Clin. exp. Immunol. (1976) 25, 389-395.
Cellular immune responses to salivary antigens in autoimmune liver disease with sicca syndrome I. G. McFARLANE, B. M. WOJCICKA, D. C. TSANTOULAS, C. FUNK, B. PORTMANN, A. L. W. F. EDDLE STON & R. W ILL IAMS Liver Unit, King's College Hospital and Medical School, London
(Received 5 April 1976) SUMMARY
Twenty-six patients with primary biliary cirrhosis (PBC) and twenty-two with active chronic hepatitis (ACH) were examined for evidence of the sicca syndrome (keratoconjunctivitis sicca, xerostomia). Measurements of tear flow and total saliva flow showed that at least one sicca feature was present in twenty (77°/%) of the patients with PBC and ten (450%) of those with ACH. Examination of cellular immune responses to a protein fraction of normal human saliva using the leucocyte migration test showed sensitization to the saliva protein in twenty-three of the thirty cases with sicca syndrome but in only two of the eighteen in whom sicca features were not detected. Antisera raised in guinea-pigs against the saliva protein gave specific immunofluorescent staining of bile duct epithelial cells in sections of normal human liver. These findings suggest that damage to structures in the liver may lead to sensitization to various self-antigens which cross-react with other tissues in which a similar disease process may consequently be initiated. INTRODUCTION A systematic survey of multi-system involvement in patients with autoimmune liver disease seen on this Unit, showed that 72% of patients with primary biliary cirrhosis and 42% of those with active chronic hepatitis had xerostomia or keratoconjunctivitis sicca or both (Golding et al., 1970). Similar observations were made by Alarcon-Segovia, Diaz-Jouanen & Fishbein, (1973), who detected at least one ocular or salivary feature of the sicca syndrome in each of fourteen consecutive cases of primary biliary cirrhosis. The association of the sicca syndrome with other diseases in which autoimmunity has been implicated, such as rheumatoid arthritis (Bloch et al., 1965) and systemic lupus erythematosis (Steinberg & Talal, 1971), would be consistent with an underlying immunological reaction probably directed at salivary and lacrymal duct epithelial antigens. To investigate this possibility, the relevant antigens must be purified, but this can be difficult when organ homogenates are used as the starting material. However, we have recently shown that antigens located in bile duct epithelial cells can be obtained from normal human bile (Tsantoulas et al., 1974a; Eddleston et al., 1973) and, by analogy, it seemed likely that antigenic material derived from salivary duct epithelium might be present in saliva. In this paper we describe studies using a protein fraction prepared from normal human saliva as antigen in the leucocyte migration test to investigate possible cellular immune reactions in patients with chronic liver disease, with and without features of the sicca syndrome. The possible occurrence of immunological cross-reactions between antigens in salivary glands and those in the liver as a basis for the development of the sicca syndrome has also been studied. PATIENTS AND METHODS Subjects. Forty-eight patients were investigated, of whom twenty-six had primary biliary cirrhosis and twenty-two had active chronic hepatitis as diagnosed by current clinical, biochemical and histological criteria. At the time of study, the Correspondence: Dr I. G. McFarlane, Liver Unit, King's College Hospital and Medical School, London SE5.
