Annexin-1 (ANXA1) is a mediator of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (GC). The mechanism of ANXA1 effects on cytokine production in macrophages is unknown and is here investigated in vivo and in vitro. In response to LPS administration, ANXA1−/− mice exhibited significantly increased serum IL-6 and TNF compared with wild-type (WT) controls. Similarly, LPS-induced IL-6 and TNF were significantly greater in ANXA1−/− than in WT peritoneal macrophages in vitro. In addition, deficiency of ANXA1 was associated with impairment of the inhibitory effects of dexamethasone (DEX) on LPS-induced IL-6 and TNF in macrophages. Increased LPS-induced cytokine expression in the absence of ANXA1 was accompanied by significantly increased LPS-induced activation of ERK and JNK MAPK and was abrogated by inhibition of either of these pathways. No differences in GC effects on MAPK or MAPK phosphatase 1 were observed in ANXA1−/− cells. In contrast, GC-induced expression of the regulatory protein GILZ was significantly reduced in ANXA1−/− cells by silencing of ANXA1 in WT cells and in macrophages of ANXA1−/− mice in vivo. GC-induced GILZ expression and GC inhibition of NF-κB activation were restored by expression of ANXA1 in ANXA1−/− cells, and GILZ overexpression in ANXA1−/− macrophages reduced ERK MAPK phosphorylation and restored sensitivity of cytokine expression and NF-κB activation to GC. These data confirm ANXA1 as a key inhibitor of macrophage cytokine expression and identify GILZ as a previously unrecognized mechanism of the anti-inflammatory effects of ANXA1.

Objective. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with established roles in a range of inflammatory conditions. However, it is not known whether MIF influences inflammation via the direct promotion of leukocyteendothelial cell interactions. Therefore, the aim of these experiments was to investigate the ability of MIF to regulate leukocyte-endothelial cell interactions in the inflamed microvasculature.Methods. Intravital microscopy was used to examine postcapillary venules in the cremaster muscle and synovium of wild-type and MIF ؊/؊ mice. Leukocyte-endothelial cell interactions (rolling, adhesion, emigration) were compared under a range of inflammatory conditions.Results. In cremasteric postcapillary venules of MIF ؊/؊ mice, lipopolysaccharide (LPS)-induced leukocyte rolling, adhesion, and emigration were significantly reduced relative to that in wild-type mice. Similar responses were observed in response to tumor necrosis factor ␣ and histamine. Examination of the synovial microvasculature following exposure to carrageenan revealed that leukocyte rolling and adhesion in synovial postcapillary venules and leukocyte entry into the joint space were also reduced significantly in MIF ؊/؊ mice. In each of these models, the level of P-selectindependent rolling was reduced in MIF ؊/؊ mice. Despite this, no difference in P-selectin expression was observed following LPS treatment. However, microvascular shear forces were elevated in MIF ؊/؊ mice, raising a possible mechanism to explain the reduced interactions in these animals.Conclusion. MIF ؊/؊ mice consistently displayed a reduction in P-selectin-dependent rolling, suggesting that MIF exerts proinflammatory effects, in part, via the promotion of P-selectin-mediated rolling. Together, these data indicate that MIF promotes interactions between leukocytes and endothelial cells, thereby enhancing the entry of leukocytes into sites of inflammation.

