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Immunosuppressive role of interleukin-10 in inflammatory bowel disease
Immunology, Microbiology, and Inflammatory Disorders A87"/
April 1995
•
DIETARY ETHANOL HAS A DISPROPORTIONATE EFFECT ON MUCOSAL COMPARED TO PERIPHERAL T CELL RESPONSES. G. Minqolelli, S. Hurat, D. Lee, J. Roeenberg, C. Waltenbaugh, T.A. Barrett.
Dept. of Medicine and Microbiology/Immunology, Lakeside VA Hospital & Northwestern Univ. Med. School, Chlcago, IL. Alcoholism is associated with an increased frequency and severity of infections, impaired meJnory T cell responses and increased risk for neoplasia. The use of oral vaccines have highlighted the importance of the mucosal immune system. The present study examined the effect of ethanol (EtOH) on T cell responses to protein antigen (Ag} using an aB T-cell receptor (TCR} tranSgenic (Tg} murlne model (DO11.10) where >80% of T cells express a clonotypic TCR specific for a peptide sequence of chicken ovalbumin (Ova). Experimental groups consisted of mice fed a nutritionally complete liquid diet (AIN76A) where 30% of total calories were derived from EtOE (LEDso), a pairfed isocaloric liquid control diet (LCD) and a solid diet of mouse chc2w (SD}. Mice were placed on the diets for 4 days and total Tg T cell numbers were assessed by flow cytometry and invaunohiatology (IH) using a murine Ab specific for the Tg TCR. Proliferative responses were measured /n vitro by activating equivalent nqmbers of purified Tg ÷ T cells with Ova peptide and measuring q4-thymidine uptake after an 18 hour culture. I~ rive responses to Ag were assessed by IH by counting Tg T cells In sections of small intestine lamina proprla of mice fed 20 mg of whole Ova or swine y-globulin (SGG, control) 24 hours earlier. NO difference in mortality or weight was detected between groups. In splenic, mesenteric lymph node, Peyer ~s patch and laminapropria tissues, Tg* T cell numbers were not stgtiatically different between SD, LCD and LEDso grou~s. The Tg T cell proliferative response to Ova was significantly impaired on LEDge in the lamina proprla, but not in the spleen. Tg T cell response to Ova in v-~vo was significantly impaired in the lamina propria compared to SGG controls (see table).
PROU~TIVE RI:SPONSE ~ ,MMUNOH,STOLOGYII GROUP~ SPLEEN ILAMINA PROPRIA[I LAMINA PROPRIA II
Ucont,~o~.
Ico,~ol
o~a N co.t~oJ
o~s
LCD
3.0
31.0
0.2
11.0
47
136
LED~o
2.5
40,0
0.3
5.0*
44
62*
II
PNOtlFEIUtTIVERESPONSE: T c e l l s uere incubated in vitro i~ith 150meg/rat of Ova peptide or media atone (control) and pulsed with aS-thysidine. DaCe expressed as CPN x lOa, ' INIgJI~IISIOLOGY: Nice fed in vivo 20mg SGG (contro() or Ova protein. Data expressed as/rig + T cet [$/NPF~;2OOx.n=4 mice/group,# experiments=2. *p
In conclusion, mucosal but not peripheral T cell responses to antigen were decreased in T~ mice on EtOH-containing diets compared to liquid or solld diet controls. The data demonstrate that mucoaal immune responses are impaired after brief periods of exposure to EtOH. This differential effect on mucosal T cells may limit responses to oral antigen.