389
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patients were adequately hydrated and were not taking any drugs known to affect salivary or lacrymal gland function. Tear flow was determined by the standard Schirmer test, and keratoconjunctivitis sicca was diagnosed if there was less than 10 mm moistening of the filter paper strips in both eyes after 5 min. Total saliva flow was measured by spitting into a beaker all saliva produced during a 10-min period while chewing mint-flavoured gum. In twenty normal subjects, the mean saliva flow was 242 ml/10 min (s.d.= 5.9). A diagnosis of xerostomia was made when the saliva flow rates were less than 12-4 ml/10 min (2 s.d. below the mean for normal subjects). Patients who were found to have keratoconjunctivitis sicca or xerostomia, or both, were considered to have the sicca syndrome. Preparation of antigens. Pooled saliva, collected as already described from five normal subjects, was dialysed overnight against ten volumes of phosphate-buffered saline, pH 7-2 (PBS) at 4VC. Insoluble material was removed by centrifugation and proteins precipitated from the supernatant with ammonium sulphate. The fraction precipitating between 30% and 50% saturation was redissolved in water, dialysed against 3 x 100 volumes of water over 72 hr at 4VC and treated again with ammonium sulphate to a saturation level of 30%. Insoluble material was removed by centrifugation and the supernatant dialysed against 5 x 100 volumes of PBS over 72 hr at 4VC. The resulting solution was designated salivary antigen and the protein concentration measured according to Lowry et al. (1951). Four separate preparations, which appeared immunologically to be identical, were used during the course of this work. Bile antigen was prepared by gel filtration over Sephadex G-50 (Eddleston et al., 1973) from pooled post-mortem samples of gall bladder bile. Homogenates of submaxillary gland, thyroid, adrenal, and kidney were prepared from post-mortem specimens of apparently normal organs as 40%Y (w/v) homogenates in 0-25 M sucrose, using a Potter homogenizer. Particulate matter was removed by centrifugation at 2000 g for 15 min and the supernatants used for immunodiffusion studies. Leucocyte migration test. The technique used for the leucocyte migration test was that described by Mitchell et al. (1972). Peripheral blood leucocytes were washed with Hanks's balanced salt solution and suspended in Waymouth's medium (Wellcome) containing 10% Bobby calf serum (Gibco-BioCult). The latter was used because we have found that it is not subject to the variations usually encountered between different batches of foetal bovine serum (Mitchell & Eddleston, 1973). The leucocytes were allowed to migrate out ofcapillary tubes for 20 hrat 37C into tissue culture chambers containing Waymouth's medium with 10%Y Bobby calf serum with, or without, the relevant test antigens. Migration indices were calculated as the ratios between the areas of migration in test and control chambers. The antigen concentrations used were the maximum which gave migration indices greater than 0-80 in three normal subjects. For the salivary antigen preparation this was found to be 50 ug/ml and for bile antigen 100 ,ug/ml of culture medium. Measurements of migration indices in twenty normal subjects were then used to determine the normal range (mean± 2 s.d.) for each antigen. Lower and upper limits for salivary antigen were 0 74 and 1-02, and for bile antigen were 0-78 and 1 01. Indices which fell below or above these limits were regarded as significant inhibition or stimulation of migration respectively. Antisera and immunofluorescence. Antisera against salivary and bile antigen preparations were raised in guinea-pigs by injecting subcutaneously 2-5 mg protein per animal in Freund's complete adjuvant. Three weeks later a similar injection, without Freund's adjuvant, was given. Animals were bled 1 week after the second injection and the sera exhaustively absorbed against freeze-dried pooled normal human plasma. Double immunodiffusion studies were performed in 1%4 agarose in PBS in polystyrene Petri dishes lightly coated with silicone grease. After incubation at 37°C for 18 hr, plates were washed with several changes of 0.9 0 sodium chloride containing 0.05%/ sodium azide, for 48 hr at 37°C, and then dried at this temperature overnight. The plates were then stained at room temperature for 30 min with 0-2y% Bromophenol Blue in methanol:acetic acid:water (43:5:52) and destained in ethanol: acetic acid: water (30:5:65). Immunofluorescence studies on cryostat sections ofliver were performed by a standard indirect technique using fluoresceinconjugated rabbit anti-guinea-pig immunoglobulin (Wellcome).
RESULTS Only eight of the forty-eight patients complained spontaneously of symptoms attributable to the sicca syndrome. These eight patients were found to have both keratoconjunctivitis sicca and xerostomia. The underlying disease in seven of these patients was primary biliary cirrhosis. Four ofthem also had rheumatoid arthritis, and could therefore be considered as cases of Sj6gren's syndrome. Testing of the remaining forty asymptomatic patients revealed that five had keratoconjunctivitis sicca, eight had xerostomia and nine had both lesions. Although there was not complete agreement between the two tests, it is of interest that the patients with keratoconjunctivitis sicca had significantly decreased (P< 0001) salivary flow rates when compared with those in whom the Schirmer test was negative (mean values of 7-8 and 15-9 ml/10 min respectively) (Fig. 1). In the complete series, sicca syndrome was found in thirty cases (Table 1), of whom twenty had primary biliary cirrhosis and ten had active chronic hepatitis. Keratoconjunctivitis sicca was found more often in patients with primary biliary cirrhosis (eighteen out of twenty-six) than in those with active chronic hepatitis (four of twenty-two) (P< 0.005).
Salivary Ag in liver disease
391
40-
30_ E:
*
0
20 _
:
0
:
._.............................
....................
..........
10 _
Normal
subjects
Negative
Sch irmer test
Positive Schirmer test
All patients
FIG. 1. Results of the measurements of total saliva flow in normal subjects and in patients with negative and ). The dotted line shows the lower positive Schirmer tests. The mean for each group is shown as ( normal limit which is 2 s.d. below the mean of the normal subjects.
