Pulmonary Chlamydia muridarum challenge activates lung interstitial macrophages which correlate with IFN-γ production and infection control in mice.

Protective immunity to the pathogen Chlamydia is dependent on a robust IFN-γ response generated by innate and adaptive lymphocytes. Here we assess the role of the macrophage in orchestrating a protective response in vivo to the murine pathogen, Chlamydia muridarum. During acute pulmonary and peritoneal infection, resident macrophages in both sites are infected with C. muridarum and adopt an inflammatory phenotype. In the lung, this activation is restricted to interstitial macrophages, which harbor higher levels of C. muridarum 16sRNA than alveolar macrophages. We examined innate and adaptive lymphocyte activation in the peritoneal cavity with macrophage depletion and with adoptive transfer of infected macrophages. These experiments demonstrate macrophage activation correlates with a protective IFN-γ response and effective control of C. muridarum. These studies suggest that a quantitative or qualitative alteration in macrophages may play a key role in the development of Chlamydia-associated diseases.

Association of an ERAP1 ERAP2 haplotype with familial ankylosing spondylitis.

OBJECTIVES: To assess whether there is excess transmission of alleles from the ERAP1 ERAP2 locus in families with ankylosing spondylitis (AS). METHODS: 199 multiplex families with AS with four non-synonymous single nucleotide polymorphisms (SNPs), three in the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene (rs27044, rs10050860 and rs30187) and one in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene (rs2549782), were genotyped and family-based association analyses were performed. RESULTS: Family-based association testing (FBAT -e; empirical variance option) analysis showed that ERAP1 rs30187[T] was associated with AS (additive model: p=0.02; dominant model: p=0.007). Haplotype permutation tests (HBAT-p) showed that a haplotype in the ERAP1 and ERAP2 locus (rs27044[G] rs30187[T] rs2549782[T]) was significantly associated with AS (two-sided p value by permutation test 0.009 for additive and 0.008 for dominant model, respectively). CONCLUSION: This study shows that one ERAP1 SNP and a haplotype in the ERAP1 and ERAP2 locus are associated with familial AS.

Activation of invariant NKT cells confers protection against Chlamydia trachomatis-induced arthritis.

The role of invariant NKT (iNKT) cells in reactive arthritis is unknown. We explored the functional role of NKT cells in reactive arthritis using an established murine model of Chlamydia trachomatis-induced arthritis (CtIA). CtIA in wild-type and CD1d knockout (KO) mice was induced by intra-articular injection of C. trachomatis. The effect of alpha-galactosylceramide (alpha-GalCer) activation of iNKT cells was investigated by intra-peritoneal administration of alpha-GalCer. Histopathological and phenotypic changes, chlamydial clearance and cytokine and chemokine production in synovial tissue of the knee joint were investigated after onset of the arthritis. The severity of CtIA was significantly increased in CD1d KO mice, which was associated with decrease in bactericidal cytokine IFN-gamma, regulatory cytokines IL-4 and IL-10 and increase in pro-inflammatory chemokines macrophage inflammatory protein-2 (MIP-2) and IFN-gamma-inducible protein-10 (IP-10). Local clearance of the pathogen from the joint was also decreased. Prior treatment of mice with alpha-GalCer, a potent activator of iNKT cells, significantly reduced the severity of CtIA in mice. The amelioration of CtIA was associated with decrease in chlamydial load and induction of cytokines IFN-gamma, IL-4 and IL-10 and significant suppression of MIP-2 and IP-10. Treatment of established CtIA with alpha-GalCer also demonstrated modulation of CtIA and decrease in chlamydial load. These results suggest that iNKT cells are protective against CtIA and alpha-GalCer-activated iNKT cells have an immunoregulatory role not only in preventing the induction of reactive arthritis but also in modulating established disease.

Intracellular survival and persistence of Chlamydia muridarum is determined by macrophage polarization.

