Proteolytic Activation of the Chemokine CCL15 by Cathepsin G from Neutrophil Granulocytes Modulates Proliferation of Primitive Hematopoietic Precursor Cells.

The human chemokine HCC-2 (MIP-1δ, small inducible cytokine subfamily A [Cys-Cys], member 15, hmrp-2b, MIP-5, NCC-3) is a potent chemoattractant for monocytes, lymphocytes and endothelial cells, and has been shown to inhibit proliferation of primitive hematopoietic precursor cells (HPC). CCL15 is constitutively expressed in gut and liver, and was identified to circulate in human plasma. The length of the N-terminus of CCL15 was found to control biological activity. We therefore aimed to investigate the processing of CCL15 in the hematopoietic system, and the effect of processed isoforms on HPC proliferation. Incubation of full-length CCL15 with cell-free bone marrow supernatant prepared from normal untreated mice revealed a decrease in molecular weight by approximately 3 kD. In contrast, the molecular weight of CCL15 remained unchanged when cell-free bone marrow supernatant of mice with regenerating hematopoiesis after 5 Gy gamma irradiation was analyzed. Incubation of full-length CCL15 with isolated neutrophils, but not monocytes, lymphocytes or thrombocytes revealed N-terminal deletions of 21 (Δ21), 23 (Δ23) and 26 (Δ26) amino acids, as assessed by Edman sequencing/mass spectrometry. Both (1) inhibition of the neutrophil-derived CCL15 proteolytic activity by protease inhibitors Suc-Ala-Ala-Pro-Phe-pNA, secretory leucoprotease inhibitor or elastatinal, and (2) addition of the recombinant serine proteases cathepsin G or elastase to full-length CCL15 revealed that the Δ23 and Δ26 isoforms are produced by cathepsin G, whereas formation of the Δ21isoform is dependent on elastase. In addition, the Δ23 and Δ26 isoforms of CCL15 was also directly isolated and sequenced from blood filtrate (hemofiltrate) collected from renal insufficiency patients. The Δ26 isoform was synthesized and analyzed in HPC proliferation assays. Compared to full length CCL15, the Δ26 isoform displayed a mean 2.5 fold increased potency to inhibit CFU-A colony formation from post 5-fluorouracil murine bone marrow. In addition, the Δ26 isoform efficiently abrogated cytosine arabinoside-induced suicide of murine spleen colony forming unit (CFU-S) in post 5-fluorouracil murine bone marrow, and inhibited growth of human cord blood CD34⁺ single cell clones induced by hematopoietic growth factors SCF, IL-3, GM-CSF and EPO after flow-cytometric cell sorting (mean 2.3 fold inhibition). Our findings indicate a role for neutrophil-derived cathepsin G in growth control of colony of primitive hematopoietic progenitor cells through the proteolytic processing of CCL15.