Clin. exp. Immunol. (1976) 24, 374-377.

BRIEF COMMUNICATION

Injurious effect on rat liver mitochondria by lymphocytes from patients with primary biliary cirrhosis A. BOOTELLO, E. FERNANDEZ-CRUZ, P. ESCARTIN, M. F. BLANCO, M. GOSALVEZ & J. M. SEGOVIA DE ARANA Departments ofImmunology, Gastroenterology and Experimental Biochemistry, Clinica Puerta de Hierro, Autonomous University, Madrid, Spain

(Received 14 November 1975)

SUMMARY

Lymphocytes from primary biliary cirrhosis (PBC) patients were shown to have an injurious effect on rat liver mitochondria, as was demonstrated by the inhibition of mitochondrial respiratory control by these cells. The incubation of the PBC patients' lymphocytes with isolated rat liver mitochondria produced a significant inhibition of mitochondrial respiration in the presence of ADP. However, no significant effect on respiration was seen with control lymphocytes of normal persons or with lymphocytes from patients with alcoholic cirrhosis and miscellaneous liver diseases. The results suggest that this injurious effect of PBC lymphocytes on mitochondria might be a consequence of sensitization in vivo of the PBC patients' lymphocytes by the mitochondrial antigens.

INTRODUCTION Primary biliary cirrhosis (PBC) is characterized by destruction of the intrahepatic biliary ductules not initiated by extrahepatic obstruction. The early histological lesion in this progressive liver disease is damage to interlobular bile ducts which are surrounded by lymphocytes and mononuclear cell infiltrates. In recent years PBC has been included as an 'autoimmune' liver disorder (Doniach & Walker, 1969). The role of an immune reaction has been suggested because of the presence of serum antibodies to bile duct cells, smooth muscle, thyroid, nuclei and mitochondria in these patients (Mackay, 1958; Paronetto, Schaffner & Popper, 1964; Doniach et al., 1966). The demonstration by immunofluorescence of mitochondrial antibodies (AMA) in almost every case of PBC (over 90%), has been a valuable advance in the differential diagnosis from extrahepatic biliary obstruction (Walker et al., 1965; Doniach et al., 1966; Goudie, MacSween & Goldberg, 1966; Doniach et al., 1970; Klatskin & Kantor, 1972). The antibody is non-organ/non-species-specific and reacts with all cells containing mitochondria. The antigen is a lipoprotein localized in the inner mitochondrial membranes (Berg et al., 1969c; Bianchi, Penfold & Roitt, 1973; Ben-Yoseph, Shapira & Doniach, 1974). Many studies have been made in an effort to discover the nature and mechanism of action of these antibodies by our group and other authors (Dumonde, Roodyn & Prose, 1965; Berg, Doniach & Roitt, 1967; Berg et al., 1969a, 1969b; FernandezCruz & Bootello, 1973), and until the present time no clear effects on mitochondria have been demonstrated. Further investigations of the mechanisms of delayed hypersensitivity in patients with PBC have revealed that they are frequently impaired (Fox et al., 1969; Fox, Dudley & Sherlock, 1973). Also lymphocytes from PBC patients show migration inhibition in leucocyte migration-inhibition tests (LMT) when cultured with mitochondria as well as with biliary proteins (Eddleston et al., 1973). Other studies have demonstrated that nearly all patients with autoimmune disorders gave a positive LMT with mitochondrial inner membrane preparations, even though the patients have no circulating antibodies to Correspondence: Dr A. Bootello, Department of Immunology, Clinica Puerta de Hierro, San Martin de Porres 4, Madrid 35, Spain.

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Effect ofPBC Iymphocytes on mitochondria

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mitochondria (Waternberg et al., 1973). Recent investigation in our laboratory have shown impairment of mitochondrial respiratory control by lymphocytes from rabbits that had previously been sensitized in vivo with rat liver mitochondrial antigen (Bootello et al., 1975). On the basis of this previous study, we wondered if lymphocytes from patients having PBC could affect the function of rat liver mitochondria. MATERIALS AND METHODS Patients. Of the patients studied, seven had primary biliary cirrhosis (PBC), five had alcoholic cirrhosis (AC), four had miscellaneous liver disease (Misc.) (two active chronic hepatitis, one extrahepatic obstructive jaundice and one idiopathic thrombocytopenic purpura) and nine were healthy controls. The diagnoses were based on current clinical, biochemical, histological and immunological criteria. Methods. Mitochondria were isolated from fresh rat liver according to the method described by Schneider (1948) with minor modifications. The livers were obtained from albino rats which had been fasted for 20 hr before sacrifice. Lymphocytes were isolated from 30-50 ml of defibrinated blood by density sedimentation on Ficoll-Triosil gradients. The suspension was centrifuged at 400 g for 45 min. The cells at the interphase were collected and washed three times in minimal essential medium (MEM). They were then incubated for 20 min at room temperature with isolated rat liver mitochondria (5-20 x 106 cells per mg mitochondrial protein). After incubation each mixture was transferred to a chamber containing 3 ml assay medium (0-25 M sucrose, 20 mm KC1, 7 mm HG, 7 mM MgC12 and 5 mm Pi, pH 7*4, 220C) and equipped with a Clark type electrode. Mitochondrial respiration, respiratory control (ratio between respiration with ADP and respiration without ADP) and ADP:O ratio was determined as described by Estabrook (1967), using glutamate plus malate (5 mM) as the substrate, and 0-2 mM ADP.

