Tumor necrosis factor–inhibiting therapy preferentially targets bone destruction but not synovial inflammation in a tumor necrosis factor–driven model of rheumatoid arthritis

Objective

To investigate how tumor necrosis factor (TNF)–inhibiting therapy affects bone destruction and inflammation in a TNF‐driven mouse model of rheumatoid arthritis.

Methods

In order to evaluate the influence of TNF on osteoclastogenesis in vitro, different concentrations of TNF were added to spleen cell–derived monocytes in the absence or presence of different concentrations of RANKL. In addition, the effects of TNF inhibition on osteoclast precursors as well as local bone destruction in vivo were assessed by treating TNF‐transgenic mice with different doses of adalimumab.

Results

TNF stimulated osteoclastogenesis mainly by increasing the number of osteoclast precursor cells in vitro. This TNF effect was independent of the presence of RANKL. In the hTNF‐transgenic mouse model of destructive arthritis, low‐dose TNF‐inhibiting therapy with adalimumab had no effect on synovial inflammation but significantly inhibited local bone destruction and the generation of osteoclasts. This inhibition was accompanied by a reduction in the number of c‐Fms–positive osteoclast precursor cells in the bone marrow and a reduction of the osteoclast precursor pools in the blood and inflamed synovial membrane of hTNF‐transgenic mice.

Conclusion

Low‐dose TNF‐inhibiting therapy significantly reduces bone erosions by reducing the number of circulating and joint‐invading osteoclast precursors. This effect is uncoupled from its antiinflammatory action.

     

Dysregulation of interleukin‐10–dependent gene expression in rheumatoid arthritis synovial macrophages

Objective

The inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin‐1 (IL‐1) are produced by activated macrophages, are key mediators of pathogenesis, and are validated therapeutic targets in rheumatoid arthritis (RA) and seronegative spondylarthritis (SpA). IL‐10 is a potent antiinflammatory cytokine that suppresses macrophage TNFα and IL‐1 production, yet is not effective in suppressing inflammatory arthritis. To gain insight into IL‐10 responses in inflammatory arthritis, we used microarray analysis to determine the patterns of IL‐10–inducible gene expression in freshly isolated RA and seronegative SpA synovial macrophages.

Methods

Macrophages from the synovial fluid of 5 patients with RA and 3 with seronegative SpA (2 with psoriatic arthritis and 1 with ankylosing spondylitis) were isolated by positive selection and stimulated ex vivo with IL‐10 or interferon‐γ (IFNγ). Gene expression was analyzed using Affymetrix microarrays and protocols. Real‐time polymerase chain reaction was used to confirm changes in gene expression.

Results

The number of genes induced by IL‐10 in arthritic macrophages was markedly smaller than that induced in control macrophages, and the strength of induction was lower in arthritic macrophages for most genes. The residual response of arthritic macrophages to IL‐10 stimulation was qualitatively altered, such that IL‐10 preferentially increased expression of IFNγ‐inducible genes. In contrast, arthritic macrophages expressed many IFNγ‐inducible genes prior to stimulation, and their response to IFNγ remained mostly intact.

Conclusion

These results demonstrate that IL‐10 responses are dysregulated in RA synovial macrophages. An altered biologic response to IL‐10, with attenuation of its antiinflammatory function and a concomitant retention of IFNγ‐like activating functions, provides a basis for the lack of efficacy of IL‐10 in suppressing inflammatory arthritis.

     

Antiinflammatory effects of tumor necrosis factor on hematopoietic cells in a murine model of erosive arthritis

Objective

To investigate the mechanisms leading to the influx of inflammatory hematopoietic cells into the synovial membrane and the role of tumor necrosis factor receptor I (TNFRI) and TNFRII in this process in an animal model of rheumatoid arthritis (RA).

Methods

We performed bone marrow transplantations in human TNF–transgenic mice using hematopoietic cells from wild‐type, TNFRI^−/−, TNFRII^−/−, and TNFRI/II^−/− mice as donors and assessed the severity of arthritis histologically. Generation of osteoclasts from the different genotypes was analyzed in vitro and in vivo. Apoptosis was analyzed using annexin V staining as well as TUNEL assays.

