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1.  Histopathological findings, phenotyping of inflammatory cells, and expression of markers of nitritative injury in joint tissue samples from calves after vaccination and intraarticular challenge with Mycoplasma bovis strain 1067 
Background
The pathogenesis of caseonecrotic lesions developing in lungs and joints of calves infected with Mycoplasma bovis is not clear and attempts to prevent M. bovis-induced disease by vaccines have been largely unsuccessful. In this investigation, joint samples from 4 calves, i.e. 2 vaccinated and 2 non-vaccinated, of a vaccination experiment with intraarticular challenge were examined. The aim was to characterize the histopathological findings, the phenotypes of inflammatory cells, the expression of class II major histocompatibility complex (MHC class II) molecules, and the expression of markers for nitritative stress, i.e. inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT), in synovial membrane samples from these calves. Furthermore, the samples were examined for M. bovis antigens including variable surface protein (Vsp) antigens and M. bovis organisms by cultivation techniques.
Results
The inoculated joints of all 4 calves had caseonecrotic and inflammatory lesions. Necrotic foci were demarcated by phagocytic cells, i.e. macrophages and neutrophilic granulocytes, and by T and B lymphocytes. The presence of M. bovis antigens in necrotic tissue lesions was associated with expression of iNOS and NT by macrophages. Only single macrophages demarcating the necrotic foci were positive for MHC class II. Microbiological results revealed that M. bovis had spread to approximately 27% of the non-inoculated joints. Differences in extent or severity between the lesions in samples from vaccinated and non-vaccinated animals were not seen.
Conclusions
The results suggest that nitritative injury, as in pneumonic lung tissue of M. bovis-infected calves, is involved in the development of caseonecrotic joint lesions. Only single macrophages were positive for MHC class II indicating down-regulation of antigen-presenting mechanisms possibly caused by local production of iNOS and NO by infiltrating macrophages.
doi:10.1186/s13028-014-0045-3
PMCID: PMC4236525  PMID: 25162202
Mycoplasma bovis; Arthritis; Vaccination; Variable surface protein antigens; MHC class II; Inducible nitric oxide; Nitrotyrosine
2.  Gene therapy for established murine collagen-induced arthritis by local and systemic adenovirus-mediated delivery of interleukin-4 
Arthritis Research  2000;2(4):293-302.
To determine whether IL-4 is therapeutic in treating established experimental arthritis, a recombinant adenovirus carrying the gene that encodes murine IL-4 (Ad-mIL-4) was used for periarticular injection into the ankle joints into mice with established collagen-induced arthritis (CIA). Periarticular injection of Ad-mIL-4 resulted in a reduction in the severity of arthritis and joint swelling compared with saline- and adenoviral control groups. Local expression of IL-4 also reduced macroscopic signs of joint inflammation and bone erosion. Moreover, injection of Ad-mIL-4 into the hind ankle joints resulted in a decrease in disease severity in the untreated front paws. Systemic delivery of murine IL-4 by intravenous injection of Ad-mIL-4 resulted in a significant reduction in the severity of early-stage arthritis.
Introduction:
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that is characterized by joint inflammation, and progressive cartilage and bone erosion. Recent research has identified certain biologic agents that appear more able than conventional therapies to halt effectively the progression of disease, as well as ameliorate disease symptoms. One potential problem with the use of biologic agents for arthritis therapy is the need for daily or weekly repeat dosing. The transfer of genes directly to the synovial lining can theoretically circumvent the need for repeat dosing and reduce potential systemic side effects [1,2]. However, although many genes have been effective in treating murine CIA if administrated at a time before disease onset, local intra-articular or periarticular gene transfer has not been highly effective in halting the progression of established disease. IL-4, similar to tumor necrosis factor (TNF)-α and IL-1 inhibitors, has been shown be therapeutic for the treatment of murine CIA when administered intravenously as a recombinant protein, either alone or in combination with IL-10. IL-4 can downregulate the production of proinflammatory and T-helper (Th)1-type cytokines by inducing mRNA degradation and upregulating the expression of inhibitors of proinflammatory cytokines such as IL-1 receptor antagonist (IL-1Ra) [3,4]. IL-4 is able to inhibit IL-2 and IFN-γ production by Th1 cells, resulting in suppression of macrophage activation and the production of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-α by monocytes and macrophages [4,5,6,7,8,9].
Objective:
In order to examine the therapeutic effects of local and systemic IL-4 expression in established CIA, an adenoviral vector carrying the gene for murine IL-4 (Ad-mIL-4) was generated. The ability of Ad-mIL-4 to treat established CIA was evaluated by local periarticular and systemic intravenous injection of Ad-mIL-4 into mice at various times after disease onset.
Materials and methods:
Male DBA/1 lacJ (H-2q) mice, aged 7-8 weeks, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were immunized intradermally at the base of tail with 100 μ g bovine type II collagen. On day 21 after priming, mice received a boost injection (intradermally) with 100 μ g type II collagen in incomplete adjuvant. For the synchronous onset of arthritis, 40 μ g lipopolysaccharide (Sigma, St Louis, MO, USA) was injected intraperitoneally on day 28. Ad-mIL-4 was injected periarticularly into the hind ankle joints of mice on day 32 or intravenously by tail vein injection on day 29. Disease severity was monitored every other day using an established macroscopic scoring system ranging from 0 to 4: 0, normal; 1, detectable arthritis with erythma; 2, significant swelling and redness; 3, severe swelling and redness from joint to digit; and 4, maximal swelling with ankylosis. The average of macroscopic score was expressed as a cumulative value for all paws, with a maximum possible score of 16 per mouse. Cytokine production by joint tissue or serum were assessed using enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA).
Results:
To examine the therapeutic effects of IL-4 gene transfer in a murine model of arthritis, 5×108 particles of Ad-mIL-4 and enhanced green fluorescent protein (Ad-eGFP) were administered by periarticular injection into the ankle joints of mice with established disease 4 days after lipopolysaccharide injection. All mice had established disease at time of injection. As shown in Figure 1, the severity of arthritis (Fig. 1a), paw thickness (Fig. 1b), and the number of arthritic paws (Fig. 1c) were all significantly reduced in the Ad-mIL-4 group, compared with the saline- and Ad-eGFP-treated groups. Analysis of the bones in the ankle joints of control arthritic mice showed evidence of erosion with an associated monocytic infiltrate around the joint space compared with the Ad-mIL-4-treated and nonarthritic control joints. In addition, injection of the ankle joints in the hind legs resulted in a therapeutic effect in the front paws. A similar contralateral effect has been observed with adenoviral-mediated delivery of viral (v)-IL-10. Interestingly, a high level of murine IL-10 also was detected from the joint lysates of Ad-mIL-4-treated naïve and arthritic mice, with the production of endogenous IL-10 correlating with the dose of Ad-mIL-4. The administration of recombinant IL-4 protein systemically has been shown to be therapeutic in murine CIA models if given before disease onset. To examine the effect of systemic IL-4 delivered by gene transfer, 1×109 particles of Ad-mIL-4 were injected via the tail vein of collagen-immunized mice the day after lipopolysaccharide injection. Whereas the immunized control mice, injected with Ad-eGFP, showed disease onset on day 3 after lipopolysaccharide injection, Ad-mIL-4-treated mice showed a delay in disease onset and as a reduction in the total number of arthritic paws. Also, systemic injection of Ad-mIL-4 suppressed the severity of arthritis in CIA mice according to arthritis index.
Discussion:
Gene therapy represents a novel approach for delivery of therapeutic agents to joints in order to treat the pathologies associated with RA and osteoarthritis, as well as other disorders of the joints. In the present study we examined the ability of local periarticular and systemic gene transfer of IL-4 to treat established and early-stage murine CIA, respectively. We have demonstrated that both local and systemic administration of Ad-mIL-4 resulted in a reduction in the severity of arthritis, as well as in the number of arthritic paws. In addition, the local gene transfer of IL-4 reduced histologic signs of inflammation and of bone erosion. Interestingly, local delivery of Ad-mIL-4 was able to confer a therapeutic effect to the untreated, front paws through a currently unknown mechanism. In addition, both local and systemic expression of IL-4 resulted in an increase in the level of endogenous IL-10, as well as of IL-1Ra (data not shown). Previous experiments have shown that gene transfer of IL-10 and IL-1 and TNF inhibitors at the time of disease initiation (day 28) is therapeutic. However, delivery of these agents after disease onset appeared to have only limited therapeutic effect. In contrast, the present results demonstrate that IL-4, resulting from local periarticular and systemic injection of Ad-mIL-4, was able partially to reverse progression of established and early-stage disease, respectively. These results, as well as those of others, support the potential application of IL-4 gene therapy for the clinical treatment of RA.
PMCID: PMC17812  PMID: 11056670
adenoviral vectors; collagen-induced arthritis; gene therapy; IL-4; IL-10; rheumatoid arthritis
3.  Role of TNF alpha in the induction of antigen induced arthritis in the rabbit and the anti-arthritic effect of species specific TNF alpha neutralising monoclonal antibodies. 
Annals of the Rheumatic Diseases  1995;54(5):366-374.
OBJECTIVE--To investigate the role of tumour necrosis factor alpha (TNF alpha) in the development of antigen induced arthritis (AIA) in rabbits. METHODS--Monoclonal antibodies to rabbit TNF alpha were developed in rats and were used to detect TNF alpha in synovial fluid by enzyme linked immunosorbent assay and to localise it in tissue sections of synovium and cartilage from rabbits up to 21 days after induction of AIA. An antibody which neutralised TNF alpha activity in vitro was injected into rabbits to block TNF alpha action in vivo in AIA. Joint swelling, leucocyte infiltration into synovium and proteoglycan loss from cartilage were measured and compared with a control group, which were injected with sterile saline. RESULTS--Monoclonal antibodies to purified rabbit TNF alpha were prepared in rats and two were selected which were able to neutralise rabbit TNF alpha in a cytotoxicity bioassay. TNF alpha was detected in significant concentrations (21.7 (SE 0.5) pg/ml) in the arthritic joint fluid of rabbits with AIA only at one day after induction and it was then also sparsely localised in cells of the synovium, but from day 3 onwards it was localised more strongly in the deep zone of articular cartilage. Injection of anti-TNF monoclonal antibody R6 over three days into rabbits with AIA reduced joint swelling and leucocyte infiltration into joint fluid and decreased the expression of CD11b and CD18 on cells in the joint fluid. However, there was no significant reduction in the loss of proteoglycan from articular cartilage, although the joint fluid at three days contained a lower glycosaminoglycan content. The antibody R6 gave most effect at a dose of 0.6 mg/kg and there was no increase in its effectiveness at a fivefold greater dose (3.0 mg/kg). Treatment over 10 days gave a more complete suppression of joint swelling, but did not result in any less proteoglycan loss from cartilage. Treatment for five days with a 16 day follow up gave a significant reduction in swelling for several days beyond the treatment, but the swelling then slowly returned, until by day 21 there was no significant difference in joint swelling and there was also no recovery of cartilage proteoglycan content. A rabbit anti-rat immunoglobulin response was detected at 21 days, which may have limited the long term effectiveness of the antibody. CONCLUSIONS--In AIA in rabbits, TNF alpha was only detected in synovial fluid at one day after induction and there was only limited cellular localisation of TNF alpha in synovium and cartilage from three days. However, neutralising TNF alpha with a monoclonal antibody was effective in suppressing inflammatory changes in the joint during the acute onset of AIA, but it had little effect on the loss of proteoglycan from cartilage. The results suggest that blocking inflammation and synovitis with anti-TNF alpha may be more easily achieved than preventing damage to articular cartilage.
