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C> 1987 S. Karger AG, Basel0014-312X/87/0l96-O388$2.75/0-Eur. surg. Res. 19: 388-394 (1987)

Decreased Reticuloendothelial Phagocytic Capacity inCirrhotic and Portacaval Shunt Rats

M. Capron-Laudereau, J. Gugenheim, M. Gigou. S. Crougneau, D. Houssin, ~m~

Groupe de Recherche de Chirurgie Hépatique et Départ~ment de Chirurgie Expérimentale,Hópital Paul-Brousse, Villejuif, France

Key Words. Reticuloendothelial systeIÍl activity .Cirrhosis .Portacaval shunt

Abstract. In cirrhosis, the phagocytic function of the reticuloendothelial system (RES) isdecreased. In order to investigate the mechanisms ofthe hepatic reduced phagocytic activitypresent in cirrhosis, the hepatic and splenic uptake of 51 Cr sheep red blood cells (SRBC) and

of colloidal carbon was measured in three groups of Sprague-Dawley rats. Group 1 consistedof 42 control rats, group 2 of 36 rats with end-to-side portacaval shunt and group 3 of 24 ratswith carbon ietrachloride-induced cirrhosis. The hepatic uptake of 51Cr SRBC and of colloi-dal carbon was significantly (p < 0.001) reduced in cirrhotic rats (group 3). Conversely, inrats with a portacaval shunt and a noncirrhotic liver (group 2), the hepatic uptake of 51CrSRBC was moderately reduced, whereas the colloidal carbon hepatic uptake was not foundto be decreased. s su est that the decreased RES ha ocytic activity observed incirrhotic rats is only partially due to portacaval s~unt and that an intrinsic defective activityOIñepatíc phagocytlc cellS l~ vJvb~el,- ~F8SI~~-

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rhosis are unknown. It can be postulated thatthis reduced phagocytic activity might bedue either to a defe~tiye activity of the he-patic phagocytic.cells or to an intra- or extra-hepatic portacavál shunting of the bloodflow allowing micFoorganisms, nonbacterialparticulates or antilens to escape from thephagocytic activity of nonparenchymal he-patic cells: The purpose of this study was toinvestigate the Tole of portacaval shunting by

Impairment of the phagocytic function ofthe reticuloendothelial system (RES) occursin cirrhosis [3, 7, 11] and plays a major Tolein predisposing cirrhotic patients to developsevere infections [14]. This impairment ismainly due to a depression of the hepaticRES which amounts to 80-90% oftotal RES

activity [17].The precise mechanisms of the hepatic

reduced phagocytic activity present in cir-

studying the hepaticantigens in rats withwith a noncirrhotic tishunt.

Material and Metbods

liver and spleen were removed, weighed, washed insaline buffer and hydrolysed in 10 mI 4 N NaOH/gtissue for 20 min at 90 .C. The hydrolysed mixturewas submitted to ultrasonic waves for l min and theoptical density was then determined at 730 nm. Theresults were expressed as percentage of carbon in-jected, either for the whole organ or for l g of theorgan.

51Cr Erythrocyte Uptake. Rats were intravenouslyinjected with 5 X 108 sheep red blood cells (SRBC)prepared as follows: 5 mI of SRBC were washed twicewith Hanks' solution using centrifugation at 2,500rpm for 10 mino The globular pellet was resuspendedin 5 mI of Hanks' solution and 200 ~Ci of 5JCr wereadded to the suspension. The mixture was incubatedat 37.C for 45 mino The pt;:llet was washed 3 timesagain with Hanks' solution in arder to eliminateunfixed radioactivity. The pellet was diluted withHanks' solution in arder to obtain a cellular suspen-sion containing 109 51Cr erythrocytes/ml. Each ratreceived 500 ~I ofthis suspension. Three hours later,the rats were sacrificed, and the liver and spleen wereremoved and weighed. Radioactivity of each organwas measured using a gamma counter. Control mea-surement of the radioactivity injected was made bycounting the radi9activity of 500 ~I of 51Cr erythro-cytes. ;Results were expressed as percentage of control,either for the whole organ or for l g ofthe organ.

AnimalsMale Sprague-Dawley rats, weighing 300-350 g,

were purchased from Charles River Lab., France.They were kept in a temperature- and light- controlledroom and received food and water ad libitum.

