Endometrial mesenchymal stem/stromal cell modulation of T cell proliferation.

Perivascular mesenchymal stem/stromal cells can be isolated from the human endometrium using the surface marker SUSD2 and are being investigated for use in tissue repair. Mesenchymal stem/stromal cells from other tissues modulate T cell responses via mechanisms including interleukin-10, prostaglandin E2, TGF-ß1 and regulatory T cells. Animal studies demonstrate that endometrial mesenchymal stem/stromal cells can also modify immune responses to implanted mesh, but the mechanism/s they employ have not been explored. We examined the immunomodulatory properties of human endometrial mesenchymal stem/stromal cells on lymphocyte proliferation using mouse splenocyte cultures. Endometrial mesenchymal stem/stromal cells inhibited mitogen-induced lymphocyte proliferation in vitro in a dose-dependent manner. Inhibition of lymphocyte proliferation was not affected by blocking the mouse interleukin-10 receptor or inhibiting prostaglandin production. Endometrial mesenchymal stem/stromal cells continued to restrain lymphocyte proliferation in the presence of an inhibitor of TGF-ß receptors, despite a reduction in regulatory T cells. Thus, the in vitro inhibition of mitogen-induced lymphocyte proliferation by endometrial mesenchymal stem/stromal cells occurs by a mechanism distinct from the interleukin-10, prostaglandin E2, TGF-ß1 and regulatory T cell-mediated mechanisms employed by MSC from other tissues. eMSCs were shown to produce interleukin-17A and Dickkopf-1 which may contribute to their immunomodulatory properties. In contrast to MSC from other sources, systemic administration of endometrial mesenchymal stem/stromal cells did not inhibit swelling in a T cell-mediated model of skin inflammation. We conclude that, while endometrial mesenchymal stem/stromal cells can modify immune responses, their immunomodulatory repertoire may not be sufficient to restrain some T cell-mediated inflammatory events.

Deficiency of annexin A1 in CD4+ T cells exacerbates T cell-dependent inflammation.

Annexin A1 (AnxA1) is recognized as an endogenous anti-inflammatory molecule. However, its effects on the adaptive immune response and, in particular, on T cells remain unclear. In this study, we investigated the actions of AnxA1 in three distinct models of T cell-mediated inflammation. In contact hypersensitivity, collagen-induced arthritis, and inflammation induced by OT-II TCR transgenic T cells responding to OVA, AnxA1 deficiency significantly increased Ag-induced T cell proliferation and the resultant level of inflammation. In the contact hypersensitivity model, this was associated with increased adhesion of CD4(+) T cells, CD8(+) T cells, and neutrophils in the dermal microvasculature, as well as increased T cell expression of RORγt and IL-17A. In collagen-induced arthritis, deficiency of endogenous AnxA1 increased susceptibility to arthritis and Ag-specific T cell activation. Deficiency of AnxA1 also increased OVA-induced cutaneous delayed-type hypersensitivity and IFN-γ and IL-17 release. Transfer experiments using CD4(+) T cells from AnxA1(-/-) mice demonstrated that the absence of AnxA1 solely in T cells resulted in increased inflammatory responses in wild-type recipients. Similarly, experiments using AnxA1(-/-) OT-II CD4(+) T cells demonstrated that the absence of AnxA1 in T cells was sufficient to induce increased Ag-specific CD4(+) T cell proliferation in vivo, augment T cell production of IFN-γ, IL-17, TNF, and IL-6, and increase Akt, ERK, and p38 activation. Together, these findings indicate that T cell-expressed AnxA1 functions to attenuate T cell-driven inflammatory responses via T cell-intrinsic effects on intracellular signaling, proliferation, and Th1/Th17 cytokine release.

A miR-19 regulon that controls NF-κB signaling.

