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Impaired specific cell-mediated immunity in experimental biliary obstruction and its reversibility by internal biliary drainage
JOURNAL
OF SURGICAL
RESEARCH
41, 113-125 (1986)
Impaired Specific Cell-Mediated Immunity in Experimental Biliary Obstruction and Its Reversibility by Internal Biliary Drainage’ PATRICK T. ROUGHNEEN, M.B., CH.B., DIRK J. GOUMA, M.D., ANIL D. KULKARNI, WILLIAM F. FANSLOW, B.S., AND BRIAN J. ROWLANDS, M.D.
MS.,
Department of Surgery, The University of Texas Medical School, Houston, Texas 77030
Submitted for publication November 22, 1985 Little is known of the effect of cholestasis on host immunity. This study evaluates lymphocytic responsivenessto PHA and LPS mitogen and to allogeneic FrU antigen in Sprague-Dawley rats 2 1 days following bile duct ligation and 3 1 days following relief of jaundice by internal biliary drainage. Serum bilirubin level was significantly elevated in the bile duct ligated animals at Day 21 (P < 0.001) and thereafter returned to preoperative levels following internal biliary drainage. Resultsdemonstratedepressed responsivenessto PHA (P < 0.00 1) and allogeneic F344antigen in vivo (P < 0.04) and in vitro (P < 0.02) in bile duct ligated animals as compared to sham, sham pair-fed, and normal control rats. The observed deficiency in responsivenessto T-cell-dependent mitogen and antigen cannot be explained on the basis of complicating nutritional, renal, or infective factors. Subsequent internal biliary drainage results in some improvement in T-cell responsivenessin the bile duct ligated group although recovery is not complete. B-Lymphocytic response to LPS mitogen is not affected by bile duct ligation. We conclude that cholestasissubsequentto extrahepatic biliary obstruction per se resultsin impairment of cell-mediated immunity in vivo. This impairment is partly reversible by internal biliary drainage. In vifro B-cell function does not appear to be affected in this model. Further study of impaired cell-mediated immunity in extrahepatic biliary obstruction will improve our understanding of the immunological status of patients with obstructive jaundice and cholestatic liver diseases. 0 1986 Academic Press. Inc.
plications and as a palliative procedure remains controversial [ 18, 27, 281. Clinical Several clinical studies have reported a high studies have demonstrated the efficacy of bilincidence of septic complications following iary drainage in improving serum bilirubin diagnostic and therapeutic procedures in pa- levels and other parameters of liver function tients with obstructive jaundice [ 1, 3, 4, 18, [27, 281 but its role in improving immuno19,27,28,3 11.The causeof this high incidence logical status of the jaundiced patient is unof septic complications is not fully understood, known. but impairment of host defense mechanisms Experimental studies have demonstrated may be implicated. Little is known of the im- impaired reticuloendothelial function in bile munological status of patients with obstructive duct ligated animals [20, 2 l] and this impairjaundice [ 11, 13, 26, 391. Such patients often ment has been confirmed in patients with obhave a malignant tumor, significant malnu- structive jaundice [8]. However, experimental trition, infection, and renal impairment, all of studies of systemic polymorphonuclear leuwhich can compromise host immunity [5, 7, kocyte, macrophage,and lymphocyte function 9, 16, 231. It remains unknown whether cho- in extrahepatic cholestasis are limited [30]. lestatic liver disease alone can result in im- The purpose of this study was to investigate pairment of systemic host immunity. lymphocytic function in the rat model followThe role of preoperative biliary drainage in ing the induction of extrahepatic biliary obreducing the incidence of postoperative com- struction, to document the contribution of nutritional, renal and infective factors to any ’ Presented at the Annual Meeting of the Association alterations observed in lymphocytic function, and to evaluate the effect of internal bilary for Academic Surgery, Cincinnati, Ohio, November lodrainage on T- and B-cell function. 13, 1985. INTRODUCTION
113
0022-4804186$1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS
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AND METHODS
Female Sprague-Dawley (SD) rats (Timco Laboratories, Houston, Tex.) weighing 200 to 250 mg were housed in metabolic cagesand subjected to 12-hr alternate light cycles daily. Throughout the study, rats were fed standard rat chow (Formulab 5008) and allowed water ad libitum. Animals were assigned to one of four groups. Group I: Bile duct ligation (BDL) (n = 26); Group II: Sham controls (n = 19); Group III: Sham pair-fed controls (n = 7); Group IV: Normal controls (n = 26). Twentyone days after initial operation, the animals either had a drainage procedure performed or were studied immunologically by assessing lymphocytic responsiveness to mitogen and F3+, allogeneic antigen (Table 1). Thirty-one days following internal or sham drainage animals were again assessedimmunologically. In those animals in which lymphocytic responsivenessto T- and B-cell mitogen was being assessed,sham animals (Group II) were pairfed with the bile duct ligated group to eliminate the possibility of a nutritional factor influencing results.
VOL. 41, NO. 2. AUGUST
1986
was identified and freed from the surrounding tissue only. Internal Biliary Drainage Twenty-one days after division of the common bile duct, the abdomen was reopened under ether anesthesia and the liver was mobilized to the anterior abdominal wall. The dilated bile duct was dissected from the surrounding soft tissue. A Silastic tube (0.03 in i.d. X 0.065 in o.d.) with a Silastic cuff was inserted into the dilated common bile duct and tied in position using two purse string 6-O silk sutures. The Silastic tube was inserted into the duodenum and secured in position with a 60 silk purse string [ 341.In rats having a sham drainage procedure, the bile duct was dissected and a purse string suture tied in the duodenum. Sacrifice of Animals The animals were sacrificed using ether anesthesia. The abdomen was opened, the spleen removed, bathed in RPM1 1640 medium, and immediately processedfor immunological assay.
Bile Duct Ligation
Hematology and Biochemistry
This was performed under ether anesthesia on the first day of the study: a central upper abdominal incision was made, the common bile duct identified, doubly ligated with two 3-O silk ligatures, and divided [24]. High bile duct ligation was performed in the same position in all animals. In those animals undergoing sham operation, the common bile duct
Total white cell count (Coulter counter S method), total serum bilirubin (modified Walters and Gerarde method) [38], and serum creatinine (Jaffee method) [25] were determined at Days 0, 7, 14, 21, 28, 35, 42, and 52. Serum electrophoretic studies (cellulose acetatepH 8.6) were performed on Day 14 of the study.
TABLE
Group Day
Sham
BDL 0
Bile duct ligation
Day 21
Drainage or immunological
Day 52
Immunological
assay
1
Normal control -
Sham operation assay
Sham, drainage, or immunological assay
Immunological
assay
Immunological
Immunologkxl
assay
assay
ROUGHNEEN
ET AL.: IMMUNOSUPPRESSION
Bacteriological Cultures
IN EXTRAHEPATIC
CHOLESTASIS
115
was measured utilizing the tritium window in a Beckman LS-8000 liquid scintillation Samples of blood and bile were taken asep counter and results were expressedasthe delta tically from BDL, sham, and normal animals value (A). at the time of sacrifice and innoculated into Mixed lymphocyte response. The one-way 3.5 ml of Schaedlersbroth (BBL, Cockeyville, mixed lymphocyte response using irradiated Md.). Samples of broth were incubated aeroF344 splenic lymphocytes as a source of allobically and anaerobically at 37°C and examgeneic effector cells was used to assaythe reined for bacterial growth 1, 2, and 7 days folsponsiveness of SD splenic lymphocytes. The lowing incubation. purified SD and F344splenic lymphocyte fractions were obtained in an identical manner to In Vitro Lymphocyte Assays that described above. SD splenic lymphocytes Mitogen assay. Rat spleen lymphocyte pro- (2 X 105)were mixed in a microtiter well plate liferative response to T- and B-cell mitogens in triplicate with 2 X lo5 F344allogeneic irrawere tested using a modified method of Strong diated cells (stimulated wells) or without Fjd4 et al. (35). Rat spleens were harvested asepti- spleen cells in a volume of 0.1 ml of complete cally, cut into small pieces, and then teased RPMI. Plateswere incubated for 96 hr at 37°C through loo-mm stainless-steel screens to in a 5% COZhumidified 95% air mixture. The make single cell suspensions in RPM1 1640 cells were then pulse labeled for 16 to 18 hr medium containing pen-strep. The cell sus- with [3H]thymidine in a volume of 50 micropensions were spun at 1200 rpm for 10 min liters. The plates were then harvested on a in a refrigerated centrifuge. Contaminating red Mash II harvester. Disks were transferred to blood cells were removed by treatment with vials containing 5 ml of scintillation cocktail Tris ammonium chloride. The cell suspen- and incorporation of [3H] thymidine was sions were further washedthree times in RPM1 measured on a Beckman LS-8000 counter. 