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T and B lymphocytes in acute and chronic hepatitis
CLINICAL
IMMUNOLOGY
T and
AND
2, 353-360
IMMUNOPATHOLOGY
B lymphocytes
in Acute
and
(1974)
Chronic
Hepatitis
RAPHAEL J. DEHORATIUS, ROBERT G. STRICKLAND, AND RALPH C. WILLIAMS, JR. of Gastroenterology, Department of Medicine, The University of New Mexico School of Medicine, Albuquerque, New Mexico, 87131
Division
Received September 13, 1973 Peripheral blood thymus-dependent (TL) and bone marrow-dependent lymphocytes (BL) were measured in healthy control subjects and patients with acute or chronic hepatitis with and without hepatitis B antigen (HBAg) and in two chronic carriers of HBAg. A significant decrease in TL occurred in patients with acute or chronic hepatitis but not in healthy HBAg carriers. The decrease occurred in all forms of acute hepatitis and was transient in patients who recovered. TL depression occurred in both chronic persistent and chronic aggressive hepatitis and did not correlate with disease activity nor mode of treatment. In both acute and chronic hepatitis, decreases in TL occurred independently of serum autoantibodies. These findings emphasize the potential importance of host responses in determining the outcome of viral hepatitis.
Acute viral hepatitis whether of short or long incubation type is generally characterized by a self-limited clinical course with complete recovery. A fulminant course, chronic hepatitis of varying severity, hepatic cirrhosis, or a carrier state with normal liver function will result in a minority of patients. The variation in clinical course may depend on both viral and host factors. Recent studies in patients with hepatitis B antigen (HBAg) positive liver disease have implicated a defect in T lymphocyte function in determining the severity of hepatic damage and development of chronic@ (1). These studies utilized phytohemagglutinin (PHA)-induced lymphocyte transformation as an index of function (2) and inhibition of leukocyte migration as a measure of cell-mediated immunity to HBAg (3). In the present study we report observations of the proportions and absolute numbers of thymus dependent (TL) and bone marrow dependent (BL) lymphocytes in the peripheral blood of patients with acute and chronic hepatitis, with and without HBAg, and in two asymptomatic carriers of HBAg.
MATERIALS Patients Acute &al
hepatitis
AND
(27 patients).
METHODS
Included were 22 patients with an acute self-limited icteric illness studied l-4 wk from the onset of symptoms. Four patients had prolonged jaundice (12-16 wk) and liver biopsy revealed persistent acute hepatitis (4) in two and subacute hepatic necrosis (5) in two. The 353 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in any form reserved.
354
DEHORATIUS,
STRICKLAND,
AND
WILLIAMS,
JR.
remaining patient had fatal fulminant hepatitis. Eleven patients were HBAg positive. Chronic hepatitis (13 patients). These included five patients with chronic persistent hepatitis (CPH) and eight with chronic aggressive hepatitis (CAH). Diagnosis was based on clinical course and liver biopsy (4). Three of the five patients with CPH and two of the eight with CAH were HBAg positive. Cirrhosis was already established in the eight patients with CAH; five were undergoing treatment with prednisone and/or azathioprine. Active disease as judged by elevated SGOT was present in three of five patients with CPH and five of eight with CAH. Chronic carriers (2 patients). Both were asymptomatic carriers of HBAg and had normal tests of liver function including serum bilirubin, SGOT, alkaline phosphatase, serum albumin, and bromsulphalein retention. Control subjects. Fifty healthy medical students, house officers and laboratory technicians of similar age and sex distribution to the patients constituted the control group.
Methods Enumeration of T and B lymphocytes. Lymphocytes were isolated from heparinized peripheral blood by separation on Hypaque-Ficoll gradients (6). BL were enumerated by direct surface immunofluorescence using monospecific conjugates for IgA, IgG, and IgM (6); the sum of these percentages represented total BL. TL were enumerated by two techniques. Indirect immunofluorescent staining of freshly isolated lymphocytes identified immunofluorescent T lymphocytes (IFC). Rabbit antihuman fetal thymocyte antiserum was used after exhaustive absorption with cells from patients with chronic lymphocytic leukemia as a source of BL (6). TL were also enumerated by rosette formation between lymphocytes and sheep erythrocytes (RFC) (7,8). After 16 h r incubation at 4”C, cells showing at least three adherent erythrocytes were scored as positive. In addition to counting relative proportions of TL and BL by the above techniques, absolute numbers of these cells were also calculated on the basis of total blood lymphocyte counts. Detection of HBAg. HBAg was determined by counterimmunoelectrophoresis (9). Serum autoantibodies. Autoantibodies to smooth muscle (SMA), mitochondria (M) and nuclei (ANA) were detected by indirect immunofluorescence (10). Statistics. Significance of differences in cell populations between the groups studied was analyzed by Student’s t test. RESULTS
Acute virul hepatitis. Eighteen of 27 patients (67%) showed decreased TL defined as values for percent IFC or RFC below one standard deviation from the mean in control subjects. Nine patients showed depression using both TL markers; isolated depressions of RFC or IFC were observed in five and four patients, respectively. There was a highly significant (P < 0.001) mean per-
T AND
B LYMPHOCYTES TABLE
IN
355
HEPATITIS
1
MEAN (k SEM) PERCENT PERIPHERAL BLOOD T- AND B-LYMPHOCYTES IN NORMAL CONTROLS AND PATIENTS WITH HEPATITIS T-lymphocytes Patient
group
Number
Normal controls Acute hepatitis
50 27
Chronic
13
hepatitis
IFC = Immunofluorescent RFC = Rosette forming NS = Not significant.
