Serum-induced Arthritis Research Articles

234: Selective regulation of CXCL1 expression and neutrophil responses by transcription repressor Hes1

Recruitment of neutrophils to sites of inflammation is essential for eliciting competent immune responses. However, uncontrolled neutrophil responses contribute to inappropriate immune activation under a number of autoimmune and inflammatory conditions. Therefore, neutrophil responses must be tightly regulated to ensure strong activation while avoiding excessive inflammation. Here we show that transcription repressor hairy and enhancer of split 1 (Hes1) suppresses toll-like receptor 4 (TLR4)-induced expression of CXCL1, a chemokine crucial for recruitment of neutrophils to sites of inflammation. Interestingly, Hes1 deficiency specifically promotes Cxcl1 expression without affecting expression of other prototypical pro-inflammatory cytokine genes such as Tnf and Ilb . Hes1 negatively regulates Cxcl1 expression and neutrophil recruitment in vivo in a model of LPS-induced peritonitis. In addition, deficiency of Hes1 exacerbated severity of K/BxN serum-induced arthritis, a model of inflammatory arthritis in which neutrophil plays a key pathogenic role. Mechanistically, regulation of Cxcl1 expression by Hes1 is at the transcriptional level but not via modification of TLR4-induced canonical signaling events such as activation of NF-kB. Instead, Hes1 represses Cxcl1 expression by inhibiting occupancy of serine 2-phosphorylated RNA polymerase II, a hallmark of efficient transcription elongation, at the Cxcl1 gene locus. In summary, these results identify Hes1 as a homeostatic negative regulator of CXCL1 expression and neutrophil responses and suggest that targeting Hes1-mediated repressive pathway may represent a novel therapeutic approach that selectively curbs neutrophil-mediated inflammation while preserving other aspects of immunity.

Evidence That CXCL16 Is a Potent Mediator of Angiogenesis and Is Involved in Endothelial Progenitor Cell Chemotaxis: Studies in Mice With K/BxN Serum–Induced Arthritis

To examine the possibility that CXCL16 recruits endothelial cells (ECs) to developing neovasculature in rheumatoid arthritis (RA) synovium. We utilized the RA synovial tissue SCID mouse chimera system to examine human microvascular EC (HMVEC) and human endothelial progenitor cell (EPC) recruitment into engrafted human synovium that was injected intragraft with CXCL16-immunodepleted RA synovial fluid (SF). CXCR6-deficient and wild-type (WT) C57BL/6 mice were primed to develop K/BxN serum-induced arthritis and evaluated for angiogenesis. HMVECs and EPCs from human cord blood were also examined for CXCR6 expression, by immunofluorescence and assessment of CXCL16 signaling activity. CXCR6 was prominently expressed on human EPCs and HMVECs, and its expression on HMVECs could be up-regulated by interleukin-1β. SCID mice injected with CXCL16-depleted RA SF exhibited a significant reduction in EPC recruitment. In experiments using the K/BxN serum-induced inflammatory arthritis model, CXCR6(-/-) mice showed profound reductions in hemoglobin levels, which correlated with reductions in monocyte and T cell recruitment to arthritic joint tissue compared to that observed in WT mice. Additionally, HMVECs and EPCs responded to CXCL16 stimulation, but exhibited unique signal transduction pathways and homing properties. These results indicate that CXCL16 and its receptor CXCR6 may be a central ligand/receptor pair that is closely associated with EPC recruitment and blood vessel formation in the RA joint.

CD44 Antibodies and Immune Thrombocytopenia in the Amelioration of Murine Inflammatory Arthritis

Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP) that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7), also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

AB0085 Murine antigen-induced arthritis is not affected by transgenic disruption of osteoblastic glucocorticoid signaling

BackgroundThe role of endogenous glucocorticoids (GC) in the initiation and maintenance of rheumatoid arthritis (RA) remains unclear. We demonstrated previously that disruption of GC signaling in osteoblasts results in a...

