Marked differences in differentiation propensity among human embryonic stem cell lines.

The differentiation potential of 17 human embryonic stem (hES) cell lines was compared. Some lines exhibit a marked propensity to differentiate into specific lineages, often with >100-fold differences in lineage-specific gene expression. For example, HUES 8 is best for pancreatic differentiation and HUES 3 for cardiomyocyte generation. These non-trivial differences in developmental potential among hES cell lines point to the importance of screening and deriving lines for lineage-specific differentiation.

Interleukin-18 induces angiogenic factors in rheumatoid arthritis synovial tissue fibroblasts via distinct signaling pathways.

OBJECTIVE: Interleukin-18 (IL-18) is a proinflammatory cytokine implicated in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to examine the role of IL-18 in up-regulating secretion of the angiogenic factors stromal cell-derived factor 1alpha (SDF-1alpha)/CXCL12, monocyte chemoattractant protein 1 (MCP-1)/CCL2, and vascular endothelial growth factor (VEGF) in RA synovial tissue (ST) fibroblasts, and the underlying signaling mechanisms involved. METHODS: We used enzyme-linked immunosorbent assays, Western blotting, and chemical inhibitors/antisense oligodeoxynucleotides to signaling intermediates to assess the role of IL-18. RESULTS: IL-18 significantly enhanced the production of SDF-1alpha/CXCL12, MCP-1/CCL2, and VEGF in RA ST fibroblasts, in a time- and concentration-dependent manner. IL-18-induced SDF-1alpha/CXCL12 up-regulation was dependent on JNK, p38 MAPK, phosphatidylinositol 3-kinase (PI3K), and NFkappaB. While IL-18-induced production of SDF-1alpha/CXCL12 was also dependent on protein kinase Cdelta (PKCdelta), production of MCP-1/CCL2 was dependent on PKCalpha, not PKCdelta. Additionally, RA ST fibroblast IL-18-induced MCP-1/CCL2 production was mediated by JNK, PI3K, and NFkappaB. In contrast, IL-18 did not induce secretion of RA ST fibroblast MCP-1/CCL2 or VEGF via p38 MAPK. IL-18-induced RA ST fibroblast production of VEGF was mediated mainly by JNK-2, PKCalpha, and NFkappaB. IL-18 induced phosphorylation of JNK, PKCdelta, p38 MAPK, and activating transcription factor 2 (ATF-2) in RA ST fibroblasts in a time-dependent manner, with JNK-2 being upstream of PKCdelta, ATF-2, and NFkappaB. CONCLUSION: These data support the notion that IL-18 has a unique role in inducing the secretion of angiogenic SDF-1alpha/CXCL12, MCP-1/CCL2, and VEGF in RA ST fibroblasts, via distinct signaling intermediates.

Macrophage migration inhibitory factor: a mediator of matrix metalloproteinase-2 production in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by destruction of bone and cartilage, which is mediated, in part, by synovial fibroblasts. Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes responsible for matrix degradation. Macrophage migration inhibitory factor (MIF) is a cytokine that induces the production of a large number of proinflammatory molecules and has an important role in the pathogenesis of RA by promoting inflammation and angiogenesis. In the present study, we determined the role of MIF in RA synovial fibroblast MMP production and the underlying signaling mechanisms. We found that MIF induces RA synovial fibroblast MMP-2 expression in a time-dependent and concentration-dependent manner. To elucidate the role of MIF in MMP-2 production, we produced zymosan-induced arthritis (ZIA) in MIF gene-deficient and wild-type mice. We found that MMP-2 protein levels were significantly decreased in MIF gene-deficient compared with wild-type mice joint homogenates. The expression of MMP-2 in ZIA was evaluated by immunohistochemistry (IHC). IHC revealed that MMP-2 is highly expressed in wild-type compared with MIF gene-deficient mice ZIA joints. Interestingly, synovial lining cells, endothelial cells, and sublining nonlymphoid mononuclear cells expressed MMP-2 in the ZIA synovium. Consistent with these results, in methylated BSA (mBSA) antigen-induced arthritis (AIA), a model of RA, enhanced MMP-2 expression was also observed in wild-type compared with MIF gene-deficient mice joints. To elucidate the signaling mechanisms in MIF-induced MMP-2 upregulation, RA synovial fibroblasts were stimulated with MIF in the presence of signaling inhibitors. We found that MIF-induced RA synovial fibroblast MMP-2 upregulation required the protein kinase C (PKC), c-jun N-terminal kinase (JNK), and Src signaling pathways. We studied the expression of MMP-2 in the presence of PKC isoform-specific inhibitors and found that the PKCdelta inhibitor rottlerin inhibits MIF-induced RA synovial fibroblast MMP-2 production. Consistent with these results, MIF induced phosphorylation of JNK, PKCdelta, and c-jun. These results indicate a potential novel role for MIF in tissue destruction in RA.

