Protective Role of the MER Tyrosine Kinase via Efferocytosis in Rheumatoid Arthritis Models.

Objective: Rheumatoid arthritis (RA) is a chronic and progressive joint disease. It appears that anti-inflammatory feedback mechanisms that could restrain joint inflammation and restore homeostasis are insufficient to perform this control. In this study, we investigated the contribution of the MER tyrosine kinase-mediated anti-inflammatory response on arthritis and whether targeting MER could be a valid approach to treat RA. Methods: KRN serum transfer arthritis (KRN STA) was induced in either Mertk-deficient mice or in mice that adenovirally overexpressed Pros1. Human synovial micromasses were treated with MER-specific antibodies or PROS1. Collagen-induced arthritis (CIA) mice were treated with MER-specific agonistic antibodies or by viral overexpression of Pros1. Results: Mertk-/- mice showed exacerbated arthritis pathology, whereas Pros1 overexpression diminished joint pathology in KRN STA. Human synovial micromasses challenged with MER-specific antibodies enhanced the secretion of inflammatory cytokines, whereas stimulating MER with PROS1 reduced the secretion of these cytokines, confirming the protective role of MER. Next, we treated CIA mice with MER-specific agonistic antibodies, and this unexpectedly resulted in exacerbated arthritis pathology. This was associated with increased numbers of apoptotic cells in their knee joints and higher serum levels of interleukin (IL)-16C, a cytokine released by secondary necrotic neutrophils. Apoptotic cell numbers and IL-16C levels were enhanced during arthritis in Mertk-/- mice and reduced in Pros1-overexpressing mice. Conclusion: MER plays a protective role during joint inflammation and activating MER by its ligand PROS1 ameliorates disease. Treatment of mice with MER receptor agonistic antibodies is deleterious due to its counterproductive effect of blocking efferocytosis in the arthritic joint.

Aberrant intestinal microbiota due to IL-1 receptor antagonist deficiency promotes IL-17- and TLR4-dependent arthritis.

BACKGROUND: Perturbation of commensal intestinal microbiota has been associated with several autoimmune diseases. Mice deficient in interleukin-1 receptor antagonist (Il1rn -/- mice) spontaneously develop autoimmune arthritis and are susceptible to other autoimmune diseases such as psoriasis, diabetes, and encephalomyelitis; however, the mechanisms of increased susceptibility to these autoimmune phenotypes are poorly understood. We investigated the role of interleukin-1 receptor antagonist (IL-1Ra) in regulation of commensal intestinal microbiota, and assessed the involvement of microbiota subsets and innate and adaptive mucosal immune responses that underlie the development of spontaneous arthritis in Il1rn -/- mice. RESULTS: Using high-throughput 16S rRNA gene sequencing, we show that IL-1Ra critically maintains the diversity and regulates the composition of intestinal microbiota in mice. IL-1Ra deficiency reduced the intestinal microbial diversity and richness, and caused specific taxonomic alterations characterized by overrepresented Helicobacter and underrepresented Ruminococcus and Prevotella. Notably, the aberrant intestinal microbiota in IL1rn -/- mice specifically potentiated IL-17 production by intestinal lamina propria (LP) lymphocytes and skewed the LP T cell balance in favor of T helper 17 (Th17) cells, an effect transferable to WT mice by fecal microbiota. Importantly, LP Th17 cell expansion and the development of spontaneous autoimmune arthritis in IL1rn -/- mice were attenuated under germ-free condition. Selective antibiotic treatment revealed that tobramycin-induced alterations of commensal intestinal microbiota, i.e., reduced Helicobacter, Flexispira, Clostridium, and Dehalobacterium, suppressed arthritis in IL1rn -/- mice. The arthritis phenotype in IL1rn -/- mice was previously shown to depend on Toll-like receptor 4 (TLR4). Using the ablation of both IL-1Ra and TLR4, we here show that the aberrations in the IL1rn -/- microbiota are partly TLR4-dependent. We further identify a role for TLR4 activation in the intestinal lamina propria production of IL-17 and cytokines involved in Th17 differentiation preceding the onset of arthritis. CONCLUSIONS: These findings identify a critical role for IL1Ra in maintaining the natural diversity and composition of intestinal microbiota, and suggest a role for TLR4 in mucosal Th17 cell induction associated with the development of autoimmune disease in mice.

Induction of Canonical Wnt Signaling by the Alarmins S100A8/A9 in Murine Knee Joints: Implications for Osteoarthritis.