In the primary biliary cirrhosis group of patients there were no apparent differences in clinical or biochemical features, or in the duration of the liver disease, between those with sicca features and those without. However, in the patients with active chronic hepatitis nine of the ten with sicca syndrome had cirrhosis compared with only five of the twelve in whom sicca features were absent (P< 0-05). There was also a significant difference in sex distribution among the patients with active chronic hepatitis. The ten patients with sicca syndrome were female compared with only five of the twelve cases in whom sicca features were not detected (P 0 2).
Characterization ofsalivary antigen Immunodiffusion studies revealed that the salivary antigen preparation contained at least four antigenic components which were not plasma proteins (Fig. 2a), two of which appeared to cross-react with
b
it
FIG. 2. Double immunodiffusion using guinea-pig antisera against: (a) salivary antigen and (b) bile antigen in centre wells. Peripheral wells contain: salivary antigen (I and II); salivary gland homogenate (III and IV); bile antigen (V) and normal human plasma (VI). Antisera were exhaustively absorbed against normal human plasma before use.
Salivary Ag in liver disease
393
FIG. 3. Sections of normal human liver showing (a) septal and (b) interlobular bile ducts stained by indirect immunofluorescence with guinea-pig antiserum against (a) salivary antigen and (b) bile antigen. (Original magnification x 320.) Staining of serial sections by H & E revealed that the dark spaces in the bile duct epithelial cells are mainly cytoplasm-the nuclei occupying only approximately one-fifth of these areas and located at the bases of the cells.
material in salivary gland homogenate. The antiserum against the salivary antigen did not react on immunodiffusion with the bile antigen, nor did it react with homogenates of thyroid, adrenal, or kidney. Conversely, antiserum against the bile antigen did not react with the salivary antigen, nor with a salivary gland homogenate (Fig. 2b). However, when indirect immunofluorescence was performed using the antiserum against the salivary antigen on sections of normal human liver, fluorescent staining of the bile duct epithelium was observed (Fig. 3a). Staining appeared to be associated with the membranes of the bile duct epithelial cells rather than with the cytoplasm, but the intensity of fluorescence was not as great as that obtained when antiserum against the bile antigen was used (Fig. 3b). Fluorescent staining with antiserum against salivary antigen was specific in that the staining pattern in liver was not observed when the antiserum was first absorbed with salivary antigen.
DISCUSSION The systemic manifestations most frequently associated with primary biliary cirrhosis and active chronic hepatitis are the sicca syndrome and renal tubular acidosis (Golding, Smith & Williams, 1973). The high frequency of these manifestations is not generally appreciated because patients may not spontaneously complain of the associated symptoms. This, indeed, was the case in the present study where diminished
394
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tear flow and/or saliva flow was revealed in twenty-two patients only after application of the appropriate tests. When the sicca syndrome is associated with other conditions, such as rheumatoid arthritis (Sjdgren's syndrome), a number of abnormal immunological features have been documented, including periductular lymphocytic infiltration of the affected glands (Chisholm & Mason, 1968; Anderson et al., 1973) and circulating antibodies to salivary and lacrymal duct antigens (Bertram & Halberg, 1964; MacSween et al., 1967; Feltkamp & van Rossum, 1968; Whaley et al., 1969). In addition, S0berg & Bertram (1968) have demonstrated cellular immune responses to a saline extract of salivary gland in 5700 of patients with Sjdgren's syndrome and, in an earlier study, we were able to show similar immune responses to a salivary gland homogenate in 42% of a group of patients in whom sicca syndrome was secondary to chronic liver disease (Smith et al., 1972). The greater frequency of sensitization (770%) in the present study might suggest a higher concentration of relevant antigens in the salivary protein fraction as compared with the organ extracts used previously. Further fractionation and purification of the salivary protein preparation is required to determine which antigen is active in the leucocyte migration test and its precise location within the salivary gland. However, the immunodiffusion studies revealed that two of the four salivary antigens were present in a salivary gland homogenate and, by analogy with the bile antigen in bile (Tsantoulas et al., 1974a), it is possible that one or both of these antigens may be derived from the salivary duct epithelium. The high frequency of sicca syndrome and renal tubular acidosis in active chronic hepatitis and primary biliary cirrhosis, both of which are thought to be autoimmune diseases, suggests that an immunological