In view of the well-established anti-inflammatory properties of latex of Calotropis procera (DL), the present study was carried out to evaluate the protective effect of its methanol extract (MeDL) against inflammation and oxidative stress in monoarthritis induced by Freund's complete adjuvant (FCA) in rats. Intra-articular injection of FCA produced inflammation of the joint with a peak effect occurring on day 4 where a maximum increase in the levels of myeloperoxidase and inflammatory mediators like PGE2, TNF-α, and nitric oxide was observed. This was associated with oxidative stress with a marked reduction in the levels of glutathione, catalase, superoxide dismutase and glutathione peroxidase and an increase in the lipid peroxidation as indicated by the higher levels of thiobarbituric acid reactive substances (TBARSs). Subsequently on day 28 the histological analysis of the joint also revealed arthritic changes. Daily treatment of rats with MeDL (50 and 500 mg/kg) and standard anti-inflammatory drug rofecoxib (20 and 100 mg/kg), produced a significant attenuation in the inflammatory response and ameliorated the arthritic changes in the joint. The protection afforded by MeDL and rofecoxib was more pronounced than that of phenylbutazone and was associated with normalization of the levels of inflammatory mediators and biochemical parameters of oxidative stress. However, the overall protection afforded by rofecoxib was better than that of MeDL.
Transcutaneous electrical nerve stimulation (TENS) has been shown to be an effective measure for pain relief. The aim of the present study was to determine the optimal intensity and interval of repeated 100 Hz TENS for the treatment of chronic inflammatory hyperalgesia in a monoarthritic pain model of the rat, and to assess the changes of the spinal substance P (SP) release in response to TENS treatment. A reliable, reproducible chronic monoarthritic pain model was produced by intra-articular injection of complete Freund's adjuvant (CFA) at single ankle joint. The efficacy of 100 Hz TENS treatments with different frequencies and intensities was compared. In the acute period (within 3 weeks) of monoarthritis, twice-a-week schedule of TENS reduced the swelling of the inflamed ankle significantly. In the stable period (4–9 weeks), however, once-a-week schedule produced a significantly better therapeutic effect on both inflammation and arthritic hyperalgesia than that of twice- or five-times-a-week schedule. Using three levels of intensity of TENS, we found that the weaker (1-1-2 mA) stimulation produced significantly better therapeutic effects. Repeated TENS produced a reduction of SP content in spinal perfusate in parallel with the progressive reduction of the arthritic pain scores. Our results suggest that (i) consecutive TENS treatments produced cumulative effect for chronic hyperalgesia, (ii) for chronic inflammatory hyperalgesia, a weaker intensity and more sparsely arranged treatment schedule may produce better therapeutic effect and (iii) a decrease in SP release may serve as one of the possible neurochemical mechanisms underlying the therapeutic effects of multiple TENS treatments on chronic inflammatory hyperalgesia.
chronic inflammatory hyperalgesia; Freund's adjuvant; monoarthritis; substance P; transcutaneous electrical nerve stimulation (TENS)
In early S‐antigen induced experimental uveitis (EAU), photoreceptor mitochondrial proteins are nitrated prior to macrophage infiltration of the retina, suggesting that oxidative stress is an initial event in the development of EAU. We attempted to detect the oxidative stress and localise it in the EAU retina.
Lewis rats were immunised with S‐antigen in complete Freund's adjuvant (CFA). Animals were injected with CFA alone and non‐immunised animals served as controls. Immunised and non‐immunised animals were killed on day 5 and subsequent days. Isolated retinas were processed for inducible nitric oxide synthase (iNOS), tumour necrosis factor (TNF)α, interferon (IFN)γ, interleukin (IL)Iα and CD28 expression by real time polymerase chain reaction. In addition, iNOS was colocalised with cytochrome c oxidase on day 5 of EAU. Oxidative stress was detected by 2′, 7′‐dichlorodihydrofluorescein diacetate and localised by a mitochondrial specific marker. Leucocyte and T cell infiltration in the retina/choroid was evaluated by immunohistochemistry.
The iNOS, TNFα, IFNγ, IL1α and CD28 transcripts were significantly upregulated on day 5 in EAU, and iNOS was colocalised with cytochrome c oxidase in the photoreceptor mitochondria. Oxidative stress was seen primarily in the photoreceptor mitochondria. Occasional T cells were present in the retina at this stage.
During early EAU, mitochondrial oxidative stress is selectively noted in the photoreceptor inner segments. The oxidative stress appears to result from iNOS upregulation in the photoreceptor mitochondria and cytokine generation in the retina by a few antigen specific infiltrating T cells.
Background: Nitrergic and prostanoid pathways have both been implicated in inflammatory processes.
Objective: To investigate their respective contributions in a rat model of chronic arthritis.