Fine-tuning of inflammatory responses by microRNAs (miRNAs) is complex, as they can both enhance and repress expression of pro-inflammatory mediators. In this study, we investigate inflammatory responses following global miRNA depletion, to better define the overall contribution of miRNAs to inflammation. We demonstrate that miRNAs positively regulate Toll-like receptor signaling using inducible Dicer1 deletion and global miRNA depletion. We establish an important contribution of miR-19b in this effect, which potentiates nuclear factor-κB (NF-κB) activity in human and mouse cells. Positive regulation of NF-κB signaling by miR-19b involves the coordinated suppression of a regulon of negative regulators of NF-κB signaling (including A20/Tnfaip3, Rnf11, Fbxl11/Kdm2a and Zbtb16). Transfection of miR-19b mimics exacerbated the inflammatory activation of rheumatoid arthritis primary fibroblast-like synoviocytes, demonstrating its physiological importance in the pathology of this disease. This study constitutes, to our knowledge, the first description of a miR-19 regulon that controls NF-κB signaling, and suggests that targeting this miRNA and linked family members could regulate the activity of NF-κB signaling in inflammation.

Regulation of lung fibroblast activation by annexin A1.

Annexin-A1 (AnxA1) is a glucocorticoid-induced protein with multiple actions in the regulation of inflammatory cell activation. The contribution of AnxA1 to human cell biology is not well understood. We investigated the contribution of AnxA1 and its receptor, formyl-peptide receptor 2 (FPR2), to the regulation of inflammatory responses in human normal lung fibroblasts (NLF). Silencing constitutive AnxA1 expression in NLF using small interfering RNA (siRNA) was associated with moderate but significant increases in tumor necrosis factor (TNF)-induced proliferation and interleukin (IL)-6 production, accompanied by reduction of ERK and NF-κB activity. AnxA1 regulation of ERK and NF-κB activation was associated with effects on proliferation. Blocking FPR2 using the specific antagonist WRW4 mimicked the effects of AnxA1 silencing on TNF-induced proliferation, IL-6, ERK, and NF-κB activation. AnxA1 silencing also impaired inhibitory effects of glucocorticoid on IL-6 production and on the expression of glucocorticoid-induced leucine zipper (GILZ), but blocking FPR2 failed to mimic these effects of AnxA1 silencing. These data suggest that AnxA1 regulates TNF-induced proliferation and inflammatory responses in lung fibroblasts, via effects on the ERK and NF-κB pathways, which depend on FPR2. AnxA1 also mediates effects of glucocorticoids and GILZ expression, but these effects appear independent of FPR2. These findings suggest that mimicking AnxA1 actions might have therapeutic potential in chronic inflammatory lung diseases.

Annexin-1 regulates macrophage IL-6 and TNF via glucocorticoid-induced leucine zipper.

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-kappaB 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-kappaB 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.

Formyl peptide receptor activation inhibits the expansion of effector T cells and synovial fibroblasts and attenuates joint injury in models of rheumatoid arthritis.

The effects of formyl peptide receptors (FPRs) on effector T cells and inflammation-causing tissue-resident cells are not well known. Here, we explored the effect of FPR activation on efferent T cell responses in models of rheumatoid arthritis (RA) and on the expansion of fibroblast-like synoviocytes (FLS). Compound 43 (Cpd43; FPR1/2 agonist) was administered to mice with collagen-induced arthritis (CIA) or antigen-induced arthritis (AIA) after disease onset. Joint inflammation/damage and immunity were assessed. FLS were cultured with Cpd43 to test its effects on cell apoptosis and proliferation. To explore the effects of endogenous FPR2 ligands on FLS proliferation, FLS FPR2 was blocked or Annexin A1 (AnxA1) expression silenced. Cpd43 reduced arthritis severity in both models. In CIA, Cpd43 decreased CD4 T cell proliferation and survival and increased the production of the protective cytokine, IFNγ, in lymph nodes. In AIA, Cpd43 increased CD4 apoptosis and production of the anti-inflammatory IL-4, while augmenting the proportion of splenic regulatory T cells and their expression of IL-2Rα. In both models, Cpd43 increased CD4 IL-17A production, without affecting humoral immunity. FPR2 inhibitors reversed Cpd43-mediated effects on AIA and T cell immunity. Cpd43 decreased TNF-induced FLS proliferation and augmented FLS apoptosis in association with intracellular FPR2 accumulation, while endogenous AnxA1 and FPR2 reduced FLS proliferation via the ERK and NFκB pathways. Overall, FPR activation inhibits the expansion of arthritogenic effector CD4 T cells and FLS, and reduces joint injury in experimental arthritis. This suggests the therapeutic potential of FPR ligation for the treatment of RA.

