Scientific Abstract
Proposal No. IBD-0056
Principal Investigator: Andrew W. Stadnyk, Ph.D.
Applicant Organization: Dalhousie University (Halifax, Nova Scotia, Canada)
Project Title: Targeting neutrophil transepithelial migration as a means of controlling inflammation
Period of Award: June 1, 2003 - May 31, 2004
Leukocyte accumulation in the inflamed bowel is a hallmark of IBD, even at preclinical stages (1). The opinion that infiltrating leukocytes are likely to exacerbate the inflammation has increased interest in controlling leukocyte recruitment into the bowel (2). Yet it has proven technically and ethically difficult to establish the relevant mechanisms in patients in order to plan to control infiltration. Human studies are typically limited to reporting elevated expression of inflammatory mediators and cell adhesion molecules. Thus, the active roles of chemoattractants and adhesion molecules in mediating infiltration are first determined from studies using rodents and human cell culture systems (3). The insights gained using these models to date have inspired some human trials aimed at blocking components of the adhesion pathways, with mixed successes. However, our knowledge of leukocyte migration into the inflamed intestine, including across the epithelium, remains incomplete.
The intestinal epithelium has been shown to act as a source of chemoattractants in IBD (4), implicating this cell type in the regulation of the early and ongoing inflammatory response. We have shown that epithelial detachment is a key stimulus leading to cytokine expression (5). We propose that neutrophil transepithelial migration provokes epithelial cell cytokine expression through a mechanism similar to detachment. Hence, preventing infiltration of the epithelium may result in less inflammation. We are therefore intent on identifying the adhesion mechanisms between these cells with the goal of specifically blocking neutrophil infiltration into the intestinal epithelium.
In order to study neutrophil/epithelial cell interactions, a Transwell preparation of neutrophils migrating across T84 colon carcinoma cells was established by others (6). These investigators described β2 integrin (CD11b/CD18)-dependent neutrophil migration using fMLP as chemoattractant (7). We adopted and refined the Transwell assay to test our hypothesis. We found that transmigration does stimulate T84 cytokine expression. Moreover, we expanded on the variety of chemoattractants and discovered that neutrophils will migrate using CD11/CD18-independent mechanisms. This new finding increases the number of potential adhesion molecules used during transmigration. In view of this development, we are concerned that the Transwell system resembles the inflamed human intestine as closely as possible, in order to ensure that new findings can be applied in the best interest of the IBD patient. We have already taken one step in this direction by using neutrophils from IBD patients. Now our goal is to establish freshly isolated (primary) human colonocytes on the Transwell, effectively replacing the immortalized cancer cells. Standardizing the use of primary intestinal epithelial cells on Transwell filters will facilitate and enhance experiments that address the problem of neutrophil exacerbation of inflammation in IBD.
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7. Parkos CA, Delp C, Arnaout A, et al. J Clin Invest 1991;88:1605.