Cell-mediated cytotoxicity in chronic active liver disease: A new test system

0016-5085/78/7405-0883$02.00/O GASTROENTEROLOCY 74:883-889, 1978 Copyright0 1978 by the AmericanGastroenterological Association

Vol. 74, No. 5, Part1 Printed in lJ.SA.

CELL-MEDIATED CYTOTOXICITY DISEASE: A NEW TEST SYSTEM

IN CHRONIC

ACTIVE

LIVER

A. J. M. VOGTEN, M.D., N. HADZIC, M.D., PH.D., R. G. SHORTER, M.D., W. H. J. SUMMERSKILL, M.D.,? AND W. F. TAYLOR, PH.D. Mayo Clinic, Mayo Foundation

and Ma.yo Medical School, Rochester, Minnesottr

An in vitro cytotoxicity system was developed for studying patients with chronic active liver disease using as the target cells “‘Cr-labeled avian erythrocytes coated with cell membrane lipoprotein extracted from human liver and, as the aggressors, mononuclear cells from peripheral venous blood. Approximately 50% of 62 patients with chronic active liver disease showed cytotoxicity in this test system as did 5% of 100 apparently healthy controls. In addition, mild cytotoxicity was shown by 2 of 8 patients with the primary biliary cirrhotic syndrome and 2 of 17 persons with other liver diseases. No specific antibody was added to the test sytem and the cytotoxicity could be inhibited by free lipoprotein, antilipoprotein, and by aggregated Ig. Cytotoxicity also was abolished by the depletion from the mononuclear cells of cells phagocytic for iron filings. The effect of depletion of these phagocytic cells was not restored by the addition of 2-mercaptoethanol. These findings add further evidence that autoimmune responses to liver tissue occur in many patients with chronic active liver disease and, importantly, suggest also that these may occur in some apparently healthy people. Despite some modest advances in knowledge, the pathogenesis of chronic active liver disease (CALD) remains obscure. Recently it has been suggested that both external and host factors are involved, that the external agents may be viruses or drugs, and that the host factors include genetically influenced immune responses. ‘-I” Also, it has been speculated that two immune mechanisms are involved in the induction and chronicity of the liver injury: (1) immune responses directed against the external agent; and (2) autoimmune responses to liver tissue. I3 In studies of patients with CALD using lymphocyte transformation or the inhibition of macrophage migration in vitro as assays of cell-mediated immunity, some investigators have demonstrated hypersensitivity to HBsAg, lcl’ to homogenates of liver’*, I9 and to liverspecific lipoprotein (LP) isolated from normal human liver,2’p”’ and it has been suggested that these immune mechanisms play a role in pathogenesis. In addition, the detection of humoral antibodies to liver cell membranes in HBsAg-negative patients with CALD,“3 and Received August 9, 1977. Accepted December 20, 1977. Address requests for reprints to: R. G. Shorter, M.D., Mayo Clinic, Rochester, Minnesota 55901. This study was supported in part by Grants AM 18634-02and AM 6908 from the United States Public Health Service, National Institutes of Health, Bethesda, Maryland. Dr. Vogten’s present address is: Department of Gastroenterology, University Hospital Utrecht, The Netherlands. Dr. Had&s present address is: Zavod Za Gastroenterologiju, Interna Klinika Rebro, Zagreb, Yugoslavia. The authors’thanks are due to Ralph E. Ellefson, Ph.D. and Mrs. S. Collyer for their invaluable assistance. t Died March 9, 1977.

the finding of immune complexes of HBsAg in the liver and extrahepatic locations in patients with HBsAgpositive disease2” have led to speculation that humoral immune mechanisms also may be involved in the pathogenesis. Geubel et al. 2shave shown that mononuclear cells (MC) from the peripheral blood of approximately 50% of their patients with CALD were cytotoxic in vitro for autologous hepatocytes, whereas other investigators,2KSo using either autologous or rabbit hepatocytes as target cells, found that MC from the majority of their patients were cytotoxic. In these studies no significant differences were found between HBsAg-positive and -negative patients. Cochrane et al.:]” suggested that antibody-dependent cell-mediated cytotoxicity was the mechanism involved in their test system. Although it is possible that these immunological findings in CALD may represent epiphenomena, in order to develop our understanding of the pathogenesis it is important to investigate further the mechanisms involved, particularly those of cytotoxicity. However, the use of autologous hepatocytes as targets for cytotoxicity testing severely restricts the number of cells available for the necessarily detailed studies. The use of other targets, such as Chang cells or rabbit hepatocytes grown in tissue culture, has a number of disadvantages, including the potential for antigenic changes in the target cells during cultural replication.“’ Because of the observations by others on cell-mediated immunity to liver-specific lipoprotein in patients with CALD,‘*-” we devised an in vitro cytotoxicity test system which uses “‘Cr-labeled avian erythrocytes, coated with liver cell membrane LP derived from normal human liverzO, 32,33 as the targets, and human MC from peripheral venous blood as the aggressors.