Results

Despite lacking responsiveness to TNF in their hematopoietic compartment, mice not only developed full‐blown erosive arthritis but even showed increased joint destruction when compared with mice with a TNF‐responsive hematopoietic compartment. We demonstrated different roles of the 2 different TNFRs in the regulation of these processes. The absence of TNFRI on hematopoietic cells did not affect joint inflammation but markedly attenuated erosive bone destruction via reduced synovial accumulation of osteoclast precursors. In contrast, the absence of TNFRII on hematopoietic cells increased joint inflammation as well as erosive bone destruction via the regulation of osteoclast precursor apoptosis.

Conclusion

Our findings indicate that selective blockade of TNFRI, leaving the antiinflammatory effects of TNFRII unaltered instead of unselectively blocking TNF, might be advantageous in patients with RA.

     

Zoledronic acid protects against local and systemic bone loss in tumor necrosis factor–mediated arthritis

Objective

Increased osteoclast activity is a key factor in bone loss in rheumatoid arthritis (RA). This suggests that osteoclast‐targeted therapies could effectively prevent skeletal damage in patients with RA. Zoledronic acid (ZA) is one of the most potent agents for blocking osteoclast function. We therefore investigated whether ZA can inhibit the bone loss associated with chronic inflammatory conditions.

Methods

Human tumor necrosis factor (TNF)–transgenic (hTNFtg) mice, which develop severe destructive arthritis as well as osteoporosis, were treated with phosphate buffered saline, single or repeated doses of ZA, calcitonin, or anti‐TNF, at the onset of arthritis.

Results

Synovial inflammation was not affected by ZA. In contrast, bone erosion was retarded by a single dose of ZA (−60%) and was almost completely blocked by repeated administration of ZA (−95%). Cartilage damage was partly inhibited, and synovial osteoclast counts were significantly reduced with ZA treatment. Systemic bone mass dramatically increased in hTNFtg mice after administration of ZA, which was attributable to an increase in trabecular number and connectivity. In addition, bone resorption parameters were significantly lowered after administration of ZA. Calcitonin had no effect on synovial inflammation, bone erosion, cartilage damage, or systemic bone mass. Anti‐TNF entirely blocked synovial inflammation, bone erosion, synovial osteoclast formation, and cartilage damage but had only minor effects on systemic bone mass.

Conclusion

ZA appears to be an effective tool for protecting bone from arthritic damage. In addition to their role in antiinflammatory drug therapy, modern bisphosphonates are promising candidates for maintaining joint integrity and reversing systemic bone loss in patients with arthritis.

     

Inhibition of interleukin‐6 receptor directly blocks osteoclast formation in vitro and in vivo

Objective

To investigate the efficacy of a murine anti–interleukin‐6 receptor (anti–IL‐6R) antibody in directly blocking tumor necrosis factor (TNF)– and RANKL‐mediated osteoclastogenesis in vitro and in vivo.

Methods

The efficacy of a murine antibody against IL‐6R in blocking osteoclast differentiation of mononuclear cells stimulated with RANKL was tested. In addition, arthritic human TNFα–transgenic mice were treated with anti–IL‐6R antibody, and osteoclast formation and bone erosion were assessed in arthritic paws.

Results

Blockade of IL‐6R dose dependently reduced osteoclast differentiation and bone resorption in monocyte cultures stimulated with RANKL or RANKL plus TNF. In human TNFα–transgenic mice, IL‐6R blockade did not inhibit joint inflammation, but it strongly reduced osteoclast formation in inflamed joints as well as bone erosion in vivo. Neither the cell influx into joints nor the synovial expression of IL‐6 and RANKL changed with IL‐6R blockade, while the synovial expression of IL‐1 was significantly reduced. In contrast, TNF‐mediated systemic bone loss was not inhibited by IL‐6R blockade.

Conclusion

These data suggest that blockade of IL‐6R directly affects osteoclast formation in vitro and in vivo, suggesting a direct and specific effect of anti–IL‐6R therapy on osteoclasts independently of its antiinflammatory effects. This effect adds significantly to the structure‐sparing potential of pharmacologic blockade of IL‐6R in arthritis.

