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Novel autocrine and paracrine loops of the stem cell factor/chymase network
A de Paulis1, G Minopoli, F Dal Piaz
1Division of Clinical Immunology and Allergy, University of Naples Federico II, Naples, Italy.
Background:
The aim of this study was to investigate whether the secretory granules of human mast cells store stem cell factor (SCF). We also addressed the question whether mast cell chymase, a chymotrypsin-like protease, also present in the secretory granules of human mast cells cleaves SCF at the peptide bound between Phe 158 and Met159.
Methods:
The skin samples were obtained from patients with mastocytosis, undergoing skin biopsy for diagnostic purposes. Mast cells were isolated and purified from human lung parenchyma (human lung mast cells, HLMC) by countercurrent elutriation followed by discontinuous Percoll density gradient. SCF contents of human mast cells were assessed for immunoreactive SCF by ELISA. Western blot analysis of SCF and its cleavage products were performed with the MoAb anti-SCF 7H6. SCF and its proteolytic fragment were characterized by electrospray mass spectrometry (ES/MS).
Results:
SCF is present in the secretory granules of human skin and lung mast cells. Immunoreactive SCF (iSCF) was detected in the cell lysates of HLMC, but not in basophils. iSCF was rapidly (3 min) released after challenge with anti-IgE, and iSCF in supernatants rapidly declined after 30 min. ES/MS analysis of rhSCF1-166 treated with recombinant human chymase showed a polypeptide of 17,977.1+/-0.6 Da and a minor component of 697.4+/-0.1 Da generated by specific cleavage at Phe159. SCF1-166 and SCF1-159 similarly activated HLMC and potentiated anti-IgE-induced activation of these cells. The cleavage product SCF160-166 had no effect on mast cells. Western blot analysis of supernatants of anti-IgE activated HLMC incubated for various intervals with rhSCF1-166 showed that rhSCF1-166 was converted to a faster-migrating form with a molecular weight compatible with SCF1-159 and to several SCF species.
Conclusion:
SCF is stored in human mast cell secretory granules and is immunologically released by mast cells. SCF1-166 is rapidly cleaved by chymase and other proteases to several SCF species.