A deficiency in the B cell response of C57BL/6 mice correlates with loss of macrophage-mediated killing of Leishmania amazonensis

Abstract

Infection of C3HeB/FeJ and C57BL/6 mice with Leishmania major stimulates a healing cell-mediated immune response, while Leishmania amazonensis infection leads to chronic disease. Here we show C3HeB/FeJ mice co-infected with both species of Leishmania heal, while co-infected C57BL/6 mice do not. Using an in vitro killing assay we determined B cells from infected C57BL/6 mice are ineffective in promoting parasite killing compared with B cells from infected C3HeB/FeJ mice. Furthermore, infected C57BL/6 mice produce less antigen-specific antibodies compared with infected C3HeB/FeJ mice. These findings suggest B cells play a required role in the cell-mediated immune response against L. amazonensis.

Fig. 1

Simultaneous co-infection with both Leishmania major (Lm) and Leishmania amazonensis (La) allows for lesion resolution in C3H but not B6 mice. A) Lesion size of co-infected C3H mice was significantly different from C3H mice infected with La alone (grey diamonds) (P < 0.001), while B) co-infected B6 mice were significantly different from the B6 mice infected with Lm alone (open squares) (P < 0.001). Lesion size was determined by measuring the infected footpad and comparing that with the non-infected footpad. Repeated measure ANOVA was used for statistical analysis. Results are representative of three separate experiments. C) The number of parasites in the lesions of co-infected C3H and B6 mice. Infected footpads were harvested and parasite suspensions were serially diluted in Complete Grace's medium and incubated at 27°C for 5 to 7 days. Different symbols (*, #) represent a statistically significant difference (P < 0.001) within the C3H infection groups and different letters (a, b) represent significant differences (P < 0.001) within the B6 infection groups. ANOVA and Scheffe pair-wise comparisons using Stat View software were used for statistical analyses. Results are the mean and standard error from three separate experiments.

Fig. 2

B cells isolated from Leishmania major (Lm)-infected C57BL/6 (B6) mice do not promote killing of Leishmania amazonensis (La) in vitro. A) CD19⁺ B cells and CD4⁺ T cells, purified from the draining lymph node (DLN) of C3H or B6 mice infected with Lm for 4 weeks, were placed into the upper chamber of a transwell plate with La-infected C3H-bone marrow-derived macrophages (BMDM) in the lower chamber as indicated above (first and second bars, respectively). B cells from B6 mice were isolated to the upper chamber and CD4⁺ T cells from C3H mice were placed in the bottom chamber (third bar). All wells contained Lm freeze-thawed antigen and were incubated for 5 days at 34°C. Black shading indicates the presence of B cells from B6 mice. B) Same as A except with BMDM derived from B6 mice and B cells from C3H mice were isolated to the upper chamber (second bar) as indicated. Black shading indicates the presence of B cells from C3H mice. * represent statistically significant differences (P < 0.001) as determined by ANOVA and Scheffe pair-wise comparison. Results are the mean and standard error from three separate experiments.

Fig. 3

Western blot analyses of parasite-specific production of total IgG and IgG isotypes IgG2a and IgG2c. Freeze-thawed Leishmania amazonensis (La) (a) or Leishmania major (Lm) (m) antigen was separated on a polyacrylamide gel and protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The blots were subsequently hybridized with mouse serum (1:25 dilution) pooled from four C3H mice or B6 mice (as indicated) that were non-infected (N) or infected for 5 or 12 weeks with Lm, La or both parasites (Co) as indicated. Following serum hybridization, the membranes were probed with goat anti-mouse antibodies to (A) total IgG or with goat anti-mouse antibodies to (B) IgG2a (C3H), or IgG2c. * identifies lanes with commercial molecular weight markers that cross-reacted with serum antibodies. Results are from one experiment at 5 weeks p.i. and representative of two experiments at 12 weeks p.i.