Neutrophils Are Essential As A Source Of Il-17 In The Effector Phase Of Arthritis

ObjectiveTh17 has been shown to have a pivotal role in the development of arthritis. However, the role of IL-17 in the T cell-independent effector phase has not fully been examined. We investigated whether IL-17 is involved in the effector phase of arthritis by using K/BxN serum-induced arthritis model.MethodsK/BxN serum was transferred into IL-17 knockout (KO) mice, SCID mice and their control mice, and arthritis was evaluated over time. In order to clarify the source of IL-17 in the effector phase, neutrophils or CD4+ T cells collected from IL-17 KO or control mice were injected into IL-17 KO recipient mice together with K/BxN serum. To examine if neutrophils secrete IL-17 upon stimulation, neutrophils were stimulated with immune complex in vitro and IL-17 in the supernatant was measured by ELISA.ResultsK/BxN serum-induced arthritis was much less severe in IL-17 KO mice than in WT mice. Since K/BxN serum-transferred SCID mice developed severe arthritis with high serum IL-17 concentration, we speculated neutrophils are the responsible player as an IL-17 source. When wild type (WT) but not IL-17 KO neutrophils were co-injected with K/BxN serum into IL-17 KO mice, arthritis was exacerbated, whereas co-injection of WT CD4+ T cells had no effect. In vitro, stimulation of neutrophils with immune complexcaused IL-17 secretion.ConclusionsNeutrophils are essential as a source of IL-17 in the effector phase of arthritis. The trigger of secreting IL-17 from neutrophils may be immune complex.

Regulatory T cell dysfunction in lipocalin 2 knockout mice exacerbates serum-induced arthritis (P1326)

Abstract Lipocalin 2 (Lcn2), a 25KDa innate immune protein, known to be dramatically up regulated in various inflammatory disorders including antibody-mediated arthritis, yet its biological role remains unclear. More recently our studies have suggested that the regulation might be necessary for the initiation and further resolution of inflammatory process. However, the exact mechanism involved in this process is still unknown. In this study, we report that Lcn2 is up regulated up to 5 fold during the course of serum-induced arthritis (SIA) in wild type (WT) mice. Likewise, Lcn2KO mice suffered a severe arthritic disease with elevated systemic proinflammatory cytokines as compared to their WT littermates. Immunological analysis revealed a considerable increase in granulocytic population (Gr1+, CD11b+, CD11c+), and a significant decrease (4.2 fold) in regulatory T cell (CD4+, CD25+ and FoxP3+) population in Lcn2KO arthritic mice as compared to their WT littermates. Accordingly, Lcn2KO mice exhibited a defect in T cell proliferation as compared to their WT counterparts, suggesting a novel role for Lcn2 in splenocyte proliferation and T-reg expansion. Collectively, our data suggests a crucial role of Lcn2 in resolution of arthritic inflammation via T-reg expansion, making it a promising target in designing better therapeutic strategies for the treatment of rheumatoid arthritis.

Differential Role of Lipocalin 2 During Immune Complex–Mediated Acute and Chronic Inflammation in Mice

Lipocalin 2 (LCN-2) is an innate immune protein that is expressed by a variety of cells and is highly up-regulated during several pathologic conditions, including immune complex (IC)-mediated inflammatory/autoimmune disorders. However, the function of LCN-2 during IC-mediated inflammation is largely unknown. Therefore, this study was undertaken to investigate the role of LCN-2 in IC-mediated diseases. The up-regulation of LCN-2 was determined by enzyme-linked immunosorbent assay in 3 different mouse models of IC-mediated autoimmune disease: systemic lupus erythematosus, collagen-induced arthritis, and serum-transfer arthritis. The in vivo role of LCN-2 during IC-mediated inflammation was investigated using LCN-2-knockout mice and their wild-type littermates. LCN-2 levels were significantly elevated in all 3 of the autoimmune disease models. Further, in an acute skin inflammation model, LCN-2-knockout mice exhibited a 50% reduction in inflammation, with histopathologic analysis revealing notably reduced immune cell infiltration as compared to wild-type mice. Administration of recombinant LCN-2 to LCN-2-knockout mice restored inflammation to levels observed in wild-type mice. Neutralization of LCN-2 using a monoclonal antibody significantly reduced inflammation in wild-type mice. In contrast, LCN-2-knockout mice developed more severe serum-induced arthritis compared to wild-type mice. Histologic analysis revealed extensive tissue and bone destruction, with significantly reduced neutrophil infiltration but considerably more macrophage migration, in LCN-2-knockout mice compared to wild-type mice. These results demonstrate that LCN-2 may regulate immune cell recruitment to the site of inflammation, a process essential for the controlled initiation, perpetuation, and resolution of inflammatory processes. Thus, LCN-2 may present a promising target in the treatment of IC-mediated inflammatory/autoimmune diseases.

