THE YALE JOURNAL OF BIOLOGY AND MEDICINE 48,47-54 (1975)
Some Immunological Aspects of Liver Function DAVID R. TRIGER' University ofSouthampton Medical School, Southampton, Hampshire, England Received October 7, 1974
Although many aspects of hepatic function have been known and studied for a number of years, little is known about the immunological function of the liver. Chronic liver disease is often accompanied by an absolute increase in serum gamma globulin concentration. All the major immunoglobulin classes may be involved, and the elevated globulin is due to increased synthesis rather than decreased metabolism (1). In this review mechanisms are examined whereby the normal liver may modify the immune response to antigens, and the damaged liver may lead to increased immunoglobulin synthesis. EXPERIMENTAL STUDIES In 1946, Chase (2) showed that guinea pigs could be rendered tolerant to intramuscular immunization with dinitrochlorobenzene (DNCB) by prior feeding with hapten. This phenomenon, subsequently named the Chase-Sulzberger effect, has been confirmed using other haptens, and tolerance may even be induced by a single feeding prior to intramuscular immunization (3). The possible role of the liver was first suggested by Cantor and Dumont (4). They examined the antibody response to intramuscular DNCB in dogs, and found that this was substantially reduced in animals fed with the hapten prior to immunization, and that this reduction was abolished if the animals were subjected to portacaval shunting before the start of the experiment. Interpretation of such feeding experiments is limited in that the rate and quantity of antigen absorption cannot be assessed. We (5) have studied the effect of the liver on the immune response in the rat by injecting sheep red blood cells directly into the portal vein and comparing the antibody response obtained with an equivalent injection via the inferior vena cava in control animals. Whereas the antibody response to a single dose of antigen was similar by either route, repeated portal venous injections of antigen produced a significantly lower antibody response than did repeated inferior vena cava injections (Table 1). The dose of antigen administered was critical; too little antigen failed to evoke a sufficient antibody response by either route for a difference to be detectable, while an excessive amount of antigen produced a large antibody response by either route of administration. Radio-labeling of the antigen with 5"Cr showed that in the nonimmunized animal the percentage uptake by the liver after a single injection was the same regardless of route of administration. By contrast, although repeated portal vein immunization resulted in the same percentage uptake by the liver, repeated inferior vena cava immunization resulted in decreased uptake by the liver. Delayed hypersensitivity was also examined in these animals by measuring the increase in ear thickness after intraauricular injection of sheep red cells. Rats immunized repeatedly via the inferior vena cava showed evidence of delayed hypersensitivity, while the animals immunized via the portal vein did not. In the 'Lecturer In Medicine.
47 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
48
DAVID R. TRIGER
TABLE I Primary, Secondary and Tertiary Immune Response to Inferior Vena Cava and Portal Vein Injection of 106 Sheep Red Cells (Haemagglutinating Antibody)
Lo92
Haemagglutinating Antibody Titre
6
4
fr
S-RBC I.V.C l10 S-RBC PV.
83
01~~~~~~
3
0
/2
1
2
4
2
~
6
4
4
85
TIME ( Days )
same animal model, damage to the liver by carbon tetrachloride led to an increased antibody response to sheep red cells, a fall in the percentage of labeled antigen taken up by the liver, and abolition of the difference in antibody response to repeated immunization between the two routes (6). An enhanced antibody response to sheep red cells after reduction in the amount of antigen taken up by the liver has also been produced by Souhami (7) by blockading the reticuloendothelial cells of the liver with colloidal carbon, although in this study the effect of immunization via the portal vein was not examined. These findings are consistent with the
theory that the intact liver is able to modify the immune response to exogenous antigens by sequestering them and rendering then nonimmunogenic and even, under certain circumstances, inducing tolerance to these antigens. Such a hypothesis presupposes that the hepatic macrophages-the Kupffer cells-behave differently from other macrophages in that antigens taken up by them lose their immunogenicity. Franzl (8) has shown that, whereas subcellular fractions of spleen containing antigen retain significant antigenicity for several days after the uptake of antigen, comparable liver fractions lost all detectable antigenic properties within 12 hr. As well as being able to sequester antigens, the liver may also influence the immune response by removing antigen/antibody complexes from the circulation. Thomas and colleagues (9) have demonstrated this in the experimental animal using 12mibovine serum albumin, and in addition they have shown that damage to the liver significantly reduces the ability to take up such complexes. CLINICAL OBSERVATIONS These animal studies may be relevant to observations in human liver disease. The elevated immunoglobulin levels found in chronic liver disease are due to increased synthesis and thus reflect increased antibody production. Since this may occur in the absence ofactivinse nsitdamage (10) the increased antibody production may reflect an abnormal response to exogenous antigens.
