r

REVIEW

Spontaneous bacterial peritonitis in patients with liver cirrhosis and

ascites - New insights using cytokine and cytokine receptor

analysis in ascites

T1 w A N nus, MD, JURl;EN SCJ l( lLM ERll'l I, M D

T ANous, J SCH6LMERICH. Spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites - New insights using cytokine and cytokine receptor analysis in ascites. Can J Gastroenterol 1992;6(3): 141-146. The cloning and expression of cytokines and their receptors has led to the development of sensitive and specific immunoassays for their determination in bio logical fluiJs such as ascites. Using such assays, the cycokines lL-lP, JL-6, IL-8 and TNFa. were found in ascitic fluid samples of patienrs with infected and malignant asci tes. IL-6 was found in high concentra tio ns even in ascites samples of not infected hepatic ascites. In adJition, high concentrations of soluble TNF receptor p55 and p75 were found in asci tes. IL-1 and IL-6 concentratio ns were increased in infected asci tes while concentrations of both TNF receptors were increased in infectcJ and malignant ascites, indicating that the combined measurement of cycokines and cytokinc receptors may be helpful for the differential diagnosis o f ascites (allowing the differentiation among infected , malignant and uncomplicated hepatic ascites). High concentrations of cytokines and soluble cyrokine recepcors in ascites, their increase during infection and malignancy, and their high potency co regulate inflammatory processes and other mediators indicate that they play an important role in the pathogenesis of ascites. Thus, measuring cytokines and soluble cytokine receptors in ascitic fluid opens a new window to study the pathogenesis of ascitcs and eventually may lead to the development of new methods of medical treatment of nscitcs using specific cytokine antagonists. (Pour resume, voir page 14 2)

Key Words: Ascites , Cytol<ines , lnterleul<in, Soluble cytol<ine receptors, Spontaneous bacterial peritonitis , Tumour 11ecrosis factor

De/>arrmenr of Internal Medicine I, U11i11er.1ity of Reg.:nslnll'g, Franz}mef Scnms., A llee, W-8400 Regemlntrg, Germany

Con·cspondence and re/mnts: Professor Dr J Sc/1/ilmcric/1. Medizinische Klinik und Poliklinik I, Universitat Regm.ihurg Fmnz ]osef Srraim Alice I I , \'v'-8400 Hcgernh11rg, Gm11w1y. Telephone 094 I /944-700 I . Fax 0941 /944-7002

Receit,ecl for /mhl,cminn April IO, 199 2. Acce/ned Ma)' 11 . J 99 2

ASCITES IS A FREQUENT COMPLl­

cauon of severe liver J i~ea~c~ with portal hypert ension and nf ma lig­nant tumnurs metastatic to the peri ­toneum ( 1,2 ). Whi le malignan t asc ites me rarely infected, r arien t~ with hepatic asc ites frequen tly develop spo ntaneous hacteria l peritoni1 is; often wi thout any ,,hvious pri mary source of infec tion (3 ) . T he prevalence of sr on­tancou~ hactcrial peritoni t is in ho~pi­rn lized patients wi th hera tic cirrhosis and ascites is arpmximatcly 15%. Sr onraneous hacterial peritoni1 is ,~ ,, severe complicatiun wi th a morta lity rate o( more than 50% and a h igh ra te of recurrence (approx imately 70% per year). T ypical symptom~ of sponta­neous hacrerial peritoni tis arc ahdomi ­nal pain , feve r, hepatic enccph­alnr athy nr unexr laincd clinical deterioration. Bacteria cultured from infccteJ ascites usuall y represent the normal acrnhic flora of the gut, bur J ue en the low num ber of bacteria in ascites (usually one/ml) , diagnosis of infec­tion by cul ture of ascit ic fl uid is often difficult or not possible ( 4 ).

T here arc several pred isposing fac­tors for 1hc manifes ta t ion of sponta-

CAN J GA:,TROENTI'RDI VOi 6 No 3 MAY/JUNE I 992 141

AN DUS AND SCI IOLMFRICI I

Peritonite bacterienne spontanee chez des patients presentant une cirrhose et de l'ascite: nouvelles approches a base de cytokines et analyse des recepteurs de cytokines dans l'ascite

