Immunoregulation of murine leishmaniasis by interleukin-12

IMMUNOREGULATION inducing amastigotes are liberated, is simply irrelevant to the biology of this organism. The ease with which IL12 release can be documented by numerous other pathogens under the same conditions underscores the unusual behaviour of Leishmania. Perhaps it is time to lay the convolutions to rest and begin the more interesting experiments that elucidate the mechanism. More difficult is the question whether IL12 is induced in vivo by promastigotes. Neutralizing antibody studies remain inconclusive, since, as pointed out by Drs. Sartori and Trinchieri, the positive amplification of the IL12IIFNy system precludes interpretation of who comes first. The authors themselves have demonstrated the exact same findings using neutralizing antibodies to IFNy. The knockout mice used by us, and the preliminary hearsay from the IL12 p40 knockout mice infected with L. major (thank you, Jeanne Magram), suggest that IL12 is substantially reduced in the IFNy knockouts (our manuscript) whereas IFNy is reduced but easily measured in the IL12 knockouts. Careful analysis of these mice at early periods will be most revealing in this issue. Our in vivo analysis, however (Reiner et al., 1994), showed that all strains of mice (including C3H) produce IL4 in vivo that peaks on day 4 after infection ; addition of IL12 at the time of infection powerfully inhibits this IL4 peak (Wang et al, 1994). The presence of IL4 in all strains following

Immunoregulation

infection with infectious metacyclics argues strongly that no IL12 is induced during this period. The kinetics of the response of the infection to neutralizing anti-IL12 demonstrated by Heinzel further reinforces a primary role for IL12 after amastigote transformation has occurred. We have always used amastigotes derived from SCID spleen preparations and performed assays in the presence of polymyxin B to avoid the contamination possibilities raised in Dr. Heinzel’s comments. Thus, we are not quite able to accept the SartorYDinchieri suggestion that IL12 is a pilot light that ignites the CM1 conflagration ; if it is, somebody must have left the gas off when Leishmania entered the house.

References Reiner, L.S., Zheng, S., Wang, Z., Stowring, L. & Locksley, R.M. (1994), Leishmania promastigotes evade interleukin 12 (B-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells during initiation of infection. J. Exp. Med., 179,

447-456. Wang, Z.E., Zheng, S., Cony, D.B., Dalton, D.K., Seder, R.A., Reiner, S.L. & Locksley, R.M. (1994), Interferon gamma-independent effects of interleukin- 12 administered during acute or established infection due to Leishmania major. Proc. Natl. Acad. Sci USA,

912, 12932-12936.

of murine leishmaniasis

F.P. Heinzel,

F. Ahmed, A.M.

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Hujer and R.M. Rerko

Division of Geographic Medicine, Case Western Reserve University School of Medicine and the VA Medical Center, 10701 East Blvd., Cleveland, OH, 44106 (USA)

Summary Distinct phenotypic outcomes following infection of mice with Leishmania major are closely linked to the emergence of functionally dissimilar Thl 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

IFNy to cure and of IL4 to diseaseexacerbation have already been well characterized. IL12 also has a defined role in shaping the immune responseagainst L. major. Early treatment with recombinant IL12, or vaccination using IL12 as an adjuvant, protects genetically susceptiblehosts from progressive infection. Protective mechanisms include both suppression of deleterious Th2 cell responsesand amplification of beneficial Thl cell activities. Although Leishmaniu are poor stimuli for macrophage-derived

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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. Introduction Cutaneous infection with L. major is a progressive visceralizing disease in susceptible BALB/c mice, whereas resistant C57BL/6 and C3H mice heal. These outcomes are highly dependent on the type of CD4+ T-cell response generated during early infection (Reiner and Locksley, 1995). The amastigote stage of this protozoan parasite infects only tissue macrophages and cure is associated with IFNydependent activation of nitric oxide and reactive products of superoxide toxic to these intracellular pathogens (Swihart et al., 1995 ; Wang et al., 1994a). Thl CD4+ T cells present in the infected tissues are the major source of IFNy, although natural killer cells can also synthesize this cytokine within the first few days of infection (Scharton and Scott, 1993). In addition to mediating crucial effector functions, IFNy is also required for the normal development of a Thl-dominant lymphocyte phenotype in naturally resistant mice (Scott, 1991). In part, this reflects differential antiproliferative effects of this cytokine mediated on Thl and Th2 cells due to the Thl-specific downregulation of the IFNy receptor p chain and consequent disruption of the inhibitory STAT-l signal transduction pathway (Per& et al., 1995). Whether direct effects of IFNy on precursor cells are required for Thl cell development is an unresolved issue. Although IFNy receptor knockout mice actually generate exaggerated Thl responses during leishmaniasis, T’hl cell development is deficient in IFNy knockout or anti-IFNy-treated mice (Scott, 1991; Swihart et al., 1995; Wang et al., 1994a). Although susceptible BALB/c mice also produce IFNy early in infection, they support the competing expansion of Th2 CD4+ T cells. IL4 produced by these cells inhibits IFNy-dependent macrophage activation and may permit unrestrained parasite growth in infected tissues. As demonstrated by the protective effect of anti-IL4 antibody therapy in other studies, Th2 cell activity essentially vetoes Thl mediated-immunity against Leishmania (Reiner et al., 1995). Studies of T-cell differentiation using TCR-transgenic mice bred on different backgrounds suggest that BALB/c T cells are uniquely and intrinsically biased towards Th2 cell maturation when cultured under normally non-selective conditions (Hsieh et al., 1995). Suceptibility may also be linked

