suppressor ratio

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

46, ? 14-220 (1988)

The Effect of Nonviral Liver Damage on the T-Lymphocyte Helper/Suppressor Ratio A. KLEIN,*

S. C. PAPPAS,? P. GORDON,? A. WONG,* J. KELLEN,* J. B. ROBINSON,+ AND A. MALKIN*

A. KOLIN,~

Departments of *Clinical Biochemistry, fGastroenterology, *Pathology, and Laboratory Hematology, Sunnybrook Medical Centre, University of Toronto Toronto, Ontario M4N 3M5. Canuda In the present investigation an attempt was made to ascertain whether nonviral liver impairment in rats affects the THelpr lTsuppnssarratio. Two hepatotoxic agents were used: (i) galactosamine (GA), which causes a drug-induced hepatitis-like damage, and (ii) orotic acid (OA), which induces fatty changes. Since these two substances act as antidotes to one another they were administered to rats either separately or simultaneously. GA caused severe liver damage documented by a 104-, 48-, and 1.6- fold rise in the plasma concentrations of ALT, AST, and ALP and by multiple foci of hepatocyte necrosis. This was followed by a drop in T&s ratio from 2.25 observed in the controls to 0.89 in the GA-treated rats. All of these phenomena were prevented by concurrent administration of GA and OA. OA alone did not show an effect on the liver with respect to changes in plasma enzyme concentrations and by light microscopic analysis. However, OA caused a drop in the T&s ratio from 2.25 to 1.55. Neither GA nor OA produced a change in T$T, ratios in in vitro experiments. 10 1988 Academic Prers. Inc.

INTRODUCTION

Human viral diseases of the liver are characterized by a low T-lymphocyte helper/suppressor ratio (CDdCD,) (I-8). This change is believed to be the result of the viral infection per se and not of liver damage (2, 3). In the present investigation, an attempt was made to ascertain whether nonviral liver impairment in rats affects the THelperjTsuppressorratio. Two different hepatotoxins were used, viz., galactosamine (GA), which induces hepatitis-like lesions (9, IO), and erotic acid (OA), which induces fatty changes (11, 12). Since liver damage is prevented when these two compounds are administered together (9) their combined administration was used to exclude the possibility that GA and OA by themselves cause the change in the T&s ratio. In addition, the T,/T, ratios of rat lymphocytes were measured after incubation in vitro in either the presence or the absence of GA or OA. We hypothesized that the liver damage per se and not the presence of the hepatotoxic substances would affect the TH/Ts ratio. MATERIALS

Preparation

AND METHODS

of Drugs

Gafactosamine. Galactosamine HCl (Sigma Chemical Co., St. Louis, MO) was dissolved in normal saline, neutralized to pH 7.4 with 0.1 NaOH, and diluted to a final concentration of 1.16 mmol/ml with normal saline. 214 0090-1229/88 $1.50 Copyright Q 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

NONVIRAL

LIVER DAMAGE

AND T,/Ts RATIO

215

Tris-orotate. Orotic acid (2,6-dihydroxypyrimidine-4-carboxylic acid, Sigma Chemical Co.) was suspended in deionized water and reacted with an excess of Tris (Tris [hydroxymethyl], Bio-Rad Laboratories, Richmond, CA). The resulting Tris-orotate was concentrated and crystallized in a Bucchi Roto-Vap and the crystals were collected in a Buchner funnel. The crude Tris-orotate for injection (1.16 mmol/ml) was prepared by suspending it in physiologic saline with the aid of a magnetic stirrer. Animal

Preparation

Male Wistar rats (400-450 g, Charles River, Quebec) were housed in individual wire-rack cages, maintained at 21°C on a 12-hr light/dark cycle, and allowed access to food (Purina Lab Chow) and water ad libitum. The rats were randomly assigned to four separate treatment groups, each group consisting of eight rats: Group 1 served as sham-treated controls and received physiologic saline (1.0 ml) by intraperitoneal (ip) injection; Group 2 received galactosamine (2.32 mmol/kg) by ip injection; Group 3 was given Tris-orotate (2.32 mmol/kg) in two divided doses 30 min apart; Group 4 rats received Tris-orotate (2.32 mmol/kg) as in Group 3 followed by neutralized galactosamine 30 min later. All groups were injected between 0700 and 0730 hr, and were sacrificed 48 hr later by exsanguination from the right ventricle of the heart while under deep halothane anaesthesia. The blood was collected in heparinized tubes (Vacutainer blood collection tubes, 143 USP Units Na heparin, Becton-Dickinson, Rutherford, NJ). Samples of liver were obtained and placed in 4% phosphate buffered formalin for histopathologic examination. T-Cell Subset Determinations

