Effect of phospholipase A2inhibitory peptide on inflammatory arthritis in a TNF transgenic mouse model: a time-course ultrastructural study
© Thwin et al.; licensee BioMed Central Ltd. 2004
Received: 19 January 2004
Accepted: 25 March 2004
Published: 28 April 2004
We evaluated the therapeutic effect of secretory phospholipase A2 (sPLA2)-inhibitory peptide at a cellular level on joint erosion, cartilage destruction, and synovitis in the human tumor necrosis factor (TNF) transgenic mouse model of arthritis. Tg197 mice (N = 18) or wild-type (N = 10) mice at 4 weeks of age were given intraperitoneal doses (7.5 mg/kg) of a selective sPLA2 inhibitory peptide, P-NT.II, or a scrambled P-NT.II (negative control), three times a week for 4 weeks. Untreated Tg197 mice (N = 10) were included as controls. Pathogenesis was monitored weekly for 4 weeks by use of an arthritis score and histologic examinations. Histopathologic analysis revealed a significant reduction after P-NT.II treatment in synovitis, bone erosion, and cartilage destruction in particular. Conspicuous ultrastructural alterations seen in articular chondrocytes (vacuolated cytoplasm and loss of nuclei) and synoviocytes (disintegrating nuclei and vacuoles, synovial adhesions) of untreated or scrambled-P-NT.II-treated Tg197 mice were absent in the P-NT.II-treated Tg197 group. Histologic scoring and ultrastructural evidence suggest that the chondrocyte appears to be the target cell mainly protected by the peptide during arthritis progression in the TNF transgenic mouse model. This is the first time ultrastructural evaluation of this model has been presented. High levels of circulating sPLA2 detected in untreated Tg197 mice at age 8 weeks of age were reduced to basal levels by the peptide treatment. Attenuation of lipopolysaccharide- and TNF-induced release of prostaglandin E2 from cultured macrophage cells by P-NT.II suggests that the peptide may influence the prostaglandin-mediated inflammatory response in rheumatoid arthritis by limiting the bioavailability of arachidonic acid through sPLA2 inhibition.
Keywordspeptide secretory phospholipase A2 inhibition rheumatoid arthritis TNF transgenic mouse model ultrastructural alterations
Secretory phospholipase A2 (sPLA2) is a key enzyme in the production of diverse mediators of inflammatory and related conditions . Because of the crucial role it plays in inflammatory diseases such as rheumatoid arthritis (RA) , sPLA2 is referred to as inflammatory PLA2 . High levels of sPLA2 have been found in synovial tissues and fluid from patients with RA [2, 4]. Purified synovial PLA2 can elicit an inflammatory arthritogenic response when injected into the joint space of healthy rabbits and rats [5, 6]. It has been reported that sPLA2 expression parallels the severity of the inflammatory process with lack of enhancement of cytosolic phospholipase A2 (cPLA2) mRNA in an adjuvant arthritis model, thus indicating the pathogenic role played by sPLA2 . Colocalization studies using primary synovial fibroblasts from RA patients have also suggested sPLA2 as a critical modulator of cytokine-mediated synovial inflammation in RA . As a result of its important role in the inflammatory response, inhibition of sPLA2 is a target for the treatment of inflammatory diseases. Inhibition of sPLA2 could result in suppression of several classes of proinflammatory lipids such as prostaglandins, leukotrienes, platelet-activating factor, and lysophospholipid .
Elevated levels of circulating sPLA2 are usually associated with high blood levels of proinflammatory cytokines , which are used as an indicator of the extent of systemic inflammation [10, 11]. sPLA2 has been shown to activate the production of proinflammatory cytokines in blood and synovial fluid monocytes , suggesting that the two can cooperate to promote inflammation by enhancing each other's secretion. sPLA2 may act on the cells stimulated with such cytokines, leading to augmentation of the inflammatory responses. The fact that cotransgenic sPLA2 and tumor necrosis factor α (TNF-α) mice show more extensive swelling than TNF-α transgenic mice  may be evidence in support of a possible synergism between sPLA2 and TNF. Hence, inhibition of sPLA2 may further help to suppress inflammation in RA by blocking the formation of proinflammatory cytokines.
