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Mesenchymal cell gene expression in up-regulated by synergistic cytokines and down-regulated by IL-1RA
April 1995
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INTERLEUKIN 15 mRNA TRANSCRIPTS IN ULCERATIVE COLITIS COLONIC SPECIMENS A. C. Stevens, W. Maslinski, A. Bitton, M.A. Peppercorn, and T.B. Strom. Center for Inflammatory Bowel Disease, Divisions of Gastroenterology and Immunology, Dept. of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, MA. INTRODUCTION: Interleukin 15 (IL-15) is a recently described T-ceU growth factor that binds to the beta and gamma chains of the interleukin 2 receptor (IL-2R). It is produced by a variety of cells and tissues and shares biological function with IL-2 (Grabstein KH et at. Science 264:965 1994). Activated T-cells are present in the colonic mucosa of inflammed Ulcerative Colitis (UC) specimens, however the potent T-cell growth factor IL-2 is not apregulated in these tissues. Enhanced mucosal IL-I5 levels might be contributing to the observed increase in activated T-cells. AIM: Therefore we measured ]].,-15 transcript levels within UC and normal colonic mucosai specimens. METHODS: Endoscopic colonic biopsies were obtained from normal and UC patients and categorized into three groups according to histology: UC active (UCA; n=20), UC inactive (UCI; n=lE), and Normal (NL; n=10). Total RNA was extracted from the biopsies, reverse transcribed to eDNA and amplified in the polymerase chain reaction (PCR) with IL-15 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide primers. Co-amplification of known quantities of respective competitive eDNA templates for each reaction permitted the densitometric measurement of size fractionated target and competitive template PCR products permitting quantitation of mRNA species. Measured amounts of IL-15 were expressed as a ratio to GAPDH to normalize any variability in the reverse transeription reaction and tissue sampling. RESULTS: IL-15 mRNA is detectable in normal and UC colonic tissues by the RT-PCR method. Quantitative IL-I5 and IL-15/GAPDH RT-PCR measurements did not vary to a statistically significant degree among UCA, UCI and NL groups. C O N C L U S I O N : IL-I5 mRNA expression determined by quantitative RT-PCR is not an accurate marker of disease activity. If IL-15 protein production correlates with steady state mRNA then mucosal IL-15 levels are unlikely to contribute to the generation of activated colonic T-cells in inflammed UC colonic tissues.
• MESENCHYMAL CELL GENE EXPRESSION IS UPREGULATED BY SYNERGISTIC CYTOKINES AND DOWNREGULATED BY IL-1RA. S.A. Strong. J.S. Klein, F. Cominelli, C. Fiocchi. The Cleveland Clinic Foundation. Cleveland, Ohio. Within the intestine's mucosal microenvironment, soluble factors and immune ceils may modulate the reactivity of non-immune mesenchymal ceils. This immune-non-immune interaction is supported by previous studies where we showed mesenchymal cells are capable of responding to and producing inflammatory mediators (GE 1992; 102:A701; 1993; 104:A784). But, it is uncertain whether inflammatory cytokine gene expression, induced by the same cytokines, can be up- or down-regulated. Therefore, the aims of this study were: determine and compare the cytokine gene expression induced by recombinant IL-1!3, IL-6, and TNFa used individually or in combination in human intestinal muscularis mucosae cell (HIMM) and fibroblast (HIF) cultures; investigate the possible modulatory effect of recombinant IL-1RA on IL-113-induced cytokine gene expression by HIMM and HIF. Autologous mesenchymal cells were derived from normal human surgical specimens. HIMM were originated by enzymatic digestion of the lamina propria and HIF grown from mucosal explants. To study upregulation, mesenchymal cell growth was arrested while cultures were still subconfluent and then activated with rIL-1[3, rIL-6, and/or rTNFm For down-regulation of rlL-113-induced gene expression, increasing concentrations of rIL-IRA (1:1 - 1:1000) were used. Northern blot hybridization was performed using cDNA probes specific for IL-I[~, IL-6, and TNFc<. In HIMM, weak constitutive expression of IL-6, IL-113, and TNF~t was differentially and substantially increased by activation with rIL-11~ alone and, to a lesser degree, rTNFc< alone. However, combinations of rIL-lJ3 and rTNFc< as well as rIL-113, rlL-6, and rTNFe~ provided a clear synergistic enhancement of the mRNA signals. Contrarily, rIL-1RA caused a blunting of the rIL-113-induced signal in a dose-dependent manner with complete inhibition of IL-6 and IL-1!3 expression at the highest rIL-1RA concentration. Autologous HIF responded in a manner similar to HIMM, except there was less gene transcript in all culture conditions. Our results suggest that HIMM and HIF potentially secrete local immune factors whose production is augmented by the synergism of inflammatory products and abated by local inhibitors. Through imbalances of this system in conditions such as inflammatory bowel disease, a cascade of events might contribute to a chronic disease state by feedback through the mesenchymal cells on the primary inflammatory response.