IN IMMUNOLOGY separately to the inability of BALB/c mouse to downregulate IL4 produced in the first three days of infection, in contrast to resistant strains of mice (Reiner et aZ., 1994). Since the accelerated IL4 production observed in vivo is inconsistent with cytokine responses typical of naive T cells, the participation of NKl. 1+ T cells that are innately primed for synthesis of IL4 has been proposed (Reiner and Locksley, 1995). Strain-specific phenotypic biases during T-cell differentiation appear to require permissive costimulatory environments in order to be fully expressed. Perturbations of BALB/c accessory cell function that can reverse susceptibility include preconditioning of antigen-pulsed macrophages with GM-CSF (Doherty and Coffman, 1993) and in vivo blockade of B7 molecules by CTLA4-Ig treatment (Carry et al., 1994). Among the many accessory cell-derived factors studied, interleukinl:! has proven especially significant in its ability to shapethe T-cell cytokine phenotype. The bioactive form of IL12 is a disulphide-linked heterodimer that interacts with high affinity receptors expressedon natural killer cells and activated T cells. Besides supporting T-cell growth and inducing antigen-independent synthesis of IFNy by these cells, IL12 triggers differentiation of Thl cells in most mouse strains, although BALB/c cultures must also be depleted of IL4 activity to permit a similar response(Hsieh et al., 1995). IL12 in combination with IFNy strongly suppresses Th2 cell development. Because IL12 critically regulates the development of T-cell subsetsknown to be causal in the healing or progression of leishmaniasis, the biology of IL12 in this diseasehas deservedly received intense scrutiny. Recombinant IL12 alters T-cell differentiation viva during leishmaniasis

in

The physiologic significance of IL12 as an immunoregulatory protein has been amply demonstrated by its unique reversal of BALB/c susceptibility to progressive infection with L. major (Heinzel et al., 1993; Sypek et al., 1993) and its effectiveness as a vaccine adjuvant in generating prophylactic immunity in these mice (Afonso et al., 1994). Although IL12 immunotherapy acutely intensifies IFNy production by L. major-infected BALB/c mice and probably accelerates parasite clearance as a direct result, sustained increases in antigen-specific Thl cell activity relative to untreated mice were not apparent (Heinzel et al., 1993). Even a prolonged three-week course of daily IL12 treatment (0.5 pg/day) did not enhance IFNy recall responses present at one week post-treatment compared to control infected BALBlc mice (fig. 1). Instead, IL4 production by antigen-stimulated lymph nodes was reduced to undetectable levels following either short

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I

IFNyl

BALBIC Fig. 1. The effect of recombinant IL12 therapy on CD4+ Thl and Th2 cell differentiation during leishmaniasis: evidence for IFNY-independent effects. BALBlc mice (n=5 mice per groups) were infected with 2~ lo6 stationary phase promastigotes of L. major and treated daily for 21 days with saline as a control group or with (rMuIL12) 0.5 pg daily doses of rMuIL12 by intraperitoneal injection. A separate group of mice (rMuIL12hIFNy) were also treated with rMuIL12 and additionally were injected with 2 mg of anti-IFNy mAb (XMG1.2) on days 0, 7 and 14 of infection. Untreated C57BW6 mice also infected with L. major are provided for comparison. At 28 days of infection, the draining lymph node cells were obtained and cultured at 5 x lo6 cells/ml in DMEM/lO% FBS containing 4 pg/ml of freeze-thawed promastigote antigen. After 48 h of incubation at 37°C in a 5% CO, incubator, the conditioned medium was recoveredand assayed by cytokine-specific ELISA. Mean values for antigen-stimulated production are shown in nglml with bars representing the standard error of the mean. Uninfected BALBk spleens produced <0.2 rig/ml of IFNy, IL4, IL2 and IL10 in response to antigen (not shown).

