFN-gamma) as well as corresponding TNP-specific immunoglobulin G2a (IgG2a) titers. In addition, the KLH-specific IL-4 was suppressed in infected mice immunized with rmIL-12. However, parasite-specific IL-4 and IgG1 production with a lack of parasite-specific IFN-gamma secretion were maintained in all infected groups of mice including those immunized with rmIL-12. These data show that despite the ongoing infection-driven Th2 response, rmIL-12 was capable of generating an antigen-specific Th1 response to an independent immunogen. Moreover, rmIL-12 administered with TNP-KLH late in infection did not alter the parasite-specific cytokine or antibody responses.

The role of recombinant murine IL-12 and IFN-gamma in the pathogenesis of a murine systemic Candida albicans infection.

Studies on murine candidiasis suggest that resistance to disease is linked to a Th1 response and production of IFN-gamma, while failure to elicit protection is associated with a Th2 response and production of IL-4 and IL-10. Experimental infection of C57BL/6 mice, IL-12 treatment of these mice, or both infection and IL-12 treatment resulted in a characteristic Th1 cytokine mRNA profile as measured by quantitative competitive PCR. Specifically, little or no IL-4 transcripts were detected, while IFN-gamma message was elevated, particularly with IL-12 treatment. Despite its role in driving increased IFN-gamma expression and production, IL-12 treatment, paradoxically, promoted disease progression in our model. Therefore, we examined the effect of IFN-gamma neutralization on IL-12-induced susceptibility to infection. None of the systemically infected mice receiving IL-12 alone survived, while IL-12- and anti-IFN-gamma-treated mice had a 70% survival rate, similar to that after infection alone. These results suggested that IFN-gamma induced by IL-12 treatment contributed to lethality. However, in separate studies, IFN-gamma knockout mice were more susceptible to infection than their wild-type counterparts, suggesting that IFN-gamma is required for resistance. Nonetheless, infected IFN-gamma knockout mice treated with recombinant murine IL-12 exhibited enhanced resistance, suggesting that the toxicities observed with IL-12 are directly attributable to IFN-gamma and that an optimal immune response to Candida infections necessitates a finely tuned balance of IFN-gamma production. Thus, we propose that although IFN-gamma can drive resistance, the overproduction of IFN-gamma during candidiasis, mediated by IL-12 administration, leads to enhanced susceptibility.

Administration of recombinant human interleukin 12 to chronically SIVmac-infected rhesus monkeys.

With the demonstration that interleukin 12 can enhance natural killer (NK) cell activity and drive CD4+ lymphocytes toward T helper type 1 (Thl) responses, there is a strong rationale for exploring the use of this cytokine as an immunomodulatory therapy in HIV-1-infected individuals. To assess its potential safety and effects on both immune and virologic aspects of HIV-1 infection, recombinant human IL-12 (rhIL-12) was assessed in rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac). The activity of rhIL-12 on rhesus monkey lymphocytes was confirmed with the demonstration that peripheral blood lymphocyte lysis of the NK-sensitive cell line Colo was enhanced by this recombinant cytokine. Further, rhIL-12 was shown to induce interferon-gamma production by rhesus monkey lymphocytes in vitro. Then, in separate studies, two treatment regimens of rhIL-12 were assessed in SIVmac-infected monkeys: a low-dose regimen (0.1 microg/kg, daily for 4 weeks) and a high-dose regimen (2.5 microg/kg, every 3-4 days, for 3 weeks). Both rhIL-12 treatment regimens were well tolerated by these virus-infected animals. The high-dose regimen of rhIL-12 induced transient decreases in circulating lymphocytes in the SIVmac-infected monkeys. Furthermore, no changes in lymphocyte-associated SIVmac DNA or SIVmac plasma RNA levels were seen in the treated monkeys. These studies indicate that short-term treatment with rhIL-12 is well tolerated and causes no measurable changes in virus load in chronically SIVmac-infected rhesus monkeys.

Interleukin-12 promotes a chronic intestinal nematode infection.

Resistance and susceptibility to the intestinal parasite Trichuris muris has been shown to be due to a dominant T helper 2 (Th2) and a dominant Th1 response, respectively. The factors determining the initial polarization of the immune response remain largely unresolved, although the cytokine environment at the time of antigen presentation clearly plays an essential role. Interleukin (IL)-12, a cytokine produced mainly by macrophages, dendritic cells, and other monocytes has been shown to be important in driving a strong Th1 response by stimulating the production of interferon (IFN)-gamma from natural killer and Th0 cells and therefore forms a link between the innate and adaptive immune system. IL-12 has been shown to play an important role in resistance to a number of intracellular pathogens, including Listeria and Leishmania. It has also been proposed as an anti-tumor agent and for use in the treatment of HIV. Conversely, IL-12 has been shown to prolong the survival of Nippostrongylus brasiliensis and to accelerate autoimmunity. Our studies demonstrate that by driving a strong Th1 response, IL-12 promotes chronic T. muris infection when given to normally resistant BALB/K mice. Parasite-specific IgG2a, a Th1 parameter of infection, was greatly up-regulated, whereas some Th2 parameters of infection were down-regulated. IL-12 treatment could be delayed until 1 week after infection had started and still promote a strong Th1 response. The actions of IL-12 in promoting a chronic infection were IFN-gamma dependent as an anti-IFN-gamma mAb abrogated the effects of IL-12.