reaction involving antigens in the liver may be related in some way to the pathogenesis of these associated conditions. One possibility is that immunologically mediated damage to structures in the liver may lead to sensitization to a number ofself-antigens which cross-react with other tissues in which a similar disease process may consequently be initiated. We have recently shown that there is material in the liver cell membrane which cross-reacts with a renal tubular antigen (Tamm-Horsfall glycoprotein) and that sensitization to this glycoprotein is associated with the presence of renal tubular acidosis (Tsantoulas et al., 1974b). Furthermore, lymphocytes from patients with chronic liver disease and renal tubular acidosis are cytotoxic to baby hamster kidney cells and Tamm-Horsfall glycoprotein appears to be the target antigen involved in this cytotoxic reaction (Cochrane et al., 1975). The present finding, by immunofluorescence, that guinea-pig anti sera to the salivary antigen preparation stain bile duct epithelial cells (in sections of normal human liver), suggests that there is cross-reaction between salivary gland material and bile duct epithelium. However, there was no correlation between sensitization to bile antigen and salivary antigen in our patients and the immunodiffusion studies indicated that the antigenic material in saliva is distinct from that found in bile. Thus, the cross-reaction detected by immunofluorescence must involve other antigens in the bile duct epithelium. If sensitization to this cross-reacting material develops as a consequence of immunologically mediated damage to bile ducts, the high frequency of the sicca syndrome in primary biliary cirrhosis would be explained. In active chronic hepatitis the primary lesion is usually regarded as being confined to the hepatocytes. However, it is recognized that there is an immunological and histological overlap between this condition and primary biliary cirrhosis. Indeed, bile duct lesions have been reported in active chronic hepatitis (Popper & Schaffner, 1970; Christoffersen et aL., 1972), particularly in the later stages of the disease. In the present study, although bile duct lesions were not seen in the needle biopsy specimens available, it is of interest that sicca features were found more frequently in those patients who had already progressed to
cirrhosis. We are grateful to the Wellcome Trust for continued support.
REFERENCES ALARC6N-SEGOVIA, D., DIAZ-JOUANEN, E. & FISHBEIN, E. (1973). Features of Sjogren's Syndrome in primary biliary cirrhosis. Ann. intern. Med. 79, 31. ANDERSON, L.G., TARPLEY, T.M., TALAL, N., CUMMINGS,
N.A., WOLF, R.O. & SCHALL, G.L. (1973) Cellularversus-humoral autoimmune responses to salivary gland in Sjogren's Syndrome. Clin. exp. Immunol. 13, 335. BERTRAM, U. & HALBERG, P. (1964) A specific antibody
Salivary Ag in liver disease against the epithelium of the salivary ducts in sera from patients with Sjbgren's Syndrome. Acta allergol. 19, 458. BLOCH, K.J., BUCHANAN, W.W., WoHL, M.J. & BUNIM, J.J. (1965) Sjogren's Syndrome. A clinical, pathological, and serological study of sixty-two cases. Medicine (Balt.), 44, 187. CHISHOLM, D.M. & MASON, D.K. (1968) Labial salivary gland biopsy in Sjogren's disease. 7. clin. Path. 21, 656. CHRISTOFFERSEN, P., PouLsEN, H. & ScHEuER, P.J. (1972) Abnormal bile duct epithelium in chronic aggressive hepatitis and primary biliary cirrhosis. Human Path. 3, 227. COCHRANE, A.M.G., TSANTOULAS, D.C., MoussouRos, A., McFARLANE, I.G., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1975) Lymphocyte cytotoxicity for kidney cells in the renal tubular acidosis ofautoimmune liver disease. Gut, 16, 828 (abstract). EDDLESTON, A.L.W.F., McFARLANE, I.G., MITCHELL, C.G., REED, W.D. & WILLIAMS, R. (1973) Cell-mediated immune response in primary biliary cirrhosis to a protein fraction from human bile. Brit. med. 3. iv, 274. FELTKAMP, T.E.W. & VAN ROSSUM, A.L. (1968) Antibodies to salivary duct cells and other autoantibodies in patients with Sjogren's Syndrome and other idiopathic autoimmune diseases. Clin. exp. Immunol. 3, 1. GOLDING, P.L., BowN, R., MASON, A.M.S. & TAYLOR, E. (1970) 'Sicca complex' in liver disease. Brit. med. ]. iv, 340. GOLDING, P.L., SMITH, M. & WILLIAMS, R. (1973) Multisystem involvement in chronic liver disease: studies on the incidence and pathogenesis. Amer. 7. Med. 55, 772. LowRy, O.H., ROSENBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) Protein measurement with the Folin phenolreagent. ]. biol. Chem. 193, 265. MACSWEEN, R.N.M., GOUDIE, R.B., ANDERSON, J.R., ARMSTRONG, E., MURRAY, M.A., MASON, D.K., JASANI, M.K., BOYLE, J.A., BUCHANAN, W.W. & WILLIAMSON, J. (1967) Occurrence of antibody to salivary duct epithelium in Siogren's disease, rheumatoid arthritis and
395