Macrophage-derived, macrophage migration inhibitory factor (MIF) is necessary to induce disease in the K/BxN serum-induced model of arthritis

Rheumatoid arthritis (RA) is characterized by the interaction of multiple mediators, among the most important of which are cytokines. In recent years, extensive studies demonstrate a pivotal role for one cytokine, macrophage migration inhibitory factor (MIF), in fundamental events in innate and adaptive immunity. MIF has now been demonstrated to be involved in the pathogenesis of many diseases, but in the case of RA the evidence for a role of MIF is very strong. MIF is abundantly expressed in the sera of RA patients and in RA synovial tissue correlating with disease activity. MIF-deficient mice were used to induce arthritis by serum transfer from K/BxN mice. K/BxN serum transfer arthritis was markedly attenuated in MIF(-) mice, with reduction in clinical index and histological severity as well as decrease in synovial cytokines. Macrophage transfers were done to investigate the specific role of macrophage-derived MIF. We show that adoptive transfer of wild-type macrophages into MIF(-) mice restores the sensitivity of MIF(-) mice to arthritis development, and this affect was associated with a restoration in serum IL-1β and IL-6 production. These results indicate that MIF plays a critical role in inflammation and joint destruction in K/BxN serum-induced arthritis and that the systemic expression of MIF by a subpopulation of macrophages is necessary and sufficient for the full development of arthritis.

Collagenase-3 (MMP-13) deficiency protects C57BL/6 mice from antibody-induced arthritis

IntroductionMatrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.MethodsFor this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.ResultsThis study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.ConclusionsMMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.

Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes

IntroductionThis study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling.MethodsThe expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation.ResultsWe found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS.ConclusionsThese data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3.

Glycoprotein 96 perpetuates the persistent inflammation of rheumatoid arthritis

The mechanisms that contribute to the persistent activation of macrophages in rheumatoid arthritis (RA) are incompletely understood. The aim of this study was to determine the contribution of endogenous gp96 in Toll-like receptor (TLR)-mediated macrophage activation in RA. RA synovial fluid was used to activate macrophages and HEK-TLR-2 and HEK-TLR-4 cells. Neutralizing antibodies to TLR-2, TLR-4, and gp96 were used to inhibit activation. RA synovial fluid macrophages were isolated by CD14 negative selection. Cell activation was measured by the expression of tumor necrosis factor α (TNFα) or interleukin-8 messenger RNA. Arthritis was induced in mice by K/BxN serum transfer. The expression of gp96 was determined by immunoblot analysis, enzyme-linked immunosorbent assay, and immunohistochemistry. Arthritis was treated with neutralizing anti-gp96 antiserum or control serum. RA synovial fluid induced the activation of macrophages and HEK-TLR-2 and HEK-TLR-4 cells. RA synovial fluid-induced macrophage and HEK-TLR-2 activation was suppressed by neutralizing anti-gp96 antibodies only in the presence of high (>800 ng/ml) rather than low (<400 ng/ml) concentrations of gp96. Neutralization of RA synovial fluid macrophage cell surface gp96 inhibited the constitutive expression of TNFα. Supporting the role of gp96 in RA, joint tissue gp96 expression was induced in mice with the K/BxN serum-induced arthritis, and neutralizing antibodies to gp96 ameliorated joint inflammation, as determined by clinical and histologic examination. These observations support the notion that gp96 plays a role as an endogenous TLR-2 ligand in RA and identify the TLR-2 pathway as a therapeutic target.