B-lymphocytes
IFC
RFC
80.4 rt 1.9 58.1k5.2 P < 0.001 69.Ok6.4 P < 0.025
63.0 r2.3 43.8k4.3 P < 0.001 46.6*6.1 P < 0.05
Id 2.1 k0.2 2.7kO.5 NS 4.3k0.6 P < 0.001
IS
IN
Total
13.1 20.6 13.421.2 NS 13.621.6 NS
6.3 kO.4 9.1k0.9 P i 0.005 8.821.2
21.5 10.9 25.322.1 P i 0.05 26.9k2.5
P < 0.025
P < 0.02
cells. cells.
cent decrease in TL measured as either IFC or RFC when compared to values in control subjects (Table 1). Decreased TL occurred in 10 of 11 HBAg positive and 8 of 16 HBAg negative patients. Mean percent TL measured as either IFC or RFC were lower in HBAg positive patients but the difference from HBAg negative patients was not significant. Hepatitis was resolving in eight patients with normal IFC and RFC. The patient with fulminant hepatitis showed markedly decreased IFC (39%) and RFC (49%). Three of four patients with prolonged acute hepatitis (two with subacute hepatic necrosis) showed decreased IFC or RFC. IgM and total BL were significantly increased in patients with acute hepatitis. The degree of increase did not however match the extent of TL depression (Table 1). Serial observations of TL and BL in two patients with biopsy proven acute viral hepatitis (both HBAg positive) are shown in Fig. 1. Initially TL enumerated by both IFC and RFC were markedly reduced. With clinical recovery the TL percentage returned to normal. Two additional patients with low initial RFC also showed increases to normal with recovery. Chronic hepatitis. Nine of 13 patients (70%) showed decreased proportions of TL. In four the decrease was in RFC, in three it was in IFC, and in two both IFC and RFC were decreased. Mean percent TL (both IFC and RFC) were significantly reduced when compared to control values (Table 1). All five HBAg positive patients and four of eight HBAg negative patients showed decreased IFC or RFC. Mean percent TL measured as either IFC or RFC were lower in HBAg positive patients but the difference from HBAg negative patients was not significant. ’ All five patients with CPH and four of eight with CAH showed TL depression. Decreased IFC or RFC occurred in five of eight patients with active disease and four of five with inactive disease. Two of five patients with CAH being treated with immunosuppressive drugs showed decreased IFC or RFC; two of three with CAH not receiving such treatment showed similar decreases.
DEHORATIUS,
B 3 i B
STRICKLAND,
AND
WILLIAMS,
JR.
21 MAR, 18 I.2
4Al=Ft. 13 0.9
5’
IO FEB. SGOT I.U. Bllwubin mg %
84 4.1
14 MAR. 12 1.2
65
23 APR SGOT 1.U. 1044 Bdirubm
FIG. 1. Serial T and B lymphocyte positive
mg %
6.9
proportions
x)APR. 580
7 MAY 125
0.4
in two patients
I.3
with
biopsy
proven
acute
HBAg
hepatitis.
