IL11 activates the placental inflammasome to drive preeclampsia.

Introduction: Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Interleukin (IL)11 is elevated in serum from pregnancies that subsequently develop early-onset preeclampsia and pharmacological elevation of IL11 in pregnant mice causes the development of early-onset preeclampsia-like features (hypertension, proteinuria, and fetal growth restriction). However, the mechanism by which IL11 drives preeclampsia is unknown. Method: Pregnant mice were administered PEGylated (PEG)IL11 or control (PEG) from embryonic day (E)10-16 and the effect on inflammasome activation, systolic blood pressure (during gestation and at 50/90 days post-natal), placental development, and fetal/post-natal pup growth measured. RNAseq analysis was performed on E13 placenta. Human 1st trimester placental villi were treated with IL11 and the effect on inflammasome activation and pyroptosis identified by immunohistochemistry and ELISA. Result: PEGIL11 activated the placental inflammasome causing inflammation, fibrosis, and acute and chronic hypertension in wild-type mice. Global and placental-specific loss of the inflammasome adaptor protein Asc and global loss of the Nlrp3 sensor protein prevented PEGIL11-induced fibrosis and hypertension in mice but did not prevent PEGIL11-induced fetal growth restriction or stillbirths. RNA-sequencing and histology identified that PEGIL11 inhibited trophoblast differentiation towards spongiotrophoblast and syncytiotrophoblast lineages in mice and extravillous trophoblast lineages in human placental villi. Discussion: Inhibition of ASC/NLRP3 inflammasome activity could prevent IL11-induced inflammation and fibrosis in various disease states including preeclampsia.

miR-23b-3p regulates human endometrial epithelial cell adhesion implying a role in implantation.

In brief: miR-23b-3p expression is increased in fertile endometrium during receptivity. This study investigates the function of miR-23b-3p on endometrial adhesion and its downstream targets. Abstract: The human endometrium undergoes dramatic remodeling throughout the menstrual cycle that is essential for successful blastocyst attachment and implantation in the mid-secretory (receptive) phase. microRNA (miR) plays a role in the preparation of endometrial receptivity. miR-23b-3p expression is increased in fertile endometrium during receptivity. Here, we aimed to investigate miR-23b-3p function during receptivity. qPCR and in situ hybridization were used to investigate the expression and localization of miR-23b-3p in human endometrium, respectively. Ishikawa cells (endometrial epithelial cell line) and endometrial organoid-derived epithelial cells were transfected with miR-23b-3p mimic, and trophoblast progenitor spheroid (blastocyst surrogate) adhesion assay was used to determine effects on blastocyst adhesion to endometrial cells. We demonstrated that miR-23b-3p was significantly upregulated in the fertile endometrium of the receptive phase compared to the non-receptive, proliferative phase. No difference was identified for the expression of miR-23b-3p between fertile and infertile mid-secretory phase endometrium. miR-23b-3p localized to the epithelium and stroma in the mid-secretory phase but was undetectable in the proliferative phase of fertile endometrium. Functionally, miR-23-3p overexpression in Ishikawa cells and fertile endometrial organoid-derived epithelial cells significantly improved their adhesive capacity to trophoblast progenitor spheroids. miR-23b-3p overexpression in infertile endometrial organoid-derived epithelial cells did not improve adhesion. Among 10 miR-predicted gene targets examined, miR-23b-3p overexpression in Ishikawa cells significantly reduced the expression of MET, secreted frizzled-related protein 4 (SFRP4) and acyl-CoA dehydrogenase short/branched chain (ACADSB) compared to control. The reduction of SFRP4 after miR23b-3p overexpression was confirmed by immunoblotting in fertile organoid-derived epithelial cells. SFRP4 expression in fertile endometrium exhibited an inverse expression pattern compared to miR-23b-3p and was higher in the proliferative phase compared to the mid-secretory phase. Overall, miR-23b-3p is likely a critical regulator of endometrial epithelial adhesion and receptivity.

Infertile human endometrial organoid apical protein secretions are dysregulated and impair trophoblast progenitor cell adhesion.

