The immune response to hepatitis B surface antigen (RBs Ag) was studied in 25 asymptomatic carriers by the leukocyte migration-inhibition (LMI) test in agarose. In the presence of purified RBs Ag, inhibition was demonstrated in only four of 25 carriers, in contrast to 24 of 28 patients who cleared the antigen after acute infection with hepatitis B. Tuberculin purified protein derivative (PPD) was also used as an antigen for the LMI test in these carriers. Inhibition was demonstrated in only 12 of 25 individuals who had positive PPD skin tests, in contrast to all of 14 normal noncarrier individuals with positive PPD skin tests and none of 12 normal noncarrier individuals with negative PPD skin tests. A nonspecific immunological investigation of the asymptomatic carriers gave normal results. The lack of an immune response to HBs Ag was thought to be responsible for the persistence of the antigen and also for the absence of symptom

Desaules, M.

Frei, P. C.

Libanska, J.

Wuilleret, B.

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THE JOURNAL OF INFECTIOUS DISEASES • VOL. 134, NO.5. NOVEMBER 1976© 1976 by the University of Chicago. All rights reserved.Lack of Leukocyte Migration Inhibition by Hepatitis B Antigenand Normal Nonspecific Immunoreactivity in Asymptomatic CarriersM. Desaules, P. C. Frei, J. Libanska, and B. Wuilleret From the Division of Immunology and Allergy, Departmentof Medicine, Centre Hospitalier Unioersitaire Vaudois; andthe Regional Blood Transfusion Center of theSwiss Red Cross, Lausanne, SwitzerlandThe immune response to hepatitis B surface antigen (RBs Ag) was studied in 25asymptomatic carriers by the leukocyte migration-inhibition (LMI) test in agarose.In the presence of purified RBs Ag, inhibition was demonstrated in only four of 25carriers, in contrast to 24 of 28 patients who cleared the antigen after acute infectionwith hepatitis B. Tuberculin purified protein derivative (PPD) was also used asan antigen for the LMI test in these carriers. Inhibition was demonstrated in only12 of 25 individuals who had positive PPD skin tests, in contrast to all of 14 normalnoncarrier individuals with positive PPD skin tests and none of 12 normal noncarrierindividuals with negative PPD skin tests. A nonspecific immunological investigationof the asymptomatic carriers gave normal results. The lack of an immune responseto HBs Ag was thought to be responsible for the persistence of the antigen and also forthe absence of symptoms.About 0.2<;'0 of the blood donors in this part ofEurope are carriers of hepatitis B surface antigen(HBs Ag). Some of these asymptomatic carriershave slightly elevated levels of transaminases,whereas others do not exhibit any biochemicalsign of hepatic cytolysis but have slight histologi-cal abnormalities of the liver. A third group ofthese carriers are normal, both biochemically andhistologically [1-3].Since HBs Ag is usually associated with acutehepatitis and disappears at the time of recovery,it is difficult to explain how the antigen can per-sist in the third (normal) group of carriers with-out damaging the liver. The fact that HBs Agcan persist in two conditions as different as thehealthy carrier state and chronic hepatitis is alsoproblematical.Received for publication February 6, 1976, and in re-vised form May II, 1976.We thank Dr. P. Kocher, Centre de Transfusion San-guine, La Chaux-de-Fonds, for allowing us to study four ofhis blood donors.This work was performed with the financial support ofthe Centre de Recherche sur les Lymphomes Malins, Lau-sanne, Switzerland.A portion of this study was presented at the EuropeanSociety for Clinical Investigation, Rotterdam, Netherlands,April 25-27, 1974.Please address requests for reprints to Dr. P. C. Frei, Di-vision of Immunology and Allergy, Department of Medi-cine, Centre Hospitalier Universitaire Vaudois, LausanneCH 1011, Switzerland.505The immune state of symptomatic and asymp-tomatic carriers has been thought to be respon-sible for the persistence of the antigen. The non-specific cellular immune response was found tobe impaired by some authors [4-6] and normalby others [1, 7, 8]. The specific immune responseto HBs Ag has