Significant increases in cells bearing surface IgA, IgM and also total BL occurred in patients with chronic hepatitis (Table 1). The degree of increase in BL did not match the extent of TL depression. Chronic carriers. The percent of TL were normal in both HBAg carriers. Percent BL were also normal in both patients. Absolute lymphocyte numbers. In addition to decreases in proportions of TL, absolute numbers of TL were calculated in 33 patients in whom absolute lymphocyte counts were available. A significant depression of total TL numbers was observed in both acute and chronic hepatitis (Table 2). In every instance in which the percentage of TL was depressed, the absolute TL numbers were also decreased. Thus in addition to a depression in relative percentages of these cells (Table I), a true decrease in absolute numbers of circulating TL was associated with both acute and chronic hepatitis. Absolut BL numbers were significantly increased in both acute and chronic hepatitrs (Table 2). Correlation of T lymphocytes, HBAg, and serum autoantibodies. SMA was not detected in chronic carriers of HBAg but was present in 8 of 27 patients (30%) with acute hepatitis (4 HBAg positive, 4 HBAg negative). SMA, M or
T AND
MEAN
(k SEM) NORMAL
B LYMPHOCYTES
ABSOLUTE CONTROLS
IN
357
HEPATITIS
TABLE 2 PERIPHERAL T- AND B-LYMPHOCYTES AND PATIENTS WITH HEPATITIS
B-lymphocytes
T-lymphocytes Patient
group
Number
Normal controls Acute hepatitis
50 22
Chronic
11
hepatitis
IFC 1747.9 1307.5 P < 1493.0 P <
IN
RFC
f 41.2 * 129.6 .ool k 150.6 .025
1369.6 985.5 P-c 1008.3 P <
Total
f 50.1 k 106.3 .OOl f 143.4 .Ol
467.4 599.4 P < 637.3 P <
” 4.3 ‘-’ 50.2 .ool + 59.1 .ool
ANA were present in 6 of 13 patients with chronic hepatitis (1 HBAg positive, 5 HBAg negative); all had CAH. Of the 14 patients in the two groups with autoantibodies TL were normal in five and decreased in nine. Amongst 26 patients without autoantibodies 18 had decreased TL and eight had normal values (Table 3). Lymphocytotoxic antibodies. Recent reports of the occurrence of lymphocytotoxic antibodies during the course of infectious mononucleosis and other viral infections (11,12) have led us to investigate this possibility in viral hepatitis. A panel of 12 normal test lymphocytes of varying HL-A phenotypes was used to screen for lymphocytotoxic antibody with methods previously described (13,14). Determinations were made using normal donor lymphocytes against sera from both groups of hepatitis patients as well as against a test panel of sera from normal donors. These studies disclosed lymphocytotoxic antibody in 39 of the 40 hepatitis patients studied. Positive and striking cytotoxicity occurred in 74% of the tests as compared to only 10% for the control panel utilizing normal sera (Table 4). No obvious correlation between degree of serum cytotoxicity and proportion or absolute numbers of TL could be demonstrated. Further characterization of these reactions and the association with clinical recovery is needed. DISCUSSION Studies utilizing strated a reversible (15,16). The present
PHA-induced lymphocyte transformation have demondefect of T lymphocyte function in acute viral hepatitis study using two direct markers of TL (namely T lympho-
TABLE 3 COMPARISON OF PRESENCE (+) OR ABSENCE (-) OF AUTOANTIBODIES WITH T LYMPHOCYTES IN 40 PATIENTS WITH ACUTE OR CHRONIC HEPATITIS T lymphocytes
.
Normal Autoantibodies
+ -
5 8
Decreased 9 18
DEHORATIUS,
358
STRICKLAND,
AND
WILLIAMS,
JR.
TABLE 4 CYTOTOXICITY OF CONTROL SERA AND SERA FROM PATIENTS WITH ACUTE OR CHRONIC HEPATITIS
Control Acute hepatitis Chronic hepatitis
No. of sera tested
No. of tests
Percent o-10%
lo-20%
30
2Q2
77.1”
12.9
27
274
0
13
137
5.2
n Tests with 20% or more L Numbers refer to actual
of test lymphocytes
killed
in cytotwic
test\” 70-80’~~
30-40’4
40-5O’i
so-m”r
fro-70%
4
2.5
2.0
0.5
0.5
0.5
0
0
26.3
17.9
13.1
12.4
4
11.7
5.5
5.8
3.3
20.3
18.2
11.7
10.2
5.2
10.9
5.2
7.3
5.8
lymphocyte
panel.
2(1-30%
kill were considered positive. percent of tested sera with cytotoxicity
of varying
degrees
for the normal
donor
8040%
go-IW%
cyte antiserum and sheep red cell rosette formation) has shown that a significant decrease in both absolute numbers and percent of circulating TL occurs in acute hepatitis. TL function has also been found to be decreased in chronic hepatitis (3,16,17). The present study demonstrates decreased proportions and absolute numbers of circulating TL in such patients. Studies of TL function in chronic carriers of HBAg have yielded conflicting results (2,18). This study has demonstrated normal proportions of TL in two HBAg carriers. In two-thirds of our patients showing TL depression, a discrepancy between IFC or RFC results occurred. In general values for IFC were slightly higher than RFC among normal subjects, however a marked discrepancy has not been observed (19). The observation is at present unexplained but may be an indication