Macrophages can display a number of distinct phenotypes, known collectively as polarized macrophages. The best defined of these phenotypes are the classically-activated, interferon gamma (IFNγ)/LPS induced (M1) and alternatively-activated, IL-4 induced (M2) macrophages. The goal of this study is to characterize macrophage-Chlamydia interactions in the context of macrophage polarization. Here we use Chlamydia muridarum and murine bone-marrow derived macrophages to show Chlamydia does not induce M2 polarization in macrophages as a survival strategy. Unexpectedly, the infection of macrophages was silent with no upregulation of M1 macrophage-associated genes. We further demonstrate that macrophages polarized prior to infection have a differential capacity to control Chlamydia. M1 macrophages harbor up to 40-fold lower inclusion forming units (IFU) than non-polarized or M2 polarized macrophages. Gene expression analysis showed an increase in 16sRNA in M2 macrophages with no change in M1 macrophages. Suppressed Chlamydia growth in M1 macrophages correlated with the induction of a bacterial gene expression profile typical of persistence as evident by increased Euo expression and decreased Omp1 and Tal expression. Observations of permissive Chlamydia growth in non-polarized and M2 macrophages and persistence in M1 macrophages were supported through electron microscopy. This work supports the importance of IFNγ in the innate immune response to Chlamydia. However, demonstration that the M1 macrophages, despite an antimicrobial signature, fail to eliminate intracellular Chlamydia supports the notion that host-pathogen co-evolution has yielded a pathogen that can evade cellular defenses against this pathogen, and persist for prolonged periods of time in the host.

Nafamostat mesylate, a serine protease inhibitor, demonstrates novel antimicrobial properties and effectiveness in Chlamydia-induced arthritis.

INTRODUCTION: Effective treatment of reactive arthritis would ideally achieve both control of inflammation and eradication of persisting arthritogenic pathogens. We use a model of experimental Chlamydia trachomatis-induced arthritis (CtIA) to evaluate the effectiveness of nafamostat mesilate (NM), a serine protease inhibitor with complement-modifying effects and anticoagulant properties. To date clinical use of NM has largely been in Asia and has been primarily confined to inflammatory states such as pancreatitis. METHODS: In vitro studies examined inhibition of Chlamydia proliferation using fibroblast cell lines as targets and phase contrast microscopy. In vivo studies used an established protocol, experimental CtIA, induced in Lewis rats by injection of synoviocyte-packaged C. trachomatis. NM was dissolved in water and administered by daily intraperitoneal injection at a dose of 10 mg/kg beginning the day prior to the administration of Chlamydia. Readouts in vivo included (i) joint swelling, (ii) histopathology scoring of severity of arthritis, (iii) host clearance of the pathogen (by ELISA, the IDEIA PCE Chlamydia). RESULTS: NM exerted a dose-dependent inhibition of chlamydial proliferation in vitro. Without NM, the mean number of inclusion bodies (IB) per well was 17,886 (± 1415). At 5 µg/mL NM, there were 8,490 (± 756) IB, at 25 µg/mL NM there were 35 IB and at 50 µg/mL NM no IB was observed. Chlamydial antigens in each well along the concentration gradient were assayed by ELISA, demonstrating that at 25 µg/mL NM inhibition of Chlamydia was almost complete. In the experimental arthritis model, joint swelling was significantly reduced with NM treatment: average joint width for the NM-treated animals was 8.55 mm (s.d. ± 0.6578, n = 10) versus 11.18 mm (s.d. ± 0.5672, n = 10) in controls (P < 0.001). Histopathology scoring indicated that NM resulted in a marked attenuation of the inflammatory infiltration and joint damage: mean pathology score in NM-treated animals was 10.9 (± 2.45, n = 11) versus 15.9 (± 1.45, n = 10) in controls (P < 0.0001). With respect to persistence of Chlamydia within the synovial tissues, NM treatment was accompanied by a reduction in the microbial load in the joint: mean optical density (O.D.) for ELISA with NM treatment was 0.05 (± 0.02, n = 4) versus 0.18 (± 0.05, n = 4) in controls (P < 0.001). CONCLUSIONS: NM is a protease inhibitor not previously recognized to possess antimicrobial properties. The present study demonstrates for the first time that NM exerts significant impact on C. trachomatis-induced arthritis and suggests that such approaches may prove clinically useful in chronic reactive arthritis.

Aberrant chondrocyte hypertrophy and activation of ß-catenin signaling precede joint ankylosis in ank/ank mice.