1640 and then finally resuspendedin complete Results were expressedas the stimulation inRPM1 containing 10% fetal bovine serum dex (S.I.) and as the delta value. (FBS), pen strep, L-glutamine, and 0.2 M Hepes. The cell suspensions were then enuIn Vivo Mixed Lymphocyte Reaction merated and the cell concentration adjusted The popliteal lymph node assay was used to 5 X lo6 cells/ml. The cells were dispersed to evaluate T-lymphocyte responsiveness in a microtiter well plate at 5 X lo5 cells/well manifested as a host versus graft response as in a volume of 0.1 ml complete RPMI. Fifty microliters of the mitogen phytohe- described by Twist and Barnes (36). Spraguemagglutinin (PHA) (Gibco) and lipopolysac- Dawley splenic lymphocytes were used as the charide (LPS) (Calbiochem) was added in source of syngeneic antigen and F344splenic triplicate wells at 1:10, 1:25, and 1:50 dilutions lymphocytes as the source of allogeneic of stock solution of PHA and 1 mg/ml LPS antigen. Syngeneic and allogeneic cells were presolution. Volume per well was adjusted to 0.2 ml total by addition of the appropriate amount pared in the manner described above and the of complete RPMI. Microtiter plates were in- enriched lymphocyte fractions obtained. Docubated for 48 hr at 37°C with humidified 5% nor cells were then irradiated at 2500 R to CO2 air mixture. Following incubation, cells eliminate graft versus host reactivity in this assay. The rats were then subjected to ether were pulse labeled with [3H]thymidine (1 &i/ well, 6.7 Ci/mole, sp act) for 16 to 18 hr. The anesthesiaand 25 X lo6 FxU cells were injected cells were then harvested on a Mash II harves- into the right (allogeneic) foot pad and 25 ter. Disks were transferred to vials and 5 ml X lo6 SD cells into the left (syngeneic) foot of scintillation cocktail was added. Incorpo- pad in 100-~1volumes using a 27-gaugeneedle ration of [3H]thymidine into stimulated cells and a 1 cc hypodermic syringe. After 1 week,
116
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the animals were sacrificed and popliteal lymph nodes were dissected from right and left popliteal fossaeand weighed. The results of the host versusgraft responsewere expressed as the weight of allogeneically stimulated lymph nodes divided by syngeneically stimulated lymph nodes (stimulation index) and as the difference in weight between allogeneic and syngeneic nodes (delta value). Statistical Analysis All study results were expressed as means + SE and subjected to the unpaired Student t test. RESULTS
I. Biochemistry and Hematology Mean bilirubin in BDL vs sham groups was 0.1 f 0 vs 0.1 + 0 mg/dl at the initiation of the study and 6.9 + 0.5 vs 0.1 + 0.02 mg/dl (P < 0.00 1) at 2 1 days, following which internal biliary drainage was performed. At the termination of the study, (Day 52), mean bilirubin was 0.1 + 0 and 0.1 f 0 mg/dl in BDL and sham groups, respectively (Table 2). Mean plasma creatinine in BDL and sham groups was 1.03 + 0.04 and 0.96 + 0.06 mg/dl at Day 0; 1.2 + 0.05 and 1.05 + 0.04 mg/dl at 2 1 days and 0.9 + 0 and 0.89 +- 0.07 mg/dl at 52 days. At 2 1 days, plasma creatinine was significantly raised in BDL animals compared to preoperative levels (P < 0.04) and postdrainage levels (P < 0.03) (Table 2). Mean WCC in the study group at the initiation of the study was 16.5 + 2.2 X lo3 cells/ mm3. Mean WCC in BDL vs sham groups at 21 days was 53.8 + 3.37 X lo3 vs 11.34 + 0.7 15 x lo3 cells mm3 (P < 0.00 1). Following internal biliary drainage, mean white cell counts in BDL and sham groups at Day 52 was 13.57+ 3.84 X lo3 and 9.75 + 0.564 X lo3 cells/mm3 (n.s.), respectively. Day 14 electrophoretic patterns demonstrated elevation of cyz-globulinand -r-globulin fractions in the BDL group with respect to sham controls.
VOL. 41, NO. 2, AUGUST
1986
ROUGHNEEN
ET AL.: IMMUNOSUPPRESSION
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II. Bacteriological Cultures
V. Responseto LPS Mitogen
All cultures of blood and bile taken from study animals showed no evidence of aerobic or anaerobic growth 1, 2, or 7 days following initial culture.
Lymphocyte response to LPS mitogen was not impaired in BDL as compared to sham pair-fed and normal controls at LPS stock solutions of 1:lO (3691 f 1191 vs 4631 + 497 and 3 177 f 546 cpm, respectively); 1:25 (4383 f1062vs2986f811and4244~1611cpm, respectively); and 1:50 dilution concentration of stock solution (3598 + 969 vs 4233 +- 7 and 6304 f 1202 cpm, respectively) (Fig. 2). Following internal biliary drainage response to LPS mitogen at 1:25 concentration showed no difference between BDL, sham, and normal control rats (3968 + 1178 vs 5998 * 1924 and 7228 f 1124 cpm, respectively) (Fig. 3).
III. Mean Weight Gain Weight gain in BDL and sham and sham pair-fed animals following bile duct ligation is shown in Fig. 1. Animals in each group gained weight at a similar rate 7, 14, and 2 1 days following bile duct ligation. IV. Responseto PHA Mitogen Figure 2 demonstratesimpaired lymphocyte responsivenessto T-cell mitogen in BDL animals as compared to sham pair-fed and normal control animals at PHA stock dilutions of 1:lO (2331 + 941 vs 50,146 f 2593 and 56,557 f 16,632 cpm P < 0.001) 1:25 (2352 + 728 vs 38,622 + 1628 and 68,014 k 15,867 cpm P < 0.001) and 1:50 dilutions (3761 + 420 vs 26,102 f 2439 and 22,847 + lo,33 1 cpm P < 0.00 1). One month following internal biliary drainage there was some recovery of lymphocytic responsiveness to PHA in the BDL group as compared with sham and normal controls at 1:10 concentration of stock PHA solution although BDL animals were still lessresponsive to PHA than sham and normal control groups (7039 f 3 187 vs 19,949 f 3440 and 22,071 + 532 cpm, respectively) (Fig. 3).
VZ. In Vitro One-Way Mixed Lymphocyte Reaction The in vitro mixed lymphocyte responseto F344allogeneic antigen was depressedin BDL as compared to sham and normal control animals 3 weeksfollowing bile duct ligation (Table3)(S.I.:0.58f0.07vs2.56~0.7(P~0.02) and 2.59 & 0.6 (P < O.Ol), respectively). Some recovery of responseto F344allogeneic antigen was seen 1 month afer internal biliary drainage in BDL rats with respect to sham and normal control animals (S.I.: 1.3 + 0.41 vs 2.16 + 0.9 and 1.59 f 0.19, respectively) (Table 4). However, recovery was not complete, as A and S.I. values were still less than those seen in the sham and normal control groups. VII. In Vivo Popliteal Lymph Node Assay
300
BDL SHAM A SHAM PAIR FED l
290
1 280 -I
n
0
T
14
7 thy
Post
21
op
FIG. 1. Weight gain in BDL, sham, and sham pair-fed rats following bile duct ligation.
Figures 4, 5, and Tables 5, 6 demonstrate depressedresponsivenessto thymic-dependent antigen (F344lymphoid cells) in vivo following bile duct ligation in comparison with sham and normal control animals (S.I.: 0.87 + 0.1 vs 1.87 f 0.4 (P < 0.04) and 3.14 f 0.4 (P < 0.002), respectively). One month after internal biliary drainage, BDL animals showed improved responsiveness to allogeneic F3u antigen as compared to predrainage responsiveness.(S.I.: BDL vs sham 1.6 f 0.48 vs 3.13 f 1.56 (n.s.); BDL vs normal: 1.6 f 0.48 vs 2.3 f 0.24 (n.s.)). These results also substan-
118
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986 90,000
8000
q Normal
80,000 n=4 mean t S.E
60.000 ? 50,000 F-t - 40,000 0 30,000 20,000 10,000 0 1:lO l
1:25
1:50
1:lO
PHA.
1:25
1:50
LPS ..-
p
sample
-cpm
unstlmuloted
sample
FIG. 2. Lymphocytic responseto PHA and LPS mitogen 2 1 days following bile duct ligation.
structive jaundice have been documented to have a high incidence of systemicendotoxemia [2, 401 presumably due to impaired Kupffer cell function [6]. Little, however, is known of the immunological status of patients with cholestasis [ 11, 13, 14, 261. Studies have suggested impaired cell-mediated immunity in patients with obstructive jaundice and diseases in which intrahepatic cholestasis is a prominent feature [ 11, 13, 14, 261. The exact basis for this impairment in cell-mediated immune mechanisms remains unknown and may be due to a combination of independent factors. Experimental studies have similarly demonstrated impaired reticuloendothelial function in extrahepatic biliary obstruction [20,21]. In addition, studies have suggestedbiliary ob-
tiate those of the in vitro MLR assay in that although improved lymphocytic responsivenessto allogeneic antigen was seenin the BDL group it was still depressedcompared to that of sham and normal control animals. DISCUSSION
Sepsis is a major cause of postoperative mortality and mobidity in patients with obstructive jaundice [ 1, 3, 3 11.The causeof this increasedsusceptibility to infection is not fully understood, but may in part reflect impairment of host immunity. Impaired reticuloendothelial function has been reported in clinical studies of patients with obstructive jaundice [8]. In addition, patients with ob-
25,000
25,000
20,000
20.000 S.E. a Y
F v 4
15000 ’ 1 o.ouo
mean t SE “2% n=3
15000 ’ 10,000
hi
5000
4
0 1
83 = cpm stimulated
-
sample -cpm
1:lO Cont. of PHA unstimubd
0
1:25 ,“s.s.
sompk
FIG. 3. Lymphocytic responseto PHA and LPS mitogen 3 1 days following internal biliary drainage.
