Cytokine Production by Skin-Derived Mast Cells: Endogenous Proteases Are Responsible for Degradation of Cytokines

Abstract

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 FcεRI. Prior to mast cell activation, mRNA only for TNF-α was detected, while after activation mRNA for IL-5, IL-6, IL-13, TNF-α, 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-α; 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-α, 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.

IL‐6 and IL‐17A degradation by mast cells is mediated by a serglycin:serine protease axis

“…Hence, these data suggest that mast cells specifically participate in the regulation of certain cytokines, whereas others may be regulated by mast cell‐independent control mechanisms. Hence, the latter finding is in line with previous studies on human mast cells suggesting that certain cytokines and chemokines, including GM‐CSF, are more resistant to degradation by mast cell‐dependent pathways 12. It should, however, be noted that we cannot exclude the possibility that some of the cytokines that appeared to be resistant to mast cell‐dependent regulation, as detected by the dot blot, could still be subject to a limited proteolysis affecting the cytokine activity without reducing the immunoreactivity.…”

“…To our knowledge, this is the first demonstration of a direct regulation of IL‐17A by mast cell granule components. Although degradation of IL‐6 by several types of mast cell proteases was reported in previous work 12, 13, the total dependency on serglycin proteoglycan demonstrated in the present study confirms that serglycin‐dependent mechanisms are crucial for mast cell‐mediated IL‐6 regulation. However, further investigations are needed to establish whether serglycin is required for limiting the in vivo levels of IL‐6 and IL‐17A during mast cell‐mediated inflammatory conditions.…”

“…In accordance with the former possibility, there are reports suggesting that mast cell serine proteases can proteolytically activate certain cytokines 31, 32, 33 and chemokines 34, 35. Conversely, protease‐dependent degradation of cytokines by human mast cells has previously been demonstrated 6, 12. In line with the latter scenario, the present report shows that fully mature peritoneal mast cells have the capacity to regulate levels of IL‐3, IL‐6, IL‐13, IL‐17A, and eotaxin.…”

“…The rationale behind this was that several different mast cell serine proteases are known to depend on serglycin for storage and there are also previous studies implicating such serine proteases in the regulation of various cytokines by proteolytic degradation 6, 11, 12, 25. To test this hypothesis, we analyzed whether a serine protease inhibitor, Pefabloc SC, could block reduction of cytokine concentrations.…”

“…It has also been demonstrated that serglycin‐associated mast cell proteases can regulate levels of tumor necrosis factor α (TNF‐α), and various venom‐derived toxins 8, 9, 10, and there are indications that serglycin‐dependent proteases can regulate the levels of IL‐13 11. Interestingly, regulation of both external and endogenously produced IL‐6 and IL‐13 by human mast cells has been shown to involve proteolytic degradation by chymase and cathepsin G 12. In contrast, murine chymase mMCP4 and murine cathepsin G were unable to degrade IL‐13 11, and degradation of IL‐6 was exerted by the tryptase mMCP6 and not by mMCP4 13.…”