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Previous Article | Next Article 
Infection and Immunity, November 2002, p. 6284-6293, Vol. 70, No. 11
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.11.6284-6293.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021,1 Department of Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304,2 Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021,3 Department of Pathology, New York University School of Medicine, New York, New York 100164
Received 11 March 2002/ Returned for modification 22 May 2002/ Accepted 11 June 2002
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ABSTRACT |
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INTRODUCTION |
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Effective defense towards visceralizing strains, including Leishmania donovani, depends strictly upon T (Th1) cells, and acquired resistance is governed by T-cell- and macrophage-activating cytokines (5, 16, 17, 27, 32, 33, 59). Among the latter (32, 33, 34, 35, 58), interleukin-12 (IL-12) and gamma interferon (IFN-
) play particularly prominent experimental roles. These cytokines initiate and/or drive the basic antileishmanial Th1 cell response and direct the assembly of tissue granulomas, structures within which intracellular parasites are killed by IFN--activated mononuclear phagocytes (17, 32, 33,36-38, 51, 57, 58). In addition, along with host T cells, endogenous IL-12 and IFN- are also required for expression of the leishmanicidal effect of conventional chemotherapy, pentavalent antimony (36, 39-41). Not surprisingly, then, employing pro-host defense cytokines in exogenous form, either alone or with antimony, represents the primary immunotherapeutic approach thus far tested experimentally and/or clinically in visceral infection (3, 4, 13, 29, 32-36, 42, 54, 55, 58).
Downregulating mechanisms have also been identified in both experimental and human visceral leishmaniasis (kala-azar), including Th2 cell-associated responses (2, 5, 11, 18, 19, 22, 27-29, 33, 50, 56). This information, coupled with immunopathogenetic lessons learned from models of cutaneous Leishmania major infection (7, 12, 20, 21, 43, 47, 60), has pointed in a separate immunotherapeutic direction — targeting (inhibiting) the effects of simultaneously induced, suppressive cytokines. While transforming growth factor beta (60) and perhaps Th2 cell-derived IL-4 and/or IL-13 may exert such effects in visceral infection (2, 43, 49, 50), considerable evidence supports a central, deactivating role for endogenous IL-10.
IL-10 is induced in both experimental and human infection, and its broad actions compromise antigen-presenting cell and T-cell costimulatory mechanisms, T-cell proliferation, and secretion of and macrophage responsiveness to activating Th1 cell-associated cytokines (5, 7-9, 11, 18, 19, 22, 28-30, 33, 43, 56, 60). Although not devoid of proinflammatory effects (24), IL-10 clearly has the potential to disable host antileishmanial defense and foster visceral infection and, in parallel, impair responsiveness to chemotherapy (antimony).
To characterize IL-10's role, we studied the behavior of L. donovani in modified mice which lack or overexpress endogenous IL-10 and then tested IL-10 receptor (IL-10R) blockade, used alone or with antimony, as immunotherapy in established infection.
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MATERIALS AND METHODS |
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Visceral infection and tissue response.
groups of three to five mice were injected via the tail vein with 1.5 x 107 hamster spleen-derived L. donovani amastigotes (1 Sudan strain) (43). Visceral infection was followed microscopically using Giemsa-stained liver imprints in which liver parasite burdens were measured by blinded counting of the number of amastigotes per 500 cell nuclei x liver weight in milligrams (Leishman-Donovan units [LDU]) (43). The histologic response to infection was assessed microscopically in liver sections stained with hematoxylin and eosin. The number of granulomas (infected Kupffer cells which had attracted 
5 mononuclear cells) was counted in 100 consecutive 40x fields, and at 100 parasitized foci, the reaction was scored as (i) none (infected Kupffer cell with no mononuclear cell infiltrate) or (ii) developing or mature granuloma (37, 38). The latter were defined as a core of fused infected Kupffer cells surrounded or well infiltrated by numerous mononuclear cells. Granulomas which contained no visible amastigotes were designated parasite-free.
Cytokine mRNA expression.
Total cellular RNA was extracted from liver homogenates from three to four mice per group, and qualitative reverse transcription-PCR using primers for IL-12 p40, IFN-, and ß-actin was carried out as described previously elsewhere (6, 25). Densitometry of amplified bands was performed with a Phosporimager (Molecular Dynamics, Sunnyvale, Calif.), and results were normalized to the density of ß-actin.
Detection of immunoreactive inducible nitric oxide synthase. Formalin-fixed, paraffin-embedded liver sections from three to four mice per group were processed for immunocytochemistry as described previously (6), using an automated immunostainer (Ventana, Tucson, Ariz.) and polyclonal rabbit anti-mouse inducible nitric oxide synthase (1:300) (Calbiochem, La Jolla, Calif.).