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ET AL.: IMMUNOSUPPRESSION
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TABLE 3 IN VITRO MIXED LYMPHOCYTE ASSAY 21 DAYS ARER BILE DUCT LIGATION (BDL) Spontaneous uptake (w-4 Normal
675 3034 7975 643 921
f + + f f
155 190 296 40 168
One-way MLR (cpm)
A (cm)
1459 + 159 8035 -t 309 12211*200 3142?515 1564 + 100
884 5001 4236 2499 643 x=
BDL
1848+ 2399 -t 2177 f 1406 + 1788 f
70 152 238 122 105
1242+ 1257+ 1708k 739* 715*
483* 40 2787-c204 881+- 78 510& 35 897? 41
2.16 2.65 1.53 4.89 1.70
2653 rf: 872**
51 58 79 15 53
x = 2.59 f 0.60*
-606 -1142 -469 -667 -1073 x= -791+
Sham
S.I.
604+ 28 6997 f 907 1750 f 140 2640? 99 16582 67
0.67 0.52 0.78 0.53 0.40 133
x = 0.58 f 0.07
121 4210 869 2130 761 x=
I .25 2.51 1.99 5.18 1.85
1618+725**
x = 2.56 k 0.68**
Note. *P < 0.01, **P < 0.02, compared with BDL group. x = means + standard error. A, Delta value = cpm allogeneic stimulated sample - cpm non-stimulated sample. Stimulation Index = cpm allogeneic stimulated sample/ cpm unstimulated sample. TABLE 4 IN VITRO MIXED LYMPH~CVTE ASSAY 3 1 DAYS AFTER INTERNAL BILIARY DRAINAGE Spontaneous uptake (cm) Normal
One-way MLR Cm-d
392k 75 743 +- 161 257+ 90 701+ 150
628-t 838+ 534& 1079*
67 78 96 159
1007+ 66 1172k 145 1275 f 157 7622 50
2516+ 744? 1390+ 758+
302 80 126 51
A (cpm) 236 95 277 378 x=
BDL
5154* 1770 488+ 26 15292+ 782
247+
1.60 1.13 2.08 1.54 59
x= 1.59kO.19
1509 -428 115 -4 x=
3683 + 126 428 5 26 3879 + 567
S.I.
2985
2.50 0.63 1.09 0.99 420
x= 1.3 eo.41
1471 60 11413
1.40 1.14 3.94
x=4315+3572
x=2.16*0.89 NS
Note. x = means + standard error. A, Delta value = cpm allogeneic stimulated sample - cpm nonstimulated Stimulation Index = cpm allogeneic stimulated sample/cpm unstimulated sample.
sample.
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986
120
L.
Ft.
BILE DUCT LIGATED ANIMALS R=Albgeneic
(F344)
L=S~wxtek
(SD) Stiiulated
Sthwlated
Node Node
FIG. 4. Popliteal lymph node assayin BDL and normal control animals 2 1 days following bile duct ligation demonstrating impaired responsivenessto allogeneic antigen in BDL animals.
L. BILE LIGATED
R. DUCT ANIMALS
R=Allogeneic
(F344)
L=Syngeneic
(SD)
Stimulated Stimulated
Node Node
FIG. 5. Popliteal lymph node assayin BDL and sham animals 2 1 days following bile duct ligation demonstrating impaired responsivenessto allogeneic antigen in BDL animals.
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ET AL.: IMMUNOSUPPRESSION
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CHOLESTASIS
TABLE 5 IN VITRO POPLITEAL LYMPH NODE ASSAY 2 I DAYS FOLLOWING BILE DUCT LIGATION (BDL)
Normal
Allogeneic (mg)
Syngeneic (w)
80.1 51.8 31.6 48.3 59.0 44.8
26.5 11.3 13.0 15.4 28.4 19.9
ok4 53.6 40.5 24.6 32.9 30.6 24.9 x=
BDL
9.6 9.1 11.1 17.0 16.3 9.9
11.3 9.9 8.5 20.2 30.9 12.3
34.6*4.5* -1.7 -0.8 2.6 -3.2 -14.6 -2.4
x = -3.4 + 2.4 Sham
13.4 35.6 12.9 27.0 47.1 17.6
20.6 15.8 20.7 21.1 22.1 12.5
7.2 19.8 52.2 5.9 24.4 5.1 x=
16.7+8.5***
S.I. 3.0 4.6 2.9 3.1 2.1 2.3 x= 3.14+0.4** 0.9 0.9 1.3 0.8 0.5 0.8 x = 0.87 f 0. I 0.7 2.3 3.5 1.3 2.1 1.4 x = 1.87 f 0.4***
Note. *P < 0.001, **P < 0.002, ***P i 0.04, compared with BDL animals. x = means + standard error. A Delta value = wt. allogeneic stimulated node - wt. syngeneic stimulated node. Stimulation Index = wt. allogeneic (Fw) stimulated node/wt. syngeneic (SD) stimulated node.
struction leads to an increased incidence of systemic endotoxemia [2,40] and that bile salt flow into the gastrointestinal tract prevents the absorption of endotoxin from the gut and its subsequent passageto the portal and systemic circulation [22]. Few experimental studies have investigated systemic polymorphonuclear and lymphocyte function in animals with extrahepatic cholestasis [30]. It remains unknown whether the impaired cell-mediated immunity reported in clinical studies of cholestatic liver diseaseis due to liver disease per se or to other associated factors such as infection, malnutrition, malignancy, impaired renal function or the proposed autoimmune basis of certain cholestatic diseases [5, 7, 9, 16, 231. The effect of extrahepatic cholestasis on B-cell function also requires investigation. If impairment of hepatobiliary
function is responsible for the reported compromise of cell-mediated immunity, the basis for this and its potential for reversibility by biliary drainage requires evaluation. Our study, documents severe T-cell dysfunction both in vivo and in vitro in mixed lymphocyte culture and mitogen assays 3 weeks following bile duct ligation. Defective lymphocytic function was seen to be most pronounced in PHA mitogen studies. Results of in vitro mitogen and mixed lymphocytic cultures show good correlation with those obtained for the in vivo popliteal lymph node assayindicating that in vitro assaysare indicative of T-lymphocyte function in vivo. The observed impairment of T-cell function appears to be due to hepatobiliary impairment rather than complicating nutritional, renal, or infective components associated with the in-
122
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986 TABLE 6 IN VIVO POPLITEAL LYMPH NODE ASSAY 3 1 DAYS FOLLOWING INTERNAL BILIARY DRAINAGE
Allogeneic (F3)4stimulated, mg)
Syngeneic (SD stimulated, mg)
Normal
23.5 22.8 38.0 22.0
14.8 9.2 16.8 8.2
8.7 13.6 21.2 13.8 x = 14.3 f 2.6
1.6 2.5 2.3 2.1 x=2.3 kO.24
BDL
15.2 21.0 18.7
11.5 8.5 21.1
3.1 12.5 -2.4 4.6 k4.32
1.3 2.5 0.9 x = 1.6 + 0.48
x= Sham
13.2 33.0 15.5 30.6
9.3 20.0 2.0 18.7
S.I.
A
3.9 13.0 13.5 11.9 x = 10.75f 2.54
1.4 1.5 1.3 1.64 x= 3.13? 1.56 NS
Note. x = means + standard error. A Delta value = wt. allogeneic stimulated node - wt. syngeneic stimulated node. Stimulation Index = wt. allogeneic (FM) stimulated node/wt. syngeneic (SD) stimulated node.
