The regulation of mast cell growth and protease expression by cytokines

Abstract

In this study the diversity of mast cell proteases and some of the factors regulating mast cell growth and protease expression were examined.

Five proteases were isolated from mouse small intestinal mucosa and characterised in terms of their substrate specificities, substrate and inhibitor kinetics and immunohistochemical localisation. These studies revealed that the isolated proteases were all of mast cell origin and that they were chymotrypsin-like in their substrate specificities. The proteases were all identified as variants of mouse mast cell protease-1 which differed only in their carbohydrate moieties. Despite the fact that these enzymes shared a common core polypeptide they all differed significantly in the rate at which they hydrolysed low molecular weight synthetic substrates and in the rates at which they were inhibited by a,-proteinase inhibitor. A similar, but distinct protease was isolated from peritoneal cavity mast cells of mice. This enzyme, also a chvmase, had N-terminal sequence consistent with identification of the enzyme as mouse mast cell protease-4 which had previously only been identified by N-terminal sequence analysis of electro-blotted protein and from cDNA sequencing. This enzyme was not inhibited by, and actually degraded a]-proteinase inhibitor.

Some of the factors which regulate mast cell growth, proliferation, survival and protease expression were examined in the rat. Stem cell factor (SCF) or cytokine-rich lymph node conditioned medium (LNCM) was administered to normal rats by intraperitoneal injection and the effects on a defined cell populations, the connective tissue mast cells (CTMC) of the peritoneal cavity were monitored. LNCM did not cause an increase in CTMC numbers but it did stimulate a switch in protease expression from rat mast cell protease I (RMCP I) alone to dual expression of both RMCP I and RMCP II. SCF alone caused a significant increase in CTMC numbers coupled with a decrease in RMCP I content and an increase in RMCP II content. Treatment with both LNCM and SCF together caused an even greater increase in both CTMC numbers and RMCP II expression than that seen with SCF alone. A second experiment was carried out where SCF was administered by daily intravenous injection for 14 days to both normal and parasitised rats and the effects of the treatment on CTMC and mucosal mast cell (MMC) populations was monitored. Intravenous SCF treatment resulted in a five-fold increase in peritoneal mast cell numbers with a concomitant decrease in RMCP I content. There was no significant expression of RMCP II in the CTMC of these animals.

An alternative approach in examining the role of SCF in regulating mast cell populations w as the use of polyclonal antibodies raised against SCF to try and block SCF activity in both normal and parasitised rats. In normal rats, treatment with anti-SCF antibodies caused an approximately 50% decrease in the number of mast cells detected in the peritoneal cavity and totally ablated the MMCs from the small intestinal mucosa. In parasitised rats treated with anti-SCF antibodies, the mucosal mast cell hyperplasia associated with infection was significantly delayed in rats infected with Nippostrongvlus brasiliensis or Trichinella .spiralis and completely ablated in rats infected with Schistosoma mansoni. The depletion of mast cells from the intestinal mucosa was accompanied by a reduction in the tissue concentration of RMCP II. When anti-SCF antibodies were administered to rats which had already developed a mast cell hyperplasia following N. brasiliensis infection, there was again a significant depletion of both mast cells and RMCP II from the small intestine.

A sensitive ELISA test was developed to measure soluble SCF in blood. This assay showed that circulating SCF levels were significantly increased by infection with both A. brasiliensis and '/'. spiralis. Taken together these results show that SCF and the cytokines in LNCM play an important role in the regulation of mast cell populations and their expression of proteases.