RESULTS In our experimental procedures (Fig. 1) control mitochondria in the absence of lymphocytes had an average respiratory control of 8-4 and this value was considered as 100% in the figure. Mitochondria incubated in the presence of lymphocytes from normal persons showed an average respiratory control of 84%. In the patients with alcoholic cirrhosis the results were closely similar to the healthy controls (average respiratory control of 80%). Furthermore, lymphocytes from patients with miscellaneous liver diseases did not produce a significant effect on the mitochondrial respiration (average respiratory control 84%). However, significant inhibition of the respiratory control occurred when lymphocytes from patients with PBC were incubated with mitochondria (average respiratory control of 11%). 100 _ 830 70 70

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500: 2040 40 20

MC

NL

ACL

PBCL Misc. L

FIG. 1. Percentage of inhibition of the respiratory control of rat liver mitochondria by lymphocytes from patients with primary biliary cirrhosis (PBC) alcoholic cirrhosis (AC) and miscellaneous liver diseases (Misc.). The bars represent the average respiratory control of mitochondria after incubation for 20 min at room temperature with different number of lymphocytes (5-20x 106 cells per mg mitochondrial protein). MC = mitochondria in the absence of lymphocytes; NL = mitochondria incubated with lymphocytes from healthy persons; ACL = mitochondria incubated with lymphocytes from AC patients; PBCL = mitochondria incubated with lymphocytes from PBC patients; Misc. L = mitochondria incubated with lymphocytes from patients with miscellaneous liver diseases.

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DISCUSSION These results clearly show that lymphocytes from patients with PBC are able to produce an injurious effect on rat liver mitochondria in vitro. This phenomenon might be a consequence of sensitization in vivo of the PBC patients' lymphocytes by the mitochondrial antigen(s). In a previous paper we reported that mitochondrial antigens were capable of priming rabbit lymphocytes in vivo, and that these sensitized lymphocytes produced a total inhibition of mitochondrial respiration (Bootello et al., 1975). The measure of respiratory control has been shown to be a very sensitive index of mitochondrial membrane integrity (Lehninger, 1964). In addition, primary biliary cirrhosis is a clearly defined entity with evidence of intense and life-long autoimmunization (Doniach, 1974). Moreover, in chronic liver diseases there is increasing data suggesting cell-mediated reactivity towards liver-specific and bile antigens (Miller et al., 1972; Eddleston, 1974; Hopf et al., 1975). Further studies are necessary to clarify the mechanisms by which this sensitization occurs and the possible relevance of this phenomenon in the pathogenesis of this autoimmune liver disease. Our grateful thanks go to Professor Deborah Doniach, for her advice and for helpful criticism of the manuscript. We acknowledge the technical assistance of Mrs Consuelo Pascau. This work was supported by a grant from the Instituto Nacional de Prevision.

REFERENCES DONIACH, D., WALKER, J.G., ROITT, I.M. & BERG, P.A. (1970) 'Autoallergic' hepatitis. New Engl. J. Med. 282, 86-89. DUMONDE, D.C., ROODYN, D.B. & PROSE, P.H. (1965) The effects of antibodies on cells. III. Studies on the interaction of rat liver mitochondria and lysosomes with antibody and complement. Immunology, 9, 177. EDDLESTON, A.L.W.F. (1974) The role of immunological mechanisms in acute and chronic hepatitis. Postgrad. med. 3. 50, 348. EDDLESTON, A.L.W.F., MACFARLANE, i.G., MITCHELL, C.G., REED, W.D. & WILLIAMs, R. (1973) Cell-mediated immune response in primary biliary cirrhosis to a protein fraction from human bile. Brit. med. ]. iv, 274. ESTABROOK, R.W. (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods in Enzymology, volume 10, p. 41. Academic Press, New York. FERNANDEZ-CRUZ, E. (1973) Studies on the mechanisms of the mitochondrial immunity. M.D. thesis. Universidad Complutense, Madrid. Fox, R.A., DUDLEY, F.J. & SHERLOCK, S. (1973) The primary immune response to haemocyanin in patients with primary biliary cirrhosis. Clin. exp. Immunol. 14, 473. Fox, R.A., JAMES, D.G., SCHEUER, P.J., SHARMA, 0. & SHERLOCK, S. (1969) Impaired delayed hypersensitivity in primary biliary cirrhosis. Lancet, i, 959. GOUDIE, R.B., MACSWEEN, N.M. & GOLDBERG, D.M. (1966) Serological and histological diagnosis of primary biliary cirrhosis. ]. clin. Path. 19, 527. HOPF, U., ARNOLD, W., MEYER ZUM BUSCHENFELDE, K.H., FOSTER, E. & BOLTE, J.P. (1975) Studies on the pathogenesis of chronic inflammatory liver diseases. I. Membrane-fixed IgG on isolated hepatocytes from patients. Clin. exp. Immunol. 22, 1. KLATSKIN, G. & KANTOR, F.S. (1972) Mitochondrial antibody in primary biliary cirrhosis and other diseases. Ann. intern. Med. 77, 533. LEHNINGER, A.L. (1964) In: The Mitochondrion: Molecular Basis of Structure and Function. Benjamin, New York. MACKAY, I.R. (1958) Primary biliary cirrhosis showing a

