Rheumatoid Arthritis Synovial Macrophages Research Articles

LIGHT is involved in the pathogenesis of rheumatoid arthritis by inducing the expression of pro‐inflammatory cytokines and MMP‐9 in macrophages

Macrophages play a crucial role in the perpetuation of inflammation and irreversible cartilage damage during the development of rheumatoid arthritis (RA). LIGHT (TNFSF14) and its receptor TR2 (TNFRSF14) are known to have pro-inflammatory activities in foam cells of atherosclerotic plaques. We tested a hypothesis that LIGHT and TR2 are involved in activation of monocyte/macrophages in RA synovium. Immunohistochemical analysis of RA synovial tissue samples revealed that both LIGHT and TR2 are expressed in CD68 positive macrophages. In contrast, synovial tissue samples from osteoarthritis (OA) patients failed to reveal the expression of LIGHT. Expression of TR2 in RA synovial macrophages was also detected using flow cytometry analysis. To identify the role of LIGHT in the functioning of macrophages in RA, we isolated macrophage enriched cells from RA synovial fluid and stimulated them with LIGHT. LIGHT induced expression of matrix metalloproteinase-9 and pro-inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8. These data indicate that LIGHT and TR2 expressed in macrophages are involved in the pathogenesis of RA by inducing the expression pro-inflammatory cytokines and matrix degrading enzymes.

Rheumatoid arthritis synovial fluid macrophages express decreased tumor necrosis factor-related apoptosis-inducing ligand R2 and increased decoy receptor tumor necrosis factor-related apoptosis-inducing ligand R3.

To characterize the expression pattern of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate receptors (TRAIL R1, R2, R3, and R4) on rheumatoid arthritis (RA) synovial fluid (SF) lymphocytes and monocyte/macrophages and on cultured RA synovial fibroblasts. The expression of TRAIL and TRAIL receptors on RA SF lymphocytes and monocyte/macrophages, normal macrophages, and RA synovial fibroblasts was examined by flow cytometry with previously characterized monoclonal antibodies. The ability of adenoviral-mediated delivery of TRAIL to induce macrophage or RA synovial fibroblast apoptosis was examined by flow cytometry. By flow cytometry, neither TRAIL nor its cognate receptors was detectable on RA SF lymphocytes or RA synovial fibroblasts. In contrast, RA SF macrophages expressed TRAIL R3, a decoy receptor (P < 0.01 versus isotype control), but not TRAIL, or TRAIL R1, R2, or R4. Normal peripheral blood-derived monocyte-differentiated macrophages expressed TRAIL R2 (P < 0.01), but not TRAIL or the other TRAIL receptors. Adenoviral-mediated delivery of TRAIL had no effect on the survival of normal macrophages or RA synovial fibroblasts but readily induced apoptosis in the prostate cancer cell line (PC-3) that expressed TRAIL R1 and R2. TRAIL R1 and R2, which are required for signal transmission by TRAIL, were not detected on RA SF lymphocytes, macrophages, or synovial fibroblasts. These observations do not support a potential therapeutic role for TRAIL in RA.

Fcgamma receptor expression levels on monocytes are elevated in rheumatoid arthritis patients with high erythrocyte sedimentation rate who do not use anti-rheumatic drugs.

Levels of immunoglobulin G (IgG) Fc receptors (FcgammaRs) affect the activity and function of monocytes/macrophages when binding IgG-containing immune complexes. Hence, the expression level of FcgammaRs on monocytic cells may influence inflammation in patients with rheumatoid arthritis (RA). In this study the expression levels of FcgammaRI, IIa and IIIa on peripheral blood monocytes of RA patients were compared with those of healthy controls and related to patient and disease characteristics and the use of disease-modifying anti-rheumatic drugs (DMARDs). In addition, FcgammaR expression levels were determined on RA synovial fluid macrophages and compared with those in RA peripheral blood. Mononuclear cells from peripheral blood and synovial fluid were isolated and FcgammaR expression levels on CD14-positive cells were analysed by flow cytometry. The effects of patient and disease characteristics and the use of DMARDs were assessed. A high expression level of FcgammaRIIa and high percentages of FcgammaRIIIa-expressing monocytes were found in RA patients with a high erythrocyte sedimentation rate. DMARD-naive early RA patients had higher FcgammaRIIa expression levels but a similar amount of FcgammaRIIIa-positive monocytes compared with RA patients using DMARDs. In synovial fluid, FcgammaRIIa expression levels were lower than in RA peripheral blood, whereas the percentage of FcgammaRIIIa-positive monocytic cells was higher in synovial fluid than in peripheral blood. These data point to the involvement of FcgammaRs, specifically FcgammaRIIa and IIIa, in the immune response of RA and suggest that FcgammaR expression levels are susceptible to modulation by DMARD therapy.

Melatonin in rheumatoid arthritis: synovial macrophages show melatonin receptors.

The pineal neurohormone melatonin is widely recognized as exerting important immunoenhancing effects that act on specific receptors in immunocompetent cells. This action results in stimulation of cytokine production in lymphocytes and macrophages. Here we report that the nocturnal plasma concentration of melatonin in rheumatoid arthritis (RA) patients is higher than in healthy controls. Furthermore, melatonin is present in the synovial fluid of RA patients and synovial macrophages express a specific binding site. We suggest that melatonin may exert a disease-promoting role in RA.

Differential expression of the angiogenic Tie receptor family in arthritic and normal synovial tissue.

Angiopoietins (Ang) are vascular endothelial cell-specific growth factors that play important roles principally during the later stages of angiogenesis. We have compared the distribution of the receptor tyrosine kinase (Tie) and the Ang ligands in synovial tissues from normal subjects and those with rheumatoid arthritis (RA) and osteoarthritis (OA). Immunohistochemical analysis was used to determine the expression of Ang-1, Ang-2, Tie1 and Tie2 in synovial tissue of normal subjects and those with RA and OA. Ang-1, Ang-2, Tie1 and Tie2 mRNA and protein expression were quantified in synovial tissues and RA synovial tissue fibroblasts with real-time reverse transcription polymerase chain reaction and western blot analysis. In RA, Ang-1 positive immunostaining on lining cells, macrophages and endothelial cells was significantly higher than in OA and normal synovial tissue. The expression pattern of Ang-2 in synovial tissue was similar in RA and OA, whereas the Ang-2 expression was low in normal tissue. Synovial tissue from subjects with RA and OA showed a significant upregulation of Tie1 on lining cells, macrophages and endothelial cells compared to that from normal subjects. Tie2 was significantly upregulated in the RA and OA synovial tissue lining cells, macrophages and smooth muscle cells compared to normal synovial tissue. Generally Ang-1, Ang-2, Tie1 and Tie2 mRNA levels were higher in RA synovial tissue compared to normal and OA synovial tissues, and RA synovial tissue fibroblasts. Western blot analysis also demonstrated greater Tie1 and Tie2 protein expression in RA and OA synovial tissue compared to RA synovial tissue fibroblasts. In conclusion, the dominance of Ang-1 mRNA and protein expression over Ang-2 is in agreement with an active neovascularization in RA synovial tissue.

Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fas-mediated apoptosis.

The chronic inflammation and progressive joint destruction observed in rheumatoid arthritis (RA) are mediated in part by macrophages. A paucity of apoptosis has been observed in RA synovial tissues, yet the mechanism remains unknown. The present study sought to characterize the expression of Fas, Fas ligand (FasL), and Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP), and to quantify the apoptosis induced by agonistic anti-Fas antibody, using mononuclear cells (MNC) isolated from the peripheral blood (PB) and synovial fluid (SF) of RA patients. The expression of Fas, FasL, and FLIP and apoptosis induced by agonistic anti-Fas antibody in MNC from the PB and SF of RA patients were determined by flow cytometry. Immunohistochemistry employing a monospecific anti-FLIP antibody was performed on RA and osteoarthritis (OA) synovial tissue. CD14-positive monocyte/macrophages from normal and RA PB and from RA SF expressed equivalent levels of Fas and FasL. Furthermore, unlike the CD14-positive PB monocytes, RA SF monocyte/macrophages were resistant to the addition of agonistic anti-Fas antibody. In contrast, both CD14-positive PB and SF monocyte/macrophages were sensitive to apoptosis mediated by a phosphatidylinositol 3-kinase inhibitor. Intracellular staining of the caspase 8 inhibitor, FLIP, in CD14-positive SF monocyte/macrophages revealed a significant up-regulation of FLIP compared with normal and RA PB monocytes. Immunohistochemical analysis of synovial tissue from RA and OA patients revealed increased FLIP expression in the RA synovial lining compared with the OA synovial lining. Furthermore, FLIP expression was observed in the CD68positive population in the RA synovial lining. Forced reduction of FLIP by a chemical inhibitor resulted in RA SF macrophage apoptosis that was enhanced by agonistic anti-Fas antibody, indicating that FLIP is necessary for SF macrophage survival. These data suggest that up-regulation of FLIP in RA macrophages may account for their persistence in the disease. Thus, the targeted suppression of FLIP may be a potential therapeutic strategy for the amelioration of RA.

