Final Progress Report
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
A. Summary of project aims.
The goal of this project was to establish primary human colonocytes in vitro, growing as monolayers on Transwell filters, to replace the T84 colon carcinoma in studies of neutrophil/epithelial cell interactions. This was an ambitious aim, as viable and sustained culture of adult human enterocytes had not yet been achieved by anyone in the world. Yet, based on our ongoing work into intestinal epithelial cell death by apoptosis following detachment (also called "anoikis"), we anticipated that certain mediators which sustain detached rat crypt epithelial cells would also support the viability of human cells. In the period of this award, we have determined this is not the case, and in the period of one year we were unable to sustain viable adult human enterocytes in culture. Nevertheless, we are confident enough in our progress and with new approaches so that we intend to continue with the project.
B. Accomplishments towards meeting those aims.
The first major achievement behind the project was the establishment of the collaborations necessary to undertake such a study. I now have a good working relationship with the surgical and anatomical pathology staff in adult and pediatric medicine. While this may seem a trivial gain, it is crucial to my obtaining human bowel resections.
Our second major accomplishment was the optimization of the protocol for handling the resections and the ultimate isolation of enterocytes. There are three stages in the isolation at which we have conducted analyses: after separating the mucosa from underlying layers, the isolated whole crypts, and after cells have grown off the crypt onto matrix-coated plastic. I will briefly describe our findings at each of these stages.
Whole mucosa Incubating the whole mucosa on the complex matrix protein mix, Matrigel™, appears to preserve the cell integrity better than incubating the mucosa on untreated plastic. In the case of mucosa on plastic, we detect molecular signs of stress (activation /phosphorylation of the p38 kinase and activated caspase 3) unlike the mucosa on Matrigel™. Then, based on our evidence that IL-1β will delay apoptosis of detached rat intestinal epithelial cells, we applied IL-β onto the mucosa incubating on plastic. To our surprise and dismay, the IL-1β was without effect. Despite the lack of an effect of IL-1 to preserve the cells, we think there is a good potential to use whole mucosa on Matrigel™ in future experiments.
Isolated crypts The next step was to isolate intact crypts from the mucosa, which we mastered. The crypts separate from the lamina propria much like a tube sock with the tip of the sock representing the crypt bottom. After filtering, we characterized the cells in this preparation using various forms of microscopy. Using phase contrast microscopy, we can readily observe the tubular shape of the isolated crypts. To confirm that enterocytes indeed comprise the greatest proportion of cells, we have stained permeabilized crypts with the epithelial cell specific antibody Ber-EP4. The crypts were incubated in defined media, which was intended to inhibit growth of fibroblasts, but encourage the growth of epithelial cells. Under these conditions, we have screened the crypts for apoptotic and activation markers, using Western blotting. The crypts incubated on collagen-coated plastic for three hours showed little change in a number of markers. In particular, a low level of caspase activation was an indication that the crypt cells were "healthy." Yet, similar to treating the whole mucosa, neither IL-1 nor TNF registered any effect on the crypts using any of multiple molecular markers. We have measured the mRNA levels of some cytokines and chemokines from the freshly isolated crypts and from crypts cultured in the presence of IL-1 and TNF. We were surprised to find several inflammatory cytokines were readily detectable, e.g., IL-8, IL-1β, TNF-α, while others increased over the three hour period of culture (interferon-γ, IL-10). Equally unexpectedly, the cells express high levels of the mRNA and protein for IL-1RII, which we consider a property of "inflamed" epithelial cells. Neither IL-1 nor TNF significantly influenced cytokine mRNA levels within three hours of treatment.