duction of extrahepatic cholestasis. Despite eating identical quantities of food and gaining weight at a rate similar to the pair-fed controls, BDL animals had a coexisting depressedresponse to PHA mitogen when compared to the former. Furthermore, another study at this institution has demonstrated that bile duct ligation does not lead to nutritional depletion in the SD rat model (Gouma et al., unpublished observations). Serum creatinine was marginally elevated in the BDL group as compared with control rats though this elevation is unlikely to indicate a degree of renal impairment sufficient to account for the observed deficiency in cell-mediated immunity in BDL animals. The leukocytosis observed following bile duct ligation was not associatedwith positive blood and bile cultures and therefore, the observed depression of cell-mediated immunity cannot be attributed to infection. The cause of the leukocytosis following bile duct ligation is not understood. It therefore appears that extrahepatic cholestasis per se can lead to depression of cellmediated immunity. This depression of T-cell
function may be due to impaired liver function or due to the lack of bile flow into the intestinal tract. The mechanism of this depression may be a cellular deficit or a circulating inhibitory plasma factor. Pinto et al. demonstrated impaired cell-mediated immunity in bile duct ligated mice in which they described suppression of plaque-forming and rosette-forming cell response following injection of sheep red blood cells (SRBC) and prolongation of survival time of skin allografts [30]. In addition, they cited evidence for an inhibitory substance in the serum of BDL mice. Other authors have proposed inhibitory plasma factors in an attempt to explain the observed T-cell changes reported in cholestasis [ 17, 32, 331. Studies demonstrated impaired responsivenessof human lymphocytes preincubated with unconjugated bilirubin prior to mitogen and mixed lymphocyte assays [32, 331. The concentrations of bilirubin used in these studies were within the range seenin patients with obstructive jaundice. It is interesting to note in our studies that although hyperbilirubinemia was immediately restored to normal levels follow-
ROUGHNEEN ET AL.: IMMUNOSUPPRESSION
ing internal drainage, T-cell responsivenessto PHA mitogen and allogeneic antigen 1 month after internal drainage was still relatively depressedwhen compared to that of sham and normal control animals. If hyperbilirubinemia is inhibitory to T lymphocytes in vivo it would appear to induce a long-lasting cellular deficit rather than one which is immediately reversible following internal biliary drainage. Lipopolysaccharide, the endotoxin of gram-negative bacteria, has also been shown to inhibit lymphocyte function [lo] and endotoxemia has been suggested to be prevalent in patients with obstructive jaundice [8]. (YZglobulin, the levels of which are elevated in obstructive jaundice, has also been documented to suppress T-cell function [29]. In this study (Ye-and -r-globulin levels were found to be elevated in BDL animals as compared with sham controls and may conceivably contribute to impaired T-cell function in this model. Gianni et al. showed that bile salts, when used in concentrations far in excessof those found clinically in obstructive jaundice, were inhibitory to T- and B-lymphocyte function in vitro [ 171.The authors stated that duration of exposure to elevated bile salt levels also may be important in leading to impaired lymphocyte function. Our study has demonstrated, however, that response to T-cell-independent B-cell mitogen is not impaired in cholestatic animals in vitro. The evidence for bile salts being the “inhibitory factor” in extrahepatic cholestasis is therefore limited. Further work is required to determine whether an inhibitory T-cell serum factor exists in obstructive jaundice and, if proven to exist, its nature needs to be characterized. Our results would indicate that the observed T-cell deficit lies, at least in part, at a cellular level; despite three washingswith RPM1 buffer solution and one treatment with chloride buffer, spleen-derived lymphocytes cleansed and free of hyperbilirubinemic serum still showed significant depression of response to T-cell-dependent antigen (PHA) and allogeneic FJd4antigen. This might be explained on the basis of an inhibitory membrane-bound factor, an alteration in the relative number of
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helper and suppressor T cells, impaired macrophage processing of antigen for T-cell presentation or an intrinsic T-cell deficit. Elucidation of the mechanism whereby cell-mediated immunity is impaired in extrahepatic cholestasis requires further investigation and the relative contribution of plasma inhibitory and cellular factors to this phenomenon requires evaluation. In their studies of the primary plaque forming response of BDL mice to thymus-independent antigen (LPS), Pinto et al. reported a response threefold in magnitude of that obtained in control mice [30]. Our studies conflict with these results and show no significant difference between the responses of BDL, sham, and normal controls to LPS at 1:10, 1: 25, and 1:50 dilutions of stock solution suggesting that B-mediated responses are unaffected in the model in vitro. The extent to which B-cell function is affected in vivo in the cholestatic animal model remains to be assessed.As B-cell function is, in part, dependent on adequate T-cell function, it may be that the in vitro assaysperformed to date have failed to reflect an impairment of T-cell-dependent B-cell response which might occur in vivo. Our results demonstrate some recovery of T-cell function in BDL animals following internal drainage though their responsivenessis still less than that of sham and normal controls. Although serum liver function indices return to normal following internal drainage there is evidence to suggest that permanent fibrotic changes occur in the liver following drainage if the original obstructive lesion persists for more than 14 days [ 151.Such residual pathology may account for the observed lymphocytic hyporesponsivenessfollowing drainage. It is of interest that Vane et al. reported that it took up to 6 months for Kupffer cell function to return to normal following internal biliary drainage in the bile duct ligated rabbit model [37]. It appears from their study that restoration of host immunological function takes a prolonged period of time to recover following relief of biliary obstruction. The clinical value of biliary drainage as a preoperative or palliative procedure is at pres-
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ent an area of much investigation and debate [ 18, 27, 281. From an immunological standpoint patients with obstructive jaundice are immunodepressed [ 11, 13, 261 and studies of percutaneous transhepatic external biliary drainage have demonstrated a high incidence of infective complications following this procedure in patients with obstructive jaundice [ 18, 27, 281. Further studies are required to determine whether biliary drainage is clinically useful in improving host immunity and if internal biliary drainage is more advantageous than external drainage in this respect. In summary, experimental biliary obstruction per se leads to impaired in viva and in vitro cell-mediated immunity not due to nutritional or infective factors. This impairment of cell-mediated immunity is partly reversible by internal biliary drainage. Whether the residual impairment of T-cell function is due to irreversible liver damage or due to prolonged T-cell recovery following drainage is undetermined. Humoral immunity does not appear to be affected by biliary obstruction in vitro. A better understanding of the mechanisms producing a deficit in cell-mediated immunity in extrahepatic biliary obstruction and its reversibility by internal drainage techniques may lead to a reduction in the incidence of infective complications associatedwith biliary obstruction. Elucidation of the mechanism of impaired T-cell function in bile duct-ligated animals may enhance our knowledge of impaired cell-mediated immunity in other liver diseases where cholestasis is a prominent feature [ 141. REFERENCES 1. Armstrong, C. P., Dixon, J. M., Taylor, T. V., and Davies, G. C. Surgical experience of deeply jaundiced patients with bile duct obstruction. &it. J. Surg. 71: 234, 1984. 2. Bailey, M. E. Endotoxin, bile saltsand renal function
in obstructive jaundice. &it. J. Surg. 63: 774, 1976. 3. Blarney, S. L., Fearon, K. C. H., Gimour, W. H., Os-
borne, D. H., and Carter, D. C. Prediction of risk in biliary surgery. &it. J. Surg. 70: 535, 1980. 4. Blenkharn, J. I., McPherson, G. A., and Blumgart, L. H. Septic complications of percutaneous transhepatic biliary drainage. Evaluation of a new closed drainage system. Amer. J. Surg. 147: 3 18, 1984. 5. Boulton-Jones, J. M., Vick, R., Cameron, J. S., and
Black, P. J. Immune responsesin uremia. C/in. Nephrol. 1: 30, 1973. 6. Brande, A. I., Carey, F. J., and Zalesky, M. Studies with radioactive endotoxin II. Correlation of physiological effects with distribution of radioactivity in rabbits injected with lethal dosesof E. coli endotoxin labeled with radioactive sodium chromate. J. Clin. Invest. 34: 858, 1955. 7. Casciani, C. U., Di Simone, C., and Bonini, S. Immunological aspects of chronic uremia. Kidney Int. (Suppl.) 8: S-49, 1978. 8. Drivas, G., James, O., and Wandle, N. Study of reticuloendothelial phagocytic capacity in patients with cholestasis.Brit. Med. J. 1: 1568, 1976. 9. Edelman, R. Cell-mediated immune responsein protein calorie malnutrition: A review. In R. M. Suskind (Ed.).Malnutrition and Immune Response.New York Raven Press, 1977. P. 265. 10. Ellner, J. J., and Spagnudo,P. J. Suppressionofantigen and mitogen induced human T lymphocyte DNA synthesis by bacterial lipopolysaccharide: Mediation by monocyte activation and production of prostaglandins. J. Immunol. 123: 2698, 1979. Il. Fargion, S. R., Podda, M., Cappellini, M. D., and Fiorelli, G. Immunita cellulare nelle celestasiintra ed extra-epatriche.Minerva Gastroenterol.22: 26 1, 1976. 12. Ford, W. H., Burr, W., and Simonsen, M. A lymph node weight assayfor the graft versus host activity of rat lymphoid cells. Transplantation 10: 258, 1970. 13 Former, J. G., Kim, D., Hopkins, L., Barrett, M. K., Pinsky, C. M., and Day, N. K. Immunological function in patients with carcinoma of the pancreas.Surg. Gynecol. Obstet. 150: 215, 1980. 14. Fox, R. A., Dudley, F. J., Samuels, M., Milligan, J., and Sherlock, S. Lymphocyte transformation in responseto phytohaemagglutinin in primary biliary cirrhosis:The searchfor a plasma inhibitory factor. Gut 14: 80, 1973.
15. France, D., Gigon, M., Szekely, A. M., and Bismuth, H. Portal hypertension after bile duct obstruction. Arch. Surg. 114: 1064, 1979. 16. Fujimoto, S., Greene, M. I., and Sehon, A. H. Regulation of the immune response to tumor antigens. J. Immunol. 116: 791, 1976. 17. Gianni, L., DiPadova, F., Zuin, M., and Podda, M. Bile acid-inducedinhibition of the lymphoproliferative response to phytohemagglutinin and pokeweed mitogen: An in vitro study. Gastroenterology 78: 231, 1980.
18. Hatfield, A. R. W., Terblanche, J., Faraar, S., Kemott, L., Tobias, R., Girchwood, A. H., Harries-Jones, R., and Marks, I. N. Preoperativeexternal biliary drainage in obstructive jaundice. Lancet 2: 896, 1982. 19. Holman, J. M., Rikkers, L. F., and Moody, F. G. Sepsisin the management of complicated biliary disorders. Amer. J. Surg. 138: 809, 1979. 20. Holman, J. M., and Rikker, L. F. Reticuloendothelial function and biliary obstruction. Curr. Surg. 37: 366, 1980.
