Immune status in mice with experimental biliary obstruction

CLINICAL

IMMUNOLOGY

AND

16, 396-405 (1980)

IMMUNOPATHOLOGY

Immune

Status in Mice with Experimental Biliary Obstruction M. PINTO AND A. KAPLUN

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Received November 27. 1979 Obstructive jaundice was induced in mice by common bile duct ligation (CBDL). High levels of total blood bilirubin (12.7 f_ 1.5 mg/lOO ml1 were observed after IO-20 days of obstruction and 15.8 ? 2.3 mg/iOO ml after l-2 months. The spleen indices in the CBDL mice were significantly elevated, whereas the thymus weights and content of lymphoid cell in thymus were drastically reduced. Immunization of biliary-obstructed mice with a thymus-dependent antigen, sheep red blood cells (SRBCl. exhibited a markedly suppressed direct plaque-forming and rosette-forming cell response, although there was no significant difference in serum hemagglutinin titers after second immunization with SRBC. The primary plaque-forming response of CBDL mice against a thymus-independent antigen, Escherichio co/i lipopolysaccharide, was threefold higher in magnitude than controls. The mean survival time of skin allografts in CBDL mice was prolonged. On the other hand, splenic lymphoid cells isolated from biliary-obstructed mice and injected in allogeneic newborn recipients gave equal magnitude of graft versus host reaction as control cells. Incubation of normal lymphocytes with bilirubinemic serum caused nonspecific blastogenesis of the cells. The results presented here indicate that biliary obstruction alters the normal immune pattern. It is suggested that severe bilirubinemia leads to disruption of the balance of cell subpopulation reactivity leading to blocked T-cell function. This impaired function is reflected in depressed graft rejection, reduced primary response to thymus-dependent antigen, and greater response to a thymusindependent antigen. Possible explanations of the mechanism(s) involved are suggested.

INTRODUCTION

Obstructive jaundice due to “backing up” or reflux of bile into the bloodstream caused by intrahepatic or extrahepatic biliary obstruction is seen under a variety of pathological conditions (1). Complete obstruction of the extrahepatic bile duct leads to jaundice with predominantly conjugated hyperbilirubinemia and bilirubinuria. In experimental animals the ducts draining at least 75% of the parenchyma must be occluded before jaundice appears (1). Common bile duct ligation (CBDL) performed in mice or rat induces jaundice (2-4). The icterus is obvious by the second day and the serum bilirubin concentration remains at a fairly steady plateau (4). Not many reports have appeared on the immune status of hyperbilirubinemic patients or experimental jaundice in animals. Najedla (5, 6) showed that the immune response of children with erythroblastasis fetalis after birth differs from that of normal children. He found that hyperbilirubinemia induces significant depression of antibody levels against diphtheria, tetanus, and pertussis antigens. Kaklamanis et al. (7) noticed that Rh incompatibility had a significant effect on phytohemagglutinin (PHA)-induced transformation. The degree of suppression, 396 0090-1229/80/080396-10$01.00/O Copyright 0 1980 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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however, did not correlate with blood levels of total bilirubin. Inhibition of allograft rejection in mice with experimental biliary obstruction was reported by Beaudoin and Rabbat (2). Comprehensive studies on the immune status of animals with experimental obstructive jaundice have not been carried out. In the present study, the effect of biliary obstruction on the primary and secondary humoral immune response, PHA-induced transformation, skin allograft survival, and graft versus host (GVH) reaction in mice was investigated. MATERIALS