Introduction: Embryo implantation failure leads to infertility. As an important approach to regulate implantation, endometrial epithelial cells produce and secrete factors apically into the uterine cavity in the receptive phase to prepare the initial blastocyst adhesion and implantation. Organoids were recently developed from human endometrial epithelium with similar apical-basal polarity compared to endometrial gland making it an ideal model to study endometrial epithelial secretions. Methods: Endometrial organoids were established using endometrial biopsies from women with primary infertility and normal fertility. Fertile and infertile organoids were treated with hormones to model receptive phase of the endometrial epithelium and intra-organoid fluid (IOF) was collected to compare the apical protein secretion profile and function on trophoblast cell adhesion. Results: Our data show that infertile organoids were dysregulated in their response to estrogen and progesterone treatment. Proteomic analysis of organoid apical secretions identified 150 dysregulated proteins between fertile and infertile groups (>1.5-fold change). Trophoblast progenitor spheroids (blastocyst surrogates) treated with infertile organoid apical secretions significantly compromised their adhesion to organoid epithelial cell monolayers compared to fertile group (P < 0.0001). Discussion: This study revealed that endometrial organoid apical secretions alter trophoblast cell adhesiveness relative to fertility status of women. It paves the way to determine the molecular mechanisms by which endometrial epithelial apical released factors regulate blastocyst initial attachment and implantation.

Fibrinogen heterogeneity in horses.

BACKGROUND: Fibrinogen heterogeneity has been observed in humans and can influence fibrinogen measurements when using the modified Clauss assay. We hypothesized that fibrinogen heterogeneity also exists in horses. OBJECTIVES: To determine whether fibrinogen heterogeneity exists in horses. ANIMALS: Five clinically healthy horses from the university equine teaching herd. METHODS: Presumed fibrinogen was purified from pooled citrated plasma and electrophoresis performed. The purified protein was subjected to Western blotting using sheep antiserum against human fibrinogen, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Gel electrophoresis of nonreduced equine purified protein yielded 2 protein bands (approximately 377 and 318 kDa) that corresponded with the molecular weights of human high molecular weight fibrinogen and low molecular weight fibrinogen fractions, respectively. Electrophoretograms of reduced purified protein, Western blots, and LC-MS/MS supported that the purified nonreduced protein bands were fibrinogen. CONCLUSION: Fibrinogen heterogeneity exists in horses.

Serum leukotriene B4 and hydroxyeicosatetraenoic acid in the prediction of pre-eclampsia.

INTRODUCTION: Pre-eclampsia (PE) affects 2-8% of pregnancies worldwide. Despite identification of numerous possible biomarkers, accurate prediction and early diagnosis of PE remain challenging. We examined the potential of leukotriene B4 (LTB4) and 15-hydroxyeicosatetraenoic acid (15(S)-HETE) as biomarkers of PE by comparing serum levels at three gestational age (GA) groups between normotensive pregnancies and asymptomatic women who subsequently developed preterm or term-PE. METHODS: This is a case-control study drawn from a prospective study of adverse pregnancy outcomes with serum samples collected at 19-24 weeks (n = 48), 30-34 weeks (n = 101) and 35-37 weeks (n = 54) GA. LTB4 and 15(S)-HETE levels were determined by ELISA. Serum level multiples of the median (MoM) were compared between normal and PE-pregnancies. Association between LTB4 and 15(S)-HETE and GA at delivery was investigated with Cox proportional-hazards models. RESULTS: Serum LTB4 levels were lower in women of East-Asian ethnicity, higher in women with PE history, and increased with GA in normotensive pregnancies, but not in PE. LTB4 was elevated at 19-24 weeks in women who developed preterm-PE. There was a negative association between LTB4 MoM and interval between sampling and delivery with PE at 19-24 weeks only. Serum 15(S)-HETE levels were not influenced by GA at testing and were elevated in women of South-Asian ethnicity. Median 15(S)-HETE levels were unchanged in preterm and term-PE at any GA. DISCUSSION: LTB4 was higher at 19-24 weeks in pregnancies that developed preterm-PE versus unaffected pregnancies, suggesting it is a potentially useful predictive marker of preterm PE in the second trimester.

Tripeptidyl peptidase I promotes human endometrial epithelial cell adhesive capacity implying a role in receptivity.

