Clin. exp. Immunol. (1977) 30, 1-3.
EDITORIAL
Lymphocyte subpopulations in chronic liver disease Perhaps the greatest clinical impact of the development of techniques for identifying thymus-dependent (T) and thymus-independent (B) lymphocytes has been in the classification of lymphomas (Lukes & Collins, 1975). However, another area in which these techniques have been widely applied is in the investigation of peripheral blood lymphocyte subpopulations in disease, in which autoimmunity is thought to be involved. The most frequent findings are changes in T-cell populations. For example, these are decreased in systemic lupus erythematosus (Messner, Lindstrdm & Williams, 1973). A reduction of circulating T cells in chronic active hepatitis has been observed by DeHoratius, Strickland & Williams (1974), and the two papers presented in this and last November's issue attempt to clarify the possible relationship between changes in lymphocyte subpopulations and the pathogenesis of chronic liver disease. Unfortunately, direct comparison of the results is not possible since the patient groupings are dissimilar and different techniques have been employed to identify the various lymphocyte subclasses. Thomas et al. (1976) have enumerated B cells by rosette formation with antibody-coated sheep erythrocytes (EA rosettes) and have, therefore, included K cells in their B-lymphocyte subpopulations, while Colombo, Vernace & Paronetto (1977) have used surface immunoglobulin markers to assess B-cell populations, and have thereby included K cells in the null cell fraction. These differences in technique may, in part, account for some of the differences in the results (Table 1). Nevertheless, some interesting new findings do emerge from these studies. TABLE 1.
Lymphocyte concentrations T cells
Disease
Active (N.Y.)
Total (N.Y.)
B cells
Total (Lond.)
B
(N.Y.)
Null cells
B+K (Lond.)
Null (Lond.)
Null+K (N.Y.)
Chronic active hepatitis 4
HBsAg-positive HBsAg-negative Without cirrhosis With cirrhosis Alcoholic liver disease Primary biliary cirrhosis Asymptomatic HBsAg carriers N.Y.
=
T n.d. n.d. n.d.
4
n.d. n.d. n.d.
i
4 n.d. n.d. 4 4
1
T T n.d. n.d. n.d.
n.d. n.d. t
T T
n.d.
n.d.
T T
Colombo et al. (New York); Lond. = Thomas et al. (London); n.d. = not done; T=increase;
n.d. n.d. n.d.
4=decrease;
=no change.
Colombo et al. have found that, in chronic active hepatitis, 'active' T cells (thought to be a functional subclass of T lymphocytes) are increased only in those who as yet have not developed cirrhosis. In view of a recent report by Felsburg, Edelman & Gilman (1976) that 'active' T-cell concentrations show a close correlation with delayed cutaneous hypersensitivity reactions, the results are suggestive of an active immunological process which diminishes as the disease progresses to its end-stage. The decreases in T-cell concentrations which Thomas et al. have observed in most of their patients with concomitant increases in null cell populations are similar to the findings of Tolentino & Astaldi (1975) in children with HBsAg-positive chronic active hepatitis. One possible explanation is that the increase in null cells is due to increased numbers of immature T cells, which also accounts for the reduction I
2
Editorial
in total T-lymphocyte populations. In support of this hypothesis, Thomas et al. have demonstrated increases in percentages of T cells in some patients after treatment with the immunopotentiating agents thymosin and levamisole. Additional evidence might have been obtained had the authors attempted to rosette null cells from these patients with neuraminidase-treated sheep erythrocytes-a procedure which has been shown to enhance E-rosette formation (Weiner, Bianco & Nussenweig, 1973). Nevertheless, it is an interesting observation, since levamisole has been shown to promote isograft rejection in mice and to enhance switching from an IgM to an IgG response (Renoux et al., 1976), and Thomas et al. (1975) havc previously suggested that patients with chronic liver disease exhibit defective switching. The effects of corticosteroid therapy on the lymphocyte subpopulations are difficult to assess from these studies. Thomas et al. investigated one patient serially before and after treatment with prednisolone and observed a decrease in the proportion of null