AND METHODS

Animals. Male and female nonsenescent mice of Swiss, C3H/eB, or RI11 strains weighing 20-22 g were used. Antigens and mitogen. Sheep erythrocytes (SRBC) were obtained in Alsever’s solution and washed three times in RPM1 1640 medium (Grand Island Biological Co., Grand Island, N.Y .) before use. Escherichia coli lipopolysaccharide-B (LPS) was obtained from Difco Laboratories, Detroit, Michigan. Phytohemagglutinin-P (PHA-P) was obtained from Difco Laboratories. Common bile duct ligation (CBDL). CBDL was performed in Swiss or C3H/eB mice under pentobarbital anesthesia. A midline upper abdominal incision was made, traction was applied on the duodenum, and the common bile duct (CBD) was identified, isolated, and ligated with silk sutures (Ospray silk yarn, England). Sham operations were conducted through the same steps omitting the ligation. Bilirubin and urea determinations. Blood samples were obtained from the heart at the time of sacrifice; the serum was stored frozen at -20°C until tested. Total serum bilirubin was measured using bilirubinometer (American Optical Co., Buffalo, N.Y.). Blood urea in serum was done by standard procedure (8). Hemolytic plaque assay (PFC). Lymphocyte suspensions were prepared by gently passing spleens through a 150-mesh metallic sieve in RPM1 medium. Cell clusters were disrupted by pipetting. Tissue fragments and debris were sedimented by gravity and the suspensions of disrupted cells were centrifuged and resuspended. PFC responses were assayed by enumerating the primary antibody-producing cells according to the Cunningham technique (9). The coating of SRBC with LPS was done according to the method of Moller (10). Assay of rosette formation (RFC). RFC was performed in a final volume of 1 ml of RPM1 1640 medium (with 10% fetal calf serum) containing 20-30 x lo6 spleen cells and 2% SRBC as described by Moav and Harris (11). The cell suspensions were incubated for 1 hr at 37°C and overnight at 4°C. The rosettes were then counted in a hemocytometer. Hemagglutination. Mice in the different groups (control and experimental) were immunized ip with 0.1 ml of a 10% suspension of washed SRBC in phosphatebuffered saline (PBS) on Days 0 and 7. Seven days from the last injection the mice were desanguinated by cardial puncture and serum agglutinin titers were determined for each animal after decomplementation at 57°C for 30 min, using serial twofold dilutions with PBS. For each test a control serum from nonimmunized normal and CBDL mice was included. Cell cultures and [3H]thymidine incorporation. Mesenteric lymph node cells

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were suspended in RPMI culture medium supplemented with L-glutamine, 10% (v/v) heat-inactivated (56”C, 30 mitt) fetal calf serum, 50 IU of penicillin/ml, and 50 pg of streptomycin/ml. The cell suspension was dispersed (1 ml) in triplicate into 10 x IOO-mm tubes, each containing 1 x IO6 cells. PHA-P (25 pg) in 0.1 ml RPM1 1640 per tube was added. One microcurie of [3H]thymidine per tube (Nuclear Research Center, Negev, Israel) was added 48 hr from the beginning of the culture period and allowed to label for 16 or 24 hr prior to counting the incorporated radioactivity. The cell cultures were incubated at 37°C in 5% CO, and 90% humidity. The method for harvesting the cultures and determining the incorporated radioactivity has been previously described (12). In additional experiments mouse bilirubinemic serum ( 1: 10 dilution) containing 1.3 mg/lOO ml total bilirubin was added to normal mesenterial lymphocytes (in RPM1 medium and 10% FCS). Simultaneously I:10 dilution of normal mouse serum was added to normal lymphocytes. The cells were then incubated for 16 hr, counted, spun down, and the supernatant was discarded. New cell cultures were set up containing IO” viable cells per milliliter in 10% FCS-RPM1 medium with PHA and in parallel without PHA. The thymidine incorporation was determined 72 hr from the PHA stimulation. Skin grufiing. Skin grafting was performed in mice as described by Billingham and Medawar (13) using the donor-recipient strain combination RI11 + C3H. Skin grafts were observed daily for signs of rejection. Survival time was taken when at least 95% of the graft was rejected. Preparation and inoculation of cell suspensions in GVH reaction test. Suspensions of viable cells from spleen of normal Swiss or CBDL Swiss mice (l.-- 2 months) were prepared in RPM1 medium and administered intravenously to the neonatal RI11 hosts via the orbital branch of the anterior facial vein according to procedures fully described by Billingham and Brent (14). The inoculum contained IO7 viable cells in 0.05 ml. All injections were carried out within 24 hr of birth and litter size was limited to a maximum of six by removal of excess newborn mice. Injected mice were killed 10 days later and the enlargement of the infants’ spleens estimated by spleen indexes. Spleen indexes and thymus weight. The spleen index for each individual mouse was calculated as follows: spleen weight of the mouse body weight x 100. The thymuses of the same mice were dissected out and weighed. The total number of the lymphoid cells per gram tissue was calculated. Enumeration of T cells. The technique used was described by Hudson and Hay (15). The tested cells were washed after the anti-T-antiserum treatment. (The anti-mouse-T-antiserum was kindly supplied by the Weizmann Institute, Rehovot, Israel.) The diluted guinea pig complement (absorbed by normal mouse mesenterial lymphoid cells) was added and the viable cells counted in hemocytometer, under a phase contrast microscope, using 0.2% nigrozin inclusion as marker of cell damage. Histological studies. Spleens and thymuses of control and CBDL mice (for 2 months) were removed, inflated, and fixed in 4% buffered paraformaldehyde at pH 7.0. The tissue was embedded in Paraplast, sectioned at 6 pm, and stained with hematoxylin and eosin.

