Immunological profiling of key inflammatory drivers of nasal polyp formation and growth in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is a chronic inflammatory condition of the upper airways, often associated with the formation of nasal polyps (CRSwNP). It is well established that macroscopically normal (non-polypoidal) sinonasal mucosa in CRSwNP patients can undergo polypoidal change over time, turning into frank polyps. However, little is known about what drives this process. This study aimed to investigate potential drivers of nasal polyp formation or growth through comparison of the immunological profiles of nasal polyps with contiguous non-polypoidal sinonasal mucosa, from the same patients. METHODS: The immune profiles of three types of tissue were compared; nasal polyps and adjacent non-polypoidal sinonasal mucosa from 10 CRSwNP patients, and sinonasal mucosa from 10 control patients undergoing trans-sphenoidal pituitary surgery. Nasal polyp and control samples were also stimulated with Staphylococcus aureus enterotoxin B (SEB) using a nasal explant model, prior to cytokine analysis. Real time quantitative polymerase chain reaction (IL-5, T-bet, IL-17A, FoxP3, TLR-4, IL-8, IL-1beta and IL-6) and Luminex (IFNgamma, IL-5 and IL-17A) were used to quantify pro-inflammatory responses. RESULTS: Nasal polyps and contiguous non-polypoidal sinonasal mucosa from CRSwNP patients displayed a very similar pro-inflammatory profile. When stimulated with SEB, nasal polyps displayed a Th2/Th17 mediated response when compared to controls. CONCLUSIONS: In CRSwNP, nasal polyps and non-polypoidal sinonasal mucosa from the same patient displayed a similar pro-inflammatory profile skewed towards the Th2/Th17 pathway in nasal polyps following SEB stimulation, with evidence of disordered bacterial clearance. These factors may contribute to enhanced survival of bacteria and development of a chronic inflammatory milieu, potentially driving new polyp formation and recurrence following surgical removal.

Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

BACKGROUND: Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. OBJECTIVES: To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. METHODS: We have injected recombinant human ßII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. RESULTS: Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. CONCLUSIONS: Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease.

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation.

Transforming growth factor beta signalling and matrix metalloproteinases in the mucosa overlying Crohn's disease strictures.

BACKGROUND AND AIMS: In addition to its crucial role in dampening tissue-damaging immune responses in the gut, transforming growth factor beta (TGFbeta) is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix-degrading matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). TGFbeta signalling was investigated by analysis of Smad proteins and MMPs/TIMPs in the mucosa overlying strictures in patients with Crohn's disease (CD). METHODS: Specimens were collected from macroscopically normal mucosa overlying strictured and non-strictured gut of patients with fibrostenosing CD. Isolated myofibroblasts were cultured with anti-TGFbeta blocking antibody or TGF beta 1. TGFbeta transcripts were analysed by quantitative reverse transcription-PCR (RT-PCR). Smad proteins and MMPs were determined by immunoblotting. MMP-12 activity was measured by a real-time MMP-12 activity assay. An in vitro wound-healing scratch assay was used to assess myofibroblast migration. RESULTS: TGFbeta transcripts, phosphorylated Smad2-Smad3 (pSmad2-3) and TIMP-1 proteins were higher in mucosa overlying strictures than in mucosa overlying non-strictured areas. In contrast, mucosa overlying strictured gut had lower expression of Smad7, MMP-12 and MMP-3. Myofibroblasts from mucosa overlying strictured gut showed higher TGFbeta transcripts, a greater pSmad2-3 response to TGFbeta, increased TIMP-1, lower Smad7, increased collagen production and reduced migration ability compared with myofibroblasts from mucosa overlying non-strictured gut. TGFbeta blockade increased myofibroblast MMP-12 production and migration, more obviously in myofibroblasts isolated from mucosa overlying non-strictured compared with strictured gut. CONCLUSIONS: Changes in TGF-beta signalling and MMP production were identified in the mucosa overlying strictures in CD which may give a window into the process of fibrosis.