A significant reduction of the inflammatory response has been reported in animals injected with natural or synthetic sPLA2 inhibitors [14, 15]. Two families of endogenous proteins, namely lipocortins and uteroglobin, have been shown to possess anti-inflammatory properties due to their ability to inhibit sPLA2. Synthetic peptides called antiflammins derived from these proteins are one of the most potent classes of anti-inflammatory agents identified to date . A recombinant protein termed PIP (phospholipase inhibitor from python), which we have expressed from the liver of a nonvenomous snake, Python reticulatus , exhibits in vivo anti-inflammatory activity that correlates well with its in vitro inhibitory potency towards sPLA2. In a clinically relevant model of postsurgical peritoneal adhesion, the peptide analog P-PB.III, which has a fragment of an anti-inflammatory protein PIP included in its sequence, exhibits stronger in vivo anti-inflammatory activity than that displayed by antiflammin . Further screening of the PIP amino acid sequence provides us with a new peptide with improved potency. This new 17-mer peptide 56LGRVDIHVWDGVYIRGR72 is a selective inhibitor of human sPLA2-IIA, with an amino acid sequence corresponding to residues 56–72 of the native protein PIP. It significantly reduces high levels of sPLA2 detected in rat hippocampal homogenates after intracerebroventricular injections of a neurotoxin, kainic acid . These findings establish that peptides or recombinant proteins that inhibit sPLA2, or their peptide derivatives, are highly attractive candidates for clinical development as anti-inflammatory agents.
The present study was designed to investigate the effect of a selective sPLA2-inhibitory peptide, P-NT.II, on ultrastructural changes of ankle-joint synovitis, cartilage degradation, and bone erosion in the Tg197 TNF transgenic mouse model of arthritis , and to assess the effects of peptide intervention on the clinical and histologic indices of RA.
Materials and methods
The generation and characterization of Tg197 human TNF transgenic mice have been previously described . Tg197 mice generated on CBA × C57BL/6 genetic backgrounds and littermate controls were bred and maintained at the animal facilities of the Biomedical Sciences Research Center, Alexander Fleming, Athens, Greece, under specific-pathogen-free conditions. All of the Tg197 mice typically developed polyarthritis 3–4 weeks after birth, whereas nontransgenic (wild-type) mice remained normal. Mice were given conventional oral food and water ad libitum. All procedures involving animals were in compliance with the Declaration of Helsinki principles.
A total of 44 weight-matched mice (34 Tg197 and 10 nontransgenic wild-type littermates) were divided into six groups for subsequent gross observations and histopathologic analyses – untreated Tg197 group (N = 10), P-NT.II-treated Tg197 group (N = 18), scrambled-P-NT.II-treated Tg197 group (N = 6), P-NT.II-treated wild-type group (N = 4), scrambled-P-NT.II-treated wild-type group (N = 4), and Tg197 baseline group – just before the treatment at 4 weeks of age (N = 4). Nontransgenic mice were given the same dose of P-NT.II or scrambled P-NT.II, and the same regimen of treatment, as the Tg197 mice.
Peptide synthesis and administration
P-NT.II (test peptide) and the scrambled P-NT.II (negative control peptide) were synthesized using the solid-phase method with 9-fluorenylmethoxy carbonyl chemistry and were purified and validated as described elsewhere . They were stored lyophilized at -20°C in sealed tubes and were dissolved freshly before use in 0.1% dimethyl sulfoxide (DMSO). Each Tg197 or wild-type mouse was given intraperitoneal injections of P-NT.II or the scrambled P-NT.II (7.5 mg/kg) in 50 μl of vehicle (0.1% final DMSO concentration), three times a week for 4 weeks (i.e. from age 4–8 weeks).
This was done by gross observations based on body-weight measurements and arthritis scoring, which were done twice weekly from 4 weeks (baseline) to 8 weeks of age (end of the study), after which all the animals were killed by CO2 inhalation. The level of severity of clinical arthritis was evaluated based on an arthritis score (AS) taken on both ankle joints. Average scores on a scale of 0–3 were used; 1 = mild arthritis (joint swelling); 2 = moderate arthritis (severe joint swelling and deformation, no grip strength); 3 = severe arthritis (ankylosis detected on flexion, and severely impaired movement) .
The whole ankle joints harvested from the right side of each mouse were fixed overnight in 10% formalin, decalcified in 30% citrate-buffered formic acid for 3 days at 4°C, dehydrated in a graded series of methanol and xylene, and then embedded in paraffin. Thin sections (6 μm thick) were stained with hematoxylin and eosin, and histopathologic scorings performed under the light microscope (Leitz Aristoplan) by a blinded observer. The histopathologic score (HS) was evaluated  using a scale of severity ranging from 1 to 4, where 1 = hyperplasia of the synovial membrane and presence of polymorphonuclear infiltrates, 2 = pannus and fibrous tissue formation and focal subchondral bone erosion, 3 = articular cartilage destruction and bone erosion, and 4 = extensive articular cartilage destruction and bone erosion.