In vivo imaging implicates CCR2+ monocytes as regulators of neutrophil recruitment during arthritis

The infiltration of neutrophils and monocytes is a prominent feature of inflammatory diseases including human rheumatoid arthritis. Understanding how neutrophil recruitment is regulated during pathogenesis is crucial for developing anti-inflammatory therapies. We optimized the K/B×N serum-induced mouse arthritis model to study neutrophil trafficking dynamics in vivo using two-photon microscopy. Arthritogenic serum was injected subcutaneously into one hind footpad to induce a local arthritis with robust neutrophil recruitment. Using this approach, we showed that the depletion of monocytes with clodronate liposomes impaired neutrophil recruitment specifically at the transendothelial migration step. The depletion of CCR2+ monocytes with the monoclonal antibody MC-21 reproduced these effects, implicating CCR2+ monocytes as key regulators of neutrophil extravasation during arthritis initiation. However, monocyte depletion did not prevent neutrophil extravasation in response to bacterial challenge. These findings suggest that anti-inflammatory therapies targeting monocytes may act in part through antagonizing neutrophil extravasation at sites of aseptic inflammation.

Lipocalin 2, an innate immune protein, plays a key role in immune‐complex (IC) mediated inflammation

Lipocalin‐2 (Lcn2), an innate immune protein predominantly secreted by neutrophils, is upregulated by several logs during inflammatory conditions, including autoimmune diseases. However, the defined role of Lcn2 in IC‐mediated inflammation is largely unknown. We investigated the role of Lcn2 in an acute model of IC‐mediated inflammation (Reverse‐Passive Arthus Reaction) and a chronic model of Serum Induced Arthritis (SIA) using Lcn2 knock out (Lcn2KO) and their wild type (WT) littermates. In the acute model of IC‐mediated inflammation, Lcn2KO mice showed approximately 50% reduced inflammation when compared to WT mice. Histopathologic analysis of skin biopsies showed substantially reduced immune cell infiltration at the site of inflammation in Lcn2KO mice. Similarly, neutralization of Lcn2 in WT mice significantly rescued from IC‐mediated inflammation and, further, supplementing rec‐Lcn2 to Lcn2KO mice resulted in inflammation similar to that seen in WT mice. In contrast, in the chronic SIA model, Lcn2KO mice suffered significantly as measured by arthritic disease severity and paw thickness. These results suggest that Lcn2‐dependent immune cell recruitment plays a role in the initiation, perpetuation and, finally, resolution of inflammation. Collectively, our studies demonstrate that targeting Lcn2 may be a promising approach for treating autoimmune diseases.

Decreased collagen‐induced arthritis severity and adaptive immunity in MKK‐6–deficient mice

The MAPK kinases MKK-3 and MKK-6 regulate p38 MAPK activation in inflammatory diseases such as rheumatoid arthritis (RA). Previous studies demonstrated that MKK-3 or MKK-6 deficiency inhibits K/BxN serum-induced arthritis. However, the role of these kinases in adaptive immunity-dependent models of chronic arthritis is not known. The goal of this study was to evaluate MKK-3 and MKK-6 deficiency in the collagen-induced arthritis (CIA) model. Wild-type (WT), MKK-3(-/-) , and MKK-6(-/-) mice were immunized with bovine type II collagen. Disease activity was evaluated by semiquantitative scoring, histologic assessment, and micro-computed tomography. Serum anticollagen antibody levels were quantified by enzyme-linked immunosorbent assay. In vitro T cell cytokine response was measured by flow cytometry and multiplex analysis. Expression of joint cytokines and matrix metalloproteinases (MMPs) was determined by quantitative polymerase chain reaction. MKK-6 deficiency markedly reduced arthritis severity compared with that in WT mice, while the absence of MKK-3 had an intermediate effect. Joint damage was minimal in arthritic MKK-6(-/-) mice and intermediate in MKK-3(-/-) mice compared with WT mice. MKK-6(-/-) mice had modestly lower levels of pathogenic anticollagen antibodies than did WT or MKK-3(-/-) mice. In vitro T cell assays showed reduced proliferation and interleukin-17 (IL-17) production by lymph node cells from MKK-6(-/-) mice in response to type II collagen. Gene expression of synovial IL-6, MMP-3, and MMP-13 was significantly inhibited in MKK-6-deficient mice. Reduced disease severity in MKK-6(-/-) mice correlated with decreased anticollagen antibody responses, indicating that MKK-6 is a crucial regulator of inflammatory joint destruction in CIA. MKK-6 is a potential therapeutic target in complex diseases involving adaptive immune responses, such as RA.