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The purpose of this report is to describe the techniques and the findings in tests involving healthy subjects and patients with CALD or other liver diseases.

Materials and Methods Patients. 1. Sixty-two patients (37 female; 25 male; aged 16 to 76 years) with biopsy-confirmed CALD, of whom 6 were HBsAg-positive in the serum and 56 were negative. All had fulfilled the criteria for entry into an ongoing therapeutic trial in CALD at our institution’-’ and, at the time of testing, 25 were receiving treatment with prednisone alone, I8 were taking prednisone in combination with azathioprine, and 19 were untreated. The dosage schedules of prednisone and azathioprine used in this therapeutic trial have been detailed previously.:” 2. Eight patients (3 male; 5 female; aged 34 to 60 years) with the primary biliary cirrhotic syndrome who were receiving penicillamine or placebo as part of a double blind therapeutic trial in progress at our institution.:<” Of these, 6 proved subsequently to have been receiving penicillamine at the time of testing. 3. Seventeen patients (9 males; 8 females; aged 16 to 74 years) with other liver diseases, including examples of Wilson’s disease, alcoholic hepatitis, hemochromatosis, BuddChiari syndrome, nonspecific reactive hepatitis, and metastatic carcinoma, and 1 patient with acute HBsAg-negative hepatitis. 4. One hundred HBsAg-negative healthy adult volunteers (54 males; 46 females; aged 19 to 54 years), 94 of whom were blood donors and were tested at the time of their donation of blood for transfusion purposes. All of these persons were screened for their fitness to serve as donors at the time of testing and also had been screened previously. The remaining 6 were laboratory personnel who admitted to no symptoms of illness either at the time of testing or in the immediate past. None of the 100 had any history of liver disease. Human blood samples. Samples (15 to 20 ml) of heparinized venous blood were collected by venipuncture from each of the patients and from the healthy volunteers. All samples were drawn between 8 and 11 AM. Appropriately informed consents were obtained. Isolation of peripheral blood MC. MC from the samples of heparinized venous blood were isolated by density gradient centrifugation using Lymphoprep (Nyegaard, Oslo, Norway), as described by Boyum. I)’The cells then were washed three times in medium TC 199 (Grand Island Biological Company, Grand Island, N. Y.) and resuspended in the same medium in a final concentration of 5 x 10” per ml. These suspensions consistently contained in excess of 99% MC, and the viabilities, determined by trypan blue exclusion, exceeded 95%. The plasma from each sample was separated, heated at 56°C for 1 hr, and stored at 4°C. In some tests, detailed later, aliquots of the MC were incubated at 37°C for 30 min with a suspension of iron filings, as described previously,“7 so that actively phagocytic cells could ingest the iron. The cells then were passed through polyethylene tubing wrapped around an electromagnet? and the effluent populations were collected and resuspended in TC 199 in a final concentration of 1 x lOtiper ml. This procedure resulted in losses of 3 to 6% of the MC. Isolation of humal liver LP. Fresh human liver tissue was obtained after therapeutic partial hepatectomy (right lobe) in a patient with a solitary metastasis from a previously resected colonic carcinoma. The metastasis and the liver tissue immediately surrounding it were carefully removed from the specimen and discarded; the remaining liver tissue (approximately