IMMUNOLOGY AND LIVER FUNCTION
49
SEQUESTRATION OF ANTIGENS BY THE LIVER Antigens from the intestinal microbial flora comprise the largest source of exogenous antigen (1 1), and the liver is uniquely situated in that it receives a large proportion of the systemic blood flow in addition to the entire portal venous system. Thus, antigens absorbed from the gastrointestinal tract must pass through the liver before they reach the antibody-producing cells in the spleen and elsewhere in the reticuloendothelial system. If the liver is damaged and the functional capacity of the Kupffer cells impaired, it is logical to expect that the antigens in the portal venous system would fail to be sequestered by the liver. As a result, increased amounts of antigen would become available to the antibody-producing cells in the spleen and other parts of the reticuloendothelial system, with consequent increased antibody production. Studies (12) of antibody titers to E. coli, and Bacteroides in liver disease support this. Antibody titers to both of these gastrointestinal tract organisms are significantly elevated in patients with chronic liver disease, in contrast to nongastrointestinal tract organisms such as Haemophilus influenzae B. Serial studies in acute hepatitis have shown that antibody titers to E. coli rise significantly some weeks after the onset of hepatitis, falling gradually to normal several months after the resolution of the liver disease. The transient loss of function of the reticuloendothelial cells of the liver may be due either to the frank destruction of the Kupffer cells accompanying the hepatitis or to a saturation of the phagocytic properties of these cells by the breakdown products of the damaged tissue. In addition to loss of functional capacity of the liver cells, a second mechanism is likely to contribute to the bypassing of the liver. Portal hypertension with the establishment of a collateral circulation is a frequent finding in many cases of chronic liver disease. Under these circumstances, antigen absorbed into the portal blood would reach the systemic circulation after bypassing the liver by way of the collateral blood vessels. This mechanism could play a significant role even in the presence of normal liver cell function. Rats with intact livers subjected to portacaval shunting have been shown to develop hyperglobulinemia and increased antibody titers to E. coli (13). Indirect evidence in support of this hypothesis is provided by the retrospective observations of Bj0rneboe and colleagues (14) that patients with portacaval anastomoses have higher antibody titers to E. coli than do those without a shunt. We2 have measured E. coli antibody titers in sera obtained from patients with cirrhosis and portal hypertension both before and at varying intervals after prophylactic portacaval anastomosis. A comparable group of patients who did not undergo portacaval anastomosis served as controls. Measurements were made using the method described previously (12). The results of the study are shown in Table 2. It will be seen that the mean E. coli antibody titer in the sera of 21 patients examined 26 mo after portacaval anastomosis is no higher than the presurgery sera. The control group sera showed titers of similar magnitude. This would suggest that the creation of a portacaval shunt has no effect on the E. coli antibody titer. It should be remembered, however, that patients with cirrhosis and portal hypertension already have severe liver damage and also a preexisting collateral circulation, thus providing two factors to encourage bypassing of the liver by antigen. Thus, the surgical creation of a portacaval shunt in these patients is unlikely to affect this 2Study performed in collaboration with Dr. H. 0. Conn, Veterans Administration Hospital, West Haven, CN.
50
DAVID R. TRIGER TABLE 2 Effect of Portacaval Anastomosis on E. coli Antibody Titers in Patients with Cirrhosis of the Liver
No. of patients studied Mean interval between 1st and 2nd serum (mo) Mean E. coli antibody titer (log2) in 1st sample Mean E. coli antibody titer (log2) in 2nd sample No. of sera showing elevated E. coli antibody titer in 1 st samplea
Cirrhosis without portacaval anastomosis 18 24 (1-97) 5.3 5.4 6/18
Cirrhosis with portacaval anastomosis 21 26 (2-71) 5.0 4.9 7/21
aE. coli antibody titer >1/32 (log25) taken as being elevated (12). situation significantly. The observation that one third of the patients have elevated E. coli antibody titers in the first sample studied lends support to this explanation. A major assumption used in interpreting these data is that bacterial antigens are absorbed from the gastrointestinal tract into the portal blood, rather than into the lymphatic system. Absorption via the latter route would bypass the liver under any circumstances, and so damage to the liver would have little or no effect. Warshaw et al. (15) have been able to study the uptake of labeled macromolecules in cannulated lymphatics and branches of the portal vein from isolated loops of bowel. These studies have shown that significant quantities of bovine serum albumin are transported directly to the lymphatic system, and this may be why antibody titers to albumin are not increased in patients with liver disease (12). Grossly elevated antibody titers to measles, rubella, and cytomegalic inclusion virus have been found in patients with chronic active hepatitis (16, 17). This increase in antibody titer appears to be highly selective, since antibodies to Herpes simplex, varicella-zoster, para-influenza 1, Coxsackie B1 and B5 and Mycoplasma pneumoniae are all within normal limits. Antibody titers to all these viruses were normal in other types of chronic liver disease, except the cytomegalovirus, to which elevated titers have been found in patients with alcoholic cirrhosis and primary biliary cirrhosis. Increased antibody titers have also been reported to some viruses in acute viral hepatitis. Alwen (18) has shown an increase in neutralizing antibody titers to adenovirus type 5 in acute hepatitis A infection, while Baals and colleagues (19) found increased titers to cytomegalovirus in patients with hepatitis, particularly those associated with the Hepatitis Bs antigen. Table 3 summarizes the elevated antibody titers to microbial and dietary protein antibodies hitherto reported in liver disease. Why should such striking elevations in viral antibody titers occur in chronic active hepatitis? The explanation here must be different from that postulated for bacterial antibodies, since none of these viruses is usually associated with the gastrointestinal tract. Increases in antibody titers to measles and rubella have been reported in systemic lupus erythematosis (22), and it has been suggested that this may be part of a phenomenon of nonspecific hyperreactivity, or that it might be associated with the finding of virus-like particles in renal biopsy material in lupus nephritis (23). The magnitude of the increase in antibody titers in chronic active hepatitis is much greater than that found in the connective tissue disorders. Normal antibody titers
51
IMMUNOLOGY AND LIVER FUNCTION TABLE 3 Elevated Microbial and Dietary Protein Antibody Titers Found in Liver Diseasea
Antigen
Acute hepatitis
Salmonella (14,20) E. coli(12,14) Bacteroides (12) Measles (16) Rubella (16) Cytomegalovirus (16,19) Adenovirus (18) Toxoplasma gondii (21) Gluten fraction III (12) o&Lactalbumin (12) Colon antigen (12) a+ Significantly increased titers; - No increase
Cirrhosis
0
-
+
+
+
+
-
-
+ +
+
+
0 + +
-
+ -
0
Chronic active hepatitis + + + + + + 0
0 +
in antibody titers; 0 Not examined.
