Secretory immunoglobulin A is the first line of immune defense, protecting the mucous membranes of the gastrointestinal and respiratory tracts against ingested and inhaled pathogens. The transport of secretory IgA into external fluids is mediated by the polymeric immunoglobulin receptor (poly- Ig-R) on the surface of mucosal epithelial cells. Because of its key role in mucosal immunity, the proposed experiments will explore the regulation of poly-Ig-R expression in a subclone of the human HT-29 colon carcinoma cell-line, HT-29.74, which undergoes enterocytic differentiation when cultured in medium devoid of glucose. Expression of poly-Ig-R by HT-29.4 cells is increased dramatically during differentiation and by treatment with cytokines such as interferon-gamma and tumor necrosis factor-alpha.
Three specific aims are proposed to determine the molecular mechanisms by which poly-Ig-R is regulated during differentiation of intestinal epithelial cells and by the action of specific immune modulators. First, the effects of the immune modulators interferon-gamma and tumor necrosis factor-alpha on steady-state levels of poly-Ig-R mRNA poly-Ig-R protein at specific stages of differentiation of HT-29.74 cells will be determined. Next, the contribution of transcriptional and post-transcriptional regulation will be assessed by measuring in vitro rates of poly-Ig-R gene transcription and stability of poly-Ig-R mRNA. Finally, specific cis- acting DNA elements in the poly-Ig-R gene which mediate its transcriptional regulation will be identified by 1) constructing chimeric plasmids in which fragments of the proximal 5'-flanking region of the poly-Ig-R gene have been inserted upstream of a reporter gene; 2) transfecting these plasmids into HT-29.74 cells; and 3) determining the effects of enterocytic differentiation and treatment with immune modulators on transcription of the reporter gene. The proposed experiments offer the potential for identifying novel regulatory mechanisms which control the differentiation- specific and cytokine-induced expression of poly-Ig-R. In addition, the proposed studies have the potential to add to our understanding of the molecular mechanisms by which cytokines in general regulate immune responses. Increased understanding of mechanisms regulating immune responses in the gut, including the effects of interferon-gamma and tumor necrosis factor-alpha, may offer new insights that could lead to better prevention and treatment of infectious diseases of mucosal surfaces, including AIDS.
