Michael W. Bennett

The Fas counterattack: cancer as a site of immune privilege

Abstract Resistance to apoptosis through the Fas receptor pathway coupled with expression of the Fas ligand might enable many cancers to deliver a pre-emptive strike or counterattack against the immune system. Therapeutic exploitation of this has exciting potential, but now seems more complex and hazardous than was first evident.

Is the mucosal route of administration essential for probiotic function? Subcutaneous administration is associated with attenuation of murine colitis and arthritis

Background: We and others have reported the prophylactic efficacy of oral consumption of probiotic lactobacilli in the interleukin 10 knockout (IL-10 KO) model of colitis. It has not been demonstrated that the oral route is essential for probiotic efficacy. Aims: (i) To determine the effect of parenteral administration (subcutaneous) of Lactobacillus salivarius 118 on colitis of IL-10 KO mice; and (ii) to determine if observed responses are disease specific. Methods: (i) IL-10 KO mice were injected subcutaneously with L salivarius 118 or saline over 19 weeks. At sacrifice, the bowels were histologically scored. Isolated splenocytes were cultured in vitro and cytokine levels measured. (ii) In the collagen induced arthritis model, DBA/1 mice were injected subcutaneously with the probiotic or saline. At sacrifice, paw thickness was measured and joints were histologically scored. Results: (i) Colonic inflammatory scores were significantly decreased in IL-10 KO mice injected with L salivarius 118 compared with controls (p<0.05). Proinflammatory cytokine production from stimulated splenocytes was significantly lower for the probiotic group whereas stimulated transforming growth factor β (TGF-β) levels were significantly increased (p<0.05). (ii) Scoring of arthritis and paw thickness were significantly improved in the group of mice injected with L salivarius 118 compared with controls. Conclusions: (1) Subcutaneous administration of L salivarius 118 significantly attenuated colitis in the IL-10 KO model and suppressed collagen induced arthritis, suggesting that the oral route may not be essential for probiotic anti-inflammatory effects and that responses are not disease specific. (2) The probiotic effect was associated with reduced production of proinflammatory (T helper 1) cytokines and maintained production of anti-TGF-β.

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.

PCR detection of Mycobacterium paratuberculosis in Crohn’s disease granulomas isolated by laser capture microdissection

Background and aims: The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn’s disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn’s disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). Methods: The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn’s granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. Results: The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn’s granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. Conclusions: LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn’s disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.

Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer

Background Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a “Fas counterattack” against antitumour immune effector cells may contribute to tumour immune escape. Aim To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. Specimens Thirty paraffin wax embedded human gastric adenocarcinomas. Methods FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Results Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. Conclusions Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.

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.

Helicobacter pylori Modulates Lymphoepithelial Cell Interactions Leading to Epithelial Cell Damage through Fas/Fas Ligand Interactions

ABSTRACT Helicobacter pylori causes a common chronic infection of humans that leads to epithelial cell damage. Studies have shown that apoptosis of the gastric epithelium is increased during infection and this response is associated with an expansion of gastric T-helper type 1 (Th1) cells. We report that gastric T cells contribute to apoptosis of the epithelium by a Fas/Fas ligand (FasL) interaction. Fas receptor expression was detected on freshly isolated gastric epithelial cells by flow cytometry and immunohistochemistry, and this level of expression was increased during infection with H. pylori. The expression of Fas receptor on three gastric epithelial cell lines was increased by H. pylori, either alone or in combination with gamma interferon or tumor necrosis factor alpha. The role of Fas in apoptosis of gastric epithelial cell lines was evidenced by DNA fragmentation after cross-linking of Fas with specific antibodies. FasL expression was detected by immunohistochemistry on mononuclear cells in gastric biopsy specimens of infected but not uninfected subjects. Gastric T-cell lines were also shown to express FasL, as evidenced by reverse transcription-PCR and killing of target cells expressing Fas receptor. Moreover, these T-cell lines were capable of killing cultured gastric epithelial target cells and antibodies that block the interaction between Fas receptor and FasL inhibited this cytotoxic activity. These observations demonstrate that local Th1 cells may contribute to the pathogenesis of gastric disease during H. pylori infection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions.

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.

Fas ligand mediates immune privilege and not inflammation in human colon cancer, irrespective of TGF-β expression

Many cancers express Fas ligand (FasL/CD95L) in vivo, and can kill lymphoid cells by Fas-mediated apoptosis in vitro. However, overexpression of recombinant FasL in murine tumour allografts revealed a potential antitumour effect of FasL, via recruitment of neutrophils. Transforming growth factor-β1 (TGF-β1) could inhibit these neutrophil-stimulatory effects of FasL. In the present study, we sought to determine directly whether FasL contributes to immune privilege or tumour rejection in human colon cancers in vivo, and whether TGF-β1 regulates FasL function. Serial tumour sections were immunostained for FasL and TGF-β1. Neutrophils and tumour infiltrating lymphocytes (TILs) were detected by immunohistochemistry for lactoferrin and CD45, respectively. Apoptotic TIL were identified by dual staining for TUNEL/CD45. FasL expression by nests of tumour cells was associated with a mean four-fold depletion of TILs (range 1.8–33-fold, n=16, P<0.001), together with a two-fold increase in TIL apoptosis (range 1.6–2.5-fold, n=14, P<0.001), relative to FasL-negative nests within the same tumours. The overall level of neutrophils present in all tumours examined was low (mean 0.3%, n=16), with FasL expression by tumour nests associated with a mean two-fold decrease in neutrophils, irrespective of TGF-β1 expression. Together, our results suggest that tumour-expressed FasL is inhibitory rather than stimulatory towards antitumour immune responses.