CC-10004 but not thalidomide or lenalidomide inhibits lamina propria mononuclear cell TNF-α and MMP-3 production in patients with inflammatory bowel disease.

BACKGROUND: Thalidomide, one of whose activities is to inhibit Tumour Necrosis Factor (TNF)-α production, has been reported to be an effective treatment for refractory inflammatory bowel disease (IBD). TNF-α driven production of matrix metalloproteinase (MMP)-3 by gut lamina propria mononuclear cells (LPMCs) is a major pathway of tissue injury in IBD; however the effect of thalidomide and newer more potent immunomodulatory derivatives on this pathway has not been studied. AIM: To investigate the effect of thalidomide, CC-4047 (pomalidomide), CC-5013 (lenalidomide), and CC-10004 (apremilast) on gut LPMC TNFα and MMP-3 production in patients with IBD. METHODS: Gut LPMCs and myofibroblasts were isolated from patients with IBD, and cultured with thalidomide, CC-4047, CC-5013, and CC-10004. MMP-3 and TIMP-1 levels were determined by western blotting and real-time PCR, and TNF-α levels by ELISA. RESULTS: CC-10004 significantly reduced both TNF-α production and MMP-3 production by cultured LPMCs. Thalidomide and CC-4047 and CC-5013 had no significant effect on the production of TNF-α or MMP-3 by LPMCs. CONCLUSION: These results provides a mechanistic rationale for both the failure of lenalidomide (CC-5013) in a recent randomised controlled trial in Crohn's disease, and for the evaluation of CC-10004 as a novel oral therapy in the treatment of CD and UC.

Systemic administration of the chemokine macrophage inflammatory protein 1alpha exacerbates inflammatory bowel disease in a mouse model.

INTRODUCTION: Exacerbations of inflammatory bowel disease are thought to be related to concurrent infections. As infections are associated with elevated local and serum concentrations of chemokines, we have determined whether systemic administration of the CC chemokine macrophage inflammatory protein 1alpha (MIP-1alpha) exacerbates colitis in a mouse model. METHODS: Colitis was induced in Balb/c mice using trinitrobenzene sulfonic acid (TNBS). Starting four days later, animals received daily intraperitoneal injections of recombinant MIP-1alpha. On day 7, mice were killed and pieces of colon taken for immunohistology and polymerase chain reaction analysis. The direct effects of MIP-1alpha on mucosal T cells and fibroblasts in vitro were also investigated. RESULTS: Systemic administration of MIP-1alpha markedly enhanced colitis with mice developing large transmural ulcers filled with granulation tissue. Treatment resulted in increased numbers of CD4 cells infiltrating the colonic lamina propria, increased interferon gamma (IFN-gamma) levels, and increased transcripts for tumour necrosis factor alpha (TNF-alpha) and matrix metalloproteinase 3 (MMP3). Isolated lamina propria lymphocytes from mice with TNBS colitis contained increased numbers of IFN-gamma and TNF-alpha transcripts when stimulated with MIP-1alpha in vitro. Colonic lamina propria fibroblasts also responded to MIP-1alpha with increased proliferation and decreased collagen 1 synthesis but fibroblast proliferation was not seen in vivo. CONCLUSIONS: These experiments show that increasing serum concentrations of a chemokine, MIP-1alpha, exacerbates immune mediated colitis. The effect seems to be due to the ability of MIP-1alpha to boost Th1 responses in the gut wall. Our findings also suggest a potential pathway by which peripheral infections can exacerbate inflammatory bowel disease.

Collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) are expressed by migrating enterocytes during intestinal wound healing.