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21. Katz, S., Grosfield, J. L., Gross, K., Plager, D. A., Ross,D., Rosenthal, R., Hull, M., and Weber, T. Impaired bacterial clearance and trapping in obstructive jaundice. Ann. Surg. 199: 14, 1984. 22. Koscar, L. T., Bertok, L., and Varteresz, V. Effect of bile acids on the intestinal absorption of endotoxin in rats. J. Bacterial. 100: 220, 1969. 23. Law, D. K., Dudrick, S. J., and Abdou, N. I. Immunocompetence in patients with protein-calorie malnutrition. Ann. Intern. Med. 79: 545, 1973. 24. Lee, E. The effect of obstructive jaundice on the migration of reticuloendothelial cells and fibroblasts into early experimental granulomata. Brit. J. Surg. 59: 875, 1979. 25. Lustgarten, J. A., and Wenk, R. E. Simple, rapid, kinetic method for serum creatinine measurement.Clin. Chem. 18: 1419, 1972. 26. Ma&ore, G., DeGiacomo, C., Scotta, M. S., Siena, S., Macario, R., and Vitiello, A. Cell mediated immunity in children with chronic cholestasis.J. Pediatr. Gastroenterol. Nutr. 1: 385, 1982. 27. McPherson, G. A., Benjamin, I. S., Hodgson, H. J. F., Bowley, N. B., Allison, D. J., and Blumgart, L. H. Preoperative percutaneous transhepatic biliary drainage: The resultsof a controlled trial. Brit. J. Surg. 71: 371, 1984. 28. McPherson, G. A., Benjamin, J. S., Habib, N. A., Bowley, N. B., and Blumgart, L. H. Percutaneous transhepatic drainage in obstructive jaundice. Advantages and problems. Brit. J. Surg. 69: 261, 1982. 29. Mowbray, J. F. Effect of large doses of an alpha 2glycoprotein fraction on the survival of rat skin homografts. Transplantation 1: 15, 1963. 30. Pinto, M., and Kaplun, A. Immune status in mice with experimental biliary obstruction. C/in. Zmmunol. Immmunopathol. 16: 396, 1980.
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3 I. Pitt, H. A., Cameron, J. L., Postier, R. G., and Gadacz, T. R. Factors affecting mortality in biliary tract surgery. Amer. J. Surg. 141: 66, 1981. 32. Rola-Pleszczynski, M., Hensen, S. A., Vincent, M. M., and Bellanti, J. A. Inhibitory effectsof bilirubin on cellular immune responsesin man. J. Pediatr. 86: 690, 1975. 33. Rubaltelli, F. F., Granati, B., Fortunato, A., Piovesan, A., Casacra, G., Colleselli, P., and Semenzato, G. Inhibitory effects of bilirubin and photobilirubin on neonatal and adult T lymphocytes and granulocytes. Biol. Neonate 42: 152, 1982. 34. Ryan, C. J., Than, T., and Blumgart, L. H. Choledochoduodenostomy in the rat with obstructive jaundice. J. Surg. Rex 23: 321, 1977. 35. Strong, D. M., Ahmed, A. A., Thurman, G. B., and Sell, K. W. In vitro stimulation of murine spleen cells using a microculture systemand a multiple automated sample harvester. J. Immunol. Methods 2: 279, 1973. 36. Twist, V. W., and Barnes, R. D. Popliteal lymph node weight gain assay for graft-versus-host reactivity in mice. Transplantation 15: 182, 1973. 37. Vane, D. W., Redlich, P., Weber, T., Leapman, S., Siddiqui, A. R., and Grosfield, J. L. Impaired immune function in obstructive jaundice. J. Surg. Rex (in press). 38. Walters, M. A., and Gerarde, H. W. An ultramicromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchem. J. 15: 23 1, 1970. 39. Wardle, E. N., and Williams, R. Polymorph leucocyte function in uraemia and jaundice. Acta Haematol. 64: 164, 1980. 40. Wardle, E. N., and Wright, N. A. Endotoxin and acute renal failure associatedwith obstructive jaundice. Brit. Med. J. 4: 472, 1970.
OF SURGICAL
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41, 113-125 (1986)
Impaired Specific Cell-Mediated Immunity in Experimental Biliary Obstruction and Its Reversibility by Internal Biliary Drainage’ PATRICK T. ROUGHNEEN, M.B., CH.B., DIRK J. GOUMA, M.D., ANIL D. KULKARNI, WILLIAM F. FANSLOW, B.S., AND BRIAN J. ROWLANDS, M.D.
MS.,
Department of Surgery, The University of Texas Medical School, Houston, Texas 77030
Submitted for publication November 22, 1985 Little is known of the effect of cholestasis on host immunity. This study evaluates lymphocytic responsivenessto PHA and LPS mitogen and to allogeneic FrU antigen in Sprague-Dawley rats 2 1 days following bile duct ligation and 3 1 days following relief of jaundice by internal biliary drainage. Serum bilirubin level was significantly elevated in the bile duct ligated animals at Day 21 (P < 0.001) and thereafter returned to preoperative levels following internal biliary drainage. Resultsdemonstratedepressed responsivenessto PHA (P < 0.00 1) and allogeneic F344antigen in vivo (P < 0.04) and in vitro (P < 0.02) in bile duct ligated animals as compared to sham, sham pair-fed, and normal control rats. The observed deficiency in responsivenessto T-cell-dependent mitogen and antigen cannot be explained on the basis of complicating nutritional, renal, or infective factors. Subsequent internal biliary drainage results in some improvement in T-cell responsivenessin the bile duct ligated group although recovery is not complete. B-Lymphocytic response to LPS mitogen is not affected by bile duct ligation. We conclude that cholestasissubsequentto extrahepatic biliary obstruction per se resultsin impairment of cell-mediated immunity in vivo. This impairment is partly reversible by internal biliary drainage. In vifro B-cell function does not appear to be affected in this model. Further study of impaired cell-mediated immunity in extrahepatic biliary obstruction will improve our understanding of the immunological status of patients with obstructive jaundice and cholestatic liver diseases. 0 1986 Academic Press. Inc.
plications and as a palliative procedure remains controversial [ 18, 27, 281. Clinical Several clinical studies have reported a high studies have demonstrated the efficacy of bilincidence of septic complications following iary drainage in improving serum bilirubin diagnostic and therapeutic procedures in pa- levels and other parameters of liver function tients with obstructive jaundice [ 1, 3, 4, 18, [27, 281 but its role in improving immuno19,27,28,3 11.The causeof this high incidence logical status of the jaundiced patient is unof septic complications is not fully understood, known. but impairment of host defense mechanisms Experimental studies have demonstrated may be implicated. Little is known of the im- impaired reticuloendothelial function in bile munological status of patients with obstructive duct ligated animals [20, 2 l] and this impairjaundice [ 11, 13, 26, 391. Such patients often ment has been confirmed in patients with obhave a malignant tumor, significant malnu- structive jaundice [8]. However, experimental trition, infection, and renal impairment, all of studies of systemic polymorphonuclear leuwhich can compromise host immunity [5, 7, kocyte, macrophage,and lymphocyte function 9, 16, 231. It remains unknown whether cho- in extrahepatic cholestasis are limited [30]. lestatic liver disease alone can result in im- The purpose of this study was to investigate pairment of systemic host immunity. lymphocytic function in the rat model followThe role of preoperative biliary drainage in ing the induction of extrahepatic biliary obreducing the incidence of postoperative com- struction, to document the contribution of nutritional, renal and infective factors to any ’ Presented at the Annual Meeting of the Association alterations observed in lymphocytic function, and to evaluate the effect of internal bilary for Academic Surgery, Cincinnati, Ohio, November lodrainage on T- and B-cell function. 13, 1985. INTRODUCTION
113
0022-4804186$1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Female Sprague-Dawley (SD) rats (Timco Laboratories, Houston, Tex.) weighing 200 to 250 mg were housed in metabolic cagesand subjected to 12-hr alternate light cycles daily. Throughout the study, rats were fed standard rat chow (Formulab 5008) and allowed water ad libitum. Animals were assigned to one of four groups. Group I: Bile duct ligation (BDL) (n = 26); Group II: Sham controls (n = 19); Group III: Sham pair-fed controls (n = 7); Group IV: Normal controls (n = 26). Twentyone days after initial operation, the animals either had a drainage procedure performed or were studied immunologically by assessing lymphocytic responsiveness to mitogen and F3+, allogeneic antigen (Table 1). Thirty-one days following internal or sham drainage animals were again assessedimmunologically. In those animals in which lymphocytic responsivenessto T- and B-cell mitogen was being assessed,sham animals (Group II) were pairfed with the bile duct ligated group to eliminate the possibility of a nutritional factor influencing results.
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was identified and freed from the surrounding tissue only. Internal Biliary Drainage Twenty-one days after division of the common bile duct, the abdomen was reopened under ether anesthesia and the liver was mobilized to the anterior abdominal wall. The dilated bile duct was dissected from the surrounding soft tissue. A Silastic tube (0.03 in i.d. X 0.065 in o.d.) with a Silastic cuff was inserted into the dilated common bile duct and tied in position using two purse string 6-O silk sutures. The Silastic tube was inserted into the duodenum and secured in position with a 60 silk purse string [ 341.In rats having a sham drainage procedure, the bile duct was dissected and a purse string suture tied in the duodenum. Sacrifice of Animals The animals were sacrificed using ether anesthesia. The abdomen was opened, the spleen removed, bathed in RPM1 1640 medium, and immediately processedfor immunological assay.
