Absence of endogeneous interleukin-10 enhances the evolution of murine type-II collagen-induced arthritis

ARTICLE

ABBREVIATIONS

NO: nitric oxide

NOS: nitric oxide synthase

ecNOS: constitutive endothelial nitric oxide synthase

iNOS: inducible nitric oxide synthase

PARS: poly (ADP-ribose) synthetase

MPO: myeloperoxidase

PMN: polymorphonuclear cell

PBS: phosphate-buffered saline

INTRODUCTION

Rheumatoid arthritis (RA) is an autoimmune disease characterised by the sequestration of various leukocyte subpopulations within both the developing pannus and synovial space. The chronic nature of this disease results in multiple joint inflammation with subsequent destruction of joint cartilage and erosion of bone. While this disease has a worldwide distribution, its pathogenesis is not clearly understood [1]. Type II collagen-induced arthritis (CIA) in the mouse has proven to be a useful model of RA, as it possesses many of the cell and humoral immunity characteristics found in human RA [2, 3]. The pathogenesis of CIA is dependent upon the host's response to type II collagen challenge and the subsequent generation of antibodies that recognises collagen-rich joint tissue [2, 3]. The chronic activities initiated by immune complexes trigger a variety of cell-mediated and humoral events. Moreover, the recruitment and activation of neutrophils, macrophages, and lymphocytes into joint tissues, and the formation of the pannus are hallmarks of the pathogenesis of both CIA and human RA. While the contribution of inflammatory leukocytes to the progression of experimental arthritis and human RA is unquestioned, the mechanisms whereby these leukocytes are recruited to the inflamed joint are still not fully known. Recently, several chemotactic cytokines (chemokines), which demonstrate a high degree of specificity for the movement of leukocyte subpopulations, have been isolated and cloned. These chemokines include, MIP-lalpha, MIP-1beta, RANTES, MCP-1, MCP-2, IL-8, and IP-10. The chemokines are chemotactic for neutrophils and for mononuclear cells. Recently, it has been demonstrated that IL-8, MIP-lalpha, MIP-1beta, and RANTES are differentially chemotactic for lymphocyte subsets [4-7]. Chemokines may play prominent roles in RA, as neutrophil and mononuclear cell elicitation and activation are prevalent in this disease.

While a number of pro-inflammatory cytokines have been studied in both human RA and murine CIA, relatively little is known regarding the production of immunomodulating cytokines, such as interleukin-10 (IL-10), during the development of this disease. Originally, IL-10 was described as a cytokine synthesis inhibitory factor produced by murine Th2 cell clones that could inhibit the synthesis of interferon-gamma by Th1 clones [8-11]. Recent studies have demonstrated that IL-10 can inhibit the synthesis of the major pro-inflammatory cytokines and chemokines, upregulate humoral immune responses and attenuate cell-mediated immune reactions [12-16]. Interestingly, because of its properties, IL-10 has the ability to modulate several infectious, immune and inflammatory diseases. In RA, IL-10 is produced not only by Th2 cells (as originally found in mice), but also by a variety of other cell types, including Th l cells, B cells, and in particular monocytes and macrophages [17-19]. IL-10 has a broad range of activities on T cells, monocyte/ macrophages, B cells, and other cell types in patients with RA. The effects of IL-10 have been studied on isolated cell populations as well as mixed cell populations such as SF and PBMC, and ST explants [20]. Although IL-10 is produced in substantial amounts in the RA joint, these levels are clearly insufficient to control inflammation [21]. Notwithstanding the fact that several studies involving neutralization of IL-10 have demonstrated a suppressive role of endogenous IL-10, since this process led to an increase in levels of IL-10, TNF-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) [21]. Administration of IL-10 to RA synovium cultures was shown to suppress production of TNF-alpha, IL-10, and IL-6 [21, 22], although stimulation of IL-10 and IL-6 production by IL-10 has also been reported [22, 23]. In addition to the data on in vitro effects on RA inflammatory activity, results of studies on experimental models of arthritis demonstrate the beneficial effects of IL-10. Aggrecan-induced arthritis in BALB/c mice was down-regulated by IL-10, in association with inhibition of the Th l response and of TNF-alpha and IL-6 production in the joints and stimulation of peripheral Th2 activity (IL-4 and IL-10 production) [24]. Administration of IL-10 to DBA/1 Lac/J mice, as well as to DA rats, led to a similar suppression of CIA [25, 29]. Also, injection of mice intravenously and intra-articularly with adenoviral vectors expressing viral IL-10, prevented induction of CIA and caused suppression of established CIA [27, 28]. Furthermore, inhibitory effects on streptococcal cell wall-induced arthritis have been observed [30]. Thus, IL-10 appears to possess certain immune-regulatory activities during inflammatory/immune responses. The purpose of this study was to investigate the role of endogenous IL-10 in a murine model of collagen-induced arthritis. To address this question, the release of the pro-inflammatory cytokines and the neutrophil infiltration were evaluated. Nitrotyrosine formation, and plasma levels of malondialdehyde were determined as indices of nitrosative and oxidative stress, respectively. Furthermore, we investigated whether genetic absence of IL-10 affects the joint expression of PARS and COX-2. We observed that absence of the IL-10 gene exaggerated the joint injury induced by collagen, whereas maintenance of endogenous IL-10 production significantly attenuated the injury, indicating that the cytokine may mediate important features of acute inflammation.