Images
PMCID: PMC1005596  PMID: 7794042
4.  Characterization of a novel and spontaneous mouse model of inflammatory arthritis 
Arthritis Research & Therapy  2011;13(4):R114.
Introduction
Mouse models of rheumatoid arthritis (RA) have proven critical for identifying genetic and cellular mechanisms of the disease. Upon discovering mice in our breeding colony that had spontaneously developed inflamed joints reminiscent of RA, we established the novel IIJ (inherited inflamed joints) strain. The purpose of this study was to characterize the histopathological, clinical, genetic and immunological properties of the disease.
Methods
To begin the IIJ strain, an arthritic male mouse was crossed with SJL/J females. Inheritance of the phenotype was then tracked by intercrossing, backcrossing and outcrossing to other inbred strains. The histopathology of the joints and extraarticular organ systems was examined. Serum cytokines and immunoglobulins (Igs) were measured by ELISA and cytometric bead array. Transfer experiments tested whether disease could be mediated by serum alone. Finally, the cellular joint infiltrate and the composition of secondary lymphoid organs were examined by immunohistochemistry and flow cytometry.
Results
After nine generations of intercrossing, the total incidence of arthritis was 33% (304 of 932 mice), with females being affected more than males (38% vs. 28%; P < 0.001). Swelling, most notably in the large distal joints, typically became evident at an early age (mean age of 52 days). In addition to the joint pathology, which included bone and cartilage erosion, synovial hyperproliferation and a robust cellular infiltration of mostly Gr-1+ neutrophils, there was also evidence of systemic inflammation. IL-6 was elevated in the sera of recently arthritic mice, and extraarticular inflammation was observed histologically in multiple organs. Total serum Ig and IgG1 levels were significantly elevated in arthritic mice, and autoantibodies such as rheumatoid factor and Ig reactive to joint components (collagen type II and joint homogenate) were also detected. Nevertheless, serum failed to transfer disease. A high percentage of double-negative (CD4-CD8-) CD3+ TCRα/β+ T cells in the lymphoid organs of arthritic IIJ mice suggested significant disruption in the T-cell compartment.
Conclusions
Overall, these data identify the IIJ strain as a new murine model of inflammatory, possibly autoimmune, arthritis. The IIJ strain is similar, both histologically and serologically, to RA and other murine models of autoimmune arthritis. It may prove particularly useful for understanding the female bias in autoimmune diseases.
doi:10.1186/ar3399
PMCID: PMC3239352  PMID: 21749708
5.  Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis. 
Journal of Clinical Investigation  1994;94(3):1012-1018.
We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs in the milieu of the inflamed joint of RA patients.
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PMCID: PMC295150  PMID: 8083342
6.  Distribution of Mycoplasma pneumoniae and Mycoplasma salivarium in the Synovial Fluid of Arthritis Patients▿  
Journal of Clinical Microbiology  2006;45(3):953-957.
By use of a very sensitive nested PCR method targeting part of the strongly conserved mycoplasmal 16S RNA genes, Mycoplasma pneumoniae was found in the synovial fluid of 19/24 (79%) of rheumatoid arthritis patients, 6/6 (100%) of patients with nonrheumatoid inflammatory arthritis, and 8/10 (80%) of osteoarthritis patients attending the rheumatology clinic for drainage of joint effusions. It was not found in the synovial exudates of 13 people attending the orthopedic clinic with traumatic knee injuries or undergoing surgery for knee replacement. However, M. pneumoniae was detected in 2/4 synovial biopsy specimens from orthopedic patients with traumatic knee injuries. M. pneumoniae was associated with the increased synovial fluids found in arthritic flares but was not found in the synovial fluids of trauma patients. Mycoplasma salivarium occurred sporadically. Mycoplasma fermentans had previously been isolated from patients with inflammatory cellular infiltrates, such as rheumatoid arthritis, but it was not detected for osteoarthritic patients from either clinic. It is possible that these organisms may contribute to chronic inflammation within the joints.
doi:10.1128/JCM.01973-06
PMCID: PMC1829110  PMID: 17122006
7.  Acute Immunologic Arthritis in Rabbits 
Mediators of acute immunologic injury have been studied in vivo by producing arthritis in rabbit knee joints. A reversed passive Arthus lesion was produced by injecting antibody into the joint space and antigen intravenously. Injury was assessed by measuring leakage of serum proteins and circulating radiolabeled proteins into the joint space and by the accumulation of neutrophils in the joint fluid. Inflammatory exudate was recovered for study by a standardized irrigation technique.
Maximal vascular permeability developed 2 hr after injection as neutrophils accumulated about immune complexes in venule walls to produce structural injury. After 5 hr the number of neutrophils in the joint space rose rapidly, followed by a second rise in permeability at 8 hr. Neutrophil depletion abolished both peaks of permeability. It was then possible to reconstitute the synovial lesion in neutrophil-depleted rabbits by intra-articular injection of purified suspensions of neutrophils.
A requirement for complement was demonstrated in development of the lesion. Rabbits genetically deficient in C6 showed delay in vascular permeability, appearance of neutrophils, and histologic lesions. The delay was longer in normal rabbits depleted of C3. In C6-deficient rabbits depleted of C3, still further reduction in injury occurred.
Evidence was obtained as well for a chemotactic attraction of neutrophils in vivo. Antigen-antibody-complement complexes in the walls of blood vessels attracted neutrophils placed in the joint space of neutrophil-depleted rabbits. Omission of either antigen or antibody from this replacement reaction prevented the migration of neutrophils.
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PMCID: PMC332928  PMID: 4257028
8.  Naturally occurring and experimentally induced mycoplasmal arthritis of cattle. 
Journal of Clinical Microbiology  1975;2(3):165-168.
Mycoplasma agalactiae subsp. bovis strain Iowa 1136 was isolated from synovial fluids of a clinical case of arthritis in cattle on pasture in Iowa. When given to calves and cows by intra-articular or intravenous injection, it caused severe and persistent joint infections with fever, lameness, and swelling of the affected joints, plus synovitis, tendonitis, and fibrinous-purulent synovial fluids of high protein content. Intramammary administration of the organism caused severe mastitis. Calves nursing the cows developed severe mycoplasmal arthritis.
PMCID: PMC274164  PMID: 1176623
9.  Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model 
World Journal of Stem Cells  2012;4(10):101-109.
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.
doi:10.4252/wjsc.v4.i10.101
PMCID: PMC3506964  PMID: 23189211
Complete freunds adjuvant-induced arthritis; Human umbilical mesenchymal stem cell; Hematopoietic stem cell; CD34+
10.  Apoptosis and p53 expression in rat adjuvant arthritis 
Arthritis Research  2000;2(3):229-235.
The kinetics of apoptosis and the apoptosis-regulating gene p53 in adjuvant arthritis (AA) were investigated to assess the value of the AA rat model for testing apoptosis-inducing therapies. Very few terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were detected during the early phases of AA, but on day 23 (chronic arthritis) the percentage of TUNEL-positive cells was significantly increased. Expression of p53 in synovial tissue gradually increased from days 5-23, which was markedly higher than p53 levels in rheumatoid arthritis (RA) synovium. Significant apoptosis only occurs late in rat AA and is concordant with marked p53 overexpression, making it useful model for testing proapoptotic therapies, but rat AA is not the best model for p53 gene therapy because dramatic p53 overexpression occurs in the latter stages of the disease.
Introduction:
RA is a chronic inflammatory disorder that is characterized by inflammation and proliferation of synovial tissue. The amount of DNA fragmentation is significantly increased in rheumatoid synovium. Only low numbers of apoptotic cells are present in rheumatoid synovial tissue, however. The proportion of cells with DNA strand breaks is so great that this disparity suggests impaired apoptosis. Therefore, the development of novel therapeutic strategies that are aimed at inducing apoptosis in rheumatoid synovial tissue is an attractive goal.
Although animal models for arthritis only approximate RA, they provide a useful test system for the evaluation of apoptosis-inducing therapies. AA in rats is among the most commonly used animal models for RA. For the interpretation of such studies, it is essential to characterize the extent to which apoptosis occurs during the natural course of the disease. Therefore, we evaluated the number of apoptotic cells and the expression of p53 in various phases of AA.
Materials and methods:
In order to generate the AA rat model, Lewis rats were immunized with Mycobacterium tuberculosis in mineral oil on day 0. Paw swelling usually started around day 10. For the temporal analysis rats were sacrificed on days 0, 5 (prearthritis), 11 (onset of arthritis), 17 (accelerating arthritis), or 23 (chronic arthritis).
For the detection of apoptotic cells, the hind paws were harvested on days 0(n=6),5 (n=6), 11 (n=6), 17 (n=6), or 23 (n=4). The right ankle joints were fixed in formalin, decalcified in ethylenediaminetetra-acetic acid, embedded in paraffin, and sectioned. The TUNEL method was applied. The percentage of TUNEL-positive cells of the total inflammatory cell infiltrate was noted.
For Western blot analysis, hind paws were harvested on days 0 (n=2), 5 (n=3), 11 (n=4), 17 (n=4), or 23 (n=4). In addition, hind paws of normal rats (n=2) were studied. The right ankle joints were snap frozen and pulverized. Synovial tissue was also obtained by arthroscopy of three patients with longstanding (>5 years) RA. After protein extraction in lysis buffer, equal amounts of protein samples from lysates were pooled and examined by Western bolt analysis using anti-p53 monoclonal antibody D07, which recognizes wild-type and mutant p53 from rodents and humans.
