Anti-TNF-α antibody allows healing of joint damage in polyarthritic transgenic mice
© Shealy et al., licensee BioMed Central Ltd 2002
Received: 14 August 2001
Accepted: 20 May 2002
Published: 28 June 2002
Anti-tumor-necrosis-factor-α (TNF-α) monoclonal antibody was used to treat Tg197 transgenic mice, which constitutively produce human TNF-α (hTNF-α) and develop a progressive polyarthritic disease. Treatment of both young (7- or 8-week-old) and aged (27- or 28-week-old) mice commenced when at least two limbs showed signs of moderate to severe arthritis. The therapeutic efficacy of anti-TNF-α antibody was assessed using various pathological indicators of disease progression. The clinical severity of arthritis in Tg197 mice was significantly reduced after anti-TNF-α treatment in comparison with saline-treated mice and in comparison with baseline assessments in both young and aged mice. The treatment with anti-TNF-α prevented loss of body weight. Inflammatory pathways as reflected by elevated circulating hTNF-α and local expression of various proinflammatory mediators were all diminished by anti-TNF-α treatment, confirming a critical role of hTNF-α in this model of progressive polyarthritis. More importantly, the amelioration of the disease was associated with reversal of existing structural damage, including synovitis and periosteal bone erosions evident on histology. Repair of cartilage was age dependent: reversal of cartilage degradation after anti-TNF-α treatment was observed in young mice but not in aged mice.
Keywordsantibody animal models cytokines rheumatoid arthritis tumor necrosis factor alpha
Rheumatoid arthritis (RA) is a significant, chronic disease that afflicts 1% of the general population in most countries . Joint damage typically occurs before patients are diagnosed, and most of the joint destruction occurs within the first 2 years of diagnosis . Therapeutic drugs such as sulfasalazine and methotrexate (MTX) only slow the progression of the disease, suggesting that these drugs fail to adequately quell the underlying pathophysiology of RA .
Tumor necrosis factor alpha (TNF-α) is elevated in sera and synovial fluid of patients with RA, suggesting that it may play a role in the pathology of the disease . Soluble TNF-α receptor or neutralizing antibodies against TNF-α have been shown to prevent collagen-induced arthritis in mice [5, 6]. Furthermore, a human TNF-α (hTNF-α) transgenic mouse (Tg197) develops a chronic, progressive polyarthritis with histologic features in common with RA . Weight loss and joint swelling in these mice are correlated with expression of hTNF-α mRNA in the joints  and hTNF-α concentrations in the serum [8, 9]. Previously, Keffer et al. showed that progression of disease in these mice could be prevented by treatment with anti-TNF-α monoclonal antibody (mAb). In this study, we utilized the Tg197 mouse model to further assess whether anti-TNF-α treatment can ameliorate established disease in both young and aged mice.
Materials and methods
Tg197 mice and anti-TNF-α mAb treatment
Heterozygous Tg197 transgenic mice, obtained from Dr George Kollias (Hellenic Pasteur Institute, Athens, Greece) , were identified by PCR analysis using primers to the 3'-modified hTNF-α gene. Two experimental groups of 25 mice were used. 'Young mice' were enrolled into the study when they developed clinical signs of arthritis at 7 or 8 weeks of age. A second group of mice was maintained disease-free with 10 mg/kg of anti-TNF-α mAb weekly starting at 4 weeks of age; when they reached 24 weeks of age, the treatment was stopped and these 'aged mice' were allowed to develop arthritis before they were enrolled into the study. The enrollment criteria for both young and aged mice required them to have a clinical score of at least 2 on two limbs. The experimental protocols were approved by Centocor's Institutional Animal Care and Use Committee.
The murine anti-TNF-α mAb used in this study binds with high affinity to hTNF-α and prevents binding to the TNF receptor . Anti-TNF-α mAb was administered by intraperitoneal injection at 10 mg/kg weekly and the animals in the control group received weekly injections of saline.
Clinical scores were based on a previously described scoring system  modified as follows: 0 (normal), 1 (edema or distortion of paw or ankle joints), 2 (distortion of paw and ankle joints), or 3 (ankylosis of wrist or ankle joints). The sum of all four paws was scored weekly, with a maximum possible score of 12 per mouse.
Histologic processing of joints
Serum samples were separated from whole blood, which was collected by intracardiac puncture at baseline and at 3, 6, and 16 weeks post-treatment. Serum levels of hTNF-α were determined by ELISA at a 1:2 dilution according to the manufacturer's instructions (Biosource International, Camarillo, CA, USA).
RNA isolation and RNase protection assay
Samples were prepared as described in the Supplementary material and analyzed in an RNase protection assay according to the manufacturer's instructions (PharMingen, San Diego, CA, USA).
All data are expressed as means ± standard deviation unless noted otherwise. Statistical significance was tested using analysis of variance for multiple groups. If significant differences were found, pair-wise testing was performed using Tukey's test. The level of significance for all comparisons was set at P < 0.05.