also been studied in vitro in bloodfrom a limited number of carriers. No inhibi-tion of leukocyte migration in the presence ofHBs Ag was found by Yeung Laiwah et al. [9] orby Dudley et al. [10], and inhibition was foundonly infrequently by Vittal et al. [11]. However,Lee et al. [12] observed inhibition in 10 of 37HBs Ag-positive blood donors. Irwin et al. foundno migration-inhibition factor (MlF) activity infive carriers tested by an indirect method [13].Koszikowski et al. [14] did not find any stimula-tion of lymphocytes by HBs Ag in 10 carriers, andalso found none in 10 patients who had recov-ered from hepatitis B infection without produc-ing detectable levels of antibody.The leukocyte migration-inhibition (LMI) testin agarose allowed us to use only very smallamounts of purified HBs Ag. With this technique,we have shown [15] the constant appearance ofa specific inhibition of leukocyte migration byHBs Ag after recovery from acute infection withhepatitis B. This inhibition was observed in al-most all patients who cleared the antigen, where-as the antibody became demonstrable by radioim-506munoassay in only half of the patients. The ab-sence of migration inhibition during and imme-diately after the acute phase of the disease wasattributed to nonspecific anergy, since migrationinhibition by purified protein derivative (PPD)disappeared temporarily at that time [16J.From these findings it was hypothesized thatspecific cell-mediated immunity might be respon-sible for the clearance of HBsAg. The presentstudy extends this investigation to asymptomaticcarriers. The results demonstrate the absence ofmigration inhibition by HBs Ag in all but four of25 asymptomatic carriers of HBs Ag.Materials and MethodsSelection of donors. HBs Ag-positive blooddonors were subjected to careful taking of theirmedical history, physical examination, and bloodtests, including levels of aspartate aminotrans-ferase (SGOT) and alanine aminotransferase(SGPT), prothrombin, alkaline phosphatase, bi-lirubin, protein, and electrophoresis. Only thosedonors who proved to be normal according tothese criteria and who had no history of hepati-tis were selected for study. This group was re-examined in the same manner nine to 12 monthslater to establish the persistence of HBs Ag inthe blood and the absence of any symptoms. Thepossibility of a donor incubating acute hepatitiscould thus be avoided. No liver biopsy was per-formed because of the absence of indication inthese asymptomatic donors. Titers of HB. Agwere measured by counterimmunoelectrophore-sis. In two carriers, HBs Ag was detectable byradioimmunoassay only. Twenty-five individuals(18 males and seven females), aged 21-63, werefinally selected for the study.Nonspecific immune state. The three mainimmunoglobulins were measured by radial im-munodiffusion (Tripartigen Behringwerke, Mar-burg, Germany). Humoral response was testedby titration of isoagglutinin, antistreptolysin(Streptolysin Isti tuto Sierterapico Milanese, Mil-an, Italy), and antistaphylolysin (StaphylolysinBehringwerke, Marburg, Germany). Delayed-typeskin reactivity was investigated by skin-testingwith a standard group of five antigens: tuber-culin PPD (5 units; Statens Seruminstitut, Co-penhagen, Denmark), streptokinase-streptodorn-Desaules et al.ase (250 units /ml: Varidase; Lederle, Pearl Riv-er, N.Y.), candidine (Bencard; Beecham, Brent-ford, Middlesex, England), trichophytine (Beech-am), and mumps skin-test antigen (Eli Lilly andCompany, Indianapolis, Ind.).Purification of HB s Ag. Sera with a titer ofHBs Ag of >1:256 by counterimmunoelectropho-resis were pooled. HBs Ag was precipitated bypolyethylene glycol 6,000; the precipitate was re-dissolved in distilled water, dialyzed against 0.05M sodium barbital buffer (pH 8.6), and furtherpurified by agarose-block electrophoresis in thesame buffer. The HBs Ag-rich fractions werepooled and concentrated by vacuum dialysisagainst phosphate buffer. The HBs Ag was thenseparated from trace amounts of contaminatingproteins by ultracentrifugation on a 10%-30%(wt/wt) sucrose gradient. The preparation wassterilized by filtration through Millex microfil-tel'S (pore size, 45 psn; Millipore