Methotrexate-induced accumulation of fluorescent annexin V in collagen-induced arthritis

We examined the accumulation of Cy5.5-labeled annexin V in the paws of mice with and without collagen-induced arthritis, with and without methotrexate (MTX) treatment, by near-infrared fluorescence imaging. Fluorescence reflectance imaging (FRI) of paws was performed 48 hr after MTX injection and at 10 min and 3 hr after the injection of Cy5.5-annexin V (1 nmol dye per mouse). With arthritic paws, MTX treatment caused a 7-fold increase in fluorescence intensity compared with the paws of untreated mice and a 4-fold increase compared to nonarthritic paws of MTX-treated mice (p < .001 each). Tissue samples of paws were examined histologically for Cy5.5 fluorescence and by TUNEL staining for apoptosis. Cy5.5-annexin V was seen in the hyperplastic synovia of MTX-treated mice, and TUNEL staining for apoptosis showed apoptotic cells in the hyperplastic synovia. Monitoring the uptake of Cy5.5-annexin V in arthritic paws by FRI provided a method of assessing a response to MTX, a response that was readily quantitated with simple instrumentation and that occurred before conventional measurements of treatment response.     

Oleyl-chitosan nanoparticles based on a dual probe for optical/MR imaging in vivo

Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could self-assemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. ION-loaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.     

Enhanced autoimmune arthritis in IFN-gamma receptor-deficient mice is conditioned by mycobacteria in Freund's adjuvant and by increased expansion of Mac-1+ myeloid cells.

Induction of experimental autoimmune diseases often relies on immunization with the organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In several of these models, including collagen-induced arthritis, endogenous IFN-gamma acts as a disease-limiting factor in the pathogenesis of the disease. Here we show that in collagen-induced arthritis the protective effect of IFN-gamma depends on the presence of mycobacteria in the adjuvant. Omission of mycobacteria inverts the role of endogenous IFN-gamma to a disease-promoting factor. Thus, the mycobacterial component of CFA opens a pathway by which endogenous IFN-gamma exerts a protective effect that supersedes its otherwise disease-promoting effect. Extramedullary hemopoiesis and expansion of the Mac-1+ cell population accompanied the accelerated and more severe disease course in the IFN-gamma receptor knockout mice immunized with CFA. Treatment of such mice with Abs against the myelopoietic cytokines IL-6 or IL-12 inhibited both disease development and the expansion of the Mac-1+ population. We postulate that mycobacteria in CFA stimulate the expansion of the Mac-1+ cell population by a hemopoietic process that is restrained by endogenous IFN-gamma. These results have important implications for the validity of animal models of autoimmunity to study the pathogenesis and to evaluate cytokine-based therapy of autoimmune diseases.     

B cell depletion delays collagen-induced arthritis in mice: arthritis induction requires synergy between humoral and cell-mediated immunity

Rheumatoid arthritis is a systemic autoimmune disease. B cells are likely to play a critical role in arthritis pathogenesis, although it is unclear whether they are necessary for disease induction, autoantibody production, or disease progression. To assess the role of B cells in inflammatory arthritis, B cells were depleted using mouse anti-mouse CD20 mAbs in a mouse model of collagen-induced arthritis. CD20 mAbs effectively depleted mature B cells from adult DBA-1 mice. When B cells were depleted using CD20 mAbs before collagen immunization, there was a delay in disease onset and autoantibody production, with significantly diminished severity of arthritis both clinically and histologically. B cell depletion further delayed disease onset if initiated before, as well as after, collagen immunization. However, in both cases, the eventual reappearance of peripheral B cells triggered autoantibody production and the subsequent development of arthritis in collagen-sensitized mice. By contrast, B cell depletion after collagen immunizations did not have a significant effect on arthritis progression or severity. Thus, disease symptoms were only induced when peripheral B cells and their autoantibody products were present in collagen-immunized mice, documenting a critical role for B cells during the elicitation phase of collagen-induced arthritis. These studies suggest that B cell depletion strategies will be most effective when initiated early in the development of inflammatory arthritis, with sustained B cell depletion required to inhibit the production of isotype-switched pathogenic Abs and the evolution of joint inflammation and destruction.     

Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.     

Identification of blood biomarkers of rheumatoid arthritis by transcript profiling of peripheral blood mononuclear cells from the rat collagen-induced arthritis model

Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA.     