OBJECTIVE: We assessed the role of Ank in the maintenance of postnatal articular cartilage using the ank/ank mouse (mice homozygous for progressive ankylosis). METHODS: We analyzed ank/ank mice and wild-type littermates (8, 12, and 18 weeks old). Sections from decalcified, paraffin-embedded joints were stained with hematoxylin and eosin. Articular chondrocyte size and cartilage thickness were determined using morphometric methods. Immuno-histochemical staining was performed with anticollagen X, antitissue nonspecific alkaline phosphatase (TNAP), and anti-ß-catenin antibodies on fixed joint sections. Axin2 expression in paw joint lysates in wild-type versus ank/ank mice were compared using Western blot analysis. RESULTS: In all age groups of normal mice studied, calcified cartilage (CC) chondrocyte areas were significantly larger than those of uncalcified cartilage (UC) chondrocytes. However, similar chondrocyte areas (UC vs CC) were found in 12-week and 18-week-old ank/ank mice, indicating that hypertrophic chondrocytes were present in the UC of these mutant mice. The ank/ank mice showed an increase in CC thickness. The ank/ank UC hypertrophic chondrocytes showed diffuse immuno-reactivity for collagen X and TNAP. Increased ß-catenin activation was demonstrated by nuclear localization of ß-catenin staining in ank/ank chondrocytes. Axin2 expression from paw lysates was downregulated in ank/ank mice. CONCLUSION: We identified a previously unrecognized phenotype in the articular cartilage of ank/ank mice: collagen X-positive hypertrophic chondrocytes in the UC. It is possible that consequent to downregulation of axin2 expression, ß-catenin signaling was activated, leading to accelerated chondrocyte maturation and eventual ankylosis in ank/ank joints. Our studies shed new light on the contribution of a key signaling pathway in this model of joint ankylosis.

Aberrant axial mineralization precedes spinal ankylosis: a molecular imaging study in ank/ank mice.

INTRODUCTION: The diagnosis of ankylosing spondylitis is made from a combination of clinical features and the presence of radiographic evidence that may be detected only after many years of inflammatory back pain. It is not uncommon to have a diagnosis confirmed 5 to 10 years after the initial onset of symptoms. Development of a more-sensitive molecular imaging technology to detect structural changes in the joints would lead to earlier diagnosis and quantitative tracking of ankylosis progression. Progressive ankylosis (ank/ank) mice have a loss of function in the Ank gene, which codes for a regulator of PPi transport. In this study, we used these ank/ank mutant mice to assess a noninvasive, quantitative measure of joint ankylosis with near-infrared (NIR) molecular imaging in vivo. METHODS: Three age groups (8, 12, and 18 weeks) of ank/ank (15 mice) and wild-type littermates (12 +/+ mice) were assessed histologically and radiographically. Before imaging, OsteoSense 750 (bisphosphonate pamidronate) was injected i.v. Whole-body images were analyzed by using the multispectral Maestro imaging system. RESULTS: OsteoSense 750 signals in the paw joints were higher in ank/ank mice in all three age groups compared with controls. In the spine, significantly higher OsteoSense 750 signals were detected early, in 8-week-old ank/ank mice compared with controls, although minimal radiographic differences were noted at this time point. The molecular imaging changes in the ank/ank spine (8 weeks) were supported by histologic changes, including calcium apatite crystals at the edge of the vertebral bodies and new syndesmophyte formation. CONCLUSIONS: Changes in joint pathology of ank/ank mice, as evaluated by histologic and radiographic means, are qualitative, but only semiquantitative. In contrast, molecular imaging provides a quantitative assessment. Ankylosis in ank/ank mice developed simultaneously in distal and axial joints, contrary to the previous notion that it is a centripetal process. NIR imaging might be feasible for early disease diagnosis and for monitoring disease progression in ankylosing spondylitis.

Serum cytokine receptors in ankylosing spondylitis: relationship to inflammatory markers and endoplasmic reticulum aminopeptidase polymorphisms.

