A role for IgG immune complexes during infection with the intracellular pathogen Leishmania

Abstract

We examined the role of immunoglobulin (Ig)G antibodies in mediating host defense to the intracellular parasite, Leishmania. We show that IgG not only fails to provide protection against this intracellular pathogen, but it actually contributes to disease progression. The J(H) strain of BALB/c mice, which lack IgG because they have a targeted deletion in the Ig heavy chain (J) locus, were more resistant to infection with Leishmania major than were normal BALB/c mice. However, the passive administration of anti-Leishmania IgG caused J(H) mice to develop large lesions containing high numbers of parasites. Antibody administration correlated with an increase in interleukin (IL) 10 production in lesions, and blocking the murine IL-10 receptor prevented antibody-mediated disease exacerbation. In human patients with active visceral leishmaniasis, high IgG levels are predictive of disease. Patients with ongoing disease had high IgG antibody titers and no delayed-type hypersensitivity (DTH) responses to Leishmania antigens. This pattern was reversed upon disease resolution after treatment, resulting in a decrease in total IgG, which was accompanied by a progressive increase in DTH responsiveness. We conclude that IgG can cause a novel form of immune enhancement due to its ability to induce IL-10 production from macrophages.

Figure 1.

Serum reconstitution of J_H mice. Lesion size of J_H mice (closed triangles) and BALB/c mice (closed circles) was compared with J_H mice (open triangles) and BALB/c mice (open circles) given 200 μl of antisera to L. major on days 1, 7, and 14. Parasite burdens (inset) in BALB/c and J_H mice that were infected with (diagonally striped bars) or without (black bars) αLm (α–L. major) antisera were determined on day 35 by limiting dilution assays as described previously (reference 3). Error bars represent the standard error of the mean of three separate experiments done with a minimum of four mice per group.

Figure 2.

Serum reconstitution of J_H mice 3 wk after infection. Lesion size of J_H mice (closed circles) were compared with those of J_H mice given 200 μl αLm (α–L. major) antiserum on day 21 after infection (open circles). Parasite burdens (inset) were determined at 42 d after infection by limiting dilution assays as described previously (reference 3). Error bars represent the standard deviation of the mean of four determinations. This experiment is representative of three.

Figure 3.

Cytokine production in mice administered anti–L. major antiserum. (A) Cytokine production by lymph node T cells from infected J_H mice (white bars) was compared with J_H mice administered 200 μl αLm (α–L. major) antiserum on days 1, 7, and 14 after infection (black bars). Lymph nodes were removed on day 21 and stimulated with soluble leishmania antigen. Supernatants were harvested 72 h later and assayed for IFN-γ and IL-4 by ELISA. (B) IL-10 production in lesions of J_H mice. IL-10 protein (left) and mRNA (right) were determined in two groups of J_H mice infected with 2 × 10⁵ L. major amastigotes. On day 21 of infection, one group was administered 600 μg of purified αLm-IgG i.p. IL-10 protein levels in the lesions of three infected mice were measured by ELISA 4 d after the administration of αLm-IgG (left axis). RNA was isolated from footpad lesions on day 2 after IgG administration, and real-time PCR was performed to determine relative IL-10 mRNA (right axis). Levels represent the average from three infected mice, and mRNA was normalized to hypoxanthine phosphoribosyltransferase levels in a single infected foot.

Figure 4.

IgG reconstitution of J_H mice and the effect of α–IL-10R mAb. Three parallel groups of J_H mice were infected with 2 × 10⁵ L. major amastigotes. One group (open circles) was administered 600 μg of purified αLm-IgG i.p. on days 1, 8, and 15. Another group (gray triangles) was given the same dose of αLm-IgG, along with α–IL-10R on days 0 (1 mg), 7 (200 μg), and 14 (200 μg). The third group (closed circles) received no treatment. Lesion size was measured at semi-weekly intervals. Parasite burdens (inset) were determined by limiting dilution. Error bars represent the standard deviation of the mean.

Figure 5.

The immunization of C57BL/6 mice with OVA. Lesion development in OVA-immunized mice (open circles) was compared with control mice given IFA alone (closed circles). Immunized mice were given 25 μg OVA in 500 μL of IFA and boosted 2 wk later. Both groups of mice were infected with L. major resuspended in PBS containing 50 μg/ml OVA. Parasite burdens (inset) were determined on day 34. Error bars represent the standard deviation of the mean.

Figure 6.

In vitro cytokine production. (A) Lesion-derived L. major amastigotes were added to bone marrow–derived macrophages in the presence of inflammatory LMW-HA. Supernatants were harvested 24 h later and cytokines IL-12 (gray bars) and IL-10 (black bars) were measured by ELISA. (B) The production of cytokines, IFN-γ and IL-4, from T cells was measured by ELISA 3 d after primary stimulation. OVA-specific TCR transgenic T cells were added to either uninfected (white bars) or L. major–infected (black bars) macrophages cultivated with OVA and LMW-HA.

Figure 7.

Flow cytometry to detect intracellular IL-10. Human monocytes were infected with a 10:1 ratio of axenic amastigotes of L. chagasi. Before infection, some amastigotes were incubated at 4°C for 15 min in 5% serum from either an uninfected volunteer (naive serum) or from a patient with visceral leishmaniasis (VL serum). Monocytes were incubated in Golgi stop for 2 h, fixed, and permeabilized and stained for intracellular IL-10 expression. Cells were gated on CD14 expression.

Figure 8.

DTH and antibody responses in patients with visceral leishmaniasis (VL). Delayed-type hypersensitivity (DTH) reactions were measured in 317 bone marrow aspirate-confirmed VL patients. The mean induration size of the DTH response (in mm diameter) was measured at 48 h. (inset table) The same VL patients were analyzed for antibody and DTH responses. Antibody levels were judged to be positive if they were >3 SD above a mean control titer. DTH responses >5 mm in diameter were judged to be positive. This figure designates a significant negative association between DTH and antibody responses. Pearson χ², df = 1; P ≤ 0.0001.

Figure 9.

DTH and Leishmania-specific antibody responses in VL patients after treatment. The mean diameter induration score of the DTH response, measured in mm, is shown by the black bars (left), and the mean antibody titers, expressed as OD reading, are shown by the white bars (right) as a function of time after treatment (abscissa).