cells with increases in percentages of T and B cells. However, the significance of this result is doubtful since the patient developed lymphopenia with absolute decreases in all cell fractions. On the other hand, Colombo et al. performed analogous studies on three patients and did not find any changes of significance. The relationship between alterations in lymphocyte subpopulations and chronic liver disease is obviously complex. The observed changes can be due to one or more of a number of factors, including differences in turnover rates of lymphocytes, homing and sequestration in various organs or alterations in membrane-associated cyclic-AMP levels-which may reflect changes in circulating hormone concentrations. As Thomas et al. suggest, the alterations in lymphocyte subpopulations are probably secondary to the liver damage. This suggestion is supported by the findings of Strickland et al. (1974), that in ulcerative colitis the percentage and number of T cells appears normal, but when this disease is associated with liver lesions there is both an absolute and a proportional decrease in T lymphocytes. A note of caution must, however, be sounded. Enumeration of the various cellular subclasses is fraught with difficulty. One problem relates to the possible presence in the patients' sera of anti-lymphocyte antibodies which, if bound to the surfaces of T lymphocytes, may result in these cells being wrongly classified as B cells. A number of other pitfalls are encountered when B lymphocytes are enumerated by immunofluorescent detection of surface immunoglobulin markers. Firstly, monocytes and macrophages bear C3 and Fc receptors on their surfaces and precautions must be taken to exclude these cells from the B-lymphocyte subpopulations. Secondly, the use of whole (as opposed to Fab2) anti-immunoglobulin antisera, or the presence of immune complexes, may give falsely high counts of B cells due to binding of the Fc fragments of antibodies to receptors on the non-B lymphocytes. Thirdly, a B cell may bear more than one surface immunoglobulin marker. This will affect not only the counts in the individual B-cell subclasses but, if summation of these counts is used to assess the total B-lymphocyte population, erroneously high values will result. In any further study it is to be hoped that at least two complementary techniques for enumeration of each lymphocyte subpopulation will be employed. I. G. McFARLANE A. L. W. F. EDDLESTON R. WILLIAMS Liver Unit,
King's College Hospital and Medical School, Denmark Hill, London S.E.5. REFERENCES COLOMBO, M., VERNACE, S.J. & PARONETTO, F. (1977) T and B lymphocytes in patients with chronic active hepatitis (CAH). Clin. exp. Immunol. 30, 4. DEHORATIUS, R.J., STRICKLAND, R.G. & WILLIAMS, R.C. JR (1974) T and B lymphocytes in acute and chronic hepatitis. Clin. Immunol. Immunopathol. 2, 353.
FELSBURG, P.J., EDELMAN, R. & GILMAN, R.H. (1976) The active E rosette test: correlation with delayed cutaneous hypersensitivity. 7. Immunol. 116, 1110. LUKES, R.J. & COLLINS, R.D. (1975) New approaches to the classification of the lymphomata. Brit. J. Cancer, 31, Suppl. II, 1.
Editorial MESSNER, R.P., LINDSTROM, F.D. & WILLIAMS, R.C., JR (1973) Peripheral blood lymphocyte cell surface markers during the course of systemic lupus erythematosus. 5. clin. Invest. 52, 3046. RENOUX, G., RENOUX, M., TELLER, M.N., MCMARION, S. & GUILLAUMIN, J.M. (1976) Potentiation of T-cell mediated immunity by levamisole. Clin. exp. immunol. 25, 288. STRICKLAND, R.G., KoisMtYER, S., SOLTIS, R.D., WILSON, I.D. & WILLIAMS, R.C., JR (1974) Peripheral blood T and B cells in chronic inflammatory bowel disease. Gastroenterology, 67, 569. THOMAS, H.C., FRENi, M., SANCHEZ-TAPIAS, J., DE
3
VILLIERS, D., JAIN, S. & SHERLOCK, S. (1976) Peripheral blood lymphocyte populations in chronic liver disease. Clin. exp. Immunol. 26, 222. THOMAS, H.C., HOLDEN, R., VERRIER-JONES, J. & PEACOCK, D. (1975) Primary and secondary immune responses to qX174 in patients with primary biliary cirrhosis and chronic active hepatitis. Gut, 16, Abstract, 836. TOLENTINO, P. & ASTALDI, A. (1975) T cells, B cells, and HL-A8 in chronic active hepatitis. Lancet, ii, 690. WEINER, N.S., BIANCO, C. & NUSSENWEIG, V. (1973) Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes. Blood, 142, 939.