IMMUNOSUPPRESSIVE ROLE OF INTERLEUKIN-10 IN INFLAMMATORY BOWEL DISEASE. K Mitsuvama. A
T o y o n a g a , K T a n i k a w a , H Ishida*. Second Department of M e d i c i n e , K u r u m e U n i v e r s i t y S c h o o l of M e d i c i n e , Kurume, Japan. *National Utano Hospital, Kyoto, Japan. B A C K G R O U N D : E n h a n c e d p r o d u c t i o n of p r o i n f l a m m a t o r y c y t o k i n e s a n d i m p a i r e d p r o d u c t i o n of IL-1 r e c e p t o r antagonist (IL-Ira) has been described in i n f l a m m a t o r y b o w e l d i s e a s e (IBD). T o g e t h e r w i t h its in vitro e f f e c t s , IL-10 may have pathogenic and therapeutic implications. METHODS: IL-10 was detected in colonlc tissue sections using the immunohistochemical tequnique. T h e e f f e c t of r e c o m b i n a n t I L - 1 0 or a n t i - I L - 1 0 on c y t o k i n e p r o d u c t i o n by c u l t u r e d c o l o n i c m u c o s a a n d blood mononuclear cells from patients with ulcerative colitis and Crohn's disease was evaluated. RESULTS: Immunohistochemical studies of serial sections indicated that T cells and macrophages w e r e a m a j o r s o u r c e of IL-10. N e u t r a l i z a t i o n of e n d o g e n o u s I L - 1 0 w a s f o u n d to a u g m e n t b o t h T N F - a a n d IL-lfl p r o d u c t i o n a n d i n h i b i t IL-ira p r o d u c t i o n . C o n v e r s e l y , t h e a d d i t i o n of r e c o m b i n a n t I L - 1 0 to the culture medium of c o l o n i c t i s s u e s or b l o o d mononuclear cells resulted in i n h i b i t i o n of b o t h IL-lfl a n d T N F - ~ p r o d u c t i o n a n d a u g m e n t a t i o n of ILIra p r o d u c t i o n . These results were statistically significant. C O N C L U S I O N S : T h e s e r e s u l t s s u g g e s t t h a t endogenous IL-10, mainly released from T cells and m a c r o p h a g e s , m a y c o n t r i b u t e to the i m m u n o r e g u l a t i o n of IBD a n d t h a t e x o g e n o u s a d m i n i s t r a t i o n of IL~I0 or its related molecules may have potential therapeutic benefits.
INCREASED INCIDENCE OF LACTOSE INTOLERANCE (LI) IN PATIENTS WITH CROHN'S DISEASE (CD) OF THE TERMINAL ILEUM (TI). Barry Mishldn, Mort7 Yalovsky, Seymour Mishkin. Division of Gastroenterology, Royal Victoria Hospital, Faculties of Medicine and Management, McGill University, Montreal, Canada, Albert Einstein College of Medicine, Bronx, N.Y. We set out to determine the incidence of LI in 160 consecutive patients with ulcerative colitis (UC) and 140 with CD. 86.4% of UC and 87.5% of CD patients underwent H2 breath testing after a 25g lactose challenge. 46.8% of UC and 58.2% of CD patients studied were LI (based on _> 20 ppm rise in end expiratory H2 plus appropriate symptoms) difference significant (p < 0.065). The averaged initial ~ response in all groups of CD patients with LI was suggestive of bacterial overgrowth. Use of ethnic origin as a predictor of LI revealed that CD patients at low risk for LI (expected historical incidence 10-15% based on -->50 g challenge) had a 40% incidence of LI - significantly greater (p < 0.01) than t h e incidence of 13.3% in the comparable group of UC patients. 67.9% of CD compared to 56.0% of UC patients at moderate risk for LI (expected incidence 60-70%) were found to be LI - difference not significant. CD involving the terminal ileum (TI) was documented in 62.5% and 61.8% respectively of low and meal risk LI patients compared to 29.2% and 46~2% in the same groups of non LI patients - differences significant (p < 0.05). Evaluation of all groups of UC patients did not reveal any significantly different clinical characteristics. In order to determine whether disease location in CD is a determining factor for LI independent of ethnic origin, we tested for a dependency between these parameters in all CD patients irrespective of ethnic origin for LI. 68.1% of patients with CD limited to the TI were LI compared to only 43.5% with Crohn's colitis (p < 0.05). The incidence of LI in this latter group could be accounted by the risk for LI based on ethnic origin. As expected 100% of patients with CD limited to the proximal small bowel were LI. These observations suggest that in UC and CD limited to the colon ethnic origin appears to account for t h e incidence of LL In CD of the small bowel with or without colonic involvement LI also appears to be dependent on disease location. The role for bacterial overgrowth as an etiologic factor in the LI of CD patients remains speculative.
SOLUBLE GPIS0, A FORM INTERLEUKIN-6 RECEPTOR INFLAMMATORY BOWEL DISEASE.