Near-infrared imaging of flare-up arthritis with native fluorochrome Cy5.5 and albumin-bound Cy5.5

The aim of the study was to visualize chronic experimental arthritis with near-infrared fluorescence imaging (NIRF) in a murine experimental arthritis model of rheumatoid arthritis (RA) (flare-up arthritis). The flare-up arthritis model is a modification of the primary antigen-induced arthritis (AIA) model. NIRF was done for two preparations of the fluorochrome Cy5.5, one native and the other albumin conjugated. Histological features of flare-up arthritis were evaluated.AIA was induced in 16 mice (strain C57/Bl6); flare-up arthritis was induced in a subgroup of eight. On day 7 after induction of flare-up arthritis, four mice received 50 nmol/kg native dye and four mice equimolar concentrations of the dye as albumin-dye conjugate intravenously. NIRF imaging was performed immediately before injection (baseline) and until 72 h thereafter. Arthritis severity was evaluated histologically for primary AIA and flare-up arthritis mice.NIRF imaging revealed higher fluorochrome uptake in all inflamed knees compared to contralateral ones. The signal intensities induced by native Cy5.5 were higher than those generated by albumin-Cy5.5 conjugate. Histological evaluation of arthritic joints showed similar abnormalities in flare-up arthritis and in primary AIA joints.Imaging of flare-up arthritis in the near-infrared range was successful for both fluorochrome preparations, but albumin conjugation prior to injection does not improve the uptake of dye in arthritic joints. Flare-up arthritis is a feasible model of chronic relapse of arthritis in human RA.     

Spectroscopically well-characterized RGD optical probe as a prerequisite for lifetime-gated tumor imaging

Labeling of RGD peptides with near-infrared fluorophores yields optical probes for noninvasive imaging of tumors overexpressing ανβ3 integrins. An important prerequisite for optimum detection sensitivity in vivo is strongly absorbing and highly emissive probes with a known fluorescence lifetime. The RGD-Cy5.5 optical probe was derived by coupling Cy5.5 to a cyclic arginine-glycine-aspartic acid-d-phenylalanine-lysine (RGDfK) peptide via an aminohexanoic acid spacer. Spectroscopic properties of the probe were studied in different matrices in comparison to Cy5.5. For in vivo imaging, human glioblastoma cells were subcutaneously implanted into nude mice, and in vivo fluorescence intensity and lifetime were measured. The fluorescence quantum yield and lifetime of Cy5.5 were found to be barely affected on RGD conjugation but dramatically changed in the presence of proteins. By time domain fluorescence imaging, we demonstrated specific binding of RGD-Cy5.5 to glioblastoma xenografts in nude mice. Discrimination of unspecific fluorescence by lifetime-gated analysis further enhanced the detection sensitivity of RGD-Cy5.5-derived signals. We characterized RGD-Cy5.5 as a strongly emissive and stable probe adequate for selective targeting of ανβ3 integrins. The specificity and thus the overall detection sensitivity in vivo were optimized with lifetime gating, based on the previous determination of the probes fluorescence lifetime under application-relevant conditions.     

The beta2-adrenergic agonist salbutamol is a potent suppressor of established collagen-induced arthritis: mechanisms of action

The therapeutic potential of salbutamol, a beta2-adrenergic agonist, was explored in collagen-induced arthritis. This study was based on a report that salbutamol, by elevating intracellular cAMP, inhibits IL-12 production by macrophages and dendritic cells, thus preventing Th1 development. Ten-week-old male DBA/1 mice were immunized by intradermal injection of type II collagen in CFA. Arthritis developed 15-30 days later and the mice were treated after onset of disease with salbutamol, 200 microgram i.p. After 10 days, the mice were sacrificed, and the hind paws were evaluated histologically. Salbutamol, 200 microgram daily or every other day, had a profound therapeutic effect on the clinical progression of arthritis, as assessed by clinical score and paw thickness. The therapeutic effect was dose dependent. Daily administration of 200 microgram of salbutamol offered the best protection against joint damage, as assessed by histology. In vitro, salbutamol reduced IL-12 and TNF-alpha release by peritoneal macrophages in a dose-dependent manner, as well as TNF release by synovial cells from arthritic mice. Ex vivo, draining lymph node cells of the salbutamol-treated arthritic mice showed a diminished CII-specific IFN-gamma production and proliferation. In vivo, salbutamol specifically blocked mast cell degranulation in joint tissues. In conclusion, salbutamol has important effects on the immunoinflammatory response and a significant therapeutic action in collagen-induced arthritis.     