MATERIALS AND METHODS

Animals

C57BLJ10 mice (4-5 weeks old, 20-22 g), with a targeted disruption of the IL-10 gene (IL-10KO) and littermate wild-type controls (IL-10WT) were purchased from Jackson Laboratories (Harlan Nossan, Italy). The animals were housed in a controlled environment and provided with a standard rodent diet and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192), as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

Induction of collagen-induced arthritis

Bovine type II collagen (CII) was dissolved in 0.01 M acetic acid at a concentration of 2 mg/ml, by stirring overnight at 4° C. Dissolved CII was frozen at - 70° C until use. Complete Freund's adjuvant (CFA) was prepared by the addition of Mycobacterium tuberculosis H37Ra at a concentration of 2 mg/ml. Before injection, CII was emulsified with an equal volume of CFA. Collagen-induced arthritis was induced as previously described [31]. On day 1, mice were injected intradermally at the base of the tail with 100 mul of the emulsion (containing 100 mug of CII). On day 21, a second injection of CII in CFA was administered.

Clinical assessment of CIA

Mice were evaluated daily for arthritis using a macroscopic scoring system: 0 = no signs of arthritis; 1 = swelling and/or redness of the paw or one digit; 2 = two joints involved; 3 = more than two joints involved; and 4 = severe arthritis of the entire paw and digits [32]. The arthritic index for each mouse was calculated by adding the four scores for the individual paws. Clinical severity was also determined by quantifying the change in the paw volume using plethysmometry (model 7140; Ugo Basile).

Assessment of arthritis damage

On day 35, animals were sacrificed under anaesthesia, and paws and knees were removed and fixed for histological examination, which was done by an investigator blind to the treatment regime. The following morphological criteria were considered: score 0, no damage; score 1, oedema; score 2, inflammatory cell presence; score 3, bone resorption.

Histological examination

For microscopic histological evaluation, paws and knees were removed and fixed in 10% formalin. The paws were then trimmed, placed in a decalcifying solution for 24 hours, embedded in paraffin, sectioned at 5 mum, stained with haematoxylin/eosin and studied using light microscopy (Dialux 22 Leitz).

Radiography

The mice were anaesthetised with sodium pentobarbital (45 mg/kg, i.p.). They were placed on a radiographic box at a distance of 90 cm from the x-ray source. Radiographic analysis of normal and arthritic hind paws was performed by x-ray machine (Philips X12 Germany), with a 40 kW exposure for 0.01 sec. An investigator blind to the treatment regime performed the radiographic scoring. The following radiograph criteria were considered: score 0, no bone damage; score 1, tissue swelling and oedema; score 2 joint erosion; 3, bone erosion and osteophyte formation.