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or prolonged courses of immunotherapy. Although IL12 therapy transiently stimulates IL10 mRNA expression in viva (Finkelman et al., 1994; Wang et al., 1994b), the long-lasting effects of IL12 are to reduce antigen-specific IL10 secretion nearly threefold. Because IL12-cured mice are protected against reinfection with L. major, we concluded that the amounts of IFNy normally produced by BALB/c mice - about 2- to 3-fold less than that produced by C57BL/6 mice in this study - were sufficient for cure and that it was the abrogation of Th2 activity that accounted for lasting resistance. On this basis, we also propose that protective vaccination with IL12 and antigen reflects more the suppression of Th2 cell priming than the amplification of early IFNy responses(Afonso et al., 1994). The mechanisms by which IL12 inhibits nascent Th2 cell responsesare of considerable interest. IFNy has been proposed as an intermediary in this response.Although there is considerable experimental support for this based on studies in helminthic infections and anti-IgD-induced immune responses (Finkelman et al., 1994; Wynn et al., 1994), findings in the leishmaniasis model appear to contradict this hypothesis. Since BALB/c mice normally pro duce IFNy early in infection, it is apparent that physiologic quantities of this cytokine alone are insufficient to overcome the bias towards Th2 cell development in this mouse strain. Although IFNy induced by IL12 immunotherapy may reach levels of production not normally achieved during infection, with greater regulatory effect, treatment with high doses of recombinant IFNy only transiently affected the developing Th2 cell response (Scott, 199 1). Instead, IL1 2 probably provides separate IFNy-dependent and -independent activities that act cooperatively to regulate Th2 cell responsesin vivo. As part of the same experiment described above, infected BALB/c mice treated for three weeks with rIL12 also received weekly injections of 2 mg of neutralizing anti-IFNy monoclonal antibody (XMG1.2). The antigen-specific cytokine response generated by draining lymph node cells was determined one week after the cessation of IL12 immunotherapy (fig. 1). The intensity with which IFNy was neutralized in vivo was clearly sufficient to inhibit both the healing of infection and the priming of IFNy recall responses, yet IL4 responses remained suppressedto approximately 2% of levels obtained in untreated BALB/c mice during infection. Decreases in IL10 production were also observed in both IL12 and IL12/anti-IFNy-treated BALB/c mice. Leishmania-specific production of IL2 was unaffected by these interventions, suggesting that combined therapy with anti-IFNy and recombinant IL12 effectively prevented T-cell differentiation beyond an early ThP-like phenotype. A more elegant illustration of the IFNy-independent, Th2-suppressive effects of IL12 were obtained in

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BALB/c mice expressing homozygous disruptions in the interferon-gamma gene (Wang et al., 1994b). Although we conclude that IL12 has direct effects on Th2 development that are independent of IFNy, we note that this may be relatively unique to the mouse model of leishmaniasis, which depends on intrinsic defects in host T-cell development for Th2 cell expansion. In contrast, helminthic infections stimulate strong Th2 immune responses in many strains of mice, consistent with immunoregulatory effects distinct from those operative in leishmaniasis. Finally, these findings may only reflect pharmacologic, as opposed to physiologic, mechanisms of IL12 action, since the amounts of recombinant IL12 administered probably far exceed that which is produced in viva during infection. Until recently, IL12 immunotherapy of L. major infection had only been effective when administered during the first week of infection ; delayed treatment was ineffective and even enhanced IL4 production under some circumstances (Sypek et al., 1993 ; Wang et al., 1994b). The molecular basis for this loss of IL12 sensitivity during Th2 cell differentiation has been attributed recently to a programmed disruption in Th2 cells of IL12-dependent activation of JAK and STAT intermediates that are otherwise preserved in ThO or Thl cell lines (Szabo et al., 1995). As a consequence, delayed IL12 therapy is more effective in models of disease, such as Leisshmania donovani infection of mice, where IFNydependent mechanisms of cure are not complicated by significant Th2 cell counter-responses (Murray and Hariprashad, 1995). The reason for the paradoxical increase in IL4 provided by IL12 in Th2-rich environments is less obvious. Since IL12 cannot directly act on differentiated Th2 cells, IL4 might be derived instead from ThO cells or represent expanded numbers of precursor T cells that rapidly acquire Th2 cytokine profiles in the presence of abundant IL4. In view of the preceding discussion, the restoration of IL12 immunoselective efficacy following Pentostam therapy of L. major-infected BALB/c mice is remarkable and suggests that parasitized macrophages are critical to maintaining Th2 cell predominance (Nabors et al., 1995). In vitro studies have shown biased activation of Th2, as compared to Thl clones, when antigen is presented by heavily parasitized BALB/c or C3H/HeN macrophages (Chakkalath and Titus, 1994). However, studies of progressive infection in resistant mice with disrupted nitric oxide synthesis suggest that merely increasing the parasite load is not in itself sufficient to trigger Th2 cell expansions. In our own unpublished studies, C57BL/6 mice treated with 400 mg/kg/day of aminoguanidine hemisulphate for four weeks were unable contain infection with L. major. Compared to control infected mice, the foot-

