British Joumal of Dermatology (1978) 99, 263.
Leukocyte migration agarose test (LMAT) in sarcoidosis using Kveim test material M.HORSMANHEIMO,*t A.HORSMANHEIMO,* H.H.FUDENBERG,* L.E.SILTZBACHt AND K.T.McKEE§ * Department of Basic and Clinical Immunology and Microbiology, and § Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29401, and t Department of Dermatology, University of Kuopio, Kuopio, Finland, and { Mount Sinai School of Medicine of The City University of New York, New York 10029, U.S.A. Accepted for publication 20 March 1978
SUMMARY
The two-Stage leukocyte migration agarose test (indirect LMAT), shown to be a sensitive in vitro assay for cell-mediated immunity, was used to study Kveim reactivity in vitro in patients with sarcoidosis. No Kveim-induced inhibition of leukocyte migration in agarose or Kveim-induced lymphocyte transformation in vitro was found in 23 patients with sarcoidosis, suggesting that the Kveim reaction is not an expression of cell-mediated immunity.
The leukocyte migration agarose test (LMAT) described by Clausen (1971) has recently been used to study cell-mediated immunity in humans (Clausen, 1971; Astor et al., 1973; Mygind & Stenbjerg, 1974; Koonakosit & Petchclai, 1975; Hof&nan et al., 1975; Frei, Erard & Zinkernagel, 1973; Passaleva, Forti & Ricci, 1974; Tautz, Khuen & Ax, 1974; Erard, 1974; Anders & Natvig, 1976; Wilson et al., 1977; Bergstrand, Kallen & Nilsson, 1974; Fleer et al., 1976) and in guinea-pigs (Hoffman, Spitler & Hsu, 1976). Using the direct technique in which antigen is incubated directly with migrating peripheral blood leukocytes, a significant correlation between the presence of migration inhibition and a delayed cutaneous hypersensitivity reaction has been observed for a number of antigens, including purified protein derivative of tuberculin (PPD) (Clausen, 1971; Astor et al., 1973; Mygind & Stenbjerg, 1974; Koonakosit & Petchclai, 1975; Hof&nan et ah, 1975; Frei, Erard & ^inkernagel, 1973; Passaleva, Forti & Ricci, 1974; Tautz, Khuen & Ax, 1974; Erard, 1974; Anders & Natvig, 1976; Wilson et al., 1977), streptokinase-streptodornase (Astor et al., 1973; Hoffman et al., 1975), Candida This is publication no. 206 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by USPHS Grants HD-09938, AI-13484, and HL13853-18 ALY; contract no. i-HR-4-2957 from the National Heart, Lung and Blood Institute; and the Finnish Antituberculosis Association. Correspondence: Dr M.Horsmanheimo, Department of Dermatology, l^niversity of Kuopio, 70210 Kuopio, Finland. 0007-0963/78/0900-0263 S02.00 © 1978 British Association of Dermatologists
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(Astor et al., 1973; Hof&nan et al., 1975), coccidiodin (Astor et al., 1973; Hoffman et al., 1975; Wilson et al., 19J7), and mumps virus antigen (Anders & Natvig, 1976). However, contradictory findings using PPD and bacillus Calmette-Guerin (BCG) in the direct LMAT have also been reported (Bergstrand, Kallen & Nilsson, 1974; Fleer et al., 1976). In the two-stage LMAT technique (indirect LMAT), supernatants from cultures of sensitive peripheral blood lymphocytes stimulated with the specific antigen are tested for migration inhibitory activity on polymorphonuclear leukocytes (or leukocytes) from a different donor. The inhibition is caused by a soluble factor which has been shown to be different from macrophage migration inhibition factor (MIF) in guinea-pigs (Hoffman, Spitler & Hsu, 1976) and in man (Rocklin, 1974, 1975; Lomnitzer, Rabson & Koornhof, 1975; Hoffman et al., 1977). This factor, called leukocyte inhibition factor (LIF) (Hoffman, Spitler & Hsu, 1975; Rocklin, 1974), specifically inhibits the migration of polymorphonuclear leukocytes (Hoffman, Spitler & Hsu, 1975; Rocklin, 1974). Clausen (1973) first reported that the indirect LMAT was more sensitive than the direct LMAT, and that the degree of migration inhibition in the indirect technique correlated not only with the presence of skin reactivity to PPD but also with the size of the skin test reaction. These findings have been confirmed by Anders & Natvig (1976), who used PPD and mumps virus as antigens. Inhibition of migration induced by Kveim material in the direct LMAT has been studied by Zweiman & Israel (1976), who found inhibition of leukocyte migration more often in patients with sarcoidosis than in controls. The differences were not striking, however, and there was considerable overlap. Hardt et al. (1976), using Danish Kveim material, observed no migration inhibition in the direct LMAT in 46 sarcoid patients. The more sensitive indirect LMAT has not been used previously to study Kveim reactivity in vitro. No Kveim-induced inhibition of leukocyte migration under agarose or Kveim-induced lymphocyte transformation in vitro was found in patients with sarcoidosis in the present study. MATERIALS AND METHODS
Patients. Of the 26 sarcoid patients studied, 18 were females and 8 males, ages 20-49 years (mean 31-0 years). The diagnosis of sarcoidosis was confirmed in all cases by biopsy. According to the duration of the disease it was classified as acute in 7 patients ( < 3 months), subacute in 7 patients (3-24 months), or chronic in 12 patients (>24 months). The disease was classified as active in cases in which the lesions were progressive or in which the pulmonary lesions had remained unchanged for less than i year, and as inactive when the lesions had not progressed during a period of more than i year. Twenty-two patients had active sarcoidosis and 4 were in an inactive stage of the disease. According to the pulmonary changes the disease was classified into stages I (6 patients), II (12 patients), and III (8 patients) (Siltzbach, 1967). Six of the patients were on steroids (15-30 mg of prednisone every second day); in these patients the blood samples were taken two days after the last dose of the steroid. Of 13 patients skin tested with 5 TU or PPD (Parke-Davis), 12 were negative to this dose. Kveim suspensions. Sarcoid spleen suspension (sarcoid spleen B lot 2A) was used as Kveim material. Spleen suspension was prepared as the standard Kveim preparation of Chase-Siltzbach Type i, but without heating and without preservative. The same sarcoid spleen suspension for in vivo studies had been shown to provide a Kveim suspension which in cutaneous tests yielded the expected proportions of positive reactions among patients at various stages of sarcoidosis and a negligible number among patients with other diseases (Siltzbach, 1976). Control test material consisted of spleen suspensions from a normal subject and from a patient with Hodgkin's disease. These suspensions were prepared in the same way as the sarcoid spleen suspensions.