For immunohistochemical analysis, six rats were sacrificed on day 23 after immunization and synovial tissue of the right ankle joints was snap frozen and evaluated by immunohistochemistry using anti-p53-pan. The sections were evaluated semi-quantitatively using a 0-4 scale.
The kruskal-Wallis test for several group means was used to compare the percentage of TUNEL-positive cells at different time points.
Results:
The percentages of TUNEL-positive cells were strongly dependent on the stage of the disease. Very few TUNEL-positive cells were detected in normal rats or in the early phases of AA; the number of TUNEL-positive cells was 1% or less of the total cell infiltrate, including neutrophils, from days 0-17 (Table 1). On day 23, however, the percentage of TUNEL-positive cells was significantly increased [15.8±5.1% (mean ± standard error of the mean); P=0.01]. TUNEL-positive cells were observed in the intimal lining layer and synovial sublining of the invasive front, as well as in the articular cartilage (Fig. 1).
Subsequently, we examined expression of the tumor suppressor gene p53, because this is a key regulator of apoptosis. Expression of p53 in pooled rat AA joint extracts gradually increased from day 0 (6 arbitrary units) to day 23 (173 arbitrary units), which was markedly higher than p53 levels in RA synovium (32 arbitrary units; Table 1). Overexpression of p53 protein on day 23 was confirmed by immunohistochemistry in a separate experiment in six rats with AA. Overexpression of p53 was observed in the intimal lining layer and synovial sublining in all rats on day 23. In all cases a semiquantitative score of 4 was assigned, indicating that 51% or more of the cells were positive, whereas control sections were negative.
Discussion:
The results presented here reveal that the number of TUNEL-positive cells remained very low until chronic arthritis developed. This indicates that, although there was sufficient DNA damage to cause an increment in p53 expression in the early phases, DNA strand breaks that can be detected by TUNEL assays only occurred in chronic AA. The observation that TUNEL-positive cells were nearly absent in early AA clearly indicates that only very few cells were undergoing programmed cell death. This is an important observation, which makes it possible to study the effects of apoptosis-inducing therapies in situ in early and accelerating AA. An effective therapy would obviously increase the number of TUNEL-positive cells.
There is already some overexpression of p53 in the preclinical phase and during the onset of the arthritis, with an additional increment in p53 expression during accelerating and chronic arthritis. Presumably, this is wild-type p53, because the disease duration is likely too short to allow for the development of p53 mutations. Transcription of p53 is probably increased in response to the toxic environment of the inflamed joint. The increased expression of p53 in the joints of rats with chronic AA was even greater than that observed in synovial tissue of RA patients with long-standing disease.
Overexpression of p53 and increased numbers of apoptotic cells did not occur simultaneously in this model; rather p53 overexpression preceded increased apoptosis. Activation of p53 leads to induction of cell growth arrest, allowing time for DNA repair. It appears that DNA damage is only extensive enough to induce apoptosis in the latter stages of AA. Factors other than p53 may also play an important role in the actual induction of apoptosis
Taken together, significant apoptosis only occurs late in AA and it follows marked p53 overexpression, making it a useful model for testing proapoptotic therapies. AA is not the best model for p53 gene therapy, however, because dramatic p53 overexpression occurs in the latter stages of the disease.
PMCID: PMC17810  PMID: 11056668
adjuvant arthritis; apoptosis; p53; rheumatoid arthritis
11.  Mast cell activation and its relation to proinflammatory cytokine production in the rheumatoid lesion 
Arthritis Research  1999;2(1):65-74.
Mast cell (MC) activation in the rheumatoid lesion provides numerous mediators that contribute to inflammatory and degradative processes, especially at sites of cartilage erosion. MC activation in rheumatoid synovial tissue has often been associated with tumour necrosis factor (TNF)-α and interleukin (IL)-1β production by adjacent cell types. By contrast, our in situ and in vitro studies have shown that the production of IL-15 was independent of MC activation, and was not related to TNF-α and IL-1β expression. Primary cultures of dissociated rheumatoid synovial cells produced all three proinflammatory cytokines, with production of IL-1β exceeding that of TNF-α, which in turn exceeded that of IL-15. In vitro cultures of synovial macrophages, synovial fibroblasts and articular chondrocytes all produced detectable amounts of free IL-15, macrophages being the most effective.
Introduction:
Increased numbers of mast cells (MCs) are found in the synovial tissues and fluids of patients with rheumatoid arthritis (RA), and at sites of cartilage erosion. MC activation has been reported for a significant proportion of rheumatoid specimens. Because the MC contains potent mediators, including histamine, heparin, proteinases, leukotrienes and multifunctional cytokines, its potential contributions to the processes of inflammation and matrix degradation have recently become evident.
Proinflammatory cytokines are important mediators of inflammation, immunity, proteolysis, cell recruitment and proliferation. Tumour necrosis factor (TNF) reportedly plays a pivotal role in the pathogenesis of RA, especially its ability to regulate interleukin (IL)-1β expression, this being important for the induction of prostanoid and matrix metalloproteinase production by synovial fibroblasts and chondrocytes. IL-15 has been assigned numerous biological effects and has been implicated as an important factor in TNF-α expression by monocyte/macrophages. Some in vitro studies have placed IL-15 upstream from TNF-α in the cytokine cascade, suggesting an interdependence between TNF, IL-1 and IL-15 for the promotion of proinflammatory cytokine expression in the rheumatoid joint.
Aims:
To examine the in situ relationships of TNF-α, IL-1β and IL-15 in relation to MC activation in rheumatoid tissues by use of immunolocalization techniques; and to compare quantitatively the proinflammatory cytokine production by specific cell cultures and rheumatoid synovial explants with and without exposure to a MC secretagogue.
Materials and methods:
Samples of rheumatoid synovial tissue and cartilage–pannus junction were obtained from patients (n = 15) with classic late-stage RA. Tissue sections were immunostained for MC (tryptase) and the proinflammatory cytokines IL-1, TNF-α and IL-15. Rheumatoid synovial tissue explants were cultured in Dulbecco's modified Eagles medium (DMEM) containing either the MC secretagogue rabbit antihuman immunoglobulin (Ig)E, or control rabbit IgG. Primary rheumatoid synovial cell cultures, human articular chondrocytes, synovial fibroblasts and synovial macrophages were prepared as described in the full article. Conditioned culture media from these cultures were collected and assayed for IL-1β, TNF-α and IL-15 using enzyme-linked immunosorbent assay methodology.
Results:
Immunohistological studies of rheumatoid synovial tissues have demonstrated local concentrations of MCs in most specimens of the rheumatoid lesion. Sites of MC activation were associated with localized oedema, and TNF-α, IL-1α and IL-1β production by a proportion of mononuclear inflammatory cells. By contrast, no evidence was found for IL-15 production in tissue sites containing either intact or activated MCs, and IL-15 expression, when observed, bore no relation to tissue sites where TNF-α and IL-1β were evident. The immunodetection of IL-15 was restricted to microfocal sites and was not typical of most junctional specimens, but was associated with a proportion of articular chondrocytes in a minority of junctional specimens.
MC activation within synovial explant cultures was induced by the addition of polyclonal antibody to human IgE. MC activation significantly reduced the levels of TNF-α and IL1β released into the medium, this representing approximately 33% of control values. By contrast, MC activation had little effect on the levels of IL-15 released into the culture medium, the average value being very low in relation to the release of TNF-α and IL-1β . Thus, induced MC activation brings about changes in the amounts of released tryptase, TNF-α and IL-1β , but not of IL-15.
Four preparations of primary rheumatoid synovial cell cultures produced more IL-1β than TNF-α, with only modest values for IL-15 production, indicating that all three cytokines are produced and released as free ligands by these cultures. Of specific cell types that produced IL-15 in vitro, macrophages produced more than fibroblasts, which in turn produced more than chondrocytes. This demonstrates that all three cell types have the potential to produce IL-15 in situ.
Discussion:
The biological consequences of MC activation in vivo are extremely complex, and in all probability relate to the release of various combinations of soluble and granular factors, as well as to the expression of appropriate receptors by neighbouring cells. The subsequent synthesis and release of cytokines such as TNF-α and IL-1 may well follow at specific stages after activation, or may be an induced cytokine response by adjacent macrophagic or fibroblastic cells. However, because no IL-15 was detectable either in or around activated or intact MCs, and the induced MC activation explant study showed no change in IL-15 production, it seems unlikely that the expression of this cytokine is regulated by MCs. The immunohistochemistry (IHC) demonstration of IL-15 at sites of cartilage erosion, and especially by some chondrocytes of articular cartilage, showed no spatial relationship with either T cells or neutrophils, and suggests other functional properties in these locations. The lack of evidence for an in situ association of IL-15 with TNF and IL-1 does not support a role for IL-15 in a proinflammatory cytokine 'cascade', as proposed by other in vitro experiments. We believe that sufficient evidence is available, however, to suggest that MC activation makes a significant contribution to the pathophysiological processes of the rheumatoid lesion.
PMCID: PMC17805  PMID: 11219391
interleukin-15; interleukin-1β; mast cells; rheumatoid arthritis; tumour necrosis factor-α
12.  c-Fms-mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis 
Introduction
Tyrosine kinases are key mediators of multiple signaling pathways implicated in rheumatoid arthritis (RA). We previously demonstrated that imatinib mesylate--a Food and Drug Administration (FDA)-approved, antineoplastic drug that potently inhibits the tyrosine kinases Abl, c-Kit, platelet-derived growth factor receptor (PDGFR), and c-Fms--ameliorates murine autoimmune arthritis. However, which of the imatinib-targeted kinases is the principal culprit in disease pathogenesis remains unknown. Here we examine the role of c-Fms in autoimmune arthritis.
Methods
We tested the therapeutic efficacy of orally administered imatinib or GW2580, a small molecule that specifically inhibits c-Fms, in three mouse models of RA: collagen-induced arthritis (CIA), anti-collagen antibody-induced arthritis (CAIA), and K/BxN serum transfer-induced arthritis (K/BxN). Efficacy was evaluated by visual scoring of arthritis severity, paw thickness measurements, and histological analysis. We assessed the in vivo effects of imatinib and GW2580 on macrophage infiltration of synovial joints in CIA, and their in vitro effects on macrophage and osteoclast differentiation, and on osteoclast-mediated bone resorption. Further, we determined the effects of imatinib and GW2580 on the ability of macrophage colony-stimulating factor (M-CSF; the ligand for c-Fms) to prime bone marrow-derived macrophages to produce tumor necrosis factor (TNF) upon subsequent Fc receptor ligation. Finally, we measured M-CSF levels in synovial fluid from patients with RA, osteoarthritis (OA), or psoriatic arthritis (PsA), and levels of total and phosphorylated c-Fms in synovial tissue from patients with RA.