Scoring of joint parameters
Arbitrary scores were used to assess the extent of synovitis, cartilage destruction, and bone erosion. Semiquantitative scores from 0 to 4 were used for each histopathologic parameter . Synovitis: 0 = normal; 1 = mild synovial hypertrophy (<5 cell layers) with few inflammatory cells; 2 = moderate synovial hypertrophy (<20 cell layers) with accumulation of inflammatory cells into intrasynovial cysts; 3 = pannus and fibrous tissue formation; and 4 = pannus and fibrous tissue formation on both sides of the ankle joint. Cartilage damage: 0 = intact; 1 = minor (<10%); 2 = moderate (10–50%); 3 = high (50–80%); and 4 = severe (80–100%). Bone erosions: 0 = normal; 1 = mild (focal subchondral erosion); 2 = moderate (multiple subchondral erosions); 3 = high (as above + focal erosion of talus); and 4 = maximum (multiple erosions of tarsal and metatarsal bones).
Transmission electron microscopy
Ankle joints dissected from the left hind leg of each mouse were split open longitudinally through the midline between the tibia and the talus, prefixed overnight with 2.5% glutaraldehyde in phosphate buffer, pH 7.4, and rinsed with the buffer. After they had been postfixed with 1% osmium tetroxide in phosphate buffer for 2 hours, they were dehydrated in a graded series of ethanol and embedded in epoxy resin (Araldite). Semithin sections (1.0 μm) were cut and stained with methylene blue to reveal their orientation for ultrathin sectioning and for histopathologic scoring under the light microscope. Ultrathin sections (80–90 nm) were then cut with an ultramicrotome (Ultracut E; Riechert-Jung, Leica, Vienna, Austria), mounted on copper grids, counterstained with uranyl acetate and lead citrate, and evaluated in the electron microscope (CM120 Biotwin; FEI Company, Electron Optics, Eindhoven, The Netherlands).
Measurement of serum PLA2
sPLA2 was measured in the serum of transgenic (Tg197) mice and nontransgenic wild-type controls, using an Escherichia coli membrane assay as described previously . In brief, [3H]arachidonate-labeled E. coli membrane suspension (5.8 μCi/μmol, PerkinElmer Life Sciences, Inc, Boston, MA, USA) was used as substrate, and 25 mM CaCl2-100 mM Tris/HCl (pH 7.5) as assay buffer. The reaction mixture, containing substrate (20 μl) and either purified human synovial sPLA2 standard (1–80 ng/ml; Cayman Chemical Company, Ann Arbor, MI, USA) or serum (10 μl), in a final volume of 250 μl in assay buffer, was incubated at 37°C for 1 hour, and the reaction was terminated with 750 μl of chilled phosphate-buffered saline containing 1% bovine serum albumin. Aliquots (500 μl) of the supernatant were then taken, for measurement of the amount of [3H]arachidonate released from the E. coli membrane using liquid scintillation counting (LS 6500 Scintillation Counter; Beckman Inc., Fullerton, CA, USA). The amount of sPLA2 present in the serum was calculated from the standard curve and is expressed as ng/ml ± SEM.
The murine macrophage cell line J774 (American Type Culture Collection, Manassas, VA, USA) was cultured at 37°C in humidified 5% CO2/95% air in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM glutamine, 20 mM HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin. After growing to confluence, the cells were dislodged by scraping, plated in 12 culture wells at a density of 5 × 105 cells/ml per well, and allowed to adhere for 2 hours. Thereafter, the medium was replaced with fresh medium containing lipopolysaccharide (LPS) (2 μg/ml) and one of the PLA2 inhibitors (P-NT.II, scrambled P-NT.II, or LY315920 [Lilly Research Laboratories, Indianapolis, IN, USA], dissolved in DMSO [final concentration 0.1% v/v]). Peptides were tested at various concentrations ranging from 0.01 to 40 μM. After incubation in 5% CO2/95% air at 37°C for 20 hours, culture medium supernatants were collected and stored frozen (-80°C) until use. In parallel experiments, cells were stimulated with mouse recombinant TNF (10 ng/ml; Sigma, St. Louis, MO, USA) for 20 hours, in the presence or absence of 10 μM P-NT.II or LY315920 dissolved in DMSO (0.1% final concentration). Culture medium supernatants were collected after centrifugation (10,000 g, 4°C, 15 min) and stored at -80°C prior to measurement of prostaglandin E2 (PGE2).
