High expression of macrophage migration inhibitory factor in the synovial tissues of rheumatoid joints

Macrophage migration inhibitory factor (MIF) plays an important role in inflammation and immunity via autocrine/paracrine and endocrine routes. We examined the presence of MIF in the synovial fluids of rheumatoid arthritis (RA) patients. The content of MIF in the synovial fluid was quantitated by enzyme-linked immunosorbent assay which revealed that the concentration of MIF for RA patients was 85. 7+/-35.2 ng/ml (mean+/-SD) (n=25). In comparison, the concentrations for osteoarthritis patients and normal volunteers were 19.5+/-5.3 ng/ml (n=12) and 10.4+/-1.1 ng/ml (n=5), respectively. The expression of MIF mRNA and presence of MIF protein in the synovial tissues of RA were demonstrated by Northern blot and Western blot analyses, respectively. Immunohistochemical analysis revealed that positive staining was largely observed in the cytoplasm of infiltrating T lymphocytes, which might be the major source of MIF detected in the synovial fluids. The pathophysiological role of MIF in RA remains to be elucidated; however, the present results for the first time suggest the possibility that MIF is involved in the potentiation of inflammatory and immunological responses in rheumatoid joints.     

Transforming growth factor-beta in synovial fluids modulates Fc gamma RII (CD16) expression on mononuclear phagocytes.

Mononuclear phagocytes in the synovium of patients with arthritis, in contrast to blood monocytes, were found to express a third receptor for the constant region of Ig (Fc gamma RIII), in addition to Fc gamma RI and Fc gamma RII. Previously identified on mature mononuclear phagocytes or phagocytes exposed to transforming growth factor-beta (TGF-beta) in vitro, this study documents the presence of Fc gamma RIII (CD16) expressing cells at an inflammatory site. Furthermore, the presence of CD16 on the majority of the LeuM3 (CD14) positive synovial monocytic cells could be mimicked by exposing blood monocytes to synovial fluids from patients with rheumatoid arthritis (17 of 19) and synovial fluids from patients with osteoarthritis (4 of 4). In additional studies, the soluble factor in inflammatory synovial fluids responsible for regulating CD16 expression was found to be consistent with the presence of TGF-beta. Inhibition of the activity in synovial fluids with a neutralizing antibody to TGF-beta confirmed a role for this peptide in synovial phagocytic cell CD16 expression. Moreover, signal transduction through CD16 on synovial phagocytes resulted in augmented extracellular release of superoxide anion that may contribute to tissue damage and other inflammatory sequelae. Identification of TGF-beta and its association with upregulation of CD16 at sites of chronic inflammation may provide insight into the destructive lesions associated with inflammatory arthropathies.     

Increased Urine and Serum Nerve Growth Factor Levels in Interstitial Cystitis Suggest Chronic Inflammation Is Involved in the Pathogenesis of Disease

Objective

Interstitial cystitis/bladder pain syndrome (IC/BPS) is considered a bladder disorder due to localized chronic inflammation. This study investigated the nerve growth factor (NGF) levels in serum and urine in patients with IC/BPS.

Materials and Methods

Thirty patients with IC/BPS and 28 normal subjects without lower urinary tract symptoms were recruited from an outpatient clinic. IC/BPS was diagnosed by frequency, bladder pain, and the presence of glomerulations during cystoscopic hydrodistention. Serum and urine were collected before any treatment was given. Serum NGF and urinary NGF/Cr levels were compared between IC/BPS and the controls.

Results

Urinary NGF levels were significantly higher in patients with IC/PBS (26.3±11.2 pg/ml) than in controls (1.40±0.63 pg) (p = 0.014). After normalization, the urinary NGF/Cr levels were significantly greater in IC/BPS (0.69±0.38 pg/mg) than controls (0.20±0.01, p = 0.011). Relative to the levels in control subjects (1.90±0.38 pg/mL), the mean serum NGF levels were higher in patients IC/BPS patients (3.48±0.55 pg/mL) (p = 0.015). No significant correlation was found between the serum and urinary NGF levels in IC/BPS patients. However, the clinical characteristics and medical co-morbidities did not show significant difference between IC/BPS patients with a higher and lower serum NGF level.

Conclusions

Increased urinary NGF levels in IC/BPS patients suggest that chronic inflammation is involved in this bladder disorder. Increased circulating serum NGF levels were noted in over half of patients with IC/BPS, however, the urinary and serum NGF were not inter-correlated and elevated serum NGF did not relate with clinical features.     

Hypoxia-Inducible Factor-1α and Interleukin 33 Form a Regulatory Circuit to Perpetuate the Inflammation in Rheumatoid Arthritis

Hyperplasia of synovial fibroblasts, infiltration with inflammatory cytokines, and tissue hypoxia are the major characteristics of rheumatoid arthritis (RA). Interleukin 33 (IL-33) is a newly identified inflammatory cytokine exacerbating the disease severity of RA. Hypoxia-inducible factor-1α (HIF-1α) showed increased expression in RA synovium and could regulate a number of inflammatory cytokine productions. Nevertheless, its correlation with IL-33 remains largely unknown. Here, we showed that elevated levels of IL-33 were demonstrated in RA patient synovial fluids, with upregulated expression of HIF-1α and IL-33 in the synovial fibroblasts. Knocking down HIF-1α compromised IL-33 expression in rheumatoid arthritis synovial fibroblasts (RASF), while enforcing HIF-1α expression in RASF substantially upregulated IL-33 levels. HIF-1α promoted the activation of the signalling pathways controlling IL-33 production, particularly the p38 and ERK pathways. Moreover, we showed for the first time that IL-33 in turn could induce more HIF-1α expression in RASF, thus forming a HIF-1α/IL-33 regulatory circuit that would perpetuate the inflammatory process in RA. Targeting this pathological pathway and HIF-1α may provide new therapeutic strategies for overcoming the persistent and chronic inflammatory disease.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.     

Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α-produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA-positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA-positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.     

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.     

Prothrombin in Normal Human Cerebrospinal Fluid Originates from the Blood

In spite of the fact that prothrombin is produced by cells within the central nervous system, its presence in the cerebrospinal fluid (CSF) has not been investigated. We determined the concentration of prothrombin in CSF with reference to the concentration in plasma in paired samples from 18 normal control patients and 4 patients with relapsing-remitting type of multiple sclerosis (MS). The newly developed ELISA was very specific (no cross-reactivity with thrombin) and sensitive (detection limit—0.7 ng/ml) with an imprecision of CV = 8.3% (intraseries) and 7.0% (interassay). The mean prothrombin concentration in normal CSF was 0.55 mg/l (CV ± 33%, range: 0.28–0.93 mg/l), in normal plasma 121.8 mg/l ± 21%, resulting in a mean CSF/plasma concentration quotient (Q_Proth)—4.5 · 10^–3 (CV ± 35%, range: 2.1–8.3 · 10^–3) corresponding to a mean albumin quotient in this group of subjects of Q_Alb = 5.8 · 10^–3. Due to the Q_Proth and the molecular weight of prothrombin (72 kDa)—similar to that of albumin—we conclude that prothrombin in normal human CSF originates predominantly (>95%) from blood. The enzymatic activity in CSF is conserved. Comparable results obtained in MS patients with only few small MRI lesions suggest that local chronic inflammatory disease of the central nervous system does not influence prothrombin concentration in the CSF if the blood-CSF barrier function is normal.     

Increased synovial fluid levels of interleukin-12, sCD25 and sTNF-RII/sTNF-RI ratio delineate a cytokine pattern characteristic of immune arthropathies

The assessment of cytokines and their soluble receptors in the synovial fluid (SF) of inflammatory arthropathies may be useful in studying pathogenetic and immunoregulatory mechanisms underlying different diseases. The aim of this work was to study the cytokine network occurring in inflammatory arthropathies and to identify a cytokine profile which is characteristic of an immune-mediated synovitis. Levels of IL-12, as well as IL-4, IL-8, IL-10, IFN-gamma, sCD25, TNF-alpha and its soluble receptors were measured in the SF of various arthropathies, i.e. non-inflammatory arthropathies: "control" meniscus pathology (n = 21), osteoarthritis (n = 22) and chronic crystal arthritis (n = 9); a non-immune inflammatory arthropathy: acute crystal arthritis (n = 11); 2 immune inflammatory arthropathies: reactive arthritis (ReA) (n = 23) and rheumatoid arthritis (RA) (n = 44). SF levels of IL-10, TNF-alpha and sTNF-RII were found to be increased in the three inflammatory arthropathies compared to the "control" meniscus group. Within the inflammatory group, acute crystal arthritis was characterized by a significantly higher sTNF-RI/TNF-alpha ratio and ReA by a significantly lower sTNF-RII/TNF-alpha ratio compared to the two other diseases. The two immune arthropathies, RA and ReA, were characterized by increased SF levels of IL-12, sCD25 and of the sTNF-RII/sTNF-RI ratio. ReA differed however from RA by showing lower IL-8 and IL-4 levels, higher IFN-gamma levels and a higher IL-12/IL-10 ratio, suggesting a more prevalent Th1 profile in ReA SF. Our data indicate that the measurement of SF cytokines and soluble receptors may discriminate between each inflammatory arthropathy and might be useful in clinical practice.     

Identification of broadly discriminatory tissue biomarkers of synovitis with binary and multicategory receiver operating characteristic analysis

Immunohistochemical synovial tissue biomarkers are used increasingly to classify arthropathies, study their pathogenesis, and to measure disease activity in clinical trials. We have used receiver operating characteristic (ROC) analysis to quantify the discriminatory abilities of markers for common inflammatory cells (subintimal CD15, CD68, CD3, CD20, CD38, and lining CD68), proliferating cells (Ki-67) and blood vessels (von Willebrand factor, vWF) among inflammatory (chronic septic arthritis, early arthritis and rheumatoid arthritis (RA)) and degenerative arthropathies (osteoarthritis (OA) and orthopedic arthropathies) and normal synovium. Six of the eight markers distinguished accurately between RA and the degenerative arthropathies (area under the curve (AUC) 0.91–0.97), whereas subintimal CD68 (AUC 0.92) and Ki-67 (AUC 0.87) distinguished best between OA and normal synovium. Fold differences in mean expression correlated only modestly with AUCs (r²?=?0.44). Multicategory ROC analysis ranked Ki-67, subintimal CD68, and CD15 as discriminating best among all six sample groups, and thus identified them as the most broadly applicable markers.     

Cytochemical investigation of cathepsin B in rheumatoid synovial membrane and fluid

Cathepsin B was cytochemically investigated in the cells of synovial membranes and in the cell pellet of synovial fluids obtained from 50 patients with rheumatoid arthritis and eight patients with various nonrheumatoid arthropathies. The activity of Cathepsin B was estimated by using the substrate N-alpha-benzