Clinical and Experimental Dermatology (1977) 2, 161.
Leukocyte migration inhibition studies in sarcoidosis with evidence of a serum blocking factor
A. E. ROSLING, BRIAN WATSON AND E.L. RHODES Department of Dermatology, St Helier Hospital, Wrythe Lane, Carshalton, Surrey SM5 ] AA
Accepted for publication 30 November 1976
Summary Washed leukocytes from sarcoid patients were not found to be significantly hyporeactive to phytohaemagglutinin in the mixed leukocyte migration test. An immune blocking factor, however, was found in the sera of these patients which suppressed the reaction of normal subject's white cells to phytohaemagglutinin.
One of the main features of sarcoidosis is the cutaneous anergy to various skin test antigens (Scadding, 1967). Hirschorn et al. (1964) and Topilsky et al. (1972) found in vitro hyporeactivity to several antigens and mitogens. Horsmanheimo & Virolainen (1974) us^'^ ^^ thymidine as a measure of blast cell transformation and found the response to phytohaemagglutinin (PHA) to be the same in a sarcoid and a control group. To explain this, Caspary & Field (1971) have suggested that there is an immunological depressant in sarcoid sera which prevents lymphocytes responding to antigens. Using the lymphocyte transformation test to PHA, Belcher, Carney & Nankervis (1974) found such a factor in the sera of 7 of 14 sarcoid patients. The mixed leukocyte migration test is now considered to be a genuine test of cellular hypersensitivity (Seborg & Bendixen, 1967), the lymphokines produced by the stimulated lymphocytes preventing the migration of leukocytes. In this study, the mixed leukocyte migration test was used to evaluate the in vitro response of washed sarcoid lymphocytes to PHA. The effect also of sera from sarcoid patients on the migration of leukocytes with and without PHA was noted. Materials and method Twenty sarcoid patients were investigated with ages ranging from 25 to 68 years. Ten of rhe i6t
i62
A.E.Rosling, B.Watson and E.L.Rhodes
twenty were classified as active and two of these had skin lesions. All were Kveim positive. Clitiically normal controls were taken from the hospital staff. The leukocyte migration inhibition test Approximately 30 ml whole blood containing 25 units ml"' mucous heparin were allowed to settle under gravity for i h at 37X. The leukocyte rich plasma was removed and the cells washed 3 times at 37 C in Hanks BSS (Wellcome). The resultant pellet of cells was resuspended in sufficient TC 199 (Wellcome), containing 10% horse serum to fill the number of capillary tubes (Microhaematocrit Gelman-Hawksley) necessary to perform the tests (10-15 tubes). The cell suspension was taken up into capillary tubes, sealed at one end with Cristaseal (Hawksley) and spun at 360 g for 10 min. The capillary tubes were cut i mm above the cell medium interface and fixed in the wells of the migration plates with a dab of silicone grease. TC 199 with 10% horse serum either alone or containing 5 /jg ml"' PHA was added, each test being set up in triplicate. The wells were sealed with cover slips and silicone grease and incubated at 37'C for 18 h. Migration areas were measured by projection microscopy and planimetry. Cross over experiments were performed using 10% horse serum, 10% control serum and 10% sarcoid serum with and without 5 //g ml"' PHA on blood group 'O' leukocytes from healthy donors.
1,2
1.0
0.8
"a O.A
Figure i. Mixed leukocytes migration test to PHA in control and sareoid patients. A migration index of less than 08 indieates hypersensitivity to the stimulus given. • , Sarcoid leukocytes; o, control leukocytes.
Leukocyte migration inhibition in sarcoidosis
163
Results All results were expressed as migration ratios. As a general test for lymphocyte function the responses of washed sarcoid and control lymphocytes, in horse serum, to PHA were measured and the results expressed: Migration ratio ^
Migration area + PHA Migration area ~ PHA
An arbitrary level of 0 8 and above was taken as showing no migration inhibition. As shown in Fig. i there was a tendency for the sarcoid leukocytes to be slightly less reactive than the control leukocytes but this difference was not significant and no ratios greater than 08 were scored. Two migration ratios were calculated for the experiments involving control and sarcoid sera added to donor group 'O' leukocytes:
16
1.2
1.0
08
0,6
0.2
(a)
(b)
Figure 2. Using blood group 'O' leukocytes, the effect of sareoid sera and eontrol sera in the mixed leukoeyte migration test (a) without PHA, (b) with the addition of PHA. Mean +1 standard deviation shown. • , Sarcoid sera; c, eontrol sera.
