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Interleukin 10 receptor blockade—pentavalent antimony treatment in experimental visceral leishmaniasis
Acta Tropica 93 (2005) 295–301
Interleukin 10 receptor blockade—pentavalent antimony treatment in experimental visceral leishmaniasis Henry W. Murray ∗ Department of Medicine, Weill Medical College of Cornell University, Box 136, 1300 York Avenue, NY 10021, USA Received 20 February 2004; received in revised form 20 July 2004; accepted 11 November 2004
Abstract Interleukin 10 (IL-10), a suppressive Th2 cell-type cytokine, promotes disease progression in experimental visceral leishmaniasis. To extend testing the therapeutic effects of applying IL-10 receptor (IL-10R) blockade with antileishmanial chemotherapy, BALB/c mice with established intracellular Leishmania donovani infection were injected once with anti-IL-10R mAb at the time low-dose, daily pentavalent antimony (Sb) therapy was initiated. In this treatment model, simultaneous administration of anti-IL-10R enhanced overall antileishmanial activity in the liver in an interferon-␥-dependent fashion, and accelerated the kinetics of Sb-associated killing, induced a >10-fold Sb dose–sparing effect and shortened the required duration of Sb treatment. These results suggest the possibility of using mAb-induced IL-10R blockade to develop low-dose and/or short-course immunochemotherapeutic regimens in visceral leishmaniasis. © 2005 Elsevier B.V. All rights reserved. Keywords: Leishmania donovani; Visceral leishmaniasis; IL-10; Anti-IL-10 receptor; Pentavalent antimony
1. Introduction Harnessing the host’s T cell-dependent immune response and applying it with antileishmanial chemotherapy represents one strategy to enhance both treatment efficacy and efficiency in visceral leishmaniasis, a disseminated intracellular protozoal disease (Murray, 2001a). In experimental Leishmania donovani infection, in which macrophages in liver, spleen ∗
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and bone marrow are targeted, successful host defense is T cell-dependent and initially regulated by multiple activating cytokines secreted by both Th1and Th2-type cells (Engwerda et al., 1998; Satoskar et al., 2000; Alexander et al., 2000; Murray, 2001a, 2001b; Stager et al., 2003). If unimpeded, this response induces tissue macrophage activation, intracellular parasite killing and control over L. donovani infection in the liver (Murray, 2001a, 2001b). In each of these expressions of acquired resistance, interleukin 12 (IL-12) and interferon-␥ (IFN-␥, Th1 cell-type cytokines, play particularly prominent roles (Engwerda
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et al., 1998; Satoskar et al., 2000; Murray, 2001a, 2001b). In L. donovani-infected mice, interventions which strengthen Th1 cell reactivity and enhance cytokine secretion and/or induce macrophage activation have been identified and combined with chemotherapy (Ghosh et al., 2003; Murray and Hariprashad, 1995; Murray et al., 1988, 2000a, 2002, 2003a, 2003b, 2003c; Murray 2001a; Murphy et al., 1998; Zubairi et al., 2004). One of these approaches, inhibiting the action of endogenous IL-10 by monoclonal antibody (mAb)-induced blockade of its receptor (Murray et al., 2003a, 2003b), has additional appeal since IL-10 is instrumental in promoting progressive L. donovani infection (Murphy et al., 2001; Murray et al., 2002). IL-10, well recognized as a suppressive-type cytokine produced by multiple cells (Moore et al., 2001), extinguishes the capacity to kill parasites in the tissues by disabling both the afferent and efferent arms of the inflammatory Th1 cell mechanism, limiting IL-12 and IFN-␥ secretion and deactivating macrophages (Moore et al., 2001; Murphy et al., 2001; Murray et al., 2002, 2003a, 2003b). The same Th1 cell cytokine response targeted by IL10 also interdigitates with pentavalent antimony (Sb), conventional antileishmanial chemotherapy (Murray et al., 1989, 1993; Murray 2001a; Escobar et al., 2001). Expression of Sb’s leishmanicidal effect in experimental visceral infection, for example, requires not only T cells but multiple cytokines, including IL-12 and IFN-␥ as well as tumor necrosis factor (TNF) and IL4 (Alexander et al., 2000; Murray et al., 1989, 2000a, 2000b; Murray and Delph-Etienne, 2000; Escobar et al., 2001). The paradoxical role of IL-4, ordinarily considered a suppressive-type cytokine, in the response to Sb appears to reflect IL-4’s less well-appreciated capacity to foster Th1 cell development and regulate initial IFN-␥ secretion (Alexander et al., 2000). In view of the regulatory role of IL-10 in the L. donovani model, BALB/c mice with established infection were injected with anti-IL-10 receptor mAb (anti-IL10R) to free up Th1 responses, including IL-12 and IFN-␥ secretion, and then 2 days later were treated with Sb (Murray et al., 2003, 2003b). The results in this model of combination therapy, in which a single injection of Sb was tested, indicated that IL-10R blockade enhanced the efficacy of chemotherapy, provided that mAb was administered before Sb (Murray et al., 2003, 2003b).
