Final Progress Report
Proposal No. IBD-0002
Principal Investigator: Matthew B. Grisham, Ph.D.
Applicant Organization: Louisiana State University (Shreveport, U.S.A.)
Project Title: Role of the collagen-binding integrin alpha 1, beta 1 in an immune-based model of chronic colitis
Period of Award: March 1, 2002 - February 29, 2004
A. Summary of project aims.
Active episodes of inflammatory bowel disease (Crohn’s disease, ulcerative colitis; IBD) are characterized by the infiltration of large numbers of monocytes, lymphocytes, and neutrophils into the intestinal and/or colonic interstitium. Leukocyte infiltration is accompanied by extensive mucosal and/or transmural injury, suggesting that the inflammatory infiltrate plays an important role in the pathophysiology of IBD. Central to inflammatory responses are the integrin-mediated adhesive interactions of cells with their extracellular matrix (ECM)-rich environment.
There is an accumulating body of evidence to suggest that interaction between the leukocyte-associated collagen-binding integrins (a1b1, a2b1) and components of the extracellular matrix promotes migration and/or activation of extravasated leukocytes. Accordingly, our laboratory has shown that that engagement of T-cell associated a1b1 with the ECM plays an important role in attenuating inflammatory tissue injury in the acute, non-immune
model of DSS colitis. However, virtually nothing is known regarding the importance of leukocyte-interstitial matrix interactions in models of chronic gut inflammation.
Therefore, the overall objective of this study is to better understand the role of the a1b1 collagen-binding integrin in the pathophysiology of chronic gut inflammation in an immune-based murine model of chronic colitis. Our working hypothesis was that T-cell and/or granulocyte-associated integrin a1b1 interacts with interstitial collagen in the colonic interstitium resulting in the activation of these cells with the upregulation of certain pro-inflammatory cytokines that may initiate and/or perpetuate chronic gut inflammation.
In order to test this hypothesis, we proposed the following specific aims:
1) Evaluate the importance of T-lymphocyte a1b1 surface expression on the promotion of chronic gut inflammation using donor T-cells from a1-deficient (a1-/-) mice.
2) Determine the importance of granulocyte (e.g., monocyte/macrophage, PMNs) a1b1 surface expression on the initiation and/or perpetuation of chronic gut inflammation using RAG-2-/- ´ a1-/- double-deficient recipient animals.
B. and C. Accomplishment of Aims and Significant Results
In the first specific aim, we wanted to directly address the role of a1b1 on disease producing T-cells in our model. Therefore, we transferred a1-deficient CD4+CD45RBhigh T-cells into RAG-1-/- recipients. After eight weeks, a1-/- injected mice showed a significant attenuation in disease compared to RAG-1-/- mice that were injected with wild type (wt) CD4+CD45RBhigh cells. Upon histopathologic scoring (0-20), the proximal and distal a1-transfered colon sections showed a significant decrease in inflammatory scores compared to that of the colon scores from wt transfer animals (11.8 ± 2.2 vs. 9.8 ± 1.3, p=0.05 and 14.0 ± 2.5 vs. 8.5 ± 1.5; p=0.0001, respectively). Interestingly, transfer of a1-/- CD45RBhigh cells did not alter the colon weight to length ratios compared to controls (0.80 ± 0.03 mg/mm vs. 0.90 ± 0.13 mg/mm). Cytokine mRNA levels were assessed using semi-quantitative RT-PCR and normalized to the housekeeping gene GAPDH. Colonic cytokine mRNA levels for TNF-α and INF-γ obtained from wt CD45RBhigh animals showed significant fold increases compared to those from CD45RBlow transfer (4.4 fold, p=0.02 and 13.7 fold, p=0.04), respectively. However, no significant alterations were evident in TGF-β levels and no difference was detected between wt and a1-tranfer tissue samples in all three cytokines tested. In conclusion, these data suggest that engagement of T-cell associated a1b1 with the ECM may play a role in mediating intestinal inflammation via promotion of cellular movement and/or activation within the inflamed interstitium.