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150 g) was histologically normal. This liver tissue was homogenized at 4°C in 150 ml of sterile 0.85% saline containing 0.25 M sucrose. The homogenate was centrifuged for 1 hr at 150,000 g at 4°C and the supernatant then was removed and immediately frozen (-20°C) in 2-ml aliquots until used. The procedure was completed within 5 hr after the resection of the liver specimen. Using the method described by Meyer zum Buschenfelde;l” as modified by Miller et al. ,LOa 2-ml aliquot of the supernatant of the liver homogenate then was applied to a Sephadex G-100 column (Pharmacia Fine Chemicals, Piscataway, N. J.) with a bed volume of 220 ml, an elution speed of 20 to 40 ml per hr, and 0.1 M Tris-HCl buffer @H 8.0). The absorbance of the eluted samples was measured at 280 nm using a Beckman DU2 spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). The first peak was concentrated lo-fold by air pressure (10 lb per sq inch) using a Diaflo ultrafilter UM-10 (Amicon Corporation, Lexington, Mass.) and then applied to a Sepharose 6B column (Pharmacia Fine Chemicals), using the same buffer, sample size, and elution speed described for the G-100 column. The first peak of the eluate from the Sepharose column, usually comprising 10 to 16 ml of LP solution, was used for the cytoxicity experiments (protein concentration approximately 1.5 mg per ml). LP was freshly prepared every week and stored at 4°C until used. The same homogenate of liver was used throughout. Colonic extract. An extract of human colonic mucosa obtained from a therapeutically resected colonic specimen was prepared as described for the LP. The protein concentration of this extract also was 1.5 mg per ml. Antisera against human LP. On day zero, male New Zealand white rabbits (body weights: 1250 to 1400 g) were injected subcutaneously in both axillae and inguinal regions with 0.7 mg of LP in 0.5 ml of Tris-HCl buffer (PH 8.0) and 0.5 ml of Freund’s complete adjuvant, the pH being adjusted to 7.45 for injection. Subsequently, on three occasions at lo-day intervals, each rabbit was injected intramuscularly with 1.5 mg of LP in 1 ml of Tris-HCl buffer (pH 8.0), the pH being adjusted to 7.45 for injection. Serum was obtained by bleeding on the 10th day after the fourth injection of LP. Each antiserum was heated at 56°C for 1 hr and stored at -20°C until used. Preparation ofLP-coated target cells. Venous blood samples were obtained from pigeons, the red cells were separated and then stored in Alsever’s solution (Microbiological Associates, Bethesda, Md.) at 4°C until used. Fresh samples were obtained weekly. Washed, packed erythrocytes, 0.2 ml, were incubated for 40 min at 37°C with 100 &i of [“Crlsodium chromate solution (Radiochemical Centre, Amersham, England, specific activity, 1 to 2 mCi per ml). Then the red cells were washed three times in 0.15 M Tris-buffer @H 7.45) and resuspended in 1 ml of TC 199 in a concentration of 2 x 10’per ml. This suspension was incubated at 37°C for 1 hr with 2 ml of LP solution on a rocking platform (Bellco Glass, Inc., Vineland, N. J.). Next, the red cells were washed once in 0.15 M Tris-buffer (PH 7.45) and resuspended in the same solution in a final concentration of 2 X 10”per ml. To demonstrate the presence of LP on the surfaces of the avian erythrocytes, an aliquot was incubated with rabbit antiLP serum (1:4) at 37°C for 1 hr, washed, and then incubated with polyvalent fluoroscein-conjugated goat antirabbit serum cl:201 for 1 hr at 37°C. The cells then were examined by fluorescence microscopy. Approximately 80% of cells treated with LP showed fluorescence, whereas untreated cells or LPtreated cells preincubated with normal rabbit serum were negative.

