Delayed clearance of zymosan-induced granuloma and depressed phagocytosis of macrophages with concomitant up-regulated kinase activities of Src-family in a human monocyte chemoattractant protein-1 transgenic mouse.

A human monocyte chemoattractant protein-1 (hMCP-1) transgenic mouse (Tgm) line which constitutively produces a large amount of hMCP-1 (7-13 ng/ml in the serum) was established. Although expression of the transgene was detected in various tissues, an accumulation of macrophages (Mphi) was seen in only lymphoid organs which might be attributed to the high concentration of hMCP-1 in these organs. A reduced phagocytosis by peritoneal Mphi in vivo and a delayed clearance of granulomas in the liver following zymosan administration were observed in these Tgm. However, peritoneal exudate cells (PEC) from Tgm exhibited normal in vitro phagocytic activity and nitric oxide (NO) production upon stimulation with IFN-gamma as compared with those from non-Tgm. In addition, high activities of src-family protein tyrosine kinases (PTK), Fgr and Hck, were also noted in the peritoneal resident cells from Tgm, whereas the level of mitogen-activated protein kinase (MAPK) activity was almost the same as that of non-Tgm. It was suggested that the low functional activities of Tgm Mphi seen in vivo were attributed to down-regulation of the unique transducing system of hMCP-1 signals under the influence of a high concentration of the hMCP-1. It seemed that the depressed functions were recovered when the peritoneal cells were released ex vivo from such a high hMCP-1 environment.

Functional modification of a murine macrophage cell line, J744A.1, transfected with rat csk genes.

In a previous study of this laboratory, a macrophage cell line, J774A.1, transfected with rat csk gene and overexpressing the Csk proteins has been established. These Csk transfectants showed depressed productions of monokines and nitric oxide (NO), but enhanced production of prostaglandin E2 (PGE2). In the present study, mechanism(s) underlying the reciprocal functions seen in NO and PGE2 productions was investigated. When aminoguanidine, an inhibitor of NO synthesis, was added to the Csk transfectants stimulated with lipopolysaccharide (LPS), not only NO production but also PGE2 production was suppressed. Exogenous NO showed no influence on PGE2 production by the transfectants stimulated with LPS. It was also shown that mitogenactivated protein kinase (MAPK) pathway was activated in the Csk transfectants as compared to parental J774A.1 or a vector control, J.pBK2, cells. Large amounts of phosphorylated MAPK were detected in the Csk transfectants compared to J774A.1. This finding appeared to be consistent with the result that MAPK inhibitor completely abolished NO production by J774A.1 cells upon stimulation with LPS + interferon-gamma (IFN-gamma), whereas the inhibitor partially blocked the NO production by J.Csk transfectants which expressed large amounts of Csk protein. The overexpressed Csk resulted in suppression of phagocytosis of latex beads and uptake of acetyl-low-density lipoprotein (LDL) by the transfectants. The present findings demonstrate that Csk regulates NO and PGE2 productions independently and suggest that introduction of csk gene may be applicable to understanding the pathogenesis of certain diseases where dysregulated macrophages are involved.