BACKGROUND: Matrix metalloproteinases (MMPs) play a crucial role in wound healing of the skin, airways, and cornea, but data on MMPs in normal intestinal wound healing is limited. The aim of this study was to clarify the role of collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) in intestinal wound repair and to determine the effect of cytokines on the expression of these MMPs in intestinal epithelial cell lines. METHODS: Surgical specimens from patients with ischemic colitis (n = 5) were used as an in vivo model of intestinal re-epithelialization. Fetal ileal explants were used as an ex vivo model. In situ hybridization for MMPs -1, -3, -7, and -10 was performed and immunohistochemical stainings were used to localize MMP-7 and -9 expressing cells. Stainings for cytokeratin and laminin-5 were performed to identify epithelial cells and migrating enterocytes, respectively. Caco-2, HT-29, and WiDr cell lines were treated for 6-48 h with different cytokines (e.g. EGF, KGF, IL-1 beta, TGF-alpha, TNF-alpha, and TGF-beta1) and Taqman real-time quantitative RT-PCR was used to investigate their effect on the expression of MMPs-1, -7, and -10. RESULTS: MMP-7, MMP-10, and MMP-1 were expressed by migrating enterocytes bordering intestinal ulcers in 5/5, 3/5, and 3/5 samples, respectively. In the fetal gut model, MMP-1 and MMP-10 were expressed by migrating enterocytes, but matrilysin-1 expression was not detected. Matrilysin-1 was up-regulated by TNF-alpha and IL-1 beta, and stromelysin-2 by TNF-alpha and EGF in Caco-2 and WiDr cell cultures. MMP-1 was up-regulated in Caco-2 cells by TGF-beta, EGF, and IL-1 beta, but only by EGF in WiDR cells. CONCLUSIONS: It is concluded that collagenase-1, stromelysin-2, and matrilysin-1 are involved in intestinal re-epithelialization in vivo and that they are up-regulated by cytokines relevant in wound repair.

Upregulation of matrix metalloproteinases in a model of T cell mediated tissue injury in the gut: analysis by gene array and in situ hybridisation.

BACKGROUND AND AIM: Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling and ulceration in inflammatory bowel disease and coeliac disease. Studies to date have concluded that stromelysin 1 is functionally involved in mucosal degradation. However, there are many other MMPs whose function in the gut is currently unknown. This work had two aims: firstly, to use gene array technology to measure changes in MMP and tissue inhibitor of metalloproteinase (TIMP) expression in a model of T cell mediated injury in the gut, and secondly, to correlate data from gene arrays with that generated by in situ hybridisation. METHODS: T cells in explants of human fetal gut were activated with pokeweed mitogen or anti-CD3 plus interleukin 12. Gene array analysis and in situ hybridisation were performed to investigate changes in MMP gene expression. RESULTS: Both gene array analysis and in situ hybridisation indicated marked upregulation of stromelysin 2 and macrophage metalloelastase expression in the explants associated with mucosal destruction. The arrays also confirmed our previous observation that interstitial collagenase (MMP-1), stromelysin 1 (MMP-3), and gelatinase B (MMP-9) are upregulated but there was no change in MMP-2, -7, -8, -9, -11, -13, -14-17, or -19. Following T cell activation, transcripts for TIMPs were reduced. CONCLUSIONS: These results show that there is differential upregulation of MMPs during T cell responses in the gut and suggest that further studies on the role of stromelysin 2 and macrophage metalloelastase may show that they have a functional role. In addition, the increase in MMPs and reduction in TIMPs suggest that the protease/antiprotease balance in the mucosa may determine the extent of mucosal degradation.

Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis.

BACKGROUND: Allergic rhinitis and asthma show many similarities in their epithelial and inflammatory responses to allergens. However, one notable difference is that disruption and desquamation of the epithelium is a characteristic feature of asthma, whereas in perennial allergic rhinitis the epithelium is intact and thickened. One reason for this might be differing expression of matrix metalloproteinases (MMPs) or their inhibitors (TIMPs). There are few published data on the presence of MMPs or TIMPs in the nasal mucosa in rhinitis. OBJECTIVE: The purpose of this study was to investigate MMP and TIMP mRNA and protein in nasal mucosa from subjects with perennial allergic rhinitis and from nonrhinitic control subjects. METHODS: Biopsy specimens of nasal mucosa were taken from 10 well-characterized subjects with perennial allergic rhinitis and 10 nonrhinitic control subjects. MMP and TIMP mRNA was quantified through use of competitive RT-PCR, and protein was detected by means of Western blotting and ELISA. RESULTS: TIMP-1 mRNA and TIMP-2 mRNA were present in nasal samples, but there was no significant difference between the 2 groups. Only small amounts of MMP-1, -2, -3, and -9 mRNA were detected in the same samples. The corresponding proteins were detected by means of Western blotting. TIMP-1 protein and TIMP-2 protein were quantified in tissue homogenates; there was no significant difference between the 2 groups. CONCLUSION: Our studies have demonstrated the presence of large amounts of TIMP-1 and TIMP-2 mRNA and protein in nasal mucosa. There is no upregulation of MMPs or changes in TIMP expression in the nasal mucosa of patients with allergic rhinitis.

Costimulatory molecules in the developing human gastrointestinal tract: a pathway for fetal allergen priming.

BACKGROUND: Antigen-specific responses can be detected in umbilical cord blood mononuclear cells. The fetal immune system must therefore attain a level of maturity compatible with the initiation of such responses as well as be exposed to antigen. OBJECTIVE: We sought to assess the expression of costimulatory molecules in fetal gut and the presence of cytokines in amniotic fluid at this time as a preliminary analysis of the suitability of the fetal gut as a site of antigen priming during intrauterine life. METHODS: Human fetal gut was analyzed for cells expressing costimulatory molecules through use of immunohistochemistry. Amniotic fluid was studied by ELISA, for cytokines regulating the nature of the response, and as a source of the common dietary antigen ovalbumin. RESULTS: MHC class II--positive cells were abundant over the period examined (11-24 weeks of gestation), other surface antigens showing spatial and temporal variation in expression. From 11 to 14 weeks of gestation, CD68-positive and CD40-positive cells, like MHC class II--positive cells, were present throughout the lamina propria; few CD3-positive cells (T cells) were observed. With the emergence of lymphoid aggregates (14-16 weeks), CD83-positive cells (dendritic cells) and CD20-positive cells (B cells) could be detected in fetal gut; however, expression was restricted to the lymphoid aggregates. In contrast, MHC class II, CD40, and CD68 continued to be expressed in the lamina propria. CD28-positive cells were also evident from 14 weeks of gestation, occurring throughout the lamina propria and lymphoid aggregates; this corresponded to the increasing numbers of CD3-positive cells. The occasional CD86-positive, CD40L-positive, or CTLA4-positive cell could be seen in or around lymphoid aggregates after 14 weeks of gestation. Lymphoid follicles forming after 16 weeks of gestation contained MHC class II--positive, CD83-positive, CD20-positive, CD40-positive, CD86-positive, CD3-positive, CD28-positive, CD40L-positive, and CTLA4-positive cells. MHC class II--positive, CD40-positive, CD68-positive, CD3-positive, and CD28-positive cells continued to be present in the lamina propria at this time. At all times studied, CD14 was not expressed in the lamina propria or lymphoid follicles. Prostaglandin E(2), TGF beta(1), and IL-10 dominated the amniotic fluid cytokine milieu, and ovalbumin was also detectable in amniotic fluid from 3 of 26 women who had detectable circulating levels. CONCLUSION: Of the costimulatory molecules studied, CD40 was the most abundant. However, both of the ligand families studied (CD40-CD40L and CD86-CD28/CD152) could provide the costimulatory signals required for the initiation of antigenspecific reactivity in the gastrointestinal tract of the human fetus as early as 16 weeks of gestation. The cytokine milieu would favor the development of T(H)2-type reactivity to antigens, such as ovalbumin, that are present at this time.

Interferon-alpha drives T cell-mediated immunopathology in the intestine.

