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Cytokine induction of inducible nitric oxide synthase (iNOS) is inhibited by mesalamine (5-ASA) in rat small intestinal epithelial cells
April 1998 triggered or followed by a relative lack of IL-13 locally in the intestinal wall. These results have important implications, as future treatment principles might involve local administration of IL-13 or inhibition of IL- 15. G4512
A CLINICAL CASE SERIES OF COLLAGENOUS COLITIS. D.Vakil, R.Batey, E.Smith, G.Radvan, E.Hewson, N.Porter, P.Wakeford. GE Department, John Hunter Hospital, Newcastle, NSW, Australia. Collagenous colitis is a chronic diarrhoeal disease characterized by a normal or near normal mucosa endoscopically and microscopic inflammation in the lamina propria, surface epithelial injury and a thick subepithelial collagen layer. We studied clinical characteristics, colonoscopic and histological findings and response to treatment in 34 patients (pts). METHOD: Outpatient records of 34 pts from the John Hunter and Tamworth Hospitals, were reviewed retrospectively. RESULTS: 25 (73%) F, 9(27%) M, with mean age of 65.2 years (36-87). Onset was insidious in 62% of pts and acute in 38%. Diarrhoea was watery with blood, mucus and pus, with nocturnal frequency in 19 (55%) pts. It was precipitated by food in 14 (41%) pts. Associated symptoms were abdominal pain 20 (58%), bloating and urgency 11 (32%) and weight loss 6 (17%) NSAID usage was noted in 20 (58%) of pts 17 (50%) had one or more associated conditions including RA, DM, hypothyroidism, myasthenia gravis, polymyalgia rheumatica, spondylolisthesis, CLL and liver transplant. INVESTIGATIONS: This showed 8/20 (40%) of pts had WBC in stool. Colonoscopy was normal in 27 pts and 7 had abnormal findings of erythema, mucosal oedema and aphthous ulcer. 8 pts had diverticular disease. Histologically 27 pts had continuous disease while 7 (21%) had patchy right sided disease. Follow-up was available in 24 pts with mean of 11.4 months (2-36). The majority of pts required treatment with multiple agents. Response rate for anti-diarrhoeals was 58% (14/24), binding agents 30% (3/10), sulphasalazine 50% (3/6), 5-ASA 55% (5/9), and Prednisolone 57% (4/7). Spontaneous resolution was noted in 6 pts, continuous diarrhoea in 9 pts, while 8 pts had a relapsing and remitting course. CONCLUSION: Collagenous colitis follows a chronic, intermittent course remitting and relapsing causing disabling symptoms. A high incidence of NSAID usage is noted in this Australian population as documented in the world literature. Histology can be patchy and at times only ® sided and multiple random biopsies should be obtained from the whole colon. Treatment can be difficult often requiring trials with multiple agents. Longterm followup is required to define the natural history of the disease. • G4513
HUMAN RECOMBINANT INTERLEUKIN-11 (rhIL-11) PROTECTS HAMSTERS AND HUMAN COLONIC MUCOSA FROM C. DIFFICILE TOXIN A (TxA) & B (TxB) EFFECTS. Valenick L, I Castagliuolo., M Riegler, *TC O'Brien, *JB Matthews, JT LaMont, & C Pothoulakis. Division of Gastroenterology and *Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA. We reported (Am. J. Physiol. 273:G333, 1997) that rhIL-11 administration in rats reduces G. diJficile TxA-enteritis and release of inflammatory mediators from intestinal macrophages (Mt~). We examined whether rhIL-11:1) protects hamsters from clindamycin-induced mortality, an animal model resembling human C. difficile infection, and 2) inhibits TxA and TxB-mediated effects on human colonic mucosa and human Mt~ in vitro. Methods: Syrian Hamsters were administered s.c. rhIL-11 (1 mg/kg) or vehicle every 2 days for a period of 10 days. C. difficile colitis was induced on day 2 by administration of clindamycin (5 mg/kg). Ussing chambered human colonic mucosae were exposed serosally and luminally to 1 ~g/ml of rhIL-11, 1 hr prior to luminal addition of TxA or B (3 nM) and changes in resistance (R) were recorded for 4.5 hr. Human M~/monocyte (THP-1) cells were incubated with rhIL-11 (1 ~g/ml) for 1-4 hr prior to addition of TxA or TxB (100 nM). TNFct and IL-8 mRNA and protein were determined after 4 hr by RT-PCR and ELISA. Results: rhIL-11 reduced C. difficile -induced mortality in hamsters by 40% (p<0.01). TxA and B caused a significant drop in human colonic R which was inhibited by rhIL-11 pretreatment [by 80% (p<0.01) and 65% (p<0.05) respectively], rhIL-11 also inhibited TxA and TxB-mediated increases of TNFt~, but not IL-8, release from THP-1 ceils at the protein and mRNA levels (p<0.01). Summary & Conclusions: IL-11 inhibits C. d!fficile toxin A and B-mediated 1) mortality in hamsters, 2) changes in resistance in human colonic mucosa, and 3) TNFct mRNA and protein increases in human monocytes/Mt~. These data suggest that the protective effects of IL-11 in human colonic mucosa may be associated with diminished upregulation of TNFct gene expression and translation and a direct action of this cytokine on colonocytes. Supported by Genetics Institute, Inc., Cambridge MA.