Treatment with antimony and/or anti-IL-10R monoclonal antibody. Two weeks after L. donovani challenge (day +14), liver parasite burdens were determined, and mice then received no treatment or a single intraperitoneal injection of antimony (42). Antimony (sodium stibogluconate; Pentostam; Wellcome Foundation Ltd., London, United Kingdom) was given at optimal (500 mg/kg) or suboptimal (25, 50, or 100 mg/kg) doses (42). Seven days later (day +21), mice were sacrificed, and liver parasite burdens were measured. Percent parasite killing was determined as [(day +14 LDU - day +21 LDU in treated mice)/(day +14 LDU)] x 100. Since IL-10 knockout mice rapidly controlled infection, single-dose antimony was given 1 week earlier on day +7, and treatment effects were determined on day +14. Differences in liver parasite burdens were analyzed using a two-tailed Student's t test.
To test IL-10R blockade, normal BALB/c mice were injected intraperitoneally with 0.5 ml of saline containing 1 mg of anti-IL-10R monoclonal antibody (1B1.3A) (1, 10) or 1 mg of an isotype control monoclonal antibody (GL117.41, anti-ß-galactosidase) (DNAX Research Institute). To determine prophylactic effects, monoclonal antibody injections were started 1 day after L. donovani challenge (day +1) and given once weekly; to test therapeutic effects, monoclonal antibody was given once on day +14 with or without a second injection on day +21. To test chemotherapy in the presence of IL-10R blockade, mice were injected once with monoclonal antibody on day +12 and then once with suboptimal antimony 2 days later. This 2-day interval was selected from a preliminary experiment (not shown) in which the efficacy of combined treatment was approximately doubled by giving anti-IL-10R 48 h before versus simultaneously with or 48 h after antimony.
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RESULTS |
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) and suppressive Th2 cell-associated cytokines (IL-4, IL-10) is induced early on in parasitized liver and spleen in normal BALB/c mice (16, 17, 27, 29, 57), their self-curing phenotype suggested little overall impact for either IL-4 or IL-10 in this model (29).
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In direct contrast, initial L. donovani replication in transgenic mice was unrestrained (Fig. 1A). While liver parasite levels then peaked and declined somewhat, there was no further progress towards resolution of infection. In one experiment carried out to week 12, liver burdens in transgenic mice remained persistently high (4,329 ± 462 LDU, n = 3 mice).
Effects on antileishmanial mechanisms.
To characterize IL-10's actions in directing outcome of infection, liver tissue was assayed for granuloma assembly and expression of IL-12 and IFN- mRNAs and inducible nitric oxide synthase reactivity. In this model, granulomas enclose parasitized tissue macrophages with influxing T cells and blood monocytes (37). IL-12 likely initiates and maintains the basic Th1 cell response (17, 36, 51), and IFN- and inducible nitric oxide synthase act together at the level of the macrophage to regulate intracellular L. donovani killing (44, 57).
(i) Granuloma assembly. Livers of both control and IL-10 knockout mice demonstrated little histologic response 1 week after challenge (not shown). By week 2, however, events accelerated markedly in knockout mice: granuloma number was increased, structural maturation with epithelioid changes was advanced, and functional activity (parasite-free granulomas) was well established (Fig. 2 and 3). Similar kinetic (data not shown) and nearly identically enhanced histologic effects were induced in normal BALB/c mice by once-weekly prophylactic treatment with anti-IL-10R (Fig. 4B).
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In IL-10 transgenic mice, early granuloma assembly was inhibited (Fig. 2 and 5). At week 2, for example, 31% ± 3% of parasitized foci showed no mononuclear cell recruitment versus 7% ± 2% in wild-type mice. However, inhibition was transient, since granulomas subsequently developed and the majority evolved to a morphologically mature appearance. Nevertheless, the suppressive effect of IL-10 on macrophage function was clear: <10% of week 4 or week 8 granulomas in transgenic mice were parasite-free, and most remained heavily infected (Fig. 2 and 5F).
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mRNA expression (Fig. 6B) and results for inducible nitric oxide synthase immunoreactivity (Fig. 7), reflecting activity of the effector arm of the antileishmanial Th1 mechanism, were consistent with the preceding correlation and also suggested impaired responses in the presence of excess IL-10.
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100% of parasitized foci in normal, knockout, and wild-type mice but at only one-third of granulomas in infected transgenic animals.
Interaction of IL-10 and chemotherapy.
In vivo expression of antimony's leishmanicidal activity is also Th1 cell dependent and specifically requires and is enhanced by IL-12 and IFN- (36, 39). Since IL-10 targets the same pathway, we treated IL-10 knockout and transgenic mice, anticipating that antimony's efficacy would be upregulated by the absence and downregulated by the overexpression of IL-10.
(i) Effect on initial response to antimony. The results in Fig. 8A confirmed the first part of the preceding hypothesis— suboptimal doses of antimony which had no (25 to 50 mg/kg) or partial static (100 mg/kg) effects in normal animals induced killing in knockout mice. However, initial responses to optimal (500 mg/kg) (Fig. 8B) as well as suboptimal (100 mg/kg) (not shown) antimony were intact in transgenic mice.