OBJECTIVE: Endoplasmic reticulum aminopeptidase (ERAP)1 is associated with ankylosing spondylitis (AS) and is known to be involved in the clipping of the cytokine receptors interleukin 1 receptor II (IL-1RII), IL-6Ralpha, and tumor necrosis factor receptor I (TNFRI). We studied the relationship of these serum cytokine receptors and their corresponding cytokines to markers of inflammation and polymorphisms in ERAP1 and ERAP2 in patients with AS. METHODS: Sera from patients with AS were assayed for TNF-alpha, IL-1, IL-6, sTNFRI, sIL-1RII, and sIL-6Ralpha by ELISA. Genotyping was performed for 3 AS-associated nonsynonymous single-nucleotide polymorphisms in the ERAP1 gene [rs27044(C/G), rs10050860(C/T), and rs30187(C/T)] and 1 in the ERAP2 gene [rs2549782(T/G)]. The serum cytokine and receptor levels were compared between the different genotype groups and correlated to markers of inflammation and disease activity. RESULTS: Eighty patients with AS (21 women) with a mean Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) of 5.3 +/- 2.4 were enrolled. There was a significant correlation of sTNFRI with C-reactive protein (CRP; R = 0.43, p < 0.001) and erythrocyte sedimentation rate (ESR; R = 0.30, p = 0.01) but not with BASDAI. Serum cytokine levels were undetectable in the majority of patients. There was no significant difference in serum cytokines or the soluble receptors between patients with the different ERAP1/ERAP2 polymorphisms and their haplotypes. Similarly, there was no relationship of the polymorphisms with the serum cytokine levels nor the cytokine-receptor ratio. CONCLUSION: Soluble TNFRI levels correlate with ESR and CRP in AS. The ERAP1 and ERAP2 polymorphisms associated with AS do not influence the serum cytokine receptor levels in patients with AS.

Heavy metal exposure reverses genetic resistance to Chlamydia-induced arthritis.

INTRODUCTION: We have previously observed that Brown Norway (BN) rats display a relative resistance to experimental Chlamydia-induced arthritis. In the present study, we examine an environmental toxin, mercuric chloride (HgCl2), as a modulator of this innate resistance to arthritis. METHODS: To assess the effect of the heavy metal exposure, one group of rats received two subcutaneous injections of HgCl2 (1 mg/kg) 48 hours apart. Seven days later, the animals received the intra-articular injection of synoviocyte-packaged Chlamydia. RESULTS: Histopathology revealed that BN rats receiving only Chlamydia had a minimal cellular infiltration in the joint, which was predominantly mononuclear in character. In contrast, mercury-exposed rats had a marked exacerbation of the histopathological severity of the arthritis, and the infiltration was predominantly neutrophilic. Mercury exposure was also associated with marked enhancement in IgE levels and an alteration in IgG2a/IgG1 ratio, reflecting a Th2 shift. The local cytokine profile in the joint was markedly altered after mercury exposure, with a suppression of tumour necrosis factor-alpha and interferon-gamma but an enhancement of vascular endothelial growth factor. This was associated with decreased host clearance capacity reflected in enhanced bacterial load in both the spleen and the joint and was accompanied by enhanced detection of microbial antigens in the synovial tissues by immunohistological staining. CONCLUSIONS: Genetically defined cytokine production in the joint defines the severity of reactive arthritis by dictating the local clearance of the pathogen. This interplay can be altered dramatically by heavy metal exposure, which results in suppression of protective cytokines in the microenvironment of the joint.

Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation.

During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic proteins like c-Src to maturing phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in phagosome maturation and microbial killing.

Retinol (vitamin A) and retinol-binding protein levels are decreased in ankylosing spondylitis: clinical and genetic analysis.

OBJECTIVE: Retinol (vitamin A) plays an important role in bone structure and function. Treatment with retinoids has been associated with bone abnormalities mimicking spondyloarthropathy and diffuse idiopathic skeletal hyperostosis. To determine whether retinol concentrations are altered in patients with ankylosing spondylitis (AS), we examined serum retinol levels in patients with AS and healthy controls. METHODS: Retinol was assessed using mass spectrometry, and retinol-binding protein levels were assessed by ELISA. Retinol levels were correlated with clinical disease activity indices. The CYP26 gene, which plays a key role in retinol metabolism, was examined to define any single-nucleotide polymorphisms (SNP) associations with AS. RESULTS: Retinol levels were significantly lower in the AS cohort than in controls (mean 2.39 +/- 0.88 micromol/l for AS, 3.34 +/- 1.01 micromol/l for controls; p < 0.0001). Retinol-binding protein levels were also lower in AS than controls (AS 4.65 +/- 2.10 microg/l; controls 7.48 +/- 4.87 microg/l; p < 0.001). Serum retinol levels did not correlate with indices of disease activity defined serologically (C-reactive protein, erythrocyte sedimentation rate) or clinically (Bath AS Disease Activity Index, Bath AS Functional Index). Genetic analysis showed that an exonic CYP26C1 SNP (rs11187265) is not associated with AS. CONCLUSION: The hallmark of AS is neo-ossification. AS is associated with abnormal serum levels of retinol, a biochemical factor linked to pathological hyperostosis. Further genetic studies are warranted into the genetic basis of the retinol-AS relationship.

