A Brief Exposure to Tryptase or Thrombin Potentiates Fibrocyte Differentiation in the Presence of Serum or Serum Amyloid P

Abstract

A key question in both wound healing and fibrosis is the trigger for the initial formation of scar tissue. To help form scar tissue, circulating monocytes enter the tissue and differentiate into fibroblast-like cells called fibrocytes, but fibrocyte differentiation is strongly inhibited by the plasma protein Serum Amyloid P (SAP), and healthy tissues contain very few fibrocytes. In wounds and fibrotic lesions, mast cells degranulate to release tryptase, and in early wounds thrombin mediates blood clotting. Tryptase and thrombin are upregulated in wound healing and fibrotic lesions, and inhibition of these proteases attenuates fibrosis. Here we report that tryptase and thrombin potentiate human fibrocyte differentiation at biologically relevant concentrations and exposure times, even in the presence of concentrations of serum and SAP that normally completely inhibit fibrocyte differentiation. The fibrocyte potentiation by thrombin and tryptase is mediated by protease-activated receptors 1 and 2, respectively. Together, these results suggest that tryptase and thrombin may be an initial trigger to override SAP inhibition of fibrocyte differentiation to initiate scar tissue formation.

Fibrocyte measurement in peripheral blood correlates with number of cultured mature fibrocytes in vitro and is a potential biomarker for interstitial lung disease in Rheumatoid Arthritis

“…[6, 7]. IL-4 level is elevated in ILD and asthma lung biopsies and in peripheral blood and synovial fluid in early RA [8, 15, 19, 26].…”

Galectin-3 Binding Protein Secreted by Breast Cancer Cells Inhibits Monocyte-Derived Fibrocyte Differentiation

“…Monocytes are recruited to wounds or fibrotic lesions by chemokines (1, 2), and in response to wound signals such as tryptase released from mast cells, or thrombin activated during blood clotting, differentiate into monocyte-derived fibrocytes (3–5). The term “fibrocyte” has been used to refer to multiple cell types, including cells of the ear (6), CD34+, CD45+, collagen+ circulating cells (4, 7), and spindle-shaped CD34+, CD45+, collagen+ cells that differentiate in a tissue or in cell culture (4, 8).…”

Protease activated-receptor 2 is necessary for neutrophil chemorepulsion induced by trypsin, tryptase, or dipeptidyl peptidase IV

“…The PAR‐1 inhibitor vorapaxar (Axon Medchem, Reston, VA, USA), PAR‐1 agonist SFLLRN‐NH2 (American Peptide Company, Sunnyvale, CA, USA), PAR2 agonists 2f‐LIGRL‐NH2 (EMD Millipore, Billerica, MD, USA), SLIGKV‐NH2 (Tocris, Minneapolis, MN, USA), AC55541 (Tocris), TPCK‐treated bovine trypsin (10,000 Nα‐benzoyl‐ l ‐arginine ethyl ester units/mg, Sigma, St Louis, MO, USA), and human thrombin (1000 NIH units/mg; Sigma) were resuspended following the manufacturer's instructions. Tryptase purified from human mast cells (70 units/mg, Fitzgerald, Acton, MA, USA) mixed with 15 kDa heparin from porcine stomach (Sigma) in a 1:10 molar ratio of tryptase to heparin immediately after thawing, and PAR2 inhibitor ENMD‐1068 (50 μg/ml; Enzo Life Sciences, Farmingdale, NY, USA) were used as previously described . Tosyl‐ l ‐lysyl‐chloromethane hydrochloride (TLCK; Sigma), an irreversible inhibitor of trypsin and trypsin‐like serine proteases, was used at 10 nM, and the PAR‐1 inhibitor vorapaxar was used at 25 μg/ ml, both in RPMI/2% BSA.…”