The endometrium undergoes cyclic remodelling throughout the menstrual cycle in preparation for embryo implantation which occurs in a short window during the mid-secretory phase. It is during this short 'receptive window' that the endometrial luminal epithelium acquires adhesive capacity permitting blastocysts firm adhesion to the endometrium to establish pregnancy. Dysregulation in any of these steps can compromise embryo implantation resulting in implantation failure and infertility. Many factors contribute to these processes including TGF-ß, LIF, IL-11 and proteases. Tripeptidyl peptidase 1 (TPP1) is a is a lysosomal serine-type protease however the contribution of the TPP1 to receptivity is unknown. We aimed to investigate the role of TPP1 in receptivity in humans.In the current study, TPP1 was expressed in both epithelial and stromal compartments of the endometrium across the menstrual cycle. Expression was confined to the cytoplasm of luminal and glandular epithelial cells and stromal cells. Staining of mid-secretory endometrial tissues of women with normal fertility and primary unexplained infertility showed reduced immunostaining intensity of TPP1 in luminal epithelial cells of infertile tissues compared to fertile tissues. By contrast, TPP1 levels in glandular epithelial and stromal cells were comparable in both groups in the mid-secretory phase. Inhibition of TPP1 using siRNA compromised HTR8/SVneo (trophoblast cell line) spheroid adhesion on siRNA-transfected Ishikawa cells (endometrial epithelial cell line) in vitro. This impairment was associated with decreased sirtuin 1 (SIRT1), BCL2 and p53 mRNA and unaltered, CD44, CDH1, CDH2, ITGB3, VEGF A, OSTEOPONTIN, MDM2, CASP4, MCL1, MMP2, ARF6, SGK1, HOXA-10, LIF, and LIF receptor gene expression between treatment groups. siRNA knockdown of TPP1 in primary human endometrial stromal cells did not affect decidualization nor the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Taken together, our data strongly suggests a role for TPP1 in endometrial receptivity via its effects on epithelial cell adhesion and suggests reduced levels associated with unexplained infertility may contribute to implantation failure.

Galectin-7 Impairs Placentation and Causes Preeclampsia Features in Mice.

Preeclampsia is a serious pregnancy-induced disorder unique to humans. The etiology of preeclampsia is poorly understood; however, poor placental formation is thought causal. Galectin-7 is produced by trophoblast and is elevated in first-trimester serum of women who subsequently develop preeclampsia. We hypothesized that elevated placental galectin-7 may be causative of preeclampsia. Here, we demonstrated increased galectin-7 production in chorionic villous samples from women who subsequently develop preterm preeclampsia compared with uncomplicated pregnancies. In vitro, galectin-7 impaired human first-trimester trophoblast outgrowth, increased placental production of the antiangiogenic sFlt-1 splice variant, sFlt-1-e15a, and reduced placental production and secretion of ADAM12 (a disintegrin and metalloproteinase12) and angiotensinogen. In vivo, galectin-7 administration (E8-E12) to pregnant mice caused elevated systolic blood pressure, albuminuria, impaired placentation (reduced labyrinth vascular branching, impaired decidual spiral artery remodeling, and a proinflammatory placental state demonstrated by elevated IL1ß, IL6 and reduced IL10), and dysregulated expression of renin-angiotensin system components in the placenta, decidua, and kidney, including angiotensinogen, prorenin, and the angiotensin II type 1 receptor. Collectively, this study demonstrates that elevated galectin-7 during placental formation contributes to abnormal placentation and suggests that it leads to the development of preeclampsia via altering placental production of sFlt-1 and renin-angiotensin system components. Targeting galectin-7 may be a new treatment option for preeclampsia.

Chloride intracellular channel 4 is dysregulated in endometrium of women with infertility and alters receptivity.

The endometrium remodels in each menstrual cycle to become receptive in preparation for embryo implantation which occurs in the mid-secretory phase of the cycle. Failure of blastocyst adhesion and implantation cause infertility. We compared chloride intracellular channel 4 (CLIC4) expression in human endometrium from women with normal fertility and primary unexplained infertility in the mid-secretory/receptive phase of the menstrual cycle. CLIC4 localised to both the epithelial and stromal regions of the endometrium of fertile tissues across the cycle. CLIC4 expression was significantly reduced in the luminal and glandular epithelium and remained unchanged in the stromal region of mid-secretory infertile endometrium compared to fertile endometrium. siRNA knockdown of CLIC4 significantly compromised adhesive capacity of Ishikawa cells (endometrial epithelial cell line). This reduced adhesion and CLIC4 expression was associated with elevated SGK1, p53, SIRT1, BCL2 and MCL1 gene expression in the Ishikawa cells. CLIC4 expression was increased in primary human endometrial stromal cells during decidualization, however, siRNA knockdown of CLIC4 did not affect decidualization. Our data provide evidence that CLIC4 may regulate receptivity and facilitate blastocyst attachment initiating implantation. Reduced CLIC4 levels may be causative of implantation failure in women.