164
A.E.Rosling, B.Watson and E.L.Rhodes Migration ratio
Migration area in test sera Migration area in horse serum
(i)
These ratios are shown in Fig. 2a and there is no difference in the migration of donor 'O' leukocytes in the presence of sarcoid or control sera. In both cases the mean (0 94) and the standard deviation (03) were the same and the distribution about i-o was similar. Migration area in test sera + PHA Migration ratio ^ Migration area in horse serum + PHA
(2)
Fig. 2b shows these ratios and there is a significant difference (P — 0-025) between the mean of the sarcoid sera group (1-22 + 0-4) and the mean of the control sera group (0-91 ±0-3). This means that there was less of a response to PHA by the donor 'O' leukocytes in the presence of sarcoid sera compared with control sera. 10/15 (67/0) of the sarcoid sera and only 7/18 (39%) of the control sera showed ratios above i. This gives a significant heterogeneity x^ result (P = o 01) and would be compatible with a blocking effect in these sera. Discussion These experiments show that washed sarcoid lymphocytes, compared with a control population, exhibit only slight hyporeactivity towards PHA. All sarcoid responses were below 08, the level beneath which inhibition is said to have occurred and were not significantly different from the controls. Washed lymphocytes from sarcoidosis patients do not appear in this case to have an in vitro defect in lymphokine production. To explain the paradox of a failure to respond in vivo to skin test antigens, the sera from sarcoid patients may be said to contain 'blocking' factors. Sarcoid sera when added without PHA to normal leukocytes behaved in a manner similar to the control sera in that there was a large spread of results with similar migration indices in both groups. When the same sarcoid sera were added to washed blood group 'O' leukocytes in the presence of 5 /jg ml" ' PHA, a significant difference in response was noted. Blocking activity was seen in some sarcoid sera compared with control sera. This was not a toxic inhibitory effect since the cells migrated further in sarcoid sera than they did in horse serum. It appears therefore, that there is some substance in sarcoid sera which prevents the interaction between PHA and the leucocytes. The blocking of the reaction to PHA could occur at three stages: (i) Lymphocyte recognition of mitogen; (2) Lymphokine synthesis; (3) Reaction of migrating cells with lymphokine. Gross (1973) injected lymphokine intradermally into sarcoid patients and found no response. This would support (3) as an explanation of the anergy in sarcoidosis. More recently, Maderazo et al. (1976) detected high levels in 19/20 sarcoid patients of a constituent found only in low levels in normal individuals which was directed against the neutrophil chemotactic factor produced by lymphocytes. This, or a similar factor, could be what was detected in sarcoid sera.
Leukocyte migration inhibition in sarcoidosis
165
Feed-back suppression by T cell products has been suggested by Kantor (1975) as an explanation for anergy in cell mediated immunity reactions where T cell numbers are normal. Anergy in infections, both bacterial and viral is known to occur. A similar situation may be present in sarcoidosis.
Acknowledgments We would like to thank the South West Metropolitan Regional Hospital Board for financial help for this project and Dr C.O.Edwards, Dr H.F.Harwood, Dr J.M.Hill and Dr E. Sanders for allowing us to use their patients.
References BELCHER, R.W., CARNEY, J.F. & NANKERVIS, G.A. (1974) The effect of sera from patients with sarcoidosis on the in vitro lymphoeyte response. International Archives of Allergy, 46, 183-190. CASPARY, E.A. & FIELD, E.J. (1971) Lymphoeyte sensitisation in sareoidosis. British Medical Journal, ii, 143-145. GROSS, N.J. (1973) The paradoxical skin response in sarcoidosis. American Review of Respiratory Diseases, IO7» 798-801. HIRSCHORN, K . , SCHREIBMAN, R . , BACH, F.H. & SII.TZBACH, L.E. (1964) In vitro studies of iymphokines
from patients with sareoidosis and lymphoproliferative diseases. Lancet, u, 842-843. HORSMANHEIMO, M . & VIROLAINEN, M . (1974) Correlation of PHA indiced lymphocyte transformation with the clinical manifestations of sarcoidosis. Acta pathologica et microbiologica scandinavica, 82, 122-126.
KANTOR, F.S. (1975) Infection, energy and cell-mediated immunity. Nezv England Journal of Medicine, 292, 629-634. MADERAZO, E.G., WARD, P.A., WORONICK, C.L., LUBIK, J. & DEGRAFF, A.C. (1976) Leucotactic dysfunc-
tion in sareoidosis. A?inals of Internal Medicine, 84, 414-419. SCADDING, J.G. (1967) Sarcoidosis, p. 363. Eyre and Spottiswoode, London. SoBORG, M. & BENDIXEN, G . (1967) Human lymphocyte migration as a parameter of hypersensitivity. Acta medica scandinavica, 181, 247-256. TOPILSKY, M . , SILTZBACH, L.E., WILLIAMS, M . & GLADE, P.R. (1972) Lymphocyte response in sar-
eoidosis. Lancet, i, 117-120.