Methods: Male Wistar rats (n = 4–6/group) received either an intra-articular injection of 2% carrageenan/4% kaolin (C/K) or intra- and periarticular injections of Freund's complete adjuvant (FCA; 10 mg/ml M tuberculosis). Joint diameter, urinary nitric oxide metabolites (NOx), and prostaglandin E2 (PGE2) levels were measured as indices of the inflammatory process. A prophylactic and therapeutic (day 5) dose ranging study of an inducible nitric oxide synthase inhibitor, L-N-(1-iminoethyl)-lysine (L-NIL), and a cyclo-oxygenase-2 (COX-2) inhibitor, SC-236, was performed with the drugs given subcutaneously. Submaximal doses were identified and used for combination studies. Appropriate vehicle controls were included.
Results: L-NIL and SC-236 dose dependently inhibited C/K induced acute joint swelling, the magnitude being greatest when they were given in combination. Both prophylactic and therapeutic administration of SC-236 in the FCA induced model of chronic arthritis produced a dose dependent reduction in all the measures assessed. However, although L-NIL demonstrated similar dose dependent inhibition of urinary NOx and PGE2 levels, joint swelling was significantly exacerbated in this model. Co-administration of the inhibitors nullified the benefits of SC-236.
Conclusion: Whereas COX-2 derived prostaglandins are proinflammatory in both acute and chronic joint inflammation, NO seems to have divergent roles, being anti-inflammatory in chronic and proinflammatory in acute joint inflammation.
AIM: To investigate the effect of human umbilical cord stem cells, both mesenchymal and hematopoietic (CD34+), in the treatment of arthritis.
METHODS: Mesenchymal stem cells (MSCs) and hematopoietic (CD34+) stem cells (HSC) were isolated from human umbilical cord blood obtained from the umbilical cord of healthy pregnant donors undergoing full-term normal vaginal delivery. MSC, HSC, methotrexate (MTX) and sterile saline were injected intra-articularly into the rat hindpaw with complete freunds adjuvant (CFA) induced arthritis after the onset of disease (day 34), when arthritis had become well established (arthritis score ≥ 2). Arthritic indices were evaluated and the levels of interleukin (IL)-1, tumor necrosis factor (TNF)-α and interferon (IFN)-γ and anti-inflammatory cytokine IL-10 in serum were determined using enzyme-linked immunosorbent assay. Animals of all groups were sacrificed 34 d after beginning treatment, except positive control (PC) which was sacrificed at 10, 21 and 34 d for microscopic observation of disease progression. We used hematoxylin, eosin and Masson’s trichrome stains for histopathological examination of cartilage and synovium.
RESULTS: The mean arthritis scores were similar in all groups at 12 and 34 d post immunization, with no statistical significant difference. Upon the injection of stem cells (hematopoietic and mesenchymal), the overall arthritis signs were significantly improved around 21 d after receiving the injection and totally disappeared at day 34 post treatment in MSC group. Mean hindpaw diameter (mm) in the MSC rats was about half that of the PC and MTX groups (P = 0.007 and P = 0.021, respectively) and 0.6 mm less than the HSC group (P = 0.047), as indicated by paw swelling. Associated with these findings, serum levels of TNF-α, IFN-γ and IL-1 decreased significantly in HSC and MSC groups compared to PC and MTX groups (P < 0.05), while the expression of IL-10 was increased. Histopathological examination with H and E stain revealed that the MTX treated group showed significant reduction of leucocytic infiltrate and hypertrophy of the synovial tissue with moderate obliteration of the joint cavity. Stem cells treated groups (both hematopoietic CD34+ and mesenchymal), showed significant reduction in leucocytic infiltrate and hypertrophy of the synovial tissue with mild obliteration of the joint cavity. With Masson’s trichrome, stain sections from the PC group showed evidence of vascular edema of almost all vessels within the synovium in nearly all arthritic rats. Vacuoles were also visible in the outer vessel wall. The vessel became hemorrhagic and finally necrotic. In addition, there was extensive fibrosis completely obliterating the joint cavity. The mean color area percentage of collagen in this group was 0.324 ± 0.096, which was significantly increased when compared to the negative control group. The mean color area percentage of collagen in hematopoietic CD34+ and mesenchymal groups was 0.176 ± 0.0137 and 0.174 ± 0.0197 respectively, which showed a marked decrement compared to the PC group, denoting a mild increase in synovial tissue collagen fibers.
CONCLUSION: MSC enhance the efficacy of CFA-induced arthritis treatment, most likely through the modulation of the expression of cytokines and amelioration of pathological changes in joints.
