Evidence against the existence of a membrane form of murine IL-1 alpha.
Previous studies have demonstrated that paraformaldehyde-treated macrophages possess IL-1 alpha activity in a variety of bioassay systems. However, no definitive biochemical data in support of the membrane IL-1 alpha concept has been reported. The purpose of the present study was to determine if the biologic activity associated with treated cells is due to a membrane form of IL-1 alpha or alternatively, to the leakage of IL-1 alpha. If the former case was true, then the exposed membrane IL-1 alpha should bind anti-IL-1 alpha antibodies or be cleaved by mild trypsin treatment. In both instances, IL-1 alpha activity should be lost when measured in a subsequent IL-1 bioassay. Our results indicate that pulsing paraformaldehyde-treated normal or cell line macrophages with anti-IL-1 alpha antibodies or treating the cells with trypsin did not affect the ability of the treated cells to function in a murine thymocyte proliferation assay. Furthermore, the standard short term treatment of cells with paraformaldehyde (15 min) did not prevent the leakage of IL-1 alpha from the cells or the processing of the precursor forms of the protein. When cells were treated with paraformaldehyde for 2 h, they no longer released IL-1 alpha or possessed thymocyte stimulatory activity. We also found that short term glutaraldehyde treatment of macrophages completely blocked the release of IL-1 alpha from cells as well as the appearance of cell-associated IL-1 alpha activity. Our results support the conclusion that the stimulatory activity of paraformaldehyde-treated macrophages is not due to a membrane form of IL-1 alpha but is, in fact, due to the continuous release of IL-1 alpha from the cells.
Department of Microbiology and Immunology, Wake Forest University Medical Center, Winston-Salem, NC 27103.
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Minnich-Carruth
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Suttles
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Mizel
D000818:Animals; D000906:Antibodies / Q000502:physiology; D005404:Fixatives; D005557:Formaldehyde; D007375:Interleukin-1 / Q000276:immunology / Q000302:isolation & purification*; D007941:Leukemia P388 / Q000378:metabolism; D008264:Macrophages / Q000032:analysis* / Q000187:drug effects; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D011108:Polymers; D011233:Precipitin Tests; D013997:Time Factors; D014357:Trypsin
D000906:Antibodies; D005404:Fixatives; D007375:Interleukin-1; D008565:Membrane Proteins; D011108:Polymers; D005557:Formaldehyde; D014357:Trypsin; C003043:paraform