EDITORIAL
Lymphocyte subpopulations in chronic liver disease Perhaps the greatest clinical impact of the development of techniques for identifying thymus-dependent (T) and thymus-independent (B) lymphocytes has been in the classification of lymphomas (Lukes & Collins, 1975). However, another area in which these techniques have been widely applied is in the investigation of peripheral blood lymphocyte subpopulations in disease, in which autoimmunity is thought to be involved. The most frequent findings are changes in T-cell populations. For example, these are decreased in systemic lupus erythematosus (Messner, Lindstrdm & Williams, 1973). A reduction of circulating T cells in chronic active hepatitis has been observed by DeHoratius, Strickland & Williams (1974), and the two papers presented in this and last November's issue attempt to clarify the possible relationship between changes in lymphocyte subpopulations and the pathogenesis of chronic liver disease. Unfortunately, direct comparison of the results is not possible since the patient groupings are dissimilar and different techniques have been employed to identify the various lymphocyte subclasses. Thomas et al. (1976) have enumerated B cells by rosette formation with antibody-coated sheep erythrocytes (EA rosettes) and have, therefore, included K cells in their B-lymphocyte subpopulations, while Colombo, Vernace & Paronetto (1977) have used surface immunoglobulin markers to assess B-cell populations, and have thereby included K cells in the null cell fraction. These differences in technique may, in part, account for some of the differences in the results (Table 1). Nevertheless, some interesting new findings do emerge from these studies. TABLE 1.
Lymphocyte concentrations T cells
Disease
Active (N.Y.)
Total (N.Y.)
B cells
Total (Lond.)
B
(N.Y.)
Null cells
B+K (Lond.)
Null (Lond.)
Null+K (N.Y.)
Chronic active hepatitis 4
HBsAg-positive HBsAg-negative Without cirrhosis With cirrhosis Alcoholic liver disease Primary biliary cirrhosis Asymptomatic HBsAg carriers N.Y.
=
T n.d. n.d. n.d.
4
n.d. n.d. n.d.
i
4 n.d. n.d. 4 4
1
T T n.d. n.d. n.d.
n.d. n.d. t
T T
n.d.
n.d.
T T
Colombo et al. (New York); Lond. = Thomas et al. (London); n.d. = not done; T=increase;
n.d. n.d. n.d.
4=decrease;
=no change.
Colombo et al. have found that, in chronic active hepatitis, 'active' T cells (thought to be a functional subclass of T lymphocytes) are increased only in those who as yet have not developed cirrhosis. In view of a recent report by Felsburg, Edelman & Gilman (1976) that 'active' T-cell concentrations show a close correlation with delayed cutaneous hypersensitivity reactions, the results are suggestive of an active immunological process which diminishes as the disease progresses to its end-stage. The decreases in T-cell concentrations which Thomas et al. have observed in most of their patients with concomitant increases in null cell populations are similar to the findings of Tolentino & Astaldi (1975) in children with HBsAg-positive chronic active hepatitis. One possible explanation is that the increase in null cells is due to increased numbers of immature T cells, which also accounts for the reduction I
2
Editorial
in total T-lymphocyte populations. In support of this hypothesis, Thomas et al. have demonstrated increases in percentages of T cells in some patients after treatment with the immunopotentiating agents thymosin and levamisole. Additional evidence might have been obtained had the authors attempted to rosette null cells from these patients with neuraminidase-treated sheep erythrocytes-a procedure which has been shown to enhance E-rosette formation (Weiner, Bianco & Nussenweig, 1973). Nevertheless, it is an interesting observation, since levamisole has been shown to promote isograft rejection in mice and to enhance switching from an IgM to an IgG response (Renoux et al., 1976), and Thomas et al. (1975) havc previously suggested that patients with chronic liver disease exhibit defective switching. The effects of corticosteroid therapy on the lymphocyte subpopulations are difficult to assess from these studies. Thomas et al. investigated one patient serially before and after treatment with prednisolone and observed a decrease in the proportion of null cells with increases in percentages