Immunohistochemical localisation of nitrotyrosine, PARS and COX-2

Tyrosine nitration, an index of the nitrosylation of proteins by peroxynitrite and/or oxygen-derived free radicals, was determined by immunohistochemistry as previously described [33]. On day 35, the joints organs were trimmed, placed in decalcifying solution for 24 hours, and 8 µm sections were prepared from paraffin-embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H₂O₂ in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Non-specific adsorption was minimised by incubating the section in 2% normal goat serum in phosphate-buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with primary anti-nitrotyrosine antibody (1:1,000), anti-COX-2 antibody or anti-poly (ADP-Ribose) (PAR) antibody (1:500), or with control solutions. Controls included buffer alone or non-specific purified rabbit IgG. Specific labelling was detected with a biotin-conjugated goat anti-rabbit IgG (for nitrotyrosine) or with a biotin-conjugated goat anti-rabbit IgG (for PAR and for COX-2) and avidin-biotin peroxidase complex.

Malondialdehyde (MDA) measurement

Plasma malondialdehyde (MDA) levels were determined as an indicator of lipid peroxidation [34]. An aliquot (100 mul) of the plasma collected at the specified time was added to a reaction mixture containing 200 mul of 8.1% SDS, 1,500 mul of 20% acetic acid (pH 3.5), 1,500 mul of 0.8% thiobarbituric acid and 700 mul distilled water. Samples were then heated for 1 hour at 95° C and centrifuged at 3,000 x g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 650 nm.

Measurement of cytokines

TNF-alpha, IL-6, IL-1beta and IL-10 levels were evaluated in the plasma from CIA mice as previously described [29]. The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-Novabiochem Corporation, Milan, Italy) with a lower detection limit of 10 pg/ml.

Measurement of chemokines

Murine chemokines (MIP-1alpha and MIP-2) were evaluated in the aqueous joint extracts. Briefly, joint tissues were prepared by first removing the skin and separating the limb below the ankle joint. Joint tissues were homogenised on ice in 3 ml lysis buffer (PBS containing: 2 mM PMSF, and 1 mg/ml [final concentration], each of aprotinin, antipain, leupeptin, and pepstatin A) using a Polytron (Brinkinarm Instruments, Westbury, NY, USA). The homogenised tissues were then centrifuged at 2,000 g for 10 min. Supernatants were sterilised with a millipore filter (0.2 mum) and stored at - 80° C until analysed. The extracts usually contained 0.2-1.5 mg protein/ml, as measured by a protein assay kit (Pierce Chemical Co., Rockford, IL, USA). The levels of MIP-1alpha and MIP-2 were quantified using a modification of a double ligand method, as previously described [36]. Briefly, flat-bottomed, 96-well microtiter plates were coated with 50 mul/well of rabbit anti-cytokine antibodies (1 mug/ml in 0.6 mol/litre NaCl, 0.26 mol/litre H₃BO₄ and 0.08 N NaOH, pH 9.6) for 16 hours at 4° C, and then washed with PBS, pH 7.5, 0.05% Tween 20 (wash buffer). Nonspecific binding sites on microtiter plates were blocked with 2% BSA in PBS and incubated for 90 min at 37° C. Plates were rinsed four times with washing buffer, and diluted aqueous joint samples (50 mul) were added, followed by incubation for 1 hour at 37° C. After washing of plates, chromogen substrate was added. The plates were incubated at room temperature to the desired extinction, and the reaction terminated with 50 mul/well of 3 M H₃SO₄ solution, and were read at 490 nm in an ELISA reader. This ELISA method consistently had a sensitivity limit of ~ 30 pg/ml.

Myeloperoxidase (MPO) assay

Neutrophil infiltration into the inflamed joints was indirectly quantified using an MPO assay, as previously described for neutrophil elicitation [37]. Tissue was prepared as described above and placed in a 50 mM phosphate buffer (pH = 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma Chemical Co.). Joint tissues were homogenised, sonicated, and centrifuged at 12,000 g for 15 min at 4° C. Supernatants were assayed for MPO activity using a spectrophotometric reaction with O-dianisidine hydrochloride (Sigma Chemical Co.) at 460 nm.

