G. Kenneth Haines

Fractalkine, a novel chemokine in rheumatoid arthritis and in rat adjuvant‐induced arthritis

OBJECTIVE To examine the expression of the novel CX3C chemokine fractalkine (Fkn) and its receptor (CX3CR1) in rheumatoid arthritis (RA) and rat adjuvant-induced arthritis (AIA), a model of RA. METHODS Immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), and chemotaxis assays were used. RESULTS In rat AIA, synovial tissue (ST) macrophages, fibroblasts, endothelial cells, and dendritic cells were Fkn immunopositive, whereas lymphocytes did not significantly express Fkn. Significant staining for CX3CR1 was found in ST macrophages, fibroblasts, and dendritic cells, whereas only a small percentage of endothelial cells stained for CX3CR1 in rat AIA. We immunolocalized Fkn to RA ST macrophages, fibroblasts, endothelial cells, and dendritic cells. We also found intense ST macrophage and dendritic cell staining for CX3CR1 in RA ST. Flow cytometry analysis of RA synovial fluid (SF) and peripheral blood revealed a greater percentage of monocytes expressing Fkn and CX3CR1 compared with T cells. By ELISA, we found significantly elevated soluble Fkn (sFkn) levels in RA SF compared with SF from patients with osteoarthritis or other forms of arthritis. By RT-PCR, we found enhanced expression of Fkn and CX3CR1 mRNA on day 18 in rat AIA, a time of pronounced inflammation in the rat joint. Soluble Fkn-depleted RA SF showed significantly decreased chemotactic activity for monocytes compared with sham-depleted RA SF. CONCLUSION These results indicate that Fkn and its receptor are both expressed in RA and in rat AIA, and that sFkn is up-regulated in RA SF. Furthermore, our data suggest a new role for Fkn in monocyte chemotaxis in the inflamed RA joint.

Combined Deficiency of Proapoptotic Regulators Bim and Fas Results in the Early Onset of Systemic Autoimmunity

Alterations in the stoichiometric balance between members of Bcl-2 and Fas apoptotic pathway could lead to the pathogenesis of systemic lupus erythematosus (SLE). We showed that patients with SLE displayed increased expression in antiapoptotic members of the Bcl-2 and Fas apoptotic pathways in isolated mononuclear cells. Further, mice (Bcl2l11(-/-)Fas(lpr/lpr)) lacking the Bcl-2 pro-apoptotic member, Bim (Bcl2l11(-/-)) and and with an lpr mutation in the gene encoding Fas (Fas(lpr/lpr)) developed severe SLE-like disease by 16 weeks of age unlike Bcl2l11(-/-) or Fas(lpr/lpr) mice. Bcl2l11(-/-)Fas(lpr/lpr) antigen-presenting cells (APCs) were markedly activated, and their numbers were increased in lymphoid tissues and in kidneys, yet numerous TUNEL-positive cells were observed in glomeruli of Bcl2l11(-/-)Fas(lpr/lpr) mice. These data demonstrate that dysregulation of the Bcl-2 or Fas pathways can alter the function of APCs, thereby leading to SLE pathogenesis.

Sensitization of IFN-γ Jak-STAT signaling during macrophage activation

A general paradigm in signal transduction is ligand-induced feedback inhibition and the desensitization of signaling. We found that subthreshold concentrations of interferon-γ (IFN-γ), which did not activate macrophages, increased their sensitivity to subsequent IFN-γ stimulation; this resulted in increased signal transducer and activator of transcription 1 (STAT1) activation and increased IFN-γ–dependent gene activation. Sensitization of IFN-γ signaling was mediated by the induction of STAT1 expression by low doses of IFN-γ that did not effectively induce feedback inhibition. IFN-γ signaling was sensitized in vivo after IFN-γ injection, and STAT1 expression was increased after injection of lipopolysaccharide and in rheumatoid arthritis synovial cells. These results identify a mechanism that sensitizes macrophages to low concentrations of IFN-γ and regulates IFN-γ responses in acute and chronic inflammation.

Temporal expression of inflammatory cytokines and chemokines in rat adjuvant-induced arthritis

OBJECTIVE To examine cytokine and chemokine production during the evolution of rat adjuvant-induced arthritis (AIA), a model of rheumatoid arthritis. METHODS Clinical and laboratory assessment of the course of AIA was performed over a 47-day period. Levels of the cytokines tumor necrosis factor a (TNFalpha), interleukin-1beta (IL-1beta), and IL-6, as well as levels of the chemokines macrophage inflammatory protein 1alpha (MIP-1alpha) and JE, the murine homolog of monocyte chemoattractant protein 1, were determined by enzyme-linked immunosorbent assay in the sera and joints of AIA and control rats. Synovia from AIA rats were (immuno)histochemically analyzed. Results of cytokine and chemokine measurements were correlated with clinical and laboratory markers of inflammation and histology. RESULTS Early (before day 14 post adjuvant injection) and later phases of AIA could be distinguished. Cytokine and chemokine production was increased in AIA versus control rats. The production of TNFalpha, IL-1beta, MIP-1alpha, and, as determined earlier, epithelial neutrophil-activating peptide 78-like protein was abundant prior to and during the course of AIA, while that of IL-6 and JE was elevated in the late phase of AIA. Cytokine and chemokine levels were correlated with the clinical symptoms of arthritis and blood neutrophil counts. Joint levels of IL-1beta showed correlation with synovial lining proliferation and neutrophil ingress into AIA synovium. CONCLUSION Cytokines and chemokines are involved in the clinical, laboratory, and histologic changes underlying AIA. The production of these mediators may be temporally and spatially regulated. These findings may be important for the optimal timing of cytokine and chemokine targeting.