OF CIRCULATING COMPONENT, IN K Mitsuvama, A
T o y o n a g a , T Nakasone*, H Yoshizaki*, M Honda*, K Tanikawa. S e c o n d D e p a r t m e n t of M e d i c i n e , K u r u m e U n i v e r s i t y S c h o o l of M e d i c i n e , Kurume, Japan. * L a b o r a t o r y of Immunology, National I n s t i t u t e of Health, Tokyo, Japan. BACKGROUND: In p r e v i o u s studies, we d e m o n s t r a t e d t h a t one f o r m of s o l u b l e IL-6 r e c e p t o r c o m p o n e n t , soluble IL-6 receptor (sIL-6R), f u n c t i o n s as an e n h a n c e r of t h e I L - 6 - m e d i a t e d immune process in i n f l a m m a t o r y b o w e l d i s e a s e (IBD) (Gastroenterology 1993;104:A747). The aim of this study was to e x a m i n e t h e in rive a p p e a r a n c e of s o l u b l e g p l 3 0 (sgpl30), a n o t h e r f o r m of s o l u b l e IL- 6 r e c e p t o r w h i c h acts as a n a t u r a l i n h i b i t o r of IL-6 action. M E T H Q D $ : S e r u m sgpl30, s I L - 6 R and IL-6 levels were measured in 27 p a t i e n t s with ulcerative colitis (UC), 20 w i t h C r o h n ' s d i s e a s e (CD) a n d in c o n t r o l s by ELISA. R E S U L T S : In c o n t r a s t to the s e r u m level of IL-6 or sIL-aR which increased maximally in t h e active phase, sgpl30 levels increased in t h e inactive phase ( m e a n ~ S D ; UC, 1 1 4 . 2 ~ 5 8 . 3 n g / m l ; CD, 7 0 . 6 ~ 37.4) as c o m p a r e d to the a c t i v e p h a s e (UC, 7 7 . 8 ~ 29.5 ng/ml; CD, 5 9 . 6 ~ 16.7) ( p < 0 . 0 5 f o r both). Fractionation of s e r u m f r o m t h e s e p a t i e n t s on a m o l e c u l a r s i z i n g c o l u m n r e v e a l e d that a part of IL6 c o e l u t e d w i t h s g p l 3 0 a n d s I L - 6 R at t h e f r a c t i o n c o r r e s p o n d i n g to 150 kDa. C O N C L U S I O N S : T h e s e r e s u l t s i n d i c a t e that t h e IL-6m e d i a t e d i m m u n e p r o c e s s in IBD is t i g h t l y r e g u l a t e d by two forms of s o l u b l e IL-6 r e c e p t o r s ; s I L - 6 R a n d sgpl30.
April 1995
•
DIETARY ETHANOL HAS A DISPROPORTIONATE EFFECT ON MUCOSAL COMPARED TO PERIPHERAL T CELL RESPONSES. G. Minqolelli, S. Hurat, D. Lee, J. Roeenberg, C. Waltenbaugh, T.A. Barrett.
Dept. of Medicine and Microbiology/Immunology, Lakeside VA Hospital & Northwestern Univ. Med. School, Chlcago, IL. Alcoholism is associated with an increased frequency and severity of infections, impaired meJnory T cell responses and increased risk for neoplasia. The use of oral vaccines have highlighted the importance of the mucosal immune system. The present study examined the effect of ethanol (EtOH) on T cell responses to protein antigen (Ag} using an aB T-cell receptor (TCR} tranSgenic (Tg} murlne model (DO11.10) where >80% of T cells express a clonotypic TCR specific for a peptide sequence of chicken ovalbumin (Ova). Experimental groups consisted of mice fed a nutritionally complete liquid diet (AIN76A) where 30% of total calories were derived from EtOE (LEDso), a pairfed isocaloric liquid control diet (LCD) and a solid diet of mouse chc2w (SD}. Mice were placed on the diets for 4 days and total Tg T cell numbers were assessed by flow cytometry and invaunohiatology (IH) using a murine Ab specific for the Tg TCR. Proliferative responses were measured /n vitro by activating equivalent nqmbers of purified Tg ÷ T cells with Ova peptide and measuring q4-thymidine uptake after an 18 hour culture. I~ rive responses to Ag were assessed by IH by counting Tg T cells In sections of small intestine lamina proprla of mice fed 20 mg of whole Ova or swine y-globulin (SGG, control) 24 hours earlier. NO difference in mortality or weight was detected between groups. In splenic, mesenteric lymph node, Peyer ~s patch and laminapropria tissues, Tg* T cell numbers were not stgtiatically different between SD, LCD and LEDso grou~s. The Tg T cell proliferative response to Ova was significantly impaired on LEDge in the lamina proprla, but not in the spleen. Tg T cell response to Ova in v-~vo was significantly impaired in the lamina propria compared to SGG controls (see table).
