Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-i (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P < 0.05) in synovial fluid from RA patients (mean 25.5±8.1 ng/ml ISEI) compared to synovial fluid from osteoarthritis (OA) patients (0.92±0.08), or from patients with other arthritides (2.9±1.5). MCP-1 levels in RA sera (8.44±2.33) were significantly greater than MCP-1 in normal sera (0.16±0.06). The quantities ofRA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P < 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1#, tumor necrosis factor-alpha (TNF-a), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50±8%) as compared to either OA macrophages (5±2) or normal macrophages (1±0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18±8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine. (J. Clin. Invest. 1992. 90:772-779.)

Summary Different subsets and/or polarized phenotypes of monocytes and macrophages may play distinct roles during the development and resolution of inflammation. Here, we demonstrate in a murine model of rheumatoid arthritis that non-classical Ly6C− monocytes are required for the initiation and progression of sterile joint inflammation. Moreover, non-classical Ly6C− monocytes differentiate into inflammatory macrophages (M1), which drive disease pathogenesis and display plasticity during the resolution phase. During the development of arthritis, these cells polarize toward an alternatively activated phenotype (M2), promoting the resolution of joint inflammation. The influx of Ly6C− monocytes and their subsequent classical and then alternative activation occurs without changes in synovial tissue-resident macrophages, which express markers of M2 polarization throughout the course of the arthritis and attenuate joint inflammation during the initiation phase. These data suggest that circulating Ly6C− monocytes recruited to the joint upon injury orchestrate the development and resolution of autoimmune joint inflammation.

We have shown that human macrophages (m4s) play an important role in the elaboration of chemotactic cytokines in rheumatoid arthritis (RA) (Koch, inflammatory protein-i (MIP-la), a cytokine with chemotactic activity for mos and neutrophils (PMNs), has been described. We have examined the production of MIP-1a using sera, synovial fluid (SF), and synovial tissue (ST) from 63 arthritic patients. MIP-la was higher in RA SF (mean, 29±8 ng/ml ISEJ) compared with other forms of arthritis (2.8±1.7), or osteoarthritis (0.7±0.4; P < 0.05). RA SF MIP-la was greater than that found in either RA or normal peripheral blood (PB) (P < 0.05). Anti-MIP-la neutralized 36±3% (mean±SE) of the chemotactic activity for mos, but not PMNs, found in RA SFs. RA SF and PB mononuclear cells produced antigenic MIP-la. Mononuclear cell MIP-la production was augmented with phytohemagglutinin or LPS. Isolated RA ST fibroblast production of antigenic MIP-la was augmented upon incubation of cells with LPS, and to a lesser extent with tumor necrosis factor-a. Isolated RA ST m4s expressed constitutive MIP-la mRNA and antigenic MIP-la.AUsing ST immunohistochemistry, MIP-la+ cells from RA compared with normal were predominantly mos and lining cells (P < 0.05). These results suggest that MIP-la plays a role in the selective recruitment of mos in synovial inflammation associated with RA. (J. Clin. Invest. 1994. 93:921-928.)

The neu (c-erbB-2) proto-oncogene encodes a tyrosine kinase receptor that is overexpressed in 20 to 30% of human breast tumors. Herein, cyclin D1 protein levels were increased in mammary tumors induced by overexpression of wild-type Neu or activating mutants of Neu in transgenic mice and in MCF7 cells overexpressing transforming Neu. Analyses of 12 Neu mutants in MCF7 cells indicated important roles for specific C-terminal autophosphorylation sites and the extracellular domain in cyclin D1 promoter activation. Induction of cyclin D1 by NeuT involved Ras, Rac, Rho, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, but not phosphatidylinositol 3-kinase. NeuT induction of the cyclin D1 promoter required the E2F and Sp1 DNA binding sites and was inhibited by dominant negative E2F-1 or DP-1. Neu-induced transformation was inhibited by a cyclin D1 antisense or dominant negative E2F-1 construct in Rat-1 cells. Growth of NeuT-transformed mammary adenocarcinoma cells in nude mice was blocked by the cyclin D1 antisense construct. These results demonstrate that E2F-1 mediates a Neu-signaling cascade to cyclin D1 and identify cyclin D1 as a critical downstream target of neu-induced transformation.The neu (c-erbB-2, HER-2) proto-oncogene encodes a receptor tyrosine kinase that is a member of a growth factor receptor family, which includes the epidermal growth factor (EGF) receptor (ErbB-1), ErbB-3, and ErbB-4. neu is overexpressed in 20 to 30% of human breast tumors (64). Both Neu and the EGF receptor stimulate proliferation of breast cancer cells, and overexpression of these two proteins correlates with progression of human breast cancer and poor patient prognosis (28,31,47). A substitution point mutation at residue 664 (Val3Glu) in the transmembrane domain of rat Neu (referred to as NeuT) encodes an activated transforming tyrosine kinase (7). Overexpression of either wild-type Neu or NeuT in transgenic mice under the control of the murine mammary tumor virus (MMTV) long terminal repeat induces mammary adenocarcinoma with high frequency (25, 41). Several independent transgenic strains bearing the identical MMTV-neuT transgene developed synchronous, multifocal mammary tumors involving all mammary glands (24), providing strong evidence that activated neu requires few if any additional genetic events to transform the epithelial cell.In mammary tumors of mice transgenic for the wild-type Neu receptor (MMTV-neu mice), the receptor's intrinsic tyrosine kinase activity was increased in association with inframe somatic mutations of the transgene (61). Introduction of these extracellular domain deletion (ECD) mutations into the wild-type Neu cDNA enhanced neu transforming potential (61). Transgenic mice expressing these Neu deletion mutants in the mammary gland (MMTV-NDL mice) developed multifocal mammary adenocarcinomas with high frequency and shorter latency compared with mice transgenic for the wildtype neu. In primary human breast tumors, a splice variant of ErbB-2 encoding a similar ECD deletion which can...

A general paradigm in signal transduction is ligand-induced feedback inhibition and the desensitization of signaling. We found that subthreshold concentrations of interferon-gamma (IFN-gamma), which did not activate macrophages, increased their sensitivity to subsequent IFN-gamma stimulation; this resulted in increased signal transducer and activator of transcription 1 (STAT1) activation and increased IFN-gamma#150;dependent gene activation. Sensitization of IFN-gamma signaling was mediated by the induction of STAT1 expression by low doses of IFN-gamma that did not effectively induce feedback inhibition. IFN-gamma signaling was sensitized in vivo after IFN-gamma injection, and STAT1 expression was increased after injection of lipopolysaccharide and in rheumatoid arthritis synovial cells. These results identify a mechanism that sensitizes macrophages to low concentrations of IFN-gamma and regulates IFN-gamma responses in acute and chronic inflammation.