Amelioration of established polyarthritis by treatment with anti-TNF-α mAb
Effects of treatment with anti-TNF-α mAb on joint histopathology
Inhibition of TNF-α and other proinflammatory cytokines
Currently, MTX is the most widely used disease-modifying antirheumatic drug for the treatment of RA. However, MTX treatment rarely results in complete disease remission  and only slows the progression of joint erosion . In addition, long-term treatment with MTX is associated with pulmonary  and liver toxicities  and other side effects. Therefore, a treatment option that facilitates reversal of joint damage and has longer effectiveness and fewer side effects is desirable.
Experimental arthritis models have contributed to the basic understanding of joint disease and to the development of effective antiarthritic agents . Several models have been used to mimic human RA, ranging from immunization with cartilage components to infection with joint trophic organisms [20, 21]. Blocking TNF-α [6, 22] or IL-1 [23, 24] in these models has routinely shown benefit, although some questions remain regarding the role of these cytokines in mitigating joint inflammation versus preventing cartilage degradation and bone erosion .
Using the Tg197 mice, we have shown that maintenance anti-TNF-α therapy, initiated after joint inflammation and erosions have occurred, allows damaged joints to heal. Specifically, anti-TNF-α mAb proved equally effective in reversing joint synovitis and erosions both in young mice, where an active repair process to damage occurs, and aged mice, where the repair process has relatively slowed. However, the repair of cartilage damage was different between young and aged mice. Cartilage in young mice treated with anti-TNF-α was significantly improved relative to saline-treated and baseline mice. By comparison, treatment of aged mice with anti-TNF-α prevented further cartilage damage but did not improve histological scores relative to baseline values. Mechanistically, the therapeutic effect of anti-TNF-α appears to be due to either neutralization of soluble hTNF-α or inhibition of hTNF-α production in the diseased joint. Additionally, other proinflammatory cytokine mRNAs were decreased in the local diseased tissues either through suppression of inflammatory cell infiltration or inhibition of cytokine production. Moreover, anti-TNF-α treatment resulted in a modest inhibition of murine IL-1β production in the diseased joint, which is consistent with previous findings that anti-TNF-α antibody inhibits the generation of IL-1 in collagen-induced arthritis and IL-1, IL-6, and IL-8 in rheumatoid synovial cultures [26, 27]. Our study provides preclinical evidence supporting the use of anti-TNF-α mAb in ameliorating arthritic pathology.
The progressive arthritis observed in Tg197 mice is similar to the pathology in patients with RA. Recent clinical data indicate that the blockade of TNF-α significantly reduces the signs and symptoms of RA [28, 29]and inhibits the progression of structural damage [30, 31]. It remains to be seen whether extended anti-TNF-α therapy might permit regeneration of articular cartilage and bone in established human disease, where multiple etiological pathways may contribute to the RA disease syndrome [32, 33]. Nevertheless, it is likely that much of the RA pathology involves TNF-α activation, and the results from the Tg197 model provide a sound scientific rationale for the therapeutic benefits observed following anti-TNF-α treatment in RA patients.
Progression of established polyarthritis in the Tg197 hTNF-α transgenic mouse can be reversed by treatment with anti-TNF-α mAb, as shown by significant improvement in clinical and histological scores.
Supplementary materials and methods
Histologic processing of joints
Joint tissues from wrists, ankles, elbows, and knees were fixed in 10% buffered formalin overnight, decalcified in 10% formic acid for 18 days, dehydrated, and then embedded in paraffin. Specimens were cut longitudinally to the midline, and 5-m sections mounted for staining with hematoxylin and eosin or toluidine blue. Joint sections stained with hematoxylin and eosin were scored for synovitis and bone erosions, as described elsewhere [12, 13]. Sections stained with toluidine blue were scored for cartilage degradation (0, normal staining; 1, some loss of staining; 2, moderate loss of staining; 3, weak staining; 4, very weak staining; 5, no visible staining). Specimens were examined using light microscopy and scored in a blinded fashion with regard to treatment.
RNA isolation and RNase protection assay
Excised ankle and paw joints were immediately frozen in liquid nitrogen and processed in a freezer/mill (SPEX Certi-Prep, Metuchen, NJ, USA) with liquid nitrogen at 2-minute pulses. RNA was isolated from 50 mg of the joint powder with 1 ml Trizol reagent according to the manufacturer's instructions (Gibco BRL, Grand Island, NY, USA). RNA was extracted with 0.2 ml chloroform, precipitated with 0.5 ml isopropyl alcohol, and resuspended in 30 l diethyl pyrocarbonate-treated water. Ten micrograms of total RNA from each sample was used for RNase protection assay (mCK -2b and -3b Multi-Probe Template Sets, PharMingen, San Diego, CA, USA), exposed to a Phosphor screen, and quantified by a Phosphorimager with the use of Image Quant software (Molecular Dynamics, Sunnyvale, CA, USA). The signal-intensity ratio of different cytokine messenger RNA (mRNA) to GAPDH was determined.
= anti-tumor necrosis factor alpha
= enzyme-linked immunosorbent assay
= human tumor necrosis factor alpha
= monoclonal antibody
= messenger ribonucleic acid
= polymerase chain reaction
= rheumatoid arthritis
= tumor necrosis factor alpha.
The authors thank Dr George Kollias for providing the Tg197 transgenic breeder mice and Dr Bernie Scallon for polymerase chain reaction analysis of the Tg197 mice.
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