British Journal of Dermatology (1976) 94, 63.
The leukocyte migration inhibition test in allergic nickel contact dermatitis T.M.MACLEOD, F.HUTCHINSON* AND EJ.RAFFLE Department of Dermatology, University of Dundee, and *Department of Medical Physics, Ninewells Hospital, Dundee Accepted for publication 22 June 1975
SUMMARY
No Statistical difference was found between the migration indices of nickel sensitive and control subjects using the leukocyte migration inhibition test with two concentrations of nickel sulphate. The results contrastwith the specific positiveresults demonstrated previously in the same subjects^ with the lymphocytetransformatioiTtesE
In nickel sensitive subjects positive lymphocyte transformation tests are obtained when nickel sulphate or acetate is used in optimum concentration as a stimulant. This lymphocyte transformation is specific for subjects who show clinical and patch test evidence of nickel sensitivity (Hutchinson, Raffle & MacLeod, 1972). In cell-mediated immune reactions a number of humoral factors are elaborated by lymphocytes in contact with antigens. Of these, macrophage inhibition factor (MIF) production is regarded as a correlate of clinical delayed hypersensitivity states. The relationship between MIF and leukocyte migration inhibition (LMI) factor is not entirely clear but LMI is commonly employed in the assessment of clinical states of delayed hypersensitivity (Mitchell et ah, 1972). We have carried out LMI tests in nickel-sensitive patients to ascertain whether this test can be used to confirm clinical sensitivity. PATIENTS AND METHODS
Ten subjects with clinical evidence of nickel dermatitis together with positive patch tests and positive lymphocyte transformation tests were studied. LMI tests were carried out by the method of Federlin et al. (1971) and Maini et al. (1973), the only modification being in the use of medium 199 instead of Waymouth medium. The nickel sensitive subjects were paired with control subjects for the study. Two diflferent concentrations of nickel were employed (as sulphate) in the tests, io"* and io~^ mEq Ni/ml of medium. In three cases and three controls peripheral blood lymphocytes were also cultured with nickel salts for 3 days prior to performing the LMI tests against independent effector cells on the basis that such prior incubation might be necessary to elaborate LMI in sufficient concentration to Correspondence: Dr F. Hutchinson, Department of Medical Physics, Ninewells Hospital, Dundee. 63
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T.M.MacLeod, F.Hutchinson and E.J.Raffle
produce a positive test. In two subjects 25% autologous serum or 25% reconstituted freeze-dried plasma were substituted for 10% horse serum as an alternative to provide a potential protein conjugate for the nickel hapten. RESULTS
There was no statistical difference between the migration indices of the nickel sensitive and control subjects for the two different concentrations of antigen used. The maximum variation was less than + 20% of unity. No consistent evidence of LMI factor elaboration under the conditions of these experiments was obtained. DISCUSSION
These results are contrary to those obtained by Mirza, Perera & Bernstein (1974) who reported positive LMI tests in patients with proven nickel dermatitis. A negligible difference would be expected between the use of bovine albumin as a nickel albumin complex and the methods used in some of our tests, i.e. 10% horse serum added to the medium in Federlin et al.'s technique or the 25% autologous serum or reconstituted freeze-dried plasma. Hence the conflicting results may be due to a concentration effect. Positive LMI results have also been reported by Thulin & Zachariae (1972) in chromium sensitive subjects when chromiimi boxmd to bovine albumin was employed as the antigen, but de Jong, Bleimiink & Nater (1974) have shown that positive LMI tests with mercuric chloride, a non-specific mitogen, resulted from a decrease in the number of migrating polymorphonuclear leukocytes, and hence were false positives. They demonstrated that a concentration of 3 x io~^ M Hg induced lysis of 30-50% of the leukocytes within 30 min of incubation. Unlike the lymphocyte transformation test the LMI test is clearly of doubtful value in the diagnosis of delayed hypersensitivity to nickel in dermatitis subjects. ACKNOWLEDGMENT
We would like to express our thanks to Mr D. Donald for his help in this work. REFERENCES DE JONG, M.C.J.M., BLEUMINK, E. & NATER, J.P. (1974) In vitro leukocyte migration inhibition studies of allergic contact dermatitis in man. gth European Congress of Allergology and Clinical Immunology (in press). FEDERLIN, K . , MAINI, R.N., RUSSELL, A . S . & DUMONDE, D . C . (1971) A micro-method for peripheral leukocyte
migration in tuberculin sensitivity. Journal of Clinical Pathology, 24, 533. HUTCHINSON, F., RAFFLE, E.J. & MACLEOD, T.M. (1972) The specificity of lymphocyte transformation in vitro by nickel salts in nickel sensitive subjects. Journal of Investigative Dermatology, 58, 362. MAINI, R.M., ROFFE, L.M., MAGRATH, I . T . & DUMONDE, D.C. (1973) Standardization of the leukocyte migration
test. International Archives of Allergy, 45, 308. MIRZA, A.M., PERERA, M . G . & BERNSTEIN, I . L . (1974) Leukocyte migration inhibition in nickel dermatitis. Federation Proceedings, 33, 728. MITCHELL, CHRISTINE G . , SMITH, M.G.M., GOLDING, P.L., EDDLESTON, A . L . W . F . & WILLIAMS, R . (1972)
Evaluation of the leukocyte migration test as a measure of delayed hypersensitivity in man. Clinical and Experimental Immunology, 11, 535. THULIN, H . & ZACHARIAE, H . (1972) The leukocyte migration test in chromium hypersensitivity. Journal of Investigative Dermatology, 58, 55.