that the two techniques identify separate lymphocyte surface membrane receptors (and perhaps different T lymphocyte subpopulations) which show a variable susceptibility to damage by viral infection. Increased BL in patients with acute and chronic hepatitis was an expected finding in view of the known alterations in serum immunoglobulins in these two diseases. However, the degree of increase in total BL in both groups did not match the extent of TL depression- indicating that compared to normal subjects a proportion of the circulating lymphocytes in patients with acute or chronic hepatitis were not identified by the B and T cell markers used. The origin of these “null” cells and their biologic significance is unknown at this time although they may be related to the shedding phenomenon (20) or to rapid endocytosis of the surface markers by these particular lymphocytes (21). Dudley et al. (1) have postulated that the clinical course of viral hepatitis may be determined by the cellular immune competence of the host. Patients who recover have a competent T lymphocyte system, those who become carriers have a defect in this system and those who develop chronic hepatitis occupy an intermediate position with respect to cellular immune functish. Their studies using inhibition of leukocyte migration of HBAg positive serum and liver extract as a measure of cell-mediated immunity to HBAg revealed a decreasing response to these antigens when patients who had recovered from acute hepatitis were compared to those with chronic hepa-
T AND
B LYMPHOCYTES
IN
HEPATITIS
359
titis or healthy carriers (3). Our findings of similar degrees of TL depression in patients with acute or chronic hepatitis of varying severity and normal percentages of TL in healthy carriers of HBAg are not entirely consistent with the above hypothesis-nor can they be explained solely by persistence or clearing of the virus. A recent study (22) showing marked decrease in rosette forming cells in a variety of acute viral infections indicates that the defect is not unique to viral hepatitis. The incidence of autoantibody to smooth muscle in our patients with acute hepatitis (30%) is lower than that reported in two previous studies (23,24) but is similar to that reported by Wright (25). It has been proposed (26) that the development of autoimmune phenomena in viral infection depends on T and B lymphocyte cooperation and that autoantibodies might arise by an increase in “helper” TL or a decrease in “suppressor” TL. Our finding of decreased TL in two-thirds of the patients with autoantibodies could be interpreted as evidence in favor of the latter mechanism. However, similar degrees of TL depression occurred in two-thirds of patients without autoantibodies. The cause of the decrease in circulating TL in the patients studied is unknown. A majority of our patients with acute or chronic hepatitis showed significant lymphocytotoxic antibody. In view of the fact that such antibodies have recently been shown to be TL specific in the case of disseminated lupus (30) it was disappointing to find no clear correlation between relative degrees of lymphocytotoxicity and TL values. No opportunity was afforded to study the patients own lymphocytes for autospecificity of lymphocytotoxins and this could explain the lack of correlation recorded here. Finally, virus may directly alter lymphocyte membrane metabolism or sterically hinder surface membrane receptors and thus prevent rosette formation or attachment of TL antisera. The present study demonstrating a decrease in proportions and absolute numbers of circulating TL in acute viral hepatitis has thus extended earlier observations of a reversible defect in TL function in this disease. Persistence of the defect has been demonstrated in protracted acute hepatitis and in chronic hepatitis of varying severity but no decrease in circulating TL was observed in two HBAg carriers without liver disease. The observations indicate that the T lymphocyte response in viral hepatitis may have relevance both to elimination or persistence of the virus and to the development of continuing liver damage. ACKNOWLEDGMENTS We thank Mrs. Carolyn Henderson and Mrs. Sally Sperry for technical assistance and Dr. H. Danemann for permission to include several of his patients in this study. This work was supported in part by Grants AM 13824-03 and TO1 A100343-03 from the U. S. Public Health Service and in part by the Lael Richards Memorial Fund.
360
DEHORATIUS,
STRICKLAND,
AND
WILLIAMS,
JR.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