CYTOTOXICITY

May 1978

Thyroglobulin-coated target cells (TGC). Thyroglobulincoated sheep erythrocytes and uncoated control cells were obtained from Ames Company (Elkhart, Ind.) and radiolabeled by the technique used for the avian erythrocytes. Viable colonic epithelial target cells. These were obtained and isolated by techniques described previously.% Aggregated Immunoglobulin (Zg) . This was prepared from normal human serum by the method used by Cochrane et a1.30 Cytotoxicity testing. Each cytotoxicity test involved the testing of MC from 1 or more patients and at least 1 healthy volunteer. MC (2.5 x 10fi) in 0.5 ml of TC 199 were added to 2 x lo5 LP-coated 5LCr-labeled avian erythrocytes (LPC) in 0.1 ml of 0.15 M Tris buffer @H 7.45) in plastic culture tubes (12 by 75 mm; Fisher Scientific Company, Chicago, Ill.). TC 199 and normal human plasma then were added so that the final volume in each tube was 1 ml and the final concentration of plasma was 10% v/v. Then the tubes were incubated for 4 hr at 37°C on a rocking platform (Bellco Glass, Inc.) in an atmosphere containing 5% carbon dioxide. As controls, LPC were incubated alone in TC 199 containing 10% v/v of the normal plasma, and MC were incubated with SICr-labeled non-LP-coated erythrocytes in the presence of 10% v/v normal plasma. All testing was done in duplicate. To estimate cytotoxicity, a modification of the method described by Henney was used.3!’ Initially, a sample (0.1 ml) of the suspension of j’Cr-labeled target cells was counted for its total radioactivity and corrected for background (gamma counter type 1185, Searle Analytic Inc., Des Plaines, Ill.). At the end of the 4-hr test period. each tube was centrifuged at 160 g (International clinical centrifuge, International Equipment Company, Needham Heights, Mass.) for 10 min and the radioactivity in each supernatant was determined by counting a 0. l-ml sample with correction for background. Cytoxicity then was expressed as follows: Cytotoxicity

=

CPM SICr release by erythrocytes incubated with MC - CPM spontaneous release CPM total red cell-associated

885

IN CHRONIC ACTIVE LIVER DISEASE

jlCr x 100

where CPM = counts per minute. For greater clarity the method of analysis of the data will be included in the Results section. Whenever positive cytotoxicity was found, the normal plasma used in the test was retested using MC from known cytotoxicity-negative healthy subjects and LP-coated “‘Cr-labeled target cells. In six tests, further assay tubes contained MC, either from patients or healthy subjects, and LPC, with the addition of 5 ng of LP. In two tests, 5 ng of the colonic extract were added instead of the LP. In four tests, rabbit anti-LP serum was added to the cytotoxicity tubes in a final dilution of 1:lO; control tubes contained 1:lO final dilutions of normal rabbit serum or rabbit antibovine serum albumin serum (Hyland Laboratories, Los Angeles, Calif.) In four tests, further tubes contained MC and LPC with the addition of 5 ng of aggregated Ig. In each instance, additional controls contained target cells, TC 199, and 10% v/v normal plasma to which LP, colonic extract, anti-LP, normal rabbit serum, rabbit antibovine serum albumin or Ig was added but no MC. In eight tests, additional tubes contained LPC, TC 199, and 10% v/v normal plasma and MC depleted of cells phagocytic for iron filings. In two of the latter tests, further tubes contained LPC, MC depleted of phagocytic cells, and 2-mercaptoethanol (ME; Sigma Chemical Company, St. Louis, MO.) in a final concen-

tration of 5 x lo-” M.“’ 41In these instances, additional control tubes contained LPC, TC 199, 10% v/v normal plasma, and ME but no MC, and others contained nondepleted MC, ME, and LPC. Cytotoxicity testing with TGC. This was done by the same techniques used for the LP-coated target cells. MC from 4 patients with CALD and 4 healthy controls were tested against thyroglobulin-coated sheep erythrocytes and uncoated cells. Controls consisted of SICr-labeled TGC and uncoated sheep red cells incubated in TC 199 containing 10% v/v normal plasma. We demonstrated that TGC were susceptible to cell-mediated lysis per se by a modification of the method described by Yust et al.12 In eight tests with “‘Cr-labeled TGC as the target cells in an antibody-dependent cell-mediated cytotoxicity test system, using antihuman thyroglobulin serum (1:lO) and MC isolated from the peripheral blood of healthy volunteers, cytotoxicity values ranging from 21 to 36% were obtained after 4 hr of incubation with a ratio of effector to target cells of 1O:l. Cytotoxicity testing with viable allogeneic colon epithelial cells as targets. The technique used has been described previously.:%

Results Characteristics

of Human

Liver LP

The major lipid components of the LP were cholesterol (0.16 mg per ml), fatty acids and phospholipids (0.05 mg per ml), and triglycerides were present in trace amounts. Lipid analysis by thin layer chromatography showed that the LP contained lysolecithin, sphingomyelin, lecithin, phosphatidyl ethanolamine, fatty acids, and cholesterol. No sialic acid was found. The LP, which was in the form of large particulate molecular aggregates, did not move in paper electrophoresis (PH 8.6) or in polyacrylamide gel electrophoresis. The sum of the molecular weights of the constituents, as determined by gel filtration (Sepharose-6B) was between 1.5 and 2.0 x 106. We concluded that this LP was a liver cell membrane component similar to that described by otherszOr32,33and differed from “F-antigen.“4:’ Cytotoxicity

