The co-regulatory effect of extracellular matrix proteins and integrins with interleukin-1beta on cytokine secretion by epithelial cells

Epithelial cells (EC) normally exist in vivo adhered to a basement membrane consisting of extracellular matrix (ECM) proteins such as collagen type IV (Col IV), laminin (LN), fibronectin (FN) and other glycoproteins. These ECM proteins mediate ECM-cell interactions through integrins and are involved in the regulation of cell adhesion and differentiation of intestinal epithelial cells (IEC). IEC have the capacity to produce a variety of pro-inflammatory cytokines such as IL-6, IL-8 and MCP-1 in response to bacterial infections, tissue injury, or other pro-inflammatory cytokines. We have studied the regulatory effects of ECM proteins, and subsequently integrin receptors, on the capacity of IEC to produce pro-inflammatory cytokines such as IL-6, IL-8, and MCP-1.; Our results revealed that IL-1β stimulated Caco-2 cells, a human colonic adenocarcinoma cell line, produced lower IL-6 and MCP-1 levels when cultured on a mixture of LN isoforms (mLN) as compared to IL-1β stimulated cells cultured on FN. RT-PCR analysis of IL-6 and MCP-1 mRNA levels revealed similar effects. The A549 cells, a lung Carcinoma cell line, were also stimulated with IL-1β and produced lower levels of IL-8 and MCP-1 when cultured on mLN as compared to IL-1β stimulated cells cultured on FN. Interestingly, similar experiments with the IEC-6 cells, a rat non-transformed cell line, did not show any significant differences in secreted cytokine levels or mRNA levels by the IEC-6 cells when cultured on mLN or FN. However, we were also able to determine that the down-regulatory effect of mLN on cytokine secretion/expression by the Caco-2 and A549 cells was due to the laminin-type 5 (LN-5) isoform of LN and its respective receptor the α3β1 integrin receptor. Additionally, EMSA analysis confirmed that the Caco-2 cells treated with the anti-α3 integrin antibody followed by IL-1β stimulation had decreased NF-κB and AP-1 activity as compared to IL-1β stimulated normal mouse IgG treated control cells. Interestingly, immunofluorescent staining results indicated that the α3 integrin subunit was expressed on the Caco-2 and A549 cells, but not the IEC-6 cells. This may explain the lack of an affect of mLN on cytokine secretion by the IEC-6 cells.; Results from these studies demonstrate that ECM proteins are able to regulate inflammatory cytokine production by EC in an immune response. A better understanding of the role of ECM/integrin in cytokine responses may further elucidate the role of IEC in mucosal immune responses as well as the development of effective and useful therapeutic drugs for the treatment of inflammatory diseases.