Antiproliferative effects of interleukin-12 treatment on human tumor colony-forming units taken directly from patients.

Interleukin-12 (IL-12) has important immunomodulatory effects on T and natural killer (NK) cells that might be exploited in anticancer treatment. Murine IL-12 models have shown antimetastatic and antitumor effects against murine tumors in vivo. Data on the effects of human IL-12 on human tumors are confined to 51Cr-release assay studies showing that IL-12 increases NK activity against cancer cells. We used a human tumor cloning assay (HTCA) to investigate the effects of human IL-12 on solid tumors taken directly from patients. The HTCA is suitable to test direct, as well as immune-mediated, antitumor effects of cytokines on heterogeneous cell preparations derived from fresh tumors. Single cell suspensions prepared from 193 tumors were continuously exposed (14 days) to 10, 100 and 1000 ng/ml of human IL-12 in a capillary HTCA. Seventy-four (38%) specimens were evaluable. Inhibition of tumor growth was observed in 35 specimens (47%; concentration-related in 33 cases), including cancers of the ovary, lung, prostate, breast, colon and kidney, as well as melanoma. Antitumor effect was observed in 10 (14%), 18 (24%) and 32 (43%) tumors, at 10, 100 and 1000 ng/ml of IL-12, respectively. One specimen (1%), a melanoma, showed stimulation of tumor proliferation only at 100 mg/ml of IL-12. Our results show that IL-12 has substantial in vitro activity against a variety of solid tumors taken directly from patients. Clinical trials of IL-12 in patients with solid tumors are warranted.

Comparison of gamma interferon, tumor necrosis factor, and direct cell contact in activation of antimycobacterial defense in murine macrophages.

We compared the abilities of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and sensitized murine lymph node lymphocytes to activate syngeneic murine peritoneal macrophages to inhibit the growth of intracellular Mycobacterium bovis BCG in vitro. IFN-gamma could activate antimycobacterial defense only when added to macrophage cultures prior to their infection with BCG. TNF-alpha was without any effect. In contrast, BCG-sensitized lymphocytes could induce antimycobacterial defenses when added after macrophages had been infected with BCG. The cell-mediated effect required direct contact between effector lymphocytes and the targets (BCG-infected macrophages), as revealed in studies in which these cell populations were separated by a semipermeable membrane. Cyclosporin A, which inhibits the production of relevant macrophage-activating lymphokines, did not abrogate the ability of sensitized lymphocytes to activate antimycobacterial effects in infected macrophages. Furthermore, only BCG-sensitized lymphocytes, and not Listeria-sensitized lymphocytes, could activate the antimycobacterial effects. These lymphocytes were not cytotoxic to the infected macrophages. The presence of anti-TNF-alpha antibody in cocultures reduced the antimicrobial effects. We propose that the activation of antimycobacterial defense in macrophages can occur by direct physical contact with sensitized lymphocytes. This process may be due to lymphocyte membrane-associated TNF-alpha, as we previously demonstrated in our studies of antileishmanial defense.

Resolution of cutaneous leishmaniasis: interleukin 12 initiates a protective T helper type 1 immune response.

Resistance to Leishmania major in mice is associated with the appearance of distinct T helper type 1 (Th1) and Th2 subsets. T cells from lymph nodes draining cutaneous lesions of resistant mice are primarily interferon gamma (IFN-gamma)-producing Th1 cells. In contrast, T cells from susceptible mice are principally Th2 cells that generate interleukin 4 (IL-4). Although existing evidence is supportive of a role for IFN-gamma in the generation of Th1 cells, additional factors may be required for a protective response to be maintained. A potential candidate is IL-12, a heterodimeric cytokine produced by monocytes and B cells that has multiple effects on T and natural killer cell function, including inducing IFN-gamma production. Using an experimental leishmanial model we have observed that daily intraperitoneal administration at the time of parasite challenge of either 0.33 micrograms IL-12 (a consecutive 5 d/wk for 5 wk) or 1.0 micrograms IL-12 per mouse (only a consecutive 5 d) caused a > 75% reduction in parasite burden at the site of infection, in highly susceptible BALB/c mice. Delay of treatment by 1 wk had less of a protective effect. Concomitant with these protective effects was an increase in IFN-gamma and a decrease in IL-4 production, as measured by enzyme-linked immunosorbent assay of supernatants generated from popliteal lymph node cells stimulated with leishmanial antigen in vitro. The reduction in parasite numbers induced by IL-12 therapy was still apparent at 10 wk postinfection. In addition, we observed that the administration of a rabbit anti-recombinant murine IL-12 polyclonal antibody (200 micrograms i.p. every other day for 25 d) at the time of infection to resistant C57Bl/6 mice exacerbated disease. These effects were accompanied by a shift in IFN-gamma production in vitro by antigen-stimulated lymph node cells indicative of a Th2-like response. These findings suggest that IL-12 has an important role in initiating a Th1 response and protective immunity.