Macrophage migration inhibitory factor is essential for osteoclastogenic mechanisms in vitro and in vivo mouse model of arthritis.

Macrophage migration inhibitory factor (MIF) enhances activation of leukocytes, endothelial cells and fibroblast-like synoviocytes (FLS), thereby contributing to the pathogenesis of rheumatoid arthritis (RA). A MIF promoter polymorphism in RA patients resulted in higher serum MIF concentration and worsens bone erosion; controversially current literature reported an inhibitory role of MIF in osteoclast formation. The controversial suggested that the precise role of MIF and its putative receptor CD74 in osteoclastogenesis and RA bone erosion, mediated by locally formed osteoclasts in response to receptor activator of NF-κB ligand (RANKL), is unclear. We reported that in an in vivo K/BxN serum transfer arthritis, reduced clinical and histological arthritis in MIF(-/-) and CD74(-/-) mice were accompanied by a virtual absence of osteoclasts at the synovium-bone interface and reduced osteoclast-related gene expression. Furthermore, in vitro osteoclast formation and osteoclast-related gene expression were significantly reduced in MIF(-/-) cells via decreasing RANKL-induced phosphorylation of NF-κB-p65 and ERK1/2. This was supported by a similar reduction of osteoclastogenesis observed in CD74(-/-) cells. Furthermore, a MIF blockade reduced RANKL-induced osteoclastogenesis via deregulating RANKL-mediated NF-κB and NFATc1 transcription factor activation. These data indicate that MIF and CD74 facilitate RANKL-induced osteoclastogenesis, and suggest that MIF contributes directly to bone erosion, as well as inflammation, in RA.

Critical role for macrophage migration inhibitory factor (MIF) in Ross River virus-induced arthritis and myositis.

Arthrogenic alphaviruses, such as Ross River virus (RRV), chikungunya, Sindbis, mayaro and o'nyong-nyong viruses circulate endemically worldwide, frequently causing outbreaks of polyarthritis. The exact mechanisms of how alphaviruses induce polyarthritis remain ill defined, although macrophages are known to play a key role. Macrophage migration inhibitory factor (MIF) is an important cytokine involved in rheumatoid arthritis pathogenesis. Here, we characterize the role of MIF in alphavirus-induced arthritides using a mouse model of RRV-induced arthritis, which has many characteristics of RRV disease in humans. RRV-infected WT mice developed severe disease associated with up-regulated MIF expression in serum and tissues, which corresponded to severe inflammation and tissue damage. MIF-deficient (MIF(-/-)) mice developed mild disease accompanied by a reduction in inflammatory infiltrates and muscle destruction in the tissues, despite having viral titers similar to WT mice. In addition, reconstitution of MIF into MIF(-/-) mice exacerbated RRV disease and treatment of mice with MIF antagonist ameliorated disease in WT mice. Collectively, these findings suggest that MIF plays a critical role in determining the clinical severity of alphavirus-induced musculoskeletal disease and may provide a target for the development of antiviral pharmaceuticals. The prospect being that early treatment with MIF-blocking pharmaceuticals may curtail the debilitating arthritis associated with alphaviral infections.

Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) facilitates multiple aspects of inflammatory arthritis, the pathogenesis of which has been significantly linked to the activity of neutrophils. The effects of MIF on neutrophil recruitment are unknown. This study was undertaken to investigate the contribution of MIF to the regulation of neutrophil chemotactic responses. METHODS: K/BxN serum-transfer arthritis was induced in wild-type (WT), MIF(-/-) , and monocyte chemotactic protein 1 (MCP-1; CCL2)-deficient mice as well as in WT mice treated with monoclonal antibodies to cytokine-induced neutrophil chemoattractant (anti-KC). Leukocyte trafficking in vivo was examined using intravital microscopy, and neutrophil function in vitro was examined using migration chambers and assessment of MAP kinase activation. RESULTS: K/BxN serum-transfer arthritis was markedly attenuated in MIF(-/-) mice, with reductions in the clinical and histologic severity of arthritis and the synovial expression of KC and interleukin-1. Arthritis was also reduced by anti-KC antibody treatment, but not in MCP-1-deficient mice. In vivo, neutrophil recruitment responses to KC were reduced in MIF(-/-) mice. Similarly, MIF(-/-) mouse neutrophils exhibited reduced chemotactic responses to KC in vitro, despite displaying unaltered chemokine receptor expression. Reduced chemotactic responses of MIF(-/-) mouse neutrophils were associated with reduced phosphorylation of p38 and ERK MAP kinases. CONCLUSION: These findings suggest that MIF promotes neutrophil trafficking in inflammatory arthritis via facilitation of chemokine-induced migratory responses and MAP kinase activation. Therapeutic MIF inhibition could limit synovial neutrophil recruitment.

Macrophage migration inhibitory factor and CD74 regulate macrophage chemotactic responses via MAPK and Rho GTPase.

Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment to sites of inflammation. However, whether this stems from a direct effect on leukocyte migration is unknown. Furthermore, the role of the MIF-binding protein CD74 in this response has not been investigated. Therefore, the aim of this study was to examine the contributions of MIF and CD74 to chemokine-induced macrophage recruitment. Intravital microscopy studies demonstrated that CCL2-induced leukocyte adhesion and transmigration were reduced in MIF(-/-) and CD74(-/-) mice. MIF(-/-) and CD74(-/-) macrophages also exhibited reduced chemotaxis in vitro, although CD74(-/-) macrophages showed increased chemokinesis. Reduced CCL2-induced migration was associated with attenuated MAPK phosphorylation, RhoA GTPase activity, and actin polymerization in MIF(-/-) and CD74(-/-) macrophages. Furthermore, in MIF(-/-) macrophages, MAPK phosphatase-1 was expressed at elevated levels, providing a potential mechanism for the reduction in MAPK phosphorylation in MIF-deficient cells. No increase in MAPK phosphatase-1 expression was observed in CD74(-/-) macrophages. In in vivo experiments assessing the link between MIF and CD74, combined administration of MIF and CCL2 increased leukocyte adhesion in both MIF(-/-) and CD74(-/-) mice, showing that CD74 was not required for this MIF-induced response. Additionally, although leukocyte recruitment induced by administration of MIF alone was reduced in CD74(-/-) mice, consistent with a role for CD74 in leukocyte recruitment induced by MIF, MIF-treated CD74(-/-) mice displayed residual leukocyte recruitment. These data demonstrate that MIF and CD74 play previously unappreciated roles in CCL2-induced macrophage adhesion and migration, and they indicate that MIF and CD74 mediate this effect via both common and independent mechanisms.

Identification of NURR1 as a mediator of MIF signaling during chronic arthritis: effects on glucocorticoid-induced MKP1.

Elucidation of factors regulating glucocorticoid (GC) sensitivity is required for the development of "steroid-sparing" therapies for chronic inflammatory diseases, including rheumatoid arthritis (RA). Accumulating evidence suggests that macrophage migration inhibitory factor (MIF) counterregulates the GC-induction of anti-inflammatory mediators, including mitogen-activated protein kinase phosphatase 1 (MKP1), a critical mitogen-activated protein kinase signaling inhibitor. This observation has yet to be extended to human disease; the molecular mechanisms remain unknown. We investigated NURR1, a GC-responsive transcription factor overexpressed in RA, as a MIF signaling target. We reveal abrogation by recombinant MIF (rMIF) of GC-induced MKP1 expression in RA fibroblast-like synoviocytes (FLS). rMIF enhanced NURR1 expression, artificial NBRE (orphan receptor DNA-binding site) reporter transactivation, and reversed GC-inhibition of NURR1. NURR1 expression was reduced during experimental arthritis in MIF-/- synovium, and silencing MIF reduced RA FLS NURR1 mRNA. Consistent with NBRE identification on the MKP1 gene, MKP1 mRNA was reduced in FLS that ectopically express NURR1, and silencing NURR1 enhanced MKP1 mRNA in RA FLS. rMIF enhanced NBRE binding on the MKP1 gene, and the absence of the NBRE prevented NURR1-repressive effects on basal and GC-induced MKP1 transactivation. This study defines NURR1 as a novel MIF target in chronic inflammation and demonstrates a role for NURR1 in regulating the anti-inflammatory mediator, MKP1. We propose a MIF-NURR1 signaling axis as a regulator of the GC sensitivity of MKP1.

