P026. Recreating the intestinal macrophage in vitro: a potential role for stromal factors
G.S.C. Lee, M.C. Grimm, University of New South Wales, St George Clinical School, Department of Medicine, Kogarah, Australia
We have previously shown that one inhibitory (LILRB1) and one activating (LILRA5) leukocyte immunoglobulin-like receptor are expressed by macrophages in the colonic lamina propria. We hypothesise that factors in the colonic stroma allow newly-recruited blood monocytes to differentiate into immune-tolerant intestinal macrophages. The aim of this study was to develop a cell culture model of intestinal-like macrophages, in order to elucidate the roles of these LILRs in the colon.
Conditioned media was generated from stroma of the colonic lamina propria. Peripheral blood monocytes were differentiated in vitro into classical macrophages with GM-CSF or into intestinal-like macrophages with GM-CSF and stromal-derived conditioned media. These cells were assessed for expression of tumour necrosis factor (TNF)-α via ELISA and qRT-PCR, and LILRB1 and LILRA5 via qRT-PCR.
Cultured monocytes produced low levels of the pro-inflammatory cytokine, TNF-α. Following stimulation with lipopolysaccharide (LPS), classically differentiated macrophages produced high levels of TNF-α. When cells were differentiated to intestinal-like macrophages with stromal-derived conditioned media, this cytokine response was down-regulated in a dose- and time-dependent manner. Interferon (IFN)-γ and interleukin (IL)-10 stimulation did not significantly affect TNF-α expression. Changes in TNF-α mRNA levels in response to cell stimulation paralleled the cytokine levels. LILRB1 mRNA was highly expressed in intestinal-like macrophages, when compared with classically differentiated macrophages or peripheral blood monocytes. Conversely, LILRA5 mRNA expression was lower in cultured cells than blood monocytes, without a significant difference between classically differentiated and intestinal-like macrophages. Stimulation of classically differentiated macrophages with LPS, IFN-γ or IL-10 resulted in higher levels of LILRB1, but not of LILRA5. LILRB1 expression, however, was not increased in intestinal-like macrophages in response to cell stimulation.
Using colonic stromal-derived conditioned media, a model of intestinal-like macrophages has been developed. These cells demonstrate a blunted TNF-α responsiveness to LPS stimulation, suggesting that colonic stromal factors are likely to hold a key to the immune tolerant state of the intestine. Of importance is the over-expression of LILRB1 in this intestinal macrophage model that correlates with its presence in colonic lamina propria macrophages. This immunoregulatory molecule is also more prevalent in inflammatory bowel disease, and our colonic macrophage model enables further determination of its role, whether protective or pro-inflammatory, in this condition.