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RESULTS

Bilirubin and urea determinations. In both CBDL mice (for lo-20 days and l-2 months) highly significant increase of the total bilirubin, 12.7 5 1.5 and 15.8 -+ 2.3 mg/lOO ml, respectively, was found; in control groups (sham operation) only 0.15 & 0.05 was noted. The blood urea in control and CBDL mice was not significantly changed; 60 ? 6 for controls and 52 + 8 mg/lOO ml for biliary-obstructed mice for l-2 months. Spleen index, thymus weight, and lymphoid cell content. The spleen index, thymus weight, and lymphoid cells per unit tissue were compared between normal and CBDL Swiss mice. Figure 1 shows the spleen index, thymus weight, and lymphoid cell content as calculated per gram tissue (mean + SE). The spleen indexes in bilirubinemic mice of both groups (lo-20 days and l-2 months of CBDL) were markedly increased, although the lymphoid cell content per unit tissue was not significantly different. In contrast the thymus weight in mice with CBDL for l-2 months decreased significantly. Furthermore the lymphoid cell content per unit tissue was only one-third of that in the normal thymus. Histological studies ofspleens and thymuses. Thymuses from CBDL mice (for

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FIG. 1. Spleen index, thymus weight, and lymphoid cell content in mice with obstructive jaundice. Values represented as mean k SE. CBDL mice, common bile duct-ligated mice. Analysis of variance test shows significant differences between groups in all cases except in spleen lymphoid cells per gram of tissue. Comparisons between the control and the experimental groups are as follows: Spleen index: t(1 vs 2) = -4.75; ?(l vs 3) = 9.49; P < 0.0001; thymus weight: t = 5.56; P 10.001; spleen lymphoid cells per gram of tissue t( 1 vs 2) = 1.04: t( 1 vs 3) = 1.30; P > 0.2; thymus lymphoid cells per gram of tissue r = 15.35: P < 0.00001.

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2 months) showed depletion of the cortical and medullary regions when compared with normal thymuses (Figs. 2A, 2A1, 2B, and 2Bl). The spleens of the CBDL mice were coupled with splenomegaly and showed no alteration when compared with spleens from control animals (Fig. 2C). Direct PFC or RFC response of mice given injections of SRBC or E. c,oli fipopolysaccharide (LPS). Normal and CBDL Swiss mice were given injections ip of 0.1 ml 20% SRBC or 50 pg of LPS. Direct PFC response in spleens of immunized animals was assayed 4 days after immunization with SRBC and 5 days after LPS. Marked suppression of direct PFC and RFC responses against SRBC were observed in biliary-obstructed mice in both groups (lo-20 days and l-2 months of CBDL). In contrast, CBDL mice showed significantly elevated direct PFC response against E. co/i LPS (Fig. 3). Hemagglutinating antibodies in sera of normal and CBDL mice Jbllowing immunization with SRBC. The antibody response after two ip injections with SRBC in the sera was measured. The mean reciprocal titer in control mice was 146 t 14 and in CBDL mice (for l-2 months), 182 + 68. Six mice were immunized in each group. Effect of biliary obstruction on skin allograft survival. To determine the effect of biliary obstruction on the rejection process, skin allograft survival across H-2 barrier was studied in inbred CBDL mice. Ten male C3H mice (CBDL for l-2 months) were grafted with RI11 tail skin. The control C3H mice were sham operated. The mean survival time for the controls was 9.3 days and for the biliaryobstructed mice 12.2 days. Comparison of the GVH-inducing capacity oj’ spleen cells Jkom normal und CBDL mice. The donor/recipient mouse strain combination was Swiss - RIII. Study has been made to evaluate the capacity of isolated lymphoid cells of normal and CBDL mice (for 2 months) to cause GVH reaction in neonatal recipients. No significant difference between the two groups was found in spite of the marked spleen enlargement (Table 1). Effect of biliary obstruction on C3H] thymidine incorporation into lymph node cells cultured with PHA. The results shown in Table 2 indicate elevated uptake of thymidine by mesenterial lymphoid cells from biliary-obstructed mice in the absence of stimulation with PHA despite three washings in medium. This increased thymidine uptake was accompanied by reduced stimulation index. Incubation of normal mesenterial lymphoid cells with diluted bilirubinemic serum (1.3 mg/lOO ml bilirubin final concentration) for 16 hr, washed three times in medium and cultured for an additional 72 hr caused elevated [3H] thymidine incorporation per se (without PHA stimulation). The stimulation index of cells incubated with bilirubinemic serum and stimulated with PHA was lower than that of cells incubated with normal mouse serum and then stimulated. Enumeration of T cells in spleens and mesenterial lymph nodes by complement-mediated cytotoxic test. The T-cell content of normal and CBDL mice in spleen and mesenterial lymph node cells was estimated by dye inclusion following anti-T-cell treatment (the count of the number of viable cells was done rather than the number of dead cells). The mean percentage I SE of T cells was 33.26 -+- 4.1 in the spleens of normal mice; 40.28 f 4 in CBDL mice for l-2