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before the response to antimony is impaired. IL-4 is not a determinant of outcome in mouse models of visceral infection (16, 29, 43, 52, 60); however, IL-4 acts synergistically with IL-10 to better suppress in vivo antileishmanial mechanisms (43, 48), and its secretion is not upregulated in these IL-10 transgenic mice (20). Therefore, antimony was tested in BALB/c mice immunized with heat-killed L. major promastigotes. This procedure conditions normal mice to cross react to L. donovani with a Th2 cell-type response, dependent upon both IL-10 and IL-4, which induces the noncure phenotype (43). Two-week-infected heat-killed L. major promastigote-stimulated mice, however, responded normally to single-dose antimony treatment (86% L. donovani killing induced by 500 mg/kg, three experiments, n = 10 mice), indicating that in the presence of increased IL-4, excess endogenous IL-10 also did not impair antimony's visceral efficacy.
(iii) Recurrence of infection posttreatment. Although antimony was active in transgenic mice at the time drug was given, we also tested whether persistent IL-10-associated deactivation could undermine the durability of the treatment response. Wild-type and transgenic mice were injected with optimal-dose antimony and left undisturbed for an additional 10 weeks. In wild-type controls, the parasite load declined further after treatment to low levels (Fig. 8B). In contrast, liver burdens increased fivefold in transgenic mice, suggesting that once the drug effect waned, sustained IL-10 can foster progression of residual infection.
Treatment of established infection with anti-IL-10R. Two of the prior findings appeared to have therapeutic application: prophylactic anti-IL-10R treatment induced rapid control over L. donovani (Fig. 1B), and antimony's activity was enhanced in the absence of IL-10 (Fig. 8A). To translate these observations into a treatment approach, we completed this study by using normal BALB/c animals with established visceral infection to test two related hypotheses — that blocking IL-10's effect would (i) induce parasite killing, and at the same time, (ii) also augment host responsiveness to chemotherapy.
(i) Effect of anti-IL-10R alone. Fourteen days after infection, mice were given a single injection of anti-IL-10R. This treatment enhanced granuloma formation (Fig. 5D) and proved surprisingly active in inducing appreciable parasite killing (65%) within 7 days (Fig. 9A). With no further treatment, this antileishmanial effect persisted for an additional week (e.g., to day +28). Injecting anti-IL-10R twice, on days +14 and +21, led to near resolution of infection by day +28, at which time parasite burdens were 11-fold lower than in mice treated twice with control monoclonal antibody.
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This high-level leishmanicidal effect was similar to the 89% killing induced in control mice treated on day +14 with 10-fold more antimony (500 mg/kg). While again indicating the activity of treatment with anti-IL-10R alone (group 3), the results in Fig. 9B also show that low-dose antimony reduced the day +21 liver burden in anti-IL-10R-treated mice by an additional 63%. In control and control monoclonal antibody-injected mice, corresponding reductions induced by the same antimony treatment were 15 and 19%, respectively. Thus, in the presence of IL-10R blockade, there was a threefold increase in the efficacy of chemotherapy.
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DISCUSSION |
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The findings described here, those recently reported in L. donovani- and L. major-infected IL-10 knockout mice (7, 21, 31) and L. major-infected IL-10 transgenic mice (20), and previous results with other diverse pathogens (30) together reemphasize the breadth of IL-10's regulatory effects in experimental infections. IL-10's actions appear particularly relevant in promoting intracellular infections in which host defense is governed by Th1 cells, mediated by cytokines including IL-12 and IFN-, and requires activated macrophages. In such infections, reducing IL-10 activity almost invariably increases resistance (30), even in the host with an established, apparently satisfactory Th1 cell response.
This effect, demonstrated in our normal BALB/c mice treated therapeutically with anti-IL-10R monoclonal antibody, has also been well illustrated in C57BL/6 mice chronically infected with L. major (7). While these animals, considered resistant to L. major, healed their primary skin lesions, persistent, low-level cutaneous infection was not eliminated until the mice were injected with anti-IL-10R (7). Thus, the benefits of inhibiting IL-10's effects also extend beyond the acute stage of infection.
Primary Th1 cell-mediated antileishmanial events — restraint of initial L. donovani replication, granuloma assembly, inducible nitric oxide synthase expression, rapid resolution of infection (acquired resistance), and responsiveness to chemotherapy (29, 33, 36, 37, 39, 44, 57) — were all more prominent and/or more efficiently induced in animals devoid of IL-10. Each of the preceding responses requires IFN-, largely induced by IL-12 (33, 36, 51). Murphy and colleagues (31) have reported that restimulated spleen cells from the same infected knockout mice show increased IFN- secretion in vitro, which in turn correlated with accelerated tissue expression of inducible nitric oxide synthase and similarly rapid L. donovani killing.