Microcytosis in ank/ank mice and the role of ANKH in promoting erythroid differentiation.

Progressive ankylosis (Ank and the human homolog, ANKH) is a transmembrane protein which regulates transport of inorganic pyrophosphate (PPi). ank/ank mice with a mutated ank gene, have calcification and bone ankylosis of the affected joints. In the course of studying these mutant mice, we found that they have microcytosis. These mutant mice have lower mean red blood cell volume (MCV) and lower hemoglobin content in red cells (mean corpuscular hemoglobin, MCH) than normal mice. Using quantitative real-time PCR analysis, we showed that Ank was expressed in the E/Meg bipotent precursor, BFU-E, CFU-E, but there was no Ank expression in the hemoglobinizing erythroblasts. Stable ANKH transfectants in K562 cells highly expressed two immature erythroid cell markers, E-cadherin and endoglin. Enhanced Erythropoietin (Epo) expression and downregulation of SHP-1 were detected in these transfectants. Consequently, the autocrine Epo-EpoR signaling pathway was activated, as evidenced by higher p-Tyr JAK2, p-Tyr EpoR and p-Tyr STAT5B in the ANKH transfectants. Our results revealed a novel function of ANKH in the promotion of early erythroid differentiation in K562 cells. We also showed that ank/ank mice have lower serum levels of Epo than the normal littermates, and this is the likely cause of microcytosis in these mutant mice.

Early cytokine profiles in the joint define pathogen clearance and severity of arthritis in Chlamydia-induced arthritis in rats.

OBJECTIVE: Although Chlamydia trachomatis-induced arthritis is among the most common rheumatic diseases having an identified infectious trigger, the pathogenesis of this arthritis is not well defined. We sought to investigate the host-microbe interactions that contribute to the severity of arthritis initiated by chlamydial infection. METHODS: We established an experimental rat model of C. trachomatis-induced arthritis that recapitulates many pathologic features of the clinical disease. The severity of the arthritis was defined using an established histopathologic scoring system. Host clearance of the pathogen and local cytokine production were examined by enzyme-linked immunosorbent assays. RESULTS: Lewis rats were susceptible to C. trachomatis-induced arthritis, whereas BN rats were relatively resistant to this disease. Significant differences in the histopathologic severity of arthritis were originally observed on day 21, and this prompted an examination of the acute phase of the arthritis. As early as day 5 after the onset of the arthritis, pathologic changes in Lewis rats were more severe than those in BN rats. An evaluation of the role of complement using cobra venom factor treatment excluded complement as being the key to differential sensitivity, because decomplementation did not eliminate the differences in arthritis severity between Lewis and BN rats. Host clearance, in contrast, was significantly different between the rat strains, with BN rats showing more prompt and effective clearance of the pathogen from both synovial tissues and spleen compared with Lewis rats. Local cytokine profiles demonstrated that host resistance was characterized by enhanced synovial expression of tumor necrosis factor alpha, interferon-gamma (IFNgamma), and interleukin-4. CONCLUSION: These studies demonstrated that cytokines thought to be proinflammatory in nature can play an important role in host defense in infection-triggered arthritis and serve to highlight the dynamic cytokine relationships that constitute effective host-pathogen interactions.

Innate immunity and arthritis: neutrophil Rac and toll-like receptor 4 expression define outcomes in infection-triggered arthritis.

