Archives of Dermatological Research

, Volume 264, Issue 2, pp 213–223 | Cite as

A new method for obtaining viable cells from dermal infiltrates

A study on 2,4 dinitrochlorobenzene induced contact dermatitis
  • L. Molnár
  • J. M. Baló-Banga
  • J. Leibinger
  • K. Kiraly
Original Works


Experimental contact dermatitis has been induced in 2,4 dinitrochlorobenzene (DNCB) sensitized guinea pigs. The developing dermal infiltrate was excised and the infiltrating cells were obtained by mechanical extraction alone as well as by the combination with collagenase and elastase treatment. The most viable cells appeared in the elastase and mechanically extracted samples and the least in those subjected to mechanical treatment alone. The most cells in the enzyme-treated samples were present 24 h after re-exposure of the sensitized animals to DNCB consisting mainly of lymphocytes and of polymorphonuclear granulocytes. The optimum conditions for the action of enzymes including optimum duration of the treatment, buffer milieu, aspecific proteolytic effect on foreign substrate and action on T and B cell receptors have been elaborated. It was concluded that 80 min of collagenase treatment with gentle mechanical extraction under specified conditions does not affect any measurable immunologic properties of the liberated cells resulting in the second best yield. A comparison of these data with earlier reports and their significance is being discussed.

Key words

Eczema Proteolytic enzymes Dermal cells Receptors 


Das experimentelle Kontaktekzem wurde an mit 2,4 Dinitrochlorbenzol (DNCB) sensibilisierten Meerschweinchen ausgelöst. Das nachfolgende dermale Infiltrat wurde herausgeschnitten und die Infiltratszellen durch ausschließlich mechanische Extraktion sowie auch durch Kombination mit Kollagenase- und Elastase-Behandlung gewonnen. Die größte Menge von lebendigen Zellen wurde in den mechanisch und Elastase behandelten, die geringste in den ausschließlich mechanisch behandelten Mustern gefunden. Die Zahl der Zellen war 24 h nach der Reexposition der mit DNCB sensibilisierten Tieren am höchsten. Der Extrakt bestand vorwiegend aus Lymphocyten und polymorphkernigen Granulocyten. Die optimalen Verhältnisse für die Enzymreaktionen, u.a. Dauer der Behandlung, Pufferkonzentration, nichtspezifische Proteolyse und die Wirkung auf T-Zellen-Rezeptoren wurden ausgearbeitet. Nach den Resultaten der Untersuchungen wurde festgestellt, daß eine 80 min lang dauernde Kollagenase-Behandlung kombiniert mit mechanischer Extraktion unter den beschriebenen Verhältnissen keine meßbare Wirkung auf die immunologischen Eigenschaften der Zellen hat.