Heparinized blood was collected from the rats and the mononuclear cells were separated by density gradient sedimentation at specific gravity 1.077. The washed separated cells were then stained by indirect immunofluorescence. The mouse monoclonal antibodies used for the first layer were clone W3/13HLK antibody against rat T lymphocytes, clone W3/25 against rat T helper cells, and clone OX8 against rat nonhelper cells (clones supplied by Sera Lab). The cells were incubated for 30 min at 4°C in an optimal antibody concentration previously determined by titration, washed twice in phosphate-buffered saline containing 0.02% sodium azide and 1.0% bovine albumin, then incubated in fluoresceinated goat anti-mouse MAb for a further 30 min at 4”C, and washed twice as above. The cells were then counted using ultraviolet fluorescence microscopy (13,- 15). Liver Lesion

Lesions to the rat livers were documented by histopathology (paraffin section H + E and PAS stain). A Greiner E-300 autoanalyzer was used to measure the concentration of the following enzymes in the plasma: aspartate aminotransferase (EC 2.6.1.1) (16), alanine aminotransferase (EC 2.6.1.2) (17), and alkaline phosphatase (EC 3.1.3.1) (18). RESULTS

Table 1 illustrates the effect of GA and OA on the enzyme levels in blood en-

216 LIVER

KLEIN

ENZYME

(NJ/liter) PRESENCE

ET AL.

TABLE I MEASURED IN RATS TREATED OR ABSENCE OF OROTIC ACID) AST

Control GA GA + OA OA

66.5 3183.0 80.0 52.2

t t t 5

WLTH EITHER GALACTOSAMINE OR OROTIC ACID ALONE ALT

7 315 11.5 8.3

44.8 2 10.2 4700.0 t 720 50.7 _t I5 36.7 k 0.9

(IN THE

ALP 124 199.0 92.0 88.0

k 2 t ”

14 30.4 5.6 5.3

zymes in plasma. Analysis of variance done on each enzyme showed a significant overall group effect of P < 0.01. Using Duncan’s multiple-range test, no significant differences among the control, GA + OA, and OA alone groups with respect to each enzyme separately were observed. However, GA treatment caused a significant elevation (P < 0.01) in the concentration of the three enzymes. The liver damage obtained by GA is shown in Fig. 1. These livers showed numerous foci of extensive hepatocyte necrosis. No histological damage was shown in livers of the control, GA + OA, and OA groups. Figure 2 demonstrates the effect of GA and OA either alone or together on the TH/TS in viva. Analysis of variance showed a significant overall group effect of P < 0.01. Using Duncan’s multiple-range test no significant difference between the control and the GA + OA groups was observed. These two groups showed a significant difference at P < 0.01 when compared with the GA group and P < 0.05 when compared with the OA groups. Duncan’s test showed no significant difference between the OA and the GA groups whereas Student’s c test showed a difference at P < 0.05. The percentages (mean +- SEM) of Tu cells were 60 t 2.8, 48.4 -C 5.3, 56.7 -+ 3.8, and 46.5 t 4.8 with respect to the control, GA, GA + OA, and OA groups, respectively, whereas the percentages of Ts were 27.6 + 2.1, 52 -t- 3.7, 26.5 -+ 3.3, and 31.1 + 2.9, respectively. The percentage of pan-T cells was 82 + 2.2 with regard to the control group. No significant differences were found among the percentages of the pan-T cells in all groups. Figure 3 shows the Tu/Ts ratio obtained in vitro by incubating lymphocytes for 48 hr in either the absence or the presence of 1 mg/ml GA. Using Student’s t test no significant difference was observed between these two groups. The percentages of Tu were 64 +- 2.2 and 63 + 1.5 with respect to the control and GA treated cells, respectively, whereas the T, percentages were 23 -+ 2.6 and 17 2 1, respectively. Similar results were obtained with lymphocytes incubated in vitro in the presence of OA. (Viability of the cells was 95% after 48 hr incubation.) DISCUSSION

Human liver diseases due to infections by various viruses such as HBV (l-5), Epstein-Barr virus (EBV) (6, 7), and cytomegalovirus (CMV) (8) are characterized by a low CD&D, ratio. This low ratio is believed to be the result of the viral infection per se and not of liver damage (2, 3). However, the fact that hepatic lesions are a common denominator in all of these disorders and that a low CDJ CDs ratio occurs in late stages of primary biliary cirrhosis as well (19, 20) raises

NONVIRAL

LIVER DAMAGE

AND T,/T, RATIO

FIG. 1. Hepatocyte necrosis (Kupfer cells missing) as a result of treating rats with GA.

217

218

KLEIN

ET AL.