IN IMMUNOLOGY pad lesion sizes in aminoguanidine-treated animals were significantly increased (3.2kO.4 mm compared to 2.320.3 mm thickness) and tissue parasite burdens were augmented by 2500-fold. Despite the observed worsening of disease, antigen-induced production of IL4 was not significantly increased (0.14eO.l compared to 0.07*0.1 rig/ml; aminoguanidine-treated and control mice, respectively), whereas IFNy production actually increased by over 3-fold in the heavily infected animals (6.5 f 1.1 compared to 2.2*05 rig/ml). Similar findings were obtained in the presence of extreme parasite loads occurring in highly resistant 129/J mice containing homozygous deletions in the inducible nitric oxide synthase gene (Wei et al., 1995). The observed resistance of the Th2 cell phenotype to IL12 intervention in heavily parasitized BALB/c mice must therefore reflect a more complicated interaction between accessory cells and T cells that could be unique to a Th2-predisposed background. Whatever the mechanisms involved, clinical applications based on these findings are obvious and worth pursuing once the issues of IL12 toxicity have been resolved. Endogenous IL12 is required for cure of leishmaniasis in normally resistant mice Several studies have demonstrated that endogenous IL12 is required for induction of Thl cell activity and cure of leishmaniasis in resistant strains of mice. Both rabbit polyclonal and rat monoclonal antibodies to mouse IL12 triggered exacerbation of disease in C57BW6 and C3H mice, with decreased antigen-specific production of IFNy apparent through at least 2 weeks of infection (Scharton-Kersten et al., 1995 ; Sypek et al., 1993). We have confirmed the IL12 dependence of Thl cell development in infected C57BW6 mice by similar means, although IFNy production recovered as circulating anti-IL12 antibody was cleared (Heinzel et aZ., 1995). Despite the emergence of IFNy responses that equaled or exceeded that of healing mice, IL12pretreated mice developed progressive infection, ultimately developing ulcerative footpad lesions and lOO,OOO-fold increases in tissue parasite burden. Rather than reflecting deficient IFNy production, progressive infection instead correlated with the appearance of strong IL4 recall responses by draining lymph node cells and was associated with enhanced expression of IL4 mRNA in vivo. These enhanced Th2 responses were required for progressive disease, as anti-IL4 antibody cotreatment of anti-IL12-injected mice prohibited Th2 cell development and restored C57BL/6 resistance without affecting IFNy production (fig. 2). We can conclude that IL12 is required for the early differentiation of Thl cells in resistant mice

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increasingly substitute for IL12 in antigen-driven responsesas disease progresses.Indeed, CTLA4-Ig blockade results in >90% suppressionof IFNy production in these same cultures. A definitive answer to the IL12 dependency of in viva Thl immunity may eventually be provided by studies in IL12 p40 knockout mice.

Production of IL12 during leishmaniasis 0 Cytoklne

*a

40

Production

(nglml)

Parasite

Load

(log,

do.1

Fig. 2. Anti-IL4 therapy reversesthe exacerbationof

gm)

L.

major infection in C57BU6 mice injectedwith anti-IL12

antibodies. Groups of 5 C57BL/6 mice each were infected with 2x lo6 promastigotesof L. major. Treatmentsincluded (a-IL12) injectionsinjectionswith neutralizing anti-IL12 mAb (100 pg eachof Cl51 and C15.6) on days 0 and 7 of infection, (a-IL4) injectionswith neutralizing anti-IL4 mAb (1 mg of llB11) on days 0 and 7 and (cc-IL12/aIL4) injectionsof both antibodieson the sameschedule. Control C57BL/6 and BALB/c mice received no treatments. Lymph node cells were harvestedat 5 weeksof infection and stimulatedwith freeze-thawedpromastigote antigenas describedin figure 1. Conditionedmediawere assayed by cytokine ELISA as described above. For determination of IL4, the culture conditions included 10 pg/ml of anti-IL4 receptor mAb (Ml, SeroteciHarlan Bioproducts, Indianapolis,IN) to prevent autoconsumption of cytokine. Footpadparasiteburdenswere determined by culture of serially diluted tissue homogenates. These data are adapted from Heinzel et al. (Heinzel, 1995).

