Modulation of cytokine production by Citrobacter rodentium

AGAA1351

April 2000

6150

6152

LOADING OF TRANSMURAL PRESSURE STIMULATES INTER· LEUKIN·6 SYNTHESIS IN INTESTINAL EPITHELIAL CELLS: ACTI VATI ON OF TRANSCRIPTION FACTORS.

ADHESION MOLECULES IN SEPTIC LEUKOCYTE RESPONSE IN THE LIVER. PROTECTIVE EFFECT OF THE IMMUNO· MODULATOR LINOMIDE.

Hiro shi Kishikawa. Soich iro Mi ura. Hirom asa Naka mizo , Hideo Yoshida, Masahik o Hirokawa, Ruri Caroline Nakatsum i, Hide tsugu Saito , Hidekazu Suzuki. Hiroshi Naga ta. Jiro Nishida. Hiromasa Ish ii, Tok yo Dent al Coli . Chiba, Japan; Nation al Defense Med Coli , Sai tama , Jap an; Sch of Med, Keio Univ , T okyo, Japa n.

Dan iel Klintman , Gunn ar Hedlund , Surg, Ma lmo , Sweden; Active Biote ch Research , Lund , Sweden.

Backgound : Intestinal moit ility and intraluminal pressure continuously produce various mechanical force s on intestinal mucosa. We previ ously reported that pressure loading on intestinal epitheli al cells (IECs ) have stimulated ce ll proliferati on (L ife Sc i. 6 1: 667 . 1997). Howe ver , it is not kno wn wheth er mechanical forces affect cy tokine production. In this study. we focused on transmura l pre ssure and inves tiga ted its effec t on IL-6 synthes is and activa tion of tran scription fac tors in IECs. Meth ods: Intestin al epithelial cells (lE C- 18) was rout inely maintained in DMEM . Pressure of 80mmHg which pro motes maxim al cell proliferat ion was loaded for 1-48 hou rs. IL-6 release int o culture media was determined by ce ll prolifera tion assay using IL-6 dependen t mouse hybri doma cell line (7T D I) af ter 24 and 48 hour s. The polarity of IL-6 release from IEC s was exa mined by using Transwell chambers (O.4 IJ.m pore size). Th e mRN A expression of IL-6 in IEC-1 8 cells was determined by RT-PCR. Activation of mitoge nactivated prot ein kinase (MAPKinase) after pre ssure loading was asse ssed by western blot analysis usin g phosphorylation of Elk I fusion protein. Activation of transcription factors, nuclear factor kappa B (NFKB) and nuclear factor IL-6 (NF-IL- 6), was determined by elect rophoretic mobility shift assay (EMSA) using FITC -conju gated speci fic primers. Phosph orylation of I-kapp a B in cy toso l of IEC cells after pressure loadin g was also determined by wes tern blot analysis. Resul ts: Exposure of IECs to pressur e (80mmHg) significantly enh anced IL-6 release into culture medi a within 24 hour s. In co ntrol condition. IL-6 secretion was dire cted to basolateral side, but after press ure loadin g it was increased in both apical and basolateral side . Pressure tre atment enhanced IL-6 mRN A expre ssion in IECs within 3 hour s and co ntinued up to 12 hou rs. Western blot analysis showe d the pressure-indu ced activation of MAPkinase in IEC -18 cells at 3 hours. Pressure loading significantly enhanc ed both NF-KB and NF-IL-6 after I hour as assessed by EMS A. Increased phosphorylation of l-kappa B was also dem onstrated within I hou r. Conclusions: Pre ssure loading stimul ated 11-6 synthesis and release from intestinal epithelial cells, possibly via activation of MAPKinase and tran scription factors, NF- KB and NF-IL-6. The pressure induced increase in IL-6 may be act ively involved in immune regulation of intestinal mucosa.

Introduction: Leuk ocyte recru itment is a mult istep pro cess orchestrate d by a coordi nated expre ssion of cytokines, chemokines and adhes ion molecul es. However, the leu kocyte-endothelial interactions in the live r have not yet been clearly described, but may play an important role in hep atic inj ury and sepsis. Linomide has been demonstrated to ameliorate several autoimmune and inflammatory diseases, but its effect in the injured liver has not yet been fully eluc idated . Objective: To de term ine the adhesive pathways of leu kocyte s in liver injury in vivo, and the effe ct of the immunomodul ator Linomide . Methods: For this purpose. we used intravital microsco py of the liver in rats pretreated for 24 hour s with TNF-a (0.5 p.g) and D- galactosamin e ( 1.1 glk g). Th is tre atment increased liver enzymes (AST. ALT, ALP and bilirubin), sinusoi dal perfusion fail ure , as well as leuk ocyte rolling and leuko cyte firm adhesion in hepatic venules. Results: Adm inistration of the polysaccharide fucoidan , whic h inhibits the function of P- and L-selectin, redu ced leukoc yte roll ing sig nificantly. The nu mber of firml y adherent leuk ocytes remained, howe ver , unchanged . On the other hand, immunization against CDI8 sign ificantly decreased leuk ocyte firm adhe sion . whereas leuk ocyte rolling was not altered . These findings suggest that leukocyte rolling is predominately selectin-depende nt and that CDI 8 medi ates firm adhesion in the recruitment process of leuk ocyt es in hepatic injury in vivo. Next, we wanted to examine the potential effec t of Linornide in hepatic injury. Rats were pretreated with Linom ide ( 100 and 300 mglk glday) for 72 h prio r to challenge with TNF-alD-gal. We found that Linomi de treatment markedl y redu ced leuk ocyte rolli ng and firm adhe sion and improve d sinusoidal perfusion . Significant redu ction s of AST and ALT were also noted with this treatment. Conclusions: Tak en togeth er, these data define the ro le of selectins and CD 18 in the leuk ocyte response in liver injury , and dem onstrate that Linom ide may be ben eficial in the treatment of liver injury in sepsis . Th is research was mad e possible by a grant fro m Active Biotech Research. Lund. Sw eden .