Complete freunds adjuvant-induced arthritis; Human umbilical mesenchymal stem cell; Hematopoietic stem cell; CD34+
FcγRIII (CD16) is a receptor expressed on immune cells that selectively binds immmunoglobulin G (IgG) molecules, IgG binding results in cellular activation and cytokine release. IgG is an important factor in arthritis and can be found in arthritic temporomandibular joints (TMJ). We hypothesize that a reduction in FcγRIII expression in the TMJ tissues will reduce the nociceptive and inflammatory response in an inflamed joint. To test this hypothesis siRNA, either naked or complexed with linear polyethylenimine (PEI) was injected into the superior joint space of the TMJ. After administration of siRNA the joint was injected with saline or with complete Freund’s adjuvant (CFA) to induce arthritis. Nociceptive responses were quantitated in the rat by measuring the animal’s meal duration. FcγRIII expression in the TMJ tissue was assayed by immunocytochemistry or western. Cleavage of FcγRIII transcript was then assayed by 5′ rapid amplification of cDNA ends method (5′ RACE). Interleukin-1β (IL-1β) and IgG content was measured in the TMJ tissue by ELISA. The results indicate that injection of FcγRIII siRNA reduced the amount of FcγRIII in the TMJ tissues and that the transcript was cleaved in a manner consistent with a RNA interference mechanism. Moreover, injection of FcγRIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of pro-inflammatory cytokine IL-1β. We conclude that FcγRIII contributes to the pain resulting from inflammatory arthritis of the TMJ and that siRNA has the potential to be an effective treatment for this disorder.
Spinal glia, particularly microglia and astrocytes, are of the utmost importance in the development and maintenance of chronic pain. A recent study from our laboratory revealed that gabapentin, a recommended first-line treatment for multiple neuropathic conditions, could also efficiently antagonize thermal hyperalgesia evoked by complete Freund's adjuvant (CFA)-induced monoarthritis (MA). In the present study, we investigated whether the spinal glia are involved in the anti-hyperalgesic effect of gabapentin and how this event occurs.
Unilateral intra-articular injection of CFA produced a robust activation of microglia and astrocytes. These cells exhibited large cell bodies, thick processes and increases in the ionized calcium binding adapter molecule 1 (Iba-1, a microglial marker) or the glia fibrillary acidic protein (GFAP, an astrocytic marker). These cells also displayed immunoreactive signals, and an upregulation of the voltage-gated calcium channels (VGCCs) α2/δ-1 subunit, CX3CL1 and CX3CR1 expression levels in the spinal cord. These changes were associated with the development of thermal hyperalgesia. Immunofluorescence staining showed that VGCC α2/δ-1 subunit, a proposed gabapentin target of action, was widely distributed in primary afferent fibers terminals and dorsal horn neurons. CX3CL1, a potential trigger to activate microglia, colocalized with VGCC α2/δ-1 subunits in the spinal dorsal horn. However, its receptor CX3CR1 was mainly expressed in the spinal microglia. Multiple intraperitoneal (i.p.) gabapentin injections (100 mg/kg, once daily for 4 days with the first injection 60 min before intra-articular CFA) suppressed the activation of spinal microglia, downregulated spinal VGCC α2/δ-1 subunits decreased CX3CL1 levels and blocked the development of thermal hyperalgesia in MA rats.
Here we provide the first evidence that gabapentin diminishes CX3CL1 signaling and spinal microglia activation induced by joint inflammation. We also show that the VGCC α2/δ-1 subunits might be involved in these events.
α2/δ-1 subunit of Voltage-gated calcium channels; CX3CL1; CX3CR1; Gabapentin; Glial activation; Monoarthritis
To examine the potential usefulness of a novel thermal imaging technique to evaluate and monitor inflammatory arthritis activity in small joints using rat models, and to determine whether thermal changes can be used to detect preclinical stages of synovitis.
Three different rat strains were studied in a model of inflammatory arthritis of the ankle induced by an intra‐articular (IA) injection of complete Freund's adjuvant (CFA), compared with the contralateral ankle injected with normal saline. Arthritis activity and severity scores, ankle diameters, pain related posture scores, and thermal images were obtained at 10 different times between 0 h (before induction) and day 7. The pristane induced arthritis (PIA) model was used to study preclinical synovitis. Thermal images were obtained at each time point using the TSA ImagIR system and were digitally analysed.
Rats developed similar ankle arthritis detected six hours after the IA injection of CFA, which persisted for seven days. All ankle clinical indices, including arthritis activity and severity scores, correlated significantly with ankle thermal imaging changes in the monoarthritis model (p<0.003). No thermal imaging changes were detected in preclinical stages of PIA. However, PIA onset coincided with increased ankle thermal signature.