Testing

Of major import to this study was the definition of a level of cytotoxicity which would be considered “unusually high” for healthy subjects and still be relatively common among CALD patients. From reviewing the methods of others26-2X,30and that used in a previous report from our laboratory,z5 we decided to examine not only the range of cytotoxicity percentages among healthy volunteers but also the entire distribution of these percentages, shown in figures 1 and 2. From this examination we defined cytotoxicity as a value of 6% or above, a value which was approximately the 95th percentile of the distribution. Thus, of the healthy volunteers, 95 were not cytotoxic for LP-coated target cells by this definition but 5 had values of 8, 14, 17, 18, and 23%, respectively. The normal plasmas used in these five tests are not believed to have contributed to the high values obtained because these same plasmas were used in tests of MC from other healthy subjects whose cytotoxicity values ranged from 0 to 5%. Review Healthy

volunteers.

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lin-coated sheep cells or uncoated sheep cells were 0 to 4%. None of these 4 showed cytotoxicity of their MC for LPC target cells. 30 CALD patients. Of the 62 patients with CALD, 30 (48%) showed cytotoxicity for LP-coated target cells with results ranging from 6 to 80% (fig. 2). However, no cytotoxicity was found when the mononuclear cells were incubated with “‘Cr-labeled avian red cells which had not been coated with LP, the values being less than 4%. No correlation could be found between the presence 0 -5 5 15 20 10 25 or degree of cytotoxicity for LP-coated cells and the age or sex of the patients, the activity or estimated duration % Cytotoxicity of disease, the liver biopsy features, or other laboratory FIG. 1. Frequency per unit of measurement and cytotoxicity (%%) including other immunological markers findings, for lipoprotein (LP)-coated target cells of mononuclear cells from the antinuclear antibody, smooth muscle antibody, antiperipheral venous blood of 100 healthy volunteers mitochondrial antibody). Two of the 6 patients who were HBsAg-positive in their sera showed modest cyto08 toxicity with values of 7 and 8%, respectively. No 8 relationship could be found between the cytotoxicity 051 and the therapy that the patient was receiving at the ?? 50 046 time. To illustrate some of these points, between 60 and 044 70% of the negative and positive patients were on therapy at the time of testing. Of the two highest values of cytotoxicity (fig. 21, 1 patient (cytotoxicity 80%) had 40

32

z.&

36

CALD 32 i

-

28

Healthy

PBC

subjects

n:6

n:lOO

Misc. liver dis

CALD "~62

n 117

FIG. 2. Cytotoxicity (%) for lipoprotein (LP)-coated target cells of mononuclear cells from the peripheral venous blood of healthy volunteers, patients with the primary biliary cirrhotic syndrome (PBC), miscellaneous liver diseases, or chronic active liver disease (CALD). The hatched area represents values that were defined as noncytotoxic (see text); n = number of patients in each group. Cytotoxicity

of the records of these 5 persons showed that they did not differ from the other 95. No cytotoxicity was found when MC from the healthy volunteers were incubated with Wr-labeled erythrocytes which were not coated with LP, the values being less than 3%. The values when MC from 4 healthy volunteers were tested against 5*Cr-labeled thvronlobu-

n=6

Cytotoxicity with

addition

of LP n=6 FIG. 3. Cytotoxicity for lipoprotem (LPI-coated target cells of mononuclear cells from 6 patients with chronic active liver disease (CALD) with and without the addition of human liver lipoprotein (LP) to the test svstem (means 2 SEM).