Author Correction: Endogenous Annexin-A1 Regulates Haematopoietic Stem Cell Mobilisation and Inflammatory Response Post Myocardial Infarction in Mice In Vivo.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice.

Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.

Endogenous Annexin-A1 Regulates Haematopoietic Stem Cell Mobilisation and Inflammatory Response Post Myocardial Infarction in Mice In Vivo.

Endogenous anti-inflammatory annexin-A1 (ANX-A1) plays an important role in preserving left ventricular (LV) viability and function after ischaemic insults in vitro, but its long-term cardioprotective actions in vivo are largely unknown. We tested the hypothesis that ANX-A1-deficiency exaggerates inflammation, haematopoietic stem progenitor cell (HSPC) activity and LV remodelling in response to myocardial ischaemia in vivo. Adult ANX - A1 -/- mice subjected to coronary artery occlusion exhibited increased infarct size and LV macrophage content after 24-48 h reperfusion compared with wildtype (WT) counterparts. In addition, ANX - A1 -/- mice exhibited greater expansion of HSPCs and altered pattern of HSPC mobilisation 8 days post-myocardial infarction, with increased circulating neutrophils and platelets, consistent with increased cardiac inflammation as a result of increased myeloid invading injured myocardium in response to MI. Furthermore, ANX - A1 -/- mice exhibited significantly increased expression of LV pro-inflammatory and pro-fibrotic genes and collagen deposition after MI compared to WT counterparts. ANX-A1-deficiency increased cardiac necrosis, inflammation, hypertrophy and fibrosis following MI, accompanied by exaggerated HSPC activity and impaired macrophage phenotype. These findings suggest that endogenous ANX-A1 regulates mobilisation and differentiation of HSPCs. Limiting excessive monocyte/neutrophil production may limit LV damage in vivo. Our findings support further development of novel ANX-A1-based therapies to improve cardiac outcomes after MI.

TNF regulates leukocyte-endothelial cell interactions and microvascular dysfunction during immune complex-mediated inflammation.

1. The aim of this study was to assess directly the role of TNF in immune complex-induced leukocyte-endothelial cell interactions and microvascular dysfunction. 2. Intravital microscopy was used to examine immune complex-induced leukocyte rolling, adhesion and emigration and microvascular permeability in cremasteric postcapillary venules in wild-type and TNF(-/-) mice. The reverse passive Arthus (RPA) reaction was used to localize immune complex formation to the cremaster muscle. 3. In wild-type mice, immune complex deposition induced a reduction in leukocyte rolling velocity and increases in leukocyte adhesion and emigration. In TNF(-/-) mice, the immune complex-induced reduction in leukocyte rolling velocity was significantly attenuated, and leukocyte adhesion and emigration were also significantly reduced relative to responses in wild-type mice. 4. The alterations in TNF(-/-) mice were associated with decreased expression of endothelial P-selectin and VCAM-1, and an absence of E-selectin-dependent rolling normally seen in wild-type mice at the peak of the response. In addition, the level of immune complex-induced microvascular permeability was attenuated in TNF(-/-) mice. 5. These findings demonstrate that in immune complex-induced inflammation, TNF promotes leukocyte rolling and adhesive interactions, and entry of leukocytes into sites of immune complex deposition, in part via the increased expression and/or function of endothelial P-selectin, E-selectin and VCAM-1. In addition, this increase in leukocyte recruitment mediated by TNF correlates directly with an increase in microvascular injury.