Corp., Bedford,Mass.) and finally was concentrated by vacuumdialysis (membrane filter SM 132000; Sartorius,Gottingen, Germany) against medium TC 199(Difco, Detroit, Mich.), until a titer of 1:16 bycounterimmunoelectrophoresis was reached. Noprecipitation line was found in agar double-diffu-sion with antisera to whole human serum. TheHBs Ag preparation was used undiluted in theLl\H test.LMI test. This test was performed under aga-rose according to the method of Clausen [17]. De-tails of this method as performed with HBsAg in our laboratory have been described [18].In brief, 2.5 X 107 leukocytes, containing at least75% polymorphonuclear leukocytes, were sus-pended in 0.1 ml of the antigen preparation. Thesuspension was incubated for 30 min at 37 C inan atmosphere of 2.5% CO 2, and 5 fJ-l were in-troduced into each of eight wells punched in anagarose layer. After incubation for 18 hr, theareas of migration were measured by planimetry.The mean surface area of the eight wells was ex-pressed as the percentage of the migration in acontrol preparation without antigen. Values of<90% were considered to indicate inhibition.Leukocytes from each individual were tested inthe presence of two antigens, purified HBs Agand tuberculin PPD (25 fJ-g/ml), without pre-servative. The statistical evaluation was made ac-cording to Student's t-test for unpaired values.Leukocyte Migration in HB 8 Ag CarriersResultsThe titers of circulating HB s Ag ranged from 1:1,024 to 1:1 by counterimmunoe1ectrophoresis,and, in two cases, antigen was detectable only byradioimmunoassay (Ausria II; Abbott Laborator-ies, North Chicago, Ill.). The titer was strikinglystable during the observation period and nevervaried by more than one dilution step. Tests ofhepatic function were normal in all of the in-dividuals selected for study. On retesting a fewmonths later, the antigen was still present andthe results of hepatic function tests were normal.The mean area of leukocyte migration in thepresence of HBs Ag was 101.9<;'0 of that in thecontrol without antigen (SE ± 3.2). Thus, no im-mune response to HBs Ag could be demonstratedin these carriers. Only in cases no. 1, 5, 16, and23 were individual values of <90% demonstra-ble.Figure 1 clearly shows the lack of leukocyteinhibition by HBs Ag in asymptomatic carriers,compared with individuals who cleared the an-tigen after acute infection with hepatitis B. Thedifference is significant (t =5.35; P < 0.001).Leukocytes from carriers did not behave differ-ently from those from normal individuals (t =0.34; 0.3 < P < 0.4) or from those from patientswith acute hepatitis B infection (t = 0.32; 0.3 <P < 0.4) in the presence of HBs Ag.Inhibition of leukocyte migration in the pres-507ence of PPD (figure 2) was demonstrable in 12carriers only, although all 25 carriers had a posi-tive skin test with PPD. In normal individuals,however, the LMI test in agarose gave a good cor-relation with the PPD skin-tests, as shown in fig-ure 2. The mean area of migration in the pres-ence of PPD was 71.0% (SE ± 2.3) in PPD-posi-tive normal individuals, 95.7% (SE ± 4.4) inPPD-positive patients with acute hepatitis B in-fection, and 91.8% (SE ± 3.2) in PPD-positivecarriers. Thus, the leukocytes from these asymp-tomatic carriers behaved, in the presence ofPPD, not like those from normal individuals (t =4.55; P < 0.001), but in the same manner asthose from acutely infected patients (t = 0.73;0.2 < P < 0.25). The reproducibility of the LMItest under agarose was satisfactory, as judged bythe eight simultaneous measurements in eachcase; in the four cases with inhibition, all eightvalues were <90%. The stimulation observed in afew cases may be without significance or may beinterpreted as a weak sensitization [18].Results of nonspecific immunological screen-ing were normal. Values obtained for the humor-al parameters were normal, except for a few iso-lated titers of antibody. This exception can alsooccur in normal individuals. Only one case hadlow values for all three antibodies tested (twodilution steps below the lowest limit) and forIgG (550 mg/100 ml). The delayed hypersen-sitivity, as judged by the skin tests, was normalin all patients..1 •-----~-----JL------- -------~---­}.Figure 1. Migration, in the presenceof purified hepatitis B surface anti-gen (RB s Ag), of leukocytes fromnormal individuals (left), patientsduring and after acute hepatitis Binfection (center), and asympto-matic carriers of RBs Ag (right).Points represent the surface of mi-gration expressed as the percentageof the migration in a control pre-paration without antigen. The shorthorizontal lines are means ± SD.Broken line represents 100% migra-tion.140120-:-...100 -------*(-------c --Ca...0.