Cytokine production by skin-derived mast cells: endogenous proteases are responsible for degradation of cytokines.
The current study characterizes the cytokine protein (ELISA) and mRNA (gene array and RT-PCR) profiles of skin-derived mast cells cultured under serum-free conditions when activated by cross-linking of Fc epsilonRI. Prior to mast cell activation, mRNA only for TNF-alpha was detected, while after activation mRNA for IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF substantially increased, and for IL-4 it minimally increased. However, at the protein level certain recombinant cytokines, as measured by ELISAs, were degraded by proteases released by these skin-derived mast cells. IL-6 and IL-13 were most susceptible, followed by IL-5 and TNF-alpha; GM-CSF was completely resistant. These observations also held for the endogenous cytokines produced by activated mast cells. By using protease inhibitors, chymase and cathepsin G, not tryptase, were identified in the mast cell releasates as the likely culprits that digest these cytokines. Their cytokine-degrading capabilities were confirmed with purified chymase and cathepsin G. Soy bean trypsin inhibitor, when added to mast cell releasates, prevented the degradation of exogenously added cytokines and, when added to mast cells prior to their activation, prevented degradation of susceptible endogenous cytokines without affecting either degranulation or GM-CSF production. Consequently, substantial levels of IL-5, IL-6, IL-13, TNF-alpha, and GM-CSF were detected 24-48 h after mast cells had been activated, while none were detected 15 min after activation, by which time preformed granule mediators had been released. IL-4 was not detected at any time point. Thus, unless cytokines are protected from degradation by endogenous proteases, cytokine production by human mast cells with chymase and cathepsin G cells may be grossly underestimated.
Zhao
Wei
W
Department of Pediatrics, Virginia Commonwealth University, Richmond 23298, USA.
Oskeritzian
Carole A
CA
Pozez
Andrea L
AL
Schwartz
Lawrence B
LB
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D056649:Cathepsin G; D002403:Cathepsins / Q000502:physiology; D015550:Cell Degranulation / Q000276:immunology; D002478:Cells, Cultured; D053818:Chymases; D016207:Cytokines / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010450:Endopeptidases / Q000502:physiology*; D006801:Humans; D015847:Interleukin-4 / Q000032:analysis / Q000235:genetics; D008407:Mast Cells / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D017455:Receptors, IgE / Q000276:immunology; D012697:Serine Endopeptidases / Q000502:physiology; D012867:Skin / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D015854:Up-Regulation / Q000276:immunology
D000911:Antibodies, Monoclonal; D016207:Cytokines; D011480:Protease Inhibitors; D012333:RNA, Messenger; D017455:Receptors, IgE; D015847:Interleukin-4; D002403:Cathepsins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D053818:Chymases