     

Estrone/17β‐estradiol conversion to,and tumor necrosis factor inhibition by,estrogen metabolites in synovial cells of patients with rheumatoid arthritis and patients with osteoarthritis

Objective

The role of estrogens in rheumatoid arthritis (RA) is debated since both proinflammatory and antiinflammatory effects have been reported. Important evidence of the dual role of estrogens is conversion to various proinflammatory or antiinflammatory metabolites. This study was undertaken to examine the downstream conversion of estrogens in synovial cells from patients with RA or osteoarthritis (OA).

Methods

We studied serum levels of estrone, estrone sulfate, and estrone sulfate membrane transporters, intracellular interconversion of estrone and 17β‐estradiol, and conversion of estrone/17β‐estradiol to various estrogen metabolites in RA and OA synovial cells. The effect of estrogen metabolites on tumor necrosis factor (TNF) secretion was also studied in RA and OA synovial cells.

Results

Serum levels of estrone sulfate were similar in healthy controls and RA patients. Estrone sulfate transporters were present in synovial tissue. Interconversion of estrone and 17β‐estradiol and the expression of converting enzymes of the cytochrome P450 family were similar in RA and OA cells. Using estrone and 17β‐estradiol as substrates, RA and OA synovial cells produced 16α‐, 4‐, and 2‐hydroxylated estrogens and their 4‐ and 2‐methylation products. The levels of 16α‐hydroxylated estrone/17β‐estradiol (16αOH‐estrone/16αOH‐17β‐estradiol) were higher than the levels of all other estrogen metabolites. RA synovial cells produced more 16αOH‐estrone than did OA synovial cells. Importantly, the 16αOH estrogens did not inhibit TNF secretion, whereas all other estrogen metabolites had marked inhibitory effects.

Conclusion

Our findings indicate that precursor estrogens are converted to proinflammatory metabolites, particularly in RA synovial cells. RA synovial cells mainly produce the proproliferative 16αOH‐estrone, which, in addition to 16αOH‐17β‐estradiol, is one of the only 2 estrogens studied that does not inhibit TNF secretion. A preponderance of 16α‐hydroxylated estrogens is an unfavorable sign in synovial inflammation.

     

Nicotine‐induced differential modulation of autoimmune arthritis in the Lewis rat involves changes in interleukin‐17 and anti–cyclic citrullinated peptide antibodies

Objective

Rheumatoid arthritis (RA) is a debilitating autoimmune disease, and smoking is an important environmental factor in a subset of RA patients. A role of the cholinergic antiinflammatory pathway in autoimmune inflammation is increasingly being realized. Nicotine is a major component of cigarette smoke, and it stimulates the α7 nicotinic acetylcholine receptors. Therefore, defining the mechanisms underlying the immunomodulatory effects of nicotine on arthritis is of high relevance. The purpose of this study was to address this issue using the rat adjuvant‐induced arthritis (AIA) model of human RA.

Methods

Lewis rats were immunized subcutaneously with heat‐killed Mycobacterium tuberculosis H37Ra for disease induction. Rats were treated with nicotine intraperitoneally either before (pretreatment) or after (posttreatment) the onset of AIA. Control rats received the vehicle (buffer) in place of nicotine. The severity of arthritis was assessed and graded. The draining lymph node cells were tested for T cell proliferative and cytokine responses against the disease‐related antigen mycobacterial heat‐shock protein 65. The sera were tested for anti–cyclic citrullinated peptide (anti‐CCP) antibodies and anti–mycobacterial Hsp65 antibodies.

Results

Nicotine pretreatment aggravated the arthritis, whereas nicotine posttreatment suppressed the disease. This altered severity of AIA directly correlated with the levels of the anti‐CCP antibodies, of the Th1/Th17 cytokines, and of the corresponding dendritic cell–derived cytokines. The majority of these effects on cellular responses could be replicated in vitro.

Conclusion

Nicotine‐induced modulation of AIA involves specific alterations in the disease‐related cellular and humoral immune responses in AIA. These results are of significance in advancing our understanding of the pathogenesis of RA.

     

Blocking ERK‐1/2 reduces tumor necrosis factor α–induced interleukin‐18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin‐18 binding protein A

Objective

To examine the mechanism of regulation of interleukin‐18 (IL‐18) bioactivity by IL‐18 binding protein (IL‐18BP) induction.