BEN-YOSEPH, Y., SHAPIRA, E. & DONIACH, D. (1974) Further purification of the mitochondrial inner membrane autoantigen reacting with primary biliary cirrhosis sera. Immunology, 26, 311. BERG, P.A., DONIACH, D. & RoITT, I.M. (1967) Mitochondrial antibodies in primary biliary cirrhosis. I. Localization of the antigen to mitochondrial membranes. .. exp. Med. 126, 277. BERG, P.A., RoITT, I.M. DONIACH, D. & HORNE, R.W. (1969a) Mitochondrial antibodies in primary biliary cirrhosis. III. Characterization of the inner-membrane complement fixing antigen. Clin. exp. Immunol. 4, 511. BERG, P.A., MUSCATELLO, U., HORNE, R.W., ROITT, I.M. & DONIACH, D. (1969b) Mitochondrial antibodies in primary biliary cirrhosis. II. The complement fixing antigen as a component of mitochondrial inner membranes. Brit. a. exp. Path. 50, 200. BERG, P.A., ROITT, I.M., DONIACH, D. & COOPER, H.M. (1969c) Mitochondrial antibodies in primary biliary cirrhosis. IV. Significance of membrane structure for the complement fixing antigen. Immunology, 17, 281. BIANCHI, F.B., PENFOLD, P. & RoITT, I.M. (1973) Mitochondrial antibodies in primary biliary cirrhosis. V. Ultrastructural localization of the antigen to the inner mitochondrial membrane using a direct peroxidase conjugate. Brit. ]. exp. Path. 54, 653. BOOTELLO, A., FERNANDEZ-CRUZ, E., KREisLER, M., ARNAIZ, A., JIMENEZ, L., GOSALVEZ, M., BLANCO, M.F. & SEGOVIA de ARANA, J.M. (1975) Impairment of mitochondrial respiratory control by activated lymphocytes. Int. Arch. Allergy appl. Immunol. 48, 4. DONIACH, D. (1974) Autoimmunity in liver diseases in relation to genes, drugs and viruses. Progress in Immunology, II, volume 4, p. 231. North-Holland Publishing Company, Amsterdam. DONIACH, D., RoITT, I.M., WALKER, J.G. & SHuRLOCK, S. (1966) Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver disease and their clinical implications. Clin. exp. Immunol. 1, 237. DONIACH, D. & WALKER, J.G. (1969) A unified concept of autoimmune hepatitis. Lancet, i, 813-815.

Effect of PBC Iymphocytes on mitochondria high titer of autoantibody. New Engl. J. Med. 258, 185. MILLER, J., SMITH, M.G.M., MITCHELL, C.G., REE, W.D., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1972) Cellmediated immunity to a human liver-specific antigen in patients with active chronic hepatitis and primary biliary biliary cirrhosis. Lancet, ii, 296. PARONETTO, F., SCHAFFNER, F. & POPPER, H. (1964) Antibodies to cytoplasmic antigens in primary biliary cirrhosis and chronic active hepatitis. I. Lab. clin. Med. 69, 979.

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SCHNNEIDER, W.C. (1948) Intracellular distribution of enzymes. J. biol. Chem. 176, 549. WALKER, J.G., RoITT, I.M., DONIACH, D. & SHERLOCK, S. (1965) Serological tests in the diagnosis of primary biliary cirrhosis. Lancet, i, 287. WATERNBERG, L., DONIACH, D., BROSTOFF, J. & RoITT, I.M. (1973) Leucocyte migration inhibition with mitochondria in human autoimmune thyroid disorders. ClGn. exp. Immunol. 14, 203.

Injurious effect on rat liver mitochondria by lymphocytes from patients with primary biliary cirrhosis.

Lymphocytes from primary biliary cirrhosis (PBC) patients were shown to have an injurious effect on rat liver mitochondria, as was demonstrated by the...
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