Suppression of collagen-induced arthritis in growth arrest and DNA damage-inducible protein 45β-deficient mice

Growth arrest and DNA damage-inducible protein 45β (GADD45β) is involved in stress responses, cell cycle regulation, and oncogenesis. Previous studies demonstrated that GADD45β deficiency exacerbates K/BxN serum-induced arthritis and experimental allergic encephalomyelitis (EAE) in mice, indicating that GADD45β plays a suppressive role in innate and adaptive immune responses. To further understand how GADD45β regulates autoimmunity, we evaluated collagen-induced arthritis in GADD45β-/- mice. Wild-type (WT) and GADD45β-/- DBA/1 mice were immunized with bovine type II collagen (CII). Serum anticollagen antibody levels were quantified by enzyme-linked immunosorbent assay. Expression of cytokines and matrix metalloproteinases in the joint and spleen was determined by quantitative polymerase chain reaction. The in vitro T cell cytokine response to CII was measured by multiplex analysis. CD4+CD25+ Treg cells and Th17 cells were quantified using flow cytometry. GADD45β-/- mice showed significantly lower arthritis severity and joint destruction compared with WT mice. MMP-3 and MMP-13 expression was also markedly reduced in GADD45β-/- mice. However, serum anti-CII antibody levels were similar in both groups. FoxP3 and interleukin-10 (IL-10) expression was increased 2-3-fold in splenocytes from arthritic GADD45β-/- mice compared with those from WT mice. Flow cytometric analysis showed greater numbers of CD4+CD25+ Treg cells in the spleen of GADD45β-/- mice than in the spleen of WT mice. In vitro studies showed that interferon-γ and IL-17 production by T cells was significantly decreased in GADD45β-/- mice. Unlike passive K/BxN arthritis and EAE, GADD45β deficiency in CIA was associated with lower arthritis severity, elevated IL-10 expression, decreased IL-17 production, and increased numbers of Treg cells. The data suggest that GADD45β plays a complex role in regulating adaptive immunity and, depending on the model, either enhances or suppresses inflammation.

JNK1, but Not JNK2, Is Required in Two Mechanistically Distinct Models of Inflammatory Arthritis

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.

Intravenous gammaglobulin suppresses inflammation through a novel TH2 pathway

High-dose intravenous immunoglobulin is a widely used therapeutic preparation of highly purified immunoglobulin G (IgG) antibodies. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings. This anti-inflammatory activity of intravenous immunoglobulin is triggered by a minor population of IgG crystallizable fragments (Fcs), with glycans terminating in α2,6 sialic acids (sFc) that target myeloid regulatory cells expressing the lectin dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN; also known as CD209). Here, to characterize this response in detail, we generated humanized DC-SIGN mice (hDC-SIGN), and demonstrate that the anti-inflammatory activity of intravenous immunoglobulin can be recapitulated by the transfer of bone-marrow-derived sFc-treated hDC-SIGN(+) macrophages or dendritic cells into naive recipients. Furthermore, sFc administration results in the production of IL-33, which, in turn, induces expansion of IL-4-producing basophils that promote increased expression of the inhibitory Fc receptor FcγRIIB on effector macrophages. Systemic administration of the T(H)2 cytokines IL-33 or IL-4 upregulates FcγRIIB on macrophages, and suppresses serum-induced arthritis. Consistent with these results, transfer of IL-33-treated basophils suppressed induced arthritic inflammation. This novel DC-SIGN-T(H)2 pathway initiated by an endogenous ligand, sFc, provides an intrinsic mechanism for maintaining immune homeostasis that could be manipulated to provide therapeutic benefit in autoimmune diseases.