Immunology, Microbiology, and Inflammatory Disorders Al103 G4514
CYTOKINE
INDUCTION
OF
INDUCIBLE
NITRIC
OXIDE
SYNTHASE (iNOS) IS INHIBITED BY MESALAMINE ($-ASA) IN
RAT SMALL INTESTINAL EPITHELIAL CELLS. JF Valentine. Dept of Medicine, University of Florida and the VAMC, Galnesville, FL 32610. The advantages and disadvantages of iNOS in intestinal inflammation has remained controversial. Previous studies have demonstrated that iNOS can be produced in vivo by the intestinal epithelium and myenteric neurons in response to colonic injury. Others have demonstrated that a combination of cytokines will induce iNOS in cultured intestinal epithelial cells. IEC-6, IRD-98 and FRI rat small intestinal epithelial cell lines were evaluated for iNOS expression and the effect of treatment with mesalamine on the expression of iNOS. The cells were grown to confluence in DMEM with 5% FBS. Confluent cells were treated for 8 hrs with IL-113 (2 ng/ml), TNFct (10 mg/ml), LPS (0.5 laM), or IFNT (100 u/ml) and iNOS mRNA levels were evaluated by Northern analysis. Other plates were treated for 8 to 24 hrs with a cytomix (IL-113; 0.5 ng/ml, TNFct; 10 ng/ml, and IFN'/; 100 u/ml) alone and in combination with 5-ASA (2 mg/ml) pretreatment; iNOS mRNA and protein levels were determined by Northern and Western analysis respectively. The induction of iNOS mRNA levels was greatest for IFNT>IL-113>>> TNFct. iNOS mRNA was not induced by LPS. The fold induction of iNOS mRNA vs control can not be calculated because iNOS mRNA is undetectable in control cells. In both the IEC-6 and IRD-98 cell lines, cytomix induction of iNOS was reduced by 50% when the cells were co-treated with 5-ASA. Protein levels of iNOS were evaluated in the IRD-98 and FRI cell lines after 24 hr cytomix treatment, iNOS protein levels were undetectable in the control and 5-ASA treated cells. Cytomix treatment resulted in a marked induction of iNOS protein level. The iNOS protein levels in response to cytomix treatment were reduced by greater than 85%. Conclusion: iNOS mRNA is induced by IL-113, TNFet or IFN'/ but the effect is greatest for IFNT. The induction of iNOS mRNA is repressed by treatment with 5-ASA at therapeutically relevant concentrations. 5-ASA treatment also inhibited iNOS protein expression in response to treatment with the cytomix. The mechanism of the inhibition of the induction of iNOS remains to be determined but may be the result of changes in the redox state of the cell or inhibition of redox sensitive transcription factors such as NF~:-B. G4515
BONE MARROW RECONSTITUTION OF MHC II DEFICIENT MICE: DEFINING THE ROLE OF CLASS II ANTIGEN PRESENTATION AND CD4+VE T CELLS IN INTESTINAL INFLAMMATION AND ENTERIC MUSCLE DYSFUNCTION. B.A. Vallance. D.P. Snider and S.M. Collins. IDRP, McMaster University, Hamilton, Ontario, Canada. We have previously shown that jejunal muscle hyper-contractility occurs during infection with the nematode parasite T. spiralis and is at least partially T cell dependent. However the responsible T cell subset is unclear, since both CD4 and CD8 +ve T cells have been shown to infiltrate the jejunal muscularis externa during infection. In the present study, we examined the role of CD4 +ve T cells, as well as the identity of the class II antigen presenting cells required for their function, by infecting normal C57BL/6 and C2D (MHC II and partially CD4 T cell deficient) mice, as well as C2D mice which had undergone bone marrow transplantation using MHC II +ve bone marrow from C57BL/6 mice, or MHC II +ve bone marrow plus purified CD4 +ve T lymphocytes. Reconstitution was confirmed by FACScan analysis. Following infection with 375 T. spiralis larvae, we examined isometric contraction of jejunal longitudinal smooth muscle, as well as intestinal inflammation as measured by myeloperoxidase (MPO) activity, histology and immuno-histochemistry for MHC II. All groups of mice developed intestinal inflammation, as measured by increased MPO activity. Muscle from MHC II deficient mice developed significantly less tension (1450 mg/mm2 than C57BL/6 mice (2500 mg/mm2). Successful bone marrow reconstitution (without CD4 T cells) failed to restore the muscle hypercontractility (1500 mg/mm2). However numerous MHC II +ve cells (of presumed bone marrow origin) appeared in the neuromuscular layers following, but not before infection. Mice which underwent both bone marrow + CD4 T cell reconstitution were found to have a restored muscle hypercontractility (2200 mg/mm2), similar to the immunocompetent C57BL/6 mice. Increased tension generation was attenuated in MHC II deficient mice, and was still absent following bone marrow transplantation. However muscle changes were restored in MHC II deficient mice that received both bone marrow and CD4 +ve T cells. Thus MHC II expression alone, is insufficient to restore altered muscle function and requires, in addition, the presence of a full complement of CD4 +ve T cells. Supported by MRC Canada.
A CLINICAL CASE SERIES OF COLLAGENOUS COLITIS. D.Vakil, R.Batey, E.Smith, G.Radvan, E.Hewson, N.Porter, P.Wakeford. GE Department, John Hunter Hospital, Newcastle, NSW, Australia. Collagenous colitis is a chronic diarrhoeal disease characterized by a normal or near normal mucosa endoscopically and microscopic inflammation in the lamina propria, surface epithelial injury and a thick subepithelial collagen layer. We studied clinical characteristics, colonoscopic and histological findings and response to treatment in 34 patients (pts). METHOD: Outpatient records of 34 pts from the John Hunter and Tamworth Hospitals, were reviewed retrospectively. RESULTS: 25 (73%) F, 9(27%) M, with mean age of 65.2 years (36-87). Onset was insidious in 62% of pts and acute in 38%. Diarrhoea was watery with blood, mucus and pus, with nocturnal frequency in 19 (55%) pts. It was precipitated by food in 14 (41%) pts. Associated symptoms were abdominal pain 20 (58%), bloating and urgency 11 (32%) and weight loss 6 (17%) NSAID usage was noted in 20 (58%) of pts 17 (50%) had one or more associated conditions including RA, DM, hypothyroidism, myasthenia gravis, polymyalgia rheumatica, spondylolisthesis, CLL and liver transplant. INVESTIGATIONS: This showed 8/20 (40%) of pts had WBC in stool. Colonoscopy was normal in 27 pts and 7 had abnormal findings of erythema, mucosal oedema and aphthous ulcer. 