They were dividéd into 3 groups: in group 1,42rats served as controls, in group 2, 36 rats underwentan end-to-side portacaval shunt, and group 3 com-prised 24 cirrhotic rats.

Portacaval Shunt RatsEnd-to-side portacaval shunts (PCS) were per-

formed as previously described [2]. Surgery was per-formed under light ether anesthesia using a cItan butnonsterile technique. One to two months after sur-gery, the .hepatic and splenic uptake of particulateantigens were studied in these animals. At this time.the ~ats weighed between 330 and 380 g.

Cirrholic RalSCirrhosis was induced by subcutaneous injections

of carbon tetrachloride (O. I mI plus an equal volumeof mineral oil per 100 g body weight) twice weekIyduring 5 months. The presence of cirrhosis was con-firmed at laparotomy on alI animals included in the

study.

Statistical AnalysisResults were expressed as means :t SEM. Com-

parison of the means was performed using Student'stest.

ResultsControl RatsTwenty-one rats had a simple laparotomy (sham

animals) and were studied 1-2 months after surgery.Twenty-one other animals received a subcutaneousinjection of 0.1 mi of mineral oil per 100 g bodyweight twice daily during 5 months.

Uptake o/ Co//oida/ CarbonThere was no difference in the hepatic

uptake of colloidal carbon between normalrats (81.0 :t 12.8%) and PCS rats (71.03 :t13.67%). On the other hand, the hepatic up-take in cirrhotic rats (25.0 :t 10.6%) was sig-nificantly decreased (p < 0.001). Thesplenic uptake of colloidal carbon increase-dfrom 4.67 :t 2.48 % in normal rats to 18.89:t 12.28 % in cirrhotic rats (p < 0.05) (fig. 1).

Assessment of the RES Phagocytic ActivityRES phagocytic activity was tested rneasuring the

hepatic and splenic uptake of either colloidal carbonor 5lCr-labeled erythrocytes.

Carbon Uptake. Rats were intravenously injectedwith 30 rng of colloidal carbon (Pelikan AG, Hanno-ver, FRG) and sacrificed 1 h and 30 rnin later. The

uptake of particulatecirrhosis and in ratsver and a portacaval

Reduced Phagocytic Activity in Cirrhosis391

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Fig. 3. Hepatic and splenicuptake of 51Cr SRBC in normal,PCS and cirrhotic rats.

When the results were expressed per gramof tissue in order to take into account liveratrophy of PCS rats and liver hypertrophy ofcirrhotic rats, the decrease inhepatic colloi-dal carbon uptake was significant (p <0.001) in cirrhotic rats. No decrease was ob-served in the PCS rats and no increasedsplenic uptake was observed due to the hy-pertrophy of the spleen in cirrhoti~ rats(fig. 2).

to 40.92 ::!: 9.40% in PCS rats (p < 0.001)and to 16..35 ::!: 12.54% in cirrhotic rats(p < 0.001). The splenic uptake of SRBCincreased from 4.17 ::!: 1.24 % in normalrats to 9.19 ::!: 1.62% in PCS rats and to30.2,0 ::!: 27.40 % in cirrhotic rats (p <0.001) (fig. 3). When the results were ex-pressed per gram of tissue, the decrease inhepatic SRBC uptake in PCS rats was mod-erate: 5.85 ::!: 1.52 % in normal rats versus4.94 ::!: 0.91 % in PCS rats (p < 0.02), butthe decrease in hepatic SRBC uptake in cir-rhotic rats was significant: 5.85 ::!: 1.52 % innormal rats versus 1.14 ::!: 0.69% in cir:-rhotic rats (p < 0.001) (fig. 4).

Uptake of 51 Cr SRBC

The nOrnlal rat retained 63.98 j: 12.42%oí injected SRBC in the liver. The hepaticuptake oí SRBC was significantly reduced

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animals [15, 18). The mechanisms of im-paired RES activity in !:irrhosis are not wellknown; responsible factors are either a re-duced blood flow to RES hepatic cells or adefective activity of RES hepatic cells. Theimpaired RES function in cirrhosis mar berelated to intra- or extrahepatic shunting ofblood escaping from the phagocytic action ofthe hepatic reticuloendothelial cells [14).Due to the reduced phagocytic activity oftheliver, gut-derived antigens might lead to anincreased stimulation of splenic and otherlymphoid tissues, re~l1lting in elevated im-munoglobulin titers [18, 19). In patients withchronic liver disease~ ¡he serum antibodytiters and immunoglobulin levels correlatewith the amount of portosystemic shunting[5). Furtherriiore, rats with a noncirrhoticliver subjected to PCS [10) or a portacaval

Discussion

In this study, we have observed a markeddecrease in the hepatic uptake of particulateantigens in cirrhotic rats, a finding whichcontrasts with the moderate reduction inPCS rats.