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Lymphocyte cultures. The method is described in detail elsewhere (Horsmanheimo et al., 1977). Peripheral blood buffy coat cells were collected after dextran sedimentation, and mononuclear cells were separated on a Ficoll-Isopaque gradient. The mononuclear cells were washed and the cell concentration was adjusted to 3 x 10' cells/ml. One ml cultures were set up in tissue culture tubes in medium RPMI 1640 with 10% inactivated FCS. Sarcoid spleen suspension was used as Kveim material. Control test material consisted of spleen suspension from a normal subject and from a patient with Hodgkin's disease. Spleen suspensions were tested first in 3 patients with sarcoidosis and in 2 healthy controls at concentrations of 10 fig/ml and 100 /^g/ml. No differences were found with the two concentrations, and in further experiments spleen suspensions were used at 10 /.ig/ml. Control cultures were set up without spleen suspensions. All cultures were set up in triplicate and harvested after 6 days. Four hours before the cultures were harvested i fiCi of [•'HldThd (New England Nuclear Corporation, specific activity 6-7 Ci/mmol) was added to the culture. The cultures were harvested with an automatic cell harvester, and [^HJdThd incorporation into DNA was coimted by scintillation spectrometer. The results are expressed as a stimulation index (SI) (mean counts per min incorporated in test cultures divided by that in control cultures). Leukocyte migration agarose test (LMA T) Direct LMA T. Buffy coat cells from peripheral blood were collected after dextran sedimentation, washed, and resuspended in medium RPMI 1640 containing 10% FCS and 100 /Ug/ml of different spleen suspensions. Controls were set up without spleen suspensions. The cell concentration was 2-4 X 10* buffy coat cells per ml and the incubation time was 30 min at 37°C. After the incubation the cell suspensions were tested immediately on agarose plates as described below. Indirect LMA T. Preparation of lymphocyte culture supernatants. Peripheral mononuclear cells were prepared as described for lymphocyte cultures. Cell concentration in cultures from which the supernatants were tested for LIF activity was 2 x 10^ mononuclear cells per ml. Spleen suspensions were used first in three patients with sarcoidosis and in two controls at concentrations of 10 and 100/(g/ml. No differences were found in the LMAT results for the two concentrations, and in further experiments the spleen suspensions were used at ioo/^g/ml. The control cultures were set up without spleen suspensions. Supernatants were harvested on the third day and used as such for incubation with indicator cells as described below. Supernatants from control cultures were used as such and after reconstitution with spleen suspensions, so that the test and control supernatants of each pair contained the same concentration of spleen suspension. Assay of supernatants. Polymorphonuclear (PMN) leukocjtes were used as indicator cells in the agarose assay. PMN leukocytes were isolated from the peripheral blood of healthy blood donors as described above. After sedimentation in a Ficoll-Isopaque gradient, the PMN cells from the bottom of the tube were washed with Dulbecco's PBS. Red blood cells were submitted to hypotonic lysis for 45 s, and the cell pellet was washed twice and resuspended in supernatants as described above. The cell concentration of PMN leukocytes was 1-5 x 10* cells/ml and the incubation time with supernatants was 30 min at 37°C. After incubation the cell suspensions were tested on agarose plates as indicated below. Preparation of agarose plates. Migration of leukocytes was assayed in 1% agarose (Indubiose A37 Agarose, L'Industrie Biologique Francaise S.A.) in medium TC 199 supplemented with 10% horse serum (Gibco, U.S.A.), and with penicillin and streptomycin (Gibco), 60 U and 60 /(g/ml
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respectively, and buffered with sodium bicarbonate. Six ml of agarose medium was poured into disposable 60 mm plastic Petri dishes (3002 Tissue Culture Dish, Falcon, USA) and allowed to solidify. Eight to nine wells 2-3 mm in diameter were punched with a stainless steel punch, and 7-/(l samples of the various cell suspensions were placed in the wells. Each cell suspension was run in triplicate or in quadruplicate.The plates were placed in inverted ioo-mm plastic Petri dishes, which were sealed with sterile water and incubated for 18 h at 37°C in an atmosphere of 5% carbon dioxide and 95% air. Two diameters of migration area were measured with a calibrated hand ocular. Migration area was calculated from the mean diameter. The bottom of the well was not included in the migration area. The migration index (MI) was calculated by dividing the mean of the migration of cells incubated with each supernatant from stimulated cultures (or in the direct LMAT with each spleen suspension) by the mean area of the migration of cells incubated in the corresponding supernatant from control cultures (or in the direct LMAT of cell suspension without spleen suspension). Results are shown as migration index+standard deviation of the mean. RESULTS
LMAT. Triphcate and quadruplicates varied less than 5% in both direct and indirect LMAT. Leukocytes from three sarcoid patients were tested with different spleen suspensions in the direct LMAT. Migration indexes were 0-97 + 0.09 (mean+SD) for normal spleen suspension, 0-91 + 0-10 for Hodgkin's spleen suspension, and 0-91 + 0-11 for sarcoid spleen suspension. One of these patients was tested in both direct and indirect assay, and no differences were found in MI values. Because of the higher sensitivity of the indirect LMAT (Anders & Natvig, 1976; Clausen, 1973), 22 additional patients were studied using this assay. The results are shown in Table i. There is no
TABLE I . Response of lymphocytes from sarcoid patients to different spleen suspensions*
LMATt Suspension used Reconstituted normal spleen Normal spleen Reconstituted Hodgkins' spleen Hodgkins' spleen Reconstituted sarcoid spleen Sarcoid spleen
No. of patients
Migration index
6 22 6 23 6 23
089 ± 0 0 8 O-87 + O-I2 0 95+ 011 0 9O±oio 0 94 + 0 04 o-89±o-io
Lymphocyte transformation No. of patients
Stimulation index
22
I 14 ±042
21J
I 08 ±044
22
1-03 + 048
* Spleen suspension prepared as a standard Kveim preparation of Chase-Siltzbach Type i, but without heating or preservative. t Indirect leukocyte migration inhibition test under agarose. J One patient with stimulation index 6-61 was not included in these results.
