Commentary
THE LEUKOCYTE MIGRATION INHIBITION TEST T. M. MACLEOD, Ph.D.
Antigenic challenge to leukocytes may result in production of a number of soluble factors or "lymphokines." Several of these lymphokines induce inhibition of migration by immunocompetent cells and in vitro tests have been developed to assay subsequent cell-mediated responses. Tests in which leukocytes are exposed to antigen may be described as "indirect," the resulting migration inhibition factor (MIF) and leukocyte inhibition factor (LIF) being separated and effects assayed independently on their respective target cells; or "direct," in which inhibitory factors produced by leukocyte-antigen interaction are allowed to effect migration on the mixed cell population in situ. This is the leukocyte migration inhibition or LMl test. The LMl test was described by Soborg and Bendixeni and later modified by Federlin et al.^
From the Department of Dermatology, University of Dundee, Dundee, Scotland
dium alone or with medium plus antigen in varying concentrations, are sealed and after incubating at 37° C for 18 hours, the zones of leukocyte migration are measured by planimetry. The "migration index" is the degree of antigen-induced inhibition of migration compared with migration in the control chamber. Effects on Results In vitro techniques used to investigate cell-mediated immunologic problems are often complicated by technical difficulties. In the method of Federlin et al.,^ the leukocyte-rich plasma layer from a gravity sedimented heparinized blood specimen is used, whereas that used by Soborg and Bendixen and other workers involve the use of dextran, PVP, or FicollHypaque. Thus, the lymphocyte content may vary from 30% upward,' and there may also be preferential T cell enrichment. The presence of a serum factor in the culture is regarded as essential and autologous serum or fetal calf serum have been used in varying concentrations. Of major importance is the concentration of antigen in the culture chamber. It has been shown that maximal cellmediated responses are often dependent on a critical concentration of antigen as, for example, in the lymphocyte trans-
Technique Leukocytes are separated from heparinized blood by one of a number of such standard techniques as dextran, FicollHypaque or gravity sedimentation. After centrifugation, the leukocytes are resuspended in a mixture of serum and culture medium at a concentration of 7 X 10" per ml: the cell suspension is transferred to microcapillaries, which are plugged at one end and centrifuged. The capillaries are then cut at the cell-fluid interface and the part with cells mounted on a silicone dab in a culture chamber. A number of such chambers, filled with me667
668
INTERNATIONAL JOURNAL OF DERMATOLOGY
formation test. In the LMI test, leukocyte-antigen contact is confined principally to the capillary interface; thus, with certain antigens in sufficiently high concentration, toxic reactions causing cell death at the interface may inhibit migration. Such an event cannot be evaluated by conventional viability tests. Other factors such as capillary diameter, pH variations, gravity, toxic products from erythrocytes, and choice of media may affect migration characteristics.t- 5 Migration indices of 0.8 or less are usually taken as significant inhibition of migration but figures greater than unity can be recorded when the antigen causes stimulation of migration. This latter phenomenon could be related to the polymorphonuclear content of the cell sample.-' Uses The LMI test has been used in the investigation of a number of immunologic problems, as a technique to study mechanisms of cellular reaction, and also as a diagnostic method free from the hazards of intracutaneous tests for sensitivity reactions. Inhibition of leukocyte migration has been conclusively demonstrated in a number of infective states, inhibition being induced, for example, by the soluble antigens tuberculoprotein, Candida extract, and coccidioidin. In sensitive subjects, skin reactivity to these antigens correlates satisfactorily with the in vitro test. The applicability of the test in Brucella abortus infection has also been demonstrated, antigenic effect on migration depending both on concentration and clinical sensitivity of the subject. The test has considerable potential in dermatology for the study of cell-mediated reactions. In eczematous contact dermatitis, it is a means of identifying
October 1977
Vol. 16