Materials

Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company (Milan, Italy). Biotin-blocking kit, biotin-conjugated goat anti-rabbit IgG, primary anti-nitrotyrosine, anti-poly(ADP-ribose), anti-COX-2 antibodies and avidin-biotin peroxidase complex were obtained from DBA (Milan, Italy). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, U.K.).

Data analysis

All values in the figures and text are expressed as mean ± standard error (s.e.m.) of the mean of n observations. For the in vivo studies, n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. Data sets were examined by one- or two-way analysis of variance, and individual group means were then compared with Student's unpaired t test. For the arthritis studies, the Mann-Whitney U test (two-tailed, independent) was used to compare medians of the arthritic indices [38]. A p-value less than 0.05 was considered significant.

RESULTS

Absence of IL-10 increases joint injury during experimental arthritis

To imitate the clinical scenario of RA, mice were subjected to collagen-induced arthritis. CIA developed rapidly in mice immunised with CII, and clinical signs (periarticular erythema and oedema) of the disease first appeared in mice hind paws between 24 and 26 days post-challenge (Figure 1A) leading to a 100% incidence of CIA at day 30. Hind paw erythema and swelling increased in frequency and severity in a time-dependent mode with maximum arthritis indices of approximately 8 observed between 29 to 35 days post-immunisation (Figure 1B) in IL-10WT mice. IL-10KO mice demonstrated a significant acceleration of joint inflammation, represented by an earlier appearance in the incidence of arthritis, and a greater arthritis index as compared to wild-type mice (Figure 1A, B).

There was no macroscopic evidence of either hind paw erythema or oedema in the sham group of mice (data not shown).

The rate and the absolute gain in body weight were comparable in normal mice and CII-immunised mice for the first week (Figure 1C). Beginning on day 25, the CII-challenged IL-10WT mice gained significantly less weight than the normal mice, and this trend continued through to day 35. The absence of endogenous IL-10 correlated with a significant increase of the weight loss caused by immunisation with CII (when compared to the respective WT mice) (Figure 1C).

The data in Figure 1D demonstrate a time-dependent increase in hind paw volume (each value represents the mean of both hind paws) in IL-10KO mice immunised with CII. The presence of IL-10 significantly attenuated hind paw swelling when compared to IL-10KO mice (Figure 1D). No increase in hind paw volume over time was observed in the sham mice group (data not shown).

The histological evaluation (at day 35) of the paws from IL-10KO mice revealed signs of severe arthritis, with bone erosion. In addition, severe or moderate necrosis was observed (Figure 2B, see Figure 3A for damage score). The bone erosion and the necrosis was more pronounced in the joints from IL-10KO mice (Figures 2C, 3A). A radiographic examination of hind paws from IL-10KO mice at 35 days post-CII immunisation revealed bone erosion (Figure 4C; see Figure 3B for radiograph score. The presence of IL-10 significantly attenuated the degree of bone resorption (Figures 4B, 3B). There was no evidence of pathology in the sham mice (Figures 2A, 4A).

Chemokine expression and neutrophil infiltration is increased in IL-10KO mice

The above histological pattern of joint pathology appeared to be correlated with the influx of leukocytes into the joint, joint space, and surrounding tissue. Therefore, we initiated studies to assess the role of IL-10 in the expression of chemokines into the inflamed joints during the development of CIA. As shown in Figure 5, the expression pattern of joint MIP-1alpha, and MIP-2 was assessed by ELISA and was found to correlate with the development of arthritis. MIP-lalpha and MIP-2 were significantly increased at 35 day after CII immunisation in the joint from IL-10WT mice (Figure 5) MIP-1alpha and MIP-2 levels in IL-10KO mice at day-35 were significantly increased in comparison to WT mice (Figure 5). Therefore, we next evaluated the neutrophil infiltration. Assessment of neutrophil infiltration in the inflamed joint tissue was performed by measurement of the activity of myeloperoxidase, an enzyme specific to granulocyte lysosomes and, therefore, directly correlated to the number of neutrophils. Myeloperoxidase activity was significantly elevated at 35 days after CII immunisation, in IL-10WT mice (Figure 6A). In IL-10KO mice, myeloperoxidase activity was markedly increased in comparison to those of IL-10WT animals (Figure 6A).