CYTOTOXICITY

May 1978

IN CHRONIC

ACTIVE

LIVER

887

DISEASE

the cytotoxicities were 43, 51, 58, and 28%, respectively (figs. 2 and 4). In the eight tests in which cells phagocytic for iron filings were depleted from aliquots of the aggressor cell suspensions before testing, the cytotoxicity values using untreated MC were 20, 19, 43, 51, 43, 58, 28, and 9% (figs. 2 and 5). In contrast, the values for the same 8 patients with in vitro depletion of the phagocytic cells were 2, 2, 0, 0, 0, 4, 5, and l%, respectively (fig. 5). In two instances (those persons with initial values of 28 and 9% and phagocyte-depleted values of 5 and l%), the addition of ME to tests of phagocyte-depleted MC yielded results of 2 and 4%, respectively. Thus, the depletion of phagocytic cells from the aggressor cell suspensions resulted in complete loss of cytotoxicity for LPC which was not restored by the addition of ME to the system. On testing MC from 4 patients with CALD for their cytotoxicity for thyroglobulin-coated “‘Cr-labeled sheep erythrocytes, the values were 0, 0, 5, and 0%, whereas those with LP-coated target cells were 34, 9, 69, and 80%, respectively. No cytotoxicity was found

CALD

IO t

51

Ot

4ol- T CALD t

I

t

I

36

OL

32 Cytotoxicity

Cytotoxicity

n:4

with of

addition

aggregated

Ig

28

nz4 FIG. 4. Cytotoxicity (%“o)for lipoprotein (LPI-coated target cells shown by mononuclear cells from 4 patients with chronic active liver disease (CALD). The mean values (+SEM) are given for tests with and without the addition of aggregated Ig.

active disease and was receiving treatment with prednisone and azathioprine. In contrast, the other (cytotoxicity 69%) was in clinical and histological remission and had ceased combined therapy 6 months previously. Only time will tell whether relapse will occur in this subject. In the six tests (fig. 3) in which free LP was added to the mixed incubates of MC and LPC, the results were 0, 9, 0, 4, 4, and 9%, in contrast to the cytotoxicity values obtained without the addition of LP, which were 10, 16, 17,43,34, and 28%, respectively, in the same 6 patients. Similar inhibition or marked reduction in cytotoxicity was not observed in the two tests in which colonic extract was substituted for the LP. In the four tests of the effect of rabbit antihuman LP serum on cytotoxicity, the results without the addition of this serum were 27, 14, 28, and 9%, but with its addition these were 3, 1, 0, and O%, respectively. Similar inhibition was not observed with the normal rabbit serum or the rabbit anti-bovine serum albumin. In the four tests in which aggregated Ig was added to the mixed incubates of aggressor cells and LPC, the results (fig. 4) were 3, 13,1, and 0%, whereas without Ig

8

Cytotoxicity (n

8)

Cytotoxicity with of

depletion

macrophages (n

8)

FIG. 5. Cytotoxicity (%) for lipoprotein (LPkoated target cells of mononuclear cells from 8 patients with chronic active liver disease (CALD). The mean values (*SEMI are given for tests with and without the depletion from the aggressor populations of cells phagocytic for iron tilings.

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VOGTEN

(values less than 2%) when the MC from these patients were incubated with uncoated sheep erythrocytes. When the MC from the patient with CALD whose cytotoxicity for LPC was 80% were tested against viable allogeneic colonic epithelial cells in vitro, the control cytotoxicity value was 7% and that with the patient’s MC was 8%. Primary biliary cirrhotic syndrome. Of the 8 patients, 2 showed cytotoxicity for LPC (10 and 12%) and 1 of these was on treatment with penicillamine (fig. 2). Miscellaneous liver diseases. Of the 17 patients in this group, 2 showed cytotoxicity for LPC (fig. 2). Of these 2 patients, 1 had hemochromatosis (cytotoxicity 10%) and the other (cytotoxicity 6%) had HBsAg-negative acute hepatitis. Discussion This study has defined another cytotoxicity system for studying patients with liver disease and has shown that MC from the peripheral venous blood of approximately half of our patients with CALD were cytotoxic for target cells coated with human LP. As with cytotoxicity systems using either autologous or xenogeneic hepatocytes or Chang cells as the targets,2”30 the cytotoxicity was shared by some patients with other liver diseases. Because our patients attended for only very short periods for their evaluations, we were unable to perform the multiple serial testing necessary to determine whether the degree of cytotoxicity varied diurnally or from day to day or from week to week. The MC were cytotoxic for LP-coated cells but not for either thyroglobulin-coated target cells, for uncoated avian or sheep erythrocytes, or for allogeneic colonic cells. In addition, their cytoxicity for LP-coated cells was inhibited or markedly reduced by the addition of free LP to the test system but not by the addition of a colonic extract similarly prepared, and the cytotoxicity also was inhibited by rabbit antihuman LP serum, so it seemed that there was some specificity of the cytotoxicity for the LP-coated targets, although this testing was not exhaustive. The results of the experiments with aggregated Ig imply that cells with Fc receptors were involved in this cytotoxicity,3”,“7 and those with MC populations depleted of cells phagocytic for iron filings indicate that macrophages were playing an important role. No specific antibody was added to the system and each normal plasma involved in positive cytotoxicity tests was associated with negative results on testing MC from other persons. The effect on the cytotoxicity produced by the depletion of phagocytic cells could not be restored by the addition of ME to the 4-hr incubates.40* 41 However, the experiments necessary to define further the mechanisms involved in the cytotoxicity, including the interaction of macrophages and lymphoid cells and the definition of the nature of the effector cells, were precluded from this study by the limited volumes of the blood samples available. Whether this cytotoxicity has any significance to the pathogenesis of CALD or other chronic liver diseases is undefined. From their experiments with assays of

