Circulating immune complexes and complement

Transcription

1 J Clin Pthol 1982;35: Circulting immune complexes nd complement concentrtions in ptients with lcoholic liver disese C GLD, H JNS From the Deprtment of Medicine, Division of Heptology, the Blood Bnk, nd Deprtment ofpthology, Hvidovre Hospitl, niversity of Copenhgen, Denmrk SMMRY prospective evlution of circulting immune complexes (CC) nd the ctivity of the complement system ws undertken in 53 lcoholic ptients just before dignostic liver biopsy. Circulting immune complexes were detected in 39 % of ptients with lcoholic stetosis (n = 26), 580% of ptients with lcoholic heptitis (n = 12), nd 600% of ptients with lcoholic cirrhosis (n = 15). No significnt difference ws found between the three group of ptients. The ctivity of the complement system ws within reference limits in the mjority of ptients nd only slight differences were detected between the three groups. No significnt differences were observed in liver biochemistry nd complement concentrtions in CC-positive nd CC-negtive ptients. Detection of CC in ptients with lcoholic liver disese does not seem to be of ny dignostic vlue or ply ny pthogenic role. The high prevlence of CC in these ptients my be due to depressing effect of ethnol on clernce of CC or to incresed immunologicl rectivity, or to both. lcoholic heptitis nd lcoholic cirrhosis hve been ssocited with n incresed prevlence of circulting immune complexes (CC).1-3 Ptients with lcoholic stetosis, however, hve been reported CC-negtive in most studies.'-3 Circulting lcoholic hyline nd ntibody to lcoholic hylin hve been demonstrted in ptients with lcoholic heptitis, but not in ptients with lcoholic stetosis nd cirrhosis.4 t hs been suggested tht lcoholic hylin (or other immunogenic mteril), being relesed into circultion s consequence of tissue destruction, led to the formtion of immune complexes.2 Furthermore, severe liver-cell dmge is supposed to cuse reduction in concentrtion of certin complement components.5 Detection of CC nd estimtion of complement concentrtions nd ctivity my therefore hve dignostic nd pthogenic implictions in ptients with lcoholic liver diseses. n order to exmine the reltion between presence of CC, the complement ctivity in serum nd degree of liver dmge, we studied chronic lcoholics just before dignostic liver biopsy. ccepted for publiction 20 ugust 1981 Ptients nd methods PTENTS fter informed consent 59 consecutive ptients dmitting consumption of more thn 50 g ethnol per dy for t lest two yers nd suspected of lcoholic liver disese were studied. Four ptients with clinicl signs of infection, one with rheumtoid rthritis nd one with gout were excluded to void ny possible effects of these diseses on the genertion of CC. The remining 53 ptients then formed the study group. Their ge rnged from 25 to 66 yr (medin 50 yr), nd ll were men. Liver biopsy showed stetosis in 26, lcoholic heptitis in 12 (with concomitnt cirrhosis in 10), nd lcoholic cirrhosis without heptitis in 15. LBORTORY NVESTGTON Routine liver test included serum bilirubin, serum sprtte-minotrnsferse (ST), serum lkline phosphtse, serum lbumin, plsm fctor + V + X, plsm gg, plsm g nd plsm gm. CC DETECTON ND COMPLEMENT NLYSS fter fsting overnight serum nd plsm were obtined by venepuncture just before dignostic 380 J Clin Pthol: first published s /jcp on 1 pril Downloded from on 17 July 2018 by guest. Protected by copyright.