Macrophage migration inhibitory factor: a key cytokine in RA, SLE and atherosclerosis.

Originally discovered and named as an in vitro inhibitor of macrophage migration, the cytokine macrophage migration inhibitory factor (MIF) has now been shown to be a key regulator of acute and chronic immuno-inflammatory conditions including rheumatoid arthritis (RA), atherosclerosis, and more recently systemic lupus erythematosus (SLE). Common inflammatory events in these diseases include activation of cells and infiltration by immune cells at the site of injury. MIF actively participates in multiple stages of the inflammatory response, acting on cells directly and/or potentiating the effects entrained by other stimuli. The overlap of inflammatory processes operating in these diseases, the known activities of MIF, and the observation of atherosclerosis as a major comorbidity of RA and SLE, make MIF a strong candidate for therapeutic targeting in these diseases. Moreover, the unique relationship between MIF and glucocorticoids, commonly used in the treatment of RA and SLE but associated with significant side effects, highlights the potential of MIF as a 'steroid sparing' therapeutic target encompassing all three conditions.

Macrophage migration inhibitory factor deficiency attenuates macrophage recruitment, glomerulonephritis, and lethality in MRL/lpr mice.

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disease of unknown etiology. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is operative in innate and adaptive immunity and important in immune-mediated diseases such as rheumatoid arthritis and atherosclerosis. The functional relevance of MIF in systemic autoimmune diseases such as SLE is unknown. Using the lupus-prone MRL/lpr mice, we aim to examine the expression and function of MIF in this murine model of systemic autoimmune disease. These experiments revealed that renal MIF expression was significantly higher in MRL/lpr mice compared with nondiseased control mice (MRL/MpJ), and MIF was also markedly up-regulated in skin lesions of MRL/lpr mice. To examine the effect of MIF on development of systemic autoimmune disease, we generated MRL/lpr mice with a targeted disruption of the MIF gene (MIF(-/-)MRL/lpr), and compared their disease manifestations to MIF(+/+)MRL/lpr littermates. MIF(-/-)MRL/lpr mice exhibited significantly prolonged survival, and reduced renal and skin manifestations of SLE. These effects occurred in the absence of major changes in T and B cell markers or alterations in autoantibody production. In contrast, renal macrophage recruitment and glomerular injury were significantly reduced in MIF(-/-)MRL/lpr mice, and this was associated with reduction in the monocyte chemokine MCP-1. Taken together, these data suggest MIF as a critical effector of organ injury in SLE.

Regulation of IL-1 and TNF receptor expression and function by endogenous macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) has a key role in regulation of innate and adaptive immunity and is implicated in sepsis, tumorigenesis, and autoimmune disease. MIF deficiency or immunoneutralization leads to protection against fatal endotoxic, exotoxic, and infective shock, and anti-inflammatory effects in other experimental models of inflammatory disease. We report a novel regulatory role of MIF in type 1 IL-1R and p55 TNFR expression and function. Compared with wild-type cells, MIF-deficient cells were hyporesponsive to IL-1- and TNF-induced MAPK activity, AP-1 activity, and cellular proliferation, while NF-kappaB function was preserved. Hyporesponsiveness of MIF-deficient cells was associated with down-regulation of cytokine receptor expression, which was restored by reconstitution of either an upstream kinase of MAPK, MAPK/ERK kinase, or MIF. These data suggest that endogenous MIF is required for cytokine activation of MAPK/AP-1 and cytokine receptor expression. This autocrine regulatory pathway defines an important amplifying role of endogenous MIF in cytokine-mediated immune and inflammatory diseases and provides further molecular evidence for the critical role of MIF in cellular activation.

IL-18 is redundant in T-cell responses and in joint inflammation in antigen-induced arthritis.