Building on the preceding result, the present study employed a different Sb treatment protocol to more fully define the beneficial effects of combination with IL-10R blockade. Treating L. donovani-infected mice with daily Sb, as the drug is used clinically in visceral leishmaniasis (kala-azar) (Murray, 2001a), permitted discrete, therapeutically relevant questions about this form of immunochemotherapy to be addressed – is pretreatment with anti-IL-10R required, what are the Sb dose–sparing effects of anti-IL-10R, does IL-10R blockade accelerate the kinetics of Sb-induced parasite killing and can Sb be combined with anti-IL-10R in a short-course, low-dose regimen?
2. Materials and methods 2.1. Mice and visceral infection Female BALB/c mice (20–30 g) (Charles Rivers Laboratories, Wilmington, MA) were injected via the tail vein with 1.5 × 107 hamster spleen-derived L. donovani amastigotes (1 Sudan strain) (Murray et al., 2002). 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 × liver weight (mg) (Leishman–Donovan units, LDU) (Murray et al., 2002). 2.2. Treatments Starting 14 days after infection (day +14), groups of three to five mice were injected i.p. once daily with Sb (sodium stibogluconate, Pentostam, Wellcome Foundation Ltd., London, UK) (Murray et al., 1988). Sb was given for up to 5 days (days +14 to +18) at 1–50 mg/kg/day in 0.2 ml of saline, and parasite burdens (LDU) were determined on day +17 (1 day after the third Sb injection) or on day +21 (3 days after the fifth injection). Day +17 or day +21 LDU were compared to day +14 LDU to determine percent parasite killing (Murray et al., 2002); differences between mean values were analyzed by a two-tailed Student’s t-test. To demonstrate the effects of combination treatment, mice were injected once i.p. on day +14 with 0.2 ml of saline containing anti-IL-10R mAb (1B1.3A)
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or an isotype control mAb (GL117.41, anti-betagalactosidase), provided by Dr. A. Beebe (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA) (Murray et al., 2002). Anti-IL-10R was used at a suboptimal dose (0.1 mg) (Murray et al., 2002, 2003b), and was given 2 h after the first injection of Sb, except where indicated. In some experiments, 1 h prior to anti-IL-10R treatment, mice were also injected once i.p. with 0.2 ml of either normal rabbit serum or rabbit anti-mouse IFN-␥ antiserum raised against murine recombinant IFN-␥. At a dilution of 1:10,000, the latter preparation neutralized the activity of 10 U of IFN-␥ (Murray et al., 2003b).
3. Results and discussion Treatment with Sb was begun on day +14, 2 weeks after L. donovani challenge. At this stage of infection in wild-type BALB/c mice, mRNA for both suppressive Th2 cell- (IL-4, IL-10) and activating Th1 cellassociated cytokines (IL-12, IFN-␥) is expressed in infected tissue and liver parasite burdens are increasing (Miralles et al., 1994; Murray et al., 2002). The data in Table 1 show the effect of treating mice with Sb at 1–50 mg/kg/day for 5 consecutive days (days +14 to +18). These dose–response results identified regimens used in subsequent experiments which on day +21 produced no (1 mg/kg), modest to moderate (5–25 mg/kg) or high-level liver parasite killing (50 mg/kg).
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3.1. Simultaneous administration of anti-IL-10R and Sb In previous studies of immunochemotherapy in this model, single-dose Sb was used for convenience and infected mice were treated with the immunoenhancing component 2 days before drug was given (Murray et al., 1988, 2000a, 2002, 2003c). This schedule induced optimal leishmanicidal activity, presumably reflecting administration of Sb in the midst of an already stimulated Th1 cell-type response (Murray et al., 1988, 2000a, 2002, 2003c). In the case of anti-IL-10R mAb, for example, giving mAb simultaneously with rather than 48 h before Sb was ∼50% less effective in enhancing parasite killing (Murray et al., 2002). Since Sb is rapidly excreted and levels in L. donovani-infected liver are low 48 h after a single i.p. injection (Murray, 1994), relatively little drug was probably still available in simultaneously-treated mice to coincide with the immunoenhancing effects of IL-10R blockade. Daily administration of Sb in the current experiments, therefore, provided the opportunity to test whether pretreatment with anti-IL-10R was actually necessary for its enhancing effect. As shown in Table 2, giving anti-IL-10R on day +14, 2 h after low-dose Sb (10 mg/kg/day) was initiated, was as effective as pretreatment with mAb on day +12 in increasing leishmanicidal activity measured on day +21. Treatment with anti-IL-10R alone contributed to some of the overall effect (25% killing), as it did in subsequent experiments. 3.2. Sb dose–sparing effect of anti-IL-10R
Table 1 Effect of daily Sb treatment* Sb treatment (mg/kg/day) 0 (control) 1 5 10 25 50
Liver parasite burden (LDU) Day +14
Day +21
1226 ± 63
1412 1259 851 748 489 178
± ± ± ± ± ±
78 69 50* 90* 27* 23*
% Killing on day +21 0 0 31 39 60 86
Results are from two to four experiments, and indicate mean + S.E.M. values for 8–17 mice per group. p < .05 vs. day +14 LDU. ∗ Fouteen days after infection, liver parasite burdens (LDU) were determined, and mice then received no treatment or once-daily i.p. injections of the indicated Sb doses on 5 consecutive days (days +14 to +18).