other arthritides. A clinical and laboratory study. Ann. rheum. Dis. 26, 402. MITCHELL, C.G. & EDDLESTON, A.L.W.F. (1973) The importance of selecting suitable foetal calf serum for use in the leucocyte migration test. Transplantation, 16, 689. MITCHELL, C.G., SMITH, M.G.M., GOLDING, P.L., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1972) Evaluation of the leucocyte migration test as a measure of delayed hypersensitivity in man. Clin. exp. Immunol. 11, 535. POPPER, H. & SCHAFFNER, F. (1970) Non-suppurative destructive chronic cholangitis and chronic hepatitis. Progress in Liver Diseases volume 3 (ed. by H. Popper & F. Schaffner), p. 336. Grune and Stratton, New York. SMITH, M.G.M., GOLDING, P.L., EDDLESTON, A.L.W.F., MITCHELL, C.G., KEMP, A. & WILLIAMS, R. (1972) Cell-mediated immune responses in chronic liver diseases. Brit. med. 3. i, 527. S0BERG, M. & BERTRAM, U. (1968) Cellular hypersensitivity in Sjogren's syndrome. Acta med. scand. 184, 319. STEINBERG, A.D. & TALAL, N. (1971) Coexistence of Sjogren's Syndrome and systemic lupus erythematosis.
Amer. intern. med., 74, 55. TSANTOULAS, D.C., McFARLANE, I.G., PORTMANN, B., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1974a) Sensitisation to human bile proteins in primary biliary cirrhosis and active chronic hepatitis: Demonstration of antigenic material in bile duct epithelial cells. Digestion, 10, 305 (abstract). TSANTOULAS, D.C., MCFARLANE, I.G., EDDLESTON, A.L.W.F. & WILLIAMS, R. (1974b) Cell-mediated immunity to human Tamm-Horsfall glycoprotein in autoimmune liver disease with renal tubular acidosis. Brit. med. 3., iv, 491. WHALEY, K., CHISHOLM, D.M., GouDIE, R.B., DOWNIE, W.W., DICK, W.C., BOYLE, J.A. & WILLIAMSON, J. (1969) Salivary duct autoantibody in Sjogren's Syndrome. Correlation with focal sialodenitis in the labial mucosa. Clin. exp. Immunol., 4, 273.
Cellular immune responses to salivary antigens in autoimmune liver disease with sicca syndrome I. G. McFARLANE, B. M. WOJCICKA, D. C. TSANTOULAS, C. FUNK, B. PORTMANN, A. L. W. F. EDDLE STON & R. W ILL IAMS Liver Unit, King's College Hospital and Medical School, London
(Received 5 April 1976) SUMMARY
Twenty-six patients with primary biliary cirrhosis (PBC) and twenty-two with active chronic hepatitis (ACH) were examined for evidence of the sicca syndrome (keratoconjunctivitis sicca, xerostomia). Measurements of tear flow and total saliva flow showed that at least one sicca feature was present in twenty (77°/%) of the patients with PBC and ten (450%) of those with ACH. Examination of cellular immune responses to a protein fraction of normal human saliva using the leucocyte migration test showed sensitization to the saliva protein in twenty-three of the thirty cases with sicca syndrome but in only two of the eighteen in whom sicca features were not detected. Antisera raised in guinea-pigs against the saliva protein gave specific immunofluorescent staining of bile duct epithelial cells in sections of normal human liver. These findings suggest that damage to structures in the liver may lead to sensitization to various self-antigens which cross-react with other tissues in which a similar disease process may consequently be initiated. INTRODUCTION A systematic survey of multi-system involvement in patients with autoimmune liver disease seen on this Unit, showed that 72% of patients with primary biliary cirrhosis and 42% of those with active chronic hepatitis had xerostomia or keratoconjunctivitis sicca or both (Golding et al., 1970). Similar observations were made by Alarcon-Segovia, Diaz-Jouanen & Fishbein, (1973), who detected at least one ocular or salivary feature of the sicca syndrome in each of fourteen consecutive cases of primary biliary cirrhosis. The association of the sicca syndrome with other diseases in which autoimmunity has been implicated, such as rheumatoid arthritis (Bloch et al., 1965) and systemic lupus erythematosis (Steinberg & Talal, 1971), would be consistent with an underlying immunological reaction probably directed at salivary and lacrymal duct epithelial antigens. To investigate this possibility, the relevant antigens must be purified, but this can be difficult when organ homogenates are used as the starting material. However, we have recently shown that antigens located in bile duct epithelial cells can be obtained from normal human bile (Tsantoulas et al., 1974a; Eddleston et al., 1973) and, by analogy, it seemed likely that antigenic material derived from salivary duct epithelium might be present in saliva. In this paper we describe studies using a protein fraction prepared from normal human saliva as antigen in the leucocyte migration test to investigate possible cellular immune reactions in patients with chronic liver disease, with and without features of the sicca syndrome. The possible occurrence of immunological cross-reactions between antigens in salivary glands and those in the liver as a basis for the development of the sicca syndrome has also been studied. PATIENTS AND METHODS Subjects. Forty-eight patients were investigated, of whom twenty-six had primary biliary cirrhosis and twenty-two had active chronic hepatitis as diagnosed by current clinical, biochemical and histological criteria. At the time of study, the Correspondence: Dr I. G. McFarlane, Liver Unit, King's College Hospital and Medical School, London SE5.