Leukocyte migration inhibition studies in sarcoidosis with evidence of a serum blocking factor
A. E. ROSLING, BRIAN WATSON AND E.L. RHODES Department of Dermatology, St Helier Hospital, Wrythe Lane, Carshalton, Surrey SM5 ] AA
Accepted for publication 30 November 1976
Summary Washed leukocytes from sarcoid patients were not found to be significantly hyporeactive to phytohaemagglutinin in the mixed leukocyte migration test. An immune blocking factor, however, was found in the sera of these patients which suppressed the reaction of normal subject's white cells to phytohaemagglutinin.
One of the main features of sarcoidosis is the cutaneous anergy to various skin test antigens (Scadding, 1967). Hirschorn et al. (1964) and Topilsky et al. (1972) found in vitro hyporeactivity to several antigens and mitogens. Horsmanheimo & Virolainen (1974) us^'^ ^^ thymidine as a measure of blast cell transformation and found the response to phytohaemagglutinin (PHA) to be the same in a sarcoid and a control group. To explain this, Caspary & Field (1971) have suggested that there is an immunological depressant in sarcoid sera which prevents lymphocytes responding to antigens. Using the lymphocyte transformation test to PHA, Belcher, Carney & Nankervis (1974) found such a factor in the sera of 7 of 14 sarcoid patients. The mixed leukocyte migration test is now considered to be a genuine test of cellular hypersensitivity (Seborg & Bendixen, 1967), the lymphokines produced by the stimulated lymphocytes preventing the migration of leukocytes. In this study, the mixed leukocyte migration test was used to evaluate the in vitro response of washed sarcoid lymphocytes to PHA. The effect also of sera from sarcoid patients on the migration of leukocytes with and without PHA was noted. Materials and method Twenty sarcoid patients were investigated with ages ranging from 25 to 68 years. Ten of rhe i6t
i62
A.E.Rosling, B.Watson and E.L.Rhodes
twenty were classified as active and two of these had skin lesions. All were Kveim positive. Clitiically normal controls were taken from the hospital staff. The leukocyte migration inhibition test Approximately 30 ml whole blood containing 25 units ml"' mucous heparin were allowed to settle under gravity for i h at 37X. The leukocyte rich plasma was removed and the cells washed 3 times at 37 C in Hanks BSS (Wellcome). The resultant pellet of cells was resuspended in sufficient TC 199 (Wellcome), containing 10% horse serum to fill the number of capillary tubes (Microhaematocrit Gelman-Hawksley) necessary to perform the tests (10-15 tubes). The cell suspension was taken up into capillary tubes, sealed at one end with Cristaseal (Hawksley) and spun at 360 g for 10 min. The capillary tubes were cut i mm above the cell medium interface and fixed in the wells of the migration plates with a dab of silicone grease. TC 199 with 10% horse serum either alone or containing 5 /jg ml"' PHA was added, each test being set up in triplicate. The wells were sealed with cover slips and silicone grease and incubated at 37'C for 18 h. Migration areas were measured by projection microscopy and planimetry. Cross over experiments were performed using 10% horse serum, 10% control serum and 10% sarcoid serum with and without 5 //g ml"' PHA on blood group 'O' leukocytes from healthy donors.
1,2
1.0
0.8
"a O.A
Figure i. Mixed leukocytes migration test to PHA in control and sareoid patients. A migration index of less than 08 indieates hypersensitivity to the stimulus given. • , Sarcoid leukocytes; o, control leukocytes.
Leukocyte migration inhibition in sarcoidosis
163
Results All results were expressed as migration ratios. As a general test for lymphocyte function the responses of washed sarcoid and control lymphocytes, in horse serum, to PHA were measured and the results expressed: Migration ratio ^
Migration area + PHA Migration area ~ PHA
An arbitrary level of 0 8 and above was taken as showing no migration inhibition. As shown in Fig. i there was a tendency for the sarcoid leukocytes to be slightly less reactive than the control leukocytes but this difference was not significant and no ratios greater than 08 were scored. Two migration ratios were calculated for the experiments involving control and sarcoid sera added to donor group 'O' leukocytes:
16
1.2
1.0
08
0,6
0.2
(a)
(b)
Figure 2. Using blood group 'O' leukocytes, the effect of sareoid sera and eontrol sera in the mixed leukoeyte migration test (a) without PHA, (b) with the addition of PHA. Mean +1 standard deviation shown. • , Sarcoid sera; c, eontrol sera.
