British Journal of Dermatology (1976) 94, 65.
A study on the clinical application of a direct leukocyte migration test in chromium contact allergy D.TIO Department of Dermatology, Binnengasthuis, Amsterdam, The Netherlands Accepted for publication 9 July 1975
SUMMARY
A capillary tube leukocyte migration inhibition assay has been adopted as an in vitro method for the demonstration of chromium hypersensitivity in twenty-two subjects with clinically proven or suspected chromium allergy. Two complexes of trivalent chromium and human serum albumin, exerting different migration inhibitory effects, have been prepared and used as the antigens. In the presence of the chromium-albumin complex with strong inhibitory activity, sensitivity to chromium was demonstrated independent of the clinical condition of the skin in all the patch test positive subjects. An additional positive response to the second chromium-albumin complex was observed only in those patients who were clinically in a state of exacerbation of an allergic contact dermatitis in which chromium allergy was an active causative factor. The results were not influenced by skin allergic reactivity to compounds other than chromium and the method was found to be of practical clinical value for diagnosing chromium allergy.
Leukocyte migration tests (LMT) have found wide employment in immunopathology and denote delayed type immunity in man. They have proved effective in bacterial (Soborg, 1967; Clausen & Soborg, 1969; Clausen, 1970; Budtz-Jergensen, 1972; Godal et al., 1972; Rauch & King, 1973) and parasitic (Gaines et al., 1971) infections, in auto-allergic diseases (Soborg & Halberg, 1968; Brostoff & Walker, 1971) and in certain contact allergic skin disorders (Nordqvist & Rorsman, 1967; Herman & Sams, 1971). Their application in investigative dermatology, however, has been limited by the difficulties often met in finding and preparing the proper antigens. Chromiimi allergy is a common cause of contact eczema and has been the subject of extensive study by many investigators. In vitro sensitivity to chromium has been demonstrated by Thulin & Zachariae (1972) using hexavalent and trivalent chromium bound to bovine albumin as antigens. The present paper reports the employment of complexes of trivalent chromiimi and human albumin as antigens in a practical application of Soborg & Bendixen's (1967) modification of the LMT in clinical allergy. MATERIALS AND METHODS
The study was carried out on five healthy controls, nine patients with clinically proved and thirteen patients with suspected chromiimi allergic contact dermatitis. 65
66
D.Tio
In cases of active eczema routine patch tests were performed as soon as the skin condition allowed such a procedure. Patients with a clinical record of chromium allergy but free of symptoms for at least 3 months were not subjected to epicutaneous testing. Antigen preparation
Two stock solutions of chromium chloride and human albumin were prepared as follows: (A) Five hundred mg of human albumin (Fraction V, Sigma) and 250 mg of chromium chloride (CrClj.eHjO, Merck) were dissolved in 15 ml of distilled water. The solution was stored overnight at 4°C and dialysed against distilled water for 72 h. (B) The same amounts of human albumin and chromium chloride were dissolved in 75 ml of distilled water and stored overnight at 4°C. Prior to dialysis the volume was adjusted to 15 ml with distilled water and processed as mentioned above. After dialysis, protein concentrations were estimated by Lowry's method (1951) and chromium contents as described by Cohen (1966a). Solution A consisted of 1-95 g% human albumin (HuA) and i g% chromium (Cr). Ten, 20, 40, and 50 lA/ml medium, corresponding with o-i mg Cr/o-i95 mg HuA, 0-2 mg Cr/o-39O mg HuA, 0-4 mg Cr/0780 mg HuA, and 0-5 mg Cr/o-975 mg HuA per ml medium respectively, were used for testing. Solution B consisted of i-8 g% human albumin (HuA) and 082 g% chromium (Cr). Preliminary studies with concentrations ranging from 2-iOfil/ml have established that 2 and 4/
0,60 0,50 0,40
1l Solution A
0-57 0-47 067
LJ
• D a
D
064
080 105
1,10 1,00
4
4^1/ml Solution B
FIGURE I. The effect of 40 //I/ml of solution A and 4 //I/ml of solution B on the leukocyte migration indices of chromium negative (squares) and chromium positive (stars) persons. Open stars, chromium positive without symptoms or with allergic contact dermatitis due to other compounds than chromium. Filled stars, chromium positive with active chromium allergic contact dermatitis.