OBJECTIVE: The role of innate immunity in Chlamydia-induced arthritis has not been defined. The purpose of this study was to examine the role of neutrophils in experimental arthritis in mice with targeted elimination of the small GTPases Rac1 and Rac2, as well as the role of Toll-like receptors (TLRs) in this model. METHODS: Arthritis was induced by intraarticular inoculation of synoviocyte-packaged Chlamydia trachomatis. The degree of arthritis was assessed according to joint swelling and pathology scores. The persistence of Chlamydia in joints was assessed by immunoassay. The expression of TLR-2 and TLR-4 in neutrophils was detected by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: In the acute phase, wild-type mice developed more severe arthritis than did Rac-deficient mice, with abundant infiltration of neutrophils into the joint. In the chronic phase, the Rac-deficient mice developed more severe arthritis and demonstrated defective clearance of the pathogen from the joint. In vitro stimulation of neutrophils with Chlamydia up-regulated the expression of TLR-4, but not TLR-2, in wild-type mice. However, neutrophils from Rac-deficient mice did not show this up-regulation of TLR-4. Sustained TLR-4 expression in neutrophils was found to be dependent on the expression of Rac. Mice genetically deficient in TLR-4 demonstrated more severe arthritis than did the controls. Thus, Rac expression plays a profound role in infection-triggered arthritis and demonstrates a bimodal influence on the disease process, exacerbating acute joint inflammation but controlling chronic arthritis. Rac deficiency was associated with diminished TLR-4 expression, impaired host clearance of the pathogen, and more severe chronic arthritis. CONCLUSION: In infection-triggered arthritis, innate immunity plays a critical role. Effective host clearance of an arthritogenic pathogen depends on intact expression of Rac and appropriate expression of TLR-4 by neutrophils.

Accumulation of diacylglycerol in the Chlamydia inclusion vacuole: possible role in the inhibition of host cell apoptosis.

Intracellular pathogens have developed strategies to survive for extended periods inside their host cells. These include avoidance of host microbicidal effectors, often by sequestration in a protected subcompartment of the host cell. In some cases, the parasites exert also an antiapoptotic effect that prolongs the life of the infected host cell. Chlamydia utilizes both strategies, but the underlying molecular mechanisms are incompletely understood. Comparatively, little is known regarding the effects that Chlamydia exerts on the metabolism and distribution of the host cell lipids. The expression of fluorescently tagged C1 domains revealed that diacylglycerol is greatly accumulated in the immediate vicinity of Chlamydia inclusion vacuoles. The concentrated diacylglycerol recruits protein kinase Cdelta (PKCdelta), a proapoptotic effector, to the immediate vicinity of the vacuole. PKCdelta normally exerts its pro-apoptotic effects at the mitochondria and in the nucleus. We speculate that Chlamydia antagonizes the pro-apoptotic effect of PKCdelta by sequestering the enzyme on the inclusion vacuole away from its conventional target sites. Accordingly, we found that the ectopic expression of a catalytic fragment of PKCdelta that cannot be recruited by the vacuole, because it lacks a functional C1 domain, overcame the anti-apoptotic effect of the bacteria. The scavenging of pro-apoptotic factors may provide a novel mechanism whereby pathogens promote their own survival by extending the life of the host cells they infect.

Breakdown of CTL tolerance to self HLA-B*2705 induced by exposure to Chlamydia trachomatis.

There is a strong association between seronegative arthritis and HLA B27, but it is still unresolved whether the contribution of B27 to disease pathogenesis is solely as a restriction element for an arthritogenic peptide, or whether B27 itself serves as an autoantigen. This study uses transgenic rats to address the question as to whether exposure to an arthritogenic pathogen can alter tolerance to B27. Unlike their nontransgenic counterparts, B27-transgenic rats are tolerant of B27 immunization using either B27(+) splenocytes or plasmid DNA and do not develop anti-B27 CTL. However, if splenocytes from such immunized animals are exposed to Chlamydia in vitro, CTL are generated that lyse B27(+) targets. No killing was seen with targets transfected with control B7, B14, B40, or B44. This phenomenon was not observed with immunization by nontransgenic splenocytes, or HLA-A2 DNA alone. Using targets expressing mutated B27, we show that the epitope for autoreactive CTL recognition of B27 involves the Lys(70) amino acid residue in the alpha(1) domain of the MHC class I molecule. The generation of CTL with specificity for B27 under these conditions demonstrates that tolerance to B27 can be subverted by CHLAMYDIA: This indicates a dynamic interrelationship between the pathogen and B27, which may have important implications for B27-related spondyloarthropathies triggered by intracellular bacteria.

Synovial fibroblasts infected with Salmonella enterica serovar Typhimurium mediate osteoclast differentiation and activation.