2 mmune complexes nd complement concentrtions in lcoholics liver biopsy. The smples were stored t - 80GC. Detection of CC ws performed by mens of the complement consumption test (CCT) performed s modifiction of the method described by Johnson et l,6 nd in the continuous flow system previously described,7 using 3-4% suspension of sheep red blood cells, optimlly sensitised with rbbit hemolytic serum (Behringwerke G). Het-inctivted serum (56 C, 60 min) ws diluted 1/10 nd 1/18 nd incubted with one CH 90 unit of guine pig complement (Behringwerke G) for 30 min t 37 C just before nlysis. The reduction in degree of hemolysis in the smples contining serum ws expressed s percentge of control contining brbitl-buffered sline insted of serum. The CCT ws registered s positive if inhibition of hemolysis in ptient serum dilutions were greter thn the inhibition in corresponding serum dilutions obtined from 100 helthy volunteer blood donors.8 The totl hemolytic ctivity of the complement system in serum (CH 50) ws estimted in the sme utonlyser system.7 Quntifiction of the complement components Clq, C4, C3 ctivtor, C5 nd C9 ws performed by mens of rocket immunoelectrophoresis,9 employing monospecific ntiser (Behringwerke G). The results were expressed s percentges of pooled stndrd. Complement conversion products (split products) of C4 nd C3 were detected in EDT-plsm by mens of crossed immunoelectrophoresis with monospecific ntiser. STTSTCL NLYSS Non-prmetric Mnn-Whitney test, Spermn rnk correltion test, nd Fischer's exct test were used in sttisticl nlysis. The type error ws fixed t the 5 % level. Results LVER BOCHEMSTRY Ptients with lcoholic heptitis nd ptients with lcoholic cirrhosis hd significntly (p < 0-05) higher concentrtions of serum ST, serum lkline phosphtse, nd plsm immunoglobulins nd significntly (p < 0 05) lower concentrtions of serum lbumin thn ptients with lcoholic stetosis (Tble 1). Ptients with lcoholic heptitis lso hd significntly (p < 0-05) higher concentrtions of serum bilirubin thn ptients with stetosis. No significnt differences were found in liver biochemistry of ptients with lcoholic heptitis nd lcoholic cirrhosis. CRCLTNG MMNE COMPLEXES The prevlence of CC ws 390%, 58 Y., nd 60% in ptients with lcoholic stetosis, heptitis nd Tble 1 Liver biochemistry in ptients with histologiclly verified lcoholic stetosis (n = 26), lcoholic heptitis (n = 12), nd lcoholic cirrhosis (n = 15). Figures re medins nd rnge Vrible lcoholic lcoholic lcoholic (reference limits) stetosis heptitis cirrhosis S-bilirubin (5-17 Lmol/l) (6-52) (9-56) (5-130) S-ST (10-40 /) (12-226) (35-184) (20-105) S-lkline phosphtse ( /) ( ) ( ) (91-853) S-lbumin ( smol/1) ( ) ( ) ( ) P-fctor ± V + X ( rbitrry ) ( ) ( ) (04-1 2) P-gG ( K/1) (69-225) ( ) (78-303) P-g ( K/1) ( ) ( ) (67-670) P-gM ( K/) (36-165) (59-205) (36-369) p < 0-05 when compred to ptients with lcoholic stetosis. S = serum, P = plsm. cirrhosis, respectively (Tble 2). No significnt difference could be found between the three groups, but when compred to controls ll groups hd significntly (p < 0 05) rised prevlence of CC. n the totl group of ptients 49 % (95 % confidence limits: %) were CGC-positive. Tble 2 The prevlence of circulting immune complexes in controls nd ptients with lcoholic liver diseses Group Number N'umber % 95% confidence tested positive positive limits (%) Controls lcoholic stetosis lcoholic heptitis lcoholic cirrhosis p < 0 05 when compred to controls. 381 COMPLEMENT CONCENTRTONS The Figure shows the complement concentrtions in the three groups of ptients with lcoholic liver disese. No significnt differences were observed between the three groups, prt from ptients with lcoholic heptitis nd cirrhosis hving significntly (p < 0-05) lower concentrtions of C3 ctivtor thn ptients with lcoholic stetosis. Further, ptients with lcoholic heptitis hd significntly (p < 0-05) higher levels of C5 thn ptients with stetosis. No significnt chnges were observed between ptients with lcoholic heptitis nd lcoholic cirrhosis. ll the medin vlues of complement components were within the reference limits of norml controls. However, number of ptients in ll groups hd vlues either bove or below reference limits. J Clin Pthol: first published s /jcp on 1 pril Downloded from on 17 July 2018 by guest. Protected by copyright.

