Reduced ratio of protectiveversusproinflammatory cytokine responses to commensal bacteria in HLA-B27 transgenic rats

Abstract

SUMMARYGerm-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T-or B-celldepleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-g , IL-12, TNF, IL-10 and TGF-b production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-g , IL-12 and TNF than did non-TG MLN cells . In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-b . T cell depletion abolished IFN-g and decreased IL-12 production, but did not affect IL-10 and TGF-b production. Conversely, neither IL-10 nor TGF-b was produced in cultures of B cell-depleted MLN. In addition, CD4 + T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-g when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-b , but not IFN-g , IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-g -producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.

Adoptive transfer of nontransgenic mesenteric lymph node cells induces colitis in athymic HLA-B27 transgenic nude rats

“…+ T cells stimulated with caecal bacterial lysates are required for production of the Th1 cytokines IFN-γ and IL-12 [7]. These findings emphasize the importance of T cells, specifically CD4…”

“…Caecal bacterial lysates were prepared as described previously [7,15]. Briefly, caecal contents from several non-TG rats were disrupted using glass beads and the lysate was filtersterilized (0·22 µ m) [7].…”

“…Briefly, caecal contents from several non-TG rats were disrupted using glass beads and the lysate was filtersterilized (0·22 µ m) [7]. Sterility was confirmed by aerobic and anaerobic bacterial culture.…”

“…MLN were removed from the rnu/rnu non-TG and TG recipients, and single cell suspensions were prepared, as described previously [7]. Unseparated MLN cells were washed twice and 4 × 10 5 cells were stimulated with caecal bacterial lysate cultured in 96 well flat bottom microplates (Costar 3595), in 0·2 ml complete medium (RPMI 1640 plus 5% heat inactivated fetal calf serum, 2 mM l -glutamine, 1 mM sodium pyruvate, 5 × 10 − 5 M 2-mercaptoethanol, and 50 µ g/ml gentamicin) for three days, as described [7].…”

“…However, intestinal inflammation does not occur in germ-free animals [6] or after monoassociation with another commensal intestinal bacterial species, E. coli [4]. Moreover, mesenteric lymph node (MLN) T cells produce interferon (IFN)-γ in response to caecal bacterial lysates [7]. Taken together, these results suggest that commensal luminal bacteria play a crucial role in the induction and perpetuation of colitis in HLA-B27 TG RATS.…”