While leishmanicidal activity in IL-10 knockout animals clearly requires both endogenous IL-12 and IFN-, treatment of knockout mice with neutralizing anti-IL-12 or anti-IFN- monoclonal antibody did not fully restore parasite replication (31). This finding may reflect incompletely neutralized, residual IL-12 or IFN- acting on sensitized or more efficiently activated effector cells no longer handcuffed by IL-10. Alternatively, the observation also suggests that the generalized immunoenhanced state in knockout animals may be supported by other antileishmanial mediators ordinarily limited by IL-10, for example, tumor necrosis factor (9, 58).
Responses in L. donovani-infected IL-10 transgenic mice appeared to be more complex than simply the result of persistent, global suppression of Th1 cell mechanisms. Induction of Th1 cell-dependent events was indeed initially suppressed (except for responsiveness to antimony), entirely consistent with IL-10's recognized effects (30), and associated with a rapid intracellular parasite replication. With time (e.g., by week 4), however, granuloma assembly emerged in transgenic mice accompanied by some inducible nitric oxide synthase expression. Thus, in this second phase, in which infection was converted from unrestrained to high-level chronic, IL-10's direct macrophage-deactivating effects (9, 30) may have assumed more prominence. The presence of heavily parasitized macrophages within structurally mature granulomas 8 weeks after infection likely reflected this state of deactivation. That IL-10 can act primarily at the level of the macrophage and inhibit the effector function of the Th1 cell response without necessarily impairing IL-12 or IFN- secretion has been reported in IL-10 transgenic mice (20, 46).
We would have predicted that if IL-10 overexpression inhibited one Th1 cell-dependent antileishmanial mechanism, it would likely inhibit all such responses. However, the intact initial response to antimony in transgenic mice and their granuloma assembly (albeit delayed) suggested compensatory mechanisms unrelated to Th1 cell cytokines or impervious to IL-10. For example, expression of ICAM-1, required for both granuloma formation and optimal antimony efficacy (45), is preserved in these transgenic mice (20). However, these observations also introduce the notion of differential in vivo sensitivity to IL-10, as previously documented in vitro for macrophage mechanisms ranging from tumor necrosis factor release to secretion of microbicidal reactive oxygen and nitrogen intermediates (9).
Thus, despite a common denominator (strict requirement for T cells, IFN-, and IL-12), the basic Th1 cell- and cytokine-mediated responses in this model showed hierarchical inhibition by sustained IL-10: (i) acquired resistance, dependent upon macrophage activation and induction of inducible nitric oxide synthase (44), appears most susceptible to deactivation, (ii) granuloma-initiating events show transient susceptibility, and (iii) the mechanism underlying expression of antimony's in vivo leishmanicidal effect appears resistant.
Since these mechanisms in L. donovani-infected mice are complex, differential in vivo sensitivity to IL-10 may well reflect the influence of more than one factor. Two possibilities include effects on endogenous tumor necrosis factor which variably contributes to each of the preceding three antileishmanmial responses (40, 58) and is readily suppressed by IL-10 (9), or different tissue thresholds (or ratios of IFN- to IL-10 [46]) which need to be reached for satisfactory expression of cytokine-induced antileishmanial events. Thus, even in the presence of excess IL-10, some Th1 cell reactivity induced in infected transgenic mice might be sufficient to exceed the threshold required for antimony responsiveness and granuloma assembly but not for induction of acquired resistance.
The preceding speculation about thresholds extends to using anti-IL-10R monoclonal antibody as treatment. Although inhibition of IL-10R signaling was globally active in enhancing the Th1 cell-dependent responses measured (parasite killing [macrophage activation], granuloma assembly, and antimony efficacy), we did not test if inflammatory host defense responses could be enhanced in a selective fashion. Narrower anti-IL-10R-induced immunostimulation might well prove critical in other infections associated with inflammation, but was not necessary in this model or in C57BL/6 mice chronically infected with L. major (7).
While anti-IL-10R treatment clearly accelerated granuloma assembly in infected livers, repeated injections did not release uncontrolled inflammation. This finding, together with the self-limited histologic response in knockout mice, indicated that IL-10 is not required to terminate the inflammatory response initiated by L. donovani. In some models of infection caused by other pathogens, however, IL-10 knockout mice have indeed developed runaway, sometimes fatal hyperinflammation — a pathological state thought to be largely induced by exaggerated and/or unopposed secretion of tumor necrosis factor, IL-12, and/or IFN- (30). In our anti-IL-10R-treated and IL-10 knockout animals, multiple factors may have limited such potential tissue damage, including other downregulating cytokines induced by L. donovani infection (transforming growth factor beta, IL-13, and possibly IL-4 [2, 33, 50, 60]), the apparent paucity of infiltrating T cells at parasitized foci, and the absence of a compensatory neutrophil response (14, 53).