contact factors in isolation in contrast with the battery patch test. It has been shown that LMI can be indiced in vitro with neomycin as the antigen using leukocytes from a neomycin-sensitive subject. Similarly, metal contact factors have been investigated with this technique. In mercury sensitivity, decrease in migration has been reported but the role of polymorphonuclear leukocytes in the test is unclear. Nickel sensitivity has been investigated by several workers'"' ^ with differing conclusions which could be related to choice of serum factors in the culture medium. The possibility that valency state and binding of antigen to a serum fraction such as albumin may be a prerequisite for LMI is indicated from studies on chromium sensitivity and the role of carrier protein in photoallergy due to salicylanilide has also been demonstrated using the indirect MIF test. A particularly interesting application of the LMI test is the investigation of the effect of hydrocortisone^ resulting in production of a factor which stimulates polymorphonuclear leukocyte migration; this could be developed further in the study of the immunosuppressive role of corticosteroids. It demonstrates how the LMI test can be adapted to evaluate cellmediated reactions not only from a mechanistic but also a therapeutic angle. Although described as the LMI test, the primary response is lymphocyte mediated and the resultant production of migratory inhibitory factors in response to antigenic challenge in vitro has recently been investigated more fully by Rocklin.** He has shown that the products of lymphocyte-antigen interaction, when separated on a Sephadex G100 column, consist of 2 distinct fractions—1 containing MIF which inhibits migration of guinea-pig macrophages and hu-
No. 8
man monocytes and another containing LIF which inhibits migration of polymorphonuclear leukocytes. These factors can be further characterized by the effect of 5-methylpentose sugars and monosaccharides on their activity toward target cells.9 Conclusion
MacLeod
LEUKOCYTE TEST
,
,
The LMI test is a useful method of investigating lymphokine production in cellular reactions. Much work is required to establish a standard methodology and to improve its reliability. Because of this, the fact that it is a timeconsuming test, and that it has not been fully investigated in a sufficient range of dermatological conditions, it has not become a routine test replacing the intracutaneous test. • • V ; ^ « References 1. Soborg, M., and Bendixen, G.: Human lymphocyte migration as a parameter of hypersensitivity. Acta Med. Scand. 181:247, : 1967. ^ ,v }.:,-.:.^
669
2. Eederlin, K., Maini, R. N., Russell, A. S., and Dumonde, D. C: A micro-method for peripheral leukocyte migration in tuberculin sensitivity. J. Clin. Pathol. 24:533, 1971. 3. Stevenson, R. D.: Hydrocortisone and the migration of human leukocytes; an indirect effect mediated by mononuclear cells. Clin. Exp. Immunol. 14:417, 1973. 4. Kaltreider, H. B., Soghar, D., Taylor, J. B., and Decker, J. L.: Capillary tube migration for detection of human delayed hypersensitivity; difficulty encountered with "buffy coat" cells and tuberculin antigen. J. Immunol. 103:179, 1969. 5. Bendixen, G., and Soborg, M.: Comments on the leukocyte migration technique as an in vitro method for demonstrating cellular hypersensitivity in man. J. Immunol. 104:1551, 1970. 6. MacLeod, T. M., Hutchinson, F., and Raffle, E. J.: The leukocyte migration inhibition test in allergic nickel contact dermatitis. Br. J. Dermatol. 94:63, 1976. 7. Mirza, A. M., Perera, M. G., and Bernstein, I. L.: Leukocyte migration inhibition In nickel dermatitis. Eed. Prod. 33:728, 1974. 8. Rocklin, R. E.: Products of activated lymphocytes: leukocyte inhibitory factor (L.I.E.) distinct from migration inhibitory factor (M.l.F.) J. Immunol. 112:1461, 1974. 9. Rocklin, R. E.: Role of monosaccharides in the interaction of two lymphocyte mediators with their target cells. J. Imrnunol. 116:816, 1976.
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Jiggers
In 1910 1 had an opportunity of studying in Uganda the sand flea (chigoe), commonly called the jigger. This flea lives in the dust of huts, and attaches itself to human beings, and other warm-blooded animals. Any person, especially children, walking about the hut or the house with bare feet may be bitten by a female jigger; the insect burrows in the skin of the sole, especially in the skin lining the clefts between the toes. When the eggs are ripe, the skin ulcerates and the flea is expelled, leaving an uncomfortable ulcer, which may be the starting point of an infection involving the loss of a toe, or fatal sepsis, and occasionally tetanus. Usually one or two of the insects exist in the skin of the foot; a score, or even a hundred, may be present. The treatment is eviction of the jigger. The flea should be turned out entire. I watched the Baganda boys at Mengo do this neatly with a pin; they prefer a safety-pin. — Bland-Sutton, /.: The Debt of Dermatology to Optical Glass. Med. Press Circ. 125:458, 1928.
