Final Progress Report
Proposal No. NICE-07
Principal Investigator: Richard Blumberg, M.D.
Applicant Organization: Brigham and Women's Hospital (Boston, Massachusetts, U.S.A.)
Project Title: Functionally characterize modulins which bind to and regulate intestinal epithelial cell function
Period of Award: January 1, 2003 - December 31, 2003
Summary of project aims:
This grant proposal focused on identifying factors from the lumen of the intestine, bacterial and otherwise, that regulate epithelial cell function and structure. The initial proposal was to screen bacterial libraries for proteins that were able to act upon epithelial barriers in model systems. Ultimately, the research group of the Principal Investigator (PI) focused on an examination of factors present in the normal luminal milieu of humans for such properties.
Accomplishments towards meeting those aims:
In a series of studies, the PI examined the presence of factors in the lumen of the human intestine that regulated expression of CD1d. CD1d is a non-classical MHC class I molecule that is expressed on epithelial cells. The PI had previously noted that whereas CD1d is expressed in significant levels on epithelial cells in vivo in humans, it was expressed at very low levels by cell lines. This raised the hypothesis that the normal luminal milieu and/or environment of the intestine contained factors that upregulated CD1d.
To this end, the PI examined aqueous components of normal human fecal material for the ability to upregulate CD1d expression. Indeed, crude components contained within the aqueous fraction of normal human fecal material when applied to multiple epithelial cell lines, including T84, CaCo2 and HT29, induced the upregulation of CD1d based upon cell surface ELISA established in the laboratory of the PI. This upregulation of CD1d occurred within the first 24 hours and was maximal within 72 hours. It was associated with upregulation of mRNA levels in epithelial cells, as well as protein levels based upon the aforementioned ELISA, as well as Western blotting and confocal microscopy.
To define the molecule in the normal human fecal material that was responsible for this function, biochemical characterization was pursued. Preliminary studies showed that the molecule responsible for this induction of CD1d was probably proteinaceous in origin in that it was heat labile. In addition, its molecular weight was discovered to be greater than 100 kDa and it segregated into an aqueous phase, as noted.
In addition, to determine whether the factor was derived from prokaryotic or eukaryotic sources, fecal material from germ-free mice was examined. The ability to upregulate CD1d on human intestinal epithelial cell lines was also evident with fecal material from germ-free mice. This indicated that the component was likely of eukaryotic rather than prokaryotic origin. In addition, the factor was most prominently present within the proximal versus the distal intestine. Based upon these properties, a biochemical purification scenario was established and a protein of greater than 100 kDa identified, which had the property of upregulating CD1d on intestinal epithelia. Microsequencing of this protein showed it to be heat shock protein 110 (Hsp110).
To prove that Hsp110 was indeed the factor responsible for this upregulation of CD1d, the following studies were performed. Recombinant Hsp110 was obtained from a collaborator at the University of Buffalo. When applied to epithelial cell lines, CD1d was induced. This induction of CD1d was blocked with an antibody specific for Hsp110, but not an antibody specific for Hsp30. In addition, Hsp110 could be identified by Western blotting in normal fecal material and the induction of CD1d by the aqueous components of normal fecal material could be blocked by an antibody specific for Hsp110, but not an antibody specific for Hsp30. In addition, immunohistochemistry was performed with an antibody specific for Hsp110 and showed that Hsp110 decorated epithelial cells as well as a subset of lamina propria mononuclear cells with a morphology consistent with either dendritic cells or macrophages. Moreover, flow cytometry of normal human small intestinal epithelial cells showed that Hsp110 could be shown to be present on the cell surface of the epithelial cell.
Significant results:
These studies suggest an autocrine pathway wherein Hsp110 from epithelial cells is passed into the lumen of the intestine, whereupon it binds the apical cell surface of epithelial cells and induces the upregulation of Cd1d. These properties of Hsp110 upregulation of CD1d are consistent with the properties of other heat shock proteins. These studies have shown that Hsp110 combined a variety of potential cell surface receptors, such as Toll receptors, to induce intracellular signals that cause secretion of a wide variety of molecules as well as the upregulation of certain cell surface molecules. It is presumed that this property of Hsp110 falls into this rubric.
Lay Summary:
Maintenance of intestinal health and the avoidance of inflammatory conditions of the intestine, such as inflammatory bowel disease, are attained by a complex communication between molecular components contained within luminal bacteria, the epithelial cells and the subjacent immune cells underneath the epithelia. This complex trialogue results in maintenance of tolerance and stability of the epithelial barrier. The epithelial cell itself, being centered between the luminal components and the subjacent immune cells, is particularly important in this regard. Understanding the regulation of the immune function of the intestine and how it relates to luminal factors is, thus, exceedingly important. One molecule that is of particular interest to the laboratory of the Principal Investigator is called CD1d. CD1d is a molecule that is known to activate specific subsets of lymphocytes. In fact, CD1d expression on epithelial cells has been linked to the maintenance of the function of the epithelial barrier. It is therefore important to understand how CD1d is regulated and, specifically, by luminal factors. During the course of the Principal Investigator's studies, it was found that a protein could be identified in the normal human fecal matter that was presumably released from the epithelial cells to signal back to the epithelial cell the upregulation of CD1d. It remains to be proven whether this complex biologic pathway identified is involved in the maintenance of epithelial health and resistance to both inflammation and infection. However, these studies identified a novel, previously unknown cellular pathway that is likely to be involved in the maintenance of mucosal integrity.