Combining anti-IL-10R with low-dose Sb (10 mg/kg/day) enhanced parasite killing in the liver (Table 2). To determine how much further the daily dose of Sb could be reduced while still preserving reasonable efficacy, mice were treated with 5 or 1 mg/kg of Sb, paired with mAb. As illustrated in Fig. 1, anti-IL-10R treatment enhanced the effect of both lower doses of Sb. For mice treated with 5 mg/kg/day, injection of anti-IL-10R increased killing on days +21 to 88%, the level (87% killing) induced by treatment with 10-fold more Sb alone (e.g., 50 mg/kg/day) (Fig. 1). In mice treated with Sb at 1 mg/kg/day, a no-effect regimen (1% killing by day +21), co-administration of anti-IL-10R also
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Table 2 Effect of Anti-IL-10R mAb given before or with Sb treatment* Treatment
mAb Injection
None Sb
Liver parasite burden (LDU)
% Killing on Day +21
Day +14
Day +21
1145 ± 112
1473 ± 102 824 ± 71
0 28
Control mAb
Day +12 Day +14
1601 ± 144 1740 ± 103
0 0
Anti-IL-10R
Day +12 Day +14
905 ± 60 859 ± 86
21 25
Sb + control mAb
Day +12 Day +14
985 ± 100 962 ± 89
14 16
Sb + anti-IL-10R
Day +12 Day +14
218 ± 48+ 149 ± 34+
81 87
Results are from two experiments, and indicate mean + S.E.M. values for six to eight mice per group. + p < 0.05 vs. day +14. ∗ Fouteen days after infection, liver parasite burdens (LDU) were measured, and mice then received no treatment or (a) 5 injections of Sb (10 mg/kg/day) on days +14 to +18, (b) a single injection of control mAb or anti-IL-10R on day +12 or +14, or (c) 5 injections of Sb plus either control mAb or anti-IL-10R given once either 2 days before (day +12) or 2 h after the first dose of Sb on day +14.
clearly increased parasite killing (e.g., to 66% by day +21). Extrapolation of these results suggested that the activity induced by Sb at 1 mg/kg/day in the presence of anti-IL-10R (66% killing) represents a ∼35-fold reduction in the effective Sb dose when compared to activity (87% killing) of Sb alone at 50 mg/kg. 3.3. Accelerated Sb-induced killing in anti-IL-10R-treated mice
Fig. 1. Anti-IL-10R enhances the effect of low-dose Sb. Fourteen days after infection, mice received no treatment (0 mg/kg/day) or 1 or 5 mg/kg/day of Sb on days +14 to +18. On day +14, untreated and Sb-treated mice also received one injection of either control mAb (open circles) or anti-IL-10R (closed circles). An additional group received Sb alone at 50 mg/kg/day on days +14 to +18 (open square). Liver parasite burden on day +14 (1257 ± 74 LDU) is shown on vertical axis at dotted line. LDU values below dotted line represent parasite killing; (n) indicates % killing (% decrease in day +21 vs. day +14 LDU). Results (mean + S.E.M.) are from 2 to 3 experiments with 6–11 mice per group. p < 0.05 for all LDU results in anti-IL-10R vs. control mAb-treated mice.
To determine if the enhanced killing induced by combining anti-IL-10R with Sb could be detected early, a higher Sb dose which produced leishmanicidal activity by itself (25 mg/kg/day, Table 1) was tested and liver burdens were measured on day +17 (24 h after the third Sb injection). As shown in Fig. 2A, killing induced by Sb plus anti-IL-10R was well established by day +17 (69% killing), at a time when the effects of giving Sb or anti-IL-10R alone were just beginning to be expressed (24–25% killing). Both the initial (day +17) and overall response to Sb plus anti-IL-10R (day +21) was largely inhibited by co-administration of anti-IFN-␥ (Fig. 2B). 3.4. Reduction in duration of Sb treatment Since IL-10R blockade permitted a >10-fold reduction in the effective dose of Sb and accelerated killing,
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Fig. 2. Anti-IL-10R accelerates Sb-induced killing and requires endogenous IFN-␥. (A) Day +14 liver parasite burden was 1341 ± 129 LDU, and mice were treated with (a) 25 mg/kg/day of Sb on days +14 to +18 plus one injection on day +14 of either control mAb (open circles) or anti-IL-10R (closed circles), or (b) one injection on day +14 of control mAb (no effect, results omitted for clarity) or anti-IL-10R alone (open squares). In (B), day +14 LDU was 1261 ± 102, and all groups of mice were treated with Sb and anti-IL-10R as in (A) with no other treatment (closed circles) or one injection on day +14 of control rabbit serum (open circles) or anti-IFN-␥ antiserum (closed squares) (see Section 2). Results in (A) and (B) are from two experiments, and indicate mean + S.E.M. values for six to nine mice per group at each time point. * p < 0.05 in (A) vs. mice treated with Sb plus control mAb, and in (B) vs. mice treated with Sb plus anti-IL-10R plus anti-IFN-␥.