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THE LEUKOCYTE MIGRATION INHIBITION TEST T. M. MACLEOD, Ph.D.
Antigenic challenge to leukocytes may result in production of a number of soluble factors or "lymphokines." Several of these lymphokines induce inhibition of migration by immunocompetent cells and in vitro tests have been developed to assay subsequent cell-mediated responses. Tests in which leukocytes are exposed to antigen may be described as "indirect," the resulting migration inhibition factor (MIF) and leukocyte inhibition factor (LIF) being separated and effects assayed independently on their respective target cells; or "direct," in which inhibitory factors produced by leukocyte-antigen interaction are allowed to effect migration on the mixed cell population in situ. This is the leukocyte migration inhibition or LMl test. The LMl test was described by Soborg and Bendixeni and later modified by Federlin et al.^
From the Department of Dermatology, University of Dundee, Dundee, Scotland
dium alone or with medium plus antigen in varying concentrations, are sealed and after incubating at 37° C for 18 hours, the zones of leukocyte migration are measured by planimetry. The "migration index" is the degree of antigen-induced inhibition of migration compared with migration in the control chamber. Effects on Results In vitro techniques used to investigate cell-mediated immunologic problems are often complicated by technical difficulties. In the method of Federlin et al.,^ the leukocyte-rich plasma layer from a gravity sedimented heparinized blood specimen is used, whereas that used by Soborg and Bendixen and other workers involve the use of dextran, PVP, or FicollHypaque. Thus, the lymphocyte content may vary from 30% upward,' and there may also be preferential T cell enrichment. The presence of a serum factor in the culture is regarded as essential and autologous serum or fetal calf serum have been used in varying concentrations. Of major importance is the concentration of antigen in the culture chamber. It has been shown that maximal cellmediated responses are often dependent on a critical concentration of antigen as, for example, in the lymphocyte trans-
Technique Leukocytes are separated from heparinized blood by one of a number of such standard techniques as dextran, FicollHypaque or gravity sedimentation. After centrifugation, the leukocytes are resuspended in a mixture of serum and culture medium at a concentration of 7 X 10" per ml: the cell suspension is transferred to microcapillaries, which are plugged at one end and centrifuged. The capillaries are then cut at the cell-fluid interface and the part with cells mounted on a silicone dab in a culture chamber. A number of such chambers, filled with me667
668
INTERNATIONAL JOURNAL OF DERMATOLOGY
formation test. In the LMI test, leukocyte-antigen contact is confined principally to the capillary interface; thus, with certain antigens in sufficiently high concentration, toxic reactions causing cell death at the interface may inhibit migration. Such an event cannot be evaluated by conventional viability tests. Other factors such as capillary diameter, pH variations, gravity, toxic products from erythrocytes, and choice of media may affect migration characteristics.t- 5 Migration indices of 0.8 or less are usually taken as significant inhibition of migration but figures greater than unity can be recorded when the antigen causes stimulation of migration. This latter phenomenon could be related to the polymorphonuclear content of the cell sample.-' Uses The LMI test has been used in the investigation of a number of immunologic problems, as a technique to study mechanisms of cellular reaction, and also as a diagnostic method free from the hazards of intracutaneous tests for sensitivity reactions. Inhibition of leukocyte migration has been conclusively demonstrated in a number of infective states, inhibition being induced, for example, by the soluble antigens tuberculoprotein, Candida extract, and coccidioidin. In sensitive subjects, skin reactivity to these antigens correlates satisfactorily with the in vitro test. The applicability of the test in Brucella abortus infection has also been demonstrated, antigenic effect on migration depending both on concentration and clinical sensitivity of the subject. The test has considerable potential in dermatology for the study of cell-mediated reactions. In eczematous contact dermatitis, it is a means of identifying
October 1977
Vol. 16