Deficiency of leukocyte NADPH oxidase increases joint inflammation in serum-induced arthritis model (44.27)

Abstract To elucidate the role of reactive oxygen species (ROS) in modulating tissue inflammation, we established an arthritis model by intraperitoneally injecting the K/BxN serum into wild type and ncf1-/- CGD mice, which have deficiency in p47phox of NOX2. By comparing the joint inflammation index ankle thickening, we found that the serum-induced arthritis were more severe in CGD mice than in wild type mice (30.2 mm ± 4.6 vs. 19.6 mm ± 0.19 on the day 10). These data suggested that lack of leukocyte-produced ROS interferes with the control of the neutrophil-dominant joint inflammation. By depleting neutrophils with anti-Ly6G antibodies, we found that serum-induced arthritis was abrogated in both ncf1-/- and wild type mice, which confirms previous reports that neutrophils play an important role in the induction and progression of serum-induced arthritis. However, the joint reaction in serum-induced diseased ncf1-/- mice, different from wild type mice, could not be reversed by depleting neutrophils. We further analyzed the proinflammatory cytokines, chemokines, and neutrophil-related proteases in ncf1-/- mice and wild type. We also analyzed the redox modulation status of redox response proteins to elucidate the possible mechanisms involved in ROS-mediated innate immunity in regulating the joint inflammation. These data suggest that ROS play an important role in modulating immune system and controlling the tissue inflammation in joints.

Cutting Edge: The Murine High-Affinity IgG Receptor FcγRIV Is Sufficient for Autoantibody-Induced Arthritis

K/BxN serum-induced passive arthritis was reported to depend on the activation of mast cells, triggered by the activating IgG receptor FcγRIIIA, when engaged by IgG1 autoantibodies present in K/BxN serum. This view is challenged by the fact that FcγRIIIA-deficient mice still develop K/BxN arthritis and because FcγRIIIA is the only activating IgG receptor expressed by mast cells. We investigated the contribution of IgG receptors, IgG subclasses, and cells in K/BxN arthritis. We found that the activating IgG2 receptor FcγRIV, expressed only by monocytes/macrophages and neutrophils, was sufficient to induce disease. K/BxN arthritis occurred not only in mast cell-deficient W(sh) mice, but also in mice whose mast cells express no activating IgG receptors. We propose that at least two autoantibody isotypes, IgG1 and IgG2, and two activating IgG receptors, FcγRIIIA and FcγRIV, contribute to K/BxN arthritis, which requires at least two cell types other than mast cells, monocytes/macrophages, and neutrophils.

Joint Tissues Amplify Inflammation and Alter Their Invasive Behavior via Leukotriene B4 in Experimental Inflammatory Arthritis

Mechanisms by which mesenchymal-derived tissue lineages participate in amplifying and perpetuating synovial inflammation in arthritis have been relatively underinvestigated and are therefore poorly understood. Elucidating these processes is likely to provide new insights into the pathogenesis of multiple diseases. Leukotriene B(4) (LTB(4)) is a potent proinflammatory lipid mediator that initiates and amplifies synovial inflammation in the K/BxN model of arthritis. We sought to elucidate mechanisms by which mesenchymal-derived fibroblast-like synoviocytes (FLSs) perpetuate synovial inflammation. We focused on the abilities of FLSs to contribute to LTB(4) synthesis and to respond to LTB(4) within the joint. Using a series of bone marrow chimeras generated from 5-lipoxygenase(-/-) and leukotriene A(4) (LTA(4)) hydrolase(-/-) mice, we demonstrate that FLSs generate sufficient levels of LTB(4) production through transcellular metabolism in K/BxN serum-induced arthritis to drive inflammatory arthritis. FLSs-which comprise the predominant lineage populating the synovial lining-are competent to metabolize exogenous LTA(4) into LTB(4) ex vivo. Stimulation of FLSs with TNF increased their capacity to generate LTB(4) 3-fold without inducing the expression of LTA(4) hydrolase protein. Moreover, LTB(4) (acting via LTB(4) receptor 1) was found to modulate the migratory and invasive activity of FLSs in vitro and also promote joint erosion by pannus tissue in vivo. Our results identify novel roles for FLSs and LTB(4) in joints, placing LTB(4) regulation of FLS biology at the center of a previously unrecognized amplification loop for synovial inflammation and tissue pathology.