Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Abstract

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether β-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of β-tryptase, and the β-tryptase-activating polyanion. The B12 mAb was used to stabilize β-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric β-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by β-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by β-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both β-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by β-tryptase in releasates of activated skin mast cells. Of further biologic interest, β-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest β-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of β-tryptase and complement anaphylatoxins are detected.

International Union of Basic and Clinical Pharmacology. LXXXVII. Complement Peptide C5a, C4a, and C3a Receptors

“…in trauma ; b-tryptase, secreted by mast cells (Fukuoka et al, 2008); and granzyme B, produced by leukocytes . The relative importance of these fragment-generation systems that lie outside the complement cascade is not yet clear.…”

Anaphylaxis as a Manifestation of Systemic Mastocytosis: A Case Report and Review of Literature

“…Mast cells are a crucial structural and functional component of the immune system; they play a key role in inflammatory reactions in allergic and nonallergic process [7,8]. Mastocytosis results from a clonal, neoplastic proliferation of morphologically and immunophenotypically abnormal MC that accumulate in one or more organ systems [9] more frequently the dermis, gastrointestinal tract, and cardiovascular system, frequently accompanied by neurologic complaints [6].…”

Human Skin Mast Cells Express Complement Factors C3 and C5

“…Secreted C3, when Western blotted, exhibited a doublet near the a subunit, likely corresponding to a and a9, indicating C3 cleavage during or after secretion. Examples of proteases that can generate C3a from C3 in serum-free medium include mast cell tryptase (26) and granzyme B (60), resulting in C3b (a9b). Mast cell proteases other than tryptase, including membrane-bound metalloproteases, also might have a role in activating C3 and C5.…”

“…Human skin mast cells, capable of proliferating when cultured in serum-free medium containing SCF, retain the functional and phenotypic characteristics from when they were freshly dispersed (25). Human mast cell b-tryptase can activate C3 and C5 to generate the corresponding anaphylatoxins (26), delineating a novel putative amplification loop for inflammation initiated by either mast cells or complement (26,27) and further linking the complement and mast cell pathways to one another in humans. In mice, expression of receptors for C3a or C5a on mast cells is essential not only for acute intradermal responses to C3a or C5a, respectively, but also for an optimal IgE-mediated passive cutaneous anaphylactic response to allergen (28).…”