Immunology, Microbiology, and Inflammatory Disorders
A923
FIBRINOLYTIC SYSTEM (FS) DISTURBANCES IN ULCERATIVE COLITIS (UC) ARE ALSO P R E S E N T IN PERIPHERAl_ BLOOD VESSELS. A. Straszak A. Nowak, E. Sierka, P. Declerck" Dept. of Gastroenterology, Silesian University School of Medical Sciences, Katowice, Poland; 1 Ctr for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium. The FS seems to play an important role in the pathogenesis of UC and its complications. Recent data have shown the prothrombotic configuration of tissue and urokinase type plasminogen activators (t-PA, uPA) and plasminogen activator inhibitor type 1 (PAl-l) in colon mucosa in active UC, as well as resolution of these changes parallel to the patient's status improvement. No such changes we have found in other parts of the gut's mucosa. The aim of current study was to answer the question whether the active stage of UC and it's subsequent effective treatment affect the FS in peripheral blood vessels of UC - patients. t-PA, u-PA and PAl -1 levels were determined in the blood plasma of 14 patients suffering from UC twice: at rest and after stimulation of FS in form of venous occlusion. Each measurement was performed in active stage, and after an effective treatment of the disease. Results were compared to those obtained in 10 healthy subjects, which served as a control group. t-PA, u-PA, and PAl-1 levels were determined using monoclonal antibodies (ELISA technique). Results: t-PA diff. u-PA diff. PAl-1 diff. before treatment 1.49 0.10 It 1.98 after treatment 3.82 [ 0.22 - 5.99 control group 16.07 0.32 3.62
p*=
0.0006 I 0.01 I 0.035 I NS
NS
INS
t-PA diff.;u-PA diff.;PAl-1 diff.: differences between values obtained after and before venous occlusion; ell the data are expressed as mean values in ng/ml of plasma; * Mann - Whitney test for non - matched pairs. Conclusions: 1. Active stage of UC is characterized by an impaired stimulated release of plasminogen activators (t-PA and u-PA) in peripheral blood vessels. 2. Impaired answer of the FS to the stimulant seems to improve parallel to the patient's condition. 3. Above observations are consistent with our earlier findings concerning prothrombotic changes of FS in inflamed colon mucosa in UC - patients.