Backcross and partial advanced intercross analysis of nonobese diabetic gene-mediated effects on collagen-induced arthritis reveals an interactive effect by two major loci

Genetic segregation analysis between NOD and C57BL strains have been used to identify loci associated with autoimmune disease. Only two loci (Cia2 and Cia9) had earlier been found to control development of arthritis, whereas none of the previously identified diabetes loci was of significance for arthritis. We have now made a high-powered analysis of a backcross of NOD genes on to the B10.Q strain for association with collagen-induced arthritis. We could confirm relevance of both Cia2 and Cia9 as well as the interaction between them, but we did not identify any other significant arthritis loci. Immune cellular subtyping revealed that Cia2 was also associated with the number of blood macrophages. Congenic strains of the Cia2 and Cia9 loci on the B10.Q background were made and used to establish a partial advanced intercross (PAI). Testing the PAI mice for development of collagen-induced arthritis confirmed the loci and the interactions and also indicated that at least two genes contribute to the Cia9 locus. Furthermore, it clearly showed that Cia2 is dominant protective but that the protection is not complete. Because these results may indicate that the Cia2 effect on arthritis is not only due to the deficiency of the complement C5, we analyzed complement functions in the Cia2 congenics as well as the PAI mice. These data show that not only arthritis but also C5-dependent complement activity is dominantly suppressed, confirming that C5 is one of the major genes explaining the Cia2 effect.     

Role of interleukin 1 and interleukin 2 in rat and mouse arthritis models

The production of interleukin 1 (IL 1) and interleukin 2 (IL 2) by macrophages and lymphocytes from three animal models commonly used for rheumatoid arthritis, viz. adjuvant-induced and type II collagen-induced rat arthritis, and MRL/1 murine arthritis was studied. Although the peritoneal macrophages from adjuvant-arthritic rats in culture produced increased amounts of prostaglandin E2 (PGE2) and lower levels of IL 1 than the control group, cells from collagen-arthritic rats released normal levels of PGE2, but increased amounts of IL 1. After activation with lipopolysaccharides, the IL 1 production by macrophages from all groups was comparable. Addition of indomethacin did not significantly change the IL 1 production in any of these groups. In the absence of any exogenous mitogen, IL 2 production by the lymphocytes of adjuvant-arthritic rats was low, but could be restored to the normal levels when phytohemagglutinin A (PHA) or concanavalin A (Con A) was added. The lymphocytes from collagen-arthritic rats were capable of producing IL 2 without the need of any T cell mitogen. The lymphocytes from MRL/1 mice seemed to lack the functionality in terms of IL 2 production. The macrophagic IL 1 production in these animals was normal. Our data suggest that the type II collagen arthritis model may closely resemble human rheumatoid arthritis in which IL 1 and IL 2 production by the mononuclear cells is significantly enhanced.     

FeCo/graphite nanocrystals for multi-modality imaging of experimental vascular inflammation

Background

FeCo/graphitic-carbon nanocrystals (FeCo/GC) are biocompatible, high-relaxivity, multi-functional nanoparticles. Macrophages represent important cellular imaging targets for assessing vascular inflammation. We evaluated FeCo/GC for vascular macrophage uptake and imaging in vivo using fluorescence and MRI.

Methods and Results

Hyperlipidemic and diabetic mice underwent carotid ligation to produce a macrophage-rich vascular lesion. In situ and ex vivo fluorescence imaging were performed at 48 hours after intravenous injection of FeCo/GC conjugated to Cy5.5 (n = 8, 8 nmol of Cy5.5/mouse). Significant fluorescence signal from FeCo/GC-Cy5.5 was present in the ligated left carotid arteries, but not in the control (non-ligated) right carotid arteries or sham-operated carotid arteries (p = 0.03 for ligated vs. non-ligated). Serial in vivo 3T MRI was performed at 48 and 72 hours after intravenous FeCo/GC (n = 6, 270 µg Fe/mouse). Significant T2* signal loss from FeCo/GC was seen in ligated left carotid arteries, not in non-ligated controls (p = 0.03). Immunofluorescence staining showed colocalization of FeCo/GC and macrophages in ligated carotid arteries.