Single enterocytes Following the isolation of whole crypts, we used a three-hour incubation on collagen-coated plastic dishes to promote cell adhesion, the first step toward establishing a monolayer. This approach is favored over dissociating the cells from the crypts using enzymes or EDTA since the rapid loss of cell to cell contact accelerates anoikis. However, after only 20 minutes following removal of the crypts from the dish, the collagen adherent cells show activated caspase 3, a sign of apoptosis. Again, in direct contrast to non-transformed rat crypt epithelial cells and the widely used human colon carcinoma cell lines in cell culture, the colonocytes adherent to the collagen appeared refractory to added IL-1 or TNF. Of course, this was not too surprising in view of the lack of responsiveness by the crypts. Our overall experience has been that following overnight culture on collagen (or other matrix protein), very few cells remain. We have systematically added growth factors intended to sustain the cells in culture. In contrast to the lack of effects of IL-1 and TNF, IL-11 seems to prevent the activation of caspases, but it is not obvious that more cells survive overnight. Neither PGE2 nor 10% human AB serum make a difference in the recovery of cells after 24 hours of culture. We used 5% fetal bovine serum and more cells seem to survive the first 24 hours of culture, but we are concerned that this condition promotes the growth of fibroblasts.
Finally, in the period that we were working with the human specimens, we began a series of experiments to better understand the effects of IL-1 treatment on intestinal epithelial cells using the rat cell line, IEC-18. This was in anticipation of making a direct comparison with the human colonocytes recovered from the resections. As was mentioned above, the human cells were unresponsive to added IL-1, but using the rat cells, we discovered that IL-1 exerts effects on multiple inflammatory molecules through multiple subcellular signaling pathways.
C. List of significant results (positive or negative).
1. Whole fresh human mucosa, isolated from normal appearing margins of bowel resected due to cancer, remains more viable if cultured on matrix-coated plastic than if cultured on bare plastic. This will facilitate the use of whole mucosa in some experiments.
2. Colonocytes isolated from fresh mucosa quickly (with 30 minutes) demonstrate signs of apoptosis. This occurs despite added IL-1, TNF, 10% human AB serum, or prostaglandin E2. The addition of IL-11 delayed the activation of apoptosis, but did not prevent cells from dying overnight.
3. Native enterocytes recovered from cancer patients are unresponsive to IL-1 or TNF, measured as a lack of activation of NFκB.
4. Native enterocytes recovered from cancer patients have high levels of mRNA for IL-1β, TNF-α, IL-8, IL-1RII, while interferon-γ and IL-10 mRNA levels increase steadily while the cells are in culture.
D. Lay summary of this report.
Crohn's disease and ulcerative colitis are chronic remitting and relapsing intestinal inflammatory diseases of unknown causes and for which there is no cure. New therapies introduced over the past decades have improved the health of many patients, but these diseases still exact a heavy toll measured in human suffering and use of heath care services. Some of these new therapies, for example, the biological agent anti-TNF monoclonal antibody, are based on information of the fundamentals of the inflammatory response. This project will provide new information about inflammatory processes relevant to the treatment of IBD.
It is presently understood that IBD is due in part to an overactive inflammatory response, possibly reacting to the bacteria in our intestines. One feature of the inflammation is the presence of neutrophils, one type of white blood cell, in the intestinal tissues. Neutrophils are normally found in the blood, but are recruited to sites of injury to battle invading pathogens. In the absence of pathogens, these cells often migrate straight across the intestinal tissues and into the lumen of the intestines, resulting in damage to the cells lining the gut. It is believed that this damage makes the inflammation worse and so stopping the neutrophils from reaching the lumen may help reduce the inflammation. We are studying how the neutrophils cross the lining cells (the epithelium) with a goal of discovering how to prevent it.
The epithelium is a formidable barrier to a cell determined to reach the lumen and the neutrophils must stick to, and push their way between adjacent epithelial cells. We can imitate these events using cells in the laboratory, in order to study the interactions. The shortcoming in the present experimental system is that human cancer cells are used as the epithelial cells and there is good reason to expect they do not resemble normal epithelium. Thus, the goal of this project was to learn to use freshly isolated human intestinal epithelial cells (from cancer surgery patients) for studies with neutrophils. We succeeded in isolating native cells, but unlike cancer cells, the native cells die soon afterwards. Our attempts to keep the cells alive, based on what we have learned about cells becoming cancerous, failed. In fact the cells we isolated seemed to not behave like normal epithelial cells and we may need to consider getting the bowel specimens from other types of patients. Meanwhile, we intend to treat the native cells so they become more like cancer cells in order to keep them alive for short term experiments with neutrophils. Establishing the use of native cells will ensure that our discoveries using neutrophils will have the greatest relevance and impact on improving the health of IBD patients.