DUDLEY, F. J., Fox, R. A., AND SHERLOCK, S., Lancet 1,723, 1972. GIUSTINO, V., DUDLEY, F. J., AND SHERLOCK, S., Lancet 2, 850, 1972. DUDLEY, F. J., GI~STINO, V., AND SHERLOCK, S., Br. Med. J. 4, 754, 1972. DEGROOTE, J., DESMET, V. J., GEDIGK, P., KORB, G., POPPER, H., POULSEN, H., SCHEUER, P. J., SCHMID, M., THALER, H., UEHLINCER, E., AND WEPLER, W., Lancet 2, 626, 1968. BOYER, J., AND KLATSIUN, G. N. Engl. /. Med. 283,1063,1970. WILLIAMS, R. C., JR., DEBORD, J. R., MELLBYE, 0. J., MESSNER, R. P., AND LINDSTR~M, F. J, J. Clin. Invest. 52,283, 1973. WYBRAN, J., AND FUDENBERG, H. H., Trans. Ass. Amer. Phys. 84,239, 1971. JONDAL, M., HOLM, G., AND WIGZELL, H.,J. Exp. Med. 136, 207, 1972. GOCKE, J. J.. AND HOWE, C. J., Immunology 104,1031, 1970. WHITTINGHAM, S., AND MACKAY, I. R., Med. J. Aust. 1, 1200, 1969. MOTTIRONI, V. D., AND TERASAKI, P. I. In “Lymphocytotoxins in Disease.” I. Infectious Mononucleosis, Rubella and Measles. Histocompatibility Testing; p. 301. Munksgaard, Copenhagen, 1970. HUANG, S-W, LATTOS, D. B., NELSON, D. B., REEB, K., AND HONG, R., J, C&n. Invest. 52, 1033, 1973. TERASAKI, P. I., AND MCCLELLAND, J. D., Nature (London) 204,998, 1964. TERASAKI, P. I., MOTTIRONI, V. D., AND BARNETT, E. V., N. Engl. J. Med. 283, 724, 1970. MARTINI, G. A., R~SSLER, R., HAVEMAN, K., AND D~LLE, W., Scund. J. Gustroent. Suppl. 7, 39, 1970. WILLEMS, F. TH. C., MELNICK, J. L., AND RAWLS, W. E., Proc. Sot. Exp. Biol. Med.130, 652, 1969. TOH, B. H., ROBERTS-THOMPSON, I. C., MATHEWS, J. D., WHITTINGHAM, S., AND MACKAY, I. R., Clin. Exp. Immunol. 14, 193, 1973. SUTNICK, A. I., BUGBEE, S. J., LONDON, W. T., LOEB, L. A., PEYRETTI, F., LITWIN, S., AND BLUMBERG, B. S., J. Lab. Clin. Med. 82,79, 1973. MESSNER, R. P., LINDSTRoM, F. D., AND WILLIAMS, R. C., JR., J. Clin. Invest. 52, 3046, 1973. WERNET, P., AND KUNKEL, H. G., Fed. PTOC. 32, (Abstr.) 975, 1973. UNANUE, E. R., PERKINS, W. D., AND KARNOVSKY, M. J., J. Immunol. 108, 569, 1972. WYBRAN, J., AND FUDENBERG, H. H., J. Clin. Invest. 52,1026, 1973. FARROW, L. J., HOLBOROW, E. J., JOHNSON, G. D., LAMBS, S. G., STEWART, J. S., TAYLOR, P. E., AND ZUCKERMAN, A. J., Br. Med. J. 2,693, 1970. AJDUKIEWICZ, A. B., DUDLEY, F. J., Fox, R. A., DONIACH, D., AND SHERLOCK, S., Luncet 1, 803, 1972. WRIGHT, R., Luncet 1, 521, 1970. ALLISON, A. C., DENMAN, A. M., AND BARNES, R. D., Luncet 2, 135, 1971. NEWELL, P. C., Cancer Res. 21, 1518, 1961. CARON, G. A., Int. Arch. Allergy32, 191, 1967. MELLA, B., AND LANG, D. J., Science 155, 80, 1967. LIES, R. B., MESSNER, R. P., AND WILLIAMS, R. C., JR., Arthritis Rheum. 16, 369, 1973.
IMMUNOLOGY
T and
AND
2, 353-360
IMMUNOPATHOLOGY
B lymphocytes
in Acute
and
(1974)
Chronic
Hepatitis
RAPHAEL J. DEHORATIUS, ROBERT G. STRICKLAND, AND RALPH C. WILLIAMS, JR. of Gastroenterology, Department of Medicine, The University of New Mexico School of Medicine, Albuquerque, New Mexico, 87131
Division
Received September 13, 1973 Peripheral blood thymus-dependent (TL) and bone marrow-dependent lymphocytes (BL) were measured in healthy control subjects and patients with acute or chronic hepatitis with and without hepatitis B antigen (HBAg) and in two chronic carriers of HBAg. A significant decrease in TL occurred in patients with acute or chronic hepatitis but not in healthy HBAg carriers. The decrease occurred in all forms of acute hepatitis and was transient in patients who recovered. TL depression occurred in both chronic persistent and chronic aggressive hepatitis and did not correlate with disease activity nor mode of treatment. In both acute and chronic hepatitis, decreases in TL occurred independently of serum autoantibodies. These findings emphasize the potential importance of host responses in determining the outcome of viral hepatitis.
Acute viral hepatitis whether of short or long incubation type is generally characterized by a self-limited clinical course with complete recovery. A fulminant course, chronic hepatitis of varying severity, hepatic cirrhosis, or a carrier state with normal liver function will result in a minority of patients. The variation in clinical course may depend on both viral and host factors. Recent studies in patients with hepatitis B antigen (HBAg) positive liver disease have implicated a defect in T lymphocyte function in determining the severity of hepatic damage and development of chronic@ (1). These studies utilized phytohemagglutinin (PHA)-induced lymphocyte transformation as an index of function (2) and inhibition of leukocyte migration as a measure of cell-mediated immunity to HBAg (3). In the present study we report observations of the proportions and absolute numbers of thymus dependent (TL) and bone marrow dependent (BL) lymphocytes in the peripheral blood of patients with acute and chronic hepatitis, with and without HBAg, and in two asymptomatic carriers of HBAg.
MATERIALS Patients Acute &al
hepatitis
AND
(27 patients).
METHODS
Included were 22 patients with an acute self-limited icteric illness studied l-4 wk from the onset of symptoms. Four patients had prolonged jaundice (12-16 wk) and liver biopsy revealed persistent acute hepatitis (4) in two and subacute hepatic necrosis (5) in two. The 353 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in any form reserved.