Results
GW2580 was as efficacious as imatinib in reducing arthritis severity in CIA, CAIA, and K/BxN models of RA. Specific inhibition of c-Fms abrogated (i) infiltration of macrophages into synovial joints of arthritic mice; (ii) differentiation of monocytes into macrophages and osteoclasts; (iii) osteoclast-mediated bone resorption; and (iv) priming of macrophages to produce TNF upon Fc receptor stimulation, an important trigger of synovitis in RA. Expression and activation of c-Fms in RA synovium were high, and levels of M-CSF were higher in RA synovial fluid than in OA or PsA synovial fluid.
Conclusions
These results suggest that c-Fms plays a central role in the pathogenesis of RA by mediating the differentiation and priming of monocyte lineage cells. Therapeutic targeting of c-Fms could provide benefit in RA.
doi:10.1186/ar2940
PMCID: PMC2875666  PMID: 20181277
13.  The effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase and prostaglandin E2 production by cells of the rheumatoid lesion 
Arthritis Research  1999;1(1):63-70.
The biologically active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], acts through vitamin D receptors, which were found in rheumatoid tissues in the present study. IL-1β-activated rheumatoid synovial fibroblasts and human articular chondrocytes were shown to respond differently to exposure to 1α,25(OH)2D3, which has different effects on the regulatory pathways of specific matrix metalloproteinases and prostaglandin E2.
Introduction:
1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the biologically active metabolite of vitamin D3, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)2D3; and cell cultures have suggested that 1α,25(OH)2D3 may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)2D3 within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)2D3 at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)2D3 modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E2 (PGE2) production in two cell types known to express chondrolytic enzymes.
Aims:
To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D3 on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE2 production.
Methods:
Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)2D3, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE2 by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.
Results:
The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)2D3 alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)2D3 whilst MMP-3 production was enhanced (Table 1). The production of PGE2 by RSFs was unaffected by 1α,25(OH)2D3 addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE2 in HACs was not affected by the simultaneous addition of 1α,25(OH)2D3 (Table 2). Pretreatment of RSFs with 1α,25(OH)2D3 for 1 h made no significant difference to IL-1β-induced stimulation of PGE2, but incubation for 16 h suppressed the expected increase in PGE2 to control values. This effect was also noted when 1α,25(OH)2D3 was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)2D3 does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE2 after IL-1β induction.
Discussion:
Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)2D3 in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)2D3 had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)2D3, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)2D3 to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)2D3 treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)2D3 had little effect on basal PGE2 production by RSF, but the enhanced PGE2 production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)2D3. As with MMP production, there are disparate effects of 1α,25(OH)2D3 on IL-1β stimulated PGE2 production by the two cell types; 1α,25(OH)2D3 added concomitantly with IL-1β had no effect on PGE2 production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)2D3 may have a functional role in the joint disease process. 1α,25(OH)2D3 does not appear to directly affect MMP or PGE2 production but does modulate cytokine-induced production.
Comparative effects of 1 α,25-dihydroxyvitamin D3 (1 α,25D3) on interleukin (IL)-1-stimulated matrix metalloproteinase (MMP)-1 and MMP-3 production by rheumatoid synovial fibroblasts and human articular chondrocytes in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
Comparative effects of 1α,25-dihydroxyvitamin D3 (1α,25D3) on Interleukin (IL)-1-stimulated prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocyte in vivo
Data given are normalized relative to control values and are expressed ± SEM for three cultures of each cell type.
PMCID: PMC17774  PMID: 11056661
1α,25-dihydroxyvitamin D3; matrix metalloproteinase; prostaglandin E2; rheumatoid arthritis
14.  Complementary action of granulocyte macrophage colony-stimulating factor and interleukin-17A induces interleukin-23, receptor activator of nuclear factor-κB ligand, and matrix metalloproteinases and drives bone and cartilage pathology in experimental arthritis: rationale for combination therapy in rheumatoid arthritis 
Introduction
Type 17 T helper cells and interleukin (IL)-17 play important roles in the pathogenesis of human and murine arthritis. Although there is a clear link between IL-17 and granulocyte macrophage colony-stimulating factor (GM-CSF) in the inflammatory cascade, details about their interaction in arthritic synovial joints are unclear. In view of the introduction of GM-CSF and IL-17 inhibitors to the clinic, we studied how IL-17 and GM-CSF orchestrate the local production of inflammatory mediators during experimental arthritis.
Methods
To allow detection of additive, complementary or synergistic effects of IL-17 and GM-CSF, we used two opposing experimental approaches: treatment of arthritic mice with neutralising antibodies to IL-17 and GM-CSF and local overexpression of these cytokines in naive synovial joints. Mice were treated for 2 weeks with antibodies against IL-17 and/or GM-CSF after onset of collagen-induced arthritis. Naive mice were injected intraarticularly with adenoviral vectors for IL-17 and/or GM-CSF, resulting in local overexpression. Joint inflammation was monitored by macroscopic scoring, X-rays and histology. Joint washouts, synovial cell and lymph node cultures were analysed for cytokines, chemokines and inflammatory mediators by Luminex analysis, flow cytometry and quantitative polymerase chain reaction.
Results
Combined therapeutic anti-IL-17 and anti-GM-CSF ameliorated arthritis progression, and joint damage was dramatically reduced compared with treatment with anti-IL-17 or anti-GM-CSF alone. Anti-IL-17 specifically reduced synovial IL-23 transcription, whereas anti-GM-CSF reduced transcription of matrix metalloproteinases (MMPs) and receptor activator of nuclear factor κB ligand (RANKL). Overexpression of IL-17 or GM-CSF in naive knee joints elicited extensive inflammatory infiltrate, cartilage damage and bone destruction. Combined overexpression revealed additive and synergistic effects on the production of MMPs, RANKL and IL-23 in the synovium and led to complete destruction of the joint structure within 7 days.
Conclusions
IL-17 and GM-CSF differentially mediate the inflammatory process in arthritic joints and show complementary and local additive effects. Combined blockade in arthritic mice reduced joint damage not only by direct inhibition of IL-17 and GM-CSF but also by indirect inhibition of IL-23 and RANKL. Our results provide a rationale for combination therapy in autoinflammatory conditions, especially for patients who do not fully respond to inhibition of the separate cytokines.
doi:10.1186/s13075-015-0683-5
PMCID: PMC4496892  PMID: 26081345
15.  Enhanced production of monocyte chemoattractant protein-1 in rheumatoid arthritis. 
Journal of Clinical Investigation  1992;90(3):772-779.
Cells within the synovial tissue may recruit mononuclear phagocytes into the synovial fluid and tissues of arthritic patients. We investigated the production of the chemotactic cytokine monocyte chemoattractant protein-1 (MCP-1) using sera, synovial fluid, synovial tissue, as well as macrophages and fibroblasts isolated from synovial tissues from 80 arthritic patients. MCP-1 levels were significantly higher (P less than 0.05) in synovial fluid from RA patients (mean 25.5 +/- 8.1 ng/ml [SE]) compared to synovial fluid from osteoarthritis (OA) patients (0.92 +/- 0.08), or from patients with other arthritides (2.9 +/- 1.5). MCP-1 levels in RA sera (8.44 +/- 2.33) were significantly greater than MCP-1 in normal sera (0.16 +/- 0.06). The quantities of RA synovial fluid IL-8, which is chemotactic for neutrophils and lymphocytes, and MCP-1 were strongly positively correlated (P less than 0.05). To examine the cellular source of MCP-1, RA synovial tissue macrophages and fibroblasts were isolated. Synovial tissue fibroblasts did not express MCP-1 mRNA, but could be induced to produce MCP-1 by stimulation with either IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), or LPS. In contrast, unlike normal peripheral blood monocytes or alveolar macrophages, RA synovial tissue macrophages constitutively expressed MCP-1 mRNA and antigen. Immunohistochemical analysis of synovial tissue showed that a significantly greater percentage of RA macrophages (50 +/- 8%) as compared to either OA macrophages (5 +/- 2) or normal macrophages (1 +/- 0.3) reacted with anti-MCP-1 antibodies. In addition, the synovial lining layer reacted with MCP-1 in both RA and OA synovial tissues. In contrast, only a minority of synovial fibroblasts (18 +/- 8%) from RA synovium were positive for immunolocalization of MCP-1. These results suggest that synovial production of MCP-1 may play an important role in the recruitment of mononuclear phagocytes during inflammation associated with RA and that synovial tissue macrophages are the dominant source of this cytokine.
Images
PMCID: PMC329929  PMID: 1522232
16.  FcgammaR expression on macrophages is related to severity and chronicity of synovial inflammation and cartilage destruction during experimental immune-complex-mediated arthritis (ICA) 
Arthritis Research  2000;2(6):489-503.
We investigated the role of Fcγ receptors (FcγRs) on synovial macrophages in immune-complex-mediated arthritis (ICA). ICA elicited in knee joints of C57BL/6 mice caused a short-lasting, florid inflammation and reversible loss of proteoglycans (PGs), moderate chondrocyte death, and minor erosion of the cartilage. In contrast, when ICA was induced in knee joints of Fc receptor (FcR) γ-chain-/- C57BL/6 mice, which lack functional FcγRI and RIII, inflammation and cartilage destruction were prevented. When ICA was elicited in DBA/1 mice, a very severe, chronic inflammation was observed, and significantly more chondrocyte death and cartilage erosion than in arthritic C57BL/6 mice. The synovial lining and peritoneal macrophages of naïve DBA/1 mice expressed a significantly higher level of FcγRs than was seen in C57BL/6 mice. Moreover, elevated and prolonged expression of IL-1 was found after stimulation of these cells with immune complexes. Zymosan or streptococcal cell walls caused comparable inflammation and only mild cartilage destruction in all strains. We conclude that FcγR expression on synovial macrophages may be related to the severity of synovial inflammation and cartilage destruction during ICA.