to cytomegalovirus have been reported in SLE, and measles, rubella, and cytomegalovirus do not appear to show any common antigenic determinants. A possible explanation for these findings may lie in the fact that infection with these three viruses is associated with a viremia, after which a low level of circulating antibody persists for life, possibly a response to repeated release of small quantities of viral antigen (16). Since the Kupffer cells are known to be capable of sequestering viral antigens (24), the intact liver could play a significant role in regulating the amount of circulating antigen. In chronic active hepatitis the macrophage activity might be so depressed that the ability to sequester these antigens is lost and the viral antigens would then become available to antibody-producing cells elsewhere with a consequent increase in antibody production. Alternatively, the viral antigens (but not necessarily the intact viruses themselves) may be sequestered in the liver macrophages and be rendered temporarily nonimmunogenic, only to be released after the liver damage which occurs in chronic active hepatitis. In contrast, infection with the other viruses studied is rarely accompanied by viremia, and although viruses such as Herpes simplex and Varicella-zoster may persist for many years, antibodies are likely to be produced in the lymph close to the virus. In such circumstances, viral antigenemia is unlikely to occur and hence damage to the liver would not affect the mechanism of antibody production. OTHER MECHANISMS FOR ENHANCED ANTIBODY PRODUCTION Although this discussion has related the increased antibody response induced by exogenous antigens in liver disease to a failure of the sequestering properties of the Kupifer cells, other mechanisms may also contribute. Adjuvant Effect of Liver Tissue The breakdown products of liver cell necrosis, such as nucleic acids, can be shown experimentally to produce an adjuvant effect (25). Such a phenomenon might explain some of the increased antibody titers which have been observed in active liver necrosis, but it is unlikely to account for the persistent hyperglobulinemia often found in established inactive hepatic cirrhosis.
52
DAVID R. TRIGER
A utoantigens There is considerable evidence (26) to suggest that antigens are produced as a direct result of liver damage, and that these may provide a stimulus to the production of antibodies, although the extent of their contribution to antibody formation in human liver disease is uncertain. Autoantibodies are commonly found in the serum of patients with both acute and chronic liver disease, but they are neither organspecific nor species-specific. They may occur in the absence of overt liver disease, and the development of clinical liver disease has been observed in such patients without any alteration in the autoantibody titers (16). Patients with chronic active hepatitis commonly have strongly positive antinuclear and antismooth muslce antibodies and we have found (17) a very significant correlation between the elevated measles and rubella antibody titers and the strongly positive autoantibodies. It is possible that the autoantibodies may represent markers for potential immunological disturbance such that damage to the liver in these people may lead to a loss of the functional ability of the Kupffer cells to sequester these antigens.
Decreased Immunological Surveillance due to T-Cell Deficiency with Normal or Enhanced B-Cell Function Cooperation between T and B lymphocytes is a crucial factor in the production of antibodies and there is evidence (27) to suggest that under certain circumstances in which T-cell function is altered, the normal regulatory mechanisms may be overcome with the production of an autoimmune response. Depressed cell-mediated immunity is known to occur in many forms of liver disease (28), and the increased antibody production may reflect a compensatory B-cell overaction to a wide variety of antigenic stimuli not necessarily related to the pathogenesis (10). This interrelationship between T- and B-cell function has been demonstrated for a number of antigens (29) in animal models, but if it is applicable to clinical liver disease, the selectivity of the exaggerated B-cell response has still to be explained. ROLE OF ENDOTOXIN Endotoxin is largely derived from the gram-negative bacilli in the gastrointestinal tract, and after absorption makes a major contribution to the high levels found in the liver. Animal studies have shown that under conditions of experimental cirrhosis the handling of endotoxin by the liver is impaired (30), and Heyman and Beeson (31) have shown that patients with cirrhosis of the liver are unusually sensitive to endotoxin. The recent finding (32) that endotoxin is a potent B-cell mitogen is of considerable interest, since the effect of increased amounts of endotoxin reaching the appropriate organs of the reticuloendothelial system would be to stimulate antibody production. Clearly this may be an important factor in the etiology of the increased antibody titers found in chronic liver disease, but much more knowledge is required before this can be accepted. It does not provide any explanation for the selective increase in antibody titer which has been described. Furthermore, although endotoxin has been shown to be an effective stimulant, in vitro its immunological role in man has yet to be established. Since, in man, using the most sensitive assay yet devised, endotoxin can be detected only in the presence of severe gram-negative shock, it is hard to assess its clinical importance. The failure to detect endotoxin in the blood does not rule out an important role, since the critical factor would be the presence of endotoxin in the antibody-producing sites, but much more work needs to
IMMUNOLOGY AND LIVER FUNCTION
53
be done on the kinetics and metabolism of endotoxin in relation to the reticuloendothelial system before its role can be adequately assessed. CONCLUSION The evidence reviewed suggests that the liver may play a significant role in the sequestering of exogenous antigen and can thus modify the immune response. Hitherto, most studies of the immune response to antigens have considered the reticuloendothelial system as a whole, but in the light of the significant antigen-sequestering properties of the liver, the time may be opportune to examine individual components of the immune system. One can imagine how the intact liver may protect the organism against possible harmful immunological effects of exogenous antigens, but whether damage to this function of the liver plays any role in the potentiation of chronic liver disease merits further investigation. Immune complex information is dependent upon the ratio of the antigen to antibody, and so disturbance in the sequestering properties of the liver may influence this. Once formed, complexes are largely cleared by the liver (9), but in situations of liver damage this may be impaired with consequent deposition in other organs such as occurs in arthritis, polyarteritis, and glomerulonephritis associated with hepatitis Bs antigen (33). Likewise, the possibility that Kupffer cell function may be impaired in other conditions of excessive antibody formation such as collagen disease deserves to be studied. In summary, yet another chapter in the long list of functions attributable to the liver appears to be unfolding.