354
DEHORATIUS,
STRICKLAND,
AND
WILLIAMS,
JR.
remaining patient had fatal fulminant hepatitis. Eleven patients were HBAg positive. Chronic hepatitis (13 patients). These included five patients with chronic persistent hepatitis (CPH) and eight with chronic aggressive hepatitis (CAH). Diagnosis was based on clinical course and liver biopsy (4). Three of the five patients with CPH and two of the eight with CAH were HBAg positive. Cirrhosis was already established in the eight patients with CAH; five were undergoing treatment with prednisone and/or azathioprine. Active disease as judged by elevated SGOT was present in three of five patients with CPH and five of eight with CAH. Chronic carriers (2 patients). Both were asymptomatic carriers of HBAg and had normal tests of liver function including serum bilirubin, SGOT, alkaline phosphatase, serum albumin, and bromsulphalein retention. Control subjects. Fifty healthy medical students, house officers and laboratory technicians of similar age and sex distribution to the patients constituted the control group.
Methods Enumeration of T and B lymphocytes. Lymphocytes were isolated from heparinized peripheral blood by separation on Hypaque-Ficoll gradients (6). BL were enumerated by direct surface immunofluorescence using monospecific conjugates for IgA, IgG, and IgM (6); the sum of these percentages represented total BL. TL were enumerated by two techniques. Indirect immunofluorescent staining of freshly isolated lymphocytes identified immunofluorescent T lymphocytes (IFC). Rabbit antihuman fetal thymocyte antiserum was used after exhaustive absorption with cells from patients with chronic lymphocytic leukemia as a source of BL (6). TL were also enumerated by rosette formation between lymphocytes and sheep erythrocytes (RFC) (7,8). After 16 h r incubation at 4”C, cells showing at least three adherent erythrocytes were scored as positive. In addition to counting relative proportions of TL and BL by the above techniques, absolute numbers of these cells were also calculated on the basis of total blood lymphocyte counts. Detection of HBAg. HBAg was determined by counterimmunoelectrophoresis (9). Serum autoantibodies. Autoantibodies to smooth muscle (SMA), mitochondria (M) and nuclei (ANA) were detected by indirect immunofluorescence (10). Statistics. Significance of differences in cell populations between the groups studied was analyzed by Student’s t test. RESULTS
Acute virul hepatitis. Eighteen of 27 patients (67%) showed decreased TL defined as values for percent IFC or RFC below one standard deviation from the mean in control subjects. Nine patients showed depression using both TL markers; isolated depressions of RFC or IFC were observed in five and four patients, respectively. There was a highly significant (P < 0.001) mean per-
T AND
B LYMPHOCYTES TABLE
IN
355
HEPATITIS
1
MEAN (k SEM) PERCENT PERIPHERAL BLOOD T- AND B-LYMPHOCYTES IN NORMAL CONTROLS AND PATIENTS WITH HEPATITIS T-lymphocytes Patient
group
Number
Normal controls Acute hepatitis
50 27
Chronic
13
hepatitis
IFC = Immunofluorescent RFC = Rosette forming NS = Not significant.
B-lymphocytes
IFC
RFC
80.4 rt 1.9 58.1k5.2 P < 0.001 69.Ok6.4 P < 0.025
63.0 r2.3 43.8k4.3 P < 0.001 46.6*6.1 P < 0.05
Id 2.1 k0.2 2.7kO.5 NS 4.3k0.6 P < 0.001
IS
IN
Total
13.1 20.6 13.421.2 NS 13.621.6 NS
6.3 kO.4 9.1k0.9 P i 0.005 8.821.2
21.5 10.9 25.322.1 P i 0.05 26.9k2.5
P < 0.025
P < 0.02
cells. cells.
cent decrease in TL measured as either IFC or RFC when compared to values in control subjects (Table 1). Decreased TL occurred in 10 of 11 HBAg positive and 8 of 16 HBAg negative patients. Mean percent TL measured as either IFC or RFC were lower in HBAg positive patients but the difference from HBAg negative patients was not significant. Hepatitis was resolving in eight patients with normal IFC and RFC. The patient with fulminant hepatitis showed markedly decreased IFC (39%) and RFC (49%). Three of four patients with prolonged acute hepatitis (two with subacute hepatic necrosis) showed decreased IFC or RFC. IgM and total BL were significantly increased in patients with acute hepatitis. The degree of increase did not however match the extent of TL depression (Table 1). Serial observations of TL and BL in two patients with biopsy proven acute viral hepatitis (both HBAg positive) are shown in Fig. 1. Initially TL enumerated by both IFC and RFC were markedly reduced. With clinical recovery the TL percentage returned to normal. Two additional patients with low initial RFC also showed increases to normal with recovery. Chronic hepatitis. Nine of 13 patients (70%) showed decreased proportions of TL. In four the decrease was in RFC, in three it was in IFC, and in two both IFC and RFC were decreased. Mean percent TL (both IFC and RFC) were significantly reduced when compared to control values (Table 1). All five HBAg positive patients and four of eight HBAg negative patients showed decreased IFC or RFC. Mean percent TL measured as either IFC or RFC were lower in HBAg positive patients but the difference from HBAg negative patients was not significant. ’ All five patients with CPH and four of eight with CAH showed TL depression. Decreased IFC or RFC occurred in five of eight patients with active disease and four of five with inactive disease. Two of five patients with CAH being treated with immunosuppressive drugs showed decreased IFC or RFC; two of three with CAH not receiving such treatment showed similar decreases.