Annexin 1 (Anx-1) is a mediator of the anti-inflammatory actions of glucocorticoids, but the mechanism of its anti-inflammatory effects is not known. We investigated the role of Anx-1 in the regulation of the proinflammatory cytokine, IL-6. Lung fibroblast cell lines derived from Anx-1−/− and wild-type (WT) mice were treated with dexamethasone and/or IL-1. IL-6 mRNA and protein were measured using real-time PCR and ELISA, and MAPK pathway activation was studied. Compared with WT cells, unstimulated Anx-1−/− cells exhibited dramatically increased basal IL-6 mRNA and protein expression. In concert with this result, Anx-1 deficiency was associated with increased basal phosphorylated p38, JNK, and ERK1/2 MAPKs. IL-1-inducible phosphorylated p38 was also increased in Anx-1−/− cells. The increase in IL-6 release in Anx-1−/− cells was inhibited by inhibition of p38 MAPK. Anx-1−/− cells were less sensitive to dexamethasone inhibition of IL-6 mRNA expression than WT cells, although inhibition by dexamethasone of IL-6 protein was similar. MAPK phosphatase-1 (MKP-1), a glucocorticoid-induced negative regulator of MAPK activation, was up-regulated by dexamethasone in WT cells, but this effect of dexamethasone was significantly impaired in Anx-1−/− cells. Treatment of Anx-1−/− cells with Anx-1 N-terminal peptide restored MKP-1 expression and inhibited p38 MAPK activity. These data demonstrate that Anx-1 is an endogenous inhibitory regulator of MAPK activation and IL-6 expression, and that Anx-1 is required for glucocorticoid up-regulation of MKP-1. Therapeutic manipulation of Anx-1 could provide glucocorticoid-mimicking effects in inflammatory disease.

SummaryMacrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine with many cellular targets in rheumatoid arthritis (RA). MIF has been reported to activate cells via mitogen-activated protein kinase and serine/threonine kinase (AKT or protein kinase B)-dependent signal transduction pathways. Its contribution to T cell activation and signalling in RA is not known. Using MIF -/-mice and a T cell-mediated model of RA, antigen-induced arthritis, we investigated the role of MIF in T cell activation and signalling. Arthritis severity was significantly reduced in MIF -/-mice compared with wildtype mice. This reduction was associated with decreased T cell activation parameters including footpad delayed type hypersensitivity, antigen-induced splenocyte proliferation and cytokine production. Splenocyte proliferation required extracellular signal-regulated kinase (ERK)1/2 phosphorylation, and decreased T cell activation in MIF -/-mice was associated with decreased phosphorylation of ERK1/2 but not AKT. Collectively, these data suggest that MIF promotes antigen-specific immune responses via regulation of ERK phosphorylation in T cells.

Summaryp53 is a transcription factor with a well-described role in the induction of apoptosis and cell cycle arrest as part of a protective response to a variety of stressful stimuli. Expansion of inflamed tissue in rheumatoid arthritis has been related to the loss of functioning p53, and the severity of collageninduced arthritis is increased in p53 -/-mice. Our objective was to assess the role of p53 in a model of adaptive immunity, antigen-induced arthritis (AIA). AIA was induced in p53-/-and wild-type mice by priming with methylated bovine serum albumin followed by intra-articular challenge. Severity of arthritis was assessed using a standardized scoring system and synovial apoptosis was detected by TdT-mediated biotin-dUTP nick-end labelling. Splenocyte proliferation was measured by [H 3 ] incorporation and interferon (IFN)-g release. Splenocyte viability was assessed using Titreglow. Splenic T cell activation status was assessed by flow cytometry. Serum cytokines were measured using enzyme-linked immunosorbent assay. Increased severity of AIA in p53 -/-mice was associated with decreased synovial apoptosis and with increased delayed-type hypersensitivity response, increased mitogen and antigen-induced splenocyte proliferation and increased IFN-g release in p53-/-mice compared with wild-type mice. Antigen-specific immunoglobulin responses were equivalent in both groups. Splenocyte viability was increased in p53 -/-mice but T cell apoptosis was equivalent. T cell activation markers were increased in p53 -/-mice compared with wild-type mice. Lipopolysaccharide-induced tumour necrosis factor release was increased in p53 -/-mice with a trend to increased interleukin-6 in p53 -/-mice compared with littermates. p53 is involved in the modulation of adaptive and innate immune responses relevant to arthritis models and is also involved in the modulation of severity of AIA by both cell-cycle dependent and cell-cycleindependent mechanisms.

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