071
Leukocyte migration test in chromium contact allergy
69
DISCUSSION
The data presented show that inhibition of migration of autologous leukocytes induced by trivalent chromium-human albumin is a sensitive in vitro parameter of chromium hypersensitivity. It is also shown that the assay may be used for different purposes. If the most important objective is to distinguish between chromium negative and chromiimi positive persons, false negative results can be avoided by using the appropriate dose of the solution A. On the other hand it may be of practical importance to know whether chromiimi compounds or other agents are actively involved in the eruptive stage of an allergic contact dermatitis, in which case solution B should be employed. As suggested in Fig. i, a quantitative estimation of the migration inhibition reaction may be obtained by performing LMT with only two test doses, the 40 ^1/ml of solution A and the 4 //I/ml of solution B. Results obtained so far seem to indicate that the principle of the method is applicable in chromium allergy and may be with other common contact allergens as well. Our results with trivalent chromium compound are in agreement with the latest concepts on the 'true' nature of the hapten in chromium hypersensitivity, as suggested by Cohen (1966a), Samitz et al. (1969), Polak & Frey (1973) and Shmunes, Katz & Samitz (1973). The difference in the migration inhibitory activity of the two test solutions is not easily explained. Since human albumin has been used as the carrier, structural changes of the protein in the presence of the chromium salt (Clark, 1959) might have influenced the physico-chemical properties of the complex. Consequently, the different procedure adopted for the preparation of solutions A and B might have resulted in the formation of two different complexes with different antigenic activity. On the other hand, the nature of the carrier may be of less importance (Cohen, 1966b, 1968) and the difference in the migration inhibitory activity might as well be attributed to the different amounts of chromium ions present in the cultures. In that case other factors, such as the presence of a specific antigen recognizing system on the cells participating in the LMT as well as the direct action of the antigens on the migrating cell population, must be kept in mind and need further evaluation. Despite the lack of detailed knowledge of the true mechanism underlying the assay, LMT has by virtue of its sensitivity become a valuable tool for fundamental and experimental research in biology and medicine. The results of this study suggest that LMT may be adopted for practical purposes in clinical dermatology as well.
REFERENCES BoYUM, A. (1968) Separation of leukocytes from blood and bone marrow. Scandinavian Journal of Clinical and Laboratory Investigation, 97, I. BROSTOFF, J. & WALKER, J.G. (1971) Leukocyte migration inhibition with Kveim antigen in Crohn's disease. Clinical and Experimental Immunology, 9, 707. BuDTZ-j0RGENSEN, E. (1972) Delayed type hypersensitivity to Candida albicans in man demonstrated in vitro: the capillary tube migration test. Acta Allergologica, 27, 41. CLARK, J . H . (1959) The denaturation of proteins by chromium salts. Archives of Industrial Health, 20, 117. CLAUSEN, J.E. (1970) Polymorphonuclear leukocytes in the specific antigen-induced inhibition of the in vitro migration of human peripheral leukocytes. Acta Medica Scandinavica, 188, 59. CLAUSEN, J.E. & SOBORG, M . (1969) In vitro detection of tuberculin hypersensitivity in man. Specific migration inhibition of white blood cells from tuberculin-positive persons. Acta Medica Scandinavica, 186, 227. COHEN, H.A. (1966a) Carrier specificity of tuberculin-type reaction to trivalent chromium. Archives of Dermatology, 93) 34COHEN, H.A. (1966b) Tuberculin-type reaction to heparin-chromium complex. Archives of Dermatology, 94, 409. COHEN, H.A. (1968) Hyaluronic acid. A specific carrier of chromium sensitivity. Archives of Dermatology, 98, 148.