Introduction:
Fcγ receptors (FcγRs) present on cells of the haematopoietic lineage communicate with IgG-containing immune complexes that are abundant in the synovial tissue of patients with rheumatoid arthritis (RA). In mice, three classes of FcγR (RI, RII, and RIII) have been described. Binding of these receptors leads to either activation (FcγRI and RIII) or deactivation (FcγRII) of intracellular transduction pathways. Together, the expression of activating and inhibitory receptors is thought to drive immune-complex-mediated diseases.
Earlier studies in our laboratory showed that macrophages of the synovial lining are of utmost importance in the onset and propagation of immune-complex-driven arthritic diseases. Selective depletion of macrophages in the joint downregulated both inflammation and cartilage destruction. As all three classes of FcγR are expressed on synovial macrophages, these cells are among the first that come in contact with immune complexes deposited in the joint. Recently, we observed that when immune complexes were injected into the knee joints of mice, strains susceptible to collagen-type-II arthritis (DBA/1, B10.RIII) developed more severe arthritis than nonsusceptible strains did, or even developed chronic arthritis. One reason why these strains are more susceptible might be their higher levels of FcγRs on macrophage membranes. To test this hypothesis, we investigated the role of FcγRs in inflammation and cartilage damage during immune-complex-mediated arthritis (ICA). First, we studied arthritis and subsequent cartilage damage in mice lacking functional FcγRI and RIII (FcR γ-chain-/- mice). Next, DBA/1 mice, which are prone to develop collagen-type-II arthritis (`collagen-induced arthritis'; CIA) and are hypersensitive to immune complexes, were compared with control C57BL/6 mice as regards cartilage damage and the expression and function of FcγRs on their macrophages.
Aims:
To examine whether FcγR expression on macrophages is related to severity of synovial inflammation and cartilage destruction during immune-complex-mediated joint inflammation.
Methods:
ICA was induced in three strains of mice (FcR γ-chain-/-, C57BL/6, and DBA/1, which have, respectively, no functional FcγRI and RIII, intermediate basal expression of FcγRs, and high basal expression of FcγRs) by passive immunisation using rabbit anti-lysozyme antibodies, followed by poly-L-lysine lysozyme injection into the right knee joint 1 day later. In other experiments, streptococcal-cell-wall (SCW)- or zymosan-induced arthritis was induced by injecting SCW (25 μg) or zymosan (180 μg) directly into the knee joint. At several time points after arthritis induction, knee joints were dissected and studied either histologically (using haematoxylin/eosin or safranin O staining) or immuno-histochemically. The arthritis severity and the cartilage damage were scored separately on an arbitrary scale of 0-3.
FcγRs were immunohistochemically detected using the monoclonal antibody 2.4G2, which detects both FcγRII and RIII. Deposition of IgG and C3c in the arthritic joint tissue was also detected immunohistochemically. Expression of FcγRs by murine peritoneal macrophages was measured using a fluorescence-activated cell sorter (FACS).
Peritoneal macrophages were stimulated using heat-aggregated gamma globulins (HAGGs), and production of IL-1 was measured using a bioassay. To assess the levels of IL-1 and its receptor antagonist (IL-1Ra) during arthritis, tissue was dissected and washed in RPMI medium. Washouts were tested for levels of IL-1 and IL-1Ra using radioimmunoassay and enzyme-linked immunosorbent assay. mRNA was isolated from the tissue, and levels of macrophage inflammatory protein (MIP)-2, monocyte chemoattractant protein (MCP)-1, IL-1, and IL-1Ra were determined using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR).
Results:
ICA induced in knee joints of C57BL/6 mice caused a florid inflammation at day 3 after induction. To investigate whether this arthritis was FcγR-mediated, ICA was induced in FcR γ-chain-/- mice, which lack functional FcγRI and RIII. At day3, virtually no inflammatory cells were found in their knee joints. Levels of mRNA of IL-1, IL-1Ra, MCP-1, and MIP-2, which are involved in the onset of this arthritis, were significantly lower in FcR γ-chain-/- mice than in control C57BL/6 mice. Levels of IL-1 protein were also measured. At 6 h after ICA induction, FcR γ-chain-/- mice and control C57BL/6 mice showed similar IL-1 production as measured by protein level. By 24 h after induction, however, IL-1 production in the FcR γ-chain-/- mice was below the detection limit, whereas the controls were still producing a significant amount. To investigate whether the difference in reaction to immune complexes between the DBA/1 and C57BL/6 mice might be due to variable expression of FcγRs in the knee joint, expression in situ of FcγRs in naïve knee joints of these mice was determined. The monoclonal antibody 2.4G2, which detects both FcγRII and RIII, stained macrophages from the synovial lining of DBA/1 mice more intensely than those from C57BL/6 mice. This finding suggests a higher constitutive expression of FcγRs by macrophages of the autoimmune-prone DBA/1 mice. To quantify the difference in FcγR expression on macrophages of the two strains, we determined the occurrence of FcγRs on peritoneal macrophages by FACS analysis. The levels of FcγR expressed by macrophages were twice as high in the DBA/1 mice as in the C57BL/6 mice (mean fluorescence, respectively, 440 ± 50 and 240 ± 30 intensity per cell). When peritoneal macrophages of both strains were stimulated with immune complexes (HAGGs), we found that the difference in basal FcγR expression was functional. The stimulated macrophages from DBA/1 mice had significantly higher IL-1α levels (120 and 135 pg/ml at 24 and 48 h, respectively) than cells from C57BL/6 mice (45 and 50 pg/ml, respectively).
When arthritis was induced using other arthritogenic triggers than immune complexes (zymosan, SCW), all the mouse strains tested (DBA/1, FcR γ-chain-/-, and C57BL/6) showed similar inflammation, indicating that the differences described above are found only when immune complexes are used to elicit arthritis.
We next compared articular cartilage damage in arthritic joints of the three mouse strains FcR γ-chain-/-, C57BL/6 (intermediate basal expression of FcγRs), and DBA/1 (high basal expression of FcγRs). Three indicators of cartilage damage were investigated: depletion of PGs, chondrocyte death, and erosion of the cartilage matrix. At day 3 after induction of ICA, there was no PG depletion in FcR γ-chain-/- mice, whereas PG depletion in the matrix of the C57BL/6 mice was marked and that in the arthritic DBA/1 mice was even greater. PG depletion was still massive at days 7 and 14 in the DBA/1 mice, whereas by day 14 the PG content was almost completely restored in knee joints of the C57BL/6 mice. Chondrocyte death and erosion of cartilage matrix, two indicators of more severe cartilage destruction, were significantly higher in the DBA/1 than in the C57BL/6 mice, while both indicators were completely absent in the FcR γ-chain-/- mice. Again, when arthritis was induced using other triggers (SCW, zymosan), all strains showed similar PG depletion and no chondrocyte death or matrix erosion. These findings underline the important role of immune complexes and FcγRs in irreversible cartilage damage.
Discussion:
Our findings indicate that inflammation and subsequent cartilage damage caused by immune complexes may be related to the occurrence of FcγRs on macrophages. The absence of functional FcγRI and RIII prevented inflammation and cartilage destruction after induction of ICA, whereas high basal expression of FcγRs on resident joint macrophages of similarly treated mice susceptible to autoimmune arthritis was correlated with markedly more synovial inflammation and cartilage destruction. The difference in joint inflammation between the three strains was not due to different susceptibilities to inflammation per se, since intra-articular injection of zymosan or SCW caused comparable inflammation. Although extensive inflammatory cell mass was found in the synovium of all strains after intra-articular injection of zymosan, no irreversible cartilage damage (chondrocyte death or matrix erosion) was found. ICA induced in C57BL/6 and DBA/1 mice did cause irreversible cartilage damage at later time points, indicating that immune complexes and FcγRs play an important role in inducing irreversible cartilage damage. Macrophages communicate with immune complexes via Fcγ receptors. Absence of functional activating receptors completely abrogates the synovial inflammation, as was shown after ICA induction in FcR γ-chain-/- mice. However, the γ-chain is essential not only in FcγRI and RIII but also for FcεRI (found on mast cells) and the T cell receptor (TcR)-CD3 (Tcells) complex of γδT cells. However, T, B, or mast cells do not play a role in this arthritis that is induced by passive immunisation. Furthermore, this effect was not caused by a difference in clearance of IgG or complement deposition in the tissue. In this study, DBA/1 mice, which are susceptible to collagen-induced autoimmune arthritis and in a recent study have been shown to react hypersensitively to immune complexes, are shown to express higher levels of FcγRs on both synovial and peritoneal macrophages. Because antibodies directed against the different subclasses of FcγR are not available, no distinction could be made between FcγRII and RIII. Genetic differences in DBA/1 mice in genes coding for or regulating FcγRs may be responsible for altered FcγR expression. If so, these mouse strains would have a heightened risk for immune-complex-mediated diseases.
To provide conclusive evidence for the roles of the various classes of FcγR during ICA, experiments are needed in which FcγRs are blocked with specific antibodies, or in which knockout mice lacking one specific class of FcγR are used. The only available specific antibody to FcγR (2.4G2) has a stimulatory effect on cells once bound to the receptor, and therefore cannot be used in blocking experiments. Experiments using specific knockout mice are now being done in our laboratory.
Macrophages are the dominant type of cell present in chronic inflammation during RA and their number has been shown to correlate well with severe cartilage destruction. Apart from that, in humans, these synovial tissue macrophages express activating FcRs, mainly FcγIIIa, which may lead to activation of these macrophages by IgG-containing immune complexes. The expression of FcRs on the surface of these cells may have important implications for joint inflammation and severe cartilage destruction and therefore FCRs may constitute a new target for therapeutic intervention.
PMCID: PMC17821  PMID: 11056679
autoimmunity; cytokines; Fc receptors; inflammation; macrophages
17.  Borrelia burgdorferi migrates into joint capsules and causes an up-regulation of interleukin-8 in synovial membranes of dogs experimentally infected with ticks. 
Infection and Immunity  1997;65(4):1273-1285.