OBJECTIVE: Both alarmins S100A8/A9 and canonical Wnt signaling have been found to play active roles in the development of experimental osteoarthritis (OA). However, what activates canonical Wnt signaling remains unknown. This study was undertaken to investigate whether S100A8 induces canonical Wnt signaling and whether S100 proteins exert their effects via activation of Wnt signaling. METHODS: Expression of the genes for S100A8/A9 and Wnt signaling pathway members was measured in an experimental OA model. Selected Wnt signaling pathway members were overexpressed, and levels of S100A8/A9 were measured. Activation of canonical Wnt signaling was determined after injection of S100A8 into naive joints and induction of collagenase-induced OA in S100A9-deficient mice. Expression of Wnt signaling pathway members was tested in macrophages and fibroblasts after S100A8 stimulation. Canonical Wnt signaling was inhibited in vivo to determine if the effects of S100A8 injections were dependent on Wnt signaling. RESULTS: The alarmins S100A8/A9 and members of the Wnt signaling pathway showed coinciding expression in synovial tissue in an experimental OA model. Synovial overexpression of selected Wnt signaling pathway members did not result in increased expression of S100 proteins. In contrast, intraarticular injection of S100A8 increased canonical Wnt signaling, whereas canonical Wnt signaling was decreased after induction of experimental OA in S100A9-deficient mice. S100A8 stimulation of macrophages, but not fibroblasts, resulted in increased expression of canonical Wnt signaling members. Overexpression of Dkk-1 to inhibit canonical Wnt signaling decreased the induction of matrix metalloproteinase 3, interleukin-6, and macrophage inflammatory protein 1α after injection of S100A8. CONCLUSION: Our findings indicate that the alarmin S100A8 induces canonical Wnt signaling in macrophages and murine knee joints. The effects of S100A8 are partially dependent on activation of canonical Wnt signaling.

Complementary action of granulocyte macrophage colony-stimulating factor and interleukin-17A induces interleukin-23, receptor activator of nuclear factor-κB ligand, and matrix metalloproteinases and drives bone and cartilage pathology in experimental arthritis: rationale for combination therapy in rheumatoid arthritis.

INTRODUCTION: Type 17 T helper cells and interleukin (IL)-17 play important roles in the pathogenesis of human and murine arthritis. Although there is a clear link between IL-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) in the inflammatory cascade, details about their interaction in arthritic synovial joints are unclear. In view of the introduction of GM-CSF and IL-17 inhibitors to the clinic, we studied how IL-17 and GM-CSF orchestrate the local production of inflammatory mediators during experimental arthritis. METHODS: To allow detection of additive, complementary or synergistic effects of IL-17 and GM-CSF, we used two opposing experimental approaches: treatment of arthritic mice with neutralising antibodies to IL-17 and GM-CSF and local overexpression of these cytokines in naive synovial joints. Mice were treated for 2 weeks with antibodies against IL-17 and/or GM-CSF after onset of collagen-induced arthritis. Naive mice were injected intraarticularly with adenoviral vectors for IL-17 and/or GM-CSF, resulting in local overexpression. Joint inflammation was monitored by macroscopic scoring, X-rays and histology. Joint washouts, synovial cell and lymph node cultures were analysed for cytokines, chemokines and inflammatory mediators by Luminex analysis, flow cytometry and quantitative polymerase chain reaction. RESULTS: Combined therapeutic anti-IL-17 and anti-GM-CSF ameliorated arthritis progression, and joint damage was dramatically reduced compared with treatment with anti-IL-17 or anti-GM-CSF alone. Anti-IL-17 specifically reduced synovial IL-23 transcription, whereas anti-GM-CSF reduced transcription of matrix metalloproteinases (MMPs) and receptor activator of nuclear factor κB ligand (RANKL). Overexpression of IL-17 or GM-CSF in naive knee joints elicited extensive inflammatory infiltrate, cartilage damage and bone destruction. Combined overexpression revealed additive and synergistic effects on the production of MMPs, RANKL and IL-23 in the synovium and led to complete destruction of the joint structure within 7 days. CONCLUSIONS: IL-17 and GM-CSF differentially mediate the inflammatory process in arthritic joints and show complementary and local additive effects. Combined blockade in arthritic mice reduced joint damage not only by direct inhibition of IL-17 and GM-CSF but also by indirect inhibition of IL-23 and RANKL. Our results provide a rationale for combination therapy in autoinflammatory conditions, especially for patients who do not fully respond to inhibition of the separate cytokines.