IL-18 is an important cofactor in Th1 immune responses and it has additional roles in inflammation. Recent reports suggest the contribution of IL-18 to immune responses may vary between mouse strains and immune contexts. We investigated the contribution of IL-18 to T-cell activation and joint inflammation in Ag-induced arthritis (AIA) in C57Bl/6 mice. AIA and cutaneous delayed-type hypersensitivity (DTH) reactions were induced in wild-type (WT) and IL-18-/- C57Bl/6 mice, and Ag-specific T-cell proliferation and IFN-gamma and IL-4 production were measured. The humoral immune response was measured as serum antibody to the disease-initiating Ag, methylated BSA (mBSA). Splenocyte production of IL-6 was measured by ELISA. To confirm the dependence of this model on Th1-cell-mediated immunity, IL-12p40-/- mice were similarly studied. WT mice developed synovitis, joint effusion, cartilage destruction and bone damage associated with induction of DTH, and in vitro Ag-specific T-cell proliferation and IFN-gamma production. Unexpectedly, IL-18-/- mice developed AIA and indices of T-cell activation were similar to those of WT mice. In contrast, IL-12p40-/- mice did not develop AIA, DTH or T-cell activation. WT and IL-18-/- mice, but not IL-12p40-/- mice, developed significantly increased serum antibody to mBSA compared with naive controls. WT and IL-18-/- splenocytes produced high levels of IL-6, whereas IL-12p40-/- cells had significantly lower IL-6 production compared with both. In conclusion, IL-18 is redundant both as a Th1 response cofactor and inflammatory cytokine, whereas IL-12p40-/- is a key cytokine, in AIA in C57Bl/6 mice.

The role of macrophage migration inhibitory factor in the inflammatory immune response and rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating disease of unknown etiology. Although the pathogenesis of RA is multifactorial, the contribution of cytokines is undoubtedly pivotal in the progression of the inflammatory process. One cytokine gaining recognition for its importance in inflammation is macrophage migration inhibitory factor (MIF). Initially described as a biological activity, a broad range of functions of MIF has emerged including induction of proinflammatory mediators as well as demonstrated roles in both innate and adaptive immunity. In RA, increased MIF levels have been demonstrated in serum, synovial fluid and tissue with the latter correlating with disease activity. In vitro, MIF induces production of key proinflammatory genes operative in arthritis, including IL-1, TNF, IL-6, IL-8, COX-2, PLA2, and MMPs. In addition, MIF regulates proliferation and apoptosis via direct effects on the tumor suppressor protein p53 implicating a role for MIF in synovial hyperplasia. In vivo, MIF antagonism or MIF deficiency result in decreased disease severity in animal models of RA further confirming a role for MIF in joint inflammation. Interestingly, MIF is induced by glucocorticoids and MIF in turn antagonises glucocorticoid effects. This unique relationship presents antagonism of MIF as a potentially effective steroid-sparing therapy.

Activation of synovial cell p38 MAP kinase by macrophage migration inhibitory factor.

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine important in animal models of rheumatoid arthritis (RA). We investigated the utilization by MIF of mitogen activated protein (MAP) kinase signalling pathways in the stimulation of fibroblast-like synoviocytes (FLS), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), and interleukin 6 (IL-6) and IL-8 expression. METHODS: Cultured human RA FLS were treated with recombinant MIF. Activation of MAPK was measured by Western blotting and blocked using specific inhibitors. The expression of COX-2, PGE(2), IL-6, and IL-8 were measured using flow cytometry, ELISA, and real-time polymerase chain reaction. RESULTS: MIF induced the phosphorylation of FLS p38 and extracellular-signal regulated kinase (ERK) MAP kinase. MIF significantly induced COX-2 and IL-6 protein and mRNA expression as well as PGE(2) and IL-8 production. Antagonism of p38 MAP kinase inhibited MIF induction of COX-2, PGE(2), and IL-6. In contrast, antagonism of ERK had no effect on COX-2, PGE(2), or IL-6. Neither antagonist inhibited MIF-induced IL-8. CONCLUSION: MIF activates RA FLS COX-2 and IL-6 expression via p38 MAP kinase activation and induces IL-8 via p38 and ERK MAP kinase-independent pathways.