Cardioprotective potential of annexin-A1 mimetics in myocardial infarction.

Myocardial infarction (MI) and its resultant heart failure remains a major cause of death in the world. The current treatments for patients with MI are revascularization with thrombolytic agents or interventional procedures. These treatments have focused on restoring blood flow to the ischemic tissue to prevent tissue necrosis and preserve organ function. The restoration of blood flow after a period of ischemia, however, may elicit further myocardial damage, called reperfusion injury. Pharmacological interventions, such as antioxidant and Ca(2+) channel blockers, have shown premises in experimental settings; however, clinical studies have shown limited success. Thus, there is a need for the development of novel therapies to treat reperfusion injury. The therapeutic potential of glucocorticoid-regulated anti-inflammatory mediator annexin-A1 (ANX-A1) has recently been recognized in a range of systemic inflammatory disorders. ANX-A1 binds to and activates the family of formyl peptide receptors (G protein-coupled receptor family) to inhibit neutrophil activation, migration and infiltration. Until recently, studies on the cardioprotective actions of ANX-A1 and its peptide mimetics (Ac2-26, CGEN-855A) have largely focused on its anti-inflammatory effects as a mechanism of preserving myocardial viability following I-R injury. Our laboratory provided the first evidence of the direct protective action of ANX-A1 on myocardium, independent of inflammatory cells in vitro. We now review the potential for ANX-A1 based therapeutics to be seen as a "triple shield" therapy against myocardial I-R injury, limiting neutrophil infiltration and preserving both cardiomyocyte viability and contractile function. This novel therapy may thus represent a valuable clinical approach to improve outcome after MI.

Macrophage migration inhibitory factor inhibits the antiinflammatory effects of glucocorticoids via glucocorticoid-induced leucine zipper.

OBJECTIVE: Glucocorticoids remain a mainstay in the treatment of rheumatoid arthritis (RA). Dose-dependent adverse effects highlight the need for therapies that regulate glucocorticoid sensitivity to enable dosage reduction. Macrophage migration inhibitory factor (MIF) is a proinflammatory protein that has been implicated in the pathogenesis of RA; it impairs glucocorticoid sensitivity via MAPK phosphatase 1 (MKP-1) inhibition. The intracellular protein glucocorticoid-induced leucine zipper (GILZ) mimics the effects of glucocorticoids in models of RA, but whether it represents a target for the modulation of glucocorticoid sensitivity remains unknown. We undertook this study to investigate whether GILZ is involved in the regulation of glucocorticoid sensitivity by MIF. METHODS: GILZ expression was studied in the presence and absence of MIF, and the role of GILZ in the MIF-dependent regulation of the glucocorticoid sensitivity mediator MKP-1 was studied at the level of expression and function. RESULTS: GILZ expression was significantly inhibited by endogenous MIF, both basally and during responses to glucocorticoid treatment. The effects of MIF on GILZ were dependent on the expression and Akt-induced nuclear translocation of the transcription factor FoxO3A. GILZ was shown to regulate the expression of MKP-1 and consequent MAPK phosphorylation and cytokine release. CONCLUSION: MIF exerts its effects on MKP-1 expression and MAPK activity through inhibitory effects on GILZ. These findings suggest a previously unsuspected interaction between MIF and GILZ and identify GILZ as a potential target for the therapeutic regulation of glucocorticoid sensitivity.

Annexin A1: potential for glucocorticoid sparing in RA.