we completed this analysis by determining if these effects could be translated into a short-course, lower-dose treatment regimen. Mice were therefore given either (a) 5 days of full-dose Sb alone (50 mg/kg/day, days +14 to +18), or (b) 2 days of half-dose Sb (25 mg/kg/day, days +14 and +15) plus anti-IL-10R once on day +14. The results measured on day +21 in the liver showed that the parasite killing (74%) induced by the abbreviated combination regimen approached the effect (84% killing) of the longer, higher-dose regimen of Sb alone (2 experiments, n = 6–8 mice per group, not shown). Thus, in this study, the use of a daily Sb regimen allowed definition of additional therapeutic features of co-induction of IL-10R blockade not identified in our previous work in which single-dose Sb was employed (Murray et al., 2002). These features, which support the appeal of this new experimental strategy in visceral leishmaniasis (Murray et al., 2002, 2003a, 2003b), suggest that anti-IL-10R mAb treatment: (a) is effective when administered simultaneously with Sb and need not be used as pretreatment, (b) accelerates the kinetics of Sb-induced killing, (c) permits an Sb dose–sparing effect through which at least >10-fold less Sb can be employed with satisfactory parasite killing, and (d) allows the duration of Sb treatment in established in-
fection to be reduced. Several of these same features of anti-IL-10R co-administration have also recently been demonstrated in livers of infected mice treated with amphotericin B as well (Murray et al., 2003a). Since daily Sb therapy in human visceral leishmaniaisis (kala-azar) is prolonged at >28 days and often arduous and toxicity is thought to be cumulative (Murray, 2000; Murray, 2000), methods to reduce treatment duration and/or drug dose are attractive. Endogenous IL10 handcuffs the curative Th1 cell-associated response (Moore et al., 2001), fosters progressive experimental infection (Murphy et al., 2001; Murray et al., 2002) and is believed to act in a similar fashion in patients with kala-azar (Ghalib et al., 1993; Karp et al., 1993; Sundar et al., 1997; Barcellar et al., 2000; Murray, 2001a). Therefore, in an antileishmanial immunochemotherapeutic regimen, specifically targeting IL-10 by neutralization or blockade of its receptor has additional appeal as well.
Acknowledgments This work was supported by National Institutes of Health grant AI 16963. Dr. Amy Beebe (DNAX Research Institute of Molecular and Cellular Biology)
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generously provided anti-IL-10R mAb, and Elaine Brooks provided technical assistance.
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maniasis: requirement for T cells and effect of lymphokines. J. Clin. Invest. 83, 1254–1259. Murray, H.W., Hariprashad, J., Fichtl, R., 1993. Treatment of experimental visceral leishmaniasis in a T cell-deficient host: response to amphotericin B and pentamidine. Antimicrob. Agents Chemother. 7, 1504–1505. Murray, H.W., 1994. Cytokines in the treatment of leishmaniasis. Bailliere’s Clin. Infect. Dis. 1, 127–144. 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. Murray, H.W., 2000. Treatment of visceral leishmaniasis (kala-azar): a decade of progress and future approaches. Int. J. Infect. Dis. 4, 158–177. Murray, H.W., Delph-Etienne, S., 2000. Role of endogenous gamma interferon and macrophage microbicidal mechanisms in host response to chemotherapy in experimental visceral leishmaniasis. Infect. Immun. 