Absence of endogenous IL-10 favours lipid peroxidation and nitrotyrosine formation

The release of free radicals and oxidant molecules during the chronic inflammation has been suggested to contribute significantly to tissue injury [39]. At day 35, all IL-10WT arthritic mice animals exhibited a substantial increase in the plasma MDA levels indicative of lipid peroxidation (Figure 6B). Furthermore, a positive staining for nitrotyrosine, a marker of nitrosative injury, was found in the joint of IL-10WT mice (Figure 7A). Targeted disruption of the IL-10 gene in mice subjected to collagen-induced arthritis exaggerated the formation of malondialdehyde (Figure 6B) and nitrotyrosine (Figure 7B), thus indicating the occurrence of a more severe oxidant-induced damage.

Endogenous IL-10 modulates production of TNF-alpha, IL-1beta and IL-6 during experimental arthritis

To test whether the endogenous IL-10 may modulate the inflammatory process through the regulation of cytokine secretion, we analysed the plasma levels of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 in IL-10-KO and wild-type mice. A substantial increase in TNF-alpha, IL-1beta, IL-6 and IL-10 production was found in IL-10WT mice at 35 days after CII immunisation (Figure 8). Levels of TNF-alpha, IL-1beta and IL-6 were significantly higher in IL-10-deficient mice in comparison to those of IL-10WT animals (Figure 8).

PARS activation and COX-2 expression during CIA is increased in the absence of a functional gene for IL-10

To investigate the cellular mechanisms, by which endogenous IL-10 may attenuate joint injury, we evaluated the activation of PARS and the expression of COX-2, two important mediators involved in the inflammatory process [40]. Immunohistochemical analysis of joint sections obtained from IL-10WT mice treated with collagen type II revealed a positive staining for PARS (Figure 9A) and COX-2 (Figure 9C). A significant increase in positive staining for PARS (Figure 9B) and COX-2 (Figure 9D) was found in the joint of CIA-treated IL-10KO mice. There was no staining for either COX-2 or PARS in joints obtained from the sham group of mice (data not shown).

DISCUSSION

Our data demonstrate that mice with a targeted deletion of the IL-10 gene are significantly more vulnerable to pathological changes in the joint associated with CIA, as compared to wild-type controls. Thus, these results suggest that the presence of a functional IL-10 gene is a major requirement to limit the magnitude and duration of CIA. Furthermore, our data provide the first evidence that the oxidative stress and PARS pathway, activated during experimental arthritis, is regulated by the endogenous secretion of IL-10.

IL-10 is a potent anti-inflammatory cytokine, which has been shown to activate a diverse array of immunomodulatory responses. To prove the crucial role of IL-10 in controlling the inflammatory process, previous experimental studies have depended on in vivo administration of exogenous IL-10. There is ample evidence that IL-10 is an important down-regulator of a number of macrophage functions, including the production of TNF-alpha and IL-1beta. IL-10 is abundantly present in joints with active RA [40], and in vitro studies with isolated synovial tissue revealed that TNF-alpha and IL-1beta production was markedly enhanced after anti-IL-10 treatment, whereas additional exogenous IL-10 was able to still further suppress this cytokine production [41, 42]. IL-4, but not IL-10, has been shown to enhance the production of IL-1beta by RA synovial cells [43]. In our study, using genetically engineered mice, we have also demonstrated that the endogenous production of IL-10 has a notable impact in determining the outcome of joint injury in experimental CIA. Interestingly, IL-10 seems to play an obligate role during the development of CIA, as demonstrated by the fact that mice lacking a functional gene for IL-10 exhibited a high arthritis score as soon as 22-26 days after collagen immunisation.