The leukocyte migration inhibition test in allergic nickel contact dermatitis T.M.MACLEOD, F.HUTCHINSON* AND EJ.RAFFLE Department of Dermatology, University of Dundee, and *Department of Medical Physics, Ninewells Hospital, Dundee Accepted for publication 22 June 1975
SUMMARY
No Statistical difference was found between the migration indices of nickel sensitive and control subjects using the leukocyte migration inhibition test with two concentrations of nickel sulphate. The results contrastwith the specific positiveresults demonstrated previously in the same subjects^ with the lymphocytetransformatioiTtesE
In nickel sensitive subjects positive lymphocyte transformation tests are obtained when nickel sulphate or acetate is used in optimum concentration as a stimulant. This lymphocyte transformation is specific for subjects who show clinical and patch test evidence of nickel sensitivity (Hutchinson, Raffle & MacLeod, 1972). In cell-mediated immune reactions a number of humoral factors are elaborated by lymphocytes in contact with antigens. Of these, macrophage inhibition factor (MIF) production is regarded as a correlate of clinical delayed hypersensitivity states. The relationship between MIF and leukocyte migration inhibition (LMI) factor is not entirely clear but LMI is commonly employed in the assessment of clinical states of delayed hypersensitivity (Mitchell et ah, 1972). We have carried out LMI tests in nickel-sensitive patients to ascertain whether this test can be used to confirm clinical sensitivity. PATIENTS AND METHODS
Ten subjects with clinical evidence of nickel dermatitis together with positive patch tests and positive lymphocyte transformation tests were studied. LMI tests were carried out by the method of Federlin et al. (1971) and Maini et al. (1973), the only modification being in the use of medium 199 instead of Waymouth medium. The nickel sensitive subjects were paired with control subjects for the study. Two diflferent concentrations of nickel were employed (as sulphate) in the tests, io"* and io~^ mEq Ni/ml of medium. In three cases and three controls peripheral blood lymphocytes were also cultured with nickel salts for 3 days prior to performing the LMI tests against independent effector cells on the basis that such prior incubation might be necessary to elaborate LMI in sufficient concentration to Correspondence: Dr F. Hutchinson, Department of Medical Physics, Ninewells Hospital, Dundee. 63
64
T.M.MacLeod, F.Hutchinson and E.J.Raffle
produce a positive test. In two subjects 25% autologous serum or 25% reconstituted freeze-dried plasma were substituted for 10% horse serum as an alternative to provide a potential protein conjugate for the nickel hapten. RESULTS
There was no statistical difference between the migration indices of the nickel sensitive and control subjects for the two different concentrations of antigen used. The maximum variation was less than + 20% of unity. No consistent evidence of LMI factor elaboration under the conditions of these experiments was obtained. DISCUSSION
These results are contrary to those obtained by Mirza, Perera & Bernstein (1974) who reported positive LMI tests in patients with proven nickel dermatitis. A negligible difference would be expected between the use of bovine albumin as a nickel albumin complex and the methods used in some of our tests, i.e. 10% horse serum added to the medium in Federlin et al.'s technique or the 25% autologous serum or reconstituted freeze-dried plasma. Hence the conflicting results may be due to a concentration effect. Positive LMI results have also been reported by Thulin & Zachariae (1972) in chromium sensitive subjects when chromiimi boxmd to bovine albumin was employed as the antigen, but de Jong, Bleimiink & Nater (1974) have shown that positive LMI tests with mercuric chloride, a non-specific mitogen, resulted from a decrease in the number of migrating polymorphonuclear leukocytes, and hence were false positives. They demonstrated that a concentration of 3 x io~^ M Hg induced lysis of 30-50% of the leukocytes within 30 min of incubation. Unlike the lymphocyte transformation test the LMI test is clearly of doubtful value in the diagnosis of delayed hypersensitivity to nickel in dermatitis subjects. ACKNOWLEDGMENT
We would like to express our thanks to Mr D. Donald for his help in this work. REFERENCES DE JONG, M.C.J.M., BLEUMINK, E. & NATER, J.P. (1974) In vitro leukocyte migration inhibition studies of allergic contact dermatitis in man. gth European Congress of Allergology and Clinical Immunology (in press). FEDERLIN, K . , MAINI, R.N., RUSSELL, A . S . & DUMONDE, D . C . (1971) A micro-method for peripheral leukocyte
migration in tuberculin sensitivity. Journal of Clinical Pathology, 24, 533. HUTCHINSON, F., RAFFLE, E.J. & MACLEOD, T.M. (1972) The specificity of lymphocyte transformation in vitro by nickel salts in nickel sensitive subjects. Journal of Investigative Dermatology, 58, 362. MAINI, R.M., ROFFE, L.M., MAGRATH, I . T . & DUMONDE, D.C. (1973) Standardization of the leukocyte migration
test. International Archives of Allergy, 45, 308. MIRZA, A.M., PERERA, M . G . & BERNSTEIN, I . L . (1974) Leukocyte migration inhibition in nickel dermatitis. Federation Proceedings, 33, 728. MITCHELL, CHRISTINE G . , SMITH, M.G.M., GOLDING, P.L., EDDLESTON, A . L . W . F . & WILLIAMS, R . (1972)
Evaluation of the leukocyte migration test as a measure of delayed hypersensitivity in man. Clinical and Experimental Immunology, 11, 535. THULIN, H . & ZACHARIAE, H . (1972) The leukocyte migration test in chromium hypersensitivity. Journal of Investigative Dermatology, 58, 55.