Methods

Levels of IL‐18 and IL‐18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme‐linked immunosorbent assays (ELISAs), followed by calculation of free IL‐18. IL‐18 and IL‐18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real‐time polymerase chain reaction and ELISA, respectively, followed by IL‐18 bioactivity determination using KG‐1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL‐18BPa/IL‐18 regulation. Tumor necrosis factor α (TNFα)–induced caspase 1 activity was determined by a colorimetric assay.

Results

IL‐18BPa was lower in RA synovial fluid than in OA synovial fluid (P < 0.05; n = 8), and free IL‐18 was higher in RA synovial fluid than in OA synovial fluid. TNFα induced RA synovial fibroblast IL‐18BPa and IL‐18 in a time‐dependent manner (P < 0.05). Evaluation of signaling pathways suggested that TNFα induced IL‐18 production through the ERK‐1/2, protein kinase Cδ (PKCδ), and Src pathways, whereas IL‐18BPa synthesis was mediated through the NFκB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL‐18BPa‐Fc reduced the RA synovial fibroblast phosphorylation of ERK‐1/2 induced by TNFα.

Conclusion

These results suggest that IL‐18BPa reduces IL‐18 bioactivity induced by TNFα, by regulating the ERK‐1/2 pathway in RA synovial fibroblasts. Targeting IL‐18 bioactivity by induction or addition of IL‐18BPa may provide another therapeutic option in the management of RA.

     

Tumor necrosis factor α and RANKL blockade cannot halt bony spur formation in experimental inflammatory arthritis

Objective

To investigate the kinetics of bony spur formation and the relationship of bony spur formation to synovial inflammation and bone erosion in 2 rat arthritis models, and to address whether bony spur formation depends on the expression of tumor necrosis factor α (TNFα) or RANKL.

Methods

Analysis of the kinetics of synovial inflammation, bone erosion, osteoclast formation, and growth of bony spurs was performed in rat collagen‐induced arthritis (CIA) and adjuvant‐induced arthritis (AIA). In addition, inhibition experiments were performed to assess whether inhibition of TNFα and RANKL by pegylated soluble TNF receptor type I (pegTNFRI) and osteoprotegerin (OPG), respectively, affected bony spur formation.

Results

Bony spurs emerged from the periosteal surface close to joints, and initial proliferation of mesenchymal cells was noted as early as 3 days and 5 days after onset of CIA and AIA, respectively. Initiation of bony spur formation occurred shortly after the onset of inflammation and bone erosion. Neither pegTNFRI nor OPG could significantly halt the osteophytic responses in CIA and AIA.

Conclusion

These results suggest that bony spur formation is triggered by inflammation and initial structural damage in these rat models of inflammatory arthritis. Moreover, emergence of bony spurs depends on periosteal proliferation and is not affected by inhibition of either TNFα or RANKL. Bony spur formation can thus be considered a process that occurs independent of TNFα and RANKL and is triggered by destructive arthritis.

     

Effectiveness of anti–folate receptor β antibody conjugated with truncated Pseudomonas exotoxin in the targeting of rheumatoid arthritis synovial macrophages

Objective

To define the distribution of folate receptor β (FRβ)–expressing cells in various tissues, including rheumatoid arthritis (RA) synovial tissues, and to verify the effects of an immunotoxin composed of an anti‐FRβ monoclonal antibody (mAb) and truncated Pseudomonas exotoxin A (PEA) on apoptosis and tumor necrosis factor α (TNFα) production by adherent synovial mononuclear cells from RA patients.

Methods

Anti‐FRβ mAb were produced by immunizing mice with FRβ‐transfected murine pre–B cells. The distribution of the FRβ antigen was examined by immunohistochemical analysis using anti‐FRβ mAb and macrophage‐specific anti‐CD163 mAb. Anti‐FRβ mAb was chemically crosslinked with truncated PEA. FRβ‐expressing macrophages were produced by the transfection of adenovirus vector containing the FRβ gene. Apoptotic cells were detected by staining with propidium iodide. TNFα was measured by enzyme‐linked immunosorbent assay.

Results

FRβ‐expressing cells were not present in peripheral blood leukocytes and their activated cells. In all of the tissues examined, most FRβ‐expressing cells were CD163+. The immunotoxin significantly induced the apoptosis of FRβ‐transfected macrophages and adherent RA synovial mononuclear cells and inhibited TNFα production by adherent RA synovial mononuclear cells.