In cirrhotic rats, the hepatic uptake ofradiolabeled erythrocytes and of colloidalcarbon was significantly (p < 0.001) re-duced. The splenic uptake was increased, butnot enough to compensate the marked de-crease of the hepatic uptake. Conversely, inPCS rats, the hepatic uptake of 51Cr erythro-cytes was moderately reduced, whereas, thecolloidal carbon hepatic uptake was notfound to be decreased.-..A reduced hepatic RES activity has beendescribed in cirrhotic patients [3, 7, 11] and

Reduced Phagocytic Activity in Cirrhosis 393

Referencestransposition [1] develop elevated immuno-globulin levels. However, humans with anoncirrhotic liver and extrahepatic portalvenous obstruction leading to chronic porto-systemic shunting have normal immuno-globulin titers [21]. In OUT stucJy, rats withPCS and a noncirrhotic liver had only amoderately reduced phagocytic activity.This result contrasts with the significant de-crease observed in cirrhotic rats. Blood flowto the liver is probably greater in cirrhosisthan after PCS [9], and intrahepatic distur-bance of blood flow is common in cirrhosisbut does not occur after PCS. These observa-tions imply that the decrease in phagocytic---activity observed in cirrhotic rats cannot beattribut~d solely to PCS and that a defectiveactlvlty of nonparenchymalliver cells is alsoresponsi~: severalstudies have ShOwn~animpaired function of peripheral monocytesin liver cirrhosis [4, 6, 8]. Although some dis-crepancies mar exist between peripheralmonocytes and hepatic Kupffer cells [16],this mar suggest an intrinsic defective activ-ity of hepatic mononuclear phagocytic cells.Whether the reduced hepatic phagocytic ac-tivity observed in cirrhotic rats is Telated toan intrinsic functional defect of RES cells orto a decrease in serum opsonin (complement[13] or fibronectin [12]) concentrations re-mains to be determined.

In conclusion, the hepatic uptake of par-ticulate antigens was -markedly reduced incirrhotic rats. On the contrary, a moderatereduction was observed in rats with a non-cirrhotic liver and PCS. These findings sug-gest that the decreased RES phagocytic ac-tivity observed in cirrhotic rats is only par-tially due to PCS and that an intrinsic defec-tive activity of hepatic phagocytic cellsand/or an intrahepatic shunting of bloodfrom the RES cells are probably presento

1 Benjamin, I.S.; Ryan, C.J.; Biol, L.I.; Mac Lay,A.L.C.; Horme, C.H. W.; Blumgart, L.H.: The ef-fects of portacaval shunting and portacaval trans-position on serum IgG levels in the rato Gastroen-tero10gy 70: 661-664 (1976).

2 Bism~th, H.; Benhamou, J.P.; Lataste, J.: L'anas-tomose portocave expérimentale chez le rat nor-mal. Techniques et résu1tats pré1iminaires. Presseméd. 71: 1853-1861 (1963).

3 Cooks1ey, W.G.E.; Powell, L.W.; Halliday, J.W.:Reticuloendothelial phagocytic function in hu-man 1iver disease and its re1ationship to hemoly-siso Br. J. Haemat. 25: 147-164 (1973).

4 Felin, E.; Gougerot, M.A.; Hakim, J.; Cramer, E.;Auclair, L.; Rueff, B.; Boivin, P.: Blood polymor-phonuclear dysfunction in patients with alcoholiccirrhosis. Eur. J. clin. Invest. 7: 571-557 (1977).

5 Galbraith, R.M.;' Eddleston, A.L.; Wil1iams, R.;Webster, A.D. W.: Enhanced antibody response inactive chronic hepatitis: relation to HLA B8, HLAB12, and portosystemic shunting. Lancet i: 930-935 (1978).