6153 GASTROINTESTINAL FUNCTION IN AN HIV·POSITIVE COHORT. Tarnsin A. Knox, Sarah C. Skinner , Donn a Spiegelman, Sherwood L. Gorbach , Tufts Uni v Sch of Medi cine, Boston. MA; Harvard Sch of Publi c Health , Boston, MA.

6151 MODULATION OF CYTOKINE PRODUCTION BY CITRO· BACTER RODENTlUM. Jan M. Klappr oth, Isabel Ca Scaletsky, Michael S. Donnenberg, Steph en P. l ame s. Mic hael S. Donnenberg, Stephe n P. lames, U of Maryl and , Balt imore. MD .

C. rodentium, a murine path ogen tha t causes colitis, is closely related to enteropathogenic E. coli (EPEC). Both pathogens encode intimin wh ich is required for colonic inflammation. Recently, we described a novel toxin in EPEC (lymphostatin, lif) that inhibits lymphocyte cytokine producti on (Klapproth et. al, Gastro 116;A74 9. 1999).AIM: To determine wheth er C. rodentium exp resses a Iymph ok ine inhibitory factor and the role of int imin in cy tokine reg ulation. METH ODS : A C. rodentium genomic DNA libr ary was co nstruc ted and scree ned for EPEC Iif. Mouse splenocy tes were cultured with bacter ial Iysates and mitogen s and superna tant was analyzed by ELI SA. RESU LTS : 3 C. rodentium clones hybrid ized with EPEC Iif. Lysa tes of wild type C. rodentium, the 3 clones and EPEC all inhibited the exp ress ion of IL-2 (table). IL-4. and IFN-y An EPEC intimin deletion muta nt that co ntai ns lif inhibite d cytokine production but recomb inant EPEC intimin alone did not. SU MMA RY: C. rodentium cos mid clo nes that cross-hy bridize with EPE C lif inhibit cy tokine produ ction. The activ ity of EPEC lif does not depend on intimi n. CONCLUSION: C. rodentium colitis may be a useful model for study of the novel lif toxin in vivo and for the role of novel E. coli strains in IBD .

Effect of bacteriallysates on mitogen stimulated IL·2 (pg/ml)production

Bacterialdose

2.5fl!!/ml

5/lglml

25flQ!ml

SO/lgfml

C.rodWT C.rod388 C. rod8C12 C.rod 10H6 EPECWT EPEC iiI del EPEC intdel recomb intimin

320 330 280 4tO t92 110 210 362

180 502 582 450 362 154 230 430

85 260 362 220 98 262 82 420

31 82 ttO 92 80 410 5 604

C.rod =C. rodent/um. WT =wild type. 388, 8C12, 10H6 =C. rodentiumcosmid clones. int del = intimindeletion. Negative control cultureshad undetectable IL-2. ConA positivecontrol. IL·2 = 124 pg/ml.

Background : With the advent of highly active antiretroviral therap y (HAA RT) for HIV , the curren t prevalence of GI dysfunction is not know n. Methods: GI function was studied cro ss-section ally in 671 per son s with HIV en rolled in the Nutrition for Life Stud y. Symptoms were determined by qu estionn aire. GI abso rptive funct ion was studied with a 25 g D-xylo se test and stool exams for path ogen s. Serum albumin and micronutrients were measured . Re sult s: 47.6% had low D-xylo se absorption; 40.3 % had a history of liver disease ; 38.9% had diarrhea; 28 .7% had chr onic diarrhea; 22.5 % had borderline or low serum vitamin BI2 levels; 12.2% had stool path ogen s: and 7.2% had low serum albumin levels. The prev alence of abn orm alities depended on soci odemographic factors . Low D-xylose absorption and diarrhe a were more pre valent in men than in wo me n (5 1.6% vs 37. 1%. p= O.OO I ; 4 1.6% vs 3 ] .9%, p=O.02). Stool pathogen s were more prevalen t in men than wo men (14 .7% vs 2% . p=O.OO \) and in those without a history of IV drug use (nonIVDUs) (15 .5 vs 4.9%, p= O.OOI ). Vitamin BI 2 deficien cy was more pre valent in nonIVDUs (5.3 % vs 1.9%, p =O.048) wher eas a history of liver disease was found in over 50 % of rVD Us (52.5% vs 34 %, p = O.OOI). Low serum albumin was more pre valent in wo men and in IVDUs (14.1 % vs 5.3%. p =O.OOI ; 11.2% vs 5.3 %, p =O.OI) . In multi vari ate analysis, diarrh ea. D-xylo se mal absorpt ion, and hypoalbu minem ia were eac h statistically unassociated with one another. The pred ictors (p
GI parameter

Predictor (OR)

D.xylose malabsorption Diarrhea Severe diarrhea (>6/day)

Female (0.59). HAART (1 .9), nelfinavir (0.32) Homosexuality (1,56), nalfinavir (2,36) Homosexuality(4,25, p=0.06), log HIV viral load (1.98), HAART(18.85) Female(2,55), current IVOU (3.71).HAART

Hypoalbuminemia

(0.38)