Conclusion

We demonstrated the limited distribution of FRβ‐expressing cells in various tissues. The immunotoxin targeting FRβ‐expressing cells will provide a therapeutic tool for rheumatoid synovitis.

     

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

Objective

Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl‐1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor α (TNFα)–induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG) in down‐regulating Mcl‐1 expression and its mechanism of RA synovial fibroblast sensitization to TNFα‐induced apoptosis.

Methods

EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP‐ribose) polymerase (PARP), Mcl‐1, Bcl‐2, Akt, and nuclear translocation of NF‐κB.

Results

In RA synovial fibroblasts, EGCG (5–50 μM) inhibited constitutive and TNFα‐induced Mcl‐1 protein expression in a concentration‐ and time‐dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl‐1 expression in RA synovial fibroblasts and affected Mcl‐1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl‐1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down‐regulation of the TNFα‐induced Akt and NF‐κB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF‐κB inhibitors. Interestingly, Mcl‐1 degradation by EGCG sensitized RA synovial fibroblasts to TNFα‐induced PARP cleavage and apoptotic cell death.

Conclusion

Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFα‐induced apoptosis by specifically blocking Mcl‐1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.

     

Evidence that anti–tumor necrosis factor therapy with both etanercept and infliximab induces apoptosis in macrophages,but not lymphocytes,in rheumatoid arthritis joints: Extended report

Objective

Treatment of rheumatoid arthritis (RA) with tumor necrosis factor (TNF) antagonists is highly effective, but their mechanisms of action are not completely clear. Since anti‐TNF therapy induces a decrease in synovial cellularity, this study focused on the modulation of RA synovial apoptosis following treatment with either soluble TNF receptor (etanercept) or TNF chimeric monoclonal antibody (infliximab).

Methods

Apoptosis (TUNEL and active caspase 3 staining) and cell surface markers were evaluated by immunohistochemistry in synovial biopsy samples obtained before and after 8 weeks of treatment with etanercept (12 patients) or infliximab (9 patients). We also determined by flow cytometry the in vitro effect of etanercept and infliximab on apoptosis of RA mononuclear cells derived from the synovial fluid (SF) and peripheral blood (PB).

Results

Eight weeks of treatment with etanercept and with infliximab significantly increased synovial apoptosis. This change was accompanied by a significant decrease in the synovial monocyte/macrophage population. The decrease in lymphocyte numbers did not reach statistical significance. In vitro, 24 hours of incubation with either etanercept or infliximab induced apoptosis of the SF monocyte/macrophage population. PB monocyte/macrophages were less susceptible to anti‐TNF–mediated apoptosis. No changes in the rate of apoptosis were observed in the lymphocyte population derived from either SF or PB.

Conclusion

In RA patients, both etanercept and infliximab are able to induce cell type–specific apoptosis in the monocyte/macrophage population. This suggests a potential pathway that would account for the diminished synovial inflammation and the decreased numbers of synovial macrophages evident after TNF blockade.

     

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen‐induced arthritis

Objective

Annexin 1 (Anx‐1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx‐1 in experimental arthritis and in GC‐mediated inhibition of inflammation, using antigen‐induced arthritis (AIA) in Anx‐1 knockout (Anx‐1^−/−) mice.

Methods

Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti‐mBSA IgG were determined by enzyme‐linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real‐time polymerase chain reaction.

Results

A significant exacerbation of arthritis was observed in the Anx‐1^−/− mice compared with wild‐type (WT) mice. This was associated with increased mRNA expression of synovial interleukin‐1β, tumor necrosis factor α, interleukin‐6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx‐1^−/− mice, and also significantly reduced the serum levels of anti‐mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx‐1^−/− mice.

Conclusion

Anx‐1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

     

The relationship between synovial lymphocyte aggregates and the clinical response to infliximab in rheumatoid arthritis: A prospective study

Objective

Some patients with rheumatoid arthritis (RA) exhibit lymphocyte aggregates in the synovium. This study was undertaken to address whether the presence of lymphocyte aggregates before treatment could serve as a biomarker for the clinical response to tumor necrosis factor (TNF) blockade, and to confirm whether the aggregation of synovial lymphocytes is reversible after anti‐TNF treatment.