70
D.Tio
GAINESJ J.D., ARAUJOJ F.G.J KRAHENBUHL, J . L . & REMONGTON, J . S . (1971) Simplified in vitro method for measur-
ing delayed type hypersensitivity to latent intracellular infection in man (toxoplasmosis). Jowrna/ of Immunology, 107, 906. GoDAL, T., MYRVANG, B., FROLAND, S.S., SHAO, J. & MELAKU, G . (1972) Evidence that the mechanism of
immunological tolerance ('central failure') is operative in the lack of host resistance in lepromatous leprosy. Scandinavian Journal of Immunology, i, 311. HERMAN, P . S . & SAMS, W . M . JR (1971) Requirement for carrier protein in salicylanilide sensitivity: the migration inhibition test in contact photo allergy. Journal of Laboratory and Clinical Medicine, 77, 572. LowRY, O.H., ROSENBROUGH, N.J., FARR, A. & RANDALL, R.J. (i9Si) Protein measurement with folin phenol reagent. Journal of Biological Chemistry, 193, 265. NORDQVIST, B. & RORSMAN, H. (1967) Leukocyte migration in vitro as an indicator of allergy in eczematous contact dermatitis. Transactions of the St John's Hospital Dermatological Society {London), 53, 154. PoLAK, J. & FREY, J.R. (1973) Studies on contact hypersensitivity to chromium in the guinea-pig. International Archives of Allergy, 44, 51. RAUCH, H . C . & KING, K . (1973) Human leukocyte migration inhibition as an indicator of cellular hypersensitivity to soluble antigens. International Archives of Allergy, 44, 862. SAMITZ, M.H., KATZ, S.A., SCHNEIDER, D . M . & GROSS, P . R . (1969) Chromium-protein interactions. Acta
dermato-venereologica, 49, 142. SHMUNES, E., KATZ, S.A. & SAMITZ, M . H . (1973) Chromium-amino acid conjugates as elicitors in chromium sensitized guinea-pigs. Jowma/ of Investigative Dermatology, 60, 193. S0BORG, M. (1967) In vitro detection of cellular hypersensitivity in man. Specific migration inhibition of white blood cells from brucella positive persons. Acta Medica Scandinavica, 182, 167. S0BORG, M. & BENDIXEN, G . (1967) Hiunan lymphocyte migration as a parameter of hypersensitivity. Acta Medica Scandinavica, 181, 247. S0BORG, M. & HALBERG, P . (1968) Cellular hypersensitivity in Hashimoto's thyroiditis. Specific action of thyroid extract upon the migration of leucocytes 'in vitro'. Acta Medica Scandinavica, 183, ioi. THULIN, H . & ZACHARIAE, H . (1972) The leucocyte migration test in chromium hypersensitivity. Journal of Investigative Dermatology, 58, 55.
A study on the clinical application of a direct leukocyte migration test in chromium contact allergy D.TIO Department of Dermatology, Binnengasthuis, Amsterdam, The Netherlands Accepted for publication 9 July 1975
SUMMARY
A capillary tube leukocyte migration inhibition assay has been adopted as an in vitro method for the demonstration of chromium hypersensitivity in twenty-two subjects with clinically proven or suspected chromium allergy. Two complexes of trivalent chromium and human serum albumin, exerting different migration inhibitory effects, have been prepared and used as the antigens. In the presence of the chromium-albumin complex with strong inhibitory activity, sensitivity to chromium was demonstrated independent of the clinical condition of the skin in all the patch test positive subjects. An additional positive response to the second chromium-albumin complex was observed only in those patients who were clinically in a state of exacerbation of an allergic contact dermatitis in which chromium allergy was an active causative factor. The results were not influenced by skin allergic reactivity to compounds other than chromium and the method was found to be of practical clinical value for diagnosing chromium allergy.