DEHORATIUS,
B 3 i B
STRICKLAND,
AND
WILLIAMS,
JR.
21 MAR, 18 I.2
4Al=Ft. 13 0.9
5’
IO FEB. SGOT I.U. Bllwubin mg %
84 4.1
14 MAR. 12 1.2
65
23 APR SGOT 1.U. 1044 Bdirubm
FIG. 1. Serial T and B lymphocyte positive
mg %
6.9
proportions
x)APR. 580
7 MAY 125
0.4
in two patients
I.3
with
biopsy
proven
acute
HBAg
hepatitis.
Significant increases in cells bearing surface IgA, IgM and also total BL occurred in patients with chronic hepatitis (Table 1). The degree of increase in BL did not match the extent of TL depression. Chronic carriers. The percent of TL were normal in both HBAg carriers. Percent BL were also normal in both patients. Absolute lymphocyte numbers. In addition to decreases in proportions of TL, absolute numbers of TL were calculated in 33 patients in whom absolute lymphocyte counts were available. A significant depression of total TL numbers was observed in both acute and chronic hepatitis (Table 2). In every instance in which the percentage of TL was depressed, the absolute TL numbers were also decreased. Thus in addition to a depression in relative percentages of these cells (Table I), a true decrease in absolute numbers of circulating TL was associated with both acute and chronic hepatitis. Absolut BL numbers were significantly increased in both acute and chronic hepatitrs (Table 2). Correlation of T lymphocytes, HBAg, and serum autoantibodies. SMA was not detected in chronic carriers of HBAg but was present in 8 of 27 patients (30%) with acute hepatitis (4 HBAg positive, 4 HBAg negative). SMA, M or
T AND
MEAN
(k SEM) NORMAL
B LYMPHOCYTES
ABSOLUTE CONTROLS
IN
357
HEPATITIS
TABLE 2 PERIPHERAL T- AND B-LYMPHOCYTES AND PATIENTS WITH HEPATITIS
B-lymphocytes
T-lymphocytes Patient
group
Number
Normal controls Acute hepatitis
50 22
Chronic
11
hepatitis
IFC 1747.9 1307.5 P < 1493.0 P <
IN
RFC
f 41.2 * 129.6 .ool k 150.6 .025
1369.6 985.5 P-c 1008.3 P <
Total
f 50.1 k 106.3 .OOl f 143.4 .Ol
467.4 599.4 P < 637.3 P <
” 4.3 ‘-’ 50.2 .ool + 59.1 .ool
ANA were present in 6 of 13 patients with chronic hepatitis (1 HBAg positive, 5 HBAg negative); all had CAH. Of the 14 patients in the two groups with autoantibodies TL were normal in five and decreased in nine. Amongst 26 patients without autoantibodies 18 had decreased TL and eight had normal values (Table 3). Lymphocytotoxic antibodies. Recent reports of the occurrence of lymphocytotoxic antibodies during the course of infectious mononucleosis and other viral infections (11,12) have led us to investigate this possibility in viral hepatitis. A panel of 12 normal test lymphocytes of varying HL-A phenotypes was used to screen for lymphocytotoxic antibody with methods previously described (13,14). Determinations were made using normal donor lymphocytes against sera from both groups of hepatitis patients as well as against a test panel of sera from normal donors. These studies disclosed lymphocytotoxic antibody in 39 of the 40 hepatitis patients studied. Positive and striking cytotoxicity occurred in 74% of the tests as compared to only 10% for the control panel utilizing normal sera (Table 4). No obvious correlation between degree of serum cytotoxicity and proportion or absolute numbers of TL could be demonstrated. Further characterization of these reactions and the association with clinical recovery is needed. DISCUSSION Studies utilizing strated a reversible (15,16). The present
PHA-induced lymphocyte transformation have demondefect of T lymphocyte function in acute viral hepatitis study using two direct markers of TL (namely T lympho-
TABLE 3 COMPARISON OF PRESENCE (+) OR ABSENCE (-) OF AUTOANTIBODIES WITH T LYMPHOCYTES IN 40 PATIENTS WITH ACUTE OR CHRONIC HEPATITIS T lymphocytes
.
Normal Autoantibodies
+ -
5 8
Decreased 9 18
DEHORATIUS,
358
STRICKLAND,
AND
WILLIAMS,
JR.
TABLE 4 CYTOTOXICITY OF CONTROL SERA AND SERA FROM PATIENTS WITH ACUTE OR CHRONIC HEPATITIS
Control Acute hepatitis Chronic hepatitis
No. of sera tested
No. of tests
Percent o-10%
lo-20%
30
2Q2
77.1”
12.9
27
274
0
13
137
5.2
n Tests with 20% or more L Numbers refer to actual
of test lymphocytes
killed
in cytotwic
test\” 70-80’~~
30-40’4
40-5O’i
so-m”r
fro-70%
4
2.5
2.0
0.5
0.5
0.5
0
0
26.3
17.9
13.1
12.4
4
11.7
5.5
5.8
3.3
20.3
18.2
11.7
10.2
5.2
10.9
5.2
7.3
5.8
lymphocyte
panel.