Methods

Synovial tissue biopsy samples were obtained from 97 patients with active RA before the initiation of infliximab treatment. Lymphocyte aggregates in the synovial tissue were counted and also graded for size. Logistic regression analysis was performed to identify whether the presence of lymphocyte aggregates could be a predictor of the clinical response at week 16. Furthermore, the effects of TNF blockade on lymphocyte aggregates were compared between patients with RA and patients with psoriatic arthritis (PsA).

Results

Fifty‐seven percent of RA synovial tissue samples contained lymphocyte aggregates, and 32% of the patients had large aggregates. Aggregates were found in 67% of clinical responders compared with 38% of nonresponders. The presence of aggregates at baseline was a highly significant predictor of the clinical response to anti‐TNF treatment (R² = 0.10, P = 0.008). Positivity for lymphocyte aggregates increased the power to predict the clinical response (R² = 0.29), when analyzed in a prediction model that included baseline disease activity evaluated by the Disease Activity Score in 28 joints, anti–cyclic citrullinated peptide antibody positivity, and synovial TNFα expression. There was a reduction in lymphocyte aggregates after anti‐TNF antibody therapy in both RA and PsA.

Conclusion

RA patients with synovial lymphocyte aggregates have, on average, a better response to infliximab treatment than those with only diffuse leukocyte infiltration. Moreover, the aggregation of synovial lymphocytes is reversible after anti‐TNF antibody treatment.

     

Inflammation is preceded by tumor necrosis factor‐dependent infiltration of mesenchymal cells in experimental arthritis

Objective

To determine the involvement of mesenchymal progenitor cells in the induction of collagen‐induced arthritis (CIA).

Methods

DBA/1 mice were immunized with type II collagen in adjuvant or adjuvant alone, and the presence of mesenchymal cells in the joints of prearthritic mice was studied by immunohistochemistry.

Results

An analysis of the joints on day 10 postimmunization (at least 10 days before the onset of arthritis) revealed synovial hyperplasia without leukocytic infiltration. Large, round cells expressing bone morphogenetic protein receptors (BMPRs), which serve as markers for primitive mesenchymal cells, were present in increased numbers in the bone marrow adjacent to the joint, in the synovium itself, and within enlarged bone canals that connect the bone marrow to the synovium. Similar changes were observed in mice given adjuvant without collagen. Adjuvant‐induced infiltration of BMPR⁺ cells and enlargement of bone canals were abrogated by anti‐tumor necrosis factor (anti‐TNF) treatment and were absent in TNFR p55/p75^−/− mice. Increased numbers of bone marrow cells and enlarged bone canals were observed in nonimmunized TNF transgenic mice (which spontaneously develop arthritis).

Conclusion

These findings suggest that in CIA, there is an antigen‐independent (innate) prearthritic phase that prepares the joint for the subsequent immune‐mediated arthritis. The induction phase involves marrow‐derived mesenchymal cells and requires the presence of TNF.

     

Exacerbation of antigen‐induced arthritis in inducible nitric oxide synthase–deficient mice

Objective

Inhibition of nitric oxide (NO) produced by inducible NO synthase (iNOS) is suggested to be beneficial in experimental arthritis. Although NO is important for the integrity of the microcirculation, the effects of inhibition of iNOS on the synovial microcirculation are not currently known. This study investigated the synovial microcirculation and leukocyte–endothelial cell interactions in iNOS‐deficient mice with antigen‐induced arthritis (AIA) and compared these findings with disease severity.

Methods

Fourteen homozygous iNOS−/− and 14 iNOS+/+ mice were used. The severity of AIA was assessed by measuring knee joint swelling and by histologic scoring. The number of rolling and adherent leukocytes was quantitatively analyzed in synovial microvessels using intravital microscopy of intraarticular synovial tissue. Nitrite/nitrate concentrations were measured, and the expression of iNOS, E‐ and P‐selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 (VCAM‐1) was assessed by immunohistochemistry.