Conclusions

FeCo/GC accumulates in vascular macrophages in vivo, allowing fluorescence and MR imaging. This multi-functional high-relaxivity nanoparticle platform provides a promising approach for cellular imaging of vascular inflammation.     

Relationship between angiogenesis and inflammation in experimental arthritis

Background. Angiogenesis is involved in rheumatoid arthritis (RA) leading to leucocyte recruitment and inflammation in the synovium. Furthermore, synovial inflammation itself further potentiates endothelial proliferation and angiogenesis. In this study, we aimed at evaluating the reciprocical relationship between synovial inflammation and angiogenesis in a RA model, namely collagen-induced arthritis (CIA). Methods. CIA was induced by immunization of DBA/1 mice with collagen type II in adjuvant. Endothelial cells were detected using a GSL-1 lectin-specific immunohistochemical staining on knee joint sections. Angiogenesis, clinical scores and histological signs of arthritis were evaluated from the induction of CIA until the end of the experiment. Angiogenesis was quantified by counting both the isolated endothelial cells and vessels stained on each section. To evaluate the effect of increased angiogenesis on CIA, VEGF gene transfer was performed using an adeno-associated virus encoding VEGF (AAV-VEGF), by intra-muscular or intra-articular injection in mice with CIA. Results. We showed an increase in synovial angiogenesis from day 6 to day 55 after CIA induction, and, moreover, joint vascularization and clinical scores of arthritis were correlated (p < 0.0001, r = 0.61). Vascularization and histological scores were also correlated (p = 0.0006, r = 0.51). Systemic VEGF overexpression in mice with CIA was followed by an aggravation of arthritis as compared to AAV-lacZ control group (p < 0.0001). In contrast, there was no difference in clinical scores between control mice and mice injected within the knee with AAV-VEGF, even if joint vascularization was higher in this group than in all other groups (p = 0,05 versus non-injected group). Intra-articular AAV-VEGF injections induced more severe signs of histological inflammation and bone destruction than AAV-Lac Z or no injection. Conclusion. Angiogenesis and joint inflammation evolve in parallel during collagen-induced arthritis. Furthermore, this work shows that exogenous VEGF can aggravate CIA. It is direct evidence that the increase in joint vascularization leads to an exacerbation of arthritis. Taken together, these results emphasize the role of angiogenesis in inflammatory arthritis. It also suggests an early involvement of angiogenesis in joint inflammation.     

Imaging tumor-induced sentinel lymph node lymphangiogenesis with LyP-1 peptide

Lymphangiogenesis in tumor-draining lymph nodes (LNs) starts before the onset of metastasis and is associated with metastasis to distant LNs and organs. In this study, we aimed to visualize tumor-induced lymphangiogenesis with a tumor lymphatics-specific peptide LyP-1. The LyP-1 peptide was labeled with a near-infrared fluorophore (Cy5.5) for optical imaging. At days 3, 7, 14 and 21 after subcutaneous 4T1 tumor inoculation, Cy5.5-LyP-1 was administered through the middle phalanges of the upper extremities of the tumor-bearing mice. At 45?min and 24?h postinjection, brachial LN fluorescence imaging was performed. Ex vivo fluorescence images were acquired for quantitative analysis of the fluorescence intensity. Tumor-induced lymphangiogenesis was confirmed by LYVE-1 immunostaining and increased size of tumor side brachial LNs. Cy5.5-LyP-1 staining in LNs co-localized with LYVE-1, suggesting lymphatics-specific binding of LyP-1 peptide. The brachial LNs were clearly visualized by optical imaging at both time points. The tumor side LNs showed significantly higher fluorescence intensities than the contralateral brachial LNs at days 7, 14, and 21, but not day 3 after tumor inoculation. At day 21 after tumor inoculation, the average signal of tumor-draining LNs was 78.0?±?2.44, 24.3?±?5.43, 25.6?±?0.25 (×10³?photon/cm²/s) using Cy5.5-LyP-1, Cy5.5-LyP-1 with blocking, and Cy5.5 only, respectively. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla. Use of LyP-1 based imaging probes with optical imaging offers a useful tool for the study of tumor-induced lymphangiogenesis. LyP-1 may serve as a marker of lymphangiogenesis useful in detecting “high risk” LNs before tumor metastasis and after micro-metastasis, as well as for screening potential anti-lymphatic therapies.     