RESUME: Le clonage ct !'expression des cytokines e t de leurs rcceptcurs ont mene ~1 la mise au poin t d'essais immunologiques sensibles ct spccifiques pour lcur detection clans !es liquidcs b iologiqucs tels que l'ascitc. A l'aidc de tc lles epreuves, lcs cyrokines lL- I B, IL-6, IL-8 et TNFa. ont ete decelees clans des echantillons J'ascice chez des pacienls presentant une ascite infectee et malignc. L'lL-6 a e te notec en concentration elevec, mcme Jans des echamillons non infecres. En outrc, des concentrations clcvees de recepteurs de TNF solubles p55 ct p75 ont etc trouvecs clans l'ascitc. Des concentrations d'IL-1 ct d'lL-6 etaicnt clcvees Jans l'ascite infectcc a lors quc des concentrations des recepteurs de TNF se trouvaicnt augmentes clans l'ascicc infcctee e t maligne, ce qui donne a penscr que la mesure combincc des cytokines ct de recepteurs de cytokincs pcuc sc revele r utile clans le diagnostic differentiel de l'ascitc ( en permettant la differenciation entre l'ascitc hepatiquc infcctce maligne et non compliqucc). Des concentrations elevees de cytokines ct des rccepteurs solubles de cytokines clans l'ascite, leur augmentation au COurs J e !'infection et Je la malignite Ct leur pOtenticl elcve a reg[cr le proccssus inflammatoirc ct d'autrcs mcdiateurs indiqucnt qu'ils joucnt un ro le important dans la pathogcnese de l'ascite. A insi, la mcsure des cytokincs ct des rcceptcurs solubles de cytokincs clans l'ascilc ouvre unc nouvelle avenue pour !'crude de la pathogcncsc de l'ascitc ct pourra mencr a la misc au point de nouvelles thcrapeutiqucs mcdicalcs contre l'ascite a !'aide d'antagonistcs specifiques de cytokines.

neous hactcrial peri tonitis: an impaired liver function weakens the immune sys­tem locally and sysLcmically (5-7); intra- and cxtrahcpalic shunt ing leads to a decreased clearance o( bacteria by the hepatic rcticulocndochclia l system and to rccurrcnL systemic bacceriemia; and pat ients with low ascitic tota l prote in concenLrat ions (8) and low op­sonic activity in a c itic fluids and scrum have a higher risk for spontaneous bac­teria l peritonitis (5,7,9).

Cyrokincs arc important med iators play ing a key role in immune system regulation and synthesis of hepatic acute phase prote ins such as comple­ment factors ( 10- 12). Most cycokincs arc glycoprotcins with a molecula r mass below 80,000 D. Cytokines usua ll y act in picomnlar concentrations via spec­ific, high affini ty cell surface receptors. Cells involved in cytokinc synthesis in­clude: blood cel ls such as monocytcs, lymphocytes and platelets; stromal cells uch as fibroblasts, enc.lorhelia l cells

and smomh muscle eel Is; and parcnchymal cells such as kcratin ­ocytcs and hcpatocytcs. Most cytokines may not only he produced hy a variety of cell L ypes as a reacLion to diffe rent

stimuli, hut a lso may have numerous biological effects on several d ifferent target cel ls.

Mo lecular cloning of cytokincs and thei r receptors led to the development of sensitive and specific assays which facilitated their measurement in blood and other body fluids such as ascitcs. T he following review summarizes the current knowledge about cytokines and their receptors in ascites and discus cs possible clinical applications of their measurement.

CYTOKINES IN ASCITES Although scrum concentrations of

cytokines have been determined in patients with liver diseases for several years ( I 3-22), cytokincs o nly recentl y have been detected in asc ites. In patients with hepatic asciLcs the cyw­kincs found in ascitic fluid were intcr­leukin(I L)- 113 (23), lL-6 (23-29), IL-8 (23) and tumour necrosis factor a lpha (TNfo) (23-25,28) . ll -6 was by forthc most abundant cytokine in ascitcs, wiLh concentrations ranging between 200 and 100,000 pg/ml (23 -27,29). ln con­trast, IL-1 13 was undcLcctablc in most ascitic flu id samples and o nly was found

in cu lture-positive asc itcs (23). Abo, lL-8 and TN Fa were only de tectable in some ascit ic nuid samples (23 ).

Ascitic concentratio n IL- I B (23 ), IL-6 (23-27,29) and T NFa (23-25) were h igher in patients with sponta­neous bacterial peritonitis than in patients with uninfected hepatic a,­c ites, and were found to be reduced tn lower concentrations after succcs ·ful an tib iotic treatment (23-2 5); finding could be used to separate culture-posi­tive from culture-negative ascitc~. Il­l B and I L-6 were found to separate the two groups with a d iagnosLic accuracy of 98% at cutoff levels of IO and 8000 pg/ml. Interestingly, patients with the culture-negative variant o( sponta­neous bacterial peri toni tis had no elevated concentrations of cyLOkines.