Glucocorticoids have broad-ranging and powerful anti-inflammatory and immunomodulatory effects. Unsurprisingly, therefore, glucocorticoids are widely and persistently used to treat a large number of inflammatory diseases, including rheumatoid arthritis (RA), despite the well-described adverse effects of these drugs. Annexin A1 is a glucocorticoid-induced molecule that is known to replicate many of the described anti-inflammatory effects of glucocorticoids. In addition to the well-documented roles of this protein in neutrophil function, emerging evidence suggests that annexin A1 is involved in the modulation of T-cell function and the adaptive immune responses relevant to RA. Interest in annexin A1 was renewed after the delineation of the receptors for this protein. This breakthrough also led to advances in our understanding of anti-inflammatory annexin A1 mimetic peptides and agonistic compounds targeting these receptors, particularly those specific for the receptor N-formyl peptide receptor 2 (FPR2). Herein, we review the current knowledge of the biological activities of annexin A1 and their relevance to RA pathogenesis. We also discuss the potential of annexin A1 mimics and strategies aimed at potentiating annexin A1 signalling to become viable approaches to minimizing glucocorticoid use in RA and other inflammatory disorders.

Reperfusion-induced myocardial dysfunction is prevented by endogenous annexin-A1 and its N-terminal-derived peptide Ac-ANX-A1(2-26).

BACKGROUND AND PURPOSE: Annexin-A1 (ANX-A1) is an endogenous, glucocorticoid-regulated anti-inflammatory protein. The N-terminal-derived peptide Ac-ANX-A1(2-26) preserves cardiomyocyte viability, but the impact of ANX-A1-peptides on cardiac contractility is unknown. We now test the hypothesis that ANX-A1 preserves post-ischaemic recovery of left ventricular (LV) function. EXPERIMENTAL APPROACH: Ac-ANX-A1(2-26) was administered on reperfusion, to adult rat cardiomyocytes as well as hearts isolated from rats, wild-type mice and mice deficient in endogenous ANX-A1 (ANX-A1(-/-)). Myocardial viability and recovery of LV function were determined. KEY RESULTS: Ischaemia-reperfusion markedly impaired both cardiomyocyte viability and recovery of LV function by 60%. Treatment with exogenous Ac-ANX-A1(2-26) at the onset of reperfusion prevented cardiomyocyte injury and significantly improved recovery of LV function, in both intact rat and wild-type mouse hearts. Ac-ANX-A1(2-26) cardioprotection was abolished by either formyl peptide receptor (FPR)-nonselective or FPR1-selective antagonists, Boc2 and cyclosporin H, but was relatively insensitive to the FPR2-selective antagonist QuinC7. ANX-A1-induced cardioprotection was associated with increased phosphorylation of the cell survival kinase Akt. ANX-A1(-/-) exaggerated impairment of post-ischaemic recovery of LV function, in addition to selective LV FPR1 down-regulation. CONCLUSIONS AND IMPLICATIONS: These data represent the first evidence that ANX-A1 affects myocardial function. Our findings suggest ANX-A1 is an endogenous regulator of post-ischaemic recovery of LV function. Furthermore, the ANX-A1-derived peptide Ac-ANX-A1(2-26) on reperfusion rescues LV function, probably via activation of FPR1. ANX-A1-based therapies may thus represent a novel clinical approach for the prevention and treatment of myocardial reperfusion injury.

Imbalanced frequencies of Th17 and Treg cells in acute coronary syndromes are mediated by IL-6-STAT3 signaling.