~ 80ClI~*'604020.-----L.During...........---A.L-···~Afterrecovery.............NORMAL ACUTE HEPATITIS B CARRIERS508140Desaules et al.120100e.~ 80-~Cl::E 60* 4020..------~--------------­.--:;-NORMAL···-----~-----...ACUTEHEPATITIS B-+________.J _.----t--...:..---------...CARRIERSFigure 2. Migration, in the presenceof purified protein derivative (PPD),of leukocytes from normal PPD-negative individuals and normalPPD-positive individuals (left), PPD-positive individuals during acutehepatitis B infection (center), andPPD-positive asymptomatic carriersof hepatitis B surface antigen (right).The short horizontal lines are means± SD. Broken line represents 100%migration.PPDskin test neg. pes. pes. pes.DiscussionThe inhibition of leukocyte migration by HBsAg in carriers occurred infrequently (four of 25carriers). This finding contrasts with the nearlyconstant inhibition we observed in patients whocleared HBs Ag (24 of 28 patients). Consequent-ly, we believe that the specific immune responsedemonstrated by the LMI test is probably respon-sible for elimination of the antigen, which oc-curs during recovery from hepatitis B infection,and that the lack of this response may explainthe carrier state.Nearly all authors consider the LMI test asan index of cell-mediated immunity. Althoughthis hypothesis has not been proven, the follow-ing findings encourage us to support it for themoment. In our experiments with HBs Ag as anantigen, migration inhibition did not correlatewith antibody production [15, 18]. In our experi-ments with PPD, the correlation with the resultsof delayed skin tests was excellent (figure 2).Furthermore, Lambert et al.' and Trepo et al. [19]found no relation between the presence of HBsAg-anti-Hfs, Ag complexes and any of the condi-tions studied here.1 P. H. Lambert, E. Tribollet, A. Celada, K. MadalinskiP. C. Frei, P. A. Miescher, "Circulating Immune Complexe~I~~ol~ingHBsAg in Patients with Acute and Chronic Hepa-tItIS III Healthy Carriers and in Polyarteritis Nodosa,"manuscript in preparation.. Our observations also contribute to explana-nons of the absence of lesions in the carriers.The lesions of hepatitis cannot, of course, beproduced by the virus itself. They might be theconsequence of the immune response demon-strated by the LMI test, and the lack of this re-sponse in carriers would explain the absence ofcytolysis in the liver. One must then consider thereason that this response could not be demon-strated during the acute phase of hepatitis (fig-ure 1), when the liver is most severely attacked.This lack of response is probably related to abroad, nonspecific anergy during the acute phase.We have shown [16, 18] that the migration ofleukocytes from PPD-positive individuals was notinhibited by PPD during hepatitis. Experimentswith PPD as antigen (figure 2).also showed thatleukocytes from some carriers behaved like thosefrom patients with acute hepatitis B infection;the mean surface areas of migration were 91.8%and 95.7%, respectively, without significant dif-ference between the two values. The anergy dem-onstrable in vitro during the acute disease exists,to a lesser extent, in healthy carriers. Thus, itseems that the circulating agent can itself pro-duce a nonspecific anergy, even in the absenceof tissue damage. Note that the anergy in thesetwo conditions was not demonstrable by the skintests. These tests may be more sensitive than theLMI test or may show a slightly different type ofresponse.Leukocyte Migration in HB s Ag CarriersThe impairment of the immune response dem-onstrable by the LMI test in carriers seems tobe an isolated defect, since the results of the non-specific immunological investigation