Bile Duct Ligation
Hematology and Biochemistry
This was performed under ether anesthesia on the first day of the study: a central upper abdominal incision was made, the common bile duct identified, doubly ligated with two 3-O silk ligatures, and divided [24]. High bile duct ligation was performed in the same position in all animals. In those animals undergoing sham operation, the common bile duct
Total white cell count (Coulter counter S method), total serum bilirubin (modified Walters and Gerarde method) [38], and serum creatinine (Jaffee method) [25] were determined at Days 0, 7, 14, 21, 28, 35, 42, and 52. Serum electrophoretic studies (cellulose acetatepH 8.6) were performed on Day 14 of the study.
TABLE
Group Day
Sham
BDL 0
Bile duct ligation
Day 21
Drainage or immunological
Day 52
Immunological
assay
1
Normal control -
Sham operation assay
Sham, drainage, or immunological assay
Immunological
assay
Immunological
Immunologkxl
assay
assay
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was measured utilizing the tritium window in a Beckman LS-8000 liquid scintillation Samples of blood and bile were taken asep counter and results were expressedasthe delta tically from BDL, sham, and normal animals value (A). at the time of sacrifice and innoculated into Mixed lymphocyte response. The one-way 3.5 ml of Schaedlersbroth (BBL, Cockeyville, mixed lymphocyte response using irradiated Md.). Samples of broth were incubated aeroF344 splenic lymphocytes as a source of allobically and anaerobically at 37°C and examgeneic effector cells was used to assaythe reined for bacterial growth 1, 2, and 7 days folsponsiveness of SD splenic lymphocytes. The lowing incubation. purified SD and F344splenic lymphocyte fractions were obtained in an identical manner to In Vitro Lymphocyte Assays that described above. SD splenic lymphocytes Mitogen assay. Rat spleen lymphocyte pro- (2 X 105)were mixed in a microtiter well plate liferative response to T- and B-cell mitogens in triplicate with 2 X lo5 F344allogeneic irrawere tested using a modified method of Strong diated cells (stimulated wells) or without Fjd4 et al. (35). Rat spleens were harvested asepti- spleen cells in a volume of 0.1 ml of complete cally, cut into small pieces, and then teased RPMI. Plateswere incubated for 96 hr at 37°C through loo-mm stainless-steel screens to in a 5% COZhumidified 95% air mixture. The make single cell suspensions in RPM1 1640 cells were then pulse labeled for 16 to 18 hr medium containing pen-strep. The cell sus- with [3H]thymidine in a volume of 50 micropensions were spun at 1200 rpm for 10 min liters. The plates were then harvested on a in a refrigerated centrifuge. Contaminating red Mash II harvester. Disks were transferred to blood cells were removed by treatment with vials containing 5 ml of scintillation cocktail Tris ammonium chloride. The cell suspen- and incorporation of [3H] thymidine was sions were further washedthree times in RPM1 measured on a Beckman LS-8000 counter. 1640 and then finally resuspendedin complete Results were expressedas the stimulation inRPM1 containing 10% fetal bovine serum dex (S.I.) and as the delta value. (FBS), pen strep, L-glutamine, and 0.2 M Hepes. The cell suspensions were then enuIn Vivo Mixed Lymphocyte Reaction merated and the cell concentration adjusted The popliteal lymph node assay was used to 5 X lo6 cells/ml. The cells were dispersed to evaluate T-lymphocyte responsiveness in a microtiter well plate at 5 X lo5 cells/well manifested as a host versus graft response as in a volume of 0.1 ml complete RPMI. Fifty microliters of the mitogen phytohe- described by Twist and Barnes (36). Spraguemagglutinin (PHA) (Gibco) and lipopolysac- Dawley splenic lymphocytes were used as the charide (LPS) (Calbiochem) was added in source of syngeneic antigen and F344splenic triplicate wells at 1:10, 1:25, and 1:50 dilutions lymphocytes as the source of allogeneic of stock solution of PHA and 1 mg/ml LPS antigen. Syngeneic and allogeneic cells were presolution. Volume per well was adjusted to 0.2 ml total by addition of the appropriate amount pared in the manner described above and the of complete RPMI. Microtiter plates were in- enriched lymphocyte fractions obtained. Docubated for 48 hr at 37°C with humidified 5% nor cells were then irradiated at 2500 R to CO2 air mixture. Following incubation, cells eliminate graft versus host reactivity in this assay. The rats were then subjected to ether were pulse labeled with [3H]thymidine (1 &i/ well, 6.7 Ci/mole, sp act) for 16 to 18 hr. The anesthesiaand 25 X lo6 FxU cells were injected cells were then harvested on a Mash II harves- into the right (allogeneic) foot pad and 25 ter. Disks were transferred to vials and 5 ml X lo6 SD cells into the left (syngeneic) foot of scintillation cocktail was added. Incorpo- pad in 100-~1volumes using a 27-gaugeneedle ration of [3H]thymidine into stimulated cells and a 1 cc hypodermic syringe. After 1 week,
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the animals were sacrificed and popliteal lymph nodes were dissected from right and left popliteal fossaeand weighed. The results of the host versusgraft responsewere expressed as the weight of allogeneically stimulated lymph nodes divided by syngeneically stimulated lymph nodes (stimulation index) and as the difference in weight between allogeneic and syngeneic nodes (delta value). Statistical Analysis All study results were expressed as means + SE and subjected to the unpaired Student t test. RESULTS
I. Biochemistry and Hematology Mean bilirubin in BDL vs sham groups was 0.1 f 0 vs 0.1 + 0 mg/dl at the initiation of the study and 6.9 + 0.5 vs 0.1 + 0.02 mg/dl (P < 0.00 1) at 2 1 days, following which internal biliary drainage was performed. At the termination of the study, (Day 52), mean bilirubin was 0.1 + 0 and 0.1 f 0 mg/dl in BDL and sham groups, respectively (Table 2). Mean plasma creatinine in BDL and sham groups was 1.03 + 0.04 and 0.96 + 0.06 mg/dl at Day 0; 1.2 + 0.05 and 1.05 + 0.04 mg/dl at 2 1 days and 0.9 + 0 and 0.89 +- 0.07 mg/dl at 52 days. At 2 1 days, plasma creatinine was significantly raised in BDL animals compared to preoperative levels (P < 0.04) and postdrainage levels (P < 0.03) (Table 2). Mean WCC in the study group at the initiation of the study was 16.5 + 2.2 X lo3 cells/ mm3. Mean WCC in BDL vs sham groups at 21 days was 53.8 + 3.37 X lo3 vs 11.34 + 0.7 15 x lo3 cells mm3 (P < 0.00 1). Following internal biliary drainage, mean white cell counts in BDL and sham groups at Day 52 was 13.57+ 3.84 X lo3 and 9.75 + 0.564 X lo3 cells/mm3 (n.s.), respectively. Day 14 electrophoretic patterns demonstrated elevation of cyz-globulinand -r-globulin fractions in the BDL group with respect to sham controls.
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II. Bacteriological Cultures
V. Responseto LPS Mitogen
All cultures of blood and bile taken from study animals showed no evidence of aerobic or anaerobic growth 1, 2, or 7 days following initial culture.
Lymphocyte response to LPS mitogen was not impaired in BDL as compared to sham pair-fed and normal controls at LPS stock solutions of 1:lO (3691 f 1191 vs 4631 + 497 and 3 177 f 546 cpm, respectively); 1:25 (4383 f1062vs2986f811and4244~1611cpm, respectively); and 1:50 dilution concentration of stock solution (3598 + 969 vs 4233 +- 7 and 6304 f 1202 cpm, respectively) (Fig. 2). Following internal biliary drainage response to LPS mitogen at 1:25 concentration showed no difference between BDL, sham, and normal control rats (3968 + 1178 vs 5998 * 1924 and 7228 f 1124 cpm, respectively) (Fig. 3).
III. Mean Weight Gain Weight gain in BDL and sham and sham pair-fed animals following bile duct ligation is shown in Fig. 1. Animals in each group gained weight at a similar rate 7, 14, and 2 1 days following bile duct ligation. IV. Responseto PHA Mitogen Figure 2 demonstratesimpaired lymphocyte responsivenessto T-cell mitogen in BDL animals as compared to sham pair-fed and normal control animals at PHA stock dilutions of 1:lO (2331 + 941 vs 50,146 f 2593 and 56,557 f 16,632 cpm P < 0.001) 1:25 (2352 + 728 vs 38,622 + 1628 and 68,014 k 15,867 cpm P < 0.001) and 1:50 dilutions (3761 + 420 vs 26,102 f 2439 and 22,847 + lo,33 1 cpm P < 0.00 1). One month following internal biliary drainage there was some recovery of lymphocytic responsiveness to PHA in the BDL group as compared with sham and normal controls at 1:10 concentration of stock PHA solution although BDL animals were still lessresponsive to PHA than sham and normal control groups (7039 f 3 187 vs 19,949 f 3440 and 22,071 + 532 cpm, respectively) (Fig. 3).