In six patients withou l pcritonilis, concentrations of I L-6 anJ TN Fa. were only slighdy h igher in ascitcs than in serum (24,25 ); however, in 21 patients without peri toni tis, ascitcs conccntra- , tions were about I 00-fold higher than corresponding plasma conccntrarions (23,26,27,29). This ratio was 500-fold h igher in patients with periton itis. T hese find ings strongly suggest Lhat JL-6 is produced locally in the pcriLOncal cavity. Furthermore, since half of the ascites fluid shifts back and fonh every hour through the large capi llary bed of the splanchn ic peri toneum (3 l,32) :md since IL-6 rapidly d iffuses into the plas­m.a (33) ( where it is cleared within a few minutes 134]), in traperiLOncal pro­duction of IL-6 must be continuously high to maintain the high concenlra­tions found in all patients with asci tes. This, in turn, suggests that there is con­tinuous timu la Li on of peritoneal in­flammatory cells even in patients with uncom.plicaccd asci Lcs. Among these cells, pcri roneal macrophages probahly arc the most importanl source of lL-6 production since isolated peri toneal macrophages from patients with ov;i­

rian carci noma (35) and from mice (36-38) have been shown to release IL- I (35, 37,38), IL-6 (35,38) and TNFa 06,37).

LipopolysacchariJes prcsumcihly arc the major st imula ling factor of cyto­kinc synthesis in hepat ic ascitcs since they strongly stimula te cytokinc syn­Lhesis ( l0,39), and h igh lipnpolys:ic-

142 CAN J GASHOENTEROL VOi 6 No 3 MAY/j UNL 1992

Cytokines and soluble cytokine receptors in ascites

charidc levels have been founJ fre­quently in ascitcs (791){,) and plasma of cirrhotic patients wiLh ascitcs (76% ) (40). The Gram-nega tive flora of the gut provide~ a huge pool of I ipopolysac­charides which mny emer systemic cir­culation and asc1uc nuid ( when hepatic clearance is impaired hy portal systemic shunting and there is decrea­sed Kupffcr cell function a~ during li ver disease) (41,42). In ,1ddition, ev idence for lymphatic uptake nf lipopolysac­charidcs and even viahle G ram-nega­tive bacteria ha~ hccn shown (43-45).

ROLE OF CYTOKINES IN A SCITES

Since cytokine~ arc ve ry potent in­flammatory mediaL,)rs affect ing a broaJ range of effector cells, the presence of cycokines in asci tic fluid may have a great impact on the pathnphysiolngy of ascitcs. Increased ascitic cy toki nc con­centrations during spontaneous bac­terial pcritoniri~ indicate that Lhcy may act locally as p,irt of the host's ddcnce system againM infectious agents. For example, IL-8 induces migrntion and act ivation ofneutrophils (46), and IL- l (47), IL-6 ( 10 ) and TN Fa. (48) incrca~c T and B cell acti vity and mmspecific resistance to infection. High concen ­trations of the anri- inflammarory cyto­kinc IL-6 (found in all asc iLcs s,1mplcs) may protect the host from the pmcn­tially d,mgcrous systemic effects tif high concentrat ion~ of pm-inflammatory cytokines (ic, IL- I , IL-8 and TNFa.), since IL-6 can inhibit the synthesb o(

IL- I and TNFa in mononuclear cells (49,50).

Besides their protective ,1ction, an animal s tudy ( 51) showed tha t cyco­kincs may he involved in ascitcs forma­tion. Cytnkincs 1rn1y induce asc iLcs for­mation hy sever.ii mechanisms. Cytokines such as I L- 1, interfcron-y and TNFa induce the production of nitrogen oxides in macrophages and endothelial cells (52,53) whi ch leads co periph eral vasodilmion a nd subsequent fluid retention. Furthermore, cytokines increase vascular pcrmeabilit y resulring in the trnnsudation nf plasma from the circulation inw extrnvascular sp,1cc (5 1,54 ).

Diffusion of cytuk ines lL- l , I L-6, IL-

8 ,rnd TNFa in to ch1: systemic c ircula­tion can cause clinical signs suc h as fever, pain, wasting, hypot ens ion and septic shock anJ can contribute co the clinical picture of spontaneous bac­ter ial peritonitis.

SOLUBLE CYTOKINE RECEPTORS

Cytokincs act specifically on target cdls hy binding lll specific surface­hound receptors which trigger Mx:ond messenger mechanisms that lead to changes in the transcription of cyto­kinc-rcgulatcd prote ins. In addition to the surface-bound receptors, there arc soluble cyLOkinc receptors.