Leukocyte migration tests (LMT) have found wide employment in immunopathology and denote delayed type immunity in man. They have proved effective in bacterial (Soborg, 1967; Clausen & Soborg, 1969; Clausen, 1970; Budtz-Jergensen, 1972; Godal et al., 1972; Rauch & King, 1973) and parasitic (Gaines et al., 1971) infections, in auto-allergic diseases (Soborg & Halberg, 1968; Brostoff & Walker, 1971) and in certain contact allergic skin disorders (Nordqvist & Rorsman, 1967; Herman & Sams, 1971). Their application in investigative dermatology, however, has been limited by the difficulties often met in finding and preparing the proper antigens. Chromiimi allergy is a common cause of contact eczema and has been the subject of extensive study by many investigators. In vitro sensitivity to chromium has been demonstrated by Thulin & Zachariae (1972) using hexavalent and trivalent chromium bound to bovine albumin as antigens. The present paper reports the employment of complexes of trivalent chromiimi and human albumin as antigens in a practical application of Soborg & Bendixen's (1967) modification of the LMT in clinical allergy. MATERIALS AND METHODS
The study was carried out on five healthy controls, nine patients with clinically proved and thirteen patients with suspected chromiimi allergic contact dermatitis. 65
66
D.Tio
In cases of active eczema routine patch tests were performed as soon as the skin condition allowed such a procedure. Patients with a clinical record of chromium allergy but free of symptoms for at least 3 months were not subjected to epicutaneous testing. Antigen preparation
Two stock solutions of chromium chloride and human albumin were prepared as follows: (A) Five hundred mg of human albumin (Fraction V, Sigma) and 250 mg of chromium chloride (CrClj.eHjO, Merck) were dissolved in 15 ml of distilled water. The solution was stored overnight at 4°C and dialysed against distilled water for 72 h. (B) The same amounts of human albumin and chromium chloride were dissolved in 75 ml of distilled water and stored overnight at 4°C. Prior to dialysis the volume was adjusted to 15 ml with distilled water and processed as mentioned above. After dialysis, protein concentrations were estimated by Lowry's method (1951) and chromium contents as described by Cohen (1966a). Solution A consisted of 1-95 g% human albumin (HuA) and i g% chromium (Cr). Ten, 20, 40, and 50 lA/ml medium, corresponding with o-i mg Cr/o-i95 mg HuA, 0-2 mg Cr/o-39O mg HuA, 0-4 mg Cr/0780 mg HuA, and 0-5 mg Cr/o-975 mg HuA per ml medium respectively, were used for testing. Solution B consisted of i-8 g% human albumin (HuA) and 082 g% chromium (Cr). Preliminary studies with concentrations ranging from 2-iOfil/ml have established that 2 and 4/
0,60 0,50 0,40
1l Solution A
0-57 0-47 067
LJ
• D a
D
064
080 105
1,10 1,00
4
4^1/ml Solution B
FIGURE I. The effect of 40 //I/ml of solution A and 4 //I/ml of solution B on the leukocyte migration indices of chromium negative (squares) and chromium positive (stars) persons. Open stars, chromium positive without symptoms or with allergic contact dermatitis due to other compounds than chromium. Filled stars, chromium positive with active chromium allergic contact dermatitis.
071
Leukocyte migration test in chromium contact allergy
69
DISCUSSION
The data presented show that inhibition of migration of autologous leukocytes induced by trivalent chromium-human albumin is a sensitive in vitro parameter of chromium hypersensitivity. It is also shown that the assay may be used for different purposes. If the most important objective is to distinguish between chromium negative and chromiimi positive persons, false negative results can be avoided by using the appropriate dose of the solution A. On the other hand it may be of practical importance to know whether chromiimi compounds or other agents are actively involved in the eruptive stage of an allergic contact dermatitis, in which case solution B should be employed. As suggested in Fig. i, a quantitative estimation of the migration inhibition reaction may be obtained by performing LMT with only two test doses, the 40 ^1/ml of solution A and the 4 //I/ml of solution B. Results obtained so far seem to indicate that the principle of the method is applicable in chromium allergy and may be with other common contact allergens as well. Our results with trivalent chromium compound are in agreement with the latest concepts on the 'true' nature of the hapten in chromium hypersensitivity, as suggested by Cohen (1966a), Samitz et al. (1969), Polak & Frey (1973) and Shmunes, Katz & Samitz (1973). The difference in the migration inhibitory activity of the two test solutions is not easily explained. Since human albumin has been used as the carrier, structural changes of the protein in the presence of the chromium salt (Clark, 1959) might have influenced the physico-chemical properties of the complex. Consequently, the different procedure adopted for the preparation of solutions A and B might have resulted in the formation of two different complexes with different antigenic activity. On the other hand, the nature of the carrier may be of less importance (Cohen, 1966b, 1968) and the difference in the migration inhibitory activity might as well be attributed to the different amounts of chromium ions present in the cultures. In that case other factors, such as the presence of a specific antigen recognizing system on the cells participating in the LMT as well as the direct action of the antigens on the migrating cell population, must be kept in mind and need further evaluation. Despite the lack of detailed knowledge of the true mechanism underlying the assay, LMT has by virtue of its sensitivity become a valuable tool for fundamental and experimental research in biology and medicine. The results of this study suggest that LMT may be adopted for practical purposes in clinical dermatology as well.