The in-vivo use of superparamagnetic iron oxide nanoparticles to detect inflammation elicits a cytokine response but does not aggravate experimental arthritis.

BACKGROUND: Superparamagnetic Iron Oxide Nanoparticles (SPION) are used in diagnostic imaging of a variety of different diseases. For such in-vivo application, an additional coating with a polymer, for example polyvinyl alcohol (PVA), is needed to stabilize the SPION and prevent aggregation. As the particles are foreign to the body, reaction against the SPION could occur. In this study we investigated the effects that SPION may have on experimental arthritis after intra-articular (i.a.) or intravenous (i.v.) injection. METHODS: PVA-coated SPION were injected either i.a. (6 or 24 µg iron) or i.v. (100 µg or 1 mg iron) into naïve Toll-like receptor-4 deficient (TLR4-/-) or wild-type C57Bl/6 mice, or C57Bl/6 mice with antigen-induced arthritis. As control, some mice were injected with PVA or PBS. MR imaging was performed at 1 and 7 days after injection. Mice were sacrificed 2 hours and 1, 2, 7, 10 and 14 days after injection of the SPION, and RNA from synovium and liver was isolated for pro-inflammatory gene expression analysis. Serum cytokine measurements and whole knee joint histology were also performed. RESULTS: Injection of a high dose of SPION or PVA into naïve knee joints resulted in an immediate upregulation of pro-inflammatory gene expression in the synovium. A similar gene expression profile was observed after SPION or PVA injection into knee joints of TLR4-/- mice, indicating that this effect is not due to LPS contamination. Histological analysis of the knee joints also revealed synovial inflammation after SPION injection. Two hours after i.v. injection of SPION or PVA into naïve mice, an upregulation of pro-inflammatory gene expression was detected in the liver. Administration of SPION or PVA into arthritic mice via i.a. injection did not result in an upregulation in gene expression and also no additional effects were observed on histology. MR imaging and histology showed long-term retention of SPION in the inflamed joint. However, 14 days after the injections no long-term effects were evident for gene expression, histology or serum cytokine concentrations. CONCLUSIONS: Injection of SPION, either locally or systemically, gives an acute inflammatory response. In the long term, up to 14 days after the injection, while the SPION reside in the joint, no further activating effects of SPION were observed. Hence, we conclude that SPION do not aggravate arthritis and can therefore be used safely to detect joint inflammation by MR imaging.

Monitoring the effects of dexamethasone treatment by MRI using in vivo iron oxide nanoparticle-labeled macrophages.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic disease causing recurring inflammatory joint attacks. These attacks are characterized by macrophage infiltration contributing to joint destruction. Studies have shown that RA treatment efficacy is correlated to synovial macrophage number. The aim of this study was to experimentally validate the use of in vivo superparamagnetic iron oxide nanoparticle (SPION) labeled macrophages to evaluate RA treatment by MRI. METHODS: The evolution of macrophages was monitored with and without dexamethasone (Dexa) treatment in rats. Two doses of 3 and 1 mg/kg Dexa were administered two and five days following induction of antigen induced arthritis. SPIONs (7 mg Fe/rat) were injected intravenously and the knees were imaged in vivo on days 6, 10 and 13. The MR images were scored for three parameters: SPION signal intensity, SPION distribution pattern and synovial oedema. Using 3D semi-automated software, the MR SPION signal was quantified. The efficacy of SPIONs and gadolinium chelate (Gd), an MR contrast agent, in illustrating treatment effects were compared. Those results were confirmed through histological measurements of number and area of macrophages and nanoparticle clusters using CD68 immunostaining and Prussian blue staining respectively. RESULTS: Results show that the pattern and the intensity of SPION-labeled macrophages on MRI were altered by Dexa treatment. While the Dexa group had a uniform elliptical line surrounding an oedema pocket, the untreated group showed a diffused SPION distribution on day 6 post-induction. Dexa reduced the intensity of SPION signal 50-60% on days 10 and 13 compared to controls (P = 0.00008 and 0.002 respectively). Similar results were found when the signal was measured by the 3D tool. On day 13, the persisting low grade arthritis progression could not be demonstrated by Gd. Analysis of knee samples by Prussian blue and CD68 immunostaining confirmed in vivo SPION uptake by macrophages. Furthermore, CD68 immunostaining revealed that Dexa treatment significantly decreased the area and number of synovial macrophages. Prussian blue quantification corresponded to the macrophage measurements and both were in agreement with the MRI findings. CONCLUSIONS: We have demonstrated the feasibility of MRI tracking of in vivo SPION-labeled macrophages to assess RA treatment effects.