Soluble TNF and membrane TNF expressed on CD4+ T lymphocytes differ in their ability to activate macrophage antileishmanial defense.

In our studies of host defense against the intracellular parasite Leishmania major, we obtained evidence for a novel mechanism of macrophage activation for antimicrobial defense that involves direct cell contact between CD4+ T lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive as it does not involve secretion of lymphokines but is apparently mediated by the membrane-anchored form of tumor necrosis factor (mTNF; approximately 50-60 kd) present on the surface of the effector T lymphocytes. Furthermore, it is not cytotoxic to the host cell and its expression is antigen specific and genetically restricted. We prepared a Leishmania-specific cloned T-T cell hybridoma line 1B6 (CD4+, TH1) that expresses membrane-bound TNF but does not secrete TNF or other macrophage activators. We now report that 1B6 cells can activate antileishmanial defense in inflammatory macrophages, whereas soluble recombinant murine TNF (sTNF) alone is unable to do so. On the other hand, both 1B6 cells and sTNF can act synergistically with recombinant murine interferon-gamma (IFN-gamma, a known soluble macrophage-activating factor) in activating antimicrobial defense and NO2- release. The effects of 1B6 alone and the synergistic effects of 1B6 and IFN-gamma or sTNF and IFN-gamma are arginine dependent. These results suggest that mTNF may be more efficient than sTNF in macrophage activation and that contact with effector CD4+ lymphocytes that express mTNF may be an important mechanism of host defense.

Antileishmanial defense in macrophages triggered by tumor necrosis factor expressed on CD4+ T lymphocyte plasma membrane.

In our studies of host defense to the intracellular protozoan Leishmania major, we uncovered a novel mechanism of antileishmanial defense that involves direct cell contact between effector CD4+ lymphocytes and Leishmania-infected macrophages. The mechanism is distinctive because it does not involve lymphokine secretion and induces no cytotoxic effects in the host cells; its expression is antigen-specific and genetically restricted. We now demonstrate that these effector CD4+ cells display tumor necrosis factor (TNF) on their surface and provide evidence that the membrane-associated TNF is involved in the activation of the antileishmanial defense. Using a Leishmania-specific cloned T-T cell hybridoma line (1B6; CD4+, T helper type 1) that activates antileishmanial defense in macrophages through cell contact and does not secrete TNF, we noted that only cells bearing surface TNF (TNF+), but not ones lacking surface TNF (TNF-), exerted these effects. Moreover, the antileishmanial effects excreted by TNF+ 1B6 cells as well as by lymph node CD4+ TNF+ lymphocytes could be blocked with anti-TNF antibody. We propose that membrane-associated TNF on CD4+ T cells may provide a mechanism of targeting activation signals to macrophages in an antigen-specific and genetically restricted manner.

Th2 lymphocyte clone can activate macrophage antileishmanial defense by a lymphokine-independent mechanism in vitro and can augment parasite attrition in vivo.

Antileishmanial defense has been ascribed to the antimicrobial effects induced by soluble macrophage-activating lymphokines (MAFs), such as interferon-gamma and granulocyte-macrophage colony-stimulating factor. Recently, we identified an additional mechanism of T cell-mediated macrophage activation of defense against Leishmania that is apparently lymphokine independent, requires cell-cell contact, and is not cytotoxic to host cells. By employing antigen-specific murine T cell hybridoma lines, we observed that this property was associated with CD4+ subpopulations possessing the characteristics of the Th1 subset. In the present study, we address the question of whether contact-mediated macrophage activation can also be induced by Th2 lymphocytes. We employed as T effector cells in antileishmanial defense assays the Th2 cell line D10.G1.4 (D10) which is specific for conalbumin. We observed that D10 cells were able to induce activation of Leishmania-infected macrophages only when the macrophages were also primed with conalbumin, and that this activation apparently occurred by a mechanism without the secretion of MAF. Moreover, when mice infected with L. major were injected into footpad lesions with conalbumin and D10 cells, in situ parasite replication was partially inhibited. The expression of this antimicrobial mechanism by Th1 as well as Th2 clones suggests that the property of contact-mediated (lymphokine-independent) activation may be shared by certain lymphocytes in both Th1 and Th2 subpopulations. We hypothesize that this activation mechanism may involve the interaction of a lymphocyte membrane-associated MAF (such as tumor necrosis factor) and its receptor on the infected macrophage, resulting in the induction of antimicrobial effects but not cytotoxicity to the host cell.