contact factors in isolation in contrast with the battery patch test. It has been shown that LMI can be indiced in vitro with neomycin as the antigen using leukocytes from a neomycin-sensitive subject. Similarly, metal contact factors have been investigated with this technique. In mercury sensitivity, decrease in migration has been reported but the role of polymorphonuclear leukocytes in the test is unclear. Nickel sensitivity has been investigated by several workers'"' ^ with differing conclusions which could be related to choice of serum factors in the culture medium. The possibility that valency state and binding of antigen to a serum fraction such as albumin may be a prerequisite for LMI is indicated from studies on chromium sensitivity and the role of carrier protein in photoallergy due to salicylanilide has also been demonstrated using the indirect MIF test. A particularly interesting application of the LMI test is the investigation of the effect of hydrocortisone^ resulting in production of a factor which stimulates polymorphonuclear leukocyte migration; this could be developed further in the study of the immunosuppressive role of corticosteroids. It demonstrates how the LMI test can be adapted to evaluate cellmediated reactions not only from a mechanistic but also a therapeutic angle. Although described as the LMI test, the primary response is lymphocyte mediated and the resultant production of migratory inhibitory factors in response to antigenic challenge in vitro has recently been investigated more fully by Rocklin.** He has shown that the products of lymphocyte-antigen interaction, when separated on a Sephadex G100 column, consist of 2 distinct fractions—1 containing MIF which inhibits migration of guinea-pig macrophages and hu-
No. 8
man monocytes and another containing LIF which inhibits migration of polymorphonuclear leukocytes. These factors can be further characterized by the effect of 5-methylpentose sugars and monosaccharides on their activity toward target cells.9 Conclusion
MacLeod
LEUKOCYTE TEST
,
,
The LMI test is a useful method of investigating lymphokine production in cellular reactions. Much work is required to establish a standard methodology and to improve its reliability. Because of this, the fact that it is a timeconsuming test, and that it has not been fully investigated in a sufficient range of dermatological conditions, it has not become a routine test replacing the intracutaneous test. • • V ; ^ « References 1. Soborg, M., and Bendixen, G.: Human lymphocyte migration as a parameter of hypersensitivity. Acta Med. Scand. 181:247, : 1967. ^ ,v }.:,-.:.^
669
2. Eederlin, K., Maini, R. N., Russell, A. S., and Dumonde, D. C: A micro-method for peripheral leukocyte migration in tuberculin sensitivity. J. Clin. Pathol. 24:533, 1971. 3. Stevenson, R. D.: Hydrocortisone and the migration of human leukocytes; an indirect effect mediated by mononuclear cells. Clin. Exp. Immunol. 14:417, 1973. 4. Kaltreider, H. B., Soghar, D., Taylor, J. B., and Decker, J. L.: Capillary tube migration for detection of human delayed hypersensitivity; difficulty encountered with "buffy coat" cells and tuberculin antigen. J. Immunol. 103:179, 1969. 5. Bendixen, G., and Soborg, M.: Comments on the leukocyte migration technique as an in vitro method for demonstrating cellular hypersensitivity in man. J. Immunol. 104:1551, 1970. 6. MacLeod, T. M., Hutchinson, F., and Raffle, E. J.: The leukocyte migration inhibition test in allergic nickel contact dermatitis. Br. J. Dermatol. 94:63, 1976. 7. Mirza, A. M., Perera, M. G., and Bernstein, I. L.: Leukocyte migration inhibition In nickel dermatitis. Eed. Prod. 33:728, 1974. 8. Rocklin, R. E.: Products of activated lymphocytes: leukocyte inhibitory factor (L.I.E.) distinct from migration inhibitory factor (M.l.F.) J. Immunol. 112:1461, 1974. 9. Rocklin, R. E.: Role of monosaccharides in the interaction of two lymphocyte mediators with their target cells. J. Imrnunol. 116:816, 1976.
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-
• '
•'•
Jiggers
In 1910 1 had an opportunity of studying in Uganda the sand flea (chigoe), commonly called the jigger. This flea lives in the dust of huts, and attaches itself to human beings, and other warm-blooded animals. Any person, especially children, walking about the hut or the house with bare feet may be bitten by a female jigger; the insect burrows in the skin of the sole, especially in the skin lining the clefts between the toes. When the eggs are ripe, the skin ulcerates and the flea is expelled, leaving an uncomfortable ulcer, which may be the starting point of an infection involving the loss of a toe, or fatal sepsis, and occasionally tetanus. Usually one or two of the insects exist in the skin of the foot; a score, or even a hundred, may be present. The treatment is eviction of the jigger. The flea should be turned out entire. I watched the Baganda boys at Mengo do this neatly with a pin; they prefer a safety-pin. — Bland-Sutton, /.: The Debt of Dermatology to Optical Glass. Med. Press Circ. 125:458, 1928.
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