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FIG. 2. Thymuses from normal mice showed normal thymic architecture (A and Al) (X 10 and x25). Thymuses from CBDL mice (for 2 months) showed decreased lymphocyte number around the cortical sinuses, in the perifolicular area, and in medullary cords (B and Bl) (X 10 and x25). Spleens from CBDL mice (for 2 months) showed normal splenic architecture (C) t x IO). There is no loss of lymphocytes in the marginal zone around the primary lymphoid follicle.

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FIG. 3. Direct PFC or RFC response of mice given injections of SRBC or E. co/i LPS. Values represented as mean + SE. Analysis of variance test shows significant differences between groups in all cases. Comparisons between the control and the experimental groups are as follows: SRBC(PFC) r(l vs 2) = 2.78, ~(1 vs 3) = 3.07, P < 0.01; SRBC(LP) t = -4.29. P < 0.01; SRBC(RFC) t(1 vs 2) = 4.29, t(1 vs 3) = 4.97, P < 0.001.

months; 49 t 9.3 in the mesenterial lymph nodes of normal and 36.04 2 6 in CBDL mice. At least six mice of each group were examined by duplicate tests. DISCUSSION

The experiments of the present study suggest that obstructive jaundice affects mostly thymus-derived antigen-sensitive cells. Similar suppression of cellular immune response has been described in uremic TABLE GRAFT

VERSUS HOST REACTION (iv) TO NEONATAL

Normal RID cells (negative control) 0.48

+ 0.05

1

TO CONTROL AND CBDL SPLEEN CELLS ADMINWERE.D RID HOSTS: SPLEEN INDEXES (MEAN + SE)”

Normal Swiss cells (positive control) 0.78

f (1.62)

0.08

CBDL Swiss cells (experimental) 0.81

f 0.09 (1.68)

” 10’ spleen cells were injected in 0.05 ml of RPM1 medium. The results represent the mean of four separate experiments involving six animals each. The difference between the reactions induced (positive control and experimental) is not statistically significant (P ) 0.2). The numbers in parentheses indicate the spleen indexes calculated on the basis of negative controls.

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TABLE [“HITHYMIDINE

INCORPORATION BILIARY-OBSTRUCTED

Mesenterial lymphocytes from Normal mice Biliary-obstructed mice (l-2 months) Normal mice incubated for 16 hr with normal serum’ Normal mice incubated for 16 hr with bilirubinemic serum’

2

INTO MESENTERIAL MICE CULTURED

[3H]Thymidine Not stimulated

403

JAUNDICE

LYMPH NODE CELLS WITH OR WITHOUT

incorporation<’ PHA stimulated

OF NORMAL

AND

PHA Stimulation index*

235 + 46

5.234 5 1,076

22.21

432 2 151

4,070 + 832

255 ? 39

7,321 zt 1,701

28.65

1,600 k 217

23,876 2 3,754

14.92

9.43

” Each value represents [“Hlthymidine incorporation by lOti cells (counts per minute f SE). Triplicate samples were cultured for each sample, and at least live or six mice used in each experiment. * Stimulation index indicates counts per minute with stimulant per counts per minute without stimulant. ” The serum was discarded and then the lymphocytes were stimulated.