absolute cut-off point in the migration inhibition test, but in most studies a migration index less than O77HD-8O has been taken as a significant response. Migration indexes between 0-76 and 079 were found in 5 of 23 patients when sarcoid spleen suspensions were used, and in one patient the MI was 073. However, when the migration indexes were calculated by dividing the migration area of the test
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supernatant by that of a control supernatant reconstituted with sarcoid spleen material, indexes of O-80-O-88 were found for all four cases in which the reconstitution was made. For two further patients, when the supernatant was not reconstituted with sarcoid spleen material, migration indexes were 0-78 and 077; in the latter patient MI to normal spleen and Hodgkin's spleen materials was also 077. Therefore, it can be concluded that no inhibition of leukocyte migration under agarose was found either in direct or indirect LMAT to sarcoid spleen material. Migration index less than 0 80 to normal spleen material was found in 5 of 22 patients with sarcoidosis (071-079). However, when control supernatants reconstituted with normal spleen material were used, migration indexes were 0-89 (vs 079), 0-91 (vs 0-67), and 076 (vs 071). In two cases in which reconstitution was not made, migration indexes were 078 and 077; in the latter patient MI to sarcoid spleen and Hodgkin's spleen materials was also the same. Migration indexes less than o-8o to Hodgkin's spleen suspension were found in 4 of 23 patients. When the MI was calculated using reconstituted control supernatant, only two patients showed MI of 077, one of those being the same patient who showed an MI of 077 in tests using sarcoid and normal spleen suspensions. In simimary, no significant inhibition of migration under agarose was found with normal or Hodgkin's spleen suspensions. Lymphocyte ^H-thymidine incorporation. In this assay, a two- or threefold increase in the response to antigen as compared with control cultures has generally been considered significant. Hodgkin's spleen material induced a stimulation index of 6-6i in one patient. The next highest stimulation index to spleen suspensions was 2-24, which was to sarcoid material. However, the same patient had a stimulation index of 2-05 to normal spleen suspension. Only one additional patient had a response with stimulation index greater than 2 (SI 2-14 to Hodgkin's spleen suspension). If a threefold response (Oppenheim et al., 1975) is taken as a significant response, no significant lymphocyte DNA synthesis in vitro was produced by lymphocytes of 22 patients with sarcoidosis by normal or sarcoid spleen suspensions. One of 22 patients responded in this assay to Hodgkin's spleen material. DISCUSSION
A handicap in using Kveim tissue suspension for skin testing is the long time required for the papule to mature at the injection site (4-6 weeks). Therefore, several attempts have been made to develop specific in vitro tests for sarcoidosis. In some studies involving few patients some increases in morphological lymphocyte transformation in the presence of Kveim material have been reported when the response was measured as the percentage of either large cells or mitoses (Hirschhorn et al., 1964) or blasts (Siltzbach et al., 1969; Mankiewicz, Kurti & Beland, 1971). Cowling et al. (1964), measuring the response as the percentage of blasts, could not find any response to Kveim material. In studies based on isotope incorporation into lymphocyte DNA, no significant response was noted (Siltzbach et al., 1969; Topilsky et al., 1972; Izumi, Nilsson & Ripe, 1973), except in one study (Zweiman & Israel, 1976) in which at least one Kveim preparation in 14 of 45 sarcoid patients and in 4 of the 30 controls induced transformation of lymphocytes, considering a stimulation index greater than 2 as a significant response. In a previous study, using three different Kveim materials in vitro, one of us found no lymphocyte transformation in 31 Kveim skin test positive patients with sarcoidosis (Horsmanheimo, 1973). Contradictory findings have been reported for the migration inhibition test in sarcoid patients. Inhibition of migration was observed by some investigators (Zweiman & Israel, 1976; Hardt & Wanstrup, 1969; Becker et al., 1972; Jones Williams et al., 1972), whereas negative results have been reported by Topilsky et al. (1972) Hardt et al. (1976), and Rocklin et al. (1972). Some Kveim materials
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have been reported to inhibit macrophage migration in diseases other than sarcoidosis (Willoughby & Mitchell, 1971; Brostoff & Walker, 1971; Pagaltsos et al, 1971). The LMAT has been used for studying Kveim reactivity in vitro only as a direct assay. The ability of sarcoid lymphocytes to release LIF in direct LMAT has been studied by Zweiman & Israel (1976) using different Kveim materials. They found significant inhibition more often in sarcoid patients than in controls. However, very small differences were found and there was considerable overlap. Hardt et al. (1976), using Danish Kveim material, found no inhibition of leukocyte migration in the direct LMAT in 46 sarcoid patients. Indirect LMAT, in which the ability of lymphocytes to release LIF in cultures is tested, has been shown to be more sensitive than the direct LMAT (Anders & Natvig, 1976; Clausen, 1973). In addition, the indirect LMAT has also been shown to correlate with lymphocyte transformation induced by the same antigens (Anders & Natvig, 1976). Therefore, these tests were used in parallel in the present study. The failure of Kveim material to induce a significant lymphocyte DNA synthesis response confirms previous studies (Siltzbach et al., 1969; Cowling, Quaglino & Barrett, 1964; Topilsky, 1972; Izumi, Nilsson & Ripe, 1973; Horsmanheimo, 1973). For the few patients tested by direct LMAT in the present study, no inhibition of leukocyte migration was seen, confirming the results of Hardt et al. (1976) and, in part, those of Zweiman & Israel (1976). In addition, in the indirect LMAT no Kveim-induced LIF release was found in 22 patients with sarcoidosis. Thus, the results of the present study, in which 3 different in vitro tests of cell-mediated immunity were used to study Kveim reactivity in vitro, strongly suggests that the Kveim reaction is not an expression of cell-mediated immunity. ACKNOWLEDGMENT
We thank Charles L.Smith for excellent editorial assistance. REFERENCES ANDERS, E.M. & NATVIG, J.B. (1976) Cell-mediated immunity to viruses measured by the indirect agarose technique of leukocyte migration inhibition. Cell Immunology, 27, 214. AsTOR, S.H.j SPITLER, L.E., FRICK, O.L. & FUDENBERG, H.H. (1973) Human leukocyte migration inhibition in agarose using four antigens: Correlation with skin reactivity. Jo«r«a/ of Immunology, n o , 1174. BncKER, F.W., DEICHER, H., KRULL, P. & KALDEN, J.R. (1972) Leukocyte migration test in sarcoidosis. Lancet, i, 120. BERGSTRAND, H . , KALLEN, B. & NILSSON, O . (1974) On the reactivity of human leukocytes to PPD in Clausen's agarose migration technique. Acta allergologica, (Kbh), 29, 117. BROSTOFF, J. & WALKER, J.G. (1971) Leukocyte migration inhibition with Kveim antigen in Crohn's disease. Clinical and Experimental Immunology, 9, 707.