Active involvement of alarmins S100A8 and S100A9 in the regulation of synovial activation and joint destruction during mouse and human osteoarthritis.

OBJECTIVE: To investigate whether alarmins S100A8 and S100A9 are involved in mediating cartilage destruction during murine and human osteoarthritis (OA). METHODS: Two different murine models of OA that differed in terms of synovial activation were compared. Cartilage destruction was measured histologically. Synovial biopsy and serum samples from OA patients were derived from the Cohort Hip and Cohort Knee (CHECK) patients with symptomatic early OA. Expression of mediators in the synovium was measured by reverse transcription-polymerase chain reaction analysis and immunolocalization. RESULTS: In collagenase-induced OA, which showed marked synovial activation, interleukin-1ß was expressed at significant levels only during the early stages of disease, whereas S100A8 and S100A9 expression remained high for a prolonged period of time (up to day 21 after induction). In S100A9-knockout mice, we found a major impact of S100A8 and S100A9 on synovial activation (62% inhibition) and OA cartilage destruction (45-73% inhibition) as compared to wild-type controls. In contrast, in the surgically induced destabilized medial meniscus model, in which synovial involvement is scant, we found no role of S100A8 and S100A9 in the focal OA cartilage destruction. Examination of arthroscopic synovial biopsy samples from patients in the early symptomatic OA CHECK cohort revealed substantial levels of S100A8 and S100A9 messenger RNA and protein, which correlated significantly with synovial lining thickness, cellularity in the subintima, and joint destruction. Levels of S100A8/A9 serum protein were significantly enhanced (19%) at baseline in patients who had pronounced progression of joint destruction after 2 years. CONCLUSION: Our data suggest that the S100A8 and S100A9 proteins are crucially involved in synovial activation and cartilage destruction during OA and that high levels may predict joint destruction in humans with OA.

Tumor necrosis factor-interleukin-17 interplay induces S100A8, interleukin-1ß, and matrix metalloproteinases, and drives irreversible cartilage destruction in murine arthritis: rationale for combination treatment during arthritis.

OBJECTIVE: To examine whether synovial interleukin-17 (IL-17) expression promotes tumor necrosis factor (TNF)-induced joint pathologic processes in vivo, and to analyze the surplus ameliorative value of neutralizing IL-17 in addition to TNF during collagen-induced arthritis (CIA). METHODS: Adenoviral vectors were used to induce overexpression of IL-17 and/or TNF in murine knee joints. In addition, mice with CIA were treated, at different stages of arthritis, with soluble IL-17 receptor (sIL-17R), TNF binding protein (TNFBP), or the combination. RESULTS: Overexpression of IL-17 and TNF resulted in joint inflammation and bone erosion in murine knees. Interestingly, IL-17 strikingly enhanced both the joint-inflammatory and joint-destructive capacity of TNF. Further analysis revealed a strongly enhanced up-regulation of S100A8, IL-1ß, and matrix metalloproteinase (MMP) messenger RNA, only when both TNF and IL-17 were present. Moreover, the increase in irreversible cartilage destruction was not merely the result of enhanced inflammation, but also was associated with a direct synergistic effect of these cytokines in the joint. S100A9 deficiency in mice protected against IL-17/TNF-induced expression of cartilage NITEGE neoepitopes. During established arthritis, the combination of sIL-17R and TNFBP was more effective than the anticytokine treatments alone, and significantly inhibited further joint inflammation and cartilage destruction. CONCLUSION: Local synovial IL-17 expression enhances the role of TNF in joint destruction. Synergy between TNF and IL-17 in vivo results in striking exaggeration of cartilage erosion, in parallel with a synergistic up-regulation of S100A8, IL-1ß, and erosive MMPs. Moreover, neutralizing IL-17 in addition to TNF further improves protection against joint damage and is still effective during late-stage CIA. Therefore, compared with anti-TNF alone, combination blocking of TNF and IL-17 may have additional therapeutic value for the treatment of destructive arthritis.

S100A8 enhances osteoclastic bone resorption in vitro through activation of Toll-like receptor 4: implications for bone destruction in murine antigen-induced arthritis.