and that it also critically regulates Th2 development in both resistant and susceptible strains. It is less clear if IL12 remains absolutely necessary for production of antigen-specific IFNy once Thl cell responseshave matured. For instance, delayed injection of anti-1112 monoclonal antibody had diminishing effects on diseaseexpression in C57BL/6 mice past one week of infection (Heinzel et al., 1995). Although antigen-specific IFNy synthesis by cultured lymph node cells can be significantly reduced in the presence of anti-IL12, up to half of the responseeventually becomesIL 12-independent after two weeks of infection (F.P. Heinzel, unpublished observations). In contrast, LPS-induced IFNy by these same cultures is SO-90% inhibited by antiIL12 throughout the course of disease. Late-phase IL12 independence in antigen-driven immunity has also been described in the mouse model of toxoplasmosis (Gazzinelli et al., 1994); Other accessory cell factors, such as B7 costimulatory proteins, may

Although the regulatory effects of IL12 are reasonably well understood in the mouse model of leishmaniasis, the source and control of IL12 synthesis in vivo remain obscure. These uncertainties arise from the inadequacy of current assay techniques. Although total p40 protein and p40 ELISPOT frequencies are measurablein lymph node cultures, the quantity of total p40 produced does not accurately reflect production of bioactive IL12. Furthermore, p40 mRNA and p35mRNA are constitutively expressedin normal spleen, but their anatomic dissociation from each other may not support the production of bioactive heterodimer (Bette et al., 1994). Monoclonal antibody capture bioassay techniques have proven useful for measurement of bioactive IL12 p70 in the serum of endotoxemic mice, but they do not reliably detect lessthan 100 pg/ml of heterodimer (Heinzel er al., 1994). Since total p40 in infected BALB/c and C57BU6 lymph nodes rarely exceeds 500 pg/ml and because ~70 is undetectable in these samples,the ability of anti-IL12 antibody to inhibit antigen-specific IFNy production at these times confirms that very small amounts of bioactive IL12 are sufficient to mediate the immunoregulatory effects observed in vivo (Heinzel et al., 1995 ; Scharton-Kerten et al., 1995). Several studies suggest that production of IL12 is delayed during infection of BALB/c and C57BL/6 mice. The reversal of susceptibility in BALB/c mice by exposure to IL12 in the first week of infection is consistent with underproduction of endogenous IL12 at this time, although the dosesused may be in excess of simple physiologic reconstitution. Expression of p40 mRNA in vivo is also delayed in both strains, suggesting that the presence or absence of IL12 is not a differentiating feature of BALB/c susceptibility compared to C57BL/6 resistance (Reiner et al., 1994). However, strongly resistant C3H mice rapidly increase production of IL12 p40 protein and sustain IL12-dependent natural killer cell hyperplasia and IFNy production in the first three days of infection (Scharton-Kersten et al., 1995). As evident from the studies of Dr. Scott and colleagues, a division of resistant strains of mice into early versus late IL12 responders seemswarranted. Macrophages are the presumed source of IL12 during leishmaniasis, but the inconsistency of

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experimental findings makes confirmation uncertain. Although cultured macrophages produce abundant IL12 upon infection with Toxoplasma gondii (Gazzinelli et al., 1994). none is synthesized following invasion by Leishmania promastigotes (Reiner et al., 1994 ; Vieira ef al., 1994). When peritoneal cells are infected by injection of promastigotes, expression of IL12 as measured by p40 ELISPOT is greatly enhanced, suggesting that additional signals provided in vivo are necessary for IL12 synthesis (Vieira et al., 1994). Others have found that only tissue-phase amastigote forms of L-eishmania induce PCR-detectable p40 mRNA expression in macrophage cultures, although these findings need to be better correlated with evidence of IL12 bioactivity (Reiner et al., 1994), since amastigotes of L.. donovani do not induce production of IFNyin SCID splenocyte cultures (Kaye and Bancroft, 1992) and infection of BALB/c mice initiated by amastigotes remains fully progressive (Sypek et al., 1993). The weak stimulation of IL12 during invasion of macrophages by Leishmania is consistent with previous reports detailing the suppressive effects of parasitederived lipophosphoglycan (Reiner et al., 1994). Although this review has focused on murine leishmaniasis, the peripheral blood mononuclear cells of human subjects with healed visceral leishmaniasis produce 200- 400 pg/ml of p40 when stimulated with antigen and IFNy release in culture is strongly IL12-dependent (Ghalib et al., 1995). Positive regulatory effects mediated by IFNy and negative effects mediated by IL10 were observed. Interestingly, a recombinant Leishmania braziliensis antigen derived from the ribosomal protein eIF4A, induces IL12 and IFNy production in human peripheral blood mononuclear cells (Skeiky et al., 1995). If comparable mechanisms regulate IL12 production by Leishmania-parasitized cells, parasite lysis and the release of these stimulatory cytoplasmic proteins may be an initiating event for IL12 synthesis. Otherwise, comparable antigen-dependent induction of IL12 synthesis in L major infected or immune mice has not been reported (Heinzel et al., 1995 ; Scharton-Kersten et al., 1995). Concluding