3 Clq C4 C3 C3.w -- :: o l 0.0 S t lcoholic stetosis lcoholic heptitis lcoholic cirrhosis Complement concentrtions (Clq, C4, C3, C3 ctivtor (C3), C5, nd C9) in ptients with lcoholic stetosis (0), lcoholic heptitis (), nd lcoholic cirrhosis (). The horizontl non-broken lines represent medin vlues nd 95% limits ofnorml controls. Broken horizontl lines represent medin vlues ofptients. The strs represent p < 0 05 when compred to ptients with stetosis. Ordinte: percentge ofstndrds. 0e - u- : v e C5 - -; O. 1-; j Gluud, Jns J Clin Pthol: first published s /jcp on 1 pril Downloded from Correlting C3 nd C9 concentrtions to serum lbumin nd plsm fctor + V + X vlues in the totl groups of ptients, showed tht C3 correlted significntly with plsmfctor + V + X (r = +0 39, p < 0 01). No other significnt correltions were found. n the totl group of ptients with lcoholic liver disese 16 ptients hd both C4 nd C3 split products (30 %, 95 % confidence limits: %) while six ptients hd either C4 or C3 split products lone (11 %, 95 % confidence limits: 4-23 %). No significnt differences were found between the three groups of ptients concerning C4 nd C3 split products, nd occurrence of split products were not significntly correlted to low concentrtions of C4 nd C3. LVER BOCHEMSTRY ND COMPLEMENT CONCENTRTONS N CC-POSTVE ND -NEGTVE PTENTS Compring CC-positive ptients (n = 26) with those without CC (n = 27) showed no significnt differences in liver biochemistry, including plsm immunoglobulin nd serum lbumin concentrtions, nd concentrtions of complement components. Nine ptients with detectble CC hd simultneous ctivtion of both C4 ndc3,suggestingin vivo ctivtion of the complement system vi the clssicl pthwy. However, seven ptients without detectble CC lso hd ctivtion of both C4 nd C3. Discussion The present study confirms nd extends previous on 17 July 2018 by guest. Protected by copyright.

4 mmune complexes nd complement concentrtions in lcoholics findings of high prevlence of CC in ptients with lcoholic liver disese. ll ptients were exmined just before dignostic liver biopsy. The ptients were referred to the deprtment becuse of symptoms ttributble to liver disese, nd ptients who exhibited symptoms of other diseses which could cuse CC were excluded from this study. prevlence of CC in 60% of ptients with lcoholic cirrhosis re in greement with erlier studies. Penner et l2 found CC in 50% of ptients with lcoholic cirrhosis using the Rji-cell ssy. n recent study, brss et l'0 found CC in 63 nd 73 % of ptients with lcoholic liver disese (mostly lcoholic heptitis nd cirrhosis) using solid-phse Clq-binding ssyndfluid-phse Cl q-binding ssy. Only limited nd conflicting dt re vilble concerning the prevlence of CC in ptients with lcoholic stetosis. Sodomnn et 13 nd Penner et 12 found no CC-positive cses in ptients with lcoholic stetosis using Clq devition nd precipittion methods nd the Rji-cell ssy. Thoms et ll reched the sme conclusion using Clq-binding to detect CC, but in five ptients with stetosis they found n incresed level of nticomplementry ctivity. n this study the prevlence of CC ws 39% in ptients with lcoholic stetosis which is significntly incresed compred to controls. The reson for these discrepncies is not known, but erlier studies concerned only limited number of subjects nd, probbly most importntly, different methods of detection of CC hve been employed. Different methods for detection of CC is bsed on vriqus biologicl properties of the complexes. Therefore, different techniques my show vrying results." The complement consumption test hs proved its bility to detect gg