Thermal measurements correlated significantly with arthritis activity and severity indices. The technique was highly sensitive and could measure directly two cardinal signs of inflammation (warmth and oedema, based on ankle diameter) in an area (ankle) that is less than half the size of a human interphalangeal joint, suggesting a potential use in drug trials or clinical practice.
autoimmunity; inflammation; rodent models; innate immunity
To study the relative contribution of various matrix degrading enzymes in the pathogenesis of arthritis, changes in the levels of various matrix metalloprtoteinases (MMPs) during the progression of collagen induced arthritis was studied in experimental animals. Arthritis was induced in male wistar rats by injecting an emulsion containing collagen type II and Freund’s complete adjuvant. The duration of the experiment was 35 days. Synovial effusate was collected at regular intervals after induction. At the end of the experiment serum and cartilage were collected and analysed. Synovial fluid of osteoarthritic patients was also analyzed. Levels of MMP-2, MMP-3, MMP-9 and MT1-MMP were found to be high in synovial effusate and cartilage of experimental animals. In synovial effusate of arthritic animals the expression of MMP-3 was found to be high during the early stages while increase in MMP-2 and MMP-9 occurred at later stages. Synovial fluid of osteoarthritic patients also showed elevated levels of MMP-2, MMP-3 and MMP-9. Our results indicated that sequential action of MMPs such as MMP-3, MMP-2 and MMP-9 can cause degradation of articular cartilage extracellular matrix.
Matrix metalloproteinases; Osteoarthritis; Synovial effusate; Synovial fluid
Rodents immunized with complete Freund's adjuvant (CFA) are resistant to subsequent attempts to induce autoimmune disease, while animals immunized with incomplete Freund's adjuvant (IFA) remain susceptible. Mycobacterial extracts can stimulate inducible nitric oxide synthase (NOS2) gene transcription. Robust expression of NOS2 has been linked to suppression of T cell proliferation and alterations in immune responses. Our studies investigated the hypothesis that the immunoprotective effect of CFA before immunization requires functional NOS2. NOS2 gene expression is chronically elevated in lymph nodes and spleens of CFA-immunized mice. Maximal expression of NOS2 after CFA immunization requires the presence of functional type I tumor necrosis factor α receptor (TNFR1) and interferon γ. Groups of nontreated and CFA-preimmunized male C57BL/6J or C57BL/6NOS2−/− mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35–55 in CFA to induce experimental allergic encephalomyelitis (EAE). Wild-type C57BL/6J mice were protected from the development of symptoms of EAE, while the NOS2−/− mice failed to be protected. NOS2-dependent effects of CFA included an augmentation of the MOG-specific IgG1 response, a decrease in interleukin 6 production by MOG-reactive lymphocytes, and a marked decrease in mononuclear cell infiltrates in the central nervous system. These studies support the hypothesis that CFA immunization modulates immune responses through a nitric oxide–dependent mechanism.
experimental allergic encephalomyelitis; Freund's adjuvant; immunosuppression; interleukin 6; tumor necrosis factor α
Rheumatoid arthritis (RA) is characterised by invasion of cartilage, bone and tendon by inflamed synovium. Previous studies in our laboratory have shown that hypoxia is a feature of RA synovitis. In the present study, we investigated the consequences of hypoxia on angiogenesis and synovial fibroblast migration in RA.
Synovial tissue was harvested from RA patients, and synovial membrane cells were cultured under conditions either of hypoxia (1% oxygen) or normoxia (21% oxygen). Protein levels of matrix metalloproteinases (MMPs) and angiogenic factors were measured, while RNA was extracted for PCR quantification of MMPs/tissue inhibitors of MMP (TIMPs) and angiogenic factors. Migration of RA synovial fibroblasts through collagen, and the effect of RA synovial cell supernatants in an in vitro angiogenesis assay, were utilised to determine the functional relevance of changes in mRNA/protein.
We observed upregulation under hypoxic conditions of MMPs responsible for collagen breakdown, specifically collagenase MMP-8, and the gelatinases MMP-2 and MMP-9, at both mRNA and protein levels. Increased MT1-MMP mRNA was also observed, but no effect on TIMP-1 or TIMP-2 was detected. RA fibroblast migration across collagen was significantly increased under hypoxic conditions, and was dependent on MMP activity. Furthermore, expression of angiogenic stimuli, such as vascular endothelial growth factor (VEGF), and VEGF/placental growth factor heterodimer, was also increased. Crucially, we show for the first time that hypoxia increased the angiogenic drive of RA cells, as demonstrated by enhanced blood vessel formation in an in vitro angiogenesis assay.
Hypoxia may be responsible for rendering RA synovial lining proangiogenic and proinvasive, thus leading to the debilitating features characteristic of RA.