Rheumatoid arthritis is one of the most common inflammatory joint diseases, having a world-wide distribution. In spite of a large research effort, the pathogenesis of this disease is not entirely clear. However, it is known that the progression of the disease is characterised by the presence of inflammatory cells in both the granuloma-like pannus and the joint fluid, followed by cartilage destruction and bone erosion. Interestingly, the active inflammatory stage of arthritis shares a number of common histological features of chronic inflammation, including the organised focal accumulation of mononuclear cells in the developing pannus, proliferation of fibroblast-like synovial cells, and injury to the surrounding tissue. While the proliferation of synovial cells and the infiltration of leukocytes are fundamental events in the development of joint inflammation, it is difficult to examine the mediators important to the initiation and maintenance of this pathological cascade in human arthritis. Therefore, it is necessary to establish and characterise experimental animal models to assess cellular and molecular events that contribute to the pathogenesis of joint inflammation. Interestingly, type-II collagen-induced arthritis in the mouse has proven to be a useful model, as it possesses many of the cellular and humoral immune events found in human rheumatoid arthritis.

While T cell and antibody responses against type II collagen are a crucial event for the initiation of CIA [2, 43], it has been demonstrated that several cytokines also appear to direct cell-to-cell communication in a cascade fashion during the progression of CIA. These cytokines include: IL-1 [44-46], TNF-alpha [47-49], IL-6 [50], TGF-beta [49], and IFN-gamma [51, 52]. The data presented in this report confirm that the cytokines (IL-1 and TNF-alpha), as well as the chemokines (MIP-1alpha and MIP-2) are expressed at the sites of inflamed joints and that these cytokines probably contribute in different capacities to the evolution of chronic joint inflammation. A number of recent studies have demonstrated that the recruitment of cells into the area of inflammation may be mediated not only by C5a, leukotrienes, platelet-activating factor, or bacterial-derived peptides, but also by a novel group of small proteins with relatively specific chemotactic activity for leukocyte subpopulations. This group includes MIP-lalpha, MIP-1beta, RANTES, MCPA, MCP-2, MCP-3, and 1-309. Recently, it has been demonstrated that MCP-1, MIP-1alpha, MIP-1beta, and RANTES are differentially chemotactic for lymphocyte subsets [4-7]. Since the recruitment of neutrophils, macrophages, and lymphocytes into joint tissue are hallmarks of both CIA and human rheumatoid arthritis, it is important to determine the contribution of chemokines in the progression of this experimental model of human rheumatoid arthritis. Recently it has been demonstrated that several chemokines, including IL-8, MCP-1, RANTES, and MIP-1alpha, are expressed in tissue from the inflamed joints in human rheumatoid arthritis [53-59]. While chemokines and other cytokines are known to play a pro-inflammatory role in the development of chronic inflammation, it has been reported that IL-10 may play an important regulatory role during the initiation and maintenance of inflammation [60, 61]. Many in vivo studies suggest that IL-10 can block the expression of pro-inflammatory cytokines, including IL-1 and TNF-alpha, which would corroborate a number of in vitro studies [13, 14, 63]. It has also been reported that IL-10 can inhibit the production and expression of chemokines, including IL-8, MIP-1alpha, and MIP-1beta in human monocytes and neutrophils [15, 16, 63]. Furthermore, IL-10 appears to increase the release of cytokine-modulating proteins, such as soluble TNF-receptor, and interleukin-1 receptor antagonist protein [21, 64]. Interestingly, using genetically engineered mice, we have also demonstrated that the endogenous production of IL-10 may regulate the release of these pro-inflammatory cytokines. Although the above studies demonstrate that endogenous IL-10 may act as a protective cytokine during the evolution of an inflammatory reaction and IL-10 is expressed by human synovium [65, 66], the contribution of chemokines and IL-10 to the arthritic response is not clear. However, it appears that the balance of pro-inflammatory cytokines, such as chemokines, and anti-inflammatory cytokines, such as IL-10, may dictate the magnitude of the arthritis response.