Inhibition of indoleamine 2,3-dioxygenase-mediated tryptophan catabolism accelerates collagen-induced arthritis in mice

Indoleamine 2,3-dioxygenase (IDO) is one of the initial and rate-limiting enzymes involved in the catabolism of the essential amino acid tryptophan. In cultured cells, the induction of IDO leads to depletion of tryptophan and tryptophan starvation. Recent studies suggest that modulation of tryptophan concentration via IDO plays a fundamental role in innate immune responses. Induction of IDO by interferon-γ in macrophages and dendritic cells results in tryptophan depletion and suppresses the immune-mediated activation of fibroblasts and T, B, and natural killer cells. To assess the role of IDO in collagen-induced arthritis (CIA), a model of rheumatoid arthritis characterized by a primarily Th1-like immune response, activity of IDO was inhibited by 1-methyl-tryptophan (1-MT) in vivo. The results showed significantly increased incidence and severity of CIA in mice treated with 1-MT. Activity of IDO, as determined by measuring the levels of kynurenine/tryptophan ratio in the sera, was increased in the acute phase of arthritis and was higher in collagen-immunized mice that did not develop arthritis. Treatment with 1-MT resulted in an enhanced cellular and humoral immune response and a more dominant polarization to Th1 in mice with arthritis compared with vehicle-treated arthritic mice. The results demonstrated that development of CIA was associated with increased IDO activity and enhanced tryptophan catabolism in mice. Blocking IDO with 1-MT aggravated the severity of arthritis and enhanced the immune responses. These findings suggest that IDO may play an important and novel role in the negative feedback of CIA and possibly in the pathogenesis of rheumatoid arthritis.     

Arthritis imaging using a near-infrared fluorescence folate-targeted probe

A recently developed near-infrared fluorescence-labeled folate probe (NIR2-folate) was tested for in vivo imaging of arthritis using a lipopolysaccharide intra-articular injection model and a KRN transgenic mice serum induction mouse model. In the lipopolysaccharide injection model, the fluorescence signal intensity of NIR2-folate (n = 12) and of free NIR2 (n = 5) was compared between lipopolysaccharide-treated and control joints. The fluorescence signal intensity of the NIR2-folate probe at the inflammatory joints was found to be significantly higher than the control normal joints (up to 2.3-fold, P < 0.001). The NIR2-free dye injection group showed a persistent lower enhancement ratio than the NIR2-folate probe injection group. Excessive folic acid was also given to demonstrate a competitive effect with the NIR2-folate. In the KRN serum transfer model (n = 4), NIR2-folate was applied at different time points after serum transfer, and the inflamed joints could be detected as early as 30 hours after arthritogenic antibody transfer (1.8-fold increase in signal intensity). Fluorescence microscopy, histology, and immunohistochemistry validated the optical imaging results. We conclude that in vivo arthritis detection was feasible using a folate-targeted near-infrared fluorescence probe. This receptor-targeted imaging method may facilitate improved arthritis diagnosis and early assessment of the disease progress by providing an in vivo characterization of active macrophage status in inflammatory joint diseases.     

Vascular endothelial growth factor expression and regulation of murine collagen-induced arthritis

We have examined the expression and function of the angiogenic factor, vascular endothelial growth factor (VEGF) during the evolution of type II collagen-induced arthritis (CIA). Biologically active VEGF was expressed along a time course that paralleled the expression of two specific VEGF receptors, Flk-1 and Flt-1, and the progression of joint disease. Moreover, levels of VEGF expression correlated with the degree of neovascularization, as defined by vWF levels, and arthritis severity. Macrophage- and fibroblast-like cells, which infiltrated inflamed sites and were then activated by other inflammatory mediators, are probably important sources of VEGF and may thus regulate angiogenesis during the development of CIA. Administration of anti-VEGF antiserum to CIA mice before the onset of arthritis delayed the onset, reduced the severity, and diminished the vWF content of arthritic joints. By contrast, administration of anti-VEGF antiserum after the onset of the disease had no effect on the progression or ultimate severity of the arthritis. These data suggest that VEGF plays a crucial role during an early stage of arthritis development, affecting both neovascularization and the progression of experimentally induced synovitis.     

A novel anti-inflammatory role for simvastatin in inflammatory arthritis

3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exert favorable effects on lipoprotein metabolism, but may also possess anti-inflammatory properties. Therefore, we explored the activities of simvastatin, a lipophilic statin, in a Th1-driven model of murine inflammatory arthritis. We report in this study that simvastatin markedly inhibited not only developing but also clinically evident collagen-induced arthritis in doses that were unable to significantly alter cholesterol concentrations in vivo. Ex vivo analysis demonstrated significant suppression of collagen-specific Th1 humoral and cellular immune responses. Moreover, simvastatin reduced anti-CD3/anti-CD28 proliferation and IFN-gamma release from mononuclear cells derived from peripheral blood and synovial fluid. Proinflammatory cytokine production in vitro by T cell contact-activated macrophages was suppressed by simvastatin, suggesting that such observations have direct clinical relevance. These data clearly illustrate the therapeutic potential of statin-sensitive pathways in inflammatory arthritis.     

In vivo administration with IL-1 accelerates the development of collagen-induced arthritis in mice

In an attempt to examine the in vivo proinflammatory properties of IL-1, the effects of rIL-1 beta on the development of collagen-induced arthritis in mice were investigated. The results presented in this paper demonstrated that the administration of rIL-1 beta via mini-osmotic pumps into DBA/1 mice which were suboptimally immunized with native chick type II collagen (NcII) markedly accelerated the onset as well as the progression of the arthritic disease. When IL-1-containing osmotic pumps were s.c. implanted onto mice 18 days post-collagen immunization, clinical signs of arthritis appeared within 3 to 4 days after the implant with the pumps. Maximal incidence of arthritis which was usually 80 to 100% occurred between the 6th and 7th day after the administration of rIL-1 beta. Histologic analyses revealed that the knee and ankle joints from mice which were treated with rIL-1 beta for 7 days were most severely and consistently affected. Furthermore, these IL-1-treated mice exhibited granulocytic hyperplasia within the marrow as well as marked peripheral blood neutrophilia. By contrast, arthritis was not observed during the 7-day course of the IL-1 study in the following control groups: 1) mice that were only immunized with NcII, and 2) collagen-immunized mice which received osmotic pumps containing PBS. A substantial number of these collagen-immunized mice which were not treated with IL-1 eventually developed arthritis but at later times after the incidence of arthritis had peaked in the IL-1-treated group. In addition, unimmunized mice failed to develop arthritis upon treatments with IL-1 beta. Moreover, the humoral responses to NcII were not altered in the IL-1-treated mice. Thus, these in vivo studies suggest that IL-1 is potentially capable of triggering the various inflammatory events of collagen-induced arthritis, and thereby, contribute to the pathogenesis of murine arthritis.     

A single early activation of invariant NK T cells confers long-term protection against collagen-induced arthritis in a ligand-specific manner

The glycosphingolipid alpha-galactosylceramide (alpha-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines upon injection in mice. The C-glycoside analog of alpha-GalCer (alpha-C-GalCer), by contrast, results in an enhanced Th1-type response upon activation of iNKT cells. We administered a single dose of these Ags to DBA/1 mice during the early induction phase of collagen-induced arthritis and demonstrated therapeutic efficacy of alpha-GalCer when administered early rather than late during the disease. Surprisingly, the Th1-polarizing analog alpha-C-GalCer also conferred protection. Furthermore, a biphasic role of IFN-gamma in the effect of iNKT cell stimulation was observed. Whereas in vivo neutralization of IFN-gamma release induced by either alpha-GalCer or alpha-C-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, blockade of IFN-gamma release later on resulted in a more rapid onset of arthritis. Although no phenotypic changes in conventional T cells, macrophages, or APCs could be detected, important functional differences in T cell cytokine production in serum were observed upon polyclonal T cell activation, 2 wk after onset of arthritis. Whereas alpha-GalCer-treated mice produced significantly higher amounts of IL-10 upon systemic anti-CD3 stimulation compared with PBS controls, T cells from alpha-C-GalCer-treated mice, by contrast, produced substantially lower levels of cytokines, suggesting the involvement of different protective mechanisms. In conclusion, these findings suggest long-term, ligand-specific, time-dependent, and partially IFN-gamma-dependent immunomodulatory effects of iNKT cells in collagen-induced arthritis.