Trypsin Potentiates Human Fibrocyte Differentiation

Abstract

Trypsin-containing topical treatments can be used to speed wound healing, although the mechanism of action is unknown. To help form granulation tissue and heal wounds, monocytes leave the circulation, enter the wound tissue, and differentiate into fibroblast-like cells called fibrocytes. We find that 20 to 200 ng/ml trypsin (concentrations similar to those used in wound dressings) potentiates the differentiation of human monocytes to fibrocytes in cell culture. Adding trypsin inhibitors increases the amount of trypsin needed to potentiate fibrocyte differentiation, suggesting that the potentiating effect is dependent on trypsin proteolytic activity. Proteases with other site specificities such as pepsin, endoprotease GluC, and chymotrypsin do not potentiate fibrocyte differentiation. This potentiation requires the presence of albumin in the culture medium, and tryptic fragments of human or bovine albumin also potentiate fibrocyte differentiation. These results suggest that topical trypsin speeds wound healing by generating tryptic fragments of albumin, which in turn potentiate fibrocyte differentiation.

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

“…Peripheral blood mononuclear cells (PBMC) and monocytes were isolated as previously described (13), and cultured as previously described (48), in either serum-free or protein-free media. Protein-free media (PFM) was Fibrolife basal media (Lifeline Cell Technology, Walkersville, MD) supplemented with 10 mM HEPES (Sigma, St. Louis, MO), 1× non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM glutamine (Lonza, Basel, Switzerland), 100 U/ml penicillin and 100 µg/ml streptomycin (Lonza).…”

“…Protein-free media (PFM) was Fibrolife basal media (Lifeline Cell Technology, Walkersville, MD) supplemented with 10 mM HEPES (Sigma, St. Louis, MO), 1× non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 2 mM glutamine (Lonza, Basel, Switzerland), 100 U/ml penicillin and 100 µg/ml streptomycin (Lonza). Serum-free media (SFM) was PFM further supplemented with 10 µg/ml recombinant human insulin (Sigma), 5 µg/ml recombinant human transferrin (Sigma), and 550 µg/ml filter-sterilized human albumin (isolated and checked for purity as previously described (48)), fish skin gelatin (Sigma), or skim milk powder (EMD Millipore, Billerica, MD). Protein supplements were checked for concentration using absorbance at 280 nm.…”

“…Where indicated, PFM was supplemented to 2.5% (v/v) with sterile filtered non-blood type specific human serum, tested negative for hepatitis A and B and HIV I and II (Lonza and Gemini Bio-products, West Sacramento, California). Monocytes were purified, tested for purity, and cultured as previously described (48, 49). Fibrocyte counts, total cell counts, and collagen staining were performed as previously described (48).…”

“…Monocytes were purified, tested for purity, and cultured as previously described (48, 49). Fibrocyte counts, total cell counts, and collagen staining were performed as previously described (48). …”

Fibroblasts secrete Slit2 to inhibit fibrocyte differentiation and fibrosis

“…Peripheral blood mononuclear cells (PBMCs) were isolated from blood using FicollPaque Plus (GE Healthcare Biosciences), as described previously (18). Monocytes were enriched from PBMCs using EasySep monocyte enrichment kits (StemCell Technologies), as described previously (57). Spleen cells from 4-to 6-wk-old male C57BL/6J mice (The Jackson Laboratory) were isolated as described previously (19).…”