389
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patients were adequately hydrated and were not taking any drugs known to affect salivary or lacrymal gland function. Tear flow was determined by the standard Schirmer test, and keratoconjunctivitis sicca was diagnosed if there was less than 10 mm moistening of the filter paper strips in both eyes after 5 min. Total saliva flow was measured by spitting into a beaker all saliva produced during a 10-min period while chewing mint-flavoured gum. In twenty normal subjects, the mean saliva flow was 242 ml/10 min (s.d.= 5.9). A diagnosis of xerostomia was made when the saliva flow rates were less than 12-4 ml/10 min (2 s.d. below the mean for normal subjects). Patients who were found to have keratoconjunctivitis sicca or xerostomia, or both, were considered to have the sicca syndrome. Preparation of antigens. Pooled saliva, collected as already described from five normal subjects, was dialysed overnight against ten volumes of phosphate-buffered saline, pH 7-2 (PBS) at 4VC. Insoluble material was removed by centrifugation and proteins precipitated from the supernatant with ammonium sulphate. The fraction precipitating between 30% and 50% saturation was redissolved in water, dialysed against 3 x 100 volumes of water over 72 hr at 4VC and treated again with ammonium sulphate to a saturation level of 30%. Insoluble material was removed by centrifugation and the supernatant dialysed against 5 x 100 volumes of PBS over 72 hr at 4VC. The resulting solution was designated salivary antigen and the protein concentration measured according to Lowry et al. (1951). Four separate preparations, which appeared immunologically to be identical, were used during the course of this work. Bile antigen was prepared by gel filtration over Sephadex G-50 (Eddleston et al., 1973) from pooled post-mortem samples of gall bladder bile. Homogenates of submaxillary gland, thyroid, adrenal, and kidney were prepared from post-mortem specimens of apparently normal organs as 40%Y (w/v) homogenates in 0-25 M sucrose, using a Potter homogenizer. Particulate matter was removed by centrifugation at 2000 g for 15 min and the supernatants used for immunodiffusion studies. Leucocyte migration test. The technique used for the leucocyte migration test was that described by Mitchell et al. (1972). Peripheral blood leucocytes were washed with Hanks's balanced salt solution and suspended in Waymouth's medium (Wellcome) containing 10% Bobby calf serum (Gibco-BioCult). The latter was used because we have found that it is not subject to the variations usually encountered between different batches of foetal bovine serum (Mitchell & Eddleston, 1973). The leucocytes were allowed to migrate out ofcapillary tubes for 20 hrat 37C into tissue culture chambers containing Waymouth's medium with 10%Y Bobby calf serum with, or without, the relevant test antigens. Migration indices were calculated as the ratios between the areas of migration in test and control chambers. The antigen concentrations used were the maximum which gave migration indices greater than 0-80 in three normal subjects. For the salivary antigen preparation this was found to be 50 ug/ml and for bile antigen 100 ,ug/ml of culture medium. Measurements of migration indices in twenty normal subjects were then used to determine the normal range (mean± 2 s.d.) for each antigen. Lower and upper limits for salivary antigen were 0 74 and 1-02, and for bile antigen were 0-78 and 1 01. Indices which fell below or above these limits were regarded as significant inhibition or stimulation of migration respectively. Antisera and immunofluorescence. Antisera against salivary and bile antigen preparations were raised in guinea-pigs by injecting subcutaneously 2-5 mg protein per animal in Freund's complete adjuvant. Three weeks later a similar injection, without Freund's adjuvant, was given. Animals were bled 1 week after the second injection and the sera exhaustively absorbed against freeze-dried pooled normal human plasma. Double immunodiffusion studies were performed in 1%4 agarose in PBS in polystyrene Petri dishes lightly coated with silicone grease. After incubation at 37°C for 18 hr, plates were washed with several changes of 0.9 0 sodium chloride containing 0.05%/ sodium azide, for 48 hr at 37°C, and then dried at this temperature overnight. The plates were then stained at room temperature for 30 min with 0-2y% Bromophenol Blue in methanol:acetic acid:water (43:5:52) and destained in ethanol: acetic acid: water (30:5:65). Immunofluorescence studies on cryostat sections ofliver were performed by a standard indirect technique using fluoresceinconjugated rabbit anti-guinea-pig immunoglobulin (Wellcome).