were indis-tinguishable from those one would obtain in nor-mal individuals. Only one carrier had low titersof antibody and a low IgG level. In this respect,our results differ from those of others [4-6]. Thisdifference is probably related to our stricter cri-teria of selection, which excluded individualswith biological signs of hepatic disease.References1. Bolin, T. D., Davis, A. E., Liddelow, A. G. Liver dis-ease and cell-mediated immunity in hepatitis-asso-ciated antigen (HAA) carriers. Gut 14:365-368, 1973.2. Reinicke, V., Dybkjaer, E., Poulsen, H., Banke, 0., Lyl-loft', K., Nordenfelt, E. A study of Australia-antigen-positive blood donors and their recipients with spe-cial reference to liver histology. N. Engl. J. Med. 286:867-870, 1972.3. Griffin, F., Jr. Hepatitis B antigenemia in apparentlyhealthy blood donors. J.A.M.A. 226:753-755, 1973.4. Giustino, V., Dudley, F.]., Sherlock, S. Thymus-depen-dent lymphocyte function in patients with hepatitis-associated antigen. Lancet 2:850-853, 1972.5. Halikowski, B., Korczowski, R., Zajaczkowski, J.Odczynskorny na dwunitrochlorobenzen (DNCB) u nosicieliantygenu Australia. Pediatr. Pol. 47:1071-1075, 1972.6. Ortona, L., Pizzigallo, E., Federico, G., Laghi, V.Richerche sui donatori di sangue au positivi: stimola-zione in vitro dei linfociti con PHA. Ann. Sclavo13:35-48, 1971.7. Nielsen, J. 0., Reinicke, V., Dietrichson, 0., Andersen,V., Thomsen, M., Andersen, E. Immunological studiesof Australia antigen carriers with and without liverdiseases. Clin. Exp. Immunol. 15:9-16,1973.8. Sutnick, A. I., Bugbee, S. J., London, W. T., Loeb, L. A.,Peyretti, F., Litwin, S., Blumberg, B. S. Lymphocytefunction in normal people with persistent Australiaantigen. J. Lab. Clin. Med. 82:79-85, 1973.9. Yeung Laiwah, A. A. C., Chaudhuri, A. K. R., Ander-son, J. R. Lymphocyte transformation and leuco-509eyre migration inhibition by Australia antigen. Clin.Exp. Immunol. 15:17-34, 1973.10. Dudley, F. J., Giustino, V., Sherlock, S. Cell-mediatedimmunity in patients positive for hepatitis-associatedantigen. Br. Med. J. 4:754-756,1972.11. Vittal, S. B. V., Dourdourekas, D., Shobassy, N., Gerber,M., Telischi, M., Szanto, P. B., Steigmann, F., Clow-dus, B. :F. Asymptomatic hepatic disease in blooddonors with hepatitis B antigenemia. Am. J. Clin.Pathol. 62:649-654, 1974.12. Lee, W. M., Reed, W. D., Mitchell, C. J. Eddleston,A. L. W. F., Dymock, I., Williams, R. Cell-mediatedimmunity to hepatitis B antigen in blood donorswith persistent antigenaemia or high titer antibody.Digestion 10:362, 1974.13. Irwin, G. R., Jr., Hierholzer, W. J., jr., Cimis, R., Mc-Collum, R. W. Delayed hypersensitivity in hepatitisB: clinical correlates of in vitro production of migra-tion inhibition factor. J. Infect. Dis. 130:580-587,1974.14. Koszinowski, D., Thomssen, R., Schober, A. In-vitro-Stimulation der Lymphozyten von H'B-Antikdrper-Tragern durch HB-Antigen (Australia-Antigen).Dtsch. Med, Wochenschr. 98:262-267,1973.15. Frei, P. C., Erard, Ph., Zinkernagel, R. Cell-mediatedimmunity to hepatitis-associated antigen (HAA)demonstrated by leukocyte migration test during andafter acute hepatitis B. Biomedicine [Express] 19:379-383, 1973.16. Erard, P., Frei, P. C., Peitrequin, R., Hofstetter, j.-R.,Magnenat, P. Etude de I'immunite cellulaire sped-fique de l'antigene Australia. Resultats preliminairesdans differentes formes d'hepatite B. Schweiz. Med.Wochenschr. 104:1882-1885, 1974.17. Clausen, J.E. Tuberculin-induced migration inhibitionof human peripheral lymphocytes in agarose medi-um. Acta Allergol. (Kbh.) 26:56-80, 1971.18. Erard, P. Technical study of the leukocyte migrationinhibition test in agarose. Application to PPD andto hepatitis B antigen. Clin. Exp. Immunol. 18:439-448,1974.19. Trepo, C. G., Zuckerman, A. J., Bird, R. C., Prince,A. M. The role of circulating hepatitis B antigen/an-tibody immune complexes in the pathogenesis of vas-cular and hepatic manifestations in polyarteritis no-dosa. J. Clin. Pathol. 27:863-868, 1974.

Lack of Leukocyte Migration Inhibition by Hepatitis B Antigen and Normal Nonspecific Immunoreactivity in Asymptomatic Carriers

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