REFERENCES BoYUM, A. (1968) Separation of leukocytes from blood and bone marrow. Scandinavian Journal of Clinical and Laboratory Investigation, 97, I. BROSTOFF, J. & WALKER, J.G. (1971) Leukocyte migration inhibition with Kveim antigen in Crohn's disease. Clinical and Experimental Immunology, 9, 707. BuDTZ-j0RGENSEN, E. (1972) Delayed type hypersensitivity to Candida albicans in man demonstrated in vitro: the capillary tube migration test. Acta Allergologica, 27, 41. CLARK, J . H . (1959) The denaturation of proteins by chromium salts. Archives of Industrial Health, 20, 117. CLAUSEN, J.E. (1970) Polymorphonuclear leukocytes in the specific antigen-induced inhibition of the in vitro migration of human peripheral leukocytes. Acta Medica Scandinavica, 188, 59. CLAUSEN, J.E. & SOBORG, M . (1969) In vitro detection of tuberculin hypersensitivity in man. Specific migration inhibition of white blood cells from tuberculin-positive persons. Acta Medica Scandinavica, 186, 227. COHEN, H.A. (1966a) Carrier specificity of tuberculin-type reaction to trivalent chromium. Archives of Dermatology, 93) 34COHEN, H.A. (1966b) Tuberculin-type reaction to heparin-chromium complex. Archives of Dermatology, 94, 409. COHEN, H.A. (1968) Hyaluronic acid. A specific carrier of chromium sensitivity. Archives of Dermatology, 98, 148.
70
D.Tio
GAINESJ J.D., ARAUJOJ F.G.J KRAHENBUHL, J . L . & REMONGTON, J . S . (1971) Simplified in vitro method for measur-
ing delayed type hypersensitivity to latent intracellular infection in man (toxoplasmosis). Jowrna/ of Immunology, 107, 906. GoDAL, T., MYRVANG, B., FROLAND, S.S., SHAO, J. & MELAKU, G . (1972) Evidence that the mechanism of
immunological tolerance ('central failure') is operative in the lack of host resistance in lepromatous leprosy. Scandinavian Journal of Immunology, i, 311. HERMAN, P . S . & SAMS, W . M . JR (1971) Requirement for carrier protein in salicylanilide sensitivity: the migration inhibition test in contact photo allergy. Journal of Laboratory and Clinical Medicine, 77, 572. LowRY, O.H., ROSENBROUGH, N.J., FARR, A. & RANDALL, R.J. (i9Si) Protein measurement with folin phenol reagent. Journal of Biological Chemistry, 193, 265. NORDQVIST, B. & RORSMAN, H. (1967) Leukocyte migration in vitro as an indicator of allergy in eczematous contact dermatitis. Transactions of the St John's Hospital Dermatological Society {London), 53, 154. PoLAK, J. & FREY, J.R. (1973) Studies on contact hypersensitivity to chromium in the guinea-pig. International Archives of Allergy, 44, 51. RAUCH, H . C . & KING, K . (1973) Human leukocyte migration inhibition as an indicator of cellular hypersensitivity to soluble antigens. International Archives of Allergy, 44, 862. SAMITZ, M.H., KATZ, S.A., SCHNEIDER, D . M . & GROSS, P . R . (1969) Chromium-protein interactions. Acta
dermato-venereologica, 49, 142. SHMUNES, E., KATZ, S.A. & SAMITZ, M . H . (1973) Chromium-amino acid conjugates as elicitors in chromium sensitized guinea-pigs. Jowma/ of Investigative Dermatology, 60, 193. S0BORG, M. (1967) In vitro detection of cellular hypersensitivity in man. Specific migration inhibition of white blood cells from brucella positive persons. Acta Medica Scandinavica, 182, 167. S0BORG, M. & BENDIXEN, G . (1967) Hiunan lymphocyte migration as a parameter of hypersensitivity. Acta Medica Scandinavica, 181, 247. S0BORG, M. & HALBERG, P . (1968) Cellular hypersensitivity in Hashimoto's thyroiditis. Specific action of thyroid extract upon the migration of leucocytes 'in vitro'. Acta Medica Scandinavica, 183, ioi. THULIN, H . & ZACHARIAE, H . (1972) The leucocyte migration test in chromium hypersensitivity. Journal of Investigative Dermatology, 58, 55.