IL-4 Adenoviral Gene Therapy Reduces Inflammation, Proinflammatory Cytokines, Vascularization, and Bony Destruction in Rat Adjuvant-Induced Arthritis

IL-4 is a cytokine with anti-inflammatory properties on activated macrophages. Rheumatoid arthritis, an autoimmune inflammatory disease, is characterized by a paucity of IL-4 and an abundance of synovial macrophage-derived mediators. Herein, the effect of a single injection of adenovirus-producing rat IL-4 (AxCAIL-4) or a control virus with no inserted gene was compared with the effect of PBS injection into rat ankles. Ankles were injected before arthritis onset or at maximal inflammation. Preventatively, AxCAIL-4 reduced adjuvant-induced arthritis (AIA)- and/or AIA/adenoviral-induced ankle inflammation, decreasing articular index scores, ankle circumferences, paw volumes, radiographic scores, mean levels of monocyte chemoattractant protein-1, the number of inflammatory cells, and the number of synovial blood vessels. Therapeutically, AxCAIL-4 also decreased ankle circumferences and paw volumes in comparison with a control virus with no inserted gene and PBS groups. After arthritis onset, mean levels of TNF-α, IL-1β, macrophage inflammatory protein-2, and RANTES were decreased in AxCAIL-4 rat ankle homogenates compared with PBS-treated homogenates. Thus, increased expression of IL-4 via gene therapy administered in a preventative and/or therapeutic manner reduced joint inflammation, synovial cellularity, levels of proinflammatory cytokines, vascularization, and bony destruction in rat AIA, suggesting that a similar treatment in humans may be beneficial.

The Contribution of Accessory Toxins of Vibrio cholerae O1 El Tor to the Proinflammatory Response in a Murine Pulmonary Cholera Model

The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model. Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera toxins.

Rheumatoid arthritis synovial macrophages express the Fas‐associated death domain–like interleukin‐1β–converting enzyme–inhibitory protein and are refractory to Fas‐mediated apoptosis

OBJECTIVE The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. METHODS The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. RESULTS CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. CONCLUSION These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.

Immunohistochemical localization of modified C-reactive protein antigen in normal vascular tissue.

BACKGROUND The prototypic acute phase reactant, C-reactive protein (CRP), is a serum soluble, cyclic pentameric protein, the concentration of which increases markedly within hours of any tissue-damaging, inflammatory event. However, upon dissociation of its pentameric quaternary structure, CRP subunits undergo a spontaneous and irreversible conformational change. The resulting molecule, termed modified CRP or mCRP, has reduced aqueous solubility and a propensity to aggregate into a matrix-like lattice structure. METHODS Using monoclonal antibodies, normal human tissues were immunohistochemically screened for the presence of CRP as well as mCRP antigens. RESULTS Significant levels of mCRP were detected in the walls of blood vessels associated with normal human tissues. These data indicate that mCRP is a naturally occurring form of CRP and that it is a tissue-based rather than serum-based molecule. SIGNIFICANCE This report describes the localization of a stable form of CRP, mCRP, in blood vessels associated with normal human tissues.

Superantigens and chronic rhinosinusitis: detection of staphylococcal exotoxins in nasal polyps.

Objective/Hypothesis: The role of infectious agents in the etiology of chronic rhinosinusitis with nasal polyposis (CRSwNP) remains unclear. Recent studies have provided indirect evidence of exposure to staphylococcal exotoxins in the blood and polyp tissue of patients with CRSwNP. These exotoxins have the capacity to act as superantigens, bypassing normal antigen processing and directly stimulating a massive inflammatory response. The objective of the study was to analyze mucus and polyp tissue samples from patients with CRSwNP for the presence of staphylococcal exotoxins.

Nonclassical Ly6C(-) Monocytes Drive the Development of Inflammatory Arthritis in Mice

Different subsets and/or polarized phenotypes of monocytes and macrophages may play distinct roles during the development and resolution of inflammation. Here, we demonstrate in a murine model of rheumatoid arthritis that nonclassical Ly6C(-) monocytes are required for the initiation and progression of sterile joint inflammation. Moreover, nonclassical Ly6C(-) monocytes differentiate into inflammatory macrophages (M1), which drive disease pathogenesis and display plasticity during the resolution phase. During the development of arthritis, these cells polarize toward an alternatively activated phenotype (M2), promoting the resolution of joint inflammation. The influx of Ly6C(-) monocytes and their subsequent classical and then alternative activation occurs without changes in synovial tissue-resident macrophages, which express markers of M2 polarization throughout the course of the arthritis and attenuate joint inflammation during the initiation phase. These data suggest that circulating Ly6C(-) monocytes recruited to the joint upon injury orchestrate the development and resolution of autoimmune joint inflammation.