The ability of interferon (IFN)-alpha to induce autoimmunity and exacerbate Th1 diseases is well known. We have recently described enhanced expression of IFN-alpha in the mucosa of patients with celiac disease (CD), a gluten-sensitive Th1-mediated enteropathy, characterized by villous atrophy and crypt cell hyperplasia. Previous studies from this laboratory have shown that T cell activation in explant cultures of human fetal gut can also result in villous atrophy and crypt cell hyperplasia. We have, therefore, examined changes that take place in explant cultures of human fetal gut after activation of T cells with anti-CD3 and/or IFN-alpha. We show that activation of T cells with anti-CD3 alone elicits a small IFN-gamma and TNF-alpha response with no tissue injury. Similarly, no changes are seen in explants cultured with IFN-alpha alone. However, addition of IFN-alpha with anti-CD3 results in enhanced Th1 response and crypt cell hyperplasia. This is associated with enhanced phosphorylation of STAT1, STAT3, and Fyn, a Src homology tyrosine kinase, which interacts with both TCR and IFN-alpha signal components. Together these data indicate that IFN-alpha can facilitate activation of Th1-reactive cells in the gut and drive immunopathology.

Parallel expression of macrophage metalloelastase (MMP-12) in duodenal and skin lesions of patients with dermatitis herpetiformis.

BACKGROUND: Dermatitis herpetiformis (DH) is a specific dermatological manifestation of coeliac disease and 80% of DH patients have gluten sensitive enteropathy manifested by crypt hyperplasia and villous atrophy. Matrix degradation mediated by collagenase 1 (MMP-1) and stromelysin 1 (MMP-3) has previously been implicated in the pathobiology of coeliac intestine and cutaneous DH blisters. AIMS: To study expression of stromelysin 2, metalloelastase, collagenase 3, and matrilysin in the intestine and skin of DH patients. METHODS: In situ hybridisation using 35S labelled cRNA probes was performed on duodenal biopsies of 15 DH patients, three samples each of control duodenal or jejunal mucosa, fetal ileal explants, lesional DH skin, and 19 serial biopsies of experimental DH blisters. Immunostaining was used to examine type IV collagen, macrophages (CD68), and 92 kDa gelatinase (MMP-9) in the specimens. RESULTS: Metalloelastase (MMP-12) was abundantly expressed by subepithelial macrophages in both coeliac intestine and spontaneous and induced DH rash. It was also upregulated in the experimental model of coeliac disease (staphylococcal endotoxin B stimulated fetal explants). The only other MMP detected was MMP-9 which did not colocalise with MMP-12. CONCLUSIONS: Upregulation of metalloelastase is associated with T cell mediated immune responses both in the intestine and skin. In addition to modulating macrophage migration, it may contribute to degradation of proteoglycans or basement membrane components in the subepithelial mucosa.

Role of interferon alpha in promoting T helper cell type 1 responses in the small intestine in coeliac disease.

Coeliac disease (CD) is caused by a CD4 T helper cell type 1 (Th1) response in the small intestinal mucosa to dietary gluten. As the major Th1 inducing cytokine, interleukin 12, is undetectable in CD gut mucosa, the mechanism by which Th1 effector cells are generated remains unknown. Interferon (IFN) alpha, a cytokine capable of promoting IFN-gamma synthesis, has been implicated in the development of Th1 mediated immune diseases. Here we report a case of CD-like enteropathy in a patient receiving IFN-alpha for chronic myeloid leukaemia. Morphological assessment of duodenal biopsies taken from the patient showed total villous atrophy, crypt cell hyperplasia, and a high number of CD3+ intraepithelial lymphocytes. Both antigliadin antibodies and antiendomysial antibodies were positive. RNA analysis revealed pronounced expression of IFN-gamma. Withdrawal of gluten from the diet resulted in a patchy improvement in intestinal morphology, normalisation of laboratory parameters, and resolution of clinical symptoms. By western blot analysis, IFN-alpha protein was seen in the duodenal mucosa from untreated CD patients but not in controls. This was associated with marked expression of IFN-gamma protein in CD mucosa. Collectively, these results suggest a role for IFN-alpha in promoting Th1 responses to gluten.