6 Ganguly, N.K.; Kingham, J.G.C.; Llyod, B.;L1yod, R.S.; Price, C.P.; Triger, D.R.; Wright, R.:Acid hydro1ases in monocytes from patients withinflammatory bowel disease, chronic 1iverdiseaseand rheumatoid arthritis. Lancet i: 1073-1075(1978).

7 Halpern, B.N.; Biozzi, G.; Pequinot, G.; Dela-haye, B.; Stiffe1, C.; Mouton, D.: Mesure de la cir-cu1ation sanguine du foie et de l'activité phagocy-taire du systeme réticu10endothé1ia1 chez le sujetnormal et le sujet cirrhotique. Path. Bio1. 7: 1367-1653 (1959).

8 Hassner, A.; KIetter, Y.; Sch1ag, D.; Yedvab, M.;Aronson, M.; Shibolet, S.: Impaired monocytefunctiQn in 1iver cirrhosis. Br. med. J. 282: 1262-1263 (1981).

9 Heer, F.W.; Si1vius, D.E.; Scipe1, R.S.; Harper,H.A.: Effect of portacaval shunts or transpositionon hepatic blood flow and function in normal andcirrhotic dogs. J. surg. Res. 3: 213-219 (1963).

10 Keraan, M.; Meyers, O.L.; Engelbrecht, G.H.C.;Hickman, R.; Saunders, S.J.; Terblanche, J.: In-creased serum irnmunoglobulin levels followingportacaval shunt in the normal rato Gut 15: 468-472 (1974).

11 Lahnborg, G.; Friman, L.; Berghern, L.: Reticulo-

Eur. surg.

1,

.I.

EffectsThrornlAn Experil

O. Lanner,

DepartmentMalmo, Swe

19 Thomas, H.C.; Mac Sween, R.N.M.; White, R.G.:Ro1e of the 1iver in contro11ing the immunogenic-ity of commensa1 bacterial of the gut. Lancet ii:

1288 (1973).20 Thomas, H.C.; Singer, C.R.J.; Tilney, N.L.; Fo1ch,

J.; Mac Sween, R.N.M.: The immune response incirrhotic rats. Antigen distribution, humoral ac-tivity, cell-mediated immunity and sp1enic sup-pressive cel1 activity. Clin. exp. Immuno1. 26:

576-582 (1976).21 Webb, L.J.; Ross, M.; Markham, L.R.; Webster,

A.D.W.; Thomas, H.C.; Sherlock, S.: Immunefunction in patients with extrahepatic portal ve-nous obstruction and the etTect of splenectomy.Gastroentero1ogy 79: 99-103 (1980).

I1

:

Abstracto"

thrombogenirinsing with !to a pressuretion and thn

Received: February 26, 1987Accepted: Apri114, 1987

endothelial' function in patients with alcoholicliver cirrhosis. Stand J. Gastroent. 70: 408-412

(1981).12 Matsuda, M.; Yamanaka, T.; Matsuda, A.: Distri-

bution of fibronectin in plasma and liver in liverdiseases. Clinica chim. Acta 98: 482-491 (1982).

13 Petz, L.D.:. Variablemechanisms for low serumcomplement in liver disease. Lancet ii: 1033-1034

(1971).14 Rimola, A.; Soto, R.; Bory, F.; Anoyo, V.; fiera,

C.; Rodes, J.: Reticuloendothelial system phago-cytic activity in cirrhosis and its rélation to bacte-rial infec.tions and prognosis. Hepatology 4: 53-58

(1984).15 Rutenburg, A.M.; Sonnemblick, E.; Koven, l., et

al.: Comparative response ofnormal and cirrhoticrats to intravenously injected bacteria. Proc. Socoexp. Biol. Med. 101: 279-282 (1959).

16 Reiner, R.G.; Tanner, A.R.; Keghani, A.M.;Wright, R.: A comparative study of Iysosomalenzyme activity in monocytes and Kupffer cellsisolated simultaneously in a rat model of liverinjury. Clin. exp. lmmunol. 43: 376-380 (1981).

17 Saba, T.M.: Physiology and physiopathology ofthe reticuloendothelial system. Archs interno Med.

126: 1031-1052 (1970).18 Souhami, R.L.: The effect of colloidal carbon on

the organ distribution of sheep RBC and the im-mune response. lmmunology 22: 685 (1972).

Jean Gugenheim, MDGroupe de Recherche deChirurgie Hépato-BiliaireHopital Paul-BrousseF-94800 Villejuif (France)

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