RESULTS Only eight of the forty-eight patients complained spontaneously of symptoms attributable to the sicca syndrome. These eight patients were found to have both keratoconjunctivitis sicca and xerostomia. The underlying disease in seven of these patients was primary biliary cirrhosis. Four ofthem also had rheumatoid arthritis, and could therefore be considered as cases of Sj6gren's syndrome. Testing of the remaining forty asymptomatic patients revealed that five had keratoconjunctivitis sicca, eight had xerostomia and nine had both lesions. Although there was not complete agreement between the two tests, it is of interest that the patients with keratoconjunctivitis sicca had significantly decreased (P< 0001) salivary flow rates when compared with those in whom the Schirmer test was negative (mean values of 7-8 and 15-9 ml/10 min respectively) (Fig. 1). In the complete series, sicca syndrome was found in thirty cases (Table 1), of whom twenty had primary biliary cirrhosis and ten had active chronic hepatitis. Keratoconjunctivitis sicca was found more often in patients with primary biliary cirrhosis (eighteen out of twenty-six) than in those with active chronic hepatitis (four of twenty-two) (P< 0.005).
Salivary Ag in liver disease
391
40-
30_ E:
*
0
20 _
:
0
:
._.............................
....................
..........
10 _
Normal
subjects
Negative
Sch irmer test
Positive Schirmer test
All patients
FIG. 1. Results of the measurements of total saliva flow in normal subjects and in patients with negative and ). The dotted line shows the lower positive Schirmer tests. The mean for each group is shown as ( normal limit which is 2 s.d. below the mean of the normal subjects.
In the primary biliary cirrhosis group of patients there were no apparent differences in clinical or biochemical features, or in the duration of the liver disease, between those with sicca features and those without. However, in the patients with active chronic hepatitis nine of the ten with sicca syndrome had cirrhosis compared with only five of the twelve in whom sicca features were absent (P< 0-05). There was also a significant difference in sex distribution among the patients with active chronic hepatitis. The ten patients with sicca syndrome were female compared with only five of the twelve cases in whom sicca features were not detected (P 0 2).
Characterization ofsalivary antigen Immunodiffusion studies revealed that the salivary antigen preparation contained at least four antigenic components which were not plasma proteins (Fig. 2a), two of which appeared to cross-react with
b
it
FIG. 2. Double immunodiffusion using guinea-pig antisera against: (a) salivary antigen and (b) bile antigen in centre wells. Peripheral wells contain: salivary antigen (I and II); salivary gland homogenate (III and IV); bile antigen (V) and normal human plasma (VI). Antisera were exhaustively absorbed against normal human plasma before use.
Salivary Ag in liver disease
393
FIG. 3. Sections of normal human liver showing (a) septal and (b) interlobular bile ducts stained by indirect immunofluorescence with guinea-pig antiserum against (a) salivary antigen and (b) bile antigen. (Original magnification x 320.) Staining of serial sections by H & E revealed that the dark spaces in the bile duct epithelial cells are mainly cytoplasm-the nuclei occupying only approximately one-fifth of these areas and located at the bases of the cells.
material in salivary gland homogenate. The antiserum against the salivary antigen did not react on immunodiffusion with the bile antigen, nor did it react with homogenates of thyroid, adrenal, or kidney. Conversely, antiserum against the bile antigen did not react with the salivary antigen, nor with a salivary gland homogenate (Fig. 2b). However, when indirect immunofluorescence was performed using the antiserum against the salivary antigen on sections of normal human liver, fluorescent staining of the bile duct epithelium was observed (Fig. 3a). Staining appeared to be associated with the membranes of the bile duct epithelial cells rather than with the cytoplasm, but the intensity of fluorescence was not as great as that obtained when antiserum against the bile antigen was used (Fig. 3b). Fluorescent staining with antiserum against salivary antigen was specific in that the staining pattern in liver was not observed when the antiserum was first absorbed with salivary antigen.