8 pts had diverticular disease. Histologically 27 pts had continuous disease while 7 (21%) had patchy right sided disease. Follow-up was available in 24 pts with mean of 11.4 months (2-36). The majority of pts required treatment with multiple agents. Response rate for anti-diarrhoeals was 58% (14/24), binding agents 30% (3/10), sulphasalazine 50% (3/6), 5-ASA 55% (5/9), and Prednisolone 57% (4/7). Spontaneous resolution was noted in 6 pts, continuous diarrhoea in 9 pts, while 8 pts had a relapsing and remitting course. CONCLUSION: Collagenous colitis follows a chronic, intermittent course remitting and relapsing causing disabling symptoms. A high incidence of NSAID usage is noted in this Australian population as documented in the world literature. Histology can be patchy and at times only ® sided and multiple random biopsies should be obtained from the whole colon. Treatment can be difficult often requiring trials with multiple agents. Longterm followup is required to define the natural history of the disease. • G4513
HUMAN RECOMBINANT INTERLEUKIN-11 (rhIL-11) PROTECTS HAMSTERS AND HUMAN COLONIC MUCOSA FROM C. DIFFICILE TOXIN A (TxA) & B (TxB) EFFECTS. Valenick L, I Castagliuolo., M Riegler, *TC O'Brien, *JB Matthews, JT LaMont, & C Pothoulakis. Division of Gastroenterology and *Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston MA. We reported (Am. J. Physiol. 273:G333, 1997) that rhIL-11 administration in rats reduces G. diJficile TxA-enteritis and release of inflammatory mediators from intestinal macrophages (Mt~). We examined whether rhIL-11:1) protects hamsters from clindamycin-induced mortality, an animal model resembling human C. difficile infection, and 2) inhibits TxA and TxB-mediated effects on human colonic mucosa and human Mt~ in vitro. Methods: Syrian Hamsters were administered s.c. rhIL-11 (1 mg/kg) or vehicle every 2 days for a period of 10 days. C. difficile colitis was induced on day 2 by administration of clindamycin (5 mg/kg). Ussing chambered human colonic mucosae were exposed serosally and luminally to 1 ~g/ml of rhIL-11, 1 hr prior to luminal addition of TxA or B (3 nM) and changes in resistance (R) were recorded for 4.5 hr. Human M~/monocyte (THP-1) cells were incubated with rhIL-11 (1 ~g/ml) for 1-4 hr prior to addition of TxA or TxB (100 nM). TNFct and IL-8 mRNA and protein were determined after 4 hr by RT-PCR and ELISA. Results: rhIL-11 reduced C. difficile -induced mortality in hamsters by 40% (p<0.01). TxA and B caused a significant drop in human colonic R which was inhibited by rhIL-11 pretreatment [by 80% (p<0.01) and 65% (p<0.05) respectively], rhIL-11 also inhibited TxA and TxB-mediated increases of TNFt~, but not IL-8, release from THP-1 ceils at the protein and mRNA levels (p<0.01). Summary & Conclusions: IL-11 inhibits C. d!fficile toxin A and B-mediated 1) mortality in hamsters, 2) changes in resistance in human colonic mucosa, and 3) TNFct mRNA and protein increases in human monocytes/Mt~. These data suggest that the protective effects of IL-11 in human colonic mucosa may be associated with diminished upregulation of TNFct gene expression and translation and a direct action of this cytokine on colonocytes. Supported by Genetics Institute, Inc., Cambridge MA.