CXCL16-mediated cell recruitment to rheumatoid arthritis synovial tissue and murine lymph nodes is dependent upon the MAPK pathway.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by profound mononuclear cell (MNC) recruitment into synovial tissue (ST), thought to be due in part to tumor necrosis factor alpha (TNFalpha), a therapeutic target for RA. Although chemokines may also be involved, the mechanisms remain unclear. We undertook this study to examine the participation of CXCL16, a novel chemokine, in recruitment of MNCs to RA ST in vivo and to determine the signal transduction pathways mediating this process. METHODS: Using a human RA ST-SCID mouse chimera, immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction, flow cytometry, and in vitro chemotaxis assays, we defined the expression and function of CXCL16 and its receptor, CXCR6, as well as the signal transduction pathways utilized by them for MNC homing in vitro and in vivo. RESULTS: CXCL16 was markedly elevated in RA synovial fluid (SF) samples, being as high as 145 ng/ml. Intense macrophage and lining cell staining for CXCL16 in RA ST correlated with increased CXCL16 messenger RNA levels in RA ST compared with those in osteoarthritis and normal ST. By fluorescence-activated cell sorting analysis, one-half of RA SF monocytes and one-third of memory lymphocytes expressed CXCR6. In vivo recruitment of human MNCs to RA ST implanted in SCID mice occurred in response to intragraft injection of human CXCL16, a response similar to that induced by TNFalpha. Lipofection of MNCs with antisense oligodeoxynucleotides for ERK-1/2 resulted in a 50% decline in recruitment to engrafted RA ST and a 5-fold decline in recruitment to regional lymph nodes. Interestingly, RA ST fibroblasts did not produce CXCL16 in response to TNFalpha in vitro, suggesting that CXCL16 protein may function in large part independently of TNFalpha. CONCLUSION: Taken together, these results point to a unique role for CXCL16 as a premier MNC recruiter in RA and suggest additional therapeutic possibilities, targeting CXCL16, its receptor, or its signaling pathways.

Chemokine receptor expression in rat adjuvant-induced arthritis.

OBJECTIVE: Chemokine receptors mediate leukocyte migration into inflamed rheumatoid arthritis (RA) synovial tissue (ST). Knowledge of their distribution is crucial for understanding the evolution of the inflammatory process. In this study, we used rat adjuvant-induced arthritis (AIA), a model for RA, to define the temporospatial expression of chemokine receptors. METHODS: ST from rats with AIA was immunostained, the percentage of cells expressing each receptor was determined, and findings were correlated with levels of inflammation. Chemokine receptor expression was evaluated on rat macrophages in vitro. RESULTS: CCR1, a receptor for macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3 and RANTES/CCL5, exhibited high constitutive expression on macrophages in AIA. CCR5, binding MIP-1alpha/CCL3 and RANTES/CCL5, was up-regulated on ST macrophages during the course of AIA, correlating with macrophage expression of CCR2, a receptor for monocyte chemoattractant protein 1/CCL2. Endothelial cell (EC) CCR2 was down-regulated as arthritis progressed, inversely correlating with inflammation. CCR3, another RANTES/CCL5 receptor, was constitutively high on macrophages in vivo and in vitro, with down-regulation during AIA. CXCR4, a receptor for stromal cell-derived factor 1/CXCL12), was prominently up-regulated on ECs, preceding the peak of inflammation. CONCLUSION: These findings show that 1) constitutive expression of CCR1 on macrophages remains high during AIA; 2) CCR2 and CCR3 may play a role in initial recruitment of leukocytes to ST in AIA; 3) macrophage expression of CCR2 and CCR5 may be important for sustaining inflammatory changes; and 4) EC CXCR4 may be a harbinger of inflammatory changes. Our results may help guide chemokine receptor blockade-targeting treatment strategies in inflammatory arthritis.

The role of COX-2 in angiogenesis and rheumatoid arthritis.

Recent evidence suggests that cyclooxygenase (COX)-2 is a mediator of angiogenesis, and COX-2 activity is known to be upregulated in the rheumatoid arthritis (RA) synovium. We examined whether mediation of angiogenesis by COX-2 was occuring in cells of the RA synovium and in microvascular endothelial cells (ECs) that are similar to those found in the RA synovium. We demonstrate that rofecoxib, a selective COX-2 inhibitor, acts directly on human dermal microvascular ECs (HMVECs) to inhibit their chemotactic and tube forming ability. Likewise, pretreatment of HMVECs with rofecoxib significantly inhibited their ability to form tubes induced by conditioned media (CM) of activated RA synovial fibroblasts. When RA synovial fibroblasts were pretreated with rofecoxib for 16 h and then stimulated with interleukin (IL)-1beta, their CM induced significantly less HMVEC tube formation when compared with CM from vehicle-treated RA synovial fibroblasts. ELISAs performed on activated RA fibroblast CM for known proangiogenic factors demonstrated a significant reduction in bFGF, in addition to the expected decrease in PGE(2). Our studies suggest that COX-2-induced angiogenic activity is an active mechanism within diseased synovium and may provide an additional rationale for the use of COX-2 inhibitors in RA.