Twenty 6-week-old specific-pathogen-free beagles were infected with Borrelia burgdorferi by tick challenge, and five uninfected dogs served as controls. During the study, all dogs were monitored for infection, clinical signs, and antibody response against B. burgdorferi. During episodes of lameness or postmortem, synovial fluids from each dog were examined for volume, cell number, polymorphonuclear leukocyte (PMN) content, cell viability, and chemotactic activity. Twenty-five tissues collected postmortem from each dog were tested for interleukin-8 (IL-8) mRNA, tumor necrosis factor alpha (TNF-alpha) mRNA, presence of live spirochetes, and histopathological changes. Thirteen infected dogs (group A), which seroconverted rapidly (maximum titers within 50 to 90 days), developed acute and severe mono- or oligoarthritis almost exclusively in the limb closest to the tick bite (median incubation period, 66 days). Synovial fluids of the arthritic joints collected during episodes of lameness had significantly elevated volume, cell count, PMN proportion, cell viability, and chemotactic activity for PMNs. The remaining joints of the same animals contained synovial fluids with elevated chemotactic activity and cell viability. Twelve dogs tested positive for IL-8 mRNA in multiple tissues (synovia, pericardium, and peritoneum), and 10 dogs expressed TNF-alpha mRNA, but only in the tributary lymph nodes of the inflamed joints. Histological examinations revealed severe poly- or oligoarthritis and moderate to severe cortical hyperplasia in draining lymph nodes of the inflamed joints in all 13 dogs. Seven infected dogs with mild or no clinical signs (group B) seroconverted slowly (peak titers after 90 days), and only some joint fluids showed chemotactic activity, which on average was lower than that in inflamed and noninflamed joints from dogs in group A. Four dogs expressed IL-8 mRNA (in the synovia and pericardium), and three dogs had TNF-alpha mRNA in tributary lymph nodes. Histologically, nonsuppurative arthritis was found in multiple joints, and mild to moderate cortical hyperplasia was found in draining lymph nodes. Five uninfected dogs without lameness (group C) had normal synovial fluids and tissues. In all infected dogs, live spirochetes were demonstrated more frequently in tissues of the somatic quadrant closest to the tick bite than in tissues further from the site of infection, suggesting that dissemination of B. burgdorferi occurs more by migration than by blood-borne spread. From these studies employing a canine model of B. burgdorferi infection, we conclude that IL-8 is involved in the pathogenesis of acute Lyme arthritis.
PMCID: PMC175128  PMID: 9119462
18.  New models of chronic synovitis in rabbits induced by mycoplasmas: microbiological, histopathological, and immunological observations on rabbits injected with Mycoplasma arthritidis and Mycoplasma pulmonis. 
Infection and Immunity  1977;16(1):382-396.
A dose-dependent chronic synovitis was induced in rabbit knees after the intra-articular injection of both Mycoplasma arthritidis and Mycoplasma pulmonis. The inflammation progressed from an initial acute phase at 1 week characterized by edema, infiltration of the synovium with monocytes and heterophils, and desquamation of lining cells, to a more chronic phase at 1 and 3 months, in which villus hyperplasia, lymph "nodules," mononuclear cell infiltration, fibroplasia, and collagen deposition were prominent. With one exception, mycoplasmas could no longer be cultivated from the joints 1 month postinoculation. Both mycoplasma species evoked a humoral antibody response that was more marked in synovial fluids than in peripheral blood. A cell-mediated immune reaction, as evidence by enhanced uptake by [3H]thymidine by sensitized blood, spleen, or node lymphocytes in the presence of homologous antigen, was detected only in rabbits injected with M. pulmonis. Lymphocytes taken from arthritic rabbits were no more cytotoxic toward synovial cells derived from normal or arthritic rabbits than were normal lymphocytes. The models of synovitis described in this study offer a convenient probe for determining the mechanisms of mycoplasma-induced inflammation, since they require only a single injection of the initiating agent and, in addition, utilize an animal host large enough for detailed investigation into the nature of mycoplasma/synovium interactions.
Images
PMCID: PMC421532  PMID: 873616
19.  Salmonella typhimurium infection in calves: cell-mediated and humoral immune reactions before and after challenge with live virulent bacteria in calves given live or inactivated vaccines. 
Infection and Immunity  1983;41(2):751-757.
Groups of six calves, 4 to 5 weeks old, were vaccinated either orally with a live auxotrophic Salmonella typhimurium (O-antigen 1,4,12) SL1479 vaccine (10(8) bacteria on day zero, 10(10) bacteria on days 7 and 14) or subcutaneously with a heat-inactivated (56 degrees C, 30 min) S. typhimurium SVA1232 vaccine (10(10) bacteria suspended in 30% [vol/vol] aluminum hydroxide on days zero, 7, and 14). The calves were then orally challenged with either 10(6) (approximately 100 X the 25% lethal dose) or 10(9) (approximately 100,000 X the 25% lethal dose) live bacteria of the calf-virulent S. typhimurium SVA44 strain. The immune reactivity of these calves and of nonvaccinated control calves was followed before and after the challenge infection up to 42 days by (i) intradermal injection of S. typhimurium crude extract, outer membrane protein preparation (porins), and lipopolysaccharide (LPS), (ii) in vitro stimulation of peripheral blood lymphocytes estimated by using uptake of [3H]thymidine, with S. typhimurium crude extract, porins, LPS, and polysaccharide (O-antigenic polysaccharide chain free of lipid A), and Salmonella sp. serotype thompson (O-antigen 6,7) strain IS40 LPS and polysaccharide, and (iii) estimation of the class-specific immunoglobulin G (IgG) and IgM antibody responses against S. typhimurium LPS and porins, and Salmonella sp. serotype thompson LPS. The immune studies showed that in calves given the live vaccine orally, the skin test reactivity and lymphocyte stimulation indices were significantly higher (P values ranging from less than 0.025 to less than 0.0005) against homologous, but not heterologous, antigens than those seen in calves given the heat-inactivated vaccine subcutaneously. In contrast, the IgG and IgM antibody titers against homologous LPS and porins were significantly higher (P less than 0.0005) in sera collected on day 21 from calves given the heat-inactivated vaccine than in calves given the live vaccine. After the oral challenge, calves given the live vaccine showed reduced cell-mediated immune reactions, in agreement with the observation that the host defense could eradicate the challenge organism, whereas calves given the heat-inactivated vaccine showed significantly increased cell-mediated immune reactions (P values ranging from less than 0.025 to less than 0.005), in agreement with the observation that in these calves, the challenge strain caused enteritis as well as systemic invasion. The increased cell-mediated immune reactivity in calves given the live vaccine correlated well with the excellent protection against challenge infection seen in these animals.
PMCID: PMC264705  PMID: 6347896
20.  Pronounced production of polyclonal immunoglobulin G1 in the synovial fluid of goats with caprine arthritis-encephalitis virus infection. 
Infection and Immunity  1983;41(2):805-815.
Infection of goats with caprine arthritis-encephalitis virus, a lentivirus, resulted in arthritis characterized by the production of intrasynovial immunoglobulin G1 concentrations that were 2 to 5.3 times the serum concentrations in the inoculated carpi at 6 months postinoculation. The intrasynovial immunoglobulin was polyclonal, and its presence was accompanied by increased albumin leakage into the joints. Synovial fluid immunoglobulin levels fluctuated temporally but remained elevated compared with medium-inoculated controls for 38 months after infection. Elevated immunoglobulin G1 concentrations correlated with focal sublumenal plasmacytic infiltrates in the synovia of inoculated carpi at 5 months postinoculation. Inflammation in the uninoculated joints of infected goats was also accompanied by increased intrasynovial immunoglobulin G1 levels. Antibody to systemically administered antigens was a greater proportion of the immunoglobulin population in sera than in synovial fluids of infected goats, suggesting that antibody production to local antigens was responsible for increased intrasynovial immunoglobulin G1 levels.
Images
PMCID: PMC264711  PMID: 6307882
21.  Ectopic Lymphoid Structures Support Ongoing Production of Class-Switched Autoantibodies in Rheumatoid Synovium 
PLoS Medicine  2009;6(1):e1.
Background
Follicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.
Methods and Findings
Using immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.
Conclusions
Our demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.
Costantino Pitzalis and colleagues show that lymphoid structures in synovial tissue of patients with rheumatoid arthritis support production of anti-citrullinated peptide antibodies, which continues following transplantation into SCID mice.
Editors' Summary
Background.
More than 1 million people in the United States have rheumatoid arthritis, an “autoimmune” condition that affects the joints. Normally, the immune system provides protection against infection by responding to foreign antigens (molecules that are unique to invading organisms) while ignoring self-antigens present in the body's own tissues. In autoimmune diseases, this ability to discriminate between self and non-self fails for unknown reasons and the immune system begins to attack human tissues. In rheumatoid arthritis, the lining of the joints (the synovium) is attacked, it becomes inflamed and thickened, and chemicals are released that damage all the tissues in the joint. Eventually, the joint may become so scarred that movement is no longer possible. Rheumatoid arthritis usually starts in the small joints in the hands and feet, but larger joints and other tissues (including the heart and blood vessels) can be affected. Its symptoms, which tend to fluctuate, include early morning joint pain, swelling, and stiffness, and feeling generally unwell. Although the disease is not always easy to diagnose, the immune systems of many people with rheumatoid arthritis make “anti-citrullinated protein/peptide antibodies” (ACPA). These “autoantibodies” (which some experts believe can contribute to the joint damage in rheumatoid arthritis) recognize self-proteins that contain the unusual amino acid citrulline, and their detection on blood tests can help make the diagnosis. Although there is no cure for rheumatoid arthritis, the recently developed biologic drugs, often used together with the more traditional disease-modifying therapies, are able to halt its progression by specifically blocking the chemicals that cause joint damage. Painkillers and nonsteroidal anti-inflammatory drugs can reduce its symptoms, and badly damaged joints can sometimes be surgically replaced.
Why Was This Study Done?
Before scientists can develop a cure for rheumatoid arthritis, they need to know how and why autoantibodies are made that attack the joints in this common and disabling disease. B cells, the immune system cells that make antibodies, mature in structures known as “germinal centers” in the spleen and lymph nodes. In the germinal centers, immature B cells are exposed to antigens and undergo two genetic processes called “somatic hypermutation” and “class-switch recombination” that ensure that each B cell makes an antibody that sticks as tightly as possible to just one antigen. The B cells then multiply and enter the bloodstream where they help to deal with infections. Interestingly, the inflamed synovium of many patients with rheumatoid arthritis contains structures that resemble germinal centers. Could these ectopic (misplaced) lymphoid structures, which are characterized by networks of immune system cells called follicular dendritic cells (FDCs), promote autoimmunity and long-term inflammation by driving the production of autoantibodies within the joint itself? In this study, the researchers investigate this possibility.
What Did the Researchers Do and Find?