ACKNOWLEDGMENTS The author gratefully acknowledges the financial assistance of the Trustees of the George Herbert Hunt Travelling Fellowship, and advice and encouragement from Professor Ralph Wright. REFERENCES 1. Havens, W. P., Dickensheets, S., Brierly, J. N., and Eberhard, T. P. The half-life of 1131 labelled normal human gammaglobulin in patients with hepatic cirrhosis. J. Immunol. 73,256-258, (1954). 2. Chase, M. W. Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc. Soc. Exp. Biol. Med. 61,257-259, (1946). 3. Pomeranz, J. R. Immunologic unresponsiveness following a single feeding of picryl chloride. J. Immunol. 104,1486-1490, (1970). 4. Cantor, H. M., and Dumont, A. E. Hepatic suppression of sensitization to antigen absorbed into the portal system. Nature (London) 215,744-745, (1967). 5. Triger, D. R., Cynamon, M. H., and Wright, R. Studies on the hepatic uptake of antigen. I. Comparison of inferior vena cava and portal vein routes of immunization. Immunology 25,941-950, (1973). 6. Triger, D. R., and Wright, R. Studies on the hepatic uptake of antigen. II. The effect of hepatotoxins on the immune response. Immunology 25,951-956, (1973). 7. Souhami, R. L. The effect of colloidal carbon on the organ distribution of sheep red cells and the immune response. Immunology 22,685-694, (1972). 8. Franzl, R. E. The primary immune response in mice. III. Retention of sheep red blood cell immunogens by the spleen and liver. Infection Immunity 6,469-482, (1972). 9. Thomas, H. C., McSween, R. N. M., and White, R. G. Role of the liver in controlling the immunogenicity of commensal bacteria in the gut. Lancet 1,1288-1291, (1973). 10. Triger, D. R., and Wright, R. Hyperglobulinaemia in liver disease. Lancet 1,1494-1496, (1973). 11. Sell, S., and Fahey, J. L. Relationship between gammaglobulin metabolism and low serum gammaglobulin in germ-free mice. J. Immunol. 93,81-87, (1964). 12. Triger, D. R., Alp, M. H., and Wright, R. Bacterial and dietary antibodies in liver disease. Lancet 1,60-63, (1972).
54
DAVID R. TRIGER
13. Keraan, M., Meyers, 0. L., Engelbrecht, G. H. C., Hickman, R., Saunders, S. J., and Terblanche, J. Increased serum immunolglobulin levels following portacaval shunt in the rat. Gut 15,468-472, (1974). 14. Bj0rneboe, M., Prytz, H., and Orskov, F. Antibodies to intestinal microbes in serum of patients with. cirrhosis of the liver. Lancet 1,58-60, (1972). 15. Warshaw, A. L., Walker, A. W., and Isselbacher, K. J. Protein uptake by the intestine: evidence for absorption of intact macromolecules. Gastroenterology 64,874, (1973). 16. Triger, D. R., Kurtz, J. B., MacCallum, F. O., and Wright, R. Raised antibody titres to measles and rubella viruses in chronic active hepatitis. Lancet 1,665-667, (1972). 17. Triger, D. R., Kurtz, J. B., and Wright, R. Viral antibodies and auto-antibodies in chronic liver disease. Gut 15,94-98, (1974). 18. Alwen, J. Antibodies to adenovirus in patients with infectious hepatitis. Lancet 1,1452-1453, (1973). 19. Baals, H., Bulow, B., Freisenhausen, H. D., and Mai, K. Nachweis von Cytomegalievirus antikorpen bei der Au (SH) -Ag positiven und der Au (SH) -Ag negativen Hepatitis. Verh. Dtch. Ges. Inn. Med. 72,926-928, (1972). 20. Protell, R. L., Soloway, R. D., Martin, W. J., Schoenfield, W. J., and Summerskill, W. H. J. AntiSalmonella agglutinins in chronic active liver disease. Lancet 2,330-332, (1971). 21. MacSween, R. N. M., Galbraith, I., Thomas, M. A., Watkinson, G., and Ludlam, G. B. Phytohaemagglutin induced lymphocyte transformation and Toxoplasma gondii antibody studies in primary biliary cirrhosis. Clin. Exp. Immunol. 15,35-42, (1973). 22. Phillips, P. E., and Christian, C. L. Myxovirus antibody increases in human connective tissue disease. Science 168,982-984, (1970). 23. Fresco, R. L. Tubular (myxovirus-like) structures in glomerular deposits form a case of lupus nephritis. Fed. Proc. 27,246, (1968). 24. Mims, C. A. Aspects of the pathogenesis of virus diseases. Bacteriol. Rev. 28,30-71, (1964). 25. Johnson, A. G., Schmidtke, J., and Merritt, K. In "Nucleic Acids in Immunology" p. 379 (0. J. Plescia and W. Braun, Eds.), p. 379 Springer-Verlag, New York, 1968. 26. Pinckard, R. N., and Weir, D. M. Antibodies against the mitochondrial fraction of liver after toxic liver damage in rats. Clin. Exp. Immunol. 1,33-43, (1966). 27. Allison, A. C., Denman, A. M., and Barnes, R. D. Co-operating and controlling functions of thymusderived lymphocytes in relation to auto-immunity. Lancet 2,135-140, (1971). 