OBJECTIVE: Rheumatoid arthritis, which is associated with elevated levels of S100A8 and S100A9, is characterized by severe bone erosions caused by enhanced osteoclast formation and activity. The aim of the present study was to investigate the role of S100A8 and S100A9 in osteoclastic bone destruction in murine antigen-induced arthritis (AIA). METHODS: Bone destruction was analyzed in the arthritic knee joints of S100A9-deficient mice in which S100A8 protein expression was also lacking, and in wild-type (WT) controls. Osteoclast precursors from S100A9-deficient and WT mice were differentiated into osteoclasts in vitro. Additionally, precursors were stimulated with S100A8, S100A9, or S100A8/A9 during osteoclastogenesis. Receptor involvement was investigated using an anti-receptor for advanced glycation end products (anti-RAGE)-blocking antibody, soluble RAGE, or Toll-like receptor 4 (TLR-4)-deficient osteoclast precursors. The formation of osteoclasts and actin rings, the regulation of osteoclast markers, and bone resorption were analyzed. RESULTS: Bone erosions and cathepsin K staining were significantly suppressed in S100A9-deficient mice after AIA induction. However, osteoclast precursors from S100A9-deficient mice developed normally into functional osteoclasts, which excludes a role for intrinsic S100A8/A9. In contrast to the results observed with S100A9 and S100A8/A9, the addition of S100A8 during osteoclastogenesis resulted in stimulation of osteoclast formation in conjunction with enhanced actin ring formation and increased bone resorption. Analysis of the putative receptor for S100A8 in osteoclastogenesis revealed that osteoclast differentiation and function could not be inhibited by blocking RAGE, whereas the increase in osteoclast numbers and enhanced bone resorption were completely abrogated using TLR-4-deficient osteoclast precursors. CONCLUSION: These results demonstrate that S100A8 stimulated osteoclast formation and activity and suggest that both S100A8 and TLR-4 are important factors in mediating osteoclastic bone destruction in experimental arthritis.

S100A8 causes a shift toward expression of activatory Fcγ receptors on macrophages via toll-like receptor 4 and regulates Fcγ receptor expression in synovium during chronic experimental arthritis.

OBJECTIVE: The levels of both Fcγ receptor (FcγR) and the alarmins S100A8 and S100A9 are correlated with the development and progression of cartilage destruction during antigen-induced arthritis (AIA). This study was undertaken to study the active involvement of S100A8, S100A9, and S100A8/S100A9 in FcγR regulation in murine macrophages and synovium during AIA. METHODS: Recombinant murine S100A8 (rS100A8) was injected into normal mouse knee joints, and the synovium was isolated for analysis of FcγR messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR). Macrophages, including bone marrow macrophages derived from Toll-like receptor 4-deficient (TLR-4(-/-)) mice, and polymorphonuclear cells (PMNs) were stimulated with S100 proteins, and levels of FcγR mRNA and protein were measured using RT-PCR and fluorescence-activated cell sorting analyses. AIA was induced in the knee joints of S100A9-deficient (S100A9(-/-)) mice, compared with wild-type (WT) controls, and the extent of cartilage destruction was determined using immunohistochemical analysis. RESULTS: Intraarticular injection of rS100A8 into the knee joints of normal mice caused a strong up-regulation of mRNA levels of activating FcγRI (64-fold increase) and FcγRIV (256-fold increase) in the synovium. Stimulation of macrophages with rS100A8 led to significant up-regulation of mRNA and protein levels of FcγRI and FcγRIV, but not FcγRIII, while the effects of S100A9 or S100A8/S100A9 complexes were less potent. Stimulation of PMNs (32Dcl3 cell line) with S100 proteins had no effect on FcγR expression. Up-regulation of FcγRI and FcγRIV was abrogated in rS100A8-stimulated macrophages from TLR-4(-/-) mice, indicating that the induction of FcγR expression by S100A8 is mediated by TLR-4. FcγR expression in the inflamed synovium of S100A9(-/-) mice was significantly lower on day 14 after arthritis induction when compared with WT controls, and these findings correlated with reduced severity of matrix metalloproteinase-mediated cartilage destruction. CONCLUSION: S100A8 is a strong promoter of activating FcγRI and FcγRIV in macrophages through the activation of TLR-4, and acts as a regulator of FcγR expression in inflamed synovium in chronic experimental arthritis.

Inflammatory role of ASC in antigen-induced arthritis is independent of caspase-1, NALP-3, and IPAF.

Because IL-1beta plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC(-/-) mice showed reduced severity of AIA, decreased levels of synovial IL-1beta, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-gamma, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC(-/-) T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC(-/-) mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.