Ekzem Proteolytische Enzyme Dermale Zellen Rezeptoren 


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  1. Askenase, P. W.: Cutaneous basophil hypersensitivity (CBH) uncovered in cell transfer of classical tuberculin hypersensitivity. J. Immunol. 117, 741–747 (1976)Google Scholar
  2. Baló, J., Banga, I.: Elastase and elastase inhibitor. Nature (Lond.) 164, 491 (1949)Google Scholar
  3. Banga, I.: Determination of elastase and elastase inhibitor by means of orcein-elastin. Acta Physiol. Acad. Sci. Hung. 24, 1–9 (1963)Google Scholar
  4. Banga, I., Baló, J.: Elastomucoproteinase and collagen mucoproteinase the mucolytic enzymes of the pancreas. Nature (Lond.) 178, 310 (1956)Google Scholar
  5. Bianco, C., Patrick, R., Nussenzweig, V.: A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization. J. Exp. Med. 132, 702–720 (1970)Google Scholar
  6. Bøyum, A.: Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Invest. 21, Suppl. 97, 51–76 (1968)Google Scholar
  7. Burg, G., Braun-Falco, O.: Morphological and functional differentiation and classification of cutaneous lymphomas. Bull. Cancer 64, 225–240 (1977)Google Scholar
  8. Dickler, H. B., Kunkel, H. G.: Interaction of aggregated γ-globulin with B lymphocytes. J. Exp. Med. 136, 191–199 (1972)Google Scholar
  9. Einbinder, J., Schubert, M.: Binding of mucopolysaccharides and dyes by collagen. J. Biol. Chem. 188, 335–341 (1951)Google Scholar
  10. Fakhri, O., Tan, R. S.-H.: The effect of trypsin on cell surface antigens. Cell. Immunol. 15, 452–456 (1975)Google Scholar
  11. Hunziker, N.: Experimental studies on guinea pig's eczema. Berlin, Heidelberg, New York: Springer 1969Google Scholar
  12. Hunziker, N., Winkelmann, R. K.: Cell populations in experimental contact dermatitis. Arch. Dermatol. 113, 1543–1549 (1977)Google Scholar
  13. Kerl, M., Burg, G., Braun-Falco, O.: Quantitative and qualitative dynamics of the epidermal and cellular inflammatory reaction in primary toxic and allergic DNCB contact dermatitis in guinea pigs. Arch. Dermatol. Res. 249, 207–226 (1974)Google Scholar
  14. Kisfaludy, S.: Bestimmung des Blutammoniaks mit der Ninhydrin Reaktion. Acta Med. Acad. Sci. Hung. 16, 175–187 (1960)Google Scholar
  15. Kolar, O., Zeman, W.: Spinal fluid cytomorphology. Description of apparatus, technique and findings. Arch. Neurol. (Chic.) 18, 44–51 (1968)Google Scholar
  16. Lay, W. H., Mendes, N. F., Bianco, C., Nussenzweig, W.: Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.) 230, 531–532 (1971)Google Scholar
  17. Magnusson, B., Kligmann, A. M.: Allergic contact dermatitis in the guinea pig. Springfield Ill.: Ch. C. Thomas 1970Google Scholar
  18. Mandl, I.: Collagenases and elastases. Adv. Enzymol. 23, 163–254 (1961)Google Scholar
  19. Mandl, I., MacLennan, J. P., Howes, E. L.: Isolation and characterization of proteinase and collagenase from Cl. histolyticum. J. Clin. Invest. 32, 1323–1329 (1953)Google Scholar
  20. Miyoshi, M., Rosenbloom, I.: General proteolytic activity of highly purified preparations of clostridial collagenase. Connective Tiss. Res. 2, 77–84 (1974)Google Scholar
  21. Molnár, L., Baló-Banga, J. M., Horváth, A., Leibinger, J., Ablonczy, E., Nováki, M.: Immune-complex phagocytosis by human polymorphonuclear granulocytes. Acta Physiol. Acad. Sci. Hung. 52, 23–29 (1978)Google Scholar
  22. Rinaldini, L. M.: An improved method for the isolation and quantitative cultivation of embryonic cells. Exp. Cell. Res. 16, 477–505 (1959)Google Scholar
  23. Silveira, N. P. A., Mendes, N. F., Tolnai, M. E. A.: Tissue localisations of two populations of human lymphocytes distinguished by membrane receptors. J. Immunol. 108, 1456–1460 (1972)Google Scholar
  24. Tan, R. S.-H., Byrom, N. A., Hayes, J. P.: A method of liberating living cells from the dermal infiltrate. (Studies on skin reticuloses and lichen planus) Br. J. Dermatol. 93, 271–276 (1975)Google Scholar
  25. Viac, J., Bustmante, R., Thivolet, J.: Characterisation of mononuclear cells in the inflammatory infiltrates of cutaneous tumours. Br. J. Dermatol. 97, 1–10 (1977)Google Scholar
  26. Walker, D. M.: Identification of subpopulations of lymphocytes and macrophages in the infiltrate of lichen planus lesions of skin and oral mucosa. Br. J. Dermatol. 94, 529–534 (1976)Google Scholar

Copyright information

© Springer-Verlag 1979

Authors and Affiliations

  • L. Molnár
    • 1
  • J. M. Baló-Banga
    • 1
  • J. Leibinger
    • 1
  • K. Kiraly
    • 1
  1. 1.The National Institute of Dermatology and Venereology of HungaryBudapestHungary

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