In the adult subject, intestinal macrophages are uniquely down-regulated for expression of innate response receptors as well as for production of inflammatory cytokines. The ‘noninflammatory’ characteristics of intestinal macrophages serve to minimize mucosal inflammation despite close proximity to bacteria. In contrast, mononuclear (and epithelial) cells from fetal/preterm intestine develop an enhanced inflammatory response following exposure to bacterial products. In this study, we sought to explain this ‘proinflammatory bias’ of the immature intestine on the basis of developmental changes in the ECM. Intestinal tissue was collected from 12- to 24 week concepti (n = 11) following elective terminations. These were compared with adult intestinal tissue from bariatric surgery (n = 3). ECM products were extracted into stroma-conditioned media (SCM) by enzymatic removal of epithelial cells and culturing the tissue for 24 hours. All SCM were assayed for endotoxin (limulus lysate), protease activity, total protein, and transforming growth factor (TGF)-β concentrations. Peripheral blood monocytes were collected from healthy adult volunteers by Ficoll-Hypaque density centrifugation followed by ferromagnetic CD14 MACS purification. Neutrophils were isolated by CD15 MACS purification. Monocytes were plated in 96-well plates and treated with SCM after attachment. Cells were treated with LPS (500 ng/mL) after 2 hours. Culture supernatants were harvested after 18 hours. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 concentrations were measured by ELISA. The ability of culture supernatants to recruit monocytes and neutrophils was tested by microchemotaxis assay. The suppressive effect of fetal SCM (25-55% reduction) on monocyte cytokine responses was significantly less than adult SCM (up to 87% reduction; p < .01). The down-regulating effect of fetal SCM was developmentally regulated in the gestational age range tested in this study. These developmentally regulated effects correlated with TGF-β concentrations in the SCM. Furthermore, monocyte IL-8 production correlated with the ability of culture supernatants to recruit leukocytes in vitro. The proinflammatory bias of the preterm intestine as seen in conditions such as necrotizing enterocolitis may be due to immaturity of the intestinal ECM. Our observations provide a possible unifying explanation for earlier reports of enhanced inflammatory responses of mononuclear as well as epithelial cells from the immature intestine.
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