Pathogenic role of B cells and antibodies in murine Leishmania amazonensis infection

Abstract

Leishmania amazonensis infection, occurring predominantly in Central and South America, can manifest itself in several forms, including those of cutaneous and diffuse cutaneous leishmaniasis. The outcome of L. amazonensis infection depends largely on host immune responses to the parasites. While CD4+ T cell activation is a prerequisite for pathogenesis in L. amazonensis-infected mice, the roles of B cells and their antibody production are unclear. In this study, we provide evidence suggesting that B cells and antibodies are involved in disease pathogenesis. We documented a correlation between B cell activation and lesion progress in immunocompetent mice. In the absence of functional B cells and antibodies, JhD mice showed a delayed onset of disease and developed small lesions. Histological examination of these mice revealed a significant reduction in CD4+ and CD8+ T cells, but not in MAC1+ macrophages, at the infection site. In contrast to the wild-type mice that showed typical tissue necrosis, L. amazonensis-infected JhD mice showed no or minimal signs of necrotic foci. A marked reduction in CD4+ T cell proliferation and cytokine (IFN-gamma and IL-10) production in infected JhD mice suggested an involvement of B cells and antibodies in the priming of parasite-specific T cells. This notion was further supported by the observations that adoptive transfer of B cells or antibodies could restore CD4+ T cell activation and migration in infected JhD mice. Moreover, antibody coating of parasites could stimulate dendritic cells to produce high levels of cytokines and increase their ability to prime nai ve CD4+ T cells. Since CD4+ T cells are crucial to disease pathogenesis, this study suggests that B cells and their antibody production enhanced L. amazonensis infection, partially by promoting T cell priming and cellular migration to the infection site.

Fig. 1

B cell outgrowth and antibody production correlated with lesion development in Leishmania amazonensis-infected mice. BALB/c and C3H mice (five per group) were left untreated or injected s.c. in the right hind foot with 2 x 10⁶ promastigotes. (A) Lesion sizes were measured weekly with a digital caliper. (B) At 10 weeks p.i., draining lymph nodes (DLNs) were collected and the total number of cells per LN was counted using a hemacytometer. (C) Cell surface markers were stained for flow cytometry and the absolute number of each cell population in DLNs was calculated. (D) The titers of total IgGs in the sera were measured via direct ELISA. Data are presented as mean ± S.D. for each group. Similar results were observed from two independent experiments. (*P < 0.05; **P < 0.01; *** P < 0.001; n.s., not significant)

Fig. 2

B cell-deficient Jh mice developed small cutaneous lesions and displayed impaired cytokine production and CD4⁺ T cell proliferative responses following Leishmania amazonensis infection. (A) Wild-type BALB/c and Jh mice (10 per group) were injected s.c. with 2 x 10⁶ amastigotes. Lesion sizes were measured weekly with a digital caliper. Parasite burden in foot tissue was determined at 7 weeks p.i.. At 7-8 weeks p.i., draining lymph node (DLN) cells were collected and restimulated with parasite antigen (Ag) for 72 h. (B–C) Cytokine profiles from the supernatants were determined with a mouse 23-Bio-Plex cytokine assay. Cytokines that were significantly different between wild-type and Jh mice are shown. (D) Splenic CD4⁺ T cells were purified from infected wild-type and Jh mice and cocultured with mitomycin C-treated splenocytes from naïve wild-type mice (1:1 ratio) in the presence or absence of parasite Ag or 2 ng/ml of Con A for 72 h. Proliferation of CD4⁺ T cells was indicated by [³H] thymidine incorporation. Data are presented as mean ± S.D. for each group. Similar results were observed from two independent experiments. (* P < 0.05; *** P < 0.001; n.s., not significant)

Fig. 3

Differential histopathology in lesions of infected Jh and wild-type mice. Mice (eight per group) were injected s.c. in the right hind foot with 2 x 10⁶ Leishmania amazonensis amastigotes. At 7–8 weeks p.i., infected foot tissues were collected for histological analysis. Representative H&E staining of tissues from wild-type mice (A, C) showed tissue necrosis (circles) and monomorphic collection of vacuolated and heavily parasitized macrophages (small arrows). Sections from Jh mice (B, D) showed no or minimal necrosis, as well as a mixture of vacuolated and non-vacuolated macrophages. Some parasites appeared to be packed in the extracellular space (big arrows).

Fig. 4

Impaired recruitment of CD4⁺ and CD8⁺ T cells, but not MAC-1⁺ cells, in Leishmania amazonensis-infected Jh mice. Mice were grouped and infected as described in Fig. 3. At 7–8 weeks p.i., foot tissues were collected and stained with anti-CD4 (A, B), anti-CD8 (D, E) or anti-MAC-1 (G, H) and then counterstained with methyl green. Representative sections show extensive infiltrates for CD4⁺ cells (A) and a significant number of CD8⁺ cells (D) in the wild-type mice. In contrast, tissues of infected Jh mice contained limited numbers of CD4⁺ (B) and CD8⁺ cells (E). MAC-1⁺ macrophages appeared distributed throughout tissues of wild-type (G) and Jh mice (H), although the staining patterns appeared differently in these two groups. Tissue sections stained side-by-side with isotype controls had no/minimal background staining (data not shown). All images were taken at 10 x magnification. The total number of CD4⁺ (C), CD8⁺ T cells (D) and MAC-1⁺ macrophages (I) on each side were counted and scored semiquantitatively under 40 x (for CD4⁺ and CD8⁺ T cells) and 10x (for MAC-1⁺ cells) (see Materials and methods). The average scores from three visual fields from each group are plotted. Data represent mean ± S.D. for each group.

Fig. 5

Ex vivo lymph node (LN) CD4⁺ T cell activation and cytokine production from amastigote (Am)-infected mice. For B cell adoptive transfer, purified spleen B cells (5 × 10⁷) were injected i.v. in to JhD mice 1 day prior to Am infection. For antibody adoptive transfer, sera (200 μl) from chronically infected BALB/c mice were adoptively transferred (i.p.) into JhD mice at 1, 3and 7 days after Am infection. (A) At 3 weeks, individual (WT) or pooled draining LNs were extracted and stained for CD4 and T cell activation markers. (B) LN cells (10⁶/ml) were restimulated with PMA/ionomycin with GolgiPlug™ for 6 h. Intracellular staining for IFN-γ and IL-10 were performed after CD4 surface marker. Percentages of positively stained CD4⁺ T cells are shown as mean ± SD from two independent repeats.

Fig. 6

Enhanced cytokine production and increased priming efficiency of CD4⁺ T cells by dendritic cells (DCs) infected with antibody-coated parasites. Bone marrow-derived dendritic cells (BM-DCs) from naive BALB/c were infected with Leishmania amazonensis axenic amastigotes at an 8:1 parasite-to-cell ratio. These parasites were untreated, previously coated with non-immune serum (NS-Am) or with serum obtained from infected BALB/c mice (immune serum, IS-Am). (A) Supernatants were collected at 24 h p.i. cytokine levels were measured using ELISA. (B) Infection rates of DC cultures were determined by counting 200 cells from each condition under a light microscope. Infected BM-DCs were then used for cocultures with purified naïve CD4⁺ T cells (2 x 10⁵ cells/well). (C) The proliferation of CD4⁺ T cells was indicated by [³H] thymidine incorporation. (D–E) Culture supernatants were collected at 72 h p.i. and cytokine levels were measured using ELISA. Data are presented as mean ± S.D. for each group. Similar results were observed from three independent experiments. (** P < 0.01; *** P < 0.001; n.s., not significant)