Our findings in transgenic mice implicated sustained IL-10 secretion in relapse after chemotherapy, correlating with clinical observations (18, 56). Nonetheless, IL-10 overexpression neither by itself nor accompanied by increased IL-4 impaired the initial response to antimony. In BALB/c mice with a polarized, IL-4-driven Th2 cell response induced by cutaneous L. major infection, responsiveness to antimony is also intact during the time drug is administered (47). However, as in our treated IL-10 transgenic mice, L. major infection also recurred once the drug was discontinued, suggesting that sustained IL-10- or IL-4-induced deactivation likely promoted progression of residual visceral or cutaneous infection posttreatment.
In the L. major model, cotreatment with anti-IL-4 monoclonal antibody plus antimony prevented recurrence of local infection and led to eventual cure (47). However, multiple studies (33), including those in IL-4 knockout, IL-4 transgenic, and anti-IL-4-treated normal BALB/c mice (29, 43, 52), have shown that IL-4 plays little or no role in experimental L. donovani infection and is therefore not a candidate for neutralization. IL-4 is also not consistently expressed in human kala-azar (2, 19, 22, 23, 56).
Our results in established visceral infection in normal mice support two potential applications for anti-IL-10R monoclonal antibody: first, as monotherapy, since injection of monoclonal antibody by itself induced rapid leishmanicidal activity; and second, in combination with chemotherapy to stimulate a synergistic effect. We do not yet know how blockade of IL-10R signaling is actually translated into L. donovani killing in vivo. Results reported by Belkaid and colleagues in anti-IL-10R-treated mice with chronic L. major infection do not point to IFN- upregulation or a more prominent immune response (7). Rather, the results suggest that macrophage responsiveness to the existing level of Th1 cell reactivity is enhanced by removing the limiting effects of IL-10 (7).
This conclusion conforms to the notion that even physiologic levels of IL-10, produced normally in response to inflammatory microbial stimuli, are capable of restraining the efficacy of the Th1 cell mechanism (30). Thus, as our results in established infection in normal mice suggest, the potential therapeutic application of anti-IL-10R monoclonal antibody extends beyond states in which IL-10 is pathologically elevated, provided the infected host has or is capable of expressing a satisfactory level of Th1 cell reactivity.
The BALB/c mice used in our anti-IL-10R treatment experiments fulfilled this requirement (29, 33, 38); studies in human visceral infection also indicate simultaneous expression of a Th1 cell cytokine response, albeit reduced or deactivated (15, 19, 22, 23, 33, 56). IL-10R blockade also appears to represent a viable alternative to administering activating Th1 cytokines in exogenous form (33, 38), the prior strategy employed to induce immunoenhancement in this L. donovani model.
Used in conjunction with antimony, conventional chemotherapy for visceral leishmaniasis in most regions (32, 33), anti-IL-10R treatment also allowed a substantial reduction (10-fold) in the dose of antimony required to kill the majority of liver amastigotes. Such an effect is similar to that achieved in the initial experimental approach to immunochemotherapy in which exogenous IFN- or IL-12 was combined with antimony (36, 42). In human visceral infection, antimony is administered daily for 28 days (33). We suspect that anti-IL-10R injection could also be used to reduce treatment duration, and experiments to test this effect are under way. Since antimony toxicity may relate to drug accumulation (32), reduction in either dose or duration of therapy permitted by coadministration of anti-IL-10R, might also diminish toxicity while making conventional treatment more effective and less arduous.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Present address: Dynavax Technologies, Berkeley, CA 94710.
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
| 1. | Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, and F. Powrie. 1999. An essential role for IL-10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190:995-1004. |
| 2. | Babaloo, Z., P. M. Kaye, and M. B. Eslami. 2001. IL-13 in Iranian patients with visceral leishmaniasis: relationship to other Th2 and Th1 cytokines. Trans. R. Soc. Trop. Med. Hyg. 95:1-4.[CrossRef][Medline] |