VZ. In Vitro One-Way Mixed Lymphocyte Reaction The in vitro mixed lymphocyte responseto F344allogeneic antigen was depressedin BDL as compared to sham and normal control animals 3 weeksfollowing bile duct ligation (Table3)(S.I.:0.58f0.07vs2.56~0.7(P~0.02) and 2.59 & 0.6 (P < O.Ol), respectively). Some recovery of responseto F344allogeneic antigen was seen 1 month afer internal biliary drainage in BDL rats with respect to sham and normal control animals (S.I.: 1.3 + 0.41 vs 2.16 + 0.9 and 1.59 f 0.19, respectively) (Table 4). However, recovery was not complete, as A and S.I. values were still less than those seen in the sham and normal control groups. VII. In Vivo Popliteal Lymph Node Assay
300
BDL SHAM A SHAM PAIR FED l
290
1 280 -I
n
0
T
14
7 thy
Post
21
op
FIG. 1. Weight gain in BDL, sham, and sham pair-fed rats following bile duct ligation.
Figures 4, 5, and Tables 5, 6 demonstrate depressedresponsivenessto thymic-dependent antigen (F344lymphoid cells) in vivo following bile duct ligation in comparison with sham and normal control animals (S.I.: 0.87 + 0.1 vs 1.87 f 0.4 (P < 0.04) and 3.14 f 0.4 (P < 0.002), respectively). One month after internal biliary drainage, BDL animals showed improved responsiveness to allogeneic F3u antigen as compared to predrainage responsiveness.(S.I.: BDL vs sham 1.6 f 0.48 vs 3.13 f 1.56 (n.s.); BDL vs normal: 1.6 f 0.48 vs 2.3 f 0.24 (n.s.)). These results also substan-
118
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986 90,000
8000
q Normal
80,000 n=4 mean t S.E
60.000 ? 50,000 F-t - 40,000 0 30,000 20,000 10,000 0 1:lO l
1:25
1:50
1:lO
PHA.
1:25
1:50
LPS ..-
p
sample
-cpm
unstlmuloted
sample
FIG. 2. Lymphocytic responseto PHA and LPS mitogen 2 1 days following bile duct ligation.
structive jaundice have been documented to have a high incidence of systemicendotoxemia [2, 401 presumably due to impaired Kupffer cell function [6]. Little, however, is known of the immunological status of patients with cholestasis [ 11, 13, 14, 261. Studies have suggested impaired cell-mediated immunity in patients with obstructive jaundice and diseases in which intrahepatic cholestasis is a prominent feature [ 11, 13, 14, 261. The exact basis for this impairment in cell-mediated immune mechanisms remains unknown and may be due to a combination of independent factors. Experimental studies have similarly demonstrated impaired reticuloendothelial function in extrahepatic biliary obstruction [20,21]. In addition, studies have suggestedbiliary ob-
tiate those of the in vitro MLR assay in that although improved lymphocytic responsivenessto allogeneic antigen was seenin the BDL group it was still depressedcompared to that of sham and normal control animals. DISCUSSION
Sepsis is a major cause of postoperative mortality and mobidity in patients with obstructive jaundice [ 1, 3, 3 11.The causeof this increasedsusceptibility to infection is not fully understood, but may in part reflect impairment of host immunity. Impaired reticuloendothelial function has been reported in clinical studies of patients with obstructive jaundice [8]. In addition, patients with ob-
25,000
25,000
20,000
20.000 S.E. a Y
F v 4
15000 ’ 1 o.ouo
mean t SE “2% n=3
15000 ’ 10,000
hi
5000
4
0 1
83 = cpm stimulated
-
sample -cpm
1:lO Cont. of PHA unstimubd
0
1:25 ,“s.s.
sompk
FIG. 3. Lymphocytic responseto PHA and LPS mitogen 3 1 days following internal biliary drainage.
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TABLE 3 IN VITRO MIXED LYMPHOCYTE ASSAY 21 DAYS ARER BILE DUCT LIGATION (BDL) Spontaneous uptake (w-4 Normal
675 3034 7975 643 921
f + + f f
155 190 296 40 168
One-way MLR (cpm)
A (cm)
1459 + 159 8035 -t 309 12211*200 3142?515 1564 + 100
884 5001 4236 2499 643 x=
BDL
1848+ 2399 -t 2177 f 1406 + 1788 f
70 152 238 122 105
1242+ 1257+ 1708k 739* 715*
483* 40 2787-c204 881+- 78 510& 35 897? 41
2.16 2.65 1.53 4.89 1.70
2653 rf: 872**
51 58 79 15 53
x = 2.59 f 0.60*
-606 -1142 -469 -667 -1073 x= -791+
Sham
S.I.
604+ 28 6997 f 907 1750 f 140 2640? 99 16582 67
0.67 0.52 0.78 0.53 0.40 133
x = 0.58 f 0.07
121 4210 869 2130 761 x=
I .25 2.51 1.99 5.18 1.85
1618+725**
x = 2.56 k 0.68**
Note. *P < 0.01, **P < 0.02, compared with BDL group. x = means + standard error. A, Delta value = cpm allogeneic stimulated sample - cpm non-stimulated sample. Stimulation Index = cpm allogeneic stimulated sample/ cpm unstimulated sample. TABLE 4 IN VITRO MIXED LYMPH~CVTE ASSAY 3 1 DAYS AFTER INTERNAL BILIARY DRAINAGE Spontaneous uptake (cm) Normal
One-way MLR Cm-d
392k 75 743 +- 161 257+ 90 701+ 150
628-t 838+ 534& 1079*
67 78 96 159
1007+ 66 1172k 145 1275 f 157 7622 50
2516+ 744? 1390+ 758+
302 80 126 51
A (cpm) 236 95 277 378 x=
BDL
5154* 1770 488+ 26 15292+ 782
247+
1.60 1.13 2.08 1.54 59
x= 1.59kO.19
1509 -428 115 -4 x=
3683 + 126 428 5 26 3879 + 567
S.I.
2985
2.50 0.63 1.09 0.99 420
x= 1.3 eo.41
1471 60 11413
1.40 1.14 3.94
x=4315+3572
x=2.16*0.89 NS
Note. x = means + standard error. A, Delta value = cpm allogeneic stimulated sample - cpm nonstimulated Stimulation Index = cpm allogeneic stimulated sample/cpm unstimulated sample.
sample.
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986
120
L.
Ft.
BILE DUCT LIGATED ANIMALS R=Albgeneic
(F344)
L=S~wxtek
(SD) Stiiulated
Sthwlated
Node Node
FIG. 4. Popliteal lymph node assayin BDL and normal control animals 2 1 days following bile duct ligation demonstrating impaired responsivenessto allogeneic antigen in BDL animals.
L. BILE LIGATED
R. DUCT ANIMALS
R=Allogeneic
(F344)
L=Syngeneic
(SD)
Stimulated Stimulated
Node Node
FIG. 5. Popliteal lymph node assayin BDL and sham animals 2 1 days following bile duct ligation demonstrating impaired responsivenessto allogeneic antigen in BDL animals.
ROUGHNEEN
ET AL.: IMMUNOSUPPRESSION
IN EXTRAHEPATIC
121
CHOLESTASIS
TABLE 5 IN VITRO POPLITEAL LYMPH NODE ASSAY 2 I DAYS FOLLOWING BILE DUCT LIGATION (BDL)
Normal
Allogeneic (mg)
Syngeneic (w)
80.1 51.8 31.6 48.3 59.0 44.8
26.5 11.3 13.0 15.4 28.4 19.9
ok4 53.6 40.5 24.6 32.9 30.6 24.9 x=
BDL
9.6 9.1 11.1 17.0 16.3 9.9
11.3 9.9 8.5 20.2 30.9 12.3
34.6*4.5* -1.7 -0.8 2.6 -3.2 -14.6 -2.4
x = -3.4 + 2.4 Sham
13.4 35.6 12.9 27.0 47.1 17.6
20.6 15.8 20.7 21.1 22.1 12.5
7.2 19.8 52.2 5.9 24.4 5.1 x=
16.7+8.5***
S.I. 3.0 4.6 2.9 3.1 2.1 2.3 x= 3.14+0.4** 0.9 0.9 1.3 0.8 0.5 0.8 x = 0.87 f 0. I 0.7 2.3 3.5 1.3 2.1 1.4 x = 1.87 f 0.4***
Note. *P < 0.001, **P < 0.002, ***P i 0.04, compared with BDL animals. x = means + standard error. A Delta value = wt. allogeneic stimulated node - wt. syngeneic stimulated node. Stimulation Index = wt. allogeneic (Fw) stimulated node/wt. syngeneic (SD) stimulated node.