Rheumatoid arthritis synovial macrophage-osteoclast differentiation is osteoprotegerin ligand-dependent.

Osteoprotegerin ligand (OPGL) is a newly discovered molecule which is essential for osteoclast differentiation. Both OPGL and its soluble decoy receptor, osteoprotegerin (OPG), which inhibits osteoclast formation, are known to be produced by osteoblasts and inflammatory cells found in the rheumatoid arthritis (RA) synovium. In this study, RA synovial macrophages were incubated in the presence or absence of OPGL, macrophage-colony stimulating factor (M-CSF), and dexamethasone for various time points. The results indicated that osteoclast formation from RA synovial macrophages is OPGL-dependent and that OPGL and M-CSF are the only humoral factors required for RA synovial macrophage-osteoclast differentiation. OPG was found to inhibit osteoclast formation by RA synovial macrophages in a dose-dependent manner. This study has shown that macrophages isolated from the synovium of RA patients are capable of differentiating into osteoclastic bone-resorbing cells; this process is OPGL- and M-CSF-dependent and is modulated by corticosteroids. Cellular (T and B cells, dendritic cells) and humoral factors in RA synovium and bone may influence osteoclast formation and bone resorption by controlling OPGL/OPG production.

Selective expression of folate receptor beta and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis.

To investigate the expression of folate receptors (FR) and reduced folate carrier (RFC) and determine their relevance to methotrexate (MTX) transport in synovial mononuclear cells (SMC) from patients with rheumatoid arthritis (RA). Levels of FR and RFC messenger RNA (mRNA) were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) in SMC from RA patients and peripheral blood mononuclear cells from healthy donors. Expression of FR-beta mRNA and protein was determined by Northern blot and Western blot analyses in RA SMC and monocyte/macrophage-lineage cells. FR-beta expression and folic acid binding capacity on the cell surface were examined by flow cytometric analysis and 3H-folic acid binding analysis. Studies of the inhibition of 3H-MTX uptake in the presence of unlabeled folic acid were performed to investigate the uptake of MTX through FR in RA SMC. RT-PCR, Northern blot, and Western blot analyses showed that FR-beta mRNA and protein were expressed selectively in activated monocytes and CD14+ RA SMC. These cells exhibited folic acid binding capacity. Furthermore, the FR-beta protein was shown to have folic acid binding capacity. Uptake of 3H-MTX through RA SMC was significantly inhibited in the presence of unlabeled folic acid. These results demonstrate that FR-beta expression is selectively elevated in RA synovial macrophages and suggest that MTX is transported through FR-beta in RA synovial macrophages. The findings suggest that folate antagonists with higher affinity for FR-beta would be useful in the treatment of RA.

Melatonin influences interleukin-12 and nitric oxide production by primary cultures of rheumatoid synovial macrophages and THP-1 cells.

Because some of the clinical symptoms related to rheumatoid arthritis (RA) synovitis, such as joint morning stiffness and gelling, might be related to the effects exerted by the diurnal rhythmicity of the neurohormone melatonin (MLT) on synovial immune cell activation, we decided to evaluate the influence of MLT on the production of IL-12 and nitric oxide (NO) on primary cultures of RA synovial macrophages. Synovial macrophages were also prestimulated with lipopolysaccaride (LPS). Results were compared with those obtained on cultured human myeloid monocytic cells (THP-1). A significant increase in IL-12 (p = 0.01) was found in media of MLT-stimulated synovial macrophages versus RMPI-treated synovial macrophage controls. Interestingly, a significant decrease of IL-12 (p < 0.0001) was observed in media of synovial macrophages previously activated with LPS and then treated with MLT, when compared to synovial macrophages treated with LPS alone. A significant increase in NO levels (p = 0.01) was found in MLT-stimulated synovial macrophages versus RMPI-treated synovial macrophage controls. Interestingly, a nonsignificant increase of NO levels was observed in media of synovial macrophages previously activated with LPS and then treated with MLT, when compared to cynovial macrophages treated with LPS alone. Finally, a significant increase in IL-12 (p = 0.03) and NO (p = 0.002) concentrations was observed in media of MLT-stimulated THP-1 cells versus RMPI-treated controls. Our results therefore show that MLT induces IL-12 secretion and NO production by unstimulated cultured RA synovial macrophages and human monocytic myeloid THP-1 cells. The unexpected and opposite effects on IL-12 and NO production in RA synovial macrophages treated with LPS may be related to dose-dependent mechanisms exerted by MLT or to altered cell priming in RA macrophages; these are matters of our further research. This study strongly supports the role of MLT in immune response modulation and in particular suggests a close relationship between diurnal rhythmicity of neuroendocrine pathways, cytokine and reactive oxygen intermediate production by monocyte/macrophages, and synovial arthritis symptomatology, at least in RA.