We demonstrated previously that local, intra-articular injection of an adenoviral vector expressing human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a rabbit knee model of inflammatory arthritis stimulated synovial apoptosis and reduced inflammation. To examine whether intra-articular injection of recombinant chimeric human TRAIL protein (rTRAIL) also induces apoptosis of proliferating rabbit synovium and reduces inflammation, we used an experimental rabbit arthritis model of rheumatoid arthritis, induced by intra-articular introduction of allogeneic fibroblasts genetically engineered to secrete human IL-1β. Analysis of synovium isolated from the rabbits treated with intra-articular injection of rTRAIL, relative to saline control, showed areas of extensive acellular debris and large fibrous regions devoid of intact cells, similar to adenoviral mediated TRAIL gene transfer. Extensive apoptosis of the synovial lining was demonstrated using TUNEL analysis of the sections, corresponding to the microscopic findings in hematoxylin and eosin staining. In addition, leukocyte infiltration into the synovial fluid of the inflamed knee joints following rTRAIL treatment was reduced more than 50% compared with the saline control. Analysis of the glycosaminoglycan synthetic rate by cultured cartilage using radiolabeled sulfur and cartilage histology demonstrated that rTRAIL did not adversely affect cartilage metabolism and structure. Analysis of serum alanine aminotransferase showed that intra-articular injection of rTRAIL did not have adverse effects on hepatic function. These results demonstrate that intra-articular injection of rTRAIL could be therapeutic for treating pathologies associated with rheumatoid arthritis.
Considerable evidence has associated the expression of matrix metalloproteinases (MMPs) with the degradation of cartilage and bone in chronic conditions such as arthritis. Direct evaluation of MMPs' role in vivo has awaited the development of MMP inhibitors with appropriate pharmacological properties. We have identified butanediamide, N4- hydroxy-2-(2-methylpropyl)-N1-[2-[[2-(morpholinyl)ethyl]-,[S- (R*,S*)] (GI168) as a potent MMP inhibitor with sufficient solubility and stability to permit evaluation in an experimental model of chronic destructive arthritis (adjuvant-induced arthritis) in rats. In this model, pronounced acute and chronic synovial inflammation, distal tibia and metatarsal marrow hyperplasia associated with osteoclasia, severe bone and cartilage destruction, and ectopic new bone growth are well developed by 3 wk after adjuvant injection. Rats were injected with Freund's adjuvant on day 0. GI168 was was administered systemically from days 8 to 21 by osmotic minipumps implanted subcutaneously. GI168 at 6, 12, and 25 mg/kg per d reduced ankle swelling in a dose-related fashion. Radiological and histological ankle joint evaluation on day 22 revealed a profound dose related inhibition of bone and cartilage destruction in treated rats relative to rats receiving vehicle alone. A significant reduction in edema, pannus formation, periosteal new bone growth and the numbers of adherent marrow osteoclasts was also noted. However, no significant decrease in polymorphonuclear and mononuclear leukocyte infiltration of synovium and marrow hematopoietic cellularity was seen. This unique profile of antiarthritic activity indicates that GI168 is osteo- and chondro-protective, and it supports a direct role for MMP in cartilage and bone damage and pannus formation in adjuvant- induced arthritis.
An acute inflammatory arthritis has been induced in 76% of rats injected intradermally with native bovine type II collagen emulsified in Freund's complete (CFA) or incomplete (ICFA) adjuvant. The arthritis became chronic in 14 out of 31 rats, and ear and tail lesions were noted in some rats. No arthritis was induced by native type I collagen, denatured type II collagen, rabbit IgG, or buffer alone injected intradermally with adjuvant. Using a solid-phase radioimmunoassay for serum antibodies we have shown that IgM and IgG levels to native bovine type II collagen were significantly higher in arthritic than nonarthritic rats. Antiglobulin antibody levels were not raised in arthritic rats. Equivalent antibody levels to native type II collgen were obtained whether this antigen was emulsified in ICFA or CFA. However, native type I collagen produced high antibody levels only when emulsified in CFA, indicating a difference in the immunogenicity of these collagens. These studies suggest that native type II collagen possesses arthritogenic properties in the rat and that humoral immunity may play a role in the induction of this arthritis.
6-Shogaol is one of the major compounds in the ginger rhizome that may contribute to its anti-inflammatory properties. Confirmation of this contribution was sought in this study in Sprague- Dawley rats (200–250 g) treated with a single injection (0.5 ml of 1 mg/ml) of a commercial preparation of complete Freund's Adjuvant (CFA) to induce monoarthritis in the right knee over a period of 28 days. During this development of arthritis, each rat received a daily oral dose of either peanut oil (0.2 ml-control) or 6-shogaol (6.2 mg/Kg in 0.2 ml peanut oil).
