Highly Selective Cleavage of TH2-Promoting Cytokines by the Human and the Mouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on TH2 Immunity.

Srinivas Akula,Lars Hellman,Michael Thorpe,Zhirong Fu

doi:10.3390/ijms20205147

Abstract

Mast cells (MC) are resident tissue cells found primarily at the interphase between tissues and the environment. These evolutionary old cells store large amounts of proteases within cytoplasmic granules, and one of the most abundant of these proteases is tryptase. To look deeper into the question of their in vivo targets, we have analyzed the activity of the human MC tryptase on 69 different human cytokines and chemokines, and the activity of the mouse tryptase (mMCP-6) on 56 mouse cytokines and chemokines. These enzymes were found to be remarkably restrictive in their cleavage of these potential targets. Only five were efficiently cleaved by the human tryptase: TSLP, IL-21, MCP3, MIP-3b, and eotaxin. This strict specificity indicates a regulatory function of these proteases and not primarily as unspecific degrading enzymes. We recently showed that the human MC chymase also had a relatively strict specificity, indicating that both of these proteases have regulatory functions. One of the most interesting regulatory functions may involve controlling excessive TH2-mediated inflammation by cleaving several of the most important TH2-promoting inflammatory cytokines, including IL-18, IL-33, TSLP, IL-15, and IL-21, indicating a potent negative feedback loop on TH2 immunity.

Highlights

Mast cells are resident tissue cells of hematopoietic origin that primarily are found at the interphase between tissues and environment such as skin, intestinal mucosa, lungs, and close to blood vessels and nerves
This protease family includes several coagulation factors, complement factors, and the pancreatic digestive enzymes. The members of this family that are expressed by hematopoietic cells have been named hematopoietic serine proteases. They are primarily found in mast cells (MCs), neutrophils, natural killer (NK) cells, and cytotoxic T cells (Tc), where they are stored in their active forms in the granules for rapid release
Ja. sMtolc. eSlcli. 2t0ry19p, t2a0s, e51, 47mMCP-6 was produced in the human cell line HEK293-EBNA, and 3aoftfe1r8 purification on Ni+2 chelating IMAC columns activated by cleavage by enterokinase, lowering the pH to 6.0, and adding heparin, as previously described (Figure 1c) [19]. using tThheesaamctievpituiersifiocfabtiootnhsetrnaztyemgyesaswtheries caonmalmyzeerdciaalgeaninzsytmtherheaevcehsrhomowongehniigchtrpyuprtiatyseasnudbtshtraattaesll th(eFidgiuvreers1ebb,da)n. ds observed on gels originate from differently glycosylated tryptase [24]

Summary

Introduction

Mast cells are resident tissue cells of hematopoietic origin that primarily are found at the interphase between tissues and environment such as skin, intestinal mucosa, lungs, and close to blood vessels and nerves. The degradation of IL-33, an IL-1-related cytokine, by mMCP-4 and HC indicates that they potentially have a role in limiting inflammation [14,15] Another of the hMC enzymes, the tryptase, has been shown to efficiently degrade the chemokine eotaxin [16]. IL-15 and IL-21, which both are efficiently cleaved by HC and the tryptase, respectively, have been indicated in either promoting TH2 immunity or inhibiting TH1 immunity [21,22,23] This indicates that one major function of the MC proteases is to limit excessive TH2 driven inflammation. They may act together as a negative feedback loop by cleaving and thereby inactivating early TH2 promoting inflammatory cytokines

Results

Analysis of the Effect of Spermine on Tryptase Activity

Cleavage of other Substrates by Human Tryptase

Enzymes and other Reagents

Analysis of the Cleavage of Human Fibrinogen and Fibronectin