28. Newberry, W. M., Shorey, J. W., Sanford, J. P., and Combes, B. Depression of lymphocyte reactivity to phytohaemagglutinin by serum from patients with liver disease. Cell. Immunol. 6,8797, (1973). 29. Parish, C. R. Immune response to chemically modified flagellin. II. Evidence for a fundamental relationship between humoral and cell-mediated immunity. J. Exp. Med. 134,21-47, (1971). 30. Rutenburg, A. M., Sonnenblick, E., Koven, I., Schweinburg, F., and Fine, J. Comparative response of normal and cirrhotic rats to intravenously injected bacteria. Proc. Soc. Exp. Biol. Med. 101, 279-281, (1959). 31. Heyman, A., and Beeson, P. B. Influence of various disease states upon the febrile response to intravenous injection of typhoid bacterial pyrogen with particular reference to malaria and cirrhosis of the liver. J. Lab. Clin. Med.34,1400-1403, (1949). 32. Bullock, W. W., and Andersson, J., In "Immunopotention." Ciba Found. Symp., Vol. 18, p. 173188, Elsevier-North-Holland, Amsterdam, 1973. 33. Australia antigen-the pace quickens. (Editorial). Lancet 1,85-86, (1973).
Some Immunological Aspects of Liver Function DAVID R. TRIGER' University ofSouthampton Medical School, Southampton, Hampshire, England Received October 7, 1974
Although many aspects of hepatic function have been known and studied for a number of years, little is known about the immunological function of the liver. Chronic liver disease is often accompanied by an absolute increase in serum gamma globulin concentration. All the major immunoglobulin classes may be involved, and the elevated globulin is due to increased synthesis rather than decreased metabolism (1). In this review mechanisms are examined whereby the normal liver may modify the immune response to antigens, and the damaged liver may lead to increased immunoglobulin synthesis. EXPERIMENTAL STUDIES In 1946, Chase (2) showed that guinea pigs could be rendered tolerant to intramuscular immunization with dinitrochlorobenzene (DNCB) by prior feeding with hapten. This phenomenon, subsequently named the Chase-Sulzberger effect, has been confirmed using other haptens, and tolerance may even be induced by a single feeding prior to intramuscular immunization (3). The possible role of the liver was first suggested by Cantor and Dumont (4). They examined the antibody response to intramuscular DNCB in dogs, and found that this was substantially reduced in animals fed with the hapten prior to immunization, and that this reduction was abolished if the animals were subjected to portacaval shunting before the start of the experiment. Interpretation of such feeding experiments is limited in that the rate and quantity of antigen absorption cannot be assessed. We (5) have studied the effect of the liver on the immune response in the rat by injecting sheep red blood cells directly into the portal vein and comparing the antibody response obtained with an equivalent injection via the inferior vena cava in control animals. Whereas the antibody response to a single dose of antigen was similar by either route, repeated portal venous injections of antigen produced a significantly lower antibody response than did repeated inferior vena cava injections (Table 1). The dose of antigen administered was critical; too little antigen failed to evoke a sufficient antibody response by either route for a difference to be detectable, while an excessive amount of antigen produced a large antibody response by either route of administration. Radio-labeling of the antigen with 5"Cr showed that in the nonimmunized animal the percentage uptake by the liver after a single injection was the same regardless of route of administration. By contrast, although repeated portal vein immunization resulted in the same percentage uptake by the liver, repeated inferior vena cava immunization resulted in decreased uptake by the liver. Delayed hypersensitivity was also examined in these animals by measuring the increase in ear thickness after intraauricular injection of sheep red cells. Rats immunized repeatedly via the inferior vena cava showed evidence of delayed hypersensitivity, while the animals immunized via the portal vein did not. In the 'Lecturer In Medicine.
47 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
48
DAVID R. TRIGER
TABLE I Primary, Secondary and Tertiary Immune Response to Inferior Vena Cava and Portal Vein Injection of 106 Sheep Red Cells (Haemagglutinating Antibody)
Lo92
Haemagglutinating Antibody Titre
6
4
fr
S-RBC I.V.C l10 S-RBC PV.