The mechanisms whereby arthritogenic organisms may induce cartilage and bone erosions in infection-triggered arthritis remain unknown. In this study, we asked whether an arthritogenic organism could contribute to osteoclast differentiation and activation through regulation of the receptor activator of NF-kappaB ligand (RANKL) in synovial fibroblasts. Rat synovial fibroblasts were infected in vitro with Salmonella enterica serovar Typhimurium and monitored over time. The expression of RANKL in resting and infected synovial fibroblasts was quantified by reverse transcription-PCR and Western blotting. Osteoclast progenitors, isolated from femurs of 8-week-old rats and cultured in the presence of macrophage colony-stimulating factor, were cocultured with either infected or noninfected synovial fibroblasts for 2 to 4 days. Differentiation and maturation of osteoclasts were determined by morphology and tartrate-resistant acid phosphatase (TRAP) staining and by a bone resorption bioassay. RANKL expression was undetectable in resting synovial fibroblasts but was dose-dependently upregulated in cells after Salmonella infection. Osteoprotegerin was constitutively expressed by synovial fibroblasts and was not upregulated by infection. Further, we observed the formation of multinucleated TRAP-positive cells and formation of bone resorption pits in cocultures of bone marrow-derived osteoclast precursors with synovial fibroblasts infected with Salmonella but not with heat-killed Salmonella or noninfected cells. Arthritogenic bacteria may alter bone structure via synovial fibroblast intermediaries, since infected synovial fibroblasts (i) upregulate RANKL expression and (ii) enhance osteoclast precursor maturation into multinucleated, TRAP-positive, bone-resorbing, osteoclast-like cells. These data provide a link between infection and osteoclastogenesis. A better understanding of infection-mediated osteoclast differentiation and activation may provide new therapeutic strategies for inflammatory joint disease.

Common aspects of human and primate seronegative arthritis.

A 27-year-old female lowland gorilla developed an asymmetric oligoarthritis 3 months post-partum. There was no evidence of an antecedent gastrointestinal or genitourinary infection. Serum was negative for rheumatoid factor and antinuclear antibody. Synovial fluid revealed 2000 white blood cells with negative cultures and polarized microscopy. Studies on synoviocytes were the following: (1) FACS analysis revealed surface expression of a B27-like epitope of the cells. (2) Analysis of intracellular clearance kinetics of arthritogenic organisms showed peak intracellular colony-forming units at 48 hours after bacterial invasion, and clearance by 13 days post-invasion. (3) Interferon-y (0.1-10.0 ng/ml)accelerated intracellular microbicidal pathways in a dose-dependent fashion. These findings closely parallel those seen in human synoviocytes of patients with spondyloarthropathy. Primate and human seronegative arthritis share clinical and immunologic features, as well as aspects of host:pathogen defense mechanisms. The interplay of genetic and microbial factors underlying this arthritis appears to be conserved across these species boundaries.

Modulation of Mac-1 (CD11b/CD18)-mediated adhesion by the leukocyte-specific protein 1 is key to its role in neutrophil polarization and chemotaxis.

Leukocyte-specific protein 1 (LSP1) is an intracellular filamentous-actin binding protein which modulates cell motility. The cellular process in which LSP1 functions to regulate motility is not yet identified. In this study, we show that LSP1 negatively regulates fMLP-induced polarization and chemotaxis of neutrophils through its function on adhesion via specific integrins. Using LSP1-deficient (Lsp1(-/-)) mice, we show increased neutrophil migration into mouse knee joints during zymosan-induced acute inflammation, an inflammatory model in which the number of resident synoviocytes are not affected by LSP1-deficiency. In vitro chemotaxis experiments performed by time-lapse videomicroscopy showed that purified Lsp1(-/-) bone-marrow neutrophils exhibit an increased migration rate toward a gradient of fMLP as compared with wild-type neutrophils. This difference was observed when cells migrated on fibrinogen, but not fibronectin, suggesting a role for LSP1 in modulating neutrophil adhesion by specific integrins. LSP1 is also a negative regulator of fMLP-induced adhesion to fibrinogen or ICAM-1, but not to ICAM-2, VCAM-1, or fibronectin. These results suggest that LSP1 regulates the function of Mac-1 (CD11b/CD18), which binds only to fibrinogen and ICAM-1 among the substrates we tested. fMLP-induced filamentous actin polarization is also increased in the absence of LSP1 when cells were layered on fibrinogen, but not on fibronectin. Our findings suggest that the increased neutrophil recruitment in Lsp1(-/-) mice during acute inflammation derives from the negative regulatory role of LSP1 on neutrophil adhesion, polarization, and migration via specific integrins, such as Mac-1, which mediate neutrophil responses to chemotactic stimuli.