TRIPLE-STAPLED ILEAL POUCH ANAL ANASTOMOSIS WITHOUT TEMPORARY ILEAL DIVERSION. Harvey J. Suqerman, M.D., Medical College of Virginia, Virginia Commonwealth University, Richmond, VA We have previously documented the efficacy of a one-stage colectomy and triple-stapled ileal pouch anal anastomosis (IPAA) in 75 patients without a temporary ileostomy in 68 (1,2). We now report a further decrease in morbidity in 32 additional patients operated upon by 1 surgeon from 5/93 to 12/94. Methods: All 32 underwent a triple-stapled [PAA without ileal diversion; 8 had acute colitis (2 fulminant toxic colitis), 2 adenocarcinoma; prednisone (28 + 3.5 mg/day) used in 19, 6-MP in 2 end cyclosporine in 1. Results: There was 1 pouch leak which healed without ileostomy, a decrease p < 0.01 ) from our previous 15% incidence (2). The average post-op length of stay (LOS), including complications, was 8.5 -+ 0.6 days [a decrease, from 28.3 + 1.7 days, p < 0.001, with the mucosectomy and hand-sewn IPAA with temporary ileostomy (1) and 12.9 4- 0.8 days, p < 0.01, in our first 68 patients with the one-stage stapled IPAA (2)], with patients discharged having excellent fecal function as early as 4, 5, 6 and 7 days after surgery in 1, 3, 3, and 9 patients, respectively. Residual colitic tissue was 0.8 _+ 0.2 cm end was at the dentate line in 10. Almost perfect fecal control was reported by 91% without active pouchitis, including those with the anastomosis at the dentate line. Ten patients (32%) developed pouchitis (watery stools, urgency and nocturnal accidents) which was controlled with repetitive courses of various oral antibiotics (metronidazole, sulfisoxazole, ciprofloxacin), 1 also required steroid enemas. No patient had postoperative sexual dysfunction. Additional complications included 1 wound infection, 1 hernia, anal fissures in 2, anastemotic stenosis in 3 (requiring dilation under anesthesia in 2), inflammation of the cuff in 2 treated with hydrocortisone suppositories, and a pouch-vaginal fistula which was asymptomatic in the absence of pouchitis. Conclusions: These data support use of the triple-stapled IPAA without temporary ileal diversion, even in steroid dependant patients, providing a marked decrease in LOS and earlier return to a functional life. Pouchitis is now the major problem with IPAA. (1) Sugerman HJ, Newsome HH, DeCosta G, Zfas$ A, Stapled ileoanal anastomosis for ulcerative colitis and familial polyposis without a temporary diverting ileostomy. Ann Surg 1991 ;213:606-19. (2) Sugerman H J, Newsome HH. Stapled iFeoanat anastomosis without a temporary ileostomy. Amer J Surg 1994;167:58-66.
•
INTERLEUKIN 15 mRNA TRANSCRIPTS IN ULCERATIVE COLITIS COLONIC SPECIMENS A. C. Stevens, W. Maslinski, A. Bitton, M.A. Peppercorn, and T.B. Strom. Center for Inflammatory Bowel Disease, Divisions of Gastroenterology and Immunology, Dept. of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, MA. INTRODUCTION: Interleukin 15 (IL-15) is a recently described T-ceU growth factor that binds to the beta and gamma chains of the interleukin 2 receptor (IL-2R). It is produced by a variety of cells and tissues and shares biological function with IL-2 (Grabstein KH et at. Science 264:965 1994). Activated T-cells are present in the colonic mucosa of inflammed Ulcerative Colitis (UC) specimens, however the potent T-cell growth factor IL-2 is not apregulated in these tissues. Enhanced mucosal IL-I5 levels might be contributing to the observed increase in activated T-cells. AIM: Therefore we measured ]].,-15 transcript levels within UC and normal colonic mucosai specimens. METHODS: Endoscopic colonic biopsies were obtained from normal and UC patients and categorized into three groups according to histology: UC active (UCA; n=20), UC inactive (UCI; n=lE), and Normal (NL; n=10). Total RNA was extracted from the biopsies, reverse transcribed to eDNA and amplified in the polymerase chain reaction (PCR) with IL-15 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) oligonucleotide primers. Co-amplification of known quantities of respective competitive eDNA templates for each reaction permitted the densitometric measurement of size fractionated target and competitive template PCR products permitting quantitation of mRNA species. Measured amounts of IL-15 were expressed as a ratio to GAPDH to normalize any variability in the reverse transeription reaction and tissue sampling. RESULTS: IL-15 mRNA is detectable in normal and UC colonic tissues by the RT-PCR method. Quantitative IL-I5 and IL-15/GAPDH RT-PCR measurements did not vary to a statistically significant degree among UCA, UCI and NL groups. C O N C L U S I O N : IL-I5 mRNA expression determined by quantitative RT-PCR is not an accurate marker of disease activity. If IL-15 protein production correlates with steady state mRNA then mucosal IL-15 levels are unlikely to contribute to the generation of activated colonic T-cells in inflammed UC colonic tissues.