3.0 -

;A-Galactosamme )A-Orotic

2.0

-

1.0

-

Acid

[1

i

:

OCONTROL

GA

I

GA+OA

OA

FIG. 2. The effect of GA and OA either alone or together on T&s measured 48 hr after the administration of the hepatotoxic agents.

in vivo. The T,/Ts ratio was

the question as to whether the effect of lymphocytes is due to the liver damage itself. We have shown that a GA-induced hepatic focal necrosis in the rat (Fig. 1, Table 1) causes the Tn/T, ratio to drop from 2.25 to 0.89 (Fig. 2). By preventing the GA effect with OA, and in the absence of GA effect on the T,/Ts ratio in vitro (Fig. 3), we were able to exclude a direct effect of GA itself on lymphocytes as being responsible for the change in the Tu/T, ratio. We wish to highlight the results obtained with OA administration. Although the histopathological examination of the liver and the plasma concentrations of ALT, AST, and ALP did not show liver damage, a change in TH/TS was observed. Fatty liver starts to occur after 4 to 8 days from the beginning of OA administration; however, electron microscopic studies indicated that after 1 day the endoplasmatic reticulum already appeared to be the first organelle affected by the OA administration (11, 12). The change in TH/TS ratio does not seem to be related to OA per se since it did not affect the ratio either in vitro or in vivo in the presence of GA. Thus, it is possible that liver impairment which is not detected by our selection of liver tests is sufftcient to affect the T,/Ts ratio. As to the mechanism by which the liver regulates this ratio, nothing is known as yet. Finally, in extrapolating to the human situation, the fact that homosexual males

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LIVER DAMAGE

AND T,/T, RATIO

219

4

3

Control

Galaclosamine

FIG. 3. The effect of GA on Tu/Ts ratio of rat lymphocytes in vitro. The cells were incubated for 48 hr in the presence or absence of GA.

and hemophiliacs frequently show evidence of having been exposed to hepatitis virus, CMV, EBV, and others (21, 22) leads one to consider the possibility that a connection exists between discrete hepatic lesion and the relatively low CD&D8 ratio characterizing these populations at risk of developing AIDS (23). REFERENCES 1. Bamaba, V., Zaccari C., Levreo, M., Ruocco, G., and Balsano, E, C/in. Immunol. Immunoparhol. 26, 83, 1983. 2. Bamaba, V., Musca, A., Cordova, C.. Levreo, M., Ruocco, G., Albertiny-Petroni, V., and Balsano, F., C/in. Exp. Immunol. 53, 281, 1983. 3. Alexander, G. J. M., Mondelli, M., Naumov, N. V., Nouraria, K. T., Vergani, D., Lowe, D., Eddelston, A. L. W. F., and Williams, R., Clin. Exp. Immunol. 63, 498, 1986. 4. Bamaba, V., Levreo, M., Van Dyke, A., Musca, A., Cordova, C., and Balsano, F., C/in. Immunol. Immunopathol. 34, 284, 1985. 5. Lemm, G., Salzer, K., and Wamatz, H., C/in. Exp. Immunol. 52, 250, 1983. 6. Reinhera, E. L., O’Brien, C., Rosenthal, P., and Schlossman, E, J. Immunol. 125, 1269, 1980. 7. De Waele, M., Thielemans, C., and Van Camp, B. K. G., N. Engl. J. Med. 304, 460, 1981. 8. Camey, W. I?, Rubin, R. H., Hoffman, R. A., Hansen, W. I?, Healey, K., and Hirsch, M. S., J. Immunol. 126, 2114, 1981. 9. Keppler, D., Rudigier, J., Reutter, W.. Lesch, R.. Decker. K.. and Hoppe-Seyler, A., Physiol. Chem. 351, 102, 1970. 10. Medline, A., Schaffner, F., and Popper, H., Exp. Mol. Pathol. 12, 210, 1970.

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19. Bhan, A. K., Dienstag, J. L., Wands, J. R.. Schlossman, S. F., and Reinhertz, E. L.. C/in. Exp. Immunol. 47, 351, 1982. 20. Miller, K. B., Elta, G. G.. Rudders, R. A., and Kaplan, M. M., Ann. Intern. Med. 100, 385, 1984. 21. Lechner, K., Niessner, H., Bettelheim, I?, Deutsch, E., Fasching, I., Fuhrmann, M., Hinterberger, H., Korninger, C., Neumann, E., Liszka, K., Knapp, W., Mayer, W. R.. Stingl, G., and Zeitlhuber, U., Thromb. Haemostasis 50, 552, 1983. 22. Drew, W. L., Mills, J.. Levy, J., Dylewski, J., Casavant, C., Amman, J., Brodie. H., and Merigan, T., Ann. Intern. Med. 103, 61, 1985. 23. Ranki, A., Valle, S. L.. Antonen. J.. Suni, J., Jokipii. L.. Jokipii, A. M. M., Saxinger, C., and Krohn, K., Cancer Res. 45, 4616. 1985. Received May 18. 1987: accepted with revisions October 6, 1987