Within 2 days of CFA injection, the control group produced maximum edematous swelling of the knee that was sustained up to the end of the investigation period. But, in the 6-shogaol treated group, significantly lower magnitudes of unsustained swelling of the knees (from 5.1 ± 0.2 mm to 1.0 ± 0.2 mm, p < 0.002, n = 6) were produced during the investigation period. Unsustained swelling of the knees (from 3.2 ± 0.6 mm to 0.8 ± 1.1 mm, p < 0.00008, n = 6) was also produced after 3 days of treatment with indomethacin (2 mg/Kg/day) as a standard anti-inflammatory drug, but during the first 2 days of drug treatment swelling of the knees was significantly larger (11.6 ± 2.0 mm, p < 0.0002, n = 6) than either the controls or the 6-shogaol treated group of rats. This exaggerated effect in the early stage of indomethacin treatment was inhibited by montelukast, a cysteinyl leukotriene receptor antagonist. Also, 6-shogaol and indomethacin were most effective in reducing swelling of the knees on day 28 when the controls still had maximum swelling. The effect of 6-shogaol compared to the controls was associated with significantly lower concentration of soluble vascular cell adhesion molecule-1 (VCAM-1) in the blood and infiltration of leukocytes, including lymphocytes and monocytes/macrophages, into the synovial cavity of the knee. There was also preservation of the morphological integrity of the cartilage lining the femur compared to damage to this tissue in the peanut oil treated control group of rats.
From these results, it is concluded that 6-shogaol reduced the inflammatory response and protected the femoral cartilage from damage produced in a CFA monoarthritic model of the knee joint of rats.
Using physiological, pharmacological, and gene disruption approaches, we demonstrate that proteinase-activated receptor-2 (PAR-2) plays a pivotal role in mediating chronic inflammation. Using an adjuvant monoarthritis model of chronic inflammation, joint swelling was substantially inhibited in PAR-2–deficient mice, being reduced by more than fourfold compared with wild-type mice, with virtually no histological evidence of joint damage. Mice heterozygous for PAR-2 gene disruption showed an intermediate phenotype. PAR-2 expression, normally limited to endothelial cells in small arterioles, was substantially upregulated 2 weeks after induction of inflammation, both in synovium and in other periarticular tissues. PAR-2 agonists showed potent proinflammatory effects as intra-articular injection of ASKH95, a novel synthetic PAR-2 agonist, induced prolonged joint swelling and synovial hyperemia. Given the absence of the chronic inflammatory response in the PAR-2–deficient mice, our findings demonstrate a key role for PAR-2 in mediating chronic inflammation, thereby identifying a novel and important therapeutic target for the management of chronic inflammatory diseases such as rheumatoid arthritis.
Chondrocyte CD44 receptors anchor hyaluronan to the cell surface, enabling the assembly and retention of proteoglycan aggregates in the pericellular matrix. Hyaluronan–CD44 interactions also provide signaling important for maintaining cartilage homeostasis. Disruption of chondrocyte–hyaluronan contact alters CD44 occupancy, initiating alternative signaling cascades. Treatment with hyaluronan oligosaccharides is one approach to uncouple CD44 receptors from its native ligand, hyaluronan. In bovine articular chondrocytes, treatment with hyaluronan oligosaccharides or purified hyaluronan hexasaccharides induced the production of nitric oxide that mirrored nitric oxide production following interleukin-1 treatment. In contrast, 120 and 1260 kDa hyaluronan did not induce production of nitric oxide. Human chondrocytes responded similarly to treatment with hyaluronan or hyaluronan oligosaccharides. Nitric oxide production from chondrocytes was mediated by activation of the inducible nitric oxide synthase, as confirmed by mRNA expression and inhibition of nitric oxide production by diphenyleneiodonium. Co-treatment of chondrocytes with hyaluronan oligosaccharides and interleukin-1 did not demonstrate additive effects. Blocking interleukin-1 receptors with an antagonist did not abolish the production of nitric oxide induced by treatment with hyaluronan oligosaccharides. Moreover, only COS-7 following transfection with a pCD44, not the CD44-null parental cells, responded to treatment with hyaluronan oligosaccharides by releasing nitric oxide. This study demonstrates a novel signaling potential by hyaluronan fragments, in lieu of endogenous hyaluronan–chondrocyte interactions, resulting in the activation of inducible nitric oxide synthase.
Chondrocytes; Hyaluronan; Interleukin-1 (IL-1); Nitric oxide (NO); CD44
The effects of treatment with a monoclonal antibody (R73 mAb) against T cell receptor alpha/beta (TCR-alpha/beta) on both established adjuvant arthritis (EAA) and established collagen-induced arthritis (ECIA) in rats have been investigated. Rats were treated with R73 mAb when arthritis reached a peak. Treatment with the anti-TCR-alpha/beta mAb markedly suppressed EAA, whereas ECIA was not affected by the mAb treatment. Histologically, R73 mAb-treated rats with EAA showed mild hyperplasia of synovial tissues, sparse infiltration of inflammatory cells, and minimal erosion of cartilage, whereas arthritic rats treated with PBS and an irrelevant control mAb against Giardia had marked hyperplasia of synovium with pannus, massive inflammatory cell infiltrate, and severe destruction of cartilage and subchondral bone. R73 mAb-treated rats with ECIA exhibited pronounced formation of pannus containing many inflammatory cells and marked cartilage and subchondral damage similar to those in arthritic rats that received the control treatments. Treatment with R73 mAb depleted markedly alpha/beta+ T cells in both peripheral blood and synovial tissues of rats with EAA and ECIA. R73 mAb treatment was associated with marked reduction in arthritogen-specific delayed-type hypersensitivity responses in both EAA and ECIA. The titers of antibodies against type II collagen produced in rats with ECIA were not affected by the mAb. Thus, alpha/beta+ T cells appear to have a central role in EAA, but not in chronic ECIA.