ggregtes of both intermedite size nd sizes > 19 S, wheres other techniques such s the Clq precipittion nd the pltelet ggregtion test only detects lrge CC (>19S).12 The complement consumption test cn give rise to flse positive results in ptients with lcoholic liver disese due to ggregted globulin or the presence of ctivtors of the complement system such s lipopolyscchride. ggregtion of immunoglobulin my occur becuse of either low serum lbumin or high plsm immunoglobulin. However, we found no significnt difference in the concentrtion of these proteins between CC-positive nd -negtive ptients. Erlier studies hve reported n incresed prevlence of endotoxin, the lipopolyscchride component of the cell wll of Grmnegtive bcteri, in ptients with cirrhosis. However, recent study, in which indvertent contmintion of specimens by endotoxin ws voided, ws not ble to detect ny endotoxin.13 t should lso be stressed tht the complement consumption test my 383 give flse-negtive results s only complement-fixing immune complexes re detected. The nture of the ntigen or ntigens involved in CC in ptients with lcoholic liver disese is not known. Severl possibilities exist. Kngsundrm et l4 demonstrted circulting lcoholic hylin nd its ntibody in ptients with lcoholic heptitis. This ntigen-ntibody system could not be detected in ptients with lcoholic stetosis or lcoholic cirrhosis. n our study, the prevlence of CC in ptients with lcoholic heptitis ws not significntly different from the prevlence in ptients with lcoholic stetosis or cirrhosis. Kehl et ll4 were not ble to confirm the findings of Kngsundrm et l.4 Our findings do not seem to support the hypothesis tht CC in ptients with lcoholic liver disese should be composed of lcoholic hylin nd ntilcoholic hylin s the prevlence of CC ws not incresed in ptients with lcoholic heptitis. n ccordnce with this ssumption is the fct tht we could find no significnt differences in liver biochemistry in CC-positive nd -negtive ptients. However, ttempts should be mde to identify the ntigen(s) of CC before this question cn be settled. Studies in ptients with primry biliry cirrhosis hve reveled significnt correltion between concentrtions of CC nd titres of ntimitochondril ntibodies.'5 n ssocition between CC nd utontibodies hs lso been described in ptients with insulin-dependent dibetes mellitus nd in norml controls.'6 Ptients with lcoholic cirrhosis hve high prevlence of utontibodies,17'19 nd it is suggested tht utontibodies re involved in t lest some of the CC found in ptients with lcoholic liver disese. n ptients with liver disese high prevlence of CC could be due to incresed formtion, but my lso be cused by decresed clernce. The heptic reticuloendothelil system is known to cler the circultion of immune complexes.20 decresed heptic clernce of ntigen hs been described in nimls with experimentl cirrhosis,2' but this would not explin the high prevlence of CC in ptients with stetosis. However, lcohol is known to depress the function of the reticuloendothelil system.22 Such n effect might explin the high prevlence of CC found in lcoholics irrespective of degree of liver dmge. Further, recent observtions hve clerly shown tht prenchyml s well s nonprenchyml liver cells tke up immune complexes.23 The uptke of CC by prenchyml liver cells my lso be depressed by lcohol. lthough mny of our ptients hd complement concentrtions either bove or below norml reference limits, ll medin vlues fell within the norml rnge, nd only minor differences were J Clin Pthol: first published s /jcp on 1 pril Downloded from on 17 July 2018 by guest. Protected by copyright.