Ligation of alpha4ss1 integrin on human intestinal mucosal mesenchymal cells selectively Up-regulates membrane type-1 matrix metalloproteinase and confers a migratory phenotype.

Human intestinal lamina propria mesenchymal cells show high surface expression of the alpha4ss1 integrin. Ligation of alpha4ss1 on mesenchymal cell lines with an activating monoclonal anti-alpha4 antibody or vascular cell adhesion molecule-immunoglobulin (VCAM-IgG) leads to the appearance of activated forms of gelatinase A in culture supernatants, and the de novo expression of activated membrane type-1-matrix metalloproteinase (MT1-MMP). In functional assays, signaling through alpha4ss1 results in an increased capacity of mesenchymal cells to migrate through an artificial extracellular matrix, an effect inhibitable by excess tissue inhibitor of metalloproteinase-2. In organ cultures of human intestine, VCAM-IgG also up-regulates MT1-MMP, and in mucosal ulcers of inflammatory bowel disease patients, MT1-MMP transcripts are abundant, coincident with expression of VCAM-1 on cells at the ulcer margin. Collectively these results suggest that alpha4ss1-induced up-regulation of MT1-MMP may be a crucial factor in the migration of mesenchymal cells into ulcer beds during restitution of diseased gut mucosa.

Butyrate upregulates stromelysin-1 production by intestinal mesenchymal cells.

Nutritional factors and resident bacteria participate in the pathogenesis of intestinal inflammation. However, the ways in which bacteria and complex diets might modulate matrix metalloproteinase (MMP) production are unknown. We hypothesized that butyrate might enhance production of MMPs, thus amplifying their response to signals in inflammatory conditions. Human mesenchymal cells were incubated with butyrate and then stimulated with cytokines. MMPs and inhibitors were studied by Western blotting and quantitative RT-PCR. Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE. We showed that butyrate selectively enhanced the protein production and mRNA expression of stromelysin-1 in tumor necrosis factor-alpha- or interleukin-1beta-stimulated mesenchymal cells. Butyrate alone did not induce any change in MMP production or mRNA expression. It increased the acetylation of histones in mesenchymal cells. Furthermore, acetylation of histones (induced by trichostatin A) reproduced the effects of butyrate. Although butyrate is a major source of nutrient for the colonic epithelial cells, it modulates intestinal inflammation through the secretion of stromelysin-1 in stimulated stromal cells via the inhibition of histone deacetylase.

Human Peyer's patch T cells are sensitized to dietary antigen and display a Th cell type 1 cytokine profile.

Animal studies have demonstrated that feeding Ags induces regulatory (Th2, Th3) cells in Peyer's patches (PP), which migrate to the periphery and produce immunomodulatory cytokines such as IL-4, IL-10, or TGF-beta. In this work we have attempted to extend this paradigm to man by analyzing the response of human PP T cells to in vitro challenge with the common dietary Ag beta-lactoglobulin (betalg) of cow's milk. PP T cells stimulated with betalg showed enhanced proliferation compared with blood T cells from the same patient. Increased expression of CD25 and the Th1-associated chemokine receptor CCR5 was also seen on CD4(+) and CD8(+) PP T cells, but not blood T cells, stimulated with betalg. By enzyme-linked immunospot assay and RT-PCR, the PP T cell recall response to betalg and casein was dominated by IFN-gamma, with negligible IL-4, IL-5, IL-10, or TGF-beta. To help explain the PP T cell response to betalg, we examined IL-12 expression. Both IL-12p40 and -p35 transcripts were abundantly expressed in PP, but not in adjacent normal ileal mucosa. Immunoreactive IL-12p40-containing cells were present below the PP dome epithelium. Furthermore, in culture, PP, but not paired PBMC, spontaneously released IL-12p70. These results suggest that the human response to oral Ags in the gut may be different from that in rodents.

Imbalance of stromelysin-1 and TIMP-1 in the mucosal lesions of children with inflammatory bowel disease.