| 3. | Badaro, R., and W. Johnson. 1993. The role of interferon- in the treatment of visceral and diffuse cutaneous leishmaniasis. J. Infect. Dis. 167(Suppl. 1):13-17.[Medline] |
| 4. | Badaro, R., C. Nascimento, J. S. Carvalho, F. Badaro, D. Russo, J. L. Ho, S. G. Reed, and T. C. Jones. 1994. Recombinant human granulocyte-macrophage colony-stimulating factor reverses neutropenia and reduces secondary infections in visceral leishmaniasis. J. Infect. Dis. 170:413-418.[Medline] |
| 5. | Barcellar, O., A. D'Oliveira, S. Jeronimo, and E. M. Carvalho. 2000. IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis. Cytokine 12:1228-1231.[CrossRef][Medline] |
| 6. | Bekker, L. G., A. L. Moreira, A. Bergtold, S. Freeman, B. Ryffel, and G. Kaplan. 2000. Immunopathologic effects of tumor necrosis factor alpha in murine mycobacterial infection are dose dependent. Infect. Immun. 68:6954-6959. |
| 7. | Belkaid, Y., K. F. Hoffmann, S. Mendez, S. Kamhawi, M. C. Udey, T. A. Wynn, and D. L. Sacks. 2001. The role of interleukin (IL)-10 in the persistence of Leishmania major in the skin after healing and the therapeutic potential of anti-IL-10 receptor antiobody for sterile cure. J. Exp. Med. 194:1497-1506. |
| 8. | Bhattacharyya, S., S. Ghosh, P. L. Jhonson, S. K. Bhattacharya, and S. Majumdar. 2001. Immunomodulatory role of interleukin-10 in visceral leishmaniasis: defective activation of protein kinase C-mediated signal transduction events. Infect. Immun. 69:1499-1507. |
| 9. | Bogdan, C., Y. Vodovotz, and C. F. Nathan. 1991. Macrophage deactivation by interleukin 10. J. Exp. Med. 174:1549-1555. |
| 10. | Castro, A. G., M. Neighbors, S. D. Hurst, F. Zonin, R. A. Silva, Y-J. Liu, and A. O'Garra. 2000. Anti-interleukin 10 receptor monoclonal antibody is an adjuvant for T helper cell type 1 responses to soluble antigen only in the presence of lipopolysaccharide. J. Exp. Med. 192:1529-1534. |
| 11. | Cenini, P., N. Berhe, A. Hailu, K. McGinnes, and D. Frommel. 1993. Mononuclear cell subpopulations and cytokine levels in human visceral leishmaniasis before and after chemotherapy. J. Infect. Dis. 168:986-993.[Medline] |
| 12. | Chatelain, R., S. Mauze, and R. L. Coffman. 1999. Experimental Leishmania major infection in mice: role of IL-10. Parasite Immunol. 21:211-217.[CrossRef][Medline] |
| 13. | Curry, A., and P. M. Kaye. 1992. Recombinant interleukin-1 alpha augments granuloma formation and cytokine production but not parasite clearance in mice infected with Leishmania donovani. Infect. Immun. 60:4422-4424. |
| 14. | Deckert, M., S. Soltek, G. Geginat, S. Lutjen, M. Montesinos-Rongen, H. Hof, and D. Schluter. 2001. Endogenous interleukin-10 is required for prevention of a hyperinflammatory intracerebral immune response in Listeria monocytogenes meningoencephalitis. Infect. Immun. 69:4561-4571. |
| 15. | de Medeiros, I., A. Castelo, and R. Saloma. 1998. Presence of circulating levels of interferon-, interleukin-10 and tumor necrosis factor- in patients with visceral leishmaniasis. Rev. Inst. Med. Trop. S. Paulo 40:31-34.[Medline] |
| 16. | Engwerda, C. R., S. C. Smelt, and P. M. Kaye. 1996. An in vivo analysis of cytokine production during Leishmania donovani infection in scid mice. Exp. Parasitol. 84:195-202.[CrossRef][Medline] |
| 17. | Engwerda, C. R., M. L. Murphy, S. E. Cotterell, S. C. Smelt, and P. M. Kaye. 1998. Neutralization of IL-12 demonstrates the existence of discrete organ-specific phases in the control of Leishmania donovani. Eur. J. Immunol. 28:669-680.[CrossRef][Medline] |
| 18. | Gassim, S., A. M. El-Hassan, E. A. G. Khalil, A. Ismail, A. M. Y. Kadarn, A. Kharazmi, and T. G. Theander. 1998. High levels of plasma IL-10 and expression of IL-10 by keratinocytes during visceral leishmaniasis predict subsequent development of post-kala-azar dermal leishmaniasis. Clin. Exp. Immunol. 111:64-69.[CrossRef][Medline] |
| 19. | Ghalib, H. W., M. R. Piuvezam, YA. W. Skeily, M. Siddig, F. A. Hasim, A. M. El-Hassan, D. M. Russo, and S. G. Reed. 1993. Interleukin-10 production correlates with pathology in human Leishmania donovani infection. J. Clin. Investig. 92:324-329. |
| 20. | Groux, H., F. Cottrez, M. Rouleau, S. Mauze, S. Aantonenko, S. Hurst, T. McNeil, M. Bigler, M-G. Roncarolo, and R. L. Coffman. 1999. A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells. J. Immunol. 62:1723-1729. |