remarks

At this time, we cannot conclude if the IL12dependent effects observed during infection represent increased production as a direct result of amastigote and/or promastigote invasion of macrophages (Reiner et al., 1994 ; Vieira et al., 1994), activation by other cytokines - which may include both Thl and Th2 type products - that enable IL12 synthesis in inflammatory macrophages (D’Andrea ef al., 1995; Flesch et al., 1995; Ghalib et al., 1995), or provision of IL12 during cognate activation of T cells by dendritic cells (Macatonia et al, 1995) and

IN IMMUNOLOGY a subset of macrophages that constitutively produce this cytokine (Bette et aI., 1994). What data are available from both mouse and human studies demonstrate that very small amounts of this cytokine critically affect the outcome of disease and that very little upregulation in IL12 synthesis is required to manifest resistance. Specifically, ELISA and ELISPOT results for p40 produced by infected C57BIJ6 lymph node increase less than two-fold during the first two weeks of infection even though anti-IL12 effectively abrogates resistance when administered during this same period of time (Scharton-Kersten et al., 1995). As a complicating issue, p40 homodimers are competitive inhibitors of p70 binding and signal transduction at the high-affinity receptor for mouse IL12, suggesting that the relative amounts of p40 and p70 produced may affect strain-specific immune responses (Gillessen et al., 1995). Because only p40-specific antibodies have been obtained against mouse IL12, a heterodimer-specific ELISA that would resolve these questions is needed. We speculate that p35-knockout mice should efficiently immunize against mouse ~35 and provide the monoclonal antibody reagents necessary for correlating p70 production with immunopathogenesis. This would allow for more precise study of the in vivo regulation of this interesting and vital component of host immunity against intracellular parasitism. Acknowledgements F.P.H. is supported by grants ROI AI35979 and from the National Institutes for Health and by Research Service, Department of Veterans Affairs.

K04 AI01229 the Medical

References Afonso, L.C.C., Scharton, Y.M., Vieira, L.Q., Wysocka, M., Trinchieri, G. & Scott, P. (1994), The adjuvant effect of interleukin-12 in a vaccine against Leishmania major. Science, 263, 235237. Bette, M., Jin, S.C., Germann, T., Schafer, M.K.H., Weihe, E., Rude, E. & Fleischer, B. (1994), Differential expression of mRNA encoding interleukin-12 ~3.5 and p40 subunits in situ. Eur. J. Immunol., 24, 24352440. Chakkalath, H.R. & Titus, R.G. (1994), Leishmania major-parasitized macrophages augment Th2-type T cell activation. J. Immunol.,153, 4378-4387. Corry, D.B., Reiner, S.L., Linsley, P.S. & Locksley, R.M. (1994), Differential effects of blockade of CD28-B7 on the development of Thl or Th2 effector cells in experimental leishmaniasis. J. Immunol., 153, 4142-4158. D’Andrea, A., Ma, X., Aste-Amezaga, M., Paganin, C. & Trinchieri, G. (1995), Stimulatory and inhibitory effects of interleukin (IL)-4 and IL-13 on the production of cytokines by human peripheral blood mononuclear cells: Priming for IL-12 and tumor necrosis factor cc production. J. Exp. Med., 181, 537-546.

