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Basophil Leukocytes in Non-vesicular Allergic Patch Test Reactions*
Preliminary and Short Report BASOPHIL LEUKOCYTES IN NON-VESICULAR ALLERGIC PATCH TEST REACTIONS* NILS ASPERGEN, M.D., SIGFRID FREGERT, M.D. AND HANS RORSMAN, M.D.
It was recently reported (1) that vesicles of allergic patch test reactions contain abundant basophil leukocytes. Many allergic test reactions
are characterized, however, by infiltration and
papules without the development of any vesicles.
In order to obtain exudate for determination of the relative number of basophils in such cases we provoked a cantharidine bulla at the site of infiltration of the area tested. This technic has been used previously by Nexmand (2) for cytologic studies of intracutaneous test reactions.
TABLE I Relative number of basophil leukoc pies in
cantharidine bullae on infiltrative test reactions and on normal skin aasnphils Basnphils in in AllergenPatient Canthari- Cantharidine dine Bulla Bulla 1%) (%)
Allergen
2 3
Chromate
MATERIAL AND METHODS
Thirty-three non-vesicular infiltrative patch Chromate
test reactions produced by 17 different allergens were studied in 16 patients. Tincture of cantharidine was applied with patch test technic (a) on
test reactions that had developed after closed
application of allergen for 24—48 hours and (b) on normal skin. The interval between application of the allergen and of the cantharidine was 24 to 120 hours. The cantharidine patches were removed
after 3 to 48 hours, by which time the bullae had appeared. Samples were obtained by puncturing the epidermis covering this bulla with a fine injection needle and gently pressing a glass slide against the drop of fluid oozing from the lesion. The slide
was then air-dried and stained in 1% toluidine blue in methanol for 5 minutes, according to Undritz (3). When possible, 1,000 nucleated cells were counted. However, in 7 bullae on infiltrative
test reactions and in 3 bullae on normal skin, less than 1,000 cells were present. Immersion objective X 100 and ocular X 12.5 were used. RESULTS
It is clear from the table that in 28 of 32 samples the basophil leukocytes represent more than 1.0%
Chromate Chromate Chromate Chromate Formaldehyde Formaldehyde Hexamethylenetetramine Balsam of pine Balsam of pine Balsam of spruce Balsam of spruce Balsam of spruce Balsam of Peru Resin beozoin Colophony Colophony Colophony Oil of turpentine Oil of turpentine Epoxy resin Epoxy resin Epoxy resin p-Phenylenediamine
of cells in the bullae produced by cantharidine at p-Aminoazobenzene the site of allergic tests. p-Toluidine The cantharidine bullae on normal skin had in p , p'-Diaminodiphenyl15 of 16 samples at most 1.0% of basophils. The methane eantharidine bulla on normal skin which contained Diphenylguanidine more than 1% basophil leukocytes was 48 hours old. Examination of a new cantharidine bulla on Neomycin normal skin in this patient after 3 hours showed Neomycin Vioform 0% basophil leukocytes.
S
9 10 11
6 15 15 7
10 1 7
9,
3.0 5.0 2.0 5.0 1.2 0.5 5.6 1.6 0.5 2.3 3.0 9.6 2
0.7 0 0 0.2 5.2 0 0 0.3 0.3 0.3 0.2 0.4 0.3 0.2
13 2 13 4
5.0 2.2 1.1 1.0 4.4 4.8 2.7 5.2 7.5
S
12.0
9 12 12 4 9
2.0
0.2
0.2
0.2
14 7
16 1 13 5
1.0 10
1.8
0
0.3 0.3 0.2 0
0.7 0
0.3 0
0.2
0.3 0.2
8.0
0 0.4 0
1
0
1.1
1.4
In all the samples studied except two, the basophil count was higher in the cantharidine bullac on test reactions than in cantharidine * From the Department of Dermatology, Uni-
versity of Lund, Lund, Sweden.