83
01~~~~~~
3
0
/2
1
2
4
2
~
6
4
4
85
TIME ( Days )
same animal model, damage to the liver by carbon tetrachloride led to an increased antibody response to sheep red cells, a fall in the percentage of labeled antigen taken up by the liver, and abolition of the difference in antibody response to repeated immunization between the two routes (6). An enhanced antibody response to sheep red cells after reduction in the amount of antigen taken up by the liver has also been produced by Souhami (7) by blockading the reticuloendothelial cells of the liver with colloidal carbon, although in this study the effect of immunization via the portal vein was not examined. These findings are consistent with the
theory that the intact liver is able to modify the immune response to exogenous antigens by sequestering them and rendering then nonimmunogenic and even, under certain circumstances, inducing tolerance to these antigens. Such a hypothesis presupposes that the hepatic macrophages-the Kupffer cells-behave differently from other macrophages in that antigens taken up by them lose their immunogenicity. Franzl (8) has shown that, whereas subcellular fractions of spleen containing antigen retain significant antigenicity for several days after the uptake of antigen, comparable liver fractions lost all detectable antigenic properties within 12 hr. As well as being able to sequester antigens, the liver may also influence the immune response by removing antigen/antibody complexes from the circulation. Thomas and colleagues (9) have demonstrated this in the experimental animal using 12mibovine serum albumin, and in addition they have shown that damage to the liver significantly reduces the ability to take up such complexes. CLINICAL OBSERVATIONS These animal studies may be relevant to observations in human liver disease. The elevated immunoglobulin levels found in chronic liver disease are due to increased synthesis and thus reflect increased antibody production. Since this may occur in the absence ofactivinse nsitdamage (10) the increased antibody production may reflect an abnormal response to exogenous antigens.
IMMUNOLOGY AND LIVER FUNCTION
49
SEQUESTRATION OF ANTIGENS BY THE LIVER Antigens from the intestinal microbial flora comprise the largest source of exogenous antigen (1 1), and the liver is uniquely situated in that it receives a large proportion of the systemic blood flow in addition to the entire portal venous system. Thus, antigens absorbed from the gastrointestinal tract must pass through the liver before they reach the antibody-producing cells in the spleen and elsewhere in the reticuloendothelial system. If the liver is damaged and the functional capacity of the Kupffer cells impaired, it is logical to expect that the antigens in the portal venous system would fail to be sequestered by the liver. As a result, increased amounts of antigen would become available to the antibody-producing cells in the spleen and other parts of the reticuloendothelial system, with consequent increased antibody production. Studies (12) of antibody titers to E. coli, and Bacteroides in liver disease support this. Antibody titers to both of these gastrointestinal tract organisms are significantly elevated in patients with chronic liver disease, in contrast to nongastrointestinal tract organisms such as Haemophilus influenzae B. Serial studies in acute hepatitis have shown that antibody titers to E. coli rise significantly some weeks after the onset of hepatitis, falling gradually to normal several months after the resolution of the liver disease. The transient loss of function of the reticuloendothelial cells of the liver may be due either to the frank destruction of the Kupffer cells accompanying the hepatitis or to a saturation of the phagocytic properties of these cells by the breakdown products of the damaged tissue. In addition to loss of functional capacity of the liver cells, a second mechanism is likely to contribute to the bypassing of the liver. Portal hypertension with the establishment of a collateral circulation is a frequent finding in many cases of chronic liver disease. Under these circumstances, antigen absorbed into the portal blood would reach the systemic circulation after bypassing the liver by way of the collateral blood vessels. This mechanism could play a significant role even in the presence of normal liver cell function. Rats with intact livers subjected to portacaval shunting have been shown to develop hyperglobulinemia and increased antibody titers to E. coli (13). Indirect evidence in support of this hypothesis is provided by the retrospective observations of Bj0rneboe and colleagues (14) that patients with portacaval anastomoses have higher antibody titers to E. coli than do those without a shunt. We2 have measured E. coli antibody titers in sera obtained from patients with cirrhosis and portal hypertension both before and at varying intervals after prophylactic portacaval anastomosis. A comparable group of patients who did not undergo portacaval anastomosis served as controls. Measurements were made using the method described previously (12). The results of the study are shown in Table 2. It will be seen that the mean E. coli antibody titer in the sera of 21 patients examined 26 mo after portacaval anastomosis is no higher than the presurgery sera. The control group sera showed titers of similar magnitude. This would suggest that the creation of a portacaval shunt has no effect on the E. coli antibody titer. It should be remembered, however, that patients with cirrhosis and portal hypertension already have severe liver damage and also a preexisting collateral circulation, thus providing two factors to encourage bypassing of the liver by antigen. Thus, the surgical creation of a portacaval shunt in these patients is unlikely to affect this 2Study performed in collaboration with Dr. H. 0. Conn, Veterans Administration Hospital, West Haven, CN.