The researchers collected synovial tissue from 55 patients with rheumatoid arthritis and used two approaches, called immunohistochemistry and real-time PCR, to investigate whether FDC-containing structures in synovium expressed an enzyme called activation-induced cytidine deaminase (AID), which is needed for both somatic hypermutation and class-switch recombination. All the FDC-containing structures that the researchers found in their samples expressed AID. Furthermore, these AID-containing structures were surrounded by mature B cells making ACPAs. To test whether these B cells were derived from AID-expressing cells resident in the synovium rather than ACPA-expressing immune system cells coming into the synovium from elsewhere in the body, the researchers transplanted synovium from patients with rheumatoid arthritis under the skin of a special sort of mouse that largely lacks its own immune system. Four weeks later, the researchers found that the transplanted human lymphoid tissue was still making AID, that the level of AID expression correlated with the amount of human ACPA in the blood of the mice, and that the B cells in the transplant were proliferating.
What Do These Findings Mean?
These findings show that the ectopic lymphoid structures present in the synovium of some patients with rheumatoid arthritis are functional and are able to make ACPA. Because ACPA may be responsible for joint damage, the survival of these structures could, therefore, be involved in the development and progression of rheumatoid arthritis. More experiments are needed to confirm this idea, but these findings may explain why drugs that effectively clear B cells from the bloodstream do not always produce a marked clinical improvement in rheumatoid arthritis. Finally, they suggest that AID might provide a new target for the development of drugs to treat rheumatoid arthritis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0060001.
This study is further discussed in a PLoS Medicine Perspective by Rene Toes and Tom Huizinga
The MedlinePlus Encyclopedia has a page on rheumatoid arthritis (in English and Spanish). MedlinePlus provides links to other information on rheumatoid arthritis (in English and Spanish)
The UK National Health Service Choices information service has detailed information on rheumatoid arthritis
The US National Institute of Arthritis and Musculoskeletal and Skin Diseases provides Fast Facts, an easy to read publication for the public, and a more detailed Handbook on rheumatoid arthritis
The US Centers for Disease Control and Prevention has an overview on rheumatoid arthritis that includes statistics about this disease and its impact on daily life
doi:10.1371/journal.pmed.0060001
PMCID: PMC2621263  PMID: 19143467
22.  Expression and function of junctional adhesion molecule-C in human and experimental arthritis 
Junctional adhesion molecule-C (JAM-C) is an adhesion molecule involved in transendothelial migration of leukocytes. In this study, we examined JAM-C expression in the synovium and investigated the role of this molecule in two experimental mouse models of arthritis. JAM-C expression was investigated by reverse transcriptase-polymerase chain reaction and immunohistochemistry. The effects of a monoclonal anti-JAM-C antibody were assessed in antigen-induced arthritis (AIA) and K/BxN serum transfer-induced arthritis. JAM-C was expressed by synovial fibroblasts in the lining layer and associated with vessels in the sublining layer in human and mouse arthritic synovial tissue. In human tissue, JAM-C expression was increased in rheumatoid arthritis (RA) as compared to osteoarthritis synovial samples (12.7 ± 1.3 arbitrary units in RA versus 3.3 ± 1.1 in OA; p < 0.05). Treatment of mice with a monoclonal anti-JAM-C antibody decreased the severity of AIA. Neutrophil infiltration into inflamed joints was selectively reduced as compared to T-lymphocyte and macrophage infiltration (0.8 ± 0.3 arbitrary units in anti-JAM-C-treated versus 2.3 ± 0.6 in isotype-matched control antibody-treated mice; p < 0.05). Circulating levels of the acute-phase protein serum amyloid A as well as antigen-specific and concanavalin A-induced spleen T-cell responses were significantly decreased in anti-JAM-C antibody-treated mice. In the serum transfer-induced arthritis model, treatment with the anti-JAM-C antibody delayed the onset of arthritis. JAM-C is highly expressed by synovial fibroblasts in RA. Treatment of mice with an anti-JAM-C antibody significantly reduced the severity of AIA and delayed the onset of serum transfer-induced arthritis, suggesting a role for JAM-C in the pathogenesis of arthritis.
doi:10.1186/ar2223
PMCID: PMC2206366  PMID: 17612407
23.  Monoarticular antigen-induced arthritis leads to pronounced bilateral upregulation of the expression of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion neurons of rats 
Arthritis Research  2000;2(5):424-427.
This study describes the upregulation of neurokinin 1 and bradykinin 2 receptors in dorsal root ganglion (DRG) neurons in the course of antigen-induced arthritis (AIA) in the rat knee. In the acute phase of AIA, which was characterized by pronounced hyperalgesia, there was a substantial bilateral increase in the proportion of lumbar DRG neurons that express neurokinin 1 receptors (activated by substance P) and bradykinin 2 receptors. In the chronic phase the upregulation of bradykinin 2 receptors persisted on the side of inflammation. The increase in the receptor expression is relevant for the generation of acute and chronic inflammatory pain.
Introduction:
Ongoing pain and hyperalgesia (enhanced pain response to stimulation of the tissue) are major symptoms of arthritis. Arthritic pain results from the activation and sensitization of primary afferent nociceptive nerve fibres ('pain fibres') supplying the tissue (peripheral sensitization) and from the activation and sensitization of nociceptive neurons in the central nervous system (central sensitization). After sensitization, nociceptive neurons respond more strongly to mechanical and thermal stimulation of the tissue, and their activation threshold is lowered. The activation and sensitization of primary afferent fibres results from the action of inflammatory mediators such as bradykinin (BK), prostaglandins and others on membrane receptors located on these neurons. BK is a potent pain-producing substance that is contained in inflammatory exudates. Up to 50% of the primary afferent nerve fibres have receptors for BK. When primary afferent nerve fibres are activated they can release neuropeptides such as substance P (SP) and calcitonin gene-related peptide from their sensory endings in the tissue. SP contributes to the inflammatory changes in the innervated tissue (neurogenic inflammation), and it might also support the sensitization of nociceptive nerve fibres by binding to neurokinin 1 (NK1) receptors. NK1 receptors are normally expressed on a small proportion of the primary afferent nerve fibres.
Aims:
Because the expression of receptors on the primary afferent neurons is essential for the pain-producing action of inflammatory mediators and neuropeptides, we investigated in the present study whether the expression of BK and NK1 receptors on primary afferent neurons is altered during the acute and chronic phases of an antigen-induced arthritis (AIA). AIA resembles in many aspects the inflammatory process of human rheumatoid arthritis. Because peptide receptors are expressed not only in the terminals of the primary afferent units but also in the cell bodies, we removed dorsal root ganglia (DRGs) of both sides from control rats and from rats with the acute or chronic phase of AIA and determined, after short-term culture of the neurons, the proportion of DRG neurons that expressed the receptors in the different phases of AIA. We also characterized the inflammatory process and the nociceptive behaviour of the rats in the course of AIA.
Materials and methods:
In 33 female Lewis rats 10 weeks old, AIA was induced in the right knee joint. First the rats were immunized in two steps with methylated bovine serum albumin (m-BSA) emulsified with Freund's complete adjuvant, and heat-inactivated Bordetella pertussis. After immunization, m-BSA was injected into the right knee joint cavity to induce arthritis. The joint swelling was measured at regular intervals. Nociceptive (pain) responses to mechanical stimulation of the injected and the contralateral knee were monitored in the course of AIA. Groups of rats were killed at different time points after the induction of AIA, and inflammation and destruction in the knee joint were graded by histological examination. The DRGs of both sides were dissected from segments L1–L5 and C1–C7 from arthritic rats, from eight immunized rats without arthritis and from ten normal control rats. Excised DRGs were dissociated into single cells which were cultured for 18 h.
The expression of the receptors was determined by assessment of the binding of SP-gold or BK-gold to the cultured neurons. For this purpose the cells were slightly fixed. Binding of SP-gold or BK-gold was detected by using enhancement with silver and subsequent densitometric analysis of the relative grey values of the neurons. Displacement controls were performed with SP, the specific NK1 receptor agonist [Sar9, Met(O2)11]-SP, BK, the specific BK 1 (B1) receptor agonist D-Arg (Hyp3-Thi5,8-D-Phe7)-BK and the specific BK 2 (B2) receptor agonist (Des-Arg10)-Lys-BK.
Results:
The inflammatory process in the injected right knee joint started on the first day after induction of AIA and persisted throughout the observation period of 84 days (Fig. 1). The initial phase of AIA was characterized by strong joint swelling and a predominantly granulocytic infiltration of the synovial membrane and the joint cavity (acute inflammatory changes). In the later phases of AIA (10–84 days after induction of AIA) the joint showed persistent swelling, and signs of chronic arthritic alterations such as infiltration of mononuclear leucocytes, hyperplasia of synovial lining layer (pannus formation) and erosions of cartilage and bone were predominant. The contralateral knee joints appeared normal at all time points. Destruction was observed only in the injected knee but some proteoglycan loss was also noted in the non-injected, contralateral knee. In the acute and initial chronic phases of AIA (1–29 days) the rats showed mechanical hyperalgesia in the inflamed knee (limping, withdrawal response to gentle pressure onto the knee). In the acute phase (up to 9 days) a pain response was also seen when gentle pressure was applied to the contralateral knee.
Figure 2 displays the changes in the receptor expression in the DRG neurons during AIA. The expression of SP–gold-binding sites in lumbar DRG neurons (Fig. 2a) was substantially increased in the acute phase of arthritis. In untreated control rats (n = 5), 7.7 ± 3.8% of the DRG neurons from the right side and 10.0 ± 1.7% of the DRG neurons from the left side showed labelling with SP–gold. The proportion of SP–gold-labelled neurons in immunized animals without knee injection (n = 3) was similar. By contrast, at days 1 (n = 2 rats) and 3 (n = 5 rats) of AIA in the right knee, approximately 50% of the DRG neurons exhibited labelling with SP–gold, and this was seen both on the side of the injected knee and on the opposite side. At day 10 of AIA (n = 3 rats), 26.3 ± 6.1% of the ipsilateral DRG neurons but only 15.7 ± 0.6% of the contralateral neurons exhibited binding of SP–gold. At days 21 (n = 5 rats), 42 (n = 3 rats) and 84 (n = 5 rats) of AIA, the proportion of SP–gold-positive neurons had returned to the control values, although the arthritis, now with signs of chronic inflammation, was still present. Compared with the DRG neurons of the untreated control rats, the increase in the proportion of labelled neurons was significant on both sides in the acute phase (days 1 and 3) and the intermediate phase (day 10) of AIA (Mann–Whitney U-test). The size distribution of the neurons was similar in the DRG neurons of all experimental groups. Under all conditions and at all time points, SP–gold binding was found mainly in small and medium-sized (less than 700 μm2) neurons. In the cervical DRGs the expression of NK1 receptors did not change in the course of AIA. The binding of SP–gold to the neurons was suppressed by the coadministration of the specific NK1 receptor agonist [Sar9, Met(O2)11]–SP in three experiments, showing that SP–gold was bound to NK1 receptors.
