Joe O'Connell

Bacterial DNA within Granulomas of Patients with Crohn's Disease—Detection by Laser Capture Microdissection and PCR

OBJECTIVES:We previously reported the use of laser capture microdissection (LCM) and PCR to detect the presence of Mycobacterium paratuberculosis DNA in granulomas of patients with Crohns disease. While this does not imply a cause-effect relationship, it may influence the disease process because bacterial DNA has immunomodulatory effects. The aim of this study was to determine whether DNA from nonmycobacterial commensals, such as Escherichia coli, is also increased in the granulomas of Crohns disease.METHODS:Archival tissue from 15 surgical cases of Crohns disease and 10 non-Crohns granulomatous bowel disease controls were examined. Granulomas were isolated using LCM, and the extracted DNA was examined for presence of E. coli DNA by nested PCR amplification of a 135 base-pair segment of the uidA gene.RESULTS:E. coli DNA was detected in microdissected granulomas in 12/15 Crohns disease patients and in 1/10 non-Crohns control granulomas (p < 0.001). Also, E. coli DNA was detected in 8/15 Crohns full-thickness sections and in 4/10 control full-thickness sections.CONCLUSIONS:E. coli DNA may be detected more frequently in Crohns granulomas than in other non-Crohns bowel granulomas. This may indicate a tendency for lumenal bacteria to colonize inflamed tissue, or may be due to increased uptake of bacterial DNA by gut antigen presenting cells. In light of previous detection of M. paratuberculosis DNA in Crohns granulomas, the nonspecific nature of the type of bacterial DNA present in granulomas is evidence against any one bacterium having a significant causative role in Crohns disease.

Immune privilege or inflammation? Insights into the Fas ligand enigma

Fas ligand (FasL) has become an enigmatic molecule: some evidence indicates that it contributes to immune privilege in tissues and tumors, whereas other data demonstrates that FasL can elicit inflammation. New findings may begin to reconcile the paradoxical effects of FasL.

Altered Mechanisms of Apoptosis in Colon Cancer: Fas Resistance and Counterattack in the Tumor‐Immune Conflict

Abstract: Fas (CD95/APO‐1) is a cell surface “death receptor” that mediates apoptosis upon engagement by its ligand, FasL. Fas‐mediated apoptosis of lymphocytes normally serves immunoregulatory roles, including tolerance acquisition, immune response termination, and maintenance of immune privilege in certain organs. Colon tumors can exploit this lymphocyte death program by expressing FasL. This may enable colon tumors to mount a “Fas counterattack” against antitumor lymphocytes, impairing antitumor immune responses. FasL‐expressing colon tumor‐derived cell lines can trigger Fas‐mediated apoptosis of cocultured T cells in vitro. FasL expressed in esophageal cancer has been significantly associated with apoptosis and depletion of tumor‐infiltrating lymphocytes (TIL) in vivo. FasL may also facilitate metastatic colonization of Fas‐sensitive organs such as the liver, by inducing apoptosis of target organ cells. Normal colonic epithelial cells express Fas and are relatively sensitive to Fas‐mediated apoptosis. By contrast, colon tumor‐derived cell lines are usually resistant to induction of Fas‐mediated apoptosis, and colon cancer cells frequently coexpress Fas and FasL. The mechanisms allowing resistance to Fasmediated apoptosis are complex, and defects have been identified at several levels of Fas signal transduction. The “Bcl‐2 rheostat” may be pitched against apoptosis in colon cancer, inasmuch as overexpression of Bcl‐2, downregulation of Bak, and mutation of Bax are common defects in colon tumors. Caspase‐1 is also downregulated in colon cancer. The high frequency of p53 mutations in late‐stage cancers may also inhibit Fas signaling. Fundamental defects in apoptosis signaling may contribute to both immuno‐ and chemoresistance in colon cancer and allow expression of FasL to counterattack antitumor lymphocytes.

Fas counter-attack--the best form of tumor defense?

New in vivo evidence confirms that tumor-expressed Fas ligand impairs immune responses to cancer by inducing apoptosis of anti-tumor immune effector cells.

Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cells sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon‐γ (IFN‐γ). We found that IFN‐γ sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95/APO‐1) ligand (FasL), short‐chain fatty acids, and chemotherapeutic drugs. The extent of IFN‐γ‐mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN‐γ‐mediated upregulation of the proapoptotic protease caspase‐1. Although IFN‐γ alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl‐2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN‐γ increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase‐1. Our findings implicate caspase‐1 and Bcl‐2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis. J. Cell. Physiol. 185:331–338, 2000.

Differential Expression of Neurokinin-1 Receptor by Human Mucosal and Peripheral Lymphoid Cells

ABSTRACT Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.