T-cell hybridomas reveal two distinct mechanisms of antileishmanial defense.

Using lymph node lymphocytes of Leishmania major-infected mice, we constructed and cloned two T-cell hybridomas that could activate macrophages to exert antileishmanial defense in vitro. One clone, 1D5, produced lymphokines (including gamma interferon) that induced these effects. Production of the macrophage-activating lymphokines and the protective effect of 1D5 were suppressed by the addition of cyclosporine A to cultures. The other clone, 1B6, produced no detectable macrophage-activating lymphokines, and its protective ability was not suppressed by cyclosporine A. Granulocyte-macrophage colony-stimulating factor (a lymphokine also known to induce antileishmanial effects in macrophages) was not detectable in culture supernatants of either clone. Furthermore, neither clone was cytotoxic to infected macrophages. Antileishmanial defense induced by 1B6 was genetically restricted; that is, infected macrophages and hybridoma cells had to be syngeneic for an antileishmanial effect to occur. In contrast, such restriction was not a property of clone 1D5, a clone that was responsive to alloantigens as well as leishmanial antigens. When incubated at a temperature (34 degrees C) at which lymphokines are relatively ineffective for antileishmanial defense, 1B6 but not 1D5 retained its antileishmanial properties. These observations provide clear evidence for the existence of two distinct mechanisms of macrophage activation: one that is lymphokine dependent, and one that is apparently lymphokine independent. The expression of these two mechanisms by cloned cells strongly suggests that they are properties of different T-cell subpopulations, extending our prior conclusions based on studies of heterogeneous T-cell populations. We hypothesize that the latter macrophage activation process involves a cell contact-dependent mechanism which might involve the interaction of a lymphocyte membrane-associated macrophage-activating factor (such as tumor necrosis factor) with its receptor on the macrophage, resulting in activation of antileishmanial effects but not host cell cytotoxicity.

Susceptibility of lymphokine-resistant Leishmania to cell contact-mediated macrophage activation.

Lymphokine-mediated activation of macrophages is considered an important host defense mechanism against Leishmania. Recently we identified an additional mechanism of T cell-mediated macrophage activation of defense against Leishmania that is apparently lymphokine-independent and cell contact-dependent but not cytotoxic to host cells. In the present study we determined that strains of Leishmania mexicana that are not susceptible to the antimicrobial effects that result from lymphokine activation are nonetheless susceptible to antileishmanial effects resulting from T cell contact-mediated macrophage activation. Lymphocytes capable of activating macrophages by the cell contact-dependent mechanism appeared transiently in C57BL/6 mice infected with one of these L. mexicana strains. Our findings provide further evidence for fundamental distinctions between lymphokine- and contact-mediated antileishmanial defense. The results suggest that in addition to resistance to lymphokine-mediated effects, suppression of cell contact-mediated defense may account for chronicity in these infections.

Macrophage activation for antileishmanial defense by an apparently novel mechanism.

Activation of macrophages by lymphokines (including interferon-gamma; IFN-gamma) is presently considered to be a major host defense mechanism against a number of intracellular microorganisms. In a series of earlier studies that made use of mice undergoing spontaneous resolution of footpad infections with Leishmania major, we obtained evidence suggesting that a subpopulation of Leishmania-sensitized lymph node T lymphocytes could activate antimicrobial effects in Leishmania-infected macrophages by an apparently lymphokine-independent mechanism. These effector lymphocytes are not cytotoxic to host cells, and their effects are antigen specific and genetically restricted. To more rigorously investigate this apparently novel mechanism of macrophage activation, we examined the effect of blocking lymphokine production with cyclosporin A (CSA) on the capacity of these effector lymphocytes to exert macrophage activating function. Although CSA blocked lymphokines that activate antileishmanial effects, it did not inhibit the antimicrobial capacity of the effector lymphocytes. We also confirmed that IFN-gamma is the major macrophage-activating lymphokine that induces antileishmanial effects; treatment of lymphokine-containing supernatants with anti-IFN-gamma antibody markedly reduced their antimicrobial effects. In contrast, treatment of effector lymphocytes with this antibody failed to reduce their macrophage-activating capacity. We conclude that there exists an apparently novel macrophage-activating mechanism for antimicrobial defense that is independent of soluble lymphokine mediators.