5 384 observed between the three groups of ptients with lcoholic liver disese. Only significnt correltion ws found between liver-cell dmge, expressed by plsm fctor + V + X, nd the concentrtion of C3. The occurrence of C4 nd C3 split products, indicting in vivo ctivtion of the complement system, did not explin the different concentrtions of the complement components either. These findings re lrgely in ccordnce with erlier reports The level of complement proteins reflects the net synthesis nd utilistion. Norml concentrtions my therefore be seen in spite of significnt ctivtion of the complement system. Rised complement concentrtions generlly reflect n cute phse rection, wheres depression reflects utilistion or decresed production. n ptients with lcoholic liver disese ll mechnisms my operte simultneously so obscuring ech other. This my explin why we re unble to demonstrte ny uniform bnormlity of either the clssicl or lterntive ctivtion pthwy of the complement system. n conclusion, CC is found in bout hlf of the ptients with lcoholic liver disese. No reltion between CC nd complement concentrtions on one hnd nd complement concentrtions nd liver function on the other could be estblished. Therefore, determintion of CC does not seem to hve dignostic or pthogenetic vlue in ptients with lcoholic liver disese. The incresed prevlence of CC in ptients with lcoholic liver disese my be due to n inhibitory effect of lcohol on clernce of CC or n incresed immunologicl rectivity, or combintion of these effects. References Thoms HC, De Villiers D, Potter B, et l. mmune complexes in cute nd chronic liver disese. Clin Exp mmunol 1978;31: Penner E, lbini B, Milgrom F. Detection of circulting immune complexes in lcoholic liver disese. Clin Exp mmunol 1978;34: Sodomnn C-P, Prokein K, Schmidt H, Mrtini G. Zirkulierende mmunkomplexe bei Leberkrnkungen. Z Gstroenterol 1978;16:501-l. 4 Kngsundrm N, Kkumu S, Chen T, Leevy CM. lcoholic hylin ntigen (Hg) nd ntibody (Hb) in lcoholic heptitis. Gstroenterology 1977;73: Chrlesworth J, Lwrence S, Worsdll P, Roy LP, Boughton CR. cute heptitis. Significnce of chnges in complement components. Clin Exp mmunol 1977; 28: Johnson H, Mowbry JF, Porter K. Detection of circulting immune complexes in pthologicl humn ser. Lncet 1975;i: Jns H, Jersild C, Tning E, Dybkjer E, Fog T, Heltberg. The occurrence of immune complexes in ptients with multiple sclerosis. n: Peeters H, ed. Protides of the biologicl fluids 26. Oxford, New York: Pergmon Press, 1979: Jns H. Circulting immune complexes in pprently helthy persons. Scnd J Rheumtol 1980;9 suppl 33:39. Gluud, Jns 9 Lurell C-B. Electroimmuno ssy. Clin Lb nvest 1972; 29 suppl 124: '0 brsse CK, Border W, Hepner G. Non-specificity of circulting immune complexes in ptients with cute nd chronic liver disese. Clin Exp mmunol 1980; 40: '1 Lmbert PH, Dixon FJ, Zubler RH, et l. WHO collbortive study for the evlution of eighteen methods for detecting immune complexes in serum. J Clin Lb mmunol 1979;1: Nielsen H, Svehg SE. Detection nd differentition of immune complexes nd gg ggregtes by complement consumption ssy. ct Pthol Microbiol Scnd 1976;84: Fulenwider JT, Sibley C, Stein SF, Evtt B, Nordlinger BM, vey GL. Endotoxemi of cirrhosis: n observtion not substntited. Gstroenterology 1980 ;78 : Kehl, Schober, Junge, Winkler K. Solid-phse rdioimmunossy for detection of lcoholic hylin ntigen (Hg) nd ntibody (nti-h). Clin Exp mmunol 1981 ;43: Gupt RC, McDuffie FC, Dickson ER, Meht S, Tn EM. The composition of the isolted soluble immune complexes in primry biliry cirrhosis (PBC). Gstroenterology 1978 ;75 : Delexpesse G, Grnsset PH, Srfti M, Bubo-Rycquoy M, Debisschop M-J, Vn Helst L. Circulting immune complexes in old people nd in dibetics: Correltion with utontibodies. 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The mononucler phgocytic functions of the liver. n: Ferguson, McSween RNM, eds. mmunologicl spects of the liver nd gstrointestinl trct. Englnd: MTP Press, 1976: Hopf, Schefer HE, Hess G, Meyer zum Buschenfelde KH. n vivo uptke of immune complexes by prenchyml nd nonprenchyml liver cells in mice. Gstroenterology 1981 ;80: Fox R, Dudley FJ, Sherlock S. The serum concentrtion of the third component of complement P1C/P1 in liver disese. Gut 1971 ;12: Teisberg P, Gjone E. Circulting conversion products of C3 in liver disese. Clin Exp mmunol 1973;14: Potter BJ, Elis E, Fyer PM, Jones E. Profiles of serum complement in ptients with heptobiliry diseses. Digestion 1978:18: Requests for reprints to: Dr Christin Gluud, Deprtment of Medicine, Division of Heptology, Hvidovre Hospitl, DK-2650 Copenhgen, Denmrk. J Clin Pthol: first published s /jcp on 1 pril Downloded from on 17 July 2018 by guest. Protected by copyright.