Immunology, Microbiology, and Inflammatory Disorders Al103 G4514
CYTOKINE
INDUCTION
OF
INDUCIBLE
NITRIC
OXIDE
SYNTHASE (iNOS) IS INHIBITED BY MESALAMINE ($-ASA) IN
RAT SMALL INTESTINAL EPITHELIAL CELLS. JF Valentine. Dept of Medicine, University of Florida and the VAMC, Galnesville, FL 32610. The advantages and disadvantages of iNOS in intestinal inflammation has remained controversial. Previous studies have demonstrated that iNOS can be produced in vivo by the intestinal epithelium and myenteric neurons in response to colonic injury. Others have demonstrated that a combination of cytokines will induce iNOS in cultured intestinal epithelial cells. IEC-6, IRD-98 and FRI rat small intestinal epithelial cell lines were evaluated for iNOS expression and the effect of treatment with mesalamine on the expression of iNOS. The cells were grown to confluence in DMEM with 5% FBS. Confluent cells were treated for 8 hrs with IL-113 (2 ng/ml), TNFct (10 mg/ml), LPS (0.5 laM), or IFNT (100 u/ml) and iNOS mRNA levels were evaluated by Northern analysis. Other plates were treated for 8 to 24 hrs with a cytomix (IL-113; 0.5 ng/ml, TNFct; 10 ng/ml, and IFN'/; 100 u/ml) alone and in combination with 5-ASA (2 mg/ml) pretreatment; iNOS mRNA and protein levels were determined by Northern and Western analysis respectively. The induction of iNOS mRNA levels was greatest for IFNT>IL-113>>> TNFct. iNOS mRNA was not induced by LPS. The fold induction of iNOS mRNA vs control can not be calculated because iNOS mRNA is undetectable in control cells. In both the IEC-6 and IRD-98 cell lines, cytomix induction of iNOS was reduced by 50% when the cells were co-treated with 5-ASA. Protein levels of iNOS were evaluated in the IRD-98 and FRI cell lines after 24 hr cytomix treatment, iNOS protein levels were undetectable in the control and 5-ASA treated cells. Cytomix treatment resulted in a marked induction of iNOS protein level. The iNOS protein levels in response to cytomix treatment were reduced by greater than 85%. Conclusion: iNOS mRNA is induced by IL-113, TNFet or IFN'/ but the effect is greatest for IFNT. The induction of iNOS mRNA is repressed by treatment with 5-ASA at therapeutically relevant concentrations. 5-ASA treatment also inhibited iNOS protein expression in response to treatment with the cytomix. The mechanism of the inhibition of the induction of iNOS remains to be determined but may be the result of changes in the redox state of the cell or inhibition of redox sensitive transcription factors such as NF~:-B. G4515
BONE MARROW RECONSTITUTION OF MHC II DEFICIENT MICE: DEFINING THE ROLE OF CLASS II ANTIGEN PRESENTATION AND CD4+VE T CELLS IN INTESTINAL INFLAMMATION AND ENTERIC MUSCLE DYSFUNCTION. B.A. Vallance. D.P. Snider and S.M. Collins. IDRP, McMaster University, Hamilton, Ontario, Canada. We have previously shown that jejunal muscle hyper-contractility occurs during infection with the nematode parasite T. spiralis and is at least partially T cell dependent. However the responsible T cell subset is unclear, since both CD4 and CD8 +ve T cells have been shown to infiltrate the jejunal muscularis externa during infection. In the present study, we examined the role of CD4 +ve T cells, as well as the identity of the class II antigen presenting cells required for their function, by infecting normal C57BL/6 and C2D (MHC II and partially CD4 T cell deficient) mice, as well as C2D mice which had undergone bone marrow transplantation using MHC II +ve bone marrow from C57BL/6 mice, or MHC II +ve bone marrow plus purified CD4 +ve T lymphocytes. Reconstitution was confirmed by FACScan analysis. Following infection with 375 T. spiralis larvae, we examined isometric contraction of jejunal longitudinal smooth muscle, as well as intestinal inflammation as measured by myeloperoxidase (MPO) activity, histology and immuno-histochemistry for MHC II. All groups of mice developed intestinal inflammation, as measured by increased MPO activity. Muscle from MHC II deficient mice developed significantly less tension (1450 mg/mm2 than C57BL/6 mice (2500 mg/mm2). Successful bone marrow reconstitution (without CD4 T cells) failed to restore the muscle hypercontractility (1500 mg/mm2). However numerous MHC II +ve cells (of presumed bone marrow origin) appeared in the neuromuscular layers following, but not before infection. Mice which underwent both bone marrow + CD4 T cell reconstitution were found to have a restored muscle hypercontractility (2200 mg/mm2), similar to the immunocompetent C57BL/6 mice. Increased tension generation was attenuated in MHC II deficient mice, and was still absent following bone marrow transplantation. However muscle changes were restored in MHC II deficient mice that received both bone marrow and CD4 +ve T cells. Thus MHC II expression alone, is insufficient to restore altered muscle function and requires, in addition, the presence of a full complement of CD4 +ve T cells. Supported by MRC Canada.