68, 288–293. Murray, H.W., Montelibano, C., Peterson, R., Sypek, J.P., 2000a. Interleukin 12 regulates the response to chemotherapy in experimental visceral leishmaniasis. J. Infect. Dis. 182, 1497– 1502. Murray, H.W., Jungbluth, A., Ritter, E., Montelibano, C., Marino, M.W., 2000b. Visceral leishmaniasis in mice devoid of tumor necrosis factor and response to treatment. Infect. Immun. 68, 6289–6293. Murray, H.W., 2001a. Clinical and experimental advances in treatment in visceral leishmaniasis. Antimicrob. Agents Chemother. 45, 2185–2197. Murray, H.W., 2001b. Tissue granuloma structure-function in experimental visceral leishmaniasis. Int. J. Exp. Pathol. 82, 249– 276. Murray, H.W., Lu, C.M., Mauze, S., Ureeman, S., Moreira, A.L., Kaplan, G., Coffman, R.L., 2002. Interleukin-10 (IL10) in experimental visceral leishmaniasis and IL-10 receptor blockade as immunotherapy. Infect. Immun. 70, 6284– 6293. Murray, H.W., Brooks, E.B., DeVecchio, J.L., Heinzel, F.P., 2003a. Immunoenhancement combined with amphotericin B as treatment in experimental visceral leishmaniasis. Antimicrob. Agents Chemother. 47, 2513–2517. Murray, H.W., Moreira, A.L., Lu, C.M., DeVecchio, J.L., Matsuhashi, M., Ma, X., Heinzel, F.P., 2003b. Determinants of response to interleukin 10 receptor blockade immunotherapy in experimental visceral leishmaniasis. J. Infect. Dis. 188, 458– 464. Murray, H.W., Lu, C.M., Brooks, E.B., Fichtl, R.E., DeVecchio, J.L., Heinzel, F.P., 2003c. Modulation of T cell costimulation as immuno- or immunochemotherapy in experimental visceral leishmaniasis. Infect. Immun. 71, 6453–6462. Satoskar, A.R., Rodig, S., Telford, S.R., Satoskar, A.A., Ghosh, S.K., von Lichtenberg, F., David, J.R., 2000. IL-12 gene-deficient C57BL/6 mice are susceptible to Leishmania donovani but have diminished hepatic pathology. Eur. J. Immunol. 30, 834–839. Stager, S., Alexander, J., Carter, K.C., Brombacher, F., Kaye, P.M., 2003. Both interleukin-4 (IL-4) and IL-4 receptor ␣ sig-
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Interleukin 10 receptor blockade—pentavalent antimony treatment in experimental visceral leishmaniasis Henry W. Murray ∗ Department of Medicine, Weill Medical College of Cornell University, Box 136, 1300 York Avenue, NY 10021, USA Received 20 February 2004; received in revised form 20 July 2004; accepted 11 November 2004
Abstract Interleukin 10 (IL-10), a suppressive Th2 cell-type cytokine, promotes disease progression in experimental visceral leishmaniasis. To extend testing the therapeutic effects of applying IL-10 receptor (IL-10R) blockade with antileishmanial chemotherapy, BALB/c mice with established intracellular Leishmania donovani infection were injected once with anti-IL-10R mAb at the time low-dose, daily pentavalent antimony (Sb) therapy was initiated. In this treatment model, simultaneous administration of anti-IL-10R enhanced overall antileishmanial activity in the liver in an interferon-␥-dependent fashion, and accelerated the kinetics of Sb-associated killing, induced a >10-fold Sb dose–sparing effect and shortened the required duration of Sb treatment. These results suggest the possibility of using mAb-induced IL-10R blockade to develop low-dose and/or short-course immunochemotherapeutic regimens in visceral leishmaniasis. © 2005 Elsevier B.V. All rights reserved. Keywords: Leishmania donovani; Visceral leishmaniasis; IL-10; Anti-IL-10 receptor; Pentavalent antimony
1. Introduction Harnessing the host’s T cell-dependent immune response and applying it with antileishmanial chemotherapy represents one strategy to enhance both treatment efficacy and efficiency in visceral leishmaniasis, a disseminated intracellular protozoal disease (Murray, 2001a). In experimental Leishmania donovani infection, in which macrophages in liver, spleen ∗
Tel.: +1 212 746 6330; fax: +1 212 746 6332. E-mail address: [email protected].