Supported by a grant from the Alfred Osterlund Foundation. Received for publication April 22, 1963.
bullae on normal skin. DISCUSSION
The present findings together with data given by Wolf-Jtirgensen (4) and Aspegren, Fregert and
107
108
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Rorsman (1) suggest that basophils may be of
importance in a]lergic eczematous contact derma-
titis. The occurrence of basophils in cantharidine builne at the site of non-vesicular allergic contact test reactions implies that all patch test reactions
value in differentiating allergic from primary irritant dermatitis. REFERENCES 1.
Basophil
can he studied for hasophils irrespective of the
presence of primary vesicles. Even if local hasophilia is not absolutely specific for allergic eczematous contact dermatitis (5, 6, 7)
contact 2.
between allergic eczematous and primary irritant dermatitis.
leukocytes in allergic eczematous
dermatitis. Tnt. Arch. Allerg., In
press. NEXMAND, P. H.: Cytologic studies of allergic reactions by means of cantharides blisters. Acta Dcrmato-vcner. (Stockholm), 34: 389,
basophil determination in test reactions may be
useful in the very important differentiation
A5PEuaEN, N., FaEOERT, S. AND RORSMAN, H.:
1954. 3.
UNDEITZ, E.: Hamatologische Tafeln Sandoz, Basel, p. 7, 23, 1950.
4. WOLF-JUROENSEN,
P.:
Communication at the
Danish Society for Allergological Research.
arnwarAlcv
By application of cantharidine on a non-vesic-
ular allergic patch test reaction an exudate is obtained which contains basophil leukocytes to
about the same extent as in primary allergic vesicles.
Determination of the relative number of baso-
phils in patch test reactions appears to be of
Copenhagen,
5.
Dcc. 7,
1962.
KLAUSNER, E. AND Knxmnicn, C.: Uber den
exsudative Mastzellen. Arch. Dermat. u.
Syph., 96: 235, 1909. 6. KLArJSNEa, E. AND KREJB!CH, C.: TJber exsuda-
tive Mastzellengehalt vesiculoscr Hautefiorescenscn. Folia Raemat., 15: 347, 1913. 7. ASPEGREN, N., Faxoawr, S. AND R0E5MAN, H.: Basophil leukocytes in lesions of various dermatoses. To be published.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
It was recently reported (1) that vesicles of allergic patch test reactions contain abundant basophil leukocytes. Many allergic test reactions
are characterized, however, by infiltration and
papules without the development of any vesicles.
In order to obtain exudate for determination of the relative number of basophils in such cases we provoked a cantharidine bulla at the site of infiltration of the area tested. This technic has been used previously by Nexmand (2) for cytologic studies of intracutaneous test reactions.
TABLE I Relative number of basophil leukoc pies in
cantharidine bullae on infiltrative test reactions and on normal skin aasnphils Basnphils in in AllergenPatient Canthari- Cantharidine dine Bulla Bulla 1%) (%)
Allergen
2 3
Chromate
MATERIAL AND METHODS
Thirty-three non-vesicular infiltrative patch Chromate
test reactions produced by 17 different allergens were studied in 16 patients. Tincture of cantharidine was applied with patch test technic (a) on
test reactions that had developed after closed
application of allergen for 24—48 hours and (b) on normal skin. The interval between application of the allergen and of the cantharidine was 24 to 120 hours. The cantharidine patches were removed
after 3 to 48 hours, by which time the bullae had appeared. Samples were obtained by puncturing the epidermis covering this bulla with a fine injection needle and gently pressing a glass slide against the drop of fluid oozing from the lesion. The slide
was then air-dried and stained in 1% toluidine blue in methanol for 5 minutes, according to Undritz (3). When possible, 1,000 nucleated cells were counted. However, in 7 bullae on infiltrative
test reactions and in 3 bullae on normal skin, less than 1,000 cells were present. Immersion objective X 100 and ocular X 12.5 were used. RESULTS
It is clear from the table that in 28 of 32 samples the basophil leukocytes represent more than 1.0%
Chromate Chromate Chromate Chromate Formaldehyde Formaldehyde Hexamethylenetetramine Balsam of pine Balsam of pine Balsam of spruce Balsam of spruce Balsam of spruce Balsam of Peru Resin beozoin Colophony Colophony Colophony Oil of turpentine Oil of turpentine Epoxy resin Epoxy resin Epoxy resin p-Phenylenediamine
of cells in the bullae produced by cantharidine at p-Aminoazobenzene the site of allergic tests. p-Toluidine The cantharidine bullae on normal skin had in p , p'-Diaminodiphenyl15 of 16 samples at most 1.0% of basophils. The methane eantharidine bulla on normal skin which contained Diphenylguanidine more than 1% basophil leukocytes was 48 hours old. Examination of a new cantharidine bulla on Neomycin normal skin in this patient after 3 hours showed Neomycin Vioform 0% basophil leukocytes.