Pathological significance of elevated soluble CD14 production in rheumatoid arthritis: in the presence of soluble CD14, lipopolysaccharides at low concentrations activate RA synovial fibroblasts.

In order to establish what contributes to elevated levels of soluble CD14 (sCD14) in rheumatoid arthritis (RA) plasma, levels of sCD14 were compared in RA-paired plasma and synovial fluids and, further, in the culture supernatants of monocyte-rich fractions from patients with RA and healthy donors, and macrophage-rich fractions from RA synovial tissues. The results showed elevated sCD14 in RA synovial fluid in 9 of 16 paired samples and in RA macrophage-rich fractions, suggesting that elevated sCD14 in RA plasma might be due to the sCD14 production by RA synovial macrophages. From the molecular analysis of elevated sCD14, the proteolytic cleavage of membranous CD14 (mCD14) was important in accelerated sCD14 production. Lipopolysaccharides (LPS) at low concentrations and sCD14 increased the ICAM-1 expression on RA synovial fibroblasts. This result implies that in vivo RA synovial fibroblasts may be sensitive to LPS in the presence of sCD14 and LPS-binding protein (LBP).

Testosterone metabolism and cyclosporin A treatment in rheumatoid arthritis.

A constant dose-dependent side-effect in cyclosporin A (CSA)-treated patients is the appearance of hypertrichosis; this occurs in both sexes and suggests an androgenizing activity. To determine the influence of CSA on peripheral androgen metabolism, we evaluated in rheumatoid arthritis (RA) patients treated with low-dose CSA (3.5 mg/kg/day), during a period of 12 months, plasma levels of testosterone (Tes) and of 5alpha-androstane-3alpha, 17beta-diol glucuronide (Adiol-G), an important peripheral Tes metabolite. Clinical and laboratory parameters of RA were also monitored. Furthermore, the metabolism of physiological concentrations of Tes (1 x 10(-8) M) was evaluated in primary cultures of RA synovial macrophages (M phi) in the presence of CSA concentrations close to the pharmacological immunosuppressive doses (100-500 ng/ml). At the final time of observation (12 months), a significant increase in the mean plasma Adiol-G level was observed in patients of both sexes. The increase was evident after 1 month of treatment in male patients (P < 0.01) and after 3 months in female patients (P < 0.05). Almost all the patients experienced the side-effect of a low-degree hypertrichosis after a mean period of 1-2 months. No significant correlations with the laboratory parameters of the disease were observed. Results from in vitro experiments on Tes metabolism by cultured synovial M phi showed at 24 and 48 h, in the presence of CSA, a significantly (P < 0.0001) greater formation of dihydrotestosterone and increased amounts of other Tes metabolites, including androstenedione, androsterone and epiandrosterone, when compared to untreated controls. In conclusion, the appearance of a dose-related hypertrichosis and the increase in plasma androgen metabolites (i.e. Adiol-G) in CSA-treated patients, as well as the hormonal metabolic effects on cultured synovial M phi, should be regarded as possible markers of the influence of CSA on peripheral androgen metabolism at the level of target cells.

Human osteoclast formation and bone resorption by monocytes and synovial macrophages in rheumatoid arthritis.

To determine whether synovial macrophages and monocytes isolated from patients with rheumatoid arthritis patients are capable of differentiating into osteoclastic bone resorbing cells; and the cellular and humoral conditions required for this to occur. Macrophages isolated from the synovium and monocytes from the peripheral blood of rheumatoid arthritis patients were cultured on bone slices and coverslips, in the presence and absence of UMR 106 rat osteoblast-like cells, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) and macrophage colony stimulating factor (M-CSF), and assessed for cytochemical and functional evidence of osteoclast differentiation. Isolated calcitonin receptor (CTR), tartrate resistant acid phosphatase (TRAP), and vitronectin receptor (VNR) negative, CD11b and CD14 positive monocytes and macrophages differentiated into CTR, TRAP, and VNR positive multinucleated cells capable of extensive lacunar bone resorption when co-cultured for 14 d with UMR 106 cells in the presence 1,25(OH)2D3 and M-CSF. Mononuclear phagocytes (monocytes and macrophages) from rheumatoid arthritis patients are capable of differentiating into multinucleated cells showing all the cytochemical and functional criteria of mature osteoclasts. Synovial macrophage-osteoclast differentiation may represent an important cellular mechanism in the bone destruction associated with rheumatoid arthritis.