0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2004.11.008
and bone marrow are targeted, successful host defense is T cell-dependent and initially regulated by multiple activating cytokines secreted by both Th1and Th2-type cells (Engwerda et al., 1998; Satoskar et al., 2000; Alexander et al., 2000; Murray, 2001a, 2001b; Stager et al., 2003). If unimpeded, this response induces tissue macrophage activation, intracellular parasite killing and control over L. donovani infection in the liver (Murray, 2001a, 2001b). In each of these expressions of acquired resistance, interleukin 12 (IL-12) and interferon-␥ (IFN-␥, Th1 cell-type cytokines, play particularly prominent roles (Engwerda
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et al., 1998; Satoskar et al., 2000; Murray, 2001a, 2001b). In L. donovani-infected mice, interventions which strengthen Th1 cell reactivity and enhance cytokine secretion and/or induce macrophage activation have been identified and combined with chemotherapy (Ghosh et al., 2003; Murray and Hariprashad, 1995; Murray et al., 1988, 2000a, 2002, 2003a, 2003b, 2003c; Murray 2001a; Murphy et al., 1998; Zubairi et al., 2004). One of these approaches, inhibiting the action of endogenous IL-10 by monoclonal antibody (mAb)-induced blockade of its receptor (Murray et al., 2003a, 2003b), has additional appeal since IL-10 is instrumental in promoting progressive L. donovani infection (Murphy et al., 2001; Murray et al., 2002). IL-10, well recognized as a suppressive-type cytokine produced by multiple cells (Moore et al., 2001), extinguishes the capacity to kill parasites in the tissues by disabling both the afferent and efferent arms of the inflammatory Th1 cell mechanism, limiting IL-12 and IFN-␥ secretion and deactivating macrophages (Moore et al., 2001; Murphy et al., 2001; Murray et al., 2002, 2003a, 2003b). The same Th1 cell cytokine response targeted by IL10 also interdigitates with pentavalent antimony (Sb), conventional antileishmanial chemotherapy (Murray et al., 1989, 1993; Murray 2001a; Escobar et al., 2001). Expression of Sb’s leishmanicidal effect in experimental visceral infection, for example, requires not only T cells but multiple cytokines, including IL-12 and IFN-␥ as well as tumor necrosis factor (TNF) and IL4 (Alexander et al., 2000; Murray et al., 1989, 2000a, 2000b; Murray and Delph-Etienne, 2000; Escobar et al., 2001). The paradoxical role of IL-4, ordinarily considered a suppressive-type cytokine, in the response to Sb appears to reflect IL-4’s less well-appreciated capacity to foster Th1 cell development and regulate initial IFN-␥ secretion (Alexander et al., 2000). In view of the regulatory role of IL-10 in the L. donovani model, BALB/c mice with established infection were injected with anti-IL-10 receptor mAb (anti-IL10R) to free up Th1 responses, including IL-12 and IFN-␥ secretion, and then 2 days later were treated with Sb (Murray et al., 2003, 2003b). The results in this model of combination therapy, in which a single injection of Sb was tested, indicated that IL-10R blockade enhanced the efficacy of chemotherapy, provided that mAb was administered before Sb (Murray et al., 2003, 2003b).
Building on the preceding result, the present study employed a different Sb treatment protocol to more fully define the beneficial effects of combination with IL-10R blockade. Treating L. donovani-infected mice with daily Sb, as the drug is used clinically in visceral leishmaniasis (kala-azar) (Murray, 2001a), permitted discrete, therapeutically relevant questions about this form of immunochemotherapy to be addressed – is pretreatment with anti-IL-10R required, what are the Sb dose–sparing effects of anti-IL-10R, does IL-10R blockade accelerate the kinetics of Sb-induced parasite killing and can Sb be combined with anti-IL-10R in a short-course, low-dose regimen?
2. Materials and methods 2.1. Mice and visceral infection Female BALB/c mice (20–30 g) (Charles Rivers Laboratories, Wilmington, MA) were injected via the tail vein with 1.5 × 107 hamster spleen-derived L. donovani amastigotes (1 Sudan strain) (Murray et al., 2002). 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 × liver weight (mg) (Leishman–Donovan units, LDU) (Murray et al., 2002). 2.2. Treatments Starting 14 days after infection (day +14), groups of three to five mice were injected i.p. once daily with Sb (sodium stibogluconate, Pentostam, Wellcome Foundation Ltd., London, UK) (Murray et al., 1988). Sb was given for up to 5 days (days +14 to +18) at 1–50 mg/kg/day in 0.2 ml of saline, and parasite burdens (LDU) were determined on day +17 (1 day after the third Sb injection) or on day +21 (3 days after the fifth injection). Day +17 or day +21 LDU were compared to day +14 LDU to determine percent parasite killing (Murray et al., 2002); differences between mean values were analyzed by a two-tailed Student’s t-test. To demonstrate the effects of combination treatment, mice were injected once i.p. on day +14 with 0.2 ml of saline containing anti-IL-10R mAb (1B1.3A)
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or an isotype control mAb (GL117.41, anti-betagalactosidase), provided by Dr. A. Beebe (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA) (Murray et al., 2002). Anti-IL-10R was used at a suboptimal dose (0.1 mg) (Murray et al., 2002, 2003b), and was given 2 h after the first injection of Sb, except where indicated. In some experiments, 1 h prior to anti-IL-10R treatment, mice were also injected once i.p. with 0.2 ml of either normal rabbit serum or rabbit anti-mouse IFN-␥ antiserum raised against murine recombinant IFN-␥. At a dilution of 1:10,000, the latter preparation neutralized the activity of 10 U of IFN-␥ (Murray et al., 2003b).
3. Results and discussion Treatment with Sb was begun on day +14, 2 weeks after L. donovani challenge. At this stage of infection in wild-type BALB/c mice, mRNA for both suppressive Th2 cell- (IL-4, IL-10) and activating Th1 cellassociated cytokines (IL-12, IFN-␥) is expressed in infected tissue and liver parasite burdens are increasing (Miralles et al., 1994; Murray et al., 2002). The data in Table 1 show the effect of treating mice with Sb at 1–50 mg/kg/day for 5 consecutive days (days +14 to +18). These dose–response results identified regimens used in subsequent experiments which on day +21 produced no (1 mg/kg), modest to moderate (5–25 mg/kg) or high-level liver parasite killing (50 mg/kg).