• MESENCHYMAL CELL GENE EXPRESSION IS UPREGULATED BY SYNERGISTIC CYTOKINES AND DOWNREGULATED BY IL-1RA. S.A. Strong. J.S. Klein, F. Cominelli, C. Fiocchi. The Cleveland Clinic Foundation. Cleveland, Ohio. Within the intestine's mucosal microenvironment, soluble factors and immune ceils may modulate the reactivity of non-immune mesenchymal ceils. This immune-non-immune interaction is supported by previous studies where we showed mesenchymal cells are capable of responding to and producing inflammatory mediators (GE 1992; 102:A701; 1993; 104:A784). But, it is uncertain whether inflammatory cytokine gene expression, induced by the same cytokines, can be up- or down-regulated. Therefore, the aims of this study were: determine and compare the cytokine gene expression induced by recombinant IL-1!3, IL-6, and TNFa used individually or in combination in human intestinal muscularis mucosae cell (HIMM) and fibroblast (HIF) cultures; investigate the possible modulatory effect of recombinant IL-1RA on IL-113-induced cytokine gene expression by HIMM and HIF. Autologous mesenchymal cells were derived from normal human surgical specimens. HIMM were originated by enzymatic digestion of the lamina propria and HIF grown from mucosal explants. To study upregulation, mesenchymal cell growth was arrested while cultures were still subconfluent and then activated with rIL-1[3, rIL-6, and/or rTNFm For down-regulation of rlL-113-induced gene expression, increasing concentrations of rIL-IRA (1:1 - 1:1000) were used. Northern blot hybridization was performed using cDNA probes specific for IL-I[~, IL-6, and TNFc<. In HIMM, weak constitutive expression of IL-6, IL-113, and TNF~t was differentially and substantially increased by activation with rIL-11~ alone and, to a lesser degree, rTNFc< alone. However, combinations of rIL-lJ3 and rTNFc< as well as rIL-113, rlL-6, and rTNFe~ provided a clear synergistic enhancement of the mRNA signals. Contrarily, rIL-1RA caused a blunting of the rIL-113-induced signal in a dose-dependent manner with complete inhibition of IL-6 and IL-1!3 expression at the highest rIL-1RA concentration. Autologous HIF responded in a manner similar to HIMM, except there was less gene transcript in all culture conditions. Our results suggest that HIMM and HIF potentially secrete local immune factors whose production is augmented by the synergism of inflammatory products and abated by local inhibitors. Through imbalances of this system in conditions such as inflammatory bowel disease, a cascade of events might contribute to a chronic disease state by feedback through the mesenchymal cells on the primary inflammatory response.