PROU~TIVE RI:SPONSE ~ ,MMUNOH,STOLOGYII GROUP~ SPLEEN ILAMINA PROPRIA[I LAMINA PROPRIA II
Ucont,~o~.
Ico,~ol
o~a N co.t~oJ
o~s
LCD
3.0
31.0
0.2
11.0
47
136
LED~o
2.5
40,0
0.3
5.0*
44
62*
II
PNOtlFEIUtTIVERESPONSE: T c e l l s uere incubated in vitro i~ith 150meg/rat of Ova peptide or media atone (control) and pulsed with aS-thysidine. DaCe expressed as CPN x lOa, ' INIgJI~IISIOLOGY: Nice fed in vivo 20mg SGG (contro() or Ova protein. Data expressed as/rig + T cet [$/NPF~;2OOx.n=4 mice/group,# experiments=2. *p
In conclusion, mucosal but not peripheral T cell responses to antigen were decreased in T~ mice on EtOH-containing diets compared to liquid or solld diet controls. The data demonstrate that mucoaal immune responses are impaired after brief periods of exposure to EtOH. This differential effect on mucosal T cells may limit responses to oral antigen.
IMMUNOSUPPRESSIVE ROLE OF INTERLEUKIN-10 IN INFLAMMATORY BOWEL DISEASE. K Mitsuvama. A
T o y o n a g a , K T a n i k a w a , H Ishida*. Second Department of M e d i c i n e , K u r u m e U n i v e r s i t y S c h o o l of M e d i c i n e , Kurume, Japan. *National Utano Hospital, Kyoto, Japan. B A C K G R O U N D : E n h a n c e d p r o d u c t i o n of p r o i n f l a m m a t o r y c y t o k i n e s a n d i m p a i r e d p r o d u c t i o n of IL-1 r e c e p t o r antagonist (IL-Ira) has been described in i n f l a m m a t o r y b o w e l d i s e a s e (IBD). T o g e t h e r w i t h its in vitro e f f e c t s , IL-10 may have pathogenic and therapeutic implications. METHODS: IL-10 was detected in colonlc tissue sections using the immunohistochemical tequnique. T h e e f f e c t of r e c o m b i n a n t I L - 1 0 or a n t i - I L - 1 0 on c y t o k i n e p r o d u c t i o n by c u l t u r e d c o l o n i c m u c o s a a n d blood mononuclear cells from patients with ulcerative colitis and Crohn's disease was evaluated. RESULTS: Immunohistochemical studies of serial sections indicated that T cells and macrophages w e r e a m a j o r s o u r c e of IL-10. N e u t r a l i z a t i o n of e n d o g e n o u s I L - 1 0 w a s f o u n d to a u g m e n t b o t h T N F - a a n d IL-lfl p r o d u c t i o n a n d i n h i b i t IL-ira p r o d u c t i o n . C o n v e r s e l y , t h e a d d i t i o n of r e c o m b i n a n t I L - 1 0 to the culture medium of c o l o n i c t i s s u e s or b l o o d mononuclear cells resulted in i n h i b i t i o n of b o t h IL-lfl a n d T N F - ~ p r o d u c t i o n a n d a u g m e n t a t i o n of ILIra p r o d u c t i o n . These results were statistically significant. C O N C L U S I O N S : T h e s e r e s u l t s s u g g e s t t h a t endogenous IL-10, mainly released from T cells and m a c r o p h a g e s , m a y c o n t r i b u t e to the i m m u n o r e g u l a t i o n of IBD a n d t h a t e x o g e n o u s a d m i n i s t r a t i o n of IL~I0 or its related molecules may have potential therapeutic benefits.