Elevated concentration~ of low ,1f­finity soluhlc IL-2 receptors have hecn found in the scra of pa tiems with herni ­titis R ( 55 ), autoimmune hepatitis ( 56), a wide variety of mhcr diseases ( 57) and after liver transpla ntation ( 58). S1,lublc reccpwrs for lL-6 and imcrforon-y can he purific1..I (mm normal human urine ( 59). The cltming and expression uf the murine IL-7 receptor led to detection of a soluble form which was secreted into the medium hy transfcc tcd cclb (60). Soluble inhibitory forms of IL- l (61, 62) and TNF (63-66) have hccn fllltnd in human scrum and urine. Recently, elevated scrum conccntrmion~ of w luhlc TNF reccptms were found in patients wi th burns o r renal failure, hut no increase was found in paticnb with c hronic polyarthritis (67).

Few daw exist ahout soluhlc cyto­kinc reccpLOrs in ascites. Soluble, high affini ty lL-4 rcccpLOrsarc present in the ascitcs fluid , scrum and urine of normal mice (68). Soluhlc IL-4 receptor con ­ccnLratio n w;is reduced in severe com­bined immuntKlcficicncy mice (68).

High conccntrntiom of ~oluhlc TN F receptor p55 and p7 5 were detec ted in all ascitcs and plasma samples of 34 patients with hcp<1 tic infected, nnnin ­fected and rmdignancy-rc latcd ascitcs (28, 30). ln all patients, molar TN F receptor cuncc ntrations were about 500-fold higher than molar TN Fa con­centrations in the asc itcs; scrum samples ranged from 2.5 to 35 ng/mL in ascitcs ,md from 1.5 to 60 ng/mL in scrum . The mean concentrations of TNF rcceptm p55 and p75 were

CAN J GASTRtlENTER,)I. Vm 6 No 3 M AY/JUN!· 1992

e levated ahout 2.5 and 3.5 times, respecLively, i.n hmh infec ted hepatic and malignant asc iLes compared wid1 uncomplicated hepatic ascitcs. The as­ci Lcs TNF receptor conccntraLions were similar in patienrt- with infected ,ind malignant ascites. Plasma con ­centrations of TNF receptor p55 and p75 were elevated in Lhcsc subgroups, but showed a large overlap wid1 die non infected hepatic ascitcs samples.

The conccntrnti.ons ofTNF receptor p55 were higher th;:in TNF receptor p75 in asciLes, whereas TNF receptor p 7 5 was h ighc r in plasma conccntra-1 inns. The e lcv.ned cuncentratitms of TNF rcccpror p55 (24.2±15.2 ng/mL) and p75 (20.2± 14.4 ng/mL) found in 111fcctcd asci tic fluid decreased to lowe r levels ( L0.7± 1.89 and 7.50±1.80 ng/mL, rc~pcctivc ly) afrcr successful antibimic treatment.

1 ligh concen trmions of ~o luhle cyto­kine receptors, which increusc or de­crease during the course of disease, misc several questions: what 1s the source of the soluble rcccprors?; hy what mcch ­a nibms a rc the receptors released ?; and htiw is the synthe~i~ and rclca~c of Lhc cytokinc receptor~ regu lated ?

ROLE OF CYTOKINE RECEPTORS IN ASCITES

High conccnwuions of soluble cyro­kine receptor~ suggest an important im­munorcgulamry role. The large amnunrs of soluble TNF receptors (500-fold molar exec~~ ahnve TNFa), for example, strongly impair TNF bio­avai lahility in ascitcs and plasma. Sllluhle JL-2 rcccpwrs found in scra of patients with inflammatory disease~ may have similar immunomodulating cffccu, on Lhe activ,1tion ofT lymphocyLcs.

These high receptor concentrations may be an important, hcncficial part of the immuno logicc1I regulatory mcch­ani~m~ by minimizing tissue destruction caused hy cxcc~sivc cywkinc rc lca~e of innammatory cells during defence against infcctiou~ agent~ and t>thcr in­juries. Al though cycokincs usually con­tribute to protection of the organism againsL infectious agents and to healing from injury, the ir act ions may become pern1cmu~ in certain patho logical ~ituatiom (cg, ~cptic shock, a more

14 l

AN11lJS AN!) SC! Jt)LMl:RICI I

hepatic infacled

~ TNFAp55

CTJ TNFAp75

malignant

! .. Q,

10000

1000

100 !BJ ll-6

10

0.1 hepalic infected malignant

Figure I) Co11centrwiom of imerlCHkin ( I L! -1. I L-6, nono,ir 11ecrosis facror (TNF) rccepwr />55 and />75 memured hy enzyme-linked i1nmrn10,orhc111 ,1s.1ay. in mcite.1 of J>utients with /ic/latic. i11fcc1ed a11d malignam lLICites. Dara an' expre.1sed as means l. scandard deviwion

~evere JesLruc tion results than rhnc in­Juced by the pathogen itself). Regu­lators of the biological activity of cyto­kincs, such as the deLccLeJ TNF receptors, which counteract potentially harmful effect~ of TNF me therefore essential to maintain physinlogic;i l homeosLasis during infection and mal ignancy.