Results

In iNOS+/+ animals with AIA, the plasma concentration of nitrite/nitrate was increased 3‐fold and iNOS expression was detected in cells of the joint. Swelling of the knee joint as well as leukocyte infiltration were enhanced in the iNOS−/− arthritic animals compared with iNOS+/+ mice with AIA. AIA‐associated leukocyte–endothelial cell interaction in synovial postcapillary venules was more pronounced in iNOS−/−, compared with iNOS+/+, arthritic mice. A strong expression of P‐selectin and VCAM‐1 was observed in the iNOS−/− arthritic mice only.

Conclusion

These data suggest that NO production by iNOS in vivo has antiinflammatory effects in experimental arthritis, by mediating a reduction in leukocyte adhesion and infiltration.

     

Proteasome inhibition aggravates tumor necrosis factor–mediated bone resorption in a mouse model of inflammatory arthritis

Objective

The proteasome inhibitor bortezomib has potent anti‐myeloma and bone‐protective activity. Recently, bortezomib was shown to directly inhibit osteoclastogenesis. The aim of this study was to analyze the influence and therapeutic effect of bortezomib in a mouse model of inflammatory arthritis.

Methods

Heterozygous human tumor necrosis factor α (hTNFα)–transgenic mice and their wild‐type (WT) littermates were intravenously injected with 0.75 mg/kg of bortezomib or phosphate buffered saline twice weekly. The mice were assessed for clinical signs of arthritis. After 6 weeks of treatment, mice were analyzed for synovial inflammation, cartilage damage, bone erosions, and systemic bone changes. Osteoclast precursors from WT and hTNF‐transgenic mice were isolated from bone marrow, treated with bortezomib, and analyzed for osteoclast differentiation, bone resorption, and expression of osteoclast‐specific genes as well as apoptosis and ubiquitination.

Results

Bortezomib‐treated hTNF‐transgenic mice showed moderately increased inflammatory activity and dramatically enhanced bone erosions associated with a significant increase in the number of synovial osteoclasts. Interestingly, bortezomib did not alter systemic bone turnover in either hTNF‐transgenic mice or WT mice. In vitro, treatment with therapeutically relevant concentrations of bortezomib resulted in increased differentiation of monocytes into osteoclasts and more resorption pits. Molecularly, bortezomib increased the expression of TNF receptor−associated factor 6, c‐Fos, and nuclear factor of activated T cells c1 in osteoclast precursors.

Conclusion

In TNF‐mediated bone destruction, bortezomib treatment increased synovial osteoclastogenesis and bone destruction. Hence, proteasome inhibition may have a direct bone‐resorptive effect via stimulation of osteoclastogenesis during chronic arthritis.

     

Decreased pain and synovial inflammation after etanercept therapy in patients with reactive and undifferentiated arthritis: An open‐label trial

Objective

Elevated levels of tumor necrosis factor α (TNFα) have been identified in the synovium of patients with reactive and undifferentiated arthritis, implicating TNFα in the pathogenesis of these disorders. This finding has provided a rationale for the use of TNFα antagonists in the treatment of reactive arthritis; however, the possibility that the triggering microorganism might persist in affected joints and become activated with use of these agents has been of concern.

Methods

The efficacy and safety of etanercept (25 mg subcutaneous twice weekly) in 16 patients with undifferentiated or reactive arthritis was assessed in a 6‐month open‐label trial. Synovial biopsies were performed before and after treatment with etanercept. Polymerase chain reaction (PCR) analysis was performed on the synovial biopsy samples to evaluate for the presence of nucleic acid material of bacterial organisms. Outcome measures including tender and swollen joint counts, pain assessment on a 10‐point visual analog scale, and functional ability as measured by the Health Assessment Questionnaire were determined before and after etanercept therapy.

Results

Ten of 16 patients completed the trial. Six patients withdrew, but none had a worsening of arthritis or infection. Of the 10 completers, 9 could be classified as treatment responders, despite the evidence of bacterial organisms on PCR analysis prior to initiating etanercept in 3 patients; 2 patients became PCR negative on etanercept. Five of 6 patients with adequate synovial biopsy specimens showed improvement, but not normalization of histology.

Conclusion

Etanercept was well‐tolerated without clinical exacerbation of any suspected underlying infections and appeared to provide therapeutic benefit in our cohort of patients with reactive and undifferentiated arthritis.