patients or experimental animals (16, 17), cachectic patients with carcinoma (18), Hodgkin’s disease (19), and primary biliary cirrhosis (20, 21). No elevated urea was found in biliary-obstructed mice in our experiments. The mechanism of the impaired T-cell function of biliary-obstructed mice can be explained in different ways. The simplest explanation would be that there exists a selective reduction of a T-cell population in CBDL mice probably due to toxic metabolic products. This explanation is not supported by our quantitative studies, which indicate that there is a disparity between the reduction of the responses and the relatively unaffected number of T cells in spleen and mesenterial lymph nodes in CBDL mice. A second explanation would be that the mechanism of obstructive jaundice immunosuppression involved the interaction of metabolic factor(s) with T cells affecting an early event at T-cell recognition stage. However, this immunosuppression did not appear to affect the priming of the animals to make a secondary response as determined by circulating hemagglutinating antibody to SRBC. The insensitivity to suppression of the primary response to a thymus-independent antigen strengthens the assumption that the thymus-derived antigen-sensitive cell is the chief target of action. Furthermore, skin graft rejection; closely related to T-cell reactivity, appears to be impaired. There are indications that this impairment is a reversible process. When lymphoid cells from biliary-obstructed mice were removed from their bilirubinemic environment and injected in allogeneic newborn recipients they evoked normal GVH reaction. It seems likely that factor(s) depressing T-cell function contribute to the impairment of cell-mediated immunity. Since a-globulin levels increased during obstructive jaundice (22) this protein could be one of the reversible factors that may operate. The immunosuppressive effect of this fraction of serum protein has

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been reported (23). The mechanism by which the cu-globulin exerts its immunosuppressive activity is not yet fully understood. However, evidence is available, which suggests that it inhibits the recognition of antigen by the T lymphocytes (24). The possibility that some of the effects are due to interference with reticuloendothelial function must be considered. Lipoprotein particles which circulate in bile duct-ligated mice are removed by Kupffer cells (3). Still another possibility is that metabolic products of biliary obstruction have some nonspecific mitogenic activity upon T-cell lymphocytes. Such activated lymphocytes in vivo would become refractive for a short period to any subsequent stimulation with SRBC or allogeneic antigen. Similar suppression by concanavalin treatment for thymus response in G.w was reported (25). The results in this communication indicate elevated mitogenic activity of normal lymphoid cells incubated with bilirubinemic serum. The possibility that the mitogenic response appears from a factor which is adherent to lymphocytes, despite three washings in medium, cannot be entirely excluded. Another explanation is that the defect may be due to a toxic effect upon lymphopoietic tissue. Obstructive jaundice in our model leads to a marked decrease in thymus weight; moreover the thymus parenchyma in CBDL mice is mostly replaced by connective tissue. Kerbel and Eidinger (26) reported results of the effect of adult thymectomy on primary antibody responses in mice to a variety of antigens. On the one hand the antibody to SRBC was markedly suppressed; the response to PVP (thymus-independent antigen) on the other hand was considerably enhanced. The enhancing effect of thymectomy provides the clearest support for the possible existence of a suppressive regulatory T-cell function (27). Another possible explanation of our results (i.e., elevated PFC response by 6. coli LPS, slight elevation of hemagglutinating antibodies by SRBC. and splenomegaly) in CBDL mice may be as follows: It may be assumed that normal liver antagonises antigenic stimulation through its well-known “filter effect”: therefore abnormalities in the liver circulation might increase the antigenic effect (28). The important fact is apparently the liver lesion, and not its etiology (29). Several investigations have evaluated the histologic and hemodynamic alterations caused by bile duct obstructions in animals (rats, rabbits, dogs, and monkeys). Most authors agree that there is a progressive and prolonged decrease in hepatic blood flow (30), impairment of intrahepatic blood flow (31), significant elevation of the portal pressure due to increased resistance to portal blood flow (32), and occasionally development of portal systemic collateral veins (30,33). No arterial-portal shunting has been observed (30, 34). The findings that elevated antibody titers in chronic active hepatitis are associated in particular with bacterial antigens (28, 29, 35) suggest that the nature of the antigen may also be an important factor in determining whether it is sequestered by the liver. These data can be consistent with our findings of increased PFC titer to E. coli LPS and with hepatosplenomegaly found in CBDL mice. Characterization of the factor(s) involved in biliary obstruction influencing the altered immune response as well as the study of their mode of action is currently being carried out.

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ACKNOWLEDGMENTS We are grateful to Professor A. Kohn for comments on the manuscript, and to Mrs. Hanna Herzberg and Miss Bilha Halperin for their excellent technical assistance.

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