Inhibition of murine splenic and mucosal lymphocyte function by enteric bacterial products

Abstract

Previously we showed that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells. The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production. Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) production by murine spleen cells, but IL-10 production was increased. The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor beta (TGF-beta). EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-gamma production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer's patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells. Lysates obtained from Shiga-like toxin-producing E. coli, E. coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells. In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-beta.

FIG. 1

Bacterial lysates inhibit IL-2 secretion of mitogen-stimulated murine splenic cells. IL-2 concentrations in supernatants from BALB/c splenic cell cultures stimulated with PMA (2.5 ng/ml) and PHA (5 μg/ml) with or without preexposure for 2 h to French press lysates of EPEC 2348-69 (50 μg/ml) were measured by ELISA 46 h after stimulation. Values shown are means for duplicate cultures for each mouse (n = 20).

FIG. 2

Exposure to EPEC does not induce increased apoptosis. Percentages of cells in various stages of apoptosis are indicated. Cells were stained with annexin V-FITC (early apoptotic) and propidium iodide (late apoptotic) and analyzed by flow cytometry. BALB/c splenic cell cultures were exposed to either 50 μg (EPEC 50) or 100 μg (EPEC 100) of EPEC lysate per ml for 1 or 18 h. After a 2-h exposure to 50 mg of EPEC lysate per ml or to PBS, indicated cultures were stimulated with PMA and PHA and stained 1 h after mitogen stimulation. Values shown are means of four replicate cultures obtained from two mice (± standard deviation).

FIG. 3

IL-4, IL-10, and IFN-γ secretion by mitogen-stimulated spleen cells preexposed to EPEC lysates. Shown are IL-4 (A), IL-10 (B), and IFN-γ (C) concentrations in BALB/c splenic cell cultures after mitogen stimulation with and without exposure to EPEC lysates as described for Fig. 1. Values shown are means for duplicate cultures for each mouse (n = 11).

FIG. 4

EPEC-inhibitory activity is independent of IL-10 and TGF-β. Shown are IL-2 (A and C) and IL-10 (B and D) concentrations of mitogen-stimulated BALB/c splenic cultures exposed to increasing concentrations of anti-IL-10 (A and B) and anti-TGF-β (C and D) with and without preexposure to EPEC lysates as described for Fig. 1. Cells were obtained from three mice (M1, M2, and M3). Values shown are means of duplicate replicate cultures for each mouse.

FIG. 5

Depletion of CD11b-expressing cells did not change the effect of EPEC lysate on cytokine secretion. IL-2 (A), IL-10 (B), and IFN-γ (C) secretion of BALB/c splenic cultures depleted of CD11b-expressing cells and stimulated with PMA and PHA was determined following exposure or not to EPEC lysate (50 μg/ml). CD11b-expressing cells were removed by using magnetic beads and anti-CD11b antibody. Results from cultures made from three separate mice are shown. Values shown are means for duplicate determinations for each mouse.

FIG. 6

IL-2 secretion of mitogen-stimulated Jurkat cells preexposed or not to EPEC lysates (50 μg/ml). Exposure to EPEC and mitogen stimulation were as described for Fig. 1. IL-2 concentration in cell culture supernatants was measured by ELISA 16 h after mitogen stimulation. Values are means of duplicate cultures for each experiment (n = 5).

FIG. 7

Preexposure to EPEC lysate inhibits IL-2 secretion but enhances IL-10 secretion by antigen-stimulated murine splenic cells. Mice were immunized with OVA (n = 9) (A and B) or KLH (n = 6) (C) by subcutaneous injection at the base of the tail with 100 μg of each antigen in 0.1 ml of complete FA. Spleens were harvested from the mice, and the cell cultures were stimulated with OVA (200 μg/ml) or KLH (25 μg/ml) with or without a 2-h preexposure to EPEC lysate (50 μg/ml). IL-2 (A and C) and IL-10 (B) concentrations in cell culture supernatants were measured by ELISA 46 h after antigen stimulation. The high-responder OVA-stimulated mouse (+) received three booster injections each containing 100 μg of OVA in incomplete FA. Each value is a mean of duplicate replicate cultures for each mouse.