BACKGROUND: Degradation of the extracellular matrix and ulceration of the mucosa are major features of inflammatory bowel disease (IBD). One of the most important enzymes in degrading the matrix and produced in excess by cytokine activated stromal cells, is stromelysin-1. The activity of stromelysin-1 is controlled by tissue inhibitor of metalloproteinase (TIMP-1), its natural inhibitor. In model systems excess stromelysin-1 produces mucosal degradation. METHODS: Quantitative competitive RT-PCR was used to analyse stromelysin-1 and TIMP-1 transcripts; western blotting was used to measure the amount of stromelysin-1 and TIMP-1 protein in biopsy samples from children with IBD. RESULTS: In biopsies from patients with active Crohn's disease (n=24), ulcerative colitis (n=23), and controls (n=16), TIMP-1 transcripts and protein were abundant and unchanged. Stromelysin-1 transcripts and protein were markedly elevated in mucosal biopsies obtained from inflamed sites of patients with active IBD but were not elevated in adjacent endoscopically normal mucosa (n=10). Elevated levels of stromelysin-1 transcripts in active Crohn's disease (n=5) returned to normal levels following treatment with enteral nutrition. CONCLUSIONS: Stromelysin-1 is markedly overexpressed at inflamed sites in patients with IBD whereas TIMP-1 remains unaltered. Excess stromelysin-1 is likely to be responsible for loss of mucosal integrity in IBD.

Recent developments in the immunology of inflammatory bowel disease.

Crohn's disease and ulcerative colitis are caused by excessive immune reactivity in the gut wall. Analysis of the type of immune responses ongoing in diseased gut has revealed important features which suggest that these conditions are different. In Crohn's disease tissue there is considerable evidence for an ongoing T helper cell type 1 response, with excess interleukin-12, interferon-gamma and TNF-alpha. There is circumstantial evidence in patients that this response is directed against the normal bacterial flora and definitive evidence in mouse models that T cell responses to the flora cause gut disease. In ulcerative colitis, the role of tissue damaging T cell responses in the gut mucosa is much less clear and there is more evidence that the lesion is owing to antibody-mediated hypersensitivity. Although different types of immune reactions initiate tissue injury in both Crohn's disease and ulcerative colitis, the downstream events which actually damage the tissue are the same in each condition. Elevated cytokine concentrations in the mucosa lead to the production of excess matrix degrading enzymes by gut fibroblasts, loss of mucosal integrity and ulceration. The same process also leads to an increased production of epithelial growth factors such as KGF Keratinocyte Growth Factor by gut fibroblasts and produces the crypt cell hyperplasia characteristic of all gut inflammatory conditions.

Enhancing Lamina propria Th1 cell responses with interleukin 12 produces severe tissue injury.

BACKGROUND & AIMS: Interleukin (IL)-12 is believed to modulate local T-cell response in human colitis. A direct functional relationship between IL-12 and tissue injury in human intestine has not been reported. The aim of this study was to examine changes that take place in explant cultures of human fetal gut after stimulation of T cells with anti-CD3 in the presence of exogenous IL-12/IL-18. METHODS: T cells in explants of fetal gut were activated with anti-CD3 antibody and/or IL-12 or IL-18. Mucosal pathology was determined by immunohistochemistry. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to determine cytokine synthesis, and the production of matrix metalloproteinases was analyzed by RT-PCR and Western blotting. RESULTS: Activation of T cells in explants with anti-CD3 antibody elicited very little interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and no tissue injury. Addition of graded doses of IL-12 with anti-CD3 resulted in a significant increase in both IFN-gamma and TNF-alpha. This change was associated with a massive increase in stromelysin-1 expression and severe tissue injury, which was inhibitable by a stromelysin-1 inhibitor. Costimulation of explants with anti-CD3 and IL-18 induced only IFN-gamma and no tissue injury. CONCLUSIONS: IL-12 can convert a physiological T-cell signal into a strong signal with the downstream effect of elevating tissue stromelysin-1 concentration and mucosal degradation.