| 21. | Kane, M. M., and D. M. Mosser. 2001. The role of IL-10 in promoting disease progression in leishmaniasis. J. Immunol. 166:1141-1147. |
| 22. | Karp, C. L., S. H. El-Safi, T. A. Wynn, M. M. H. Satti, A. N. Kordofani, F. A. Hashim, M. Hag-Ali, F. A. Neva, T. B. Nutman, and D. L. Sacks. 1993. In vivo cytokine profiles in patients with kala-azar. Marked elevation of both interleukin-10 and interferon-gamma. J. Clin. Investig. 91:1644-1648. |
| 23. | Kenney, R. T., D. L. Sacks, A. A. Gam, H. W. Murray, and S. Sundar. 1998. Splenic cytokine responses in Indian kala-azar before and after treatment. Clin. Infect. Dis. 27:215-219.[Medline] |
| 24. | Kuhn, R., J. Lohler, R. Rennick, K. Rajewsky, and W. Muller. 1993. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 75:263-274.[CrossRef][Medline] |
| 25. | Laochumroonvorapong, P., J. Wang, C.-C. Liu, W. Ye, A. L. Moreira, K. B. Elkon, V. H. Freedman, and G. Kaplan. 1997. Perforin, a cytotoxic molecule which mediates cell necrosis, is not required for the early control of mycobacterial infection in mice. Infect. Immun. 65:127-132.[Abstract] |
| 26. | McElrath, J. J., H. W. Murray, and Z. A. Cohn. 1988. The dynamics of granuloma formation in experimental visceral leishmaniasis. J. Exp. Med. 167:1927-1937. |
| 27. | Melby, P. C., Y-Z. Yang, J. Cheng, and W. Zhao. 1998. Regional differences in the cellular immune response to experimental cutaneous or visceral infection with Leishmania donovani. Infect. Immun. 66:18-27. |
| 28. | Melby, P. C., V. V. Tyron, B. Chandrasekar, and G. L. Freeman. 1998. Cloning of Syrian hamster (Merocricetus auratus) cytokine cDNAs and analysis of cytokine mRNA expression in experimental visceral leishmaniasis. Infect. Immun. 66:2135-2142. |
| 29. | Miralles, G. D., M. Y. Stoeckle, D. F. McDermott, F. D. Finkelman, and H. W. Murray. 1994. Induction of Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis. Infect. Immun. 62:1058-1063. |
| 30. | Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O'Garra. 2001. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19:683-765.[CrossRef][Medline] |
| 31. | Murphy, M. L., U. Wille, E. N. Villegas, C. A. Hunter, and J. P. Farrell. 2001. IL-10 mediates suscpetibility to Leishmania donovani infection. Eur. J. Immunol. 31:2848-2856.[CrossRef][Medline] |
| 32. | Murray, H. W. 2000. Treatment of visceral leishmaniasis (kala-azar): a decade of progress and future approaches. Int. J. Infect. Dis. 4:158-177.[CrossRef][Medline] |
| 33. | Murray, H. W. 2001. Clinical and experimental advances in treatment of visceral leishmaniasis. Antimicrob. Agents Chemother. 45:2185-2197. |
| 34. | Murray, H. W., G. D. Miralles, M. Y. Stoeckle, and D. F. McDermott. 1993. Role and effect of interleukin-2 in experimental visceral leishmaniasis. J. Immunol. 151:929-934.[Abstract] |
| 35. | Murray, H. W., J. Cervia, J. Hariprashad, A. P. Taylor, M. Y. Stoeckle, and H. Hockman. 1995. Effect of granulocyte-macrophage colony-stimulating factor in experimental visceral leishmaniasis. J. Clin. Investig. 95:1183-1189. |
| 36. | Murray, H. W., C. Montelibano, R. Peterson, and J. P. Sypek. 2000. Interleukin 12 regulates the response to chemotherapy in experimental visceral leishmaniasis. J. Infect. Dis. 182:1497-1502.[CrossRef][Medline] |
| 37. | Murray, H. W. 2001. Tissue granuloma structure-function in experimental visceral leishmaniasis. Int. J. Exp. Pathol. 82:249-276.[CrossRef][Medline] |
| 38. | Murray, H. W. 1999. Granulomatous inflammation: host antimicrobial defense in the tissues in visceral leishmaniasis, p. 977-994. In J. Gallin, R. Synderman, D. Fearon, B. Haynes, and C. Nathan (ed.), Inflammation: basic principles and clinical correlates, 3rd ed. Lippincott-Raven, Philadelphia, Pa. |
| 39. | Murray, H. W., and S. Delph-Etienne. 2000. Role of endogenous gamma interferon and macrophage microbicidal mechanisms in host response to chemotherapy in experimental visceral leishmaniasis. Infect. Immun. 68:288-293. |
| 40. | Murray, H. W., A. Jungbluth, E. Ritter, C. Montelibano, and M. W. Marino. 2000. Visceral leishmaniasis in mice devoid of tumor necrosis factor and response to treatment. Infect. Immun. 68:6289-6293. |