50
DAVID R. TRIGER TABLE 2 Effect of Portacaval Anastomosis on E. coli Antibody Titers in Patients with Cirrhosis of the Liver
No. of patients studied Mean interval between 1st and 2nd serum (mo) Mean E. coli antibody titer (log2) in 1st sample Mean E. coli antibody titer (log2) in 2nd sample No. of sera showing elevated E. coli antibody titer in 1 st samplea
Cirrhosis without portacaval anastomosis 18 24 (1-97) 5.3 5.4 6/18
Cirrhosis with portacaval anastomosis 21 26 (2-71) 5.0 4.9 7/21
aE. coli antibody titer >1/32 (log25) taken as being elevated (12). situation significantly. The observation that one third of the patients have elevated E. coli antibody titers in the first sample studied lends support to this explanation. A major assumption used in interpreting these data is that bacterial antigens are absorbed from the gastrointestinal tract into the portal blood, rather than into the lymphatic system. Absorption via the latter route would bypass the liver under any circumstances, and so damage to the liver would have little or no effect. Warshaw et al. (15) have been able to study the uptake of labeled macromolecules in cannulated lymphatics and branches of the portal vein from isolated loops of bowel. These studies have shown that significant quantities of bovine serum albumin are transported directly to the lymphatic system, and this may be why antibody titers to albumin are not increased in patients with liver disease (12). Grossly elevated antibody titers to measles, rubella, and cytomegalic inclusion virus have been found in patients with chronic active hepatitis (16, 17). This increase in antibody titer appears to be highly selective, since antibodies to Herpes simplex, varicella-zoster, para-influenza 1, Coxsackie B1 and B5 and Mycoplasma pneumoniae are all within normal limits. Antibody titers to all these viruses were normal in other types of chronic liver disease, except the cytomegalovirus, to which elevated titers have been found in patients with alcoholic cirrhosis and primary biliary cirrhosis. Increased antibody titers have also been reported to some viruses in acute viral hepatitis. Alwen (18) has shown an increase in neutralizing antibody titers to adenovirus type 5 in acute hepatitis A infection, while Baals and colleagues (19) found increased titers to cytomegalovirus in patients with hepatitis, particularly those associated with the Hepatitis Bs antigen. Table 3 summarizes the elevated antibody titers to microbial and dietary protein antibodies hitherto reported in liver disease. Why should such striking elevations in viral antibody titers occur in chronic active hepatitis? The explanation here must be different from that postulated for bacterial antibodies, since none of these viruses is usually associated with the gastrointestinal tract. Increases in antibody titers to measles and rubella have been reported in systemic lupus erythematosis (22), and it has been suggested that this may be part of a phenomenon of nonspecific hyperreactivity, or that it might be associated with the finding of virus-like particles in renal biopsy material in lupus nephritis (23). The magnitude of the increase in antibody titers in chronic active hepatitis is much greater than that found in the connective tissue disorders. Normal antibody titers
51
IMMUNOLOGY AND LIVER FUNCTION TABLE 3 Elevated Microbial and Dietary Protein Antibody Titers Found in Liver Diseasea
Antigen
Acute hepatitis
Salmonella (14,20) E. coli(12,14) Bacteroides (12) Measles (16) Rubella (16) Cytomegalovirus (16,19) Adenovirus (18) Toxoplasma gondii (21) Gluten fraction III (12) o&Lactalbumin (12) Colon antigen (12) a+ Significantly increased titers; - No increase
Cirrhosis
0
-
+
+
+
+
-
-
+ +
+
+
0 + +
-
+ -
0
Chronic active hepatitis + + + + + + 0
0 +
in antibody titers; 0 Not examined.
to cytomegalovirus have been reported in SLE, and measles, rubella, and cytomegalovirus do not appear to show any common antigenic determinants. A possible explanation for these findings may lie in the fact that infection with these three viruses is associated with a viremia, after which a low level of circulating antibody persists for life, possibly a response to repeated release of small quantities of viral antigen (16). Since the Kupffer cells are known to be capable of sequestering viral antigens (24), the intact liver could play a significant role in regulating the amount of circulating antigen. In chronic active hepatitis the macrophage activity might be so depressed that the ability to sequester these antigens is lost and the viral antigens would then become available to antibody-producing cells elsewhere with a consequent increase in antibody production. Alternatively, the viral antigens (but not necessarily the intact viruses themselves) may be sequestered in the liver macrophages and be rendered temporarily nonimmunogenic, only to be released after the liver damage which occurs in chronic active hepatitis. In contrast, infection with the other viruses studied is rarely accompanied by viremia, and although viruses such as Herpes simplex and Varicella-zoster may persist for many years, antibodies are likely to be produced in the lymph close to the virus. In such circumstances, viral antigenemia is unlikely to occur and hence damage to the liver would not affect the mechanism of antibody production. OTHER MECHANISMS FOR ENHANCED ANTIBODY PRODUCTION Although this discussion has related the increased antibody response induced by exogenous antigens in liver disease to a failure of the sequestering properties of the Kupifer cells, other mechanisms may also contribute. Adjuvant Effect of Liver Tissue The breakdown products of liver cell necrosis, such as nucleic acids, can be shown experimentally to produce an adjuvant effect (25). Such a phenomenon might explain some of the increased antibody titers which have been observed in active liver necrosis, but it is unlikely to account for the persistent hyperglobulinemia often found in established inactive hepatic cirrhosis.
52
DAVID R. TRIGER
A utoantigens There is considerable evidence (26) to suggest that antigens are produced as a direct result of liver damage, and that these may provide a stimulus to the production of antibodies, although the extent of their contribution to antibody formation in human liver disease is uncertain. Autoantibodies are commonly found in the serum of patients with both acute and chronic liver disease, but they are neither organspecific nor species-specific. They may occur in the absence of overt liver disease, and the development of clinical liver disease has been observed in such patients without any alteration in the autoantibody titers (16). Patients with chronic active hepatitis commonly have strongly positive antinuclear and antismooth muslce antibodies and we have found (17) a very significant correlation between the elevated measles and rubella antibody titers and the strongly positive autoantibodies. It is possible that the autoantibodies may represent markers for potential immunological disturbance such that damage to the liver in these people may lead to a loss of the functional ability of the Kupffer cells to sequester these antigens.