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ET AL.

macrophage migration inhibition factor, Miller et al.“” speculated that the demonstration of cell-mediated immunity to LP in approximately 70% of their patients with CALD supports a role for autoimmune processes in the pathogenesis, and a similar suggestion could be made for our observations. However, Miller et al.‘” found positive results in some patients with the primary biliary cirrhotic syndrome as we did using this cytotoxicity system, and we noted mild cytotoxicity in 2 patients with other liver diseases. If our findings in vitro do indeed reflect a specific cytotoxic response to LP, it could be speculated that such an immunological reaction in vivo to a surface lipoprotein of hepatocytes might lead to liver damage, and, by immunization with LP, some workers?” have induced histological changes in the livers of rabbits similar to those seen in the liver in CALD in human beings. Thus, our findings, and those of Miller et al. ,9”may not represent epiphenomena but could be of importance to the pathogenesis of chronic liver disease, perhaps supporting the hypothesis developed by others to explain the induction and perpetuation of liver injury.:‘, 45 However, much further work is needed to test this suggestion, and the finding of cytotoxicity of MC for LP-coated cells in 5 apparently healthy volunteers is important and remains to be explained. One question is: were they healthy? By the criteria used for the selection of the 100 subjects classified as healthy in this study, they did not differ from the other 95 and were judged fit to be donors for blood transfusion purposes by an excellent blood bank. Because of the possible sociological effects, particularly as the significance of positive cytotoxicity is not known, we did not consider it prudent to subject them to further extensive evaluations. However, all have remained well for periods of 6 to 12 months since testing and continue to serve uneventfully as blood donors. As indicated earlier, much further work needs to be done to evaluate the mechanisms involved and whether this in vitro cytotoxicity for LP-coated target cells is anything other than an epiphenomenon. However, as both Eddlestone45 and Paronetto4” have stressed, immunological mechanisms may be of importance in the pathogenesis of certain chronic liver diseases and a consensus of current opinion favors the importance of cell-mediated immunity. 46 The cytotoxicity system we have described provides an additional valuable tool for studies to explore this concept. REFERENCES 1. Wright R, McCollum RW, Klatskin G: Australia antigen in acute and chronic liver disease. Lancet 2:117-121, 1969 2. Popper H, Mackay IR: Relation between Australia antigen and autoimmune hepatitis. Lancet 1:1161-1163, 1972 3. Eddleston ALWF, Williams R: Inadequate antibody response to HB Ag or suppressor T-cell defect in development of active chronic hepatitis. Lancet 2:X143-1545, 1974 4. Edgington TS, Chisari FV: Immunological aspects of hepatitis B virus infection. Am J Med Sci 270:213-227, 1975 5. Klatskin G: Toxic and drug induced hepatitis. In Diseases of the Liver. Edited by L Schiff. Philadelphia, JB Lippincott, 1975, p 604

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6. Mistilis

SP, Blackburn Med 48:484-495, 1970

CYTOTOXICITY CRB: Active

chronic

IN CHRONIC ACTIVE LIVER DISEASE

hepatitis.

Am J

7. Sherlock S: Chronic hepatitis.

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