Selectively induced death of macrophages in the synovial lining of murine knee joints using 10B-liposomes and boron neutron capture synovectomy.

OBJECTIVE: To investigate whether macrophages in the synovial lining can be selectively eliminated by local administration of an improved boron-10 ((10)B) containing liposome formulation combined with neutron irradiation (boron neutron capture synovectomy [BNCS]). METHODS: Disodium dodecahydrododecaborate (Na(2)(10)B(12)H(12)) was encapsulated into unilamellar liposomes ((10)B-liposomes). (10)B-liposomes were injected into the mouse knee joint. Amounts of (10)B in synovial tissue were measured over time using inductively coupled plasma-optical emission spectrometry (ICP-OES). Arthritis was induced in knee joints of mice. Joint inflammation and cartilage destruction was measured using histology. RESULTS: When a 10 microl (10)B-liposome solution (containing 40 microg (10)B) was injected into the murine knee joint, high concentrations of (10)B were measured in macrophages in the synovial lining (At 24 h 306+/-226 microg. g(-1) macrophages). Completing the BNCS by neutron irradiation of the legs 24 h after (10)B-liposome injection showed a clear selective depletion of macrophages in synovial lining of the knee joints. An estimated total physical dose of 13+/-9 Gy was given to the macrophages. When arthritis was induced in the macrophage-depleted joints, swelling of the knee was significantly lower as compared to the controls (53% and 79% lower at days 1 and 3, respectively). Histology confirmed the influx of inflammatory cells was strongly decreased and severe cartilage destruction was almost completely prevented. CONCLUSION: BNCS using an improved (10)B-containing liposome formulation can cause selective depletion of macrophages in the synovial lining of murine knee joints. As a result of this proof-of principle, future applications are recommended.

Oral administration of the NADPH-oxidase inhibitor apocynin partially restores diminished cartilage proteoglycan synthesis and reduces inflammation in mice.

Apocynin, an inhibitor of NADPH-oxidase, is known to partially reverse the inflammation-mediated cartilage proteoglycan synthesis in chondrocytes. More recently, it was reported that apocynin prevents cyclooxygenase (COX)-2 expression in monocytes. The present study aimed to investigate whether these in vitro features of apocynin could be confirmed in vivo. In a mouse model of zymosan-induced acute arthritis apocynin was administered orally (0, 3.2, 16 and 80 microg/ml in the drinking water) and the effects on cartilage proteoglycan synthesis were monitored. In a mouse model of zymosan-induced inflammation of the ears apocynin was administered orally (14 mg/kg/day by gavage) and the effects on ear swelling and ex vivo produced prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated blood cells were measured. In this study, ibuprofen was used as a positive control (50 mg/kg/day by gavage) and animals received vehicle as a negative control. Apocynin dose-dependently reversed the inhibition of proteoglycan synthesis in articular cartilage of the arthritic joint. A statistically significant increase in proteoglycan synthesis was found at a dose of 80 microg/ml apocynin. Apocynin did not affect the proteoglycan synthesis of the control knee joints. Apocynin significantly decreased the zymosan-induced ear swelling at 1, 2 and 4 h (hours) after zymosan injection versus the vehicle treated group at 14 mg/kg/day. The ex vivo production of PGE2 by LPS-stimulated blood cells was significantly decreased after in vivo apocynin treatment. Ibuprofen decreased ear swelling at the same time-points as apocynin and inhibited the ex vivo produced PGE2. In conclusion, the present study confirmed two important features of apocynin in vivo: (1) oral administration of apocynin can partially reverse the inflammation-induced inhibition of cartilage proteoglycan synthesis, and (2) oral administration of apocynin has COX inhibitory effects similar to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen. Therefore, apocynin might be of potential use during the treatment of chronic inflammatory joint diseases like osteoarthritis or rheumatoid arthritis.

Influence of heme oxygenase 1 modulation on the progression of murine collagen-induced arthritis.