| 41. | Murray, H. W., M. J. Oca, A. M. Granger, and R. D. Schreiber. 1989. Successful response to chemotherapy in experimental visceral leishmaniasis: requirement for T cells and effect of lymphokines. J. Clin. Investig. 83:1254-1259. |
| 42. | Murray, H. W., J. D. Berman, and S. D. Wright. 1988. Immunochemotherapy for intracellular Leishmania donovani infection: interferon-gamma plus pentavalent antimony. J. Infect. Dis. 157:973-978.[Medline] |
| 43. | Murray, H. W., J. Hariprashad, and R. L. Coffman. 1997. Behavior of visceral Leishmania donovani in an experimentally induced Th2 cell-associated response model. J. Exp. Med. 185:867-874. |
| 44. | Murray, H. W., and C. F. Nathan. 1999. Macrophage microbicidal mechanisms in vivo: reactive nitrogen versus oxygen intermediates in the killing of intracellular visceral Leishmania donovani. J. Exp. Med. 189:741-746. |
| 45. | Murray, H. W. 2000. Mononuclear cell recruitment, granuloma assembly, and response to treatment in experimental visceral leishmaniasis: intracellular adhesion molecule 1-dependent and -independent regulation. Infect. Immun. 68:6294-6299. |
| 46. | Murray, P. J., L. Wang, C. Onufryk, R. I. Tepper, and R. A. Young. 1997. T-cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J. Immunol. 158:315-321.[Abstract] |
| 47. | Nabors, G. S. 1997. Modulating ongoing Th2-cell responses in experimental leishmaniasis. Parasitol. Today 13:76-79.[CrossRef][Medline] |
| 48. | Powrie, F., S. Menon, and R. L. Coffman. 1993. IL-4 and IL-10 synergize to inhibit cell-mediated immunity in vivo. Eur. J. Immunol. 23:2223-2229.[Medline] |
| 49. | Quinnell, R. J., O. Courtenay, M-A. Shaw, M. J. day, L. M. Garcez, C. Dye, and P. M. Kaye. 2001. Tissue cytokine responses in canine visceral leishmaniasis. J. Infect. Dis. 183:1421-1424.[CrossRef][Medline] |
| 50. | Sang, D. K., J. H. Ouma, C. C. John, C. C. Whalen, C. L. King, A. A. Mahmoud, and F. P. Heinzel. 1999. Increased levels of soluble interleukin-4 receptor in the sera of patients with visceral leishmaniasis. J. Infect. Dis. 179:743-746.[CrossRef][Medline] |
| 51. | Satoskar, A. R., S. Rodig, S. R. Telford, A. A. Satoskar, S. K. Ghosh, F. von Lichtenberg, and J. R. David. 2000. IL-12 gene-deficient C57BL/6 mice are susceptible to Leishmania donovani but have diminished hepatic pathology. Eur. J. Immunol. 30:834-839.[CrossRef][Medline] |
| 52. | Satoskar, A., H. Bluethmann, and J. Alexander. 1995. Disruption of the murine interleukin-4 gene inhibits disease progession during Leishmania mexicana infection but does not increase control of Leishmania donovani infection. Infect. Immun. 63:4894-4899.[Abstract] |
| 53. | Sewnath, M. E., D. P. Olszyna, R. Birjmohun, F. J. W. ten Kate, D. J. Gouma, and T. van der Poll. 2001. IL-10-deficient mice demonstrate multiple organ failure and increased mortality during Escherichia coli peritonitis despite an accelerated bacterial clearance. J. Immunol. 166:6323-6331. |
| 54. | Sundar, S., and H. W. Murray. 1997. Response to interferon- plus antimony in Indian visceral leishmaniasis. J. Infect. Dis. 176:1117-1121.[Medline] |
| 55. | Sundar, S., and H. W. Murray. 1995. Effect of treatment with interferon-gamma alone in Indian visceral leishmaniasis. J. Infect. Dis. 172:1627-1629.[Medline] |
| 56. | Sundar, S., S. G. Reed, S. Sharma, A. Mehrota, and H. W. Murray. 1997. Circulating Th1 cell- and Th2 cell-associated cytokines in Indian patients with visceral leishmaniasis. Am. J. Trop. Med. Hyg. 56:522-526. |
| 57. | Taylor, A., and H. W. Murray. 1997. Intracellular antimicrobial activity in the absence of interferon-: effect of interleukin 12 in experimental visceral leishmanaisis in interferon- gene-disrupted mice. J. Exp. Med. 185:1231-1239. |
| 58. | Tumang, M., C. Keogh, L. L. Moldawer, R. Teitelbaum, J. Hariprashad, and H. W. Murray. 1994. The role and effect of tumor necrosis factor-alpha in experimental visceral leishmaniasis. J. Immunol. 153:768-775.[Abstract] |
| 59. | Wilson, M. E., M. Sandor, A. M. Blum, B. M. Young, A. Metwali, D. Elliott, R. G. Lynch, and J. V. Weinstick. 1996. Local suppression of IFN- in hepatic granulomas correlates with tissue-specific replication of Leishmania chagasi. J. Immunol. 156:2231-2239.[Abstract] |
| 60. | Wilson, M. E., B. M. Young, B. L. Davidson, K. A. Mente, and S. E. McGowan. 1998. The importance of transforming growth factor beta-ß in murine visceral leishmaniasis. J. Immunol. 161:6148-6155. |
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