CLAUSEN, J.E. (1971) Tuberculin-induced migration inhibition of human peripheral leukocytes in agarose medium. Acta allergologica, (Kbh), 26, 56.
CLAUSEN, J.E. (1973) Migration inhibitory effect of cell-free supernatants from tuberculin-stimulated cultures of human mononuclear leukocytes demonstrated by two-step MIF agarose !&sa.y. Journal of Immunology, n o , 546. COWLING, D.C, QUAGLINO, D . & BARRETT, P . K . M . (1964) Effect of Kveim antigen and old tuberculin on lymphocytes in cultures from sarcoid patients. British Medical Journal, i, 1481. ERARD, P . (1974) Technical study of the leukocyte migration inhibition test in agarose. Application to PPD and to hepatitis B antigen. Clinical and Experimental Immunology, 18, 439. FLEER, A., VAN DER HART, M . , BLOK-SCHUT, B . J . T . & SCHELLEKENS, P . T . A . (1976) Correlation of PPD and BCG-
induced leukocyte migration inhibition, delayed cutaneous hypersensitivity, lymphocyte transformation in vitro and humoral antibodies to PPD in man. European Journal of Immunology, 6, 163. FREI, P.C., ERARD, P. & ZINKERNAGEL, R . (1973) Cell-mediated immunity to hepatitis-associated antigen (HAA) demonstrated by leukocyte migration test during and after acute B hepatitis. Biomedicine, 19, 379.
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HARDT, F . , VEIEN, N . , BENDIXEN, G . , BRODTHAGEN, H . , FARBER, V., GENNER, J., HECKSCHER, T . , RINGSTED, J.,
FREIESLEBEN SORENSEN, S., WANSTRUP, J. & WIIK, A. (1976) Immunological studies in sarcoidosis. A com-
parison of in vivo and in vitro Kveim tests. Annals of the New York Academy of Science, 278, 711. HARDT, F . & WANSTRUP, J. (1969) Sarcoidosis. An in vitro Kveim reaction based on the leukocyte migration test. Acta pathologica et microbiologica Scandinavica, 76, 493. HiRSCHHORN, K., SCHREIBMAN, R.K., BACH, F . H . & SiLTZBACH, L.E. (1964) In vitro Studies of lymphocytes from patients with sarcoidosis and lymphoproliferative diseases. Lancet, ii, 842. HOFFMAN, P.M., SPITLER, L.E., Hsu, M. & FUDENBERG, H . (1975) Leukocyte migration inhibition in agarose. Cellular Immunology, 18, 21. HOFFMAN, P.M., SPITLER, L.E. & Hsu, M. (1976) Leukocyte migration inhibition in guinea-pigs. L Correlation with skin test reactivity and macrophage migration inhibition. Cellular Immunology, 21, 358. HORSMANHEIMO, M . (1973) Lymphocyte transformation and the Kveim test. Lancet, i, 1120. HORSMANHEIMO, M . , HORSMANHEIMO, A., BANOV, C.H., AINSWORTH, S . K . & FUDENBERG, H.H. (1977) Cell-
mediated immunity in vitro in atopic dermatitis. Archives of Dermatology, in press. IZUMI, T . , NILSSON, B.S. & RIPE, E . (1973) In vitro lymphocyte reactivity to different Kveim preparations in patients with sarcoidosis. Scandinavian Journal of Respiratory Disease, 54, 123. JONES WILLIAMS, W . , PIOLI, E . , JONES, D.J. & DIGHERO, M . (1972) The Kmif (Kveim-induced macrophage
migration inhibition factor) test in sarcoidosis. ^ow^aZ of Clinical Pathology, 25, 951. KooNAKOSiT, R. & PETCHCLAI, B. (1975) Leukocyte migration test in a modified agarose medium. American Journal of Clinical Pathology, 64, 531. LOMNITZER, R., RABSON, A.R. & KooRNHOF, H.J. (i975) Production of leukocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes. Clinical and Experimental Immunology, 22, 522. MANKIEWICZ, E., KURTI, V. & BELAND, J. (1971) Diagnostic procedures in sarcoidosis. Canadian Medical Association Journal, 104, 684. MYGIND, K . & STENBJERG, S. (1974) Leukocyte migration test in agarose. The use of puromycin in disclosure of non-immunological inhibition. Acta Pathologica et microbiologica Scandinavica, Sect. B., 82, 277. OPPENHEIM, J.J., DOUGHERTY, S., CHAN, S.P. & BAKER, J. (1975) tJse of lymphocyte transformation to assess
clinical disorders. In: Laboratory Diagnosis of Immunologic Disorders (Ed. by G.N.Vyas, D.P.Stites and G. Brecher), p. 87. Grune and Stratton, Inc., New York. PAGALTSOS, A.S., WILLOUGHBY, J.M.T., KUMAN, P.J. & DAWSON, A.M. (1971) In vitro inhibition of leukocyte
migration by sarcoid spleen suspension in coeliac disease and dermatitis herpetiformis. Lancet, ii, 1179. PASSALEVA, A., FORTI, A. & Ricci, M. (1974) Inhibition of leukocyte migration in man by the agarose plate technique using protein purified derivations (PPD) from three different sources. Acta allergologica (Kbh), 29, 209. ROCKLIN, R.E. (1974) Products of activated lymphocytes: Leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor {M.W). Journal of Immunology, 112, 1461. ROCKLIN, R.E. (1975) Partial characterization of leukocyte inhibitory factor by concanavalin A-stimulated human lymphocytes (LIF ConA). Journal of Immunology, 114, 1161. ROCKLIN, R.B., SHEFFER, A.L. & DAVID, J . K . (1972) Sarcoidosis: A clinical and in vitro immunologic study. In: Proceedings of the Sixth Leukocyte Culture Conference (Ed. by M.R. Schwarz), p. 743. Academic Press, New York. SILTZBACH, L.E. (1967) Sarcoidosis: Clinical features and management. Medical Clinics of North America, 51,483. SILTZBACH, L.E. (1976) Qualities and behaviour of satisfactory Kveim suspensions. Annals of the New York Academy of Science, 278, 665. SILTZBACH, L.E., GLADE, P.R., HIRSHAUT, Y . , VIEIRA, L.O.B.D., CELIKOGLU, J.S. & HIRSCHHORN, K . (1969)
In vitro stimulation of peripheral lymphocytes in sarcoidosis. In: Fifth International Conference on Sarcoidosis (Ed. by L. Levinsky and F. Macholda), p. 217. State Printing House, Prague. TAUTZ, C , KHUEN, F . & Ax, W.Z. (1974) Human leukocyte migration under agarose: Migration inhibition by soluble and tumor antigens with special reference to antimelanoma activity in healthy blacks. Zeitschrift fiir Immunitdtsforschung und experimentelle Therapie, 147, 155. TOPILSKY, M . , WILLIAMS, M . , SILTZBASCH, L.E. & GLADE, P . R . (1972) Lymphocyte response in sarcoidosis.