This study was aimed at examining the effect of an ointment containing essential oils (EO) on the severity of adjuvant arthritis (AA), an experimental model of human rheumatoid arthritis (RA), in Lewis rats and to define the underlying mechanisms. At the onset of AA, rats received topical application twice daily of ointment containing 20% EO or placebo ointment. The synovial fluid (SF) and synovium-infiltrating cells (SIC) of rats were tested for pro-inflammatory cytokines TNF-α and IL-1β. The hind paws and skin were examined histologically. The activity/level of matrix metalloproteinases (MMPs) and anti-mycobacterial heat-shock protein 65 (Bhsp65) antibodies was tested. Arthritic rats treated with ointment containing EO developed less severe clinical arthritis compared to the controls, and this activity was attributable to EO and not the carrier oil. The levels of TNF-α and IL-1β, and the activity of MMPs in SF and SIC-lysate were significantly (p<0.05) reduced in EO-treated arthritic rats compared to the controls. However, the levels of anti-Bhsp65 antibodies were unaffected by treatment. Thus, topical dermal delivery of EO-containing ointment downmodulates the severity of AA in Lewis rats by inhibiting defined mediators of inflammation. Such ointments should be tested in patients with RA and other arthritic conditions.
Essential oils; Arthritis; Cytokines; Topical application; MMPs; Inflammation
In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.
Therapies directed against tumour necrosis factor (TNF) are effective for the treatment of rheumatoid arthritis and reduce pain scores in this condition. In this study, we sought to explore mechanisms by which TNF contributes to inflammatory pain in an experimental model of arthritis. The effects of an anti-TNF agent, etanercept, on behavioural pain responses arising from rat monoarthritis induced by complete Freund's adjuvant were assessed and compared with expression of TNF receptors (TNFRs) by dorsal root ganglion (DRG) cells at corresponding time points. Etanercept had no effect on evoked pain responses in normal animals but exerted a differential effect on the thermal and mechanical hyperalgesia associated with rat arthritis induced by complete Freund's adjuvant (CFA). Joint inflammation was associated with increased TNFR1 and TNFR2 expression on DRG cells, which was maintained throughout the time course of the model. TNFR1 expression was increased in neuronal cells of the DRG bilaterally after arthritis induction. In contrast, TNFR2 expression occurred exclusively on non-neuronal cells of the macrophage–monocyte lineage, with cell numbers increasing in a TNF-dependent fashion during CFA-induced arthritis. A strong correlation was observed between numbers of macrophages and the development of mechanical hyperalgesia in CFA-induced arthritis. These results highlight the potential for TNF to play a vital role in inflammatory hyperalgesia, both by a direct action on neurons via TNFR1 and by facilitating the accumulation of macrophages in the DRG via a TNFR2-mediated pathway.
Translocator protein 18kDa (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is predominantly located in the mitochondrial outer membrane and plays an important role in steroidogenesis, immunomodulation, cell survival and proliferation. Previous studies have shown an increased expression of TSPO centrally in neuropathology, as well as in injured nerves. TSPO has also been implicated in modulation of nociception. In the present study, we examined the hypothesis that TSPO is involved in the initiation and maintenance of inflammatory pain using a rat model of Complete Freund’s Adjuvant (CFA)-induced monoarthritis of the tibio-tarsal joint. Immunohistochemistry was performed using Iba-1 (microglia), NeuN (neurons), anti-Glial Fibrillary Acidic Protein, GFAP (astrocytes) and anti-PBR (TSPO) on day 1, 7 and 14 after CFA-induced arthritis. Rats with CFA-induced monoarthritis showed mechanical allodynia and thermal hyperalgesia on the ipsilateral hindpaw, which correlated with the increased TSPO expression in ipsilateral lamina I-II on all experimental days. Iba-1 expression in the ipsilateral dorsal horn was also increased on Day 7 and 14. Moreover, TSPO was co-localized with Iba-1, GFAP and NeuN within the spinal cord dorsal horn. The TSPO agonist Ro5-4864, given intrathecally, dose-dependently retarded or prevented the development of mechanical allodynia and thermal hyperalgesia in rats with CFA-induced monoarthritis. These findings provide evidence that spinal TSPO is involved in the development and maintenance of inflammatory pain behaviors in rats. Thus, spinal TSPO may present a central target as a complementary therapy to reduce inflammatory pain.