INCREASED INCIDENCE OF LACTOSE INTOLERANCE (LI) IN PATIENTS WITH CROHN'S DISEASE (CD) OF THE TERMINAL ILEUM (TI). Barry Mishldn, Mort7 Yalovsky, Seymour Mishkin. Division of Gastroenterology, Royal Victoria Hospital, Faculties of Medicine and Management, McGill University, Montreal, Canada, Albert Einstein College of Medicine, Bronx, N.Y. We set out to determine the incidence of LI in 160 consecutive patients with ulcerative colitis (UC) and 140 with CD. 86.4% of UC and 87.5% of CD patients underwent H2 breath testing after a 25g lactose challenge. 46.8% of UC and 58.2% of CD patients studied were LI (based on _> 20 ppm rise in end expiratory H2 plus appropriate symptoms) difference significant (p < 0.065). The averaged initial ~ response in all groups of CD patients with LI was suggestive of bacterial overgrowth. Use of ethnic origin as a predictor of LI revealed that CD patients at low risk for LI (expected historical incidence 10-15% based on -->50 g challenge) had a 40% incidence of LI - significantly greater (p < 0.01) than t h e incidence of 13.3% in the comparable group of UC patients. 67.9% of CD compared to 56.0% of UC patients at moderate risk for LI (expected incidence 60-70%) were found to be LI - difference not significant. CD involving the terminal ileum (TI) was documented in 62.5% and 61.8% respectively of low and meal risk LI patients compared to 29.2% and 46~2% in the same groups of non LI patients - differences significant (p < 0.05). Evaluation of all groups of UC patients did not reveal any significantly different clinical characteristics. In order to determine whether disease location in CD is a determining factor for LI independent of ethnic origin, we tested for a dependency between these parameters in all CD patients irrespective of ethnic origin for LI. 68.1% of patients with CD limited to the TI were LI compared to only 43.5% with Crohn's colitis (p < 0.05). The incidence of LI in this latter group could be accounted by the risk for LI based on ethnic origin. As expected 100% of patients with CD limited to the proximal small bowel were LI. These observations suggest that in UC and CD limited to the colon ethnic origin appears to account for t h e incidence of LL In CD of the small bowel with or without colonic involvement LI also appears to be dependent on disease location. The role for bacterial overgrowth as an etiologic factor in the LI of CD patients remains speculative.
SOLUBLE GPIS0, A FORM INTERLEUKIN-6 RECEPTOR INFLAMMATORY BOWEL DISEASE.
OF CIRCULATING COMPONENT, IN K Mitsuvama, A
T o y o n a g a , T Nakasone*, H Yoshizaki*, M Honda*, K Tanikawa. S e c o n d D e p a r t m e n t of M e d i c i n e , K u r u m e U n i v e r s i t y S c h o o l of M e d i c i n e , Kurume, Japan. * L a b o r a t o r y of Immunology, National I n s t i t u t e of Health, Tokyo, Japan. BACKGROUND: In p r e v i o u s studies, we d e m o n s t r a t e d t h a t one f o r m of s o l u b l e IL-6 r e c e p t o r c o m p o n e n t , soluble IL-6 receptor (sIL-6R), f u n c t i o n s as an e n h a n c e r of t h e I L - 6 - m e d i a t e d immune process in i n f l a m m a t o r y b o w e l d i s e a s e (IBD) (Gastroenterology 1993;104:A747). The aim of this study was to e x a m i n e t h e in rive a p p e a r a n c e of s o l u b l e g p l 3 0 (sgpl30), a n o t h e r f o r m of s o l u b l e IL- 6 r e c e p t o r w h i c h acts as a n a t u r a l i n h i b i t o r of IL-6 action. M E T H Q D $ : S e r u m sgpl30, s I L - 6 R and IL-6 levels were measured in 27 p a t i e n t s with ulcerative colitis (UC), 20 w i t h C r o h n ' s d i s e a s e (CD) a n d in c o n t r o l s by ELISA. R E S U L T S : In c o n t r a s t to the s e r u m level of IL-6 or sIL-aR which increased maximally in t h e active phase, sgpl30 levels increased in t h e inactive phase ( m e a n ~ S D ; UC, 1 1 4 . 2 ~ 5 8 . 3 n g / m l ; CD, 7 0 . 6 ~ 37.4) as c o m p a r e d to the a c t i v e p h a s e (UC, 7 7 . 8 ~ 29.5 ng/ml; CD, 5 9 . 6 ~ 16.7) ( p < 0 . 0 5 f o r both). Fractionation of s e r u m f r o m t h e s e p a t i e n t s on a m o l e c u l a r s i z i n g c o l u m n r e v e a l e d that a part of IL6 c o e l u t e d w i t h s g p l 3 0 a n d s I L - 6 R at t h e f r a c t i o n c o r r e s p o n d i n g to 150 kDa. C O N C L U S I O N S : T h e s e r e s u l t s i n d i c a t e that t h e IL-6m e d i a t e d i m m u n e p r o c e s s in IBD is t i g h t l y r e g u l a t e d by two forms of s o l u b l e IL-6 r e c e p t o r s ; s I L - 6 R a n d sgpl30.