The second specific aim of this proposal focuses on the role of the leukocyte- associated a1b1 integrin in chronic colitis. In order to address a1b1 on invading leukocytes, mice had to be generated with the genotype of a1-/- and RAG-1-/-. These mice were generated by crossing BALB/c a1-/- mice with RAG-1-/- mice. After four generations, these mice have now been generated. Figure1 shows three representative animal (#1, 2 and 3) genotypes for both VLA-1 and RAG-1. As shown for both genes, the knockout PCR product (upper band, KO) migrates slower compared to the lower wild type (WT) product. These mice have no abnormal gross phenotype and the colony is being expanded to generate the desired amount of recipient animals.
D. Additional Studies
It is well known that the interaction of the integrin aLb2 (CD11a/CD18 or LFA-1) with its ligands Intracellular Adhesion Molecule (ICAM-1 or -2) is important for lymphocyte homing as well as lymphocyte activation. We wished to assess the role of T cell-associated LFA-1 in initiating and/or perpetuating chronic colitis in vivo. Transfer of CD4+CD45RBhigh T-cells isolated from either wild type (wt) or CD11a (aL) deficient (LFA-1-/-) C57Bl/6 mice, into Recombinase Activating Gene-1-deficient (RAG-1-/-) C57Bl/6 recipients was used to induce chronic colitis. Blinded histopathology and differences in colon weight/length ratios were used to quantify colonic inflammation. Cytokine protein levels produced by lamina propria lymphocytes (LPLs) were quantified using Cytometric Bead Array. We found that at eight-ten weeks following T-cell transfer, we observed a significant decrease in colon weight/length ratios in LFA-1-/- injected mice compared to wt T-cell injected colitic mice (4.08 ± 0.42 vs. 6.64 ± 0.64, respectively; p=0.007). This apparent decrease in colonic inflammation was confirmed by blinded histopathological scoring (14 ± 2.53 vs. 1.86 ± 0.36 for wt vs. LFA-1-/- T-cell injected mice, respectively; p=0.008). In addition, the number of colonic IELs and LPLs from LFA-1-/- injected mice were reduced by 11- and 5-fold, respectively. Isolated colonic LPLs from RAG-1 mice (injected with wt CD4+CD45RBhigh T-cells) and stimulated with CD3/CD28 antibodies expressed large amounts of TNF-a and IFN-γ (3.23 and 3.55 ng/ml, respectively), whereas LPLs from RAG-1-/- reconstituted with LFA-1-/- CD4+CD45Rbhigh T-cells produced three-fold less protein levels of these two cytokines (1.18 and 1.02 ng/ml, respectively). LFA-1 deficiency on T-cells attenuates chronic colitis in an immune-based model, which is associated with a decrease in colonic CD3+CD4+ lymphocytes in the IEL and LPL compartments. Additionally, the capacity for LFA-1-/- colonic LPLs to produce inflammatory cytokines is reduced. Taken together, our preliminary data suggest that LFA-1 is critical for T-cell activation, polarization, and/or recruitment into the colonic interstitium where they induce chronic colitis.
E. Lay summary of this report
The inflammatory bowel diseases (IBD) are known to be associated with a large influx of white blood cells (leukocytes) into the intestinal and/or colonic tissue. These leukocytes are thought to be responsible for producing diarrhea, rectal bleeding, cramping, abdominal pain and fever that is observed in patients with active episodes of IBD. For these cells to invade the tissue, they must leave the blood stream and enter the tissue using specific proteins. Once in the tissue, these cells bind to the proteins that hold together the tissue, which activates the white cells to produce products that have deleterious effects on the gut. The purpose of this proposal was to better understand the interaction between the invading white cells and the surrounding gut tissue in animals with chronic colitis. The findings obtained from the experiments listed in our first specific aim indicate that the white blood cells need the a1b1 surface protein in order to properly migrate in the gut tissue. Lack of this surface protein on certain white blood cells resulted in the attenuation of disease in a mouse model of chronic colitis. However, we hypothesized that a1b1 was involved in boosting the activity of the cells by the release of inflammatory factors. This, however, was not the case such that lack of a1b1 on specific white blood cells did not influence the expression of the inflammatory factors in the colon tissue of the experimental mice. We have only begun to address the role of this surface protein on other white blood cells involved in the initiation and perpetuation of chronic colitis.