AIMS: Extensive evidence suggests inflammatory components participate in the pathogenic processes of acute coronary syndromes (ACS). In this study, we aimed to elucidate the role and mechanism underlying the imbalance of Th17 and Treg cell peripheral populations in the pathogenesis of ACS. METHODS AND RESULTS: Using a flow cytometric analysis, we observed a significantly increased frequency of Th17 cells and a concurrently decreased CD4(+)CD25(+)Foxp3(+) Treg cells in patients with ACS. To elucidate the mechanism of Th17/Treg imbalance in ACS, 22 inflammatory cytokines were measured using multiplexed immunobead-based assays. Of six elevated cytokines in ACS patients, only IL-6 was positively correlated with a higher Th17 cell level (r = 0.39, P<0.01). Relying on IL-6 stimulating and neutralizing studies, we demonstrated a direct role for IL-6 in sera from ACS patients with an increased frequency of Th17 cells. IL-6 induces the differentiation of Th17 cells from naïve CD4(+) T cells through STAT3 activation and RORγt induction. However, we observed that high levels of TGF-ß1 inhibited IL-6-dependent Th17 cell differentiation, indicating a complex interplay between the two cytokines in the control of Th17 and Treg cell populations. CONCLUSIONS: Our results demonstrate the role of IL-6-STAT3 signaling in ACS through increased Th17 cell differentiation. These findings indicate that IL-6 neutralizing strategies could present novel therapeutic avenues in the treatment of ACS.

Increased CD45RA+ FoxP3(low) regulatory T cells with impaired suppressive function in patients with systemic lupus erythematosus.

BACKGROUND: The role of naturally occurring regulatory T cells (Treg) in the control of the development of systemic lupus erythematosus (SLE) has not been well defined. Therefore, we dissect the phenotypically heterogeneous CD4(+)FoxP3(+) T cells into subpopulations during the dynamic SLE development. METHODLOGY/PRINCIPAL FINDINGS: To evaluate the proliferative and suppressive capacities of different CD4(+) T cell subgroups between active SLE patients and healthy donors, we employed CD45RA and CD25 as surface markers and carboxyfluorescein diacetatesuccinimidyl ester (CFSE) dilution assay. In addition, multiplex cytokines expression in active SLE patients was assessed using Luminex assay. Here, we showed a significant increase in the frequency of CD45RA(+)FoxP3(low) naive Treg cells (nTreg cells) and CD45RA(-)FoxP3(low) (non-Treg) cells in patients with active SLE. In active SLE patients, the increased proportions of CD45RA(+)FoxP3(low) nTreg cells were positively correlated with the disease based on SLE disease activity index (SLEDAI) and the status of serum anti-dsDNA antibodies. We found that the surface marker combination of CD25(+)CD45RA(+) can be used to defined CD45RA(+)FoxP3(low) nTreg cells for functional assays, wherein nTreg cells from active SLE patients demonstrated defective suppression function. A significant correlation was observed between inflammatory cytokines, such as IL-6, IL-12 and TNFα, and the frequency of nTreg cells. Furthermore, the CD45RA(+)FoxP3(low) nTreg cell subset increased when cultured with SLE serum compared to healthy donor serum, suggesting that the elevated inflammatory cytokines of SLE serum may promote nTreg cell proliferation/expansion. CONCLUSIONS/SIGNIFICANCE: Our results indicate that impaired numbers of functional CD45RA(+)FoxP3(low) naive Treg cell and CD45RA(-)FoxP3(low) non-suppressive T cell subsets in inflammatory conditions may contribute to SLE development. Therefore, analysis of subsets of FoxP3(+) T cells, using a combination of FoxP3, CD25 and CD45RA, rather than whole FoxP3(+) T cells, will help us to better understand the pathogenesis of SLE and may lead to the development of new therapeutic strategies.

Regulation of annexin I in rheumatoid synovial cells by glucocorticoids and interleukin-1.

The glucocorticoid (GC)-induced antiinflammatory molecule annexin I is expressed in leukocytes and has antiinflammatory effects in animal models of arthritis, but the expression of annexin I in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) is unknown. We report the constitutive and dexamethasone (DEX)-inducible expression of annexin I in RA FLS. DEX increased FLS annexin I protein translocation and mRNA expression. Interleukin (IL)-1beta also induced annexin I translocation and mRNA but also increased intracellular protein. DEX and IL-1 had additive effects on annexin I mRNA, but DEX inhibited the inducing effect of IL-1beta on cell surface annexin I. These results indicate that glucocorticoids and IL-1beta upregulate the synthesis and translocation of annexin I in RA FLS, but interdependent signalling pathways are involved.