Lancet, i, 117. WILLOUGHBY, J . M . T . & MITCHELL, D . N . (1971) In vitro inhibition of leukocyte migration in Crohn's disease by a sarcoid spleen suspension. British Medical Journal, iii, 155.
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WILSON, G.B., HORSMANHEIMO, M . & FUDENBERG, H.H. (1978) Effects of dialyzable transfer factor (TFd) on leukocyte migration in vitro. I. Antigen-independent enhancement of migration. Submitted for publication. ZWEIMAN, B. & ISRAEL, H.L. (1976) Comparative in vitro reactivities of leukocytes from sarcoid and normals to different Kveim preparations. Annals of the New York Academy of Science, 278, 700.
Leukocyte migration agarose test (LMAT) in sarcoidosis using Kveim test material M.HORSMANHEIMO,*t A.HORSMANHEIMO,* H.H.FUDENBERG,* L.E.SILTZBACHt AND K.T.McKEE§ * Department of Basic and Clinical Immunology and Microbiology, and § Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29401, and t Department of Dermatology, University of Kuopio, Kuopio, Finland, and { Mount Sinai School of Medicine of The City University of New York, New York 10029, U.S.A. Accepted for publication 20 March 1978
SUMMARY
The two-Stage leukocyte migration agarose test (indirect LMAT), shown to be a sensitive in vitro assay for cell-mediated immunity, was used to study Kveim reactivity in vitro in patients with sarcoidosis. No Kveim-induced inhibition of leukocyte migration in agarose or Kveim-induced lymphocyte transformation in vitro was found in 23 patients with sarcoidosis, suggesting that the Kveim reaction is not an expression of cell-mediated immunity.
The leukocyte migration agarose test (LMAT) described by Clausen (1971) has recently been used to study cell-mediated immunity in humans (Clausen, 1971; Astor et al., 1973; Mygind & Stenbjerg, 1974; Koonakosit & Petchclai, 1975; Hof&nan et al., 1975; Frei, Erard & Zinkernagel, 1973; Passaleva, Forti & Ricci, 1974; Tautz, Khuen & Ax, 1974; Erard, 1974; Anders & Natvig, 1976; Wilson et al., 1977; Bergstrand, Kallen & Nilsson, 1974; Fleer et al., 1976) and in guinea-pigs (Hoffman, Spitler & Hsu, 1976). Using the direct technique in which antigen is incubated directly with migrating peripheral blood leukocytes, a significant correlation between the presence of migration inhibition and a delayed cutaneous hypersensitivity reaction has been observed for a number of antigens, including purified protein derivative of tuberculin (PPD) (Clausen, 1971; Astor et al., 1973; Mygind & Stenbjerg, 1974; Koonakosit & Petchclai, 1975; Hof&nan et ah, 1975; Frei, Erard & ^inkernagel, 1973; Passaleva, Forti & Ricci, 1974; Tautz, Khuen & Ax, 1974; Erard, 1974; Anders & Natvig, 1976; Wilson et al., 1977), streptokinase-streptodornase (Astor et al., 1973; Hoffman et al., 1975), Candida This is publication no. 206 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by USPHS Grants HD-09938, AI-13484, and HL13853-18 ALY; contract no. i-HR-4-2957 from the National Heart, Lung and Blood Institute; and the Finnish Antituberculosis Association. Correspondence: Dr M.Horsmanheimo, Department of Dermatology, l^niversity of Kuopio, 70210 Kuopio, Finland. 0007-0963/78/0900-0263 S02.00 © 1978 British Association of Dermatologists
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(Astor et al., 1973; Hof&nan et al., 1975), coccidiodin (Astor et al., 1973; Hoffman et al., 1975; Wilson et al., 19J7), and mumps virus antigen (Anders & Natvig, 1976). However, contradictory findings using PPD and bacillus Calmette-Guerin (BCG) in the direct LMAT have also been reported (Bergstrand, Kallen & Nilsson, 1974; Fleer et al., 1976). In the two-stage LMAT technique (indirect LMAT), supernatants from cultures of sensitive peripheral blood lymphocytes stimulated with the specific antigen are tested for migration inhibitory activity on polymorphonuclear leukocytes (or leukocytes) from a different donor. The inhibition is caused by a soluble factor which has been shown to be different from macrophage migration inhibition factor (MIF) in guinea-pigs (Hoffman, Spitler & Hsu, 1976) and in man (Rocklin, 1974, 1975; Lomnitzer, Rabson & Koornhof, 1975; Hoffman et al., 1977). This factor, called leukocyte inhibition factor (LIF) (Hoffman, Spitler & Hsu, 1975; Rocklin, 1974), specifically inhibits the migration of polymorphonuclear leukocytes (Hoffman, Spitler & Hsu, 1975; Rocklin, 1974). Clausen (1973) first reported that the indirect LMAT was more sensitive than the direct LMAT, and that the degree of migration inhibition in the indirect technique correlated not only with the presence of skin reactivity to PPD but also with the size of the skin test reaction. These findings have been confirmed by Anders & Natvig (1976), who used PPD and mumps virus as antigens. Inhibition of migration induced by Kveim material in the direct LMAT has been studied by Zweiman & Israel (1976), who found inhibition of leukocyte migration more often in patients with sarcoidosis than in controls. The differences were not striking, however, and there was considerable overlap. Hardt et al. (1976), using Danish Kveim material, observed no migration inhibition in the direct LMAT in 46 sarcoid patients. The more sensitive indirect LMAT has not been used previously to study Kveim reactivity in vitro. No Kveim-induced inhibition of leukocyte migration under agarose or Kveim-induced lymphocyte transformation in vitro was found in patients with sarcoidosis in the present study. MATERIALS AND METHODS