On the other hand, release of TNF recepwrs in malignant ascites may be a defe nsive mechanism of malignant cclb to proLCCt them from cyto tox ic effects ofTNF. High concentrations of TNF recepturs in malignant asc ites -which a lso can strongly Jiminish hioavailability of locally <1dminisLered TNF in the trcatmenL o f cancer patient~ (65) - should be considered in such clinica l trials.

CLINICAL APPLICATIONS The finding of cytokines and soluble

cytok ine receptors in ascitic fluid is an important sLep co unJerstand the parhophysiology of asciLes. It also 1m1y lead ro several c linical applications. The fi rst studies suggest that measure-

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144

ment of cytokincs and soluble cytokine receptors can help differentiate be­tween asc itcs of different origins; for example, of IL- IP and IL-6 in the as­c iLic flu id separated with a diagnost ic accuracy of 98% herween cuhure-pt)Si­tivc and culture-negat ive spontaneous bac terial peritonitis (this can be espe­cially important when antibinLic t reat­ment is interfering with Jiagnosis by bacLerial cultures) (23,27,29,30). As­ciLic fluid concentrations of both TNF receptor p5 5 and p 75 differentiated with d iagnostic accuracie· of 94 and 89%, respectively, between patients with malignant or infec ted ascites and patients with uncomplicatcJ hepatic asc ites.

A lthough IL- lp and IL-6 did not separate between uncomplicated hepa­tic asc ites and malignant asc itcs, and soluhlc TNF receptor concentrations did nnL differ hetween infected and malignant asciLes, the comhination may be helpful. Whereas in infected ascires, IL- l p, IL-6 and both soluble TNF receptors a rc higher than in un­complicated ascites, in malignant as-

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cites o nly the conccntrnt ion.s of the soluble TNF rcceptnr arc e levated (Fig­ure I ). Although these first result, are very promising, further studies arc re­quired for confirmation. The inclusion of other cytnkinc, and snluble cytokinc receptors in future studies 1m1y detect additional helpful p,irameters for dif ­ferential diagnosis.

The hacterial culture of asc ite~ flurd remains the gold standard for chc diag­nosis of spontanc\lus bacterial peri­tonitis, bur measuremenL of cytokine, a lso may he helpful when patients have been prcLreated with an t ihio1 ics and tn

study the response to antibi tltic trcar­menL.

The study of cytokincs nnd their soluble receplors wi II lead to a hetrcr understanding of the parhophysiology and paLhogencsis of ascit es. If cyttikinc, play an essenLial role in the pathogene­sis of ascites, an understand ing of their actions probably wi II lead tn develop­ment of new therapie, using specifil cywkincs or cyrok ine an tagonists ;inJ may he lp assess the prognosis nl paticnls with asc it es.

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Cyto kines and soluble cytokine receptors in asc ites

I lepa1nlog\ llJ91;14:97A. (Ah,t) 24 l)l'' 1crc J. C.>n1en1 .f, ))11p1111t E.

lntrnpn11nm·,1l ,ccrct1,m ,if TNFu and IL-6 , ltmng ,p,>1ll,111,·,>11, ha, renal pernun11 "· ( ,,istmcnll'n>logy 1990;98:A '>H2 ( Ah,1)

25. l)e\'lcr,· J, l \intent J, l 'nN,1u, A, I \1prnll E. II -6 ,md TN Fu 111 asut ll il111d durmg 'l'<llll,lllt'<>u, baol'n,11 pern,111111,. 1)1g 1)1, ~LI 1991; '6: 121 4. ( Lw \

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2H. And11, T. (,n1" V, 11,ibtegl' ;\, e1 :d l'rc,cnc,• ofh1gh c,Hlll.'lllra111>11s 111 ,olublc t1111u>r nccrn,is f,Klnr reccp1,>r, (p55 .md p75 m .i,c1tl''· I k·p:11,1l11g1 ( In pre")

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, I. Prrnllcl' T( ', :Sin W, J,>inl.'r l·E. Quanllt.Hl\'l' ,tud1<'' ,>f ,i-,111t fluid urutl,1111u1 \\'1t h tnt1um-L1h,·led 11':lln.

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