Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line.

Abstract

The capacity ofvarious cell types to infiltrate inflammatory sites suggests the existence of specific chemotactic cytokines for each cell type. Snyderman et al. (1) first described that leukocytes from human peripheral blood when stimulated in vitro with a specific antigen (purified protein derivative [PPD]) or a nonspecific mitogen (PHA) produced a soluble factor that was chemotactic for homologous monocytes (2) . The production of this lymphokine proved to be a sensitive in vitro correlate of delayed hypersensitivity (1) . We have previously reported the identification, purification, and cDNA cloning ofa novel neutrophil chemotactic factor (NCF) produced by LPS-stimulated human monocytes (3-5) . In the course of identifying NCF, we detected in the same LPS-stimulated human monocyte-conditioned media a chemotactic activity for monocytes that was distinct in molecular weight from NCF. Since the level of monocyte chemotactic factor (MCF) in these conditioned media was at least 10 times less than that of NCF, we used conditioned media from a human myelomonocytic cell line (THP-1) as a source of MCF The identical conditioned media were previously used for the purification of human IL-1/3 (6). We now report the purification of human MCF produced by THP-1 cells to homogeneity and describe the potent monocyte activating factor (MAF) activity of this MCE Materials and MethodsChromatography Proceduresfor the Purification ofMCF. (a) For Heparin Sepharose chromatography, the 200-ml DEAE Sephacel breakthrough fraction was applied to 50 ml Heparin Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden), that had been equilibrated with 0.05 M Tris-HCl, pH 8.0, with 0.2 M NaCl. After washing the column extensively, MCF was eluted with 50 mM Tris HCI, pH 8.0, with 0.75 M NaCI. (b) For gel filtration, the 4-ml concentrate from Heparin Sepharose was loaded onto a 2.6 x 90-cm column of Sephacryl S-200 (Pharmacia Fine Chemicals) equilibrated with D-PBS at 4°C. The flow rate was adjusted to 15 ml/h and 8-ml fractions were collected . (c) For cation HPLC, fractions corresponding 10-25 kD in gel filtration were concentrated and dialyzed against 0.02 MOPS, pH 6.5, and applied to a 7.5 x 150-mm CM-3SW column (Toyo Soda, Tokyo, Japan) equipped to an HPLC system (model 2150; LKB Instruments, Inc., Sweden) that had been equilibrated with the same buffer. The starting buffer was 0.02 M MOPS, pH 6.5, and the limiting buffer

IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice

“…Activated macrophages are toxic to pancreatic islets in vitro [58] and macrophage depletion or inactivation prevent diabetes in BB rats and NOD mice [59,60]. IP-10 was reported to attract monocytes [61], and it is therefore conceivable that the early expression of IP-10 (current data) and MCP-1 [8] by NOD islet cells contributes to the recruitment of macrophages in the early stages of insulitis. In this context, it is puzzling that IP-10 expression seems to precede a detectable increase in IFN-γ, the main IP-10 inducer.…”

Migration of cytotoxic T lymphocytes toward melanoma cells in three‐dimensional organotypic culture is dependent on CCL2 and CCR4

“…CCL2 was identified as a monocyte-specific chemoattractant [22,23] that was later shown to attract T lymphocytes and NK cells [24,25].…”

Expression of monocyte chemotactic and activating factor in rheumatoid arthritis. regulation of its production in synovial cells by interleukin‐1 and tumor necrosis factor

“…Chemotaxis for monocytes was assayed using 48-well micro-chemotaxis chambers (Neuro Probe, Cabin John, MD) and a 5-pm pore-size polycarbonate filter as described previously (7). Human peripheral blood mononuclear cells were prepared from the peripheral blood of normal volunteers by Ficoll-Hypaque density gradient centrifugation.…”

“…A novel cytokine, monocyte chemotactic and activating factor (MCAF) or monocyte chemoattractant protein 1 (MCP-l), has been recently identified (7,8). MCAF is a peptide that consists of 76 amino acids and possesses structural homology with a group of putative polypeptide mediators known as macrophagederived inflammatory protein 1 (MIP-l), LD-78, RANTES, and JE.…”