struction leads to an increased incidence of systemic endotoxemia [2,40] and that bile salt flow into the gastrointestinal tract prevents the absorption of endotoxin from the gut and its subsequent passageto the portal and systemic circulation [22]. Few experimental studies have investigated systemic polymorphonuclear and lymphocyte function in animals with extrahepatic cholestasis [30]. It remains unknown whether the impaired cell-mediated immunity reported in clinical studies of cholestatic liver diseaseis due to liver disease per se or to other associated factors such as infection, malnutrition, malignancy, impaired renal function or the proposed autoimmune basis of certain cholestatic diseases [5, 7, 9, 16, 231. The effect of extrahepatic cholestasis on B-cell function also requires investigation. If impairment of hepatobiliary
function is responsible for the reported compromise of cell-mediated immunity, the basis for this and its potential for reversibility by biliary drainage requires evaluation. Our study, documents severe T-cell dysfunction both in vivo and in vitro in mixed lymphocyte culture and mitogen assays 3 weeks following bile duct ligation. Defective lymphocytic function was seen to be most pronounced in PHA mitogen studies. Results of in vitro mitogen and mixed lymphocytic cultures show good correlation with those obtained for the in vivo popliteal lymph node assayindicating that in vitro assaysare indicative of T-lymphocyte function in vivo. The observed impairment of T-cell function appears to be due to hepatobiliary impairment rather than complicating nutritional, renal, or infective components associated with the in-
122
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986 TABLE 6 IN VIVO POPLITEAL LYMPH NODE ASSAY 3 1 DAYS FOLLOWING INTERNAL BILIARY DRAINAGE
Allogeneic (F3)4stimulated, mg)
Syngeneic (SD stimulated, mg)
Normal
23.5 22.8 38.0 22.0
14.8 9.2 16.8 8.2
8.7 13.6 21.2 13.8 x = 14.3 f 2.6
1.6 2.5 2.3 2.1 x=2.3 kO.24
BDL
15.2 21.0 18.7
11.5 8.5 21.1
3.1 12.5 -2.4 4.6 k4.32
1.3 2.5 0.9 x = 1.6 + 0.48
x= Sham
13.2 33.0 15.5 30.6
9.3 20.0 2.0 18.7
S.I.
A
3.9 13.0 13.5 11.9 x = 10.75f 2.54
1.4 1.5 1.3 1.64 x= 3.13? 1.56 NS
Note. x = means + standard error. A Delta value = wt. allogeneic stimulated node - wt. syngeneic stimulated node. Stimulation Index = wt. allogeneic (FM) stimulated node/wt. syngeneic (SD) stimulated node.
duction of extrahepatic cholestasis. Despite eating identical quantities of food and gaining weight at a rate similar to the pair-fed controls, BDL animals had a coexisting depressedresponse to PHA mitogen when compared to the former. Furthermore, another study at this institution has demonstrated that bile duct ligation does not lead to nutritional depletion in the SD rat model (Gouma et al., unpublished observations). Serum creatinine was marginally elevated in the BDL group as compared with control rats though this elevation is unlikely to indicate a degree of renal impairment sufficient to account for the observed deficiency in cell-mediated immunity in BDL animals. The leukocytosis observed following bile duct ligation was not associatedwith positive blood and bile cultures and therefore, the observed depression of cell-mediated immunity cannot be attributed to infection. The cause of the leukocytosis following bile duct ligation is not understood. It therefore appears that extrahepatic cholestasis per se can lead to depression of cellmediated immunity. This depression of T-cell
function may be due to impaired liver function or due to the lack of bile flow into the intestinal tract. The mechanism of this depression may be a cellular deficit or a circulating inhibitory plasma factor. Pinto et al. demonstrated impaired cell-mediated immunity in bile duct ligated mice in which they described suppression of plaque-forming and rosette-forming cell response following injection of sheep red blood cells (SRBC) and prolongation of survival time of skin allografts [30]. In addition, they cited evidence for an inhibitory substance in the serum of BDL mice. Other authors have proposed inhibitory plasma factors in an attempt to explain the observed T-cell changes reported in cholestasis [ 17, 32, 331. Studies demonstrated impaired responsivenessof human lymphocytes preincubated with unconjugated bilirubin prior to mitogen and mixed lymphocyte assays [32, 331. The concentrations of bilirubin used in these studies were within the range seenin patients with obstructive jaundice. It is interesting to note in our studies that although hyperbilirubinemia was immediately restored to normal levels follow-
ROUGHNEEN ET AL.: IMMUNOSUPPRESSION
ing internal drainage, T-cell responsivenessto PHA mitogen and allogeneic antigen 1 month after internal drainage was still relatively depressedwhen compared to that of sham and normal control animals. If hyperbilirubinemia is inhibitory to T lymphocytes in vivo it would appear to induce a long-lasting cellular deficit rather than one which is immediately reversible following internal biliary drainage. Lipopolysaccharide, the endotoxin of gram-negative bacteria, has also been shown to inhibit lymphocyte function [lo] and endotoxemia has been suggested to be prevalent in patients with obstructive jaundice [8]. (YZglobulin, the levels of which are elevated in obstructive jaundice, has also been documented to suppress T-cell function [29]. In this study (Ye-and -r-globulin levels were found to be elevated in BDL animals as compared with sham controls and may conceivably contribute to impaired T-cell function in this model. Gianni et al. showed that bile salts, when used in concentrations far in excessof those found clinically in obstructive jaundice, were inhibitory to T- and B-lymphocyte function in vitro [ 171.The authors stated that duration of exposure to elevated bile salt levels also may be important in leading to impaired lymphocyte function. Our study has demonstrated, however, that response to T-cell-independent B-cell mitogen is not impaired in cholestatic animals in vitro. The evidence for bile salts being the “inhibitory factor” in extrahepatic cholestasis is therefore limited. Further work is required to determine whether an inhibitory T-cell serum factor exists in obstructive jaundice and, if proven to exist, its nature needs to be characterized. Our results would indicate that the observed T-cell deficit lies, at least in part, at a cellular level; despite three washingswith RPM1 buffer solution and one treatment with chloride buffer, spleen-derived lymphocytes cleansed and free of hyperbilirubinemic serum still showed significant depression of response to T-cell-dependent antigen (PHA) and allogeneic FJd4antigen. This might be explained on the basis of an inhibitory membrane-bound factor, an alteration in the relative number of
IN EXTRAHEPATIC
CHOLESTASIS
123
helper and suppressor T cells, impaired macrophage processing of antigen for T-cell presentation or an intrinsic T-cell deficit. Elucidation of the mechanism whereby cell-mediated immunity is impaired in extrahepatic cholestasis requires further investigation and the relative contribution of plasma inhibitory and cellular factors to this phenomenon requires evaluation. In their studies of the primary plaque forming response of BDL mice to thymus-independent antigen (LPS), Pinto et al. reported a response threefold in magnitude of that obtained in control mice [30]. Our studies conflict with these results and show no significant difference between the responses of BDL, sham, and normal controls to LPS at 1:10, 1: 25, and 1:50 dilutions of stock solution suggesting that B-mediated responses are unaffected in the model in vitro. The extent to which B-cell function is affected in vivo in the cholestatic animal model remains to be assessed.As B-cell function is, in part, dependent on adequate T-cell function, it may be that the in vitro assaysperformed to date have failed to reflect an impairment of T-cell-dependent B-cell response which might occur in vivo. Our results demonstrate some recovery of T-cell function in BDL animals following internal drainage though their responsivenessis still less than that of sham and normal controls. Although serum liver function indices return to normal following internal drainage there is evidence to suggest that permanent fibrotic changes occur in the liver following drainage if the original obstructive lesion persists for more than 14 days [ 151.Such residual pathology may account for the observed lymphocytic hyporesponsivenessfollowing drainage. It is of interest that Vane et al. reported that it took up to 6 months for Kupffer cell function to return to normal following internal biliary drainage in the bile duct ligated rabbit model [37]. It appears from their study that restoration of host immunological function takes a prolonged period of time to recover following relief of biliary obstruction. The clinical value of biliary drainage as a preoperative or palliative procedure is at pres-
124
JOURNAL OF SURGICAL RESEARCH: VOL. 41, NO. 2, AUGUST 1986
ent an area of much investigation and debate [ 18, 27, 281. From an immunological standpoint patients with obstructive jaundice are immunodepressed [ 11, 13, 261 and studies of percutaneous transhepatic external biliary drainage have demonstrated a high incidence of infective complications following this procedure in patients with obstructive jaundice [ 18, 27, 281. Further studies are required to determine whether biliary drainage is clinically useful in improving host immunity and if internal biliary drainage is more advantageous than external drainage in this respect. In summary, experimental biliary obstruction per se leads to impaired in viva and in vitro cell-mediated immunity not due to nutritional or infective factors. This impairment of cell-mediated immunity is partly reversible by internal biliary drainage. Whether the residual impairment of T-cell function is due to irreversible liver damage or due to prolonged T-cell recovery following drainage is undetermined. Humoral immunity does not appear to be affected by biliary obstruction in vitro. A better understanding of the mechanisms producing a deficit in cell-mediated immunity in extrahepatic biliary obstruction and its reversibility by internal drainage techniques may lead to a reduction in the incidence of infective complications associatedwith biliary obstruction. Elucidation of the mechanism of impaired T-cell function in bile duct-ligated animals may enhance our knowledge of impaired cell-mediated immunity in other liver diseases where cholestasis is a prominent feature [ 141. REFERENCES 1. Armstrong, C. P., Dixon, J. M., Taylor, T. V., and Davies, G. C. Surgical experience of deeply jaundiced patients with bile duct obstruction. &it. J. Surg. 71: 234, 1984. 2. Bailey, M. E. Endotoxin, bile saltsand renal function
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