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3.1. Simultaneous administration of anti-IL-10R and Sb In previous studies of immunochemotherapy in this model, single-dose Sb was used for convenience and infected mice were treated with the immunoenhancing component 2 days before drug was given (Murray et al., 1988, 2000a, 2002, 2003c). This schedule induced optimal leishmanicidal activity, presumably reflecting administration of Sb in the midst of an already stimulated Th1 cell-type response (Murray et al., 1988, 2000a, 2002, 2003c). In the case of anti-IL-10R mAb, for example, giving mAb simultaneously with rather than 48 h before Sb was ∼50% less effective in enhancing parasite killing (Murray et al., 2002). Since Sb is rapidly excreted and levels in L. donovani-infected liver are low 48 h after a single i.p. injection (Murray, 1994), relatively little drug was probably still available in simultaneously-treated mice to coincide with the immunoenhancing effects of IL-10R blockade. Daily administration of Sb in the current experiments, therefore, provided the opportunity to test whether pretreatment with anti-IL-10R was actually necessary for its enhancing effect. As shown in Table 2, giving anti-IL-10R on day +14, 2 h after low-dose Sb (10 mg/kg/day) was initiated, was as effective as pretreatment with mAb on day +12 in increasing leishmanicidal activity measured on day +21. Treatment with anti-IL-10R alone contributed to some of the overall effect (25% killing), as it did in subsequent experiments. 3.2. Sb dose–sparing effect of anti-IL-10R
Table 1 Effect of daily Sb treatment* Sb treatment (mg/kg/day) 0 (control) 1 5 10 25 50
Liver parasite burden (LDU) Day +14
Day +21
1226 ± 63
1412 1259 851 748 489 178
± ± ± ± ± ±
78 69 50* 90* 27* 23*
% Killing on day +21 0 0 31 39 60 86
Results are from two to four experiments, and indicate mean + S.E.M. values for 8–17 mice per group. p < .05 vs. day +14 LDU. ∗ Fouteen days after infection, liver parasite burdens (LDU) were determined, and mice then received no treatment or once-daily i.p. injections of the indicated Sb doses on 5 consecutive days (days +14 to +18).
Combining anti-IL-10R with low-dose Sb (10 mg/kg/day) enhanced parasite killing in the liver (Table 2). To determine how much further the daily dose of Sb could be reduced while still preserving reasonable efficacy, mice were treated with 5 or 1 mg/kg of Sb, paired with mAb. As illustrated in Fig. 1, anti-IL-10R treatment enhanced the effect of both lower doses of Sb. For mice treated with 5 mg/kg/day, injection of anti-IL-10R increased killing on days +21 to 88%, the level (87% killing) induced by treatment with 10-fold more Sb alone (e.g., 50 mg/kg/day) (Fig. 1). In mice treated with Sb at 1 mg/kg/day, a no-effect regimen (1% killing by day +21), co-administration of anti-IL-10R also
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Table 2 Effect of Anti-IL-10R mAb given before or with Sb treatment* Treatment
mAb Injection
None Sb
Liver parasite burden (LDU)
% Killing on Day +21
Day +14
Day +21
1145 ± 112
1473 ± 102 824 ± 71
0 28
Control mAb
Day +12 Day +14
1601 ± 144 1740 ± 103
0 0
Anti-IL-10R
Day +12 Day +14
905 ± 60 859 ± 86
21 25
Sb + control mAb
Day +12 Day +14
985 ± 100 962 ± 89
14 16
Sb + anti-IL-10R
Day +12 Day +14
218 ± 48+ 149 ± 34+
81 87
Results are from two experiments, and indicate mean + S.E.M. values for six to eight mice per group. + p < 0.05 vs. day +14. ∗ Fouteen days after infection, liver parasite burdens (LDU) were measured, and mice then received no treatment or (a) 5 injections of Sb (10 mg/kg/day) on days +14 to +18, (b) a single injection of control mAb or anti-IL-10R on day +12 or +14, or (c) 5 injections of Sb plus either control mAb or anti-IL-10R given once either 2 days before (day +12) or 2 h after the first dose of Sb on day +14.
clearly increased parasite killing (e.g., to 66% by day +21). Extrapolation of these results suggested that the activity induced by Sb at 1 mg/kg/day in the presence of anti-IL-10R (66% killing) represents a ∼35-fold reduction in the effective Sb dose when compared to activity (87% killing) of Sb alone at 50 mg/kg. 3.3. Accelerated Sb-induced killing in anti-IL-10R-treated mice
Fig. 1. Anti-IL-10R enhances the effect of low-dose Sb. Fourteen days after infection, mice received no treatment (0 mg/kg/day) or 1 or 5 mg/kg/day of Sb on days +14 to +18. On day +14, untreated and Sb-treated mice also received one injection of either control mAb (open circles) or anti-IL-10R (closed circles). An additional group received Sb alone at 50 mg/kg/day on days +14 to +18 (open square). Liver parasite burden on day +14 (1257 ± 74 LDU) is shown on vertical axis at dotted line. LDU values below dotted line represent parasite killing; (n) indicates % killing (% decrease in day +21 vs. day +14 LDU). Results (mean + S.E.M.) are from 2 to 3 experiments with 6–11 mice per group. p < 0.05 for all LDU results in anti-IL-10R vs. control mAb-treated mice.