Patients. Of the 26 sarcoid patients studied, 18 were females and 8 males, ages 20-49 years (mean 31-0 years). The diagnosis of sarcoidosis was confirmed in all cases by biopsy. According to the duration of the disease it was classified as acute in 7 patients ( < 3 months), subacute in 7 patients (3-24 months), or chronic in 12 patients (>24 months). The disease was classified as active in cases in which the lesions were progressive or in which the pulmonary lesions had remained unchanged for less than i year, and as inactive when the lesions had not progressed during a period of more than i year. Twenty-two patients had active sarcoidosis and 4 were in an inactive stage of the disease. According to the pulmonary changes the disease was classified into stages I (6 patients), II (12 patients), and III (8 patients) (Siltzbach, 1967). Six of the patients were on steroids (15-30 mg of prednisone every second day); in these patients the blood samples were taken two days after the last dose of the steroid. Of 13 patients skin tested with 5 TU or PPD (Parke-Davis), 12 were negative to this dose. Kveim suspensions. Sarcoid spleen suspension (sarcoid spleen B lot 2A) was used as Kveim material. Spleen suspension was prepared as the standard Kveim preparation of Chase-Siltzbach Type i, but without heating and without preservative. The same sarcoid spleen suspension for in vivo studies had been shown to provide a Kveim suspension which in cutaneous tests yielded the expected proportions of positive reactions among patients at various stages of sarcoidosis and a negligible number among patients with other diseases (Siltzbach, 1976). Control test material consisted of spleen suspensions from a normal subject and from a patient with Hodgkin's disease. These suspensions were prepared in the same way as the sarcoid spleen suspensions.
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Lymphocyte cultures. The method is described in detail elsewhere (Horsmanheimo et al., 1977). Peripheral blood buffy coat cells were collected after dextran sedimentation, and mononuclear cells were separated on a Ficoll-Isopaque gradient. The mononuclear cells were washed and the cell concentration was adjusted to 3 x 10' cells/ml. One ml cultures were set up in tissue culture tubes in medium RPMI 1640 with 10% inactivated FCS. Sarcoid spleen suspension was used as Kveim material. Control test material consisted of spleen suspension from a normal subject and from a patient with Hodgkin's disease. Spleen suspensions were tested first in 3 patients with sarcoidosis and in 2 healthy controls at concentrations of 10 fig/ml and 100 /^g/ml. No differences were found with the two concentrations, and in further experiments spleen suspensions were used at 10 /.ig/ml. Control cultures were set up without spleen suspensions. All cultures were set up in triplicate and harvested after 6 days. Four hours before the cultures were harvested i fiCi of [•'HldThd (New England Nuclear Corporation, specific activity 6-7 Ci/mmol) was added to the culture. The cultures were harvested with an automatic cell harvester, and [^HJdThd incorporation into DNA was coimted by scintillation spectrometer. The results are expressed as a stimulation index (SI) (mean counts per min incorporated in test cultures divided by that in control cultures). Leukocyte migration agarose test (LMA T) Direct LMA T. Buffy coat cells from peripheral blood were collected after dextran sedimentation, washed, and resuspended in medium RPMI 1640 containing 10% FCS and 100 /Ug/ml of different spleen suspensions. Controls were set up without spleen suspensions. The cell concentration was 2-4 X 10* buffy coat cells per ml and the incubation time was 30 min at 37°C. After the incubation the cell suspensions were tested immediately on agarose plates as described below. Indirect LMA T. Preparation of lymphocyte culture supernatants. Peripheral mononuclear cells were prepared as described for lymphocyte cultures. Cell concentration in cultures from which the supernatants were tested for LIF activity was 2 x 10^ mononuclear cells per ml. Spleen suspensions were used first in three patients with sarcoidosis and in two controls at concentrations of 10 and 100/(g/ml. No differences were found in the LMAT results for the two concentrations, and in further experiments the spleen suspensions were used at ioo/^g/ml. The control cultures were set up without spleen suspensions. Supernatants were harvested on the third day and used as such for incubation with indicator cells as described below. Supernatants from control cultures were used as such and after reconstitution with spleen suspensions, so that the test and control supernatants of each pair contained the same concentration of spleen suspension. Assay of supernatants. Polymorphonuclear (PMN) leukocjtes were used as indicator cells in the agarose assay. PMN leukocytes were isolated from the peripheral blood of healthy blood donors as described above. After sedimentation in a Ficoll-Isopaque gradient, the PMN cells from the bottom of the tube were washed with Dulbecco's PBS. Red blood cells were submitted to hypotonic lysis for 45 s, and the cell pellet was washed twice and resuspended in supernatants as described above. The cell concentration of PMN leukocytes was 1-5 x 10* cells/ml and the incubation time with supernatants was 30 min at 37°C. After incubation the cell suspensions were tested on agarose plates as indicated below. Preparation of agarose plates. Migration of leukocytes was assayed in 1% agarose (Indubiose A37 Agarose, L'Industrie Biologique Francaise S.A.) in medium TC 199 supplemented with 10% horse serum (Gibco, U.S.A.), and with penicillin and streptomycin (Gibco), 60 U and 60 /(g/ml