Decreased Immunological Surveillance due to T-Cell Deficiency with Normal or Enhanced B-Cell Function Cooperation between T and B lymphocytes is a crucial factor in the production of antibodies and there is evidence (27) to suggest that under certain circumstances in which T-cell function is altered, the normal regulatory mechanisms may be overcome with the production of an autoimmune response. Depressed cell-mediated immunity is known to occur in many forms of liver disease (28), and the increased antibody production may reflect a compensatory B-cell overaction to a wide variety of antigenic stimuli not necessarily related to the pathogenesis (10). This interrelationship between T- and B-cell function has been demonstrated for a number of antigens (29) in animal models, but if it is applicable to clinical liver disease, the selectivity of the exaggerated B-cell response has still to be explained. ROLE OF ENDOTOXIN Endotoxin is largely derived from the gram-negative bacilli in the gastrointestinal tract, and after absorption makes a major contribution to the high levels found in the liver. Animal studies have shown that under conditions of experimental cirrhosis the handling of endotoxin by the liver is impaired (30), and Heyman and Beeson (31) have shown that patients with cirrhosis of the liver are unusually sensitive to endotoxin. The recent finding (32) that endotoxin is a potent B-cell mitogen is of considerable interest, since the effect of increased amounts of endotoxin reaching the appropriate organs of the reticuloendothelial system would be to stimulate antibody production. Clearly this may be an important factor in the etiology of the increased antibody titers found in chronic liver disease, but much more knowledge is required before this can be accepted. It does not provide any explanation for the selective increase in antibody titer which has been described. Furthermore, although endotoxin has been shown to be an effective stimulant, in vitro its immunological role in man has yet to be established. Since, in man, using the most sensitive assay yet devised, endotoxin can be detected only in the presence of severe gram-negative shock, it is hard to assess its clinical importance. The failure to detect endotoxin in the blood does not rule out an important role, since the critical factor would be the presence of endotoxin in the antibody-producing sites, but much more work needs to
IMMUNOLOGY AND LIVER FUNCTION
53
be done on the kinetics and metabolism of endotoxin in relation to the reticuloendothelial system before its role can be adequately assessed. CONCLUSION The evidence reviewed suggests that the liver may play a significant role in the sequestering of exogenous antigen and can thus modify the immune response. Hitherto, most studies of the immune response to antigens have considered the reticuloendothelial system as a whole, but in the light of the significant antigen-sequestering properties of the liver, the time may be opportune to examine individual components of the immune system. One can imagine how the intact liver may protect the organism against possible harmful immunological effects of exogenous antigens, but whether damage to this function of the liver plays any role in the potentiation of chronic liver disease merits further investigation. Immune complex information is dependent upon the ratio of the antigen to antibody, and so disturbance in the sequestering properties of the liver may influence this. Once formed, complexes are largely cleared by the liver (9), but in situations of liver damage this may be impaired with consequent deposition in other organs such as occurs in arthritis, polyarteritis, and glomerulonephritis associated with hepatitis Bs antigen (33). Likewise, the possibility that Kupffer cell function may be impaired in other conditions of excessive antibody formation such as collagen disease deserves to be studied. In summary, yet another chapter in the long list of functions attributable to the liver appears to be unfolding.
ACKNOWLEDGMENTS The author gratefully acknowledges the financial assistance of the Trustees of the George Herbert Hunt Travelling Fellowship, and advice and encouragement from Professor Ralph Wright. REFERENCES 1. Havens, W. P., Dickensheets, S., Brierly, J. N., and Eberhard, T. P. The half-life of 1131 labelled normal human gammaglobulin in patients with hepatic cirrhosis. J. Immunol. 73,256-258, (1954). 2. Chase, M. W. Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc. Soc. Exp. Biol. Med. 61,257-259, (1946). 3. Pomeranz, J. R. Immunologic unresponsiveness following a single feeding of picryl chloride. J. Immunol. 104,1486-1490, (1970). 4. Cantor, H. M., and Dumont, A. E. Hepatic suppression of sensitization to antigen absorbed into the portal system. Nature (London) 215,744-745, (1967). 5. Triger, D. R., Cynamon, M. H., and Wright, R. Studies on the hepatic uptake of antigen. I. Comparison of inferior vena cava and portal vein routes of immunization. Immunology 25,941-950, (1973). 6. Triger, D. R., and Wright, R. Studies on the hepatic uptake of antigen. II. The effect of hepatotoxins on the immune response. Immunology 25,951-956, (1973). 7. Souhami, R. L. The effect of colloidal carbon on the organ distribution of sheep red cells and the immune response. Immunology 22,685-694, (1972). 8. Franzl, R. E. The primary immune response in mice. III. Retention of sheep red blood cell immunogens by the spleen and liver. Infection Immunity 6,469-482, (1972). 9. Thomas, H. C., McSween, R. N. M., and White, R. G. Role of the liver in controlling the immunogenicity of commensal bacteria in the gut. Lancet 1,1288-1291, (1973). 10. Triger, D. R., and Wright, R. Hyperglobulinaemia in liver disease. Lancet 1,1494-1496, (1973). 11. Sell, S., and Fahey, J. L. Relationship between gammaglobulin metabolism and low serum gammaglobulin in germ-free mice. J. Immunol. 93,81-87, (1964). 12. Triger, D. R., Alp, M. H., and Wright, R. Bacterial and dietary antibodies in liver disease. Lancet 1,60-63, (1972).
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