Neurokinin‐1 receptor (NK‐1R) expression is induced in human colonic epithelial cells by proinflammatory cytokines and mediates proliferation in response to substance P

We have previously shown that the receptor for substance P (SP), neurokinin‐1 receptor (NK‐1R), is a marker of human mucosal but not peripheral mononuclear cells. In the present study, we investigate NK‐1R expression in the human colonic mucosa in vivo, particularly in the epithelial cells. We investigate the influence of proinflammatory Th1 cytokines and SP on expression and function of NK‐1R in colonic epithelial cells in vitro. Using in situ hybridization to detect NK‐1R mRNA, and immunohistochemistry to detect NK‐1R protein, colonic epithelial cells were found to express NK‐1R in vivo. In contrast, colon epithelial cell lines (Caco‐2, HT29, SW620, T84) were negative for NK‐1R mRNA and protein. However, stimulation with a proinflammatory cytokine cocktail containing IFN‐γ, TNF‐α, and IL‐1β, caused induction of NK‐1R expression. Expression of NK‐1R in human colonic epithelial cells in vivo may therefore reflect cytokine conditioning by the mucosal microenvironment. SP did not alter ion transport in monolayers of cytokine‐treated T84 cells. While SP stimulated epithelial ion transport in colonic mucosae ex vivo, this was not a direct effect of SP on the epithelial cells, and appeared to be neurally mediated. However, SP (10−10–10−8 M) elicited a dose‐dependent proliferative effect on cytokine‐stimulated, but not unstimulated, SW620 cells. Proliferation of the epithelial cells in response to SP was mediated specifically via cytokine‐induced NK‐1R, since an NK‐1R‐specific antagonist (Spantide 1) completely blocked SP‐mediated proliferation in the cytokine‐treated cells. Our results therefore demonstrate that proinflammatory cytokines induce expression of NK‐1R in human colonic epithelial cell lines, and that SP induces proliferation of the epithelial cells via cytokine‐induced NK‐1R. J. Cell. Physiol. 197: 30–41, 2003© 2003 Wiley‐Liss, Inc.

Fas Ligand Promotes Tumor Immune Evasion of Colon Cancer In Vivo

The study of the role of Fas ligand (FasL/CD95L) in tumor immune evasion has been complicated by the discovery that FasL may trigger cytokine secretion and induce inflammation. Antisense suppression of FasL expression by colon tumor cells was used to investigate if a reduction in endogenously expressed FasL in tumors resulted in reduced tumor development and improved anti-tumor immune challenge in vivo. Downregulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immune-competent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells was associated with increased lymphocyte infiltration. Moreover, constitutively expressed FasL was not pro-inflammatory. This study indicates that upregulation of FasL expression by colon tumor cells results in an improved anti-tumor immune challenge in vivo, providing functional evidence in favor of the ‘Fas counterattack’ as a mechanism of tumor immune evasion.

Bcl-x(L) expression in vivo in rheumatoid synovium

To examine the expression of the apoptosis regulatory protein, Bcl-xL, in the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunohistochemistry for Bcl-xL was carried out on synovial samples from patients with RA and OA. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis were performed to qualitatively examine the expression of Bcl-xL. Bcl-xL expression was detected in the lining, endothelium and inflammatory cells of both RA (n=20) and OA (n=10) samples. However, there was significantly more expression in the lining of RA synovium compared to OA (77 vs 61%, p<0.05). Many of the positive cells in the RA subsynovium were noted to be plasma cells. There was a significant correlation between Bcl-xL expression and the number of inflammatory cells in the subsynovium of RA and OA patients (rs=0.376, p<0.05, n=30). Age and disease duration did not correlate with Bcl-xL expression in rheumatoid patients. Bcl-xL may play a role in the extended survival of synoviocytes and inflammatory cells in rheumatoid synovium.

Fas ligand expressed in colon cancer is not associated with increased apoptosis of tumor cells in vivo

Expression of Fas ligand (FasL/CD95L) may help to maintain colon cancers in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Colon tumor‐derived cell lines appear to be relatively insensitive to apoptosis mediated by their own or exogenous FasL in vitro, despite expression of cell surface Fas. In our present study, we sought to investigate if FasL upregulated in human colon cancers leads to any increase in apoptosis of the tumor cells in vivo. FasL and Fas receptor (APO‐1/CD95) expression by tumor cells were detected immunohistochemically. Apoptotic tumor cell death was detected by immunohistochemistry for caspase‐cleaved cytokeratin‐18. FasL expression did not correlate with the extent of apoptosis of tumor cells. There was no significant local difference in the frequency of apoptosis of tumor cells between tumor nests that expressed FasL (mean = 2.4%) relative to those that did not (mean = 2.6%) (p = 0.625, n = 10; Wilcoxon signed rank). FasL expressed by the tumor cells appeared to be functional, since FasL expression in tumor nests correlated with diminished infiltration of tumor‐infiltrating lymphocytes (TILs). TILs were detected using immunohistochemistry for CD45. Expression of FasL by tumor nests was associated with a mean 4‐fold fewer TILs relative to FasL‐negative nests (range 2.4–33‐fold, n = 10, p < 0.003). Together, our results indicate that colon tumors are insensitive to FasL‐mediated apoptosis in vivo.