DISCUSSION The systemic manifestations most frequently associated with primary biliary cirrhosis and active chronic hepatitis are the sicca syndrome and renal tubular acidosis (Golding, Smith & Williams, 1973). The high frequency of these manifestations is not generally appreciated because patients may not spontaneously complain of the associated symptoms. This, indeed, was the case in the present study where diminished
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tear flow and/or saliva flow was revealed in twenty-two patients only after application of the appropriate tests. When the sicca syndrome is associated with other conditions, such as rheumatoid arthritis (Sjdgren's syndrome), a number of abnormal immunological features have been documented, including periductular lymphocytic infiltration of the affected glands (Chisholm & Mason, 1968; Anderson et al., 1973) and circulating antibodies to salivary and lacrymal duct antigens (Bertram & Halberg, 1964; MacSween et al., 1967; Feltkamp & van Rossum, 1968; Whaley et al., 1969). In addition, S0berg & Bertram (1968) have demonstrated cellular immune responses to a saline extract of salivary gland in 5700 of patients with Sjdgren's syndrome and, in an earlier study, we were able to show similar immune responses to a salivary gland homogenate in 42% of a group of patients in whom sicca syndrome was secondary to chronic liver disease (Smith et al., 1972). The greater frequency of sensitization (770%) in the present study might suggest a higher concentration of relevant antigens in the salivary protein fraction as compared with the organ extracts used previously. Further fractionation and purification of the salivary protein preparation is required to determine which antigen is active in the leucocyte migration test and its precise location within the salivary gland. However, the immunodiffusion studies revealed that two of the four salivary antigens were present in a salivary gland homogenate and, by analogy with the bile antigen in bile (Tsantoulas et al., 1974a), it is possible that one or both of these antigens may be derived from the salivary duct epithelium. The high frequency of sicca syndrome and renal tubular acidosis in active chronic hepatitis and primary biliary cirrhosis, both of which are thought to be autoimmune diseases, suggests that an immunological reaction involving antigens in the liver may be related in some way to the pathogenesis of these associated conditions. One possibility is that immunologically mediated damage to structures in the liver may lead to sensitization to a number ofself-antigens which cross-react with other tissues in which a similar disease process may consequently be initiated. We have recently shown that there is material in the liver cell membrane which cross-reacts with a renal tubular antigen (Tamm-Horsfall glycoprotein) and that sensitization to this glycoprotein is associated with the presence of renal tubular acidosis (Tsantoulas et al., 1974b). Furthermore, lymphocytes from patients with chronic liver disease and renal tubular acidosis are cytotoxic to baby hamster kidney cells and Tamm-Horsfall glycoprotein appears to be the target antigen involved in this cytotoxic reaction (Cochrane et al., 1975). The present finding, by immunofluorescence, that guinea-pig anti sera to the salivary antigen preparation stain bile duct epithelial cells (in sections of normal human liver), suggests that there is cross-reaction between salivary gland material and bile duct epithelium. However, there was no correlation between sensitization to bile antigen and salivary antigen in our patients and the immunodiffusion studies indicated that the antigenic material in saliva is distinct from that found in bile. Thus, the cross-reaction detected by immunofluorescence must involve other antigens in the bile duct epithelium. If sensitization to this cross-reacting material develops as a consequence of immunologically mediated damage to bile ducts, the high frequency of the sicca syndrome in primary biliary cirrhosis would be explained. In active chronic hepatitis the primary lesion is usually regarded as being confined to the hepatocytes. However, it is recognized that there is an immunological and histological overlap between this condition and primary biliary cirrhosis. Indeed, bile duct lesions have been reported in active chronic hepatitis (Popper & Schaffner, 1970; Christoffersen et aL., 1972), particularly in the later stages of the disease. In the present study, although bile duct lesions were not seen in the needle biopsy specimens available, it is of interest that sicca features were found more frequently in those patients who had already progressed to
cirrhosis. We are grateful to the Wellcome Trust for continued support.
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