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respectively, and buffered with sodium bicarbonate. Six ml of agarose medium was poured into disposable 60 mm plastic Petri dishes (3002 Tissue Culture Dish, Falcon, USA) and allowed to solidify. Eight to nine wells 2-3 mm in diameter were punched with a stainless steel punch, and 7-/(l samples of the various cell suspensions were placed in the wells. Each cell suspension was run in triplicate or in quadruplicate.The plates were placed in inverted ioo-mm plastic Petri dishes, which were sealed with sterile water and incubated for 18 h at 37°C in an atmosphere of 5% carbon dioxide and 95% air. Two diameters of migration area were measured with a calibrated hand ocular. Migration area was calculated from the mean diameter. The bottom of the well was not included in the migration area. The migration index (MI) was calculated by dividing the mean of the migration of cells incubated with each supernatant from stimulated cultures (or in the direct LMAT with each spleen suspension) by the mean area of the migration of cells incubated in the corresponding supernatant from control cultures (or in the direct LMAT of cell suspension without spleen suspension). Results are shown as migration index+standard deviation of the mean. RESULTS
LMAT. Triphcate and quadruplicates varied less than 5% in both direct and indirect LMAT. Leukocytes from three sarcoid patients were tested with different spleen suspensions in the direct LMAT. Migration indexes were 0-97 + 0.09 (mean+SD) for normal spleen suspension, 0-91 + 0-10 for Hodgkin's spleen suspension, and 0-91 + 0-11 for sarcoid spleen suspension. One of these patients was tested in both direct and indirect assay, and no differences were found in MI values. Because of the higher sensitivity of the indirect LMAT (Anders & Natvig, 1976; Clausen, 1973), 22 additional patients were studied using this assay. The results are shown in Table i. There is no
TABLE I . Response of lymphocytes from sarcoid patients to different spleen suspensions*
LMATt Suspension used Reconstituted normal spleen Normal spleen Reconstituted Hodgkins' spleen Hodgkins' spleen Reconstituted sarcoid spleen Sarcoid spleen
No. of patients
Migration index
6 22 6 23 6 23
089 ± 0 0 8 O-87 + O-I2 0 95+ 011 0 9O±oio 0 94 + 0 04 o-89±o-io
Lymphocyte transformation No. of patients
Stimulation index
22
I 14 ±042
21J
I 08 ±044
22
1-03 + 048
* Spleen suspension prepared as a standard Kveim preparation of Chase-Siltzbach Type i, but without heating or preservative. t Indirect leukocyte migration inhibition test under agarose. J One patient with stimulation index 6-61 was not included in these results.
absolute cut-off point in the migration inhibition test, but in most studies a migration index less than O77HD-8O has been taken as a significant response. Migration indexes between 0-76 and 079 were found in 5 of 23 patients when sarcoid spleen suspensions were used, and in one patient the MI was 073. However, when the migration indexes were calculated by dividing the migration area of the test
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supernatant by that of a control supernatant reconstituted with sarcoid spleen material, indexes of O-80-O-88 were found for all four cases in which the reconstitution was made. For two further patients, when the supernatant was not reconstituted with sarcoid spleen material, migration indexes were 0-78 and 077; in the latter patient MI to normal spleen and Hodgkin's spleen materials was also 077. Therefore, it can be concluded that no inhibition of leukocyte migration under agarose was found either in direct or indirect LMAT to sarcoid spleen material. Migration index less than 0 80 to normal spleen material was found in 5 of 22 patients with sarcoidosis (071-079). However, when control supernatants reconstituted with normal spleen material were used, migration indexes were 0-89 (vs 079), 0-91 (vs 0-67), and 076 (vs 071). In two cases in which reconstitution was not made, migration indexes were 078 and 077; in the latter patient MI to sarcoid spleen and Hodgkin's spleen materials was also the same. Migration indexes less than o-8o to Hodgkin's spleen suspension were found in 4 of 23 patients. When the MI was calculated using reconstituted control supernatant, only two patients showed MI of 077, one of those being the same patient who showed an MI of 077 in tests using sarcoid and normal spleen suspensions. In simimary, no significant inhibition of migration under agarose was found with normal or Hodgkin's spleen suspensions. Lymphocyte ^H-thymidine incorporation. In this assay, a two- or threefold increase in the response to antigen as compared with control cultures has generally been considered significant. Hodgkin's spleen material induced a stimulation index of 6-6i in one patient. The next highest stimulation index to spleen suspensions was 2-24, which was to sarcoid material. However, the same patient had a stimulation index of 2-05 to normal spleen suspension. Only one additional patient had a response with stimulation index greater than 2 (SI 2-14 to Hodgkin's spleen suspension). If a threefold response (Oppenheim et al., 1975) is taken as a significant response, no significant lymphocyte DNA synthesis in vitro was produced by lymphocytes of 22 patients with sarcoidosis by normal or sarcoid spleen suspensions. One of 22 patients responded in this assay to Hodgkin's spleen material. DISCUSSION
A handicap in using Kveim tissue suspension for skin testing is the long time required for the papule to mature at the injection site (4-6 weeks). Therefore, several attempts have been made to develop specific in vitro tests for sarcoidosis. In some studies involving few patients some increases in morphological lymphocyte transformation in the presence of Kveim material have been reported when the response was measured as the percentage of either large cells or mitoses (Hirschhorn et al., 1964) or blasts (Siltzbach et al., 1969; Mankiewicz, Kurti & Beland, 1971). Cowling et al. (1964), measuring the response as the percentage of blasts, could not find any response to Kveim material. In studies based on isotope incorporation into lymphocyte DNA, no significant response was noted (Siltzbach et al., 1969; Topilsky et al., 1972; Izumi, Nilsson & Ripe, 1973), except in one study (Zweiman & Israel, 1976) in which at least one Kveim preparation in 14 of 45 sarcoid patients and in 4 of the 30 controls induced transformation of lymphocytes, considering a stimulation index greater than 2 as a significant response. In a previous study, using three different Kveim materials in vitro, one of us found no lymphocyte transformation in 31 Kveim skin test positive patients with sarcoidosis (Horsmanheimo, 1973). Contradictory findings have been reported for the migration inhibition test in sarcoid patients. Inhibition of migration was observed by some investigators (Zweiman & Israel, 1976; Hardt & Wanstrup, 1969; Becker et al., 1972; Jones Williams et al., 1972), whereas negative results have been reported by Topilsky et al. (1972) Hardt et al. (1976), and Rocklin et al. (1972). Some Kveim materials