OBJECTIVE: Heme oxygenase 1 (HO-1) can be induced by inflammatory mediators as an adaptive response. The objective of the present study was to determine the consequences of HO-1 modulation in the murine collagen-induced arthritis (CIA) model. METHODS: DBA/1J mice were treated with an inhibitor of HO-1, tin protoporphyrin IX (SnPP), or with an inducer of HO-1, cobalt protoporphyrin IX (CoPP), from day 22 to day 29 after CIA induction. The clinical evolution of disease was monitored visually. At the end of the experiment, joints were examined for histopathologic changes. Cytokine levels in paws were measured by enzyme-linked immunosorbent assay. Levels of HO-1, cyclooxygenase 2 (COX-2), and prostaglandin E2 (PGE2) were determined. Effects of treatments on the early phase of disease and after prophylactic administration were also assessed. RESULTS: CoPP strongly induced HO-1, resulting in the inhibition of cartilage erosion accompanied by extensive fibrosis in the joint. Levels of tumor necrosis factor alpha (TNFalpha), interleukin-2 (IL-2), and IL-10 were inhibited by CoPP, whereas levels of vascular endothelial growth factor were increased. Treatment with SnPP significantly reduced the severity of CIA, with inhibition of joint inflammation and cartilage destruction. The levels of PGE2, IL-1beta, and TNFalpha were also significantly reduced by SnPP treatment, which did not modify COX-2 protein expression. SnPP was more effective than CoPP in preventing the development of CIA (prophylactic administration). CONCLUSION: HO-1 is induced during CIA. Although overexpression of this protein causes some beneficial effects, strategies aimed at HO-1 overexpression cannot slow the progression of the chronic inflammatory disease, whereas treatment with SnPP, which inhibits HO-1, exerts prophylactic and therapeutic effects.

Selective COX-2 inhibition by a Pterocarpus marsupium extract characterized by pterostilbene, and its activity in healthy human volunteers.

In this study, an extract of Pterocarpus marsupium Roxb. containing pterostilbene has been evaluated for its PGE2-inhibitory activity in LPS-stimulated PBMC. In addition, the COX-1/2 selective inhibitory activity of P. marsupium (PM) extract was investigated. Biological activity, as well as safety of PM extract was evaluated in healthy human volunteers. PM extract, pterostilbene and resveratrol inhibited PGE2 production from LPS-stimulated human peripheral blood mononuclear cells (PBMC) with IC50 values of 3.2 +/- 1.3 microg/mL, 1.0 +/- 0.6 microM and 3.2 +/- 1.4 microM, respectively. When pterostilbene content of PM extract is calculated, PGE2 production inhibition of PM extract is comparable to PGE2 production inhibition of purified pterostilbene. Furthermore, in a COX-1 whole blood assay (WBA) PM extract was not effective while in a COX-2 WBA, PM extract decreased PGE2 production indicating COX-2 specific inhibition. In healthy human volunteers, the oral use of 450 mg PM extract did not decrease PGE2 production ex vivo in a WBA. Pterostilbene levels in serum were increased, but were 5-fold lower than the observed IC50 for PGE2 inhibition in LPS-stimulated PBMC. No changes from base-line of the safety parameters were observed and no extract-related adverse events occurred during the study. In conclusion, this is the first study to describe the selective COX-2 inhibitory activity of a Pterocarpus marsupium extract. Moreover, the PGE2 inhibitory activity of PM extract was related to its pterostilbene content. In humans, 450 mg PM extract resulted in elevated pterostilbene levels in serum, which were below the active concentration observed in vitro. In addition, short-term supplementation of 450 mg PM extract is considered to be a safe dose based on the long history of use, the absence of abnormal blood cell counts and blood chemistry values and the absence of extract-related adverse events. This strongly argues for a dose-finding study of PM extract in humans to corroborate the in vitro observed inhibitory activity on PGE2 production in order to resolve the potential use of PM extract in inflammatory disorders and/or inflammatory pain.

Male IL-6 gene knock out mice developed more advanced osteoarthritis upon aging.

OBJECTIVE: Interleukin-6 (IL-6) is expressed in osteoarthritic joints but its function in osteoarthritis (OA) is unknown. To study this, spontaneous and experimental OA were evaluated in IL-6 deficient (IL-6(-/-)) mice. DESIGN: Histology of knees of 18-23-month-old wild type (wt) and IL-6(-/-) mice was compared for signs of OA. Cartilage proteoglycan (PG) density was measured by image analysis on safranin-O stained whole knee sections. Chondrocyte PG synthesis was measured ex vivo by (35)S-sulfate incorporation. Knee bone mineral density (BMD) was measured by dual energy x-ray absorptiometry. In young mice (3 months), OA was induced by intra-articular injection of collagenase. RESULTS: The incidence of extensive cartilage loss at both lateral and medial sides was markedly higher in old IL-6(-/-) males, but not in females, as compared to their wt controls. Compared to age-matched wt mice, reduced ex vivo PG synthesis was found during aging in IL-6(-/-) males, without affecting their cartilage PG density. IL-6(-/-) males showed more extensive extracellular matrix deposition in the collateral ligaments and subchondral bone sclerosis, predominantly at the medial side. Total knee BMD decreased more in IL-6(-/-) (-23%) than in wt (-10%) males during aging. Collagenase-induced OA showed a similar degree of joint pathology in both strains, implying that OA susceptibility was not different at younger age. CONCLUSIONS: Upon aging, IL-6(-/-) male mice developed more severe spontaneous OA. Reduced PG synthesis and BMD values might be indicative for an impaired repair response in IL-6(-/-) mice. This suggests a protective role for IL-6 in age-related OA in male mice.