Macrophage migration inhibitory factor induces macrophage recruitment via CC chemokine ligand 2.

Macrophage migration inhibitory factor (MIF) was originally identified for its ability to inhibit the random migration of macrophages in vitro. MIF is now recognized as an important mediator in a range of inflammatory disorders. We recently observed that the absence of MIF is associated with a reduction in leukocyte-endothelial cell interactions induced by a range of inflammatory mediators, suggesting that one mechanism whereby MIF acts during inflammatory responses is by promoting leukocyte recruitment. However, it is unknown whether MIF is capable of inducing leukocyte recruitment independently of additional inflammatory stimuli. In this study, we report that MIF is capable of inducing leukocyte adhesion and transmigration in postcapillary venules in vivo. Moreover, leukocytes recruited in response to MIF were predominantly CD68(+) cells of the monocyte/macrophage lineage. Abs against the monocyte-selective chemokine CCL2 (JE/MCP-1) and its receptor CCR2, but not CCL3 and CXCL2, significantly inhibited MIF-induced monocyte adhesion and transmigration. CCL2(-/-) mice displayed a similar reduction in MIF-induced recruitment indicating a critical role of CCL2 in the MIF-induced response. This hypothesis was supported by findings that MIF induced CCL2 release from primary microvascular endothelial cells. These data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues. This function may be critical to the ability of MIF to promote diseases such as atherosclerosis and rheumatoid arthritis, in which macrophages are key participants.

Annexin 1 negatively regulates IL-6 expression via effects on p38 MAPK and MAPK phosphatase-1.

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.

Reduction of arthritis severity in protease-activated receptor-deficient mice.

OBJECTIVE: Protease-activated receptor 1 (PAR-1) is the cell surface receptor for thrombin. It is unclear whether thrombin contributes to inflammation other than by effects on coagulation. We investigated the proinflammatory participation of PAR-1 in antigen-induced arthritis (AIA). METHODS: Arthritis was induced by intraarticular injection of methylated bovine serum albumin (mBSA) in preimmunized PAR-1-deficient (PAR-1(-/-)) and wild-type (WT) mice. The disease was assessed after 7 days by histologic examination of knee joints after decalcification and Safranin O/toluidine blue staining. Serum levels of anti-mBSA IgG, interferon-gamma, and interleukin-4 (IL-4) were determined by enzyme-linked immunosorbent assay. T cell proliferation response was determined by measuring the incorporation of (3)H-thymidine. Cytokine messenger RNA (mRNA) expression was detected in synovial tissues and peritoneal cells by real-time polymerase chain reaction. RESULTS: Arthritis severity was significantly reduced in PAR-1(-/-) mice compared with WT mice (P = 0.017). Analysis of individual aspects of joint histology revealed significant reductions in synovial exudates (P < 0.001), cartilage degradation (P < 0.01), and bone damage (P = 0.05) in PAR-1(-/-) mice. Synovial IL-1, IL-6, and matrix metalloproteinase 13 (MMP-13) mRNA was significantly reduced in PAR-1(-/-) mice. The titers of antigen-specific serum anti-mBSA total IgG, IgG1, and IgG2a were significantly reduced, and serum IL-4 was significantly increased in arthritic PAR-1(-/-) mice. In contrast, no difference was observed in antigen-induced T cell proliferation between PAR-1(-/-) and WT mice. In vitro, thrombin-induced (but not lipopolysaccharide-induced) IL-1, IL-6, and MMP-13 mRNA expression was significantly impaired in PAR-1(-/-) mice compared with WT controls. CONCLUSION: These data demonstrate the requirement of PAR-1 for the expression of AIA, the development of an antigen-specific Ig response, thrombin-induced macrophage cytokine and MMP expression, and the inhibitory effect of PAR-1 on serum IL-4. We conclude that PAR-1 plays a significant role in this model of arthritis.