Immunology, Microbiology, and Inflammatory Disorders
A923
FIBRINOLYTIC SYSTEM (FS) DISTURBANCES IN ULCERATIVE COLITIS (UC) ARE ALSO P R E S E N T IN PERIPHERAl_ BLOOD VESSELS. A. Straszak A. Nowak, E. Sierka, P. Declerck" Dept. of Gastroenterology, Silesian University School of Medical Sciences, Katowice, Poland; 1 Ctr for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium. The FS seems to play an important role in the pathogenesis of UC and its complications. Recent data have shown the prothrombotic configuration of tissue and urokinase type plasminogen activators (t-PA, uPA) and plasminogen activator inhibitor type 1 (PAl-l) in colon mucosa in active UC, as well as resolution of these changes parallel to the patient's status improvement. No such changes we have found in other parts of the gut's mucosa. The aim of current study was to answer the question whether the active stage of UC and it's subsequent effective treatment affect the FS in peripheral blood vessels of UC - patients. t-PA, u-PA and PAl -1 levels were determined in the blood plasma of 14 patients suffering from UC twice: at rest and after stimulation of FS in form of venous occlusion. Each measurement was performed in active stage, and after an effective treatment of the disease. Results were compared to those obtained in 10 healthy subjects, which served as a control group. t-PA, u-PA, and PAl-1 levels were determined using monoclonal antibodies (ELISA technique). Results: t-PA diff. u-PA diff. PAl-1 diff. before treatment 1.49 0.10 It 1.98 after treatment 3.82 [ 0.22 - 5.99 control group 16.07 0.32 3.62
p*=
0.0006 I 0.01 I 0.035 I NS
NS
INS
t-PA diff.;u-PA diff.;PAl-1 diff.: differences between values obtained after and before venous occlusion; ell the data are expressed as mean values in ng/ml of plasma; * Mann - Whitney test for non - matched pairs. Conclusions: 1. Active stage of UC is characterized by an impaired stimulated release of plasminogen activators (t-PA and u-PA) in peripheral blood vessels. 2. Impaired answer of the FS to the stimulant seems to improve parallel to the patient's condition. 3. Above observations are consistent with our earlier findings concerning prothrombotic changes of FS in inflamed colon mucosa in UC - patients.
TRIPLE-STAPLED ILEAL POUCH ANAL ANASTOMOSIS WITHOUT TEMPORARY ILEAL DIVERSION. Harvey J. Suqerman, M.D., Medical College of Virginia, Virginia Commonwealth University, Richmond, VA We have previously documented the efficacy of a one-stage colectomy and triple-stapled ileal pouch anal anastomosis (IPAA) in 75 patients without a temporary ileostomy in 68 (1,2). We now report a further decrease in morbidity in 32 additional patients operated upon by 1 surgeon from 5/93 to 12/94. Methods: All 32 underwent a triple-stapled [PAA without ileal diversion; 8 had acute colitis (2 fulminant toxic colitis), 2 adenocarcinoma; prednisone (28 + 3.5 mg/day) used in 19, 6-MP in 2 end cyclosporine in 1. Results: There was 1 pouch leak which healed without ileostomy, a decrease p < 0.01 ) from our previous 15% incidence (2). The average post-op length of stay (LOS), including complications, was 8.5 -+ 0.6 days [a decrease, from 28.3 + 1.7 days, p < 0.001, with the mucosectomy and hand-sewn IPAA with temporary ileostomy (1) and 12.9 4- 0.8 days, p < 0.01, in our first 68 patients with the one-stage stapled IPAA (2)], with patients discharged having excellent fecal function as early as 4, 5, 6 and 7 days after surgery in 1, 3, 3, and 9 patients, respectively. Residual colitic tissue was 0.8 _+ 0.2 cm end was at the dentate line in 10. Almost perfect fecal control was reported by 91% without active pouchitis, including those with the anastomosis at the dentate line. Ten patients (32%) developed pouchitis (watery stools, urgency and nocturnal accidents) which was controlled with repetitive courses of various oral antibiotics (metronidazole, sulfisoxazole, ciprofloxacin), 1 also required steroid enemas. No patient had postoperative sexual dysfunction. Additional complications included 1 wound infection, 1 hernia, anal fissures in 2, anastemotic stenosis in 3 (requiring dilation under anesthesia in 2), inflammation of the cuff in 2 treated with hydrocortisone suppositories, and a pouch-vaginal fistula which was asymptomatic in the absence of pouchitis. Conclusions: These data support use of the triple-stapled IPAA without temporary ileal diversion, even in steroid dependant patients, providing a marked decrease in LOS and earlier return to a functional life. Pouchitis is now the major problem with IPAA. (1) Sugerman HJ, Newsome HH, DeCosta G, Zfas$ A, Stapled ileoanal anastomosis for ulcerative colitis and familial polyposis without a temporary diverting ileostomy. Ann Surg 1991 ;213:606-19. (2) Sugerman H J, Newsome HH. Stapled iFeoanat anastomosis without a temporary ileostomy. Amer J Surg 1994;167:58-66.