Other studies have also demonstrated that IL-10 has a homeostatic role on leukocyte-endothelial cell interactions in response to endotoxin through regulation of endothelial adhesion molecules [67, 68]. Similarly, it has recently been demonstrated that endogenous IL-10 protects the ischaemic and reperfused myocardium through the suppression of ICAM-I expression and neutrophil recruitment [69]. In the present study, the neutrophil infiltration which took place to lesser degree in wild-type mice correlated well with the moderation of joint tissue damage. Furthermore, we found that the joint damage induced by CIA in IL-10-deficient mice was associated with high levels of plasma thiobarbituric acid-reactant malondialdehyde, which is considered a good indicator of lipid peroxidation [34, 71]. An intense immunostaining of nitrotyrosine formation also suggested that a structural alteration of the joint had occurred, most probably due to the formation of highly reactive nitrogen-derivatives. Recent evidence indicates in fact, that several chemical reactions, involving nitrite, peroxynitrite, hypochlorous acid and peroxidases can induce tyrosine nitration and may contribute to tissue damage [72-75].

There is a large amount of evidence that the production of ROS such as hydrogen peroxide, superoxide and hydroxyl radicals at the site of inflammation contributes to tissue damage [76-78]. Inhibitors of NOS activity reduce the development of arthritis, and these findings support a role for NO in the pathophysiology associated with this model of inflammation [79, 80]. In addition to NO, peroxynitrite is also generated in collagen-induced arthritis [32]. The biological activity and decomposition of peroxynitrite is very much dependent on the cellular or chemical environment (presence of proteins, thiols, glucose, the ratio of NO and superoxide, carbon dioxide levels and other factors), and these factors influence its toxic potential [81, 82]. ROS and peroxynitrite produce cellular injury and necrosis via several mechanisms including peroxidation of membrane lipids, protein denaturation and DNA damage. ROS produce strand breaks in DNA which triggers energy-consuming DNA repair mechanisms and activates the nuclear enzyme PARS resulting in the depletion of its substrate NAD in vitro and a reduction in the rate of glycolysis. As NAD functions as a cofactor in glycolysis and the tricarboxylic acid cycle, NAD depletion leads to a rapid fall in intracellular ATP. This process has been termed "the PARS suicide hypothesis" [83-85]. There is recent evidence that the activation of PARS may also play an important role in inflammation [32, 83, 84]. We demonstrate here that endogenous IL-10 modulated the activation of PARS during collagen-induced arthritis in the joint. Thus, we propose that the anti-inflammatory effects of IL-10 may be, at least in part, due to the prevention of the activation of PARS.

Several cellular mechanisms, including the mode of gene regulation and signal transduction, may account for the role of IL-10 in the modulation of joint injury. In vitro and in vivo studies have reported that exogenously administered IL-10 inhibited NF-kappaB activation, thus suppressing the pro-inflammatory cytokine production in human monocyte [85], the development of immune complex-induced lung injury in rats and hepatic ischaemia and reperfusion in mice [86]. NF-kappaB has been shown to activate, via transcription, the genes encoding pro-inflammatory cytokines (TNF-alpha, IL-lbeta and IL-12), cell adhesion molecules (VCAM-1 and ICAM-1), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). Several studies also support the conclusion that pro-inflammatory cytokines, cell adhesion molecules and NO and prostaglandin E₂ (PGE₂) derived from iNOS and COX-2, respectively, play important roles in the pathogenesis of acute and chronic inflammation.

CONCLUSION

In the current study, we have obtained evidence that expression of COX-2 is reduced in the joint tissue of wild-type mice when compared to IL-10KO mice. This suggests that endogenous IL-10 may also target the COX-2 signalling pathway. Taken together, our studies demonstrate that chronic joint inflammation is a multi-factorial response, which is dependent upon both pro-inflammatory chemokines, such as MIP-1alpha and MIP-2, as well as regulatory cytokines, such as IL-10. This latter cytokine appears to be particularly important as a modulating cytokine during the progression of experimental arthritis and may play a similar role during the pathogenesis of autoimune responses in rheumatoid arthritis.

Acknowledgements. This study was supported by a grant from Consiglio Nazionale delle ricerche. The authors would like to thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study, Mrs Caterina Cutrona for secretarial assistance and Miss Valentina Malvagni for editorial assistance with the manuscript.

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