S
9 10 11
6 15 15 7
10 1 7
9,
3.0 5.0 2.0 5.0 1.2 0.5 5.6 1.6 0.5 2.3 3.0 9.6 2
0.7 0 0 0.2 5.2 0 0 0.3 0.3 0.3 0.2 0.4 0.3 0.2
13 2 13 4
5.0 2.2 1.1 1.0 4.4 4.8 2.7 5.2 7.5
S
12.0
9 12 12 4 9
2.0
0.2
0.2
0.2
14 7
16 1 13 5
1.0 10
1.8
0
0.3 0.3 0.2 0
0.7 0
0.3 0
0.2
0.3 0.2
8.0
0 0.4 0
1
0
1.1
1.4
In all the samples studied except two, the basophil count was higher in the cantharidine bullac on test reactions than in cantharidine * From the Department of Dermatology, Uni-
versity of Lund, Lund, Sweden.
Supported by a grant from the Alfred Osterlund Foundation. Received for publication April 22, 1963.
bullae on normal skin. DISCUSSION
The present findings together with data given by Wolf-Jtirgensen (4) and Aspegren, Fregert and
107
108
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Rorsman (1) suggest that basophils may be of
importance in a]lergic eczematous contact derma-
titis. The occurrence of basophils in cantharidine builne at the site of non-vesicular allergic contact test reactions implies that all patch test reactions
value in differentiating allergic from primary irritant dermatitis. REFERENCES 1.
Basophil
can he studied for hasophils irrespective of the
presence of primary vesicles. Even if local hasophilia is not absolutely specific for allergic eczematous contact dermatitis (5, 6, 7)
contact 2.
between allergic eczematous and primary irritant dermatitis.
leukocytes in allergic eczematous
dermatitis. Tnt. Arch. Allerg., In
press. NEXMAND, P. H.: Cytologic studies of allergic reactions by means of cantharides blisters. Acta Dcrmato-vcner. (Stockholm), 34: 389,
basophil determination in test reactions may be
useful in the very important differentiation
A5PEuaEN, N., FaEOERT, S. AND RORSMAN, H.:
1954. 3.
UNDEITZ, E.: Hamatologische Tafeln Sandoz, Basel, p. 7, 23, 1950.
4. WOLF-JUROENSEN,
P.:
Communication at the
Danish Society for Allergological Research.
arnwarAlcv
By application of cantharidine on a non-vesic-
ular allergic patch test reaction an exudate is obtained which contains basophil leukocytes to
about the same extent as in primary allergic vesicles.
Determination of the relative number of baso-
phils in patch test reactions appears to be of
Copenhagen,
5.
Dcc. 7,
1962.
KLAUSNER, E. AND Knxmnicn, C.: Uber den
exsudative Mastzellen. Arch. Dermat. u.
Syph., 96: 235, 1909. 6. KLArJSNEa, E. AND KREJB!CH, C.: TJber exsuda-
tive Mastzellengehalt vesiculoscr Hautefiorescenscn. Folia Raemat., 15: 347, 1913. 7. ASPEGREN, N., Faxoawr, S. AND R0E5MAN, H.: Basophil leukocytes in lesions of various dermatoses. To be published.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.