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have been reported to inhibit macrophage migration in diseases other than sarcoidosis (Willoughby & Mitchell, 1971; Brostoff & Walker, 1971; Pagaltsos et al, 1971). The LMAT has been used for studying Kveim reactivity in vitro only as a direct assay. The ability of sarcoid lymphocytes to release LIF in direct LMAT has been studied by Zweiman & Israel (1976) using different Kveim materials. They found significant inhibition more often in sarcoid patients than in controls. However, very small differences were found and there was considerable overlap. Hardt et al. (1976), using Danish Kveim material, found no inhibition of leukocyte migration in the direct LMAT in 46 sarcoid patients. Indirect LMAT, in which the ability of lymphocytes to release LIF in cultures is tested, has been shown to be more sensitive than the direct LMAT (Anders & Natvig, 1976; Clausen, 1973). In addition, the indirect LMAT has also been shown to correlate with lymphocyte transformation induced by the same antigens (Anders & Natvig, 1976). Therefore, these tests were used in parallel in the present study. The failure of Kveim material to induce a significant lymphocyte DNA synthesis response confirms previous studies (Siltzbach et al., 1969; Cowling, Quaglino & Barrett, 1964; Topilsky, 1972; Izumi, Nilsson & Ripe, 1973; Horsmanheimo, 1973). For the few patients tested by direct LMAT in the present study, no inhibition of leukocyte migration was seen, confirming the results of Hardt et al. (1976) and, in part, those of Zweiman & Israel (1976). In addition, in the indirect LMAT no Kveim-induced LIF release was found in 22 patients with sarcoidosis. Thus, the results of the present study, in which 3 different in vitro tests of cell-mediated immunity were used to study Kveim reactivity in vitro, strongly suggests that the Kveim reaction is not an expression of cell-mediated immunity. ACKNOWLEDGMENT
We thank Charles L.Smith for excellent editorial assistance. REFERENCES ANDERS, E.M. & NATVIG, J.B. (1976) Cell-mediated immunity to viruses measured by the indirect agarose technique of leukocyte migration inhibition. Cell Immunology, 27, 214. AsTOR, S.H.j SPITLER, L.E., FRICK, O.L. & FUDENBERG, H.H. (1973) Human leukocyte migration inhibition in agarose using four antigens: Correlation with skin reactivity. Jo«r«a/ of Immunology, n o , 1174. BncKER, F.W., DEICHER, H., KRULL, P. & KALDEN, J.R. (1972) Leukocyte migration test in sarcoidosis. Lancet, i, 120. BERGSTRAND, H . , KALLEN, B. & NILSSON, O . (1974) On the reactivity of human leukocytes to PPD in Clausen's agarose migration technique. Acta allergologica, (Kbh), 29, 117. BROSTOFF, J. & WALKER, J.G. (1971) Leukocyte migration inhibition with Kveim antigen in Crohn's disease. Clinical and Experimental Immunology, 9, 707.
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CLAUSEN, J.E. (1973) Migration inhibitory effect of cell-free supernatants from tuberculin-stimulated cultures of human mononuclear leukocytes demonstrated by two-step MIF agarose !&sa.y. Journal of Immunology, n o , 546. COWLING, D.C, QUAGLINO, D . & BARRETT, P . K . M . (1964) Effect of Kveim antigen and old tuberculin on lymphocytes in cultures from sarcoid patients. British Medical Journal, i, 1481. ERARD, P . (1974) Technical study of the leukocyte migration inhibition test in agarose. Application to PPD and to hepatitis B antigen. Clinical and Experimental Immunology, 18, 439. FLEER, A., VAN DER HART, M . , BLOK-SCHUT, B . J . T . & SCHELLEKENS, P . T . A . (1976) Correlation of PPD and BCG-
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migration inhibition factor) test in sarcoidosis. ^ow^aZ of Clinical Pathology, 25, 951. KooNAKOSiT, R. & PETCHCLAI, B. (1975) Leukocyte migration test in a modified agarose medium. American Journal of Clinical Pathology, 64, 531. LOMNITZER, R., RABSON, A.R. & KooRNHOF, H.J. (i975) Production of leukocyte inhibitory factor (LIF) and macrophage inhibitory factor (MIF) by PHA-stimulated lymphocytes. Clinical and Experimental Immunology, 22, 522. MANKIEWICZ, E., KURTI, V. & BELAND, J. (1971) Diagnostic procedures in sarcoidosis. Canadian Medical Association Journal, 104, 684. MYGIND, K . & STENBJERG, S. (1974) Leukocyte migration test in agarose. The use of puromycin in disclosure of non-immunological inhibition. Acta Pathologica et microbiologica Scandinavica, Sect. B., 82, 277. OPPENHEIM, J.J., DOUGHERTY, S., CHAN, S.P. & BAKER, J. (1975) tJse of lymphocyte transformation to assess
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In vitro stimulation of peripheral lymphocytes in sarcoidosis. In: Fifth International Conference on Sarcoidosis (Ed. by L. Levinsky and F. Macholda), p. 217. State Printing House, Prague. TAUTZ, C , KHUEN, F . & Ax, W.Z. (1974) Human leukocyte migration under agarose: Migration inhibition by soluble and tumor antigens with special reference to antimelanoma activity in healthy blacks. Zeitschrift fiir Immunitdtsforschung und experimentelle Therapie, 147, 155. TOPILSKY, M . , WILLIAMS, M . , SILTZBASCH, L.E. & GLADE, P . R . (1972) Lymphocyte response in sarcoidosis.
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