To determine if the enhanced killing induced by combining anti-IL-10R with Sb could be detected early, a higher Sb dose which produced leishmanicidal activity by itself (25 mg/kg/day, Table 1) was tested and liver burdens were measured on day +17 (24 h after the third Sb injection). As shown in Fig. 2A, killing induced by Sb plus anti-IL-10R was well established by day +17 (69% killing), at a time when the effects of giving Sb or anti-IL-10R alone were just beginning to be expressed (24–25% killing). Both the initial (day +17) and overall response to Sb plus anti-IL-10R (day +21) was largely inhibited by co-administration of anti-IFN-␥ (Fig. 2B). 3.4. Reduction in duration of Sb treatment Since IL-10R blockade permitted a >10-fold reduction in the effective dose of Sb and accelerated killing,
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Fig. 2. Anti-IL-10R accelerates Sb-induced killing and requires endogenous IFN-␥. (A) Day +14 liver parasite burden was 1341 ± 129 LDU, and mice were treated with (a) 25 mg/kg/day of Sb on days +14 to +18 plus one injection on day +14 of either control mAb (open circles) or anti-IL-10R (closed circles), or (b) one injection on day +14 of control mAb (no effect, results omitted for clarity) or anti-IL-10R alone (open squares). In (B), day +14 LDU was 1261 ± 102, and all groups of mice were treated with Sb and anti-IL-10R as in (A) with no other treatment (closed circles) or one injection on day +14 of control rabbit serum (open circles) or anti-IFN-␥ antiserum (closed squares) (see Section 2). Results in (A) and (B) are from two experiments, and indicate mean + S.E.M. values for six to nine mice per group at each time point. * p < 0.05 in (A) vs. mice treated with Sb plus control mAb, and in (B) vs. mice treated with Sb plus anti-IL-10R plus anti-IFN-␥.
we completed this analysis by determining if these effects could be translated into a short-course, lower-dose treatment regimen. Mice were therefore given either (a) 5 days of full-dose Sb alone (50 mg/kg/day, days +14 to +18), or (b) 2 days of half-dose Sb (25 mg/kg/day, days +14 and +15) plus anti-IL-10R once on day +14. The results measured on day +21 in the liver showed that the parasite killing (74%) induced by the abbreviated combination regimen approached the effect (84% killing) of the longer, higher-dose regimen of Sb alone (2 experiments, n = 6–8 mice per group, not shown). Thus, in this study, the use of a daily Sb regimen allowed definition of additional therapeutic features of co-induction of IL-10R blockade not identified in our previous work in which single-dose Sb was employed (Murray et al., 2002). These features, which support the appeal of this new experimental strategy in visceral leishmaniasis (Murray et al., 2002, 2003a, 2003b), suggest that anti-IL-10R mAb treatment: (a) is effective when administered simultaneously with Sb and need not be used as pretreatment, (b) accelerates the kinetics of Sb-induced killing, (c) permits an Sb dose–sparing effect through which at least >10-fold less Sb can be employed with satisfactory parasite killing, and (d) allows the duration of Sb treatment in established in-
fection to be reduced. Several of these same features of anti-IL-10R co-administration have also recently been demonstrated in livers of infected mice treated with amphotericin B as well (Murray et al., 2003a). Since daily Sb therapy in human visceral leishmaniaisis (kala-azar) is prolonged at >28 days and often arduous and toxicity is thought to be cumulative (Murray, 2000; Murray, 2000), methods to reduce treatment duration and/or drug dose are attractive. Endogenous IL10 handcuffs the curative Th1 cell-associated response (Moore et al., 2001), fosters progressive experimental infection (Murphy et al., 2001; Murray et al., 2002) and is believed to act in a similar fashion in patients with kala-azar (Ghalib et al., 1993; Karp et al., 1993; Sundar et al., 1997; Barcellar et al., 2000; Murray, 2001a). Therefore, in an antileishmanial immunochemotherapeutic regimen, specifically targeting IL-10 by neutralization or blockade of its receptor has additional appeal as well.
Acknowledgments This work was supported by National Institutes of Health grant AI 16963. Dr. Amy Beebe (DNAX Research Institute of Molecular and Cellular Biology)
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generously provided anti-IL-10R mAb, and Elaine Brooks provided technical assistance.
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