IMMUNOREGULATION Doherty, T. & Coffman, R. (1993), Leishmania antigens presented by GM-CSF-derived macrophages protect susceptible mice against challenge with Leishmania major. J. Immunol., 150, 54765483. Finkelman, F.D., Madden, K.B., Cheever, A.W., Katona, I.M., Morris, S.C., Gately, M.K., Hubbard, B.R., Gause, W.C. & Urban, J.F., (1994), Effects of interleukin 12 on immune responses and host protection in mice infected with intestinal nematode parasites. J. Exp. Med., 179, 1563-1572. Flesch, I.E.A., Hess, J.H., Huang, S., Aguet, M., Rothe, J., Bluethmann, H. & Kaufmann, S.H.E. (1995), Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon y and tumor necrosis factor cc. J. Bxp. Med., 181, 1615-1621. Gazzinelli, R.T., Wysocka, M., Hayashi, S., Denkers, E.Y., Hieny, S., Caspar, P., Trinchieri, G. & Sher, A. (1994), Parasite-induced IL-12 stimulates early IFNy synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol., 153, 2533-2543. Ghalib, H.W., Whittle, J.A., Kubin, M., Hashim, F.A., ElHassan, A.M., Grabstein, K.H. & Reed, S.G. (1995), IL-12 enhances Thl-type responses in human Leishmania abnovani infections. J. Immunal., 154,46234629. Gillessen, S., Carvajal, D., Ling, P., Podlaski, F.J., Stremlo, D.L., Familletti, P.C., Gubler, U., Presky, D.H., Stern, A.S. & Gately, M.K. (1995), Mouse interleukin-12 (IL- 12) p40 homodimer : a potent IL12 antagonist. Eur. J. Immunol., 25, 200-206. Heinzel, F.P., Rerko, R.M., Ahmed, F. & Pearlman, E. (1995), Endogenous interleukin-12 (IL-12) is required for control of Th2 CD4+ T cell responses capable of exacerbating leishmaniasis in normally resistant C57BLf6 mice. J. Immunoi., 155, 730-739. Heinzel, F.P., Rerko, R.M., Ling, P., Hakimi, J. & Schoenhaut, D.S. (1994), Interleukin-12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon. Infect. Immun., 62, 4244-4249. Heinzel, F.P., Schoenhaut, D.S., Rerko, R.M., Rosser, L.E. & Gately, M.K. (1993), Recombinant interleukin 12 cures mice infected with Leishmania major. J. Exp. Med., 177, 1505-1509. Hsieh, C.S., Macatonia, S.E., O’Garra, A. & Murphy, K.M. (1995), T cell genetic background determines default T helper phenotype development in vitro. J. Exp. Med., 181, 713-721. Kaye, P.M. & Bancroft, G.J. (1992), Leishmania donovani infection in scid mice: Lack of tissue response and in vivo macrophage activation correlates with failure to trigger natural killer cell-derived gamma interferon production in vitro. Infect. Immun., 60,4335-4342. Macatonia, S.E., Hosken, N.A., Vierea, P., Hsieh, C., Culpepper, J.A., Trinchieri, G., Murphy, K. & G’Garra, A. (1995), Dendritic cells produce IL-12 and direct the development of Thl cells from naive CD4+ T cells. J. Immunol., 154, 5071-5079. Murray, H.W. & Hariprashad, J. (1995) Interleukin 12 is effective treatment for an established systemic intracellular infection: Experimental visceral leishmaniasis. J. Exp. Med., 181, 387-391. Nabors, G.S., Afonso, L.C.C., Farrel, J.P. & Scott, P. (1995), Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy

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with interleukin 12 and Pentostam. Proc. Natl. Acad. Sci., USA, 92, 3142-3146. Pemis, A., Gupta, S., Gollob, K.J., Garfein, E., Coffman, R.L., Schindler, C. & Rothman, P. (1995), Lack of interferon-y receptor B chain and the prevention of interferon y signaling in Tul cells. Science, 269, 245-247. Reiner, S.L. & Locksley, R.M. (1995), The regulation of immunity to Leishmania major. Annu. Rev. Immunol., 13, 151-177. Reiner, S.L., Zheng, S., Wang, Z.E., Stowring, L. & Locksley, R.M. (1994) Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4+ T cells durmg initiation of infection. J. Exp. Med., 179, 447-456. Scharton, T.M. & Scott, P. (1993), Natural killer cells are a source of interferon y that drives differentiation of CD4+ T cell subsets and induces early resistance to Leishmania major in mice. J. Exp. Med., 178, 567577. Scharton-Kersten, T., Afonso, L.C.C., Wysocka, M., Trinchieri, G. & Scott, P. (1995), IL-12 is required for natural killer cell activation and subsequent T helper 1 cell development in experimental leishmaniasis. J. Immunol., 154, 5320-5330. Scott, P. (1991), IFN-y modulates the early development of Thl and Th2 responses in a murine model of cutaneous leishmaniasis. J. Immunol., 147, 3149-3155. Skeiky, Y.A.W., Guderian, J.A., Benson, D.R., Bacelar, O., Carvalho, E.M., Kubin, M., Badaro, R., Trinchieri, G. & Reed, S.G. (1995), A recombinant Leishmania antigen that stimulates human peripheral blood mononuclear cells to express a Thl-type cytokine profile and to produce interleukin 12. J. Exp. Med., 181, 1527-1537. Swihart, K., Fmth, U., Messmer, N., Hug, K., Behin, R., Huang, S., Del Giudice, G., Aguet, M. & Louis, J.A. (1995), Mice from a genetically resistant background lacking the interferon y receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell l-type CD4+ T cell response. J. Enp. Med., 181, 961-971. Sypek, J.P., Chung, C.L., Mayor, S., Subramanyam, J., Goldman, S., Sieburth, D., Wolf, S. & Schaub, R.G. (1993), Resolution of cutaneous leishmaniasis : Interleukin 12 initiates a protective T helper type 1 immune response. J. Exp. Med., 177, 1797-1802. Szabo, S.J., Jacobson, N.G., Dighe, A.S., Gubler, U. & Murphy, K.M. (1995), Developmental commitment to the Th2 lineage by extinction of IL-12 signaling. Immunity, 2, 665-675. Vieira, L.Q., Hondowicz, B.D., Afonso, L.C.C., Wysocka, M., Trinchieri, G. & Scott, P. (1994), Infection with Leishmania major induces interleukin-12 production in vivo. Immunol. Len., 40, 157-161. Wang, Z.E., Reiner, S.L., Zheng, S., Dalton, D.K. & Locksley, R.M. (1994a), CD4+ effector cells defaut to the Th2 pathway in interferon y-deficient mice infected with Leishmania major. J. Exp. Med., 179, 1367-1372. Wang, Z.E., Zheng, S., Cony, D.B., Kalton, D.K., Seder, R.A., Reiner, S.L. & Locksley, R.M. (1994b), Interferon y-independent effects of interleukin 12 administered during acute or established infection due to