of T and B cells. However, the significance of this result is doubtful since the patient developed lymphopenia with absolute decreases in all cell fractions. On the other hand, Colombo et al. performed analogous studies on three patients and did not find any changes of significance. The relationship between alterations in lymphocyte subpopulations and chronic liver disease is obviously complex. The observed changes can be due to one or more of a number of factors, including differences in turnover rates of lymphocytes, homing and sequestration in various organs or alterations in membrane-associated cyclic-AMP levels-which may reflect changes in circulating hormone concentrations. As Thomas et al. suggest, the alterations in lymphocyte subpopulations are probably secondary to the liver damage. This suggestion is supported by the findings of Strickland et al. (1974), that in ulcerative colitis the percentage and number of T cells appears normal, but when this disease is associated with liver lesions there is both an absolute and a proportional decrease in T lymphocytes. A note of caution must, however, be sounded. Enumeration of the various cellular subclasses is fraught with difficulty. One problem relates to the possible presence in the patients' sera of anti-lymphocyte antibodies which, if bound to the surfaces of T lymphocytes, may result in these cells being wrongly classified as B cells. A number of other pitfalls are encountered when B lymphocytes are enumerated by immunofluorescent detection of surface immunoglobulin markers. Firstly, monocytes and macrophages bear C3 and Fc receptors on their surfaces and precautions must be taken to exclude these cells from the B-lymphocyte subpopulations. Secondly, the use of whole (as opposed to Fab2) anti-immunoglobulin antisera, or the presence of immune complexes, may give falsely high counts of B cells due to binding of the Fc fragments of antibodies to receptors on the non-B lymphocytes. Thirdly, a B cell may bear more than one surface immunoglobulin marker. This will affect not only the counts in the individual B-cell subclasses but, if summation of these counts is used to assess the total B-lymphocyte population, erroneously high values will result. In any further study it is to be hoped that at least two complementary techniques for enumeration of each lymphocyte subpopulation will be employed. I. G. McFARLANE A. L. W. F. EDDLESTON R. WILLIAMS Liver Unit,
King's College Hospital and Medical School, Denmark Hill, London S.E.5. REFERENCES COLOMBO, M., VERNACE, S.J. & PARONETTO, F. (1977) T and B lymphocytes in patients with chronic active hepatitis (CAH). Clin. exp. Immunol. 30, 4. DEHORATIUS, R.J., STRICKLAND, R.G. & WILLIAMS, R.C. JR (1974) T and B lymphocytes in acute and chronic hepatitis. Clin. Immunol. Immunopathol. 2, 353.
FELSBURG, P.J., EDELMAN, R. & GILMAN, R.H. (1976) The active E rosette test: correlation with delayed cutaneous hypersensitivity. 7. Immunol. 116, 1110. LUKES, R.J. & COLLINS, R.D. (1975) New approaches to the classification of the lymphomata. Brit. J. Cancer, 31, Suppl. II, 1.
Editorial MESSNER, R.P., LINDSTROM, F.D. & WILLIAMS, R.C., JR (1973) Peripheral blood lymphocyte cell surface markers during the course of systemic lupus erythematosus. 5. clin. Invest. 52, 3046. RENOUX, G., RENOUX, M., TELLER, M.N., MCMARION, S. & GUILLAUMIN, J.M. (1976) Potentiation of T-cell mediated immunity by levamisole. Clin. exp. immunol. 25, 288. STRICKLAND, R.G., KoisMtYER, S., SOLTIS, R.D., WILSON, I.D. & WILLIAMS, R.C., JR (1974) Peripheral blood T and B cells in chronic inflammatory bowel disease. Gastroenterology, 67, 569. THOMAS, H.C., FRENi, M., SANCHEZ-TAPIAS, J., DE
3
VILLIERS, D., JAIN, S. & SHERLOCK, S. (1976) Peripheral blood lymphocyte populations in chronic liver disease. Clin. exp. Immunol. 26, 222. THOMAS, H.C., HOLDEN, R., VERRIER-JONES, J. & PEACOCK, D. (1975) Primary and secondary immune responses to qX174 in patients with primary biliary cirrhosis and chronic active hepatitis. Gut, 16, Abstract, 836. TOLENTINO, P. & ASTALDI, A. (1975) T cells, B cells, and HL-A8 in chronic active hepatitis. Lancet, ii, 690. WEINER, N.S., BIANCO, C. & NUSSENWEIG, V. (1973) Enhanced binding of neuraminidase-treated sheep erythrocytes to human T lymphocytes. Blood, 142, 939.