164
A.E.Rosling, B.Watson and E.L.Rhodes Migration ratio
Migration area in test sera Migration area in horse serum
(i)
These ratios are shown in Fig. 2a and there is no difference in the migration of donor 'O' leukocytes in the presence of sarcoid or control sera. In both cases the mean (0 94) and the standard deviation (03) were the same and the distribution about i-o was similar. Migration area in test sera + PHA Migration ratio ^ Migration area in horse serum + PHA
(2)
Fig. 2b shows these ratios and there is a significant difference (P — 0-025) between the mean of the sarcoid sera group (1-22 + 0-4) and the mean of the control sera group (0-91 ±0-3). This means that there was less of a response to PHA by the donor 'O' leukocytes in the presence of sarcoid sera compared with control sera. 10/15 (67/0) of the sarcoid sera and only 7/18 (39%) of the control sera showed ratios above i. This gives a significant heterogeneity x^ result (P = o 01) and would be compatible with a blocking effect in these sera. Discussion These experiments show that washed sarcoid lymphocytes, compared with a control population, exhibit only slight hyporeactivity towards PHA. All sarcoid responses were below 08, the level beneath which inhibition is said to have occurred and were not significantly different from the controls. Washed lymphocytes from sarcoidosis patients do not appear in this case to have an in vitro defect in lymphokine production. To explain the paradox of a failure to respond in vivo to skin test antigens, the sera from sarcoid patients may be said to contain 'blocking' factors. Sarcoid sera when added without PHA to normal leukocytes behaved in a manner similar to the control sera in that there was a large spread of results with similar migration indices in both groups. When the same sarcoid sera were added to washed blood group 'O' leukocytes in the presence of 5 /jg ml" ' PHA, a significant difference in response was noted. Blocking activity was seen in some sarcoid sera compared with control sera. This was not a toxic inhibitory effect since the cells migrated further in sarcoid sera than they did in horse serum. It appears therefore, that there is some substance in sarcoid sera which prevents the interaction between PHA and the leucocytes. The blocking of the reaction to PHA could occur at three stages: (i) Lymphocyte recognition of mitogen; (2) Lymphokine synthesis; (3) Reaction of migrating cells with lymphokine. Gross (1973) injected lymphokine intradermally into sarcoid patients and found no response. This would support (3) as an explanation of the anergy in sarcoidosis. More recently, Maderazo et al. (1976) detected high levels in 19/20 sarcoid patients of a constituent found only in low levels in normal individuals which was directed against the neutrophil chemotactic factor produced by lymphocytes. This, or a similar factor, could be what was detected in sarcoid sera.
Leukocyte migration inhibition in sarcoidosis
165
Feed-back suppression by T cell products has been suggested by Kantor (1975) as an explanation for anergy in cell mediated immunity reactions where T cell numbers are normal. Anergy in infections, both bacterial and viral is known to occur. A similar situation may be present in sarcoidosis.
Acknowledgments We would like to thank the South West Metropolitan Regional Hospital Board for financial help for this project and Dr C.O.Edwards, Dr H.F.Harwood, Dr J.M.Hill and Dr E. Sanders for allowing us to use their patients.
References BELCHER, R.W., CARNEY, J.F. & NANKERVIS, G.A. (1974) The effect of sera from patients with sarcoidosis on the in vitro lymphoeyte response. International Archives of Allergy, 46, 183-190. CASPARY, E.A. & FIELD, E.J. (1971) Lymphoeyte sensitisation in sareoidosis. British Medical Journal, ii, 143-145. GROSS, N.J. (1973) The paradoxical skin response in sarcoidosis. American Review of Respiratory Diseases, IO7» 798-801. HIRSCHORN, K . , SCHREIBMAN, R . , BACH, F.H. & SII.TZBACH, L.E. (1964) In vitro studies of iymphokines
from patients with sareoidosis and lymphoproliferative diseases. Lancet, u, 842-843. HORSMANHEIMO, M . & VIROLAINEN, M . (1974) Correlation of PHA indiced lymphocyte transformation with the clinical manifestations of sarcoidosis. Acta pathologica et microbiologica scandinavica, 82, 122-126.
KANTOR, F.S. (1975) Infection, energy and cell-mediated immunity. Nezv England Journal of Medicine, 292, 629-634. MADERAZO, E.G., WARD, P.A., WORONICK, C.L., LUBIK, J. & DEGRAFF, A.C. (1976) Leucotactic dysfunc-
tion in sareoidosis. A?inals of Internal Medicine, 84, 414-419. SCADDING, J.G. (1967) Sarcoidosis, p. 363. Eyre and Spottiswoode, London. SoBORG, M. & BENDIXEN, G . (1967) Human lymphocyte migration as a parameter of hypersensitivity. Acta medica scandinavica, 181, 247-256. TOPILSKY, M . , SILTZBACH, L.E., WILLIAMS, M . & GLADE, P.R. (1972) Lymphocyte response in sar-
eoidosis. Lancet, i, 117-120.