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62nd FORUM L.eishmania major. Proc. Natl. Acad. Sci. USA, 91,

12932-12936. Wei, X.Q., Charles, I.G., Smith, A., Ure, J., Feng, G., Huang, F., Xu, D., Muller, W., Moncada & Liew, F.Y. (1995), Altered immune responses in mice lacking inducible nitric oxide syntase. Nature (Lond.), 375, 408-411.

IN IMMUNOLOGY Wynn, T.A., Eltoum, I., Oswald, I.P., Cheever, A.W. & Sher, A. (1994), Endogenous interleukin 12 (IL12) regulates granuloma formation induced by eggs of Schistosoma mansoni and exogenous IL- 12 both inhibits and prophylactically immunizes against egg pathology. J. Exp. Med., 179, 15511561.

COMMENTARY ON HEINZELETAL. By P. Scott:

Heinzel and colleagues conclude that the protective effects of IL12 are primarily related to the suppression of IL4, rather than enhancement of IFNy, and proposes that the protective vaccination with IL12 and SLA that we observed in BALB/c mice functions by suppression of Th2 cell priming. Since Thl and Th2 cells (or IL4 and IFN$ regulate each other so closely in this model, it is sometimes difficult to determine whether suppression of IL4 or enhancement of IFNy production is responsible for alterations in disease outcome. Heinzel’s results suggest that administration of IL12 in BALB/c mice during the infection promoted resistance primarily by inhibiting IL4. However, in the vaccination model, we found that IL12 enhanced both NK cell responses and IFNy production, and that abrogation of either of these inhibited the Thl promoting effects of IL12. An important issue raised by Heinzel is

whether an established cell-mediated type of immune response is dependent upon continued IL12 production. He notes that as the infection progresses the ability of lymph node cells to make IFNy becomes largely lL12-independent. We have observed similar results, although interestingly the ability of Thl clones derived from L. major infected mice to produce IFNy in vitro still remains largely dependent on IL12. The conclusion of Heinzel et a/., that other accessory factors may play a more predominant role during later stages of the infection, seems likely. If this is the case, the continued dependency of the Thl clones on IL12 could be due to the absence of such accessory factors in the antigen presenting cells used for in vitro restimulation of the clones. As pointed out by Heinzel, the role of endogenous IL12 required for cure of leishmaniasis remains controversial. However, we agree with Heinzel’s statement that IL12 is a necessary component for Thl cell development in leishmaniasis.

IL12 as an adjuvant

for vaccines designed to prevent infection and immunopathology by schistosomes T.A. Wynn (*) and A. Sher Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda (MD) 20892 (USA)

Introduction With many infectious diseases,the induction of a pathogen-specific CD4+ T helper cell responsecharacterized by the production of Thl (IFNy) or Th2 (*) To whom

correspondence

should

he addressed.

(IL4, IL5, IL13) type cytokines often dictates the resistance or susceptibility of the host to the infecting organism. Consequently, numerous investigators have sought to dissect the factors which influence Th cell differentiation. The genetic background of