Cell viability assays
XTT (sodium 3'-[(phenyl amine carboxyl)-3,4-tetrazolium]-bis(4-methoxy-nitro) benzene sulfonic acid hydrate) Cell Proliferation Kit II (Roche Applied Science) was used to assess the possible cytoxic effect of the peptide P-NT.II on the mouse macrophage J774 cell line.
Measurement of PGE2
PGE2 (EIA kit-monoclonal; Cayman) concentrations were measured in the cell-culture supernatants in accordance with the manufacturer's instructions.
Statistical analyses were performed using GraphPad Prism software to calculate the means and SEMs. Group means were compared by using one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison post test to identify statistically significant differences (i.e. P < 0.05).
Gross histologic findings
Histopathologic assessment of ankle joints
% of total at indicated HS
Time course (weeks)
HS (Mean ± SEM)
3.30 ± 0.11 a
2.12 ± 0.12
2.37 ± 0.12
2.50 ± 0.20
2.62 ± 0.31 b
Scrambled-P-NT.II-treated (negative control peptide)
3.25 ± 0.17 c
Ultrastructural changes in articular cartilage
Ultrastructural changes in synovium
Serum levels of sPLA2
PGE2release from cultured macrophages
Here we report the beneficial effect of peptide treatment, and the ultrastructural changes seen at the cellular level in the articular cartilage and synovium of the ankle joints of TNF transgenic Tg197 mice treated with the anti-inflammatory peptide P-NT.II. While several studies have previously been carried out on the early ultrastructural changes in other animal models of experimental arthritis [23–25], no morphological evaluations in this TNF transgenic mouse model of RA have yet been available, in either the absence or the presence of therapeutic intervention.
The lesions in the TNF transgenic mouse model of arthritis we used in the present study histologically and ultrastructurally resemble RA lesions , with synovial proliferation along the articular surface and subsequent invasion with erosion of the articular cartilage and subchondral bone. Although visual disease scores (ASs) did not show any significant difference between P-NT.II-treated and control (scrambled-P-NT.II-treated or untreated) groups, the results obtained from gross histologic analysis (Table 1) and semiquantitative analysis of pathologic parameters (Fig. 3) clearly demonstrate the beneficial effect of peptide treatment in preventing synovitis, cartilage destruction, and bone erosion. Similar discrepancies between AS and HS have also been reported in TNF-transgenic and other experimental models of arthritis. Redlich and colleagues  recently reported a protective effect of osteoprotegerin treatment on bone damage in Tg197 mice, with lack of any beneficial effect on the clinical symptoms. In another experimental model of passive collagen-induced arthritis using JNK2-deficient mice, it has been shown that clinical symptoms appear to be slightly more severe than HS despite significant reductions in joint destruction due to preservation of the articular cartilage . It seems, therefore, that preservation of the bone structure may not always correlate with the clinical symptoms. The striking difference observed in the ultrastructural features of the articular cartilage and synovial membrane in our animal model before and after peptide treatment did confirm that P-NT.II administered as an exogenous drug in this TNF transgenic mouse model of RA was able to improve the overall morphology and the cellular component of the synovium, and of the cartilage in particular.
Ultrastructural changes of ankle articular cartilage and synovium in Tg197 mice were evaluated using transmission electron microscopy, before and during the 4-week course of treatment. Histologically, we observed an apparent suppression of pannus formation and minimal erosive damage to the articular cartilage and subchondral bone. At 1–4 weeks post-treatment with peptide (i.e. at age 5–8 weeks), the number of inflammatory cells in the synovial tissue was reduced as early as 1 week after initiation of treatment, and the structural organization of the synovial membrane of the ankle joint appeared less modified. In the P-NT.II-treated group, lesions such as synovial adhesions, cell fragmentation due to degeneration of synoviocytes, and dilation of the r-ER and distorted cristae of type B cells were less obvious than in the untreated or scrambled-P-NT.II-treated groups. In our cell-culture experiments using mouse macrophages, P-NT.II has been found to dose-dependently inhibit LPS- or TNF-induced PGE2 production, with a potency equal to that of a potent and selective sPLA2 inhibitor, LY315920 . It is possible that P-NT.II may modulate ultrastructural modifications to the synovium by reducing the bioavailability of arachidonic acid (AA) through sPLA2 inhibition, and suppress the severity of the prostaglandin-mediated inflammatory response in the synovium.