Translocator protein 18kDa; TSPO; Peripheral benzodiazepine receptor; pain; inflammation; inflammatory pain; arthritis; spinal cord
Antigen presenting cells (APCs) play an important role in arthritis and APC specific gene therapeutic targeting will enable intracellular modulation of cell activity. Viral mediated overexpression is a potent approach to achieve adequate transgene expression levels and lentivirus (LV) is useful for sustained expression in target cells. Therefore, we studied the feasibility of lentiviral mediated targeting of APCs in experimental arthritis. Third generation VSV-G pseudotyped self-inactivating (SIN)-LV were injected intravenously and spleen cells were analyzed with flow cytometry for green fluorescent protein (GFP) transgene expression and cell surface markers. Collagen-induced arthritis (CIA) was induced by immunization with bovine collagen type II in complete Freund's adjuvant. Effect on inflammation was monitored macroscopically and T-cell subsets in spleen were analyzed by flow cytometry. Synovium from arthritic knee joints were analyzed for proinflammatory cytokine expression. Lentiviruses injected via the tail vein preferentially infected the spleen and transduction peaks at day 10. A dose escalating study showed that 8% of all spleen cells were targeted and further analysis showed that predominantly Ly6C+ and F4/80+ cells in spleen were targeted by the LV. To study the feasibility of blocking TAK1-dependent pathways by this approach, a catalytically inactive mutant of TAK1 (TAK1-K63W) was overexpressed during CIA. LV-TAK1-K63W significantly reduced incidence and arthritis severity macroscopically. Further histological analysis showed a significant decrease in bone erosion in LV-TAK1-K63W treated animals. Moreover, systemic Th17 levels were decreased by LV-TAK1-K63W treatment in addition to diminished IL-6 and KC production in inflamed synovium. In conclusion, systemically delivered LV efficiently targets monocytes and macrophages in spleen that are involved in autoimmune arthritis. Moreover, this study confirms efficacy of TAK1 targeting in arthritis. This approach may provide a valuable tool in targeting splenic APCs, to unravel their role in autoimmune arthritis and to identify and validate APC specific therapeutic targets.
Nitric oxide is one of many proinflammatory mediators that are involved in temporomandibular joint (TMJ) inflammatory disorder and is synthesized by inducible nitric oxide synthase (iNOS). iNOS is transcriptionally regulated by nuclear factor-κB (NF-κB) in cases of inflammation, proliferation, and apoptosis. It has also been reported that nitric oxide is positively regulated by carrageenan and negatively regulated by hyaluronan in the knee joint. The aim of this study was to histologically evaluate how inflammation and cell proliferation of the synovial membrane are affected by the exogenous administration of carrageenan and hyaluronan in the rat TMJ by investigating iNOS, NF-κB, and anti proliferating cell nuclear antigen (PCNA) immunoreactivity. As results, immunoreactive cells to iNOS, NF-κB, and PCNA were normally localized only in the synovial membrane of wild type TMJs. The numbers of immunoreactive cells were extensively larger in the carrageenan-injected synovial membranes exhibiting excessive folding, and smaller in the hyaluronan-injected synovial membranes showing a few folds. These results indicate that a carrageenan injection induced inflammation and cell proliferation especially in the synovial membrane and that hyaluronan relieved the inflammation by decreasing inflammatory molecules in the synovial membrane.
Hyaluronan; Carrageenan; TMJ; iNOS; NF-κB
Oxidative stress such as reactive oxygen species (ROS) within the inflamed joint have been indicated as being involved as inflammatory mediators in the induction of arthritis. Correlations between extracellular-superoxide dismutase (EC-SOD) and inflammatory arthritis have been shown in several animal models of RA. However, there is a question whether the over-expression of EC-SOD on arthritic joint also could suppress the progression of disease or not. In the present study, the effect on the synovial tissue of experimental arthritis was investigated using EC-SOD over-expressing transgenic mice. The over-expression of EC-SOD in joint tissue was confirmed by RT-PCR and immunohistochemistry. The degree of the inflammation in EC-SOD transgenic mice was suppressed in the collagen-induced arthritis model. In a cytokine assay, the production of pro-inflammatory cytokines such as, IL-1β, TNFα, and matrix metalloproteinases (MMPs) was decreased in fibroblast-like synoviocyte (FLS) but not in peripheral blood. Histological examination also showed repressed cartilage destruction and bone in EC-SOD transgenic mice. In conclusion, these data suggest that the over-expression of EC-SOD in FLS contributes to the activation of FLS and protection from joint destruction by depressing the production of the pro-inflammatory cytokines and MMPs. These results provide EC-SOD transgenic mice with a useful animal model for inflammatory arthritis research.
arthritis, experimental; reactive oxygen species; rheumatoid arthritis; superoxide dismutase; synovial membrane