Androgen and estrogen receptors are present in primary cultures of human synovial macrophages.

Macrophages, as antigen-processing and -presenting cells to T lymphocytes, play a key role in the immune system and are suspected to be target cells of the sex hormone-related dimorphism in the immune response peculiar to rheumatoid arthritis (RA) pathology. In the present study, the use of specific monoclonal antibodies revealed immunostaining for androgen and estrogen receptors in primary cultures of macrophages obtained from synovial tissues of patients affected by RA and controls without RA disease. Soluble and nuclear type I (high affinity, low capacity) and type II (lower affinity, greater capacity) sites of androgen or estrogen binding were detected in primary cultures of RA macrophages using radioligand binding assay. Higher levels of type I and type II estrogen receptor compared to those of androgen receptor were found, particularly in the soluble fraction; however, contrary to what was observed in whole synovial tissues, higher steroid receptor concentrations were found in the soluble than in the nuclear fraction of RA synovial macrophages. Binding affinities and receptor contents of cultured synovial macrophages were comparable to those previously reported in other well established sex hormone-responsive cells and tissues. Further, specific messenger ribonucleic acids for sex hormone receptors, encoding for a sequence of the DNA-binding domain of the receptor proteins were revealed by RT-PCR.

Tumor Necrosis Factor α and Interleukin-1α Stimulate Late Shedding of p75 TNF Receptors but Not p55 TNF Receptors from Human Monocytes

Soluble receptors for TNF (sTNF-R) are present at elevated concentrations in the synovial fluid of patients with rheumatoid arthritis. They are presumably released by cells of the synovial membrane, including the monocyte-derived synovial macrophages. Cytokines from the synovium, including IL-1 and TNF-alpha, may stimulate release. We therefore examined the release of sTNF-R from monocytes exposed to IL-1 and TNF-alpha. Elutriator-purified human blood monocytes spontaneously released both the p75 and the p55 sTNF-R (1011 +/- 199 and 177 +/- 20 pg/10(6) cells, respectively, mean +/- SEM) during 48 h of in vitro culture. TNF-alpha and IL-1 alpha induced time- and concentration-dependent increases in the release of sTNF-R75 from monocytes, but neither had a measurable effect on the release of sTNF-R55. The release of sTNF-R75 was inhibited by cycloheximide. Neither lymphocytes nor polymorphonuclear leukocytes (PMN) released measurable sTNF-R spontaneously or in response to stimulation with IL-1 alpha, but TNF-alpha stimulated the release of small amounts of sTNF-R75 by PMN. The timing, cycloheximide sensitivity, and selectivity of stimulated release of TNF-R75 by monocytes are consistent with previous observations on other cell types of late (8-20 h) increased synthesis and turnover of cell surface TNF-R75, but not TNF-R55, after stimulation with TNF-alpha or IL-1. These observations help to explain why elevated levels of sTNF-R in synovial fluid coexist with enhanced expression of cell surface TNF-R on synovial macrophages in rheumatoid arthritis.

Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis.

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.

Expression of interleukin-1 and interleukin-1 receptor antagonist by human rheumatoid synovial tissue macrophages

Interleukin-1 (IL-1) has protean effects in the pathogenesis of rheumatoid arthritis (RA). These effects include production of prostaglandins and collagenase from rheumatoid fibroblasts as well as upregulation of adhesion molecule expression on these cells. IL-1 can activate monocytes and neutrophils, as well as promote the growth of fibroblasts and endothelial cells. Recently, a novel interleukin-1 receptor antagonist protein (IRAP) has been isolated, purified, cloned, and expressed, which may modulate the effects of IL-1. In this study, we present data demonstrating that macrophages isolated from human RA synovial tissues express both IL-1 and IRAP genes. In addition, RA synovial tissue macrophages and lining cells display IL-1 and IRAP antigenic expression by immunohistochemistry. In contrast, osteoarthritis synovial tissues, as compared to RA, have fewer IL-1 and IRAP-positive macrophages. Thus, the production of IL-1 balanced by IRAP may affect the joint destruction found in these diseases.