The expression of BK–gold-binding sites in the lumbar DRG neurons showed also changes in the course of AIA, but the pattern was different (Fig. 2b). In untreated control rats (n = 5), 42.3 ± 3.1% of the DRG neurons of the right side and 39.6 ± 2.6% of the DRG neurons of the left side showed binding of BK–gold. At days 1 (n = 2 rats) and 3 (n = 5 rats) of AIA, approximately 80% of the DRG neurons on the side of the knee injection (ipsilateral) and approximately 70% on the opposite side were labelled. In comparison with the untreated control group, the increase in the proportion of labelled neurons was significant on both sides. The proportion of labelled neurons in the ipsilateral DRGs remained significantly increased in both the intermediate phase (day 10, n = 3 rats) and chronic phase (days 21, n = 5 rats, and 42, n = 3 rats) of inflammation. At 84 days after the induction of AIA (n = 5 rats), 51.0 ± 12.7% of the neurons showed an expression of BK–gold-binding sites and this was close to the prearthritic values. However, in the contralateral DRG of the same animals the proportion of BK–gold-labelled neurons declined in the intermediate phase (day 10) and chronic phase (days 21–84) of AIA and was not significantly different from the control value. Thus the increase in BK–gold-labelled neurons was persistent on the side where the inflammation had been induced, and transient on the opposite side. The size distribution of the DRG neurons of the different experimental groups was similar. In the cervical DRGs the expression of BK receptors did not change in the course of AIA. In another series of experiments, we determined the subtype(s) of BK receptor(s) that were expressed in DRGs L1–L5 in different experimental groups. In neither untreated control animals (n = 5) nor immunized rats without knee injection (n = 5) nor in rats at 3 days (n = 5) and 42 days (n = 5) of AIA was the binding of BK–gold decreased by the coadministration of BK–gold and the B1 agonist. By contrast, in these experimental groups the binding of BK–gold was suppressed by the coadministration of the B2 agonist. These results show that B2 receptors, but not B1 receptors, were expressed in both normal animals and in animals with AIA.
Discussion:
These results show that in AIA in the rat the expression of SP-binding and BK-binding sites in the perikarya of DRGs L1–L5 is markedly upregulated in the course of knee inflammation. Although the inflammation was induced on one side only, the initial changes in the binding sites were found in the lumbar DRGs of both sides. No upregulation of SP-binding or BK-binding sites was observed in the cervical DRGs. The expression of SP-binding sites was upregulated only in the first days of AIA, that is, in the acute phase, in which the pain responses to mechanical stimulation were most pronounced. By contrast, the upregulation of BK-binding sites on the side of AIA persisted for up to 42 days, that is, in the acute and chronic phase of AIA. Only the B2 receptor, not the B1 receptor, was upregulated. The coincidence of the enhanced expression of NK1 and BK receptors on sensory neurons and the pain behaviour suggests that the upregulation of these receptors is relevant for the generation and maintenance of arthritic pain.
In the acute phase of AIA, approximately 50% of the lumbar DRG neurons showed an expression of SP-binding sites. Because peptide receptors are transported to the periphery, the marked upregulation of SP-binding receptors probably leads to an enhanced density of receptors in the sensory endings of the primary afferent units. This will permit SP to sensitize more neurons under inflammatory conditions than under normal conditions. However, the expression of NK1 receptors was upregulated only in the acute phase of inflammation, suggesting that SP and NK1 receptors are less important for the generation of hyperalgesia in the chronic phase of AIA.
Because BK is one of the most potent algesic compounds, the functional consequence of the upregulation of BK receptors is likely to be of immediate importance for the generation and maintenance of inflammatory pain. The persistence of the upregulation of BK receptors on the side of inflammation suggests that BK receptors should be an interesting target for pain treatment in the acute and chronic phases. Only B2 receptors were identified in normal animals and in rats with AIA. This is surprising because previous pharmacological studies have provided evidence that, during inflammation, B1 receptors can be newly expressed.
Receptor upregulation in the acute phase of AIA was bilateral and almost symmetrical. However, hyperalgesia was much more pronounced on the inflamed side. It is most likely that receptors on the contralateral side were not readily activated because in the absence of gross inflammation the local concentration of the ligands BK and SP was probably quite low. We hypothesize that the bilateral changes in receptor expression are generated at least in part by mechanisms involving the nervous system. Symmetrical segmental changes can be produced only by the symmetrical innervation, involving either the sympathetic nervous system or the primary afferent fibres. Under inflammatory conditions, primary afferent fibres can be antidromically activated bilaterally in the entry zone of afferent fibres in the spinal cord, and it was proposed that this antidromic activation might release neuropeptides and thus contribute to neurogenic inflammation. Because both sympathetic efferent fibres and primary afferent nerve fibres can aggravate inflammatory symptoms, it is also conceivable that they are involved in the regulation of receptor expression in primary afferent neurons. A neurogenic mechanism might also have been responsible for the bilateral degradation of articular cartilage in the present study.
PMCID: PMC17819  PMID: 11056677
antigen-induced arthritis; bradykinin receptor; dorsal root ganglion neurons; neurokinin 1 receptor; pain
24.  Regulation of Peripheral Inflammation by Spinal p38 MAP Kinase in Rats 
PLoS Medicine  2006;3(9):e338.
Background
Somatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP) kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.
Methods and Findings
Selective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT) catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and −6 and matrix metalloproteinase (MMP3) gene expression. Activation of p38 required tumor necrosis factor α (TNFα) in the nervous system because IT etanercept (a TNF inhibitor) given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.
Conclusions
These data suggest that peripheral inflammation is sensed by the central nervous system (CNS), which subsequently activates stress-induced kinases in the spinal cord via a TNFα-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses.
Inhibition of p38 MAP kinase in the CNS reduces peripheral inflammation and joint destruction in arthritic rats.
Editors' Summary
Background.
Rheumatoid arthritis is a disease marked by chronic inflammation, leading to joint pain and destruction. Pain and inflammation in the joints as well as other locations in the body (i.e., the “periphery”) are constantly monitored by the central nervous system (i.e., the brain and spinal cord). Scientists have long suspected that the central nervous system (CNS) can regulate inflammation and immune responses, but little is known about how the CNS does this. One potential player is a protein called p38 that is involved in a number of cellular processes critical to the development of rheumatoid arthritis. Several substances that block the action of p38 are effective in animal models of arthritis and are currently being tested in clinical trials in patients with rheumatoid arthritis. Originally, p38 was considered as a drug target that should mainly be blocked in the joints. But recent work has shown that pain in the periphery can lead to activation of p38 in the spinal cord, and that blocking p38 in the spinal cord might reduce peripheral pain.
Why Was This Study Done?
Based on the observation that p38 is activated in the CNS in response to peripheral pain, the researchers who did this study wondered whether it might be involved in the interaction between inflammation in the joints and the CNS.
What Did the Researchers Do and Find?
They induced inflammation in the joints of rats and then looked for responses in the spinal cord. They found that p38 was indeed activated in the spinal cord of these rats. This activation depended on another protein, called TNFα, which is another major regulator of inflammation. The scientists then blocked either p38 or the TNFα with drugs directly delivered to the spinal cord of the arthritic rats, they could substantially reduce inflammation, arthritis, and destruction of the joints, compared with rats that had undergone the same treatment but received no active drug. Treatment of arthritic rats with the same amount of drugs given directly under the skin (this is called “systemic treatment”) did not have any effect on the joints.
What Do These Findings Mean?
Blocking p38 and TNFα by giving drugs systemically is known to have beneficial effects in animal models and human patients with rheumatoid arthritis. However, the drugs tested in patients to date also have side effects. Given that much lower doses were needed to achieve beneficial effects in the rats when the drugs were administered directly into the spinal cord, it is possible that spinal cord administration might reduce the side effects (and possibly the costs) of the drugs without compromising the benefits to the patients. If future studies confirm that the action of these drugs on the CNS is essential to achieve a response even when administered as a systemic treatment, designing drugs that get into the CNS easier might improve the effectiveness and/or make it possible to use lower doses systemically.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030338.
MedlinePlus entry on rheumatoid arthritis
Rheumatoid arthritis pages from the US National Institute of Arthritis and Musculoskeletal and Skin Diseases
Rheumatoid Arthritis fact sheet from the American College of Rheumatology Description
Wikipedia entry on rheumatoid arthritis (note: Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030338
PMCID: PMC1560929  PMID: 16953659
25.  Citrullination of fibronectin modulates synovial fibroblast behavior 
Arthritis Research & Therapy  2012;14(6):R240.
Introduction
Rheumatoid arthritis is an autoimmune arthritis characterized by joint destruction. Anti-citrullinated protein antibodies are pathologic in rheumatoid arthritis, but the role of the citrullinated proteins themselves is much less clear. Citrullination is the conversion of the arginine residues of a protein to citrulline. In the inflamed rheumatoid joint there is increased protein citrullination. Several proteins are citrullinated in rheumatoid arthritis, including collagen type II, fibrinogen, and fibronectin. Fibronectin is thought to mediate the adhesion of joint-invading synovial fibroblasts to the rheumatoid cartilage in addition to regulating other synovial fibroblast functions. However, the effect of citrullinated fibronectin on synovial fibroblasts is unknown.
Methods
To investigate the effect of citrullinated fibronectin on synovial fibroblast behavior, we cultured normal murine, arthritic murine, and human rheumatoid synovial fibroblasts. We then compared several synovial fibroblast functions in the presence of fibronectin versus citrullinated fibronectin. We assessed adhesion with time-lapse microscopy, migration with transwell assays, focal adhesion kinase and paxillin phosphorylation by western blot, and focal matrix degradation by fluorescent gelatin degradation.
Results
Normal synovial fibroblasts have impaired adhesion, spreading, migration, and integrin-mediated phosphorylation of focal adhesion kinase and paxillin on citrullinated fibronectin. Murine arthritic and human rheumatoid synovial fibroblasts also have impaired adhesion and spreading on citrullinated fibronectin, but focal matrix degradation is unaffected by citrullinated fibronectin.
Conclusion
Citrullination of fibronectin alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of rheumatoid arthritis.
doi:10.1186/ar4083
PMCID: PMC3674601  PMID: 23127210

Results 1-25 (376959)