2(1-30%
kill were considered positive. percent of tested sera with cytotoxicity
of varying
degrees
for the normal
donor
8040%
go-IW%
cyte antiserum and sheep red cell rosette formation) has shown that a significant decrease in both absolute numbers and percent of circulating TL occurs in acute hepatitis. TL function has also been found to be decreased in chronic hepatitis (3,16,17). The present study demonstrates decreased proportions and absolute numbers of circulating TL in such patients. Studies of TL function in chronic carriers of HBAg have yielded conflicting results (2,18). This study has demonstrated normal proportions of TL in two HBAg carriers. In two-thirds of our patients showing TL depression, a discrepancy between IFC or RFC results occurred. In general values for IFC were slightly higher than RFC among normal subjects, however a marked discrepancy has not been observed (19). The observation is at present unexplained but may be an indication that the two techniques identify separate lymphocyte surface membrane receptors (and perhaps different T lymphocyte subpopulations) which show a variable susceptibility to damage by viral infection. Increased BL in patients with acute and chronic hepatitis was an expected finding in view of the known alterations in serum immunoglobulins in these two diseases. However, the degree of increase in total BL in both groups did not match the extent of TL depression- indicating that compared to normal subjects a proportion of the circulating lymphocytes in patients with acute or chronic hepatitis were not identified by the B and T cell markers used. The origin of these “null” cells and their biologic significance is unknown at this time although they may be related to the shedding phenomenon (20) or to rapid endocytosis of the surface markers by these particular lymphocytes (21). Dudley et al. (1) have postulated that the clinical course of viral hepatitis may be determined by the cellular immune competence of the host. Patients who recover have a competent T lymphocyte system, those who become carriers have a defect in this system and those who develop chronic hepatitis occupy an intermediate position with respect to cellular immune functish. Their studies using inhibition of leukocyte migration of HBAg positive serum and liver extract as a measure of cell-mediated immunity to HBAg revealed a decreasing response to these antigens when patients who had recovered from acute hepatitis were compared to those with chronic hepa-
T AND
B LYMPHOCYTES
IN
HEPATITIS
359
titis or healthy carriers (3). Our findings of similar degrees of TL depression in patients with acute or chronic hepatitis of varying severity and normal percentages of TL in healthy carriers of HBAg are not entirely consistent with the above hypothesis-nor can they be explained solely by persistence or clearing of the virus. A recent study (22) showing marked decrease in rosette forming cells in a variety of acute viral infections indicates that the defect is not unique to viral hepatitis. The incidence of autoantibody to smooth muscle in our patients with acute hepatitis (30%) is lower than that reported in two previous studies (23,24) but is similar to that reported by Wright (25). It has been proposed (26) that the development of autoimmune phenomena in viral infection depends on T and B lymphocyte cooperation and that autoantibodies might arise by an increase in “helper” TL or a decrease in “suppressor” TL. Our finding of decreased TL in two-thirds of the patients with autoantibodies could be interpreted as evidence in favor of the latter mechanism. However, similar degrees of TL depression occurred in two-thirds of patients without autoantibodies. The cause of the decrease in circulating TL in the patients studied is unknown. A majority of our patients with acute or chronic hepatitis showed significant lymphocytotoxic antibody. In view of the fact that such antibodies have recently been shown to be TL specific in the case of disseminated lupus (30) it was disappointing to find no clear correlation between relative degrees of lymphocytotoxicity and TL values. No opportunity was afforded to study the patients own lymphocytes for autospecificity of lymphocytotoxins and this could explain the lack of correlation recorded here. Finally, virus may directly alter lymphocyte membrane metabolism or sterically hinder surface membrane receptors and thus prevent rosette formation or attachment of TL antisera. The present study demonstrating a decrease in proportions and absolute numbers of circulating TL in acute viral hepatitis has thus extended earlier observations of a reversible defect in TL function in this disease. Persistence of the defect has been demonstrated in protracted acute hepatitis and in chronic hepatitis of varying severity but no decrease in circulating TL was observed in two HBAg carriers without liver disease. The observations indicate that the T lymphocyte response in viral hepatitis may have relevance both to elimination or persistence of the virus and to the development of continuing liver damage. ACKNOWLEDGMENTS We thank Mrs. Carolyn Henderson and Mrs. Sally Sperry for technical assistance and Dr. H. Danemann for permission to include several of his patients in this study. This work was supported in part by Grants AM 13824-03 and TO1 A100343-03 from the U. S. Public Health Service and in part by the Lael Richards Memorial Fund.
360
DEHORATIUS,
STRICKLAND,
AND
WILLIAMS,
JR.
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