The ultrastructural features of the articular cartilage observed in this human TNF transgenic mouse model of RA suggest that the chondrocyte may be one of the important targets of the peptide intervention in modulating the progression of the joint erosion. Our extensive histopathologic analysis of joints in the Tg197 TNF model in this study (Fig. 3) has revealed both articular cartilage destruction and subchondral bone erosion at the advanced stages of disease (i.e. 8 weeks of age). Similar severe cartilage destruction in Tg197 mice at 7–8 weeks of age has previously been shown as evidenced by the loss of safranin-O staining . Massive cartilage and subchondral bone erosion in the joints is the hallmark of inflammatory arthritis in the TNF transgenic mouse model . At 3–4 weeks post-treatment (i.e. at 7–8 weeks of age), P-NT.II significantly reduced chondrocyte necrobiosis, which was frequently seen in the proximity of invading synovium in untreated controls at same age. It is possible that sPLA2 might be involved in cartilage destruction in the TNF-transgenic model. sPLA2 found in the synovial fluid has been reported to originate from chondrocytes and not from the synovial lining or inflammatory cells . Human articular chondrocytes synthesize and constitutively release sPLA2, and are therefore suggested to be responsible for the high concentration of sPLA2 present in articular cartilage . cPLA2 is also reported to be involved in PGE2 production by osteoblast cells , while there are reports indicating that sPLA2 augments cPLA2 expression in mouse osteoblasts via endogenous PGE [13, 34]. Because of the significant functional coupling and/or synergism that can exist between cPLA2 and sPLA2 in various cells [3, 13, 33–35], sPLA2 could conceivably be involved in chondrocyte destruction in RA by playing a role in bone resorption through crosstalk with cPLA2.
We have found significantly elevated levels of circulating sPLA2 in Tg197 mice at 8 weeks of age as compared with the much lower baseline levels detected at 4 weeks of age. Elevated levels of sPLA2 have been reported in the plasma of patients with acute and chronic inflammatory diseases . sPLA2 can mobilize AA to induce the de novo synthesis of eicosanoids in a variety of inflammatory cells , leading to subsequent release of proinflammatory mediators. Recently, sPLA2 has been shown to amplify TNF-induced PGE2 synthesis in human rheumatoid synoviocytes , a process that is blocked by cyclic peptide inhibitors of human sPLA2 . The use of a low-molecular-weight peptide, such as P-NT.II, that effectively lowers sPLA2 could be of clear clinical benefit in similar situations. Our results obtained with P-NT.II-treated Tg197 mice demonstrated that this new peptide inhibitor significantly suppressed the circulating sPLA2 activity in those mice, whereas scrambled P-NT.II (negative control peptide) was without any effect.
The data obtained from the present study suggest that P-NT.II ameliorates synovitis and bone and cartilage erosions in the joints through modulation of circulatory and localized sPLA2, which might otherwise amplify TNF-dependent pathways in rheumatoid synovium. Although the mode of action of sPLA2 in this animal model is not exactly known, the potential mechanism may involve binding to a receptor , followed by internalization  and transfer of sPLA2 to intracellular pools of phospholipids enriched in AA . Further catalysis by sPLA2 through surface interactions can then initiate and promote pathology by releasing AA, which can subsequently be converted to proinflammatory prostaglandins and leukotrienes. There are no published reports of sPLA2 inhibitors showing benefit on bone erosion. The ultrastructural evidence of the beneficial effect of the peptide on joint destruction as shown here suggests a possible use of sPLA2 inhibitors in the treatment of inflammatory bone loss diseases such as RA. However, some caution is advisable in the interpretation of the findings, since the nature of the arthritis in a purely TNF-driven disease, such as that observed in TNF transgenic mice, may not truly reflect the situation in human inflammatory joint diseases.
The present study provides ultrastructural demonstration of the modulatory effect of the P-NT.II peptide on synovial inflammation and joint destruction in TNF-driven Tg197 mouse model of human RA. The results suggest that sPLA2 seems to play a significant role in inflammatory arthritis, and sPLA2 inhibitors may be useful for the development of novel agents to treat RA and other inflammatory diseases.
analysis of variance
cytosolic phospholipase A2
phospholipase inhibitor from python
rough endoplasmic reticulum
standard error of the mean
secretory phospholipase A2
tumor necrosis factor.
We are grateful to Mr Spiros Lalos and Ms Alexia Giannakopoulou, Institute of Immunology, Biomedical Sciences Research Centre, Al Fleming, Greece, and Ms Ng Geok Lan and Ms Chan Yee Gek, Department of Anatomy, Faculty of Medicine, National University of Singapore, for their excellent technical assistance.
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