Citrullination of synovial proteins in murine models of rheumatoid arthritis.

OBJECTIVE: Antibodies directed to citrulline-containing proteins are highly specific for rheumatoid arthritis (RA) and can be detected in up to 80% of patients with RA. Citrulline is a nonstandard amino acid that can be incorporated into proteins only by posttranslational modification of arginine by peptidylarginine deiminase (PAD) enzymes. The objective of this study was to investigate the presence of anticitrulline antibodies, PAD enzymes, and citrullinated antigens in mouse models of both acute and chronic destructive arthritis: streptococcal cell wall (SCW)-induced arthritis and collagen-induced arthritis (CIA), respectively. METHODS: Synovial tissue biopsy specimens were obtained from naive mice, mice with CIA, and mice with SCW-induced arthritis. The expression of messenger RNA (mRNA) for PAD enzymes was analyzed by reverse transcriptase-polymerase chain reaction; the presence of PAD proteins and their products (citrullinated proteins) was analyzed by Western blotting and by immunolocalization. The presence of anticitrullinated protein antibodies was investigated by an anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) and an ELISA using in vitro citrullinated fibrinogen. RESULTS: In both mouse models, PAD type 2 (PAD2) mRNA was present in the synovium but was not translated into PAD2 protein. In contrast, PAD4 mRNA, although absent from healthy synovium, was readily transcribed and translated by polymorphonuclear neutrophils infiltrating the synovial tissue during inflammation. As a consequence, several synovial proteins were subjected to citrullination. One of these proteins was identified as fibrin, which has been reported to be citrullinated also in synovium of patients with RA. Although generation of citrullinated antigens during synovial inflammation in the mice was eminent, no anti-CCP antibodies could be detected. CONCLUSION: Citrullination of synovial antigens is an active process during joint inflammation in both mice and humans, but the induction of autoantibodies directed to these proteins is a more specific phenomenon, detectable only in human RA patients.

Deficiency of NADPH oxidase components p47phox and gp91phox caused granulomatous synovitis and increased connective tissue destruction in experimental arthritis models.

Recent studies indicated that the nicotinamide dinucleotide phosphate oxidase (NADPH) oxidase-derived oxygen radicals plays a deleterious role in arthritis. To study this in more detail, gonarthritis was induced in NADPH oxidase-deficient mice. Mice received an intraarticular injection of either zymosan, to elicit an irritant-induced inflammation, or poly-L-lysine coupled lysozyme, to evoke an immune-complex mediated inflammation in passively immunized mice. In contrast to wild-type mice, arthritis elicited in both p47phox(-/-) and gp91(-/-) mice showed more severe joint inflammation, which developed into a granulomatous synovitis. Treatment with either Zileuton or cobra venom factor showed that the chemokines LTB4 and complement C3 were not the driving force behind the aggravated inflammation in these mice. Arthritic NADPH oxidase-deficient mice showed irreversible cartilage damage as judged by the enhanced aggrecan VDIPEN expression, and chondrocyte death. Furthermore, only in the absence of NADPH oxidase-derived oxygen radicals, the arthritic joints showed osteoclast-like cells, tartrate-resistant acid phosphatase (TRAP)-positive/multinucleated cells, extensive bone erosion, and osteolysis. The enhanced synovial gene expression of tumor necrosis factor-alpha, interleukin-1alpha, matrix metalloproteinase (MMP)-3, MMP-9 and receptor activator of NF-kappaB ligand (RANKL) might contribute to the aggravated arthritis in the NADPH oxidase-deficient mice. This showed that the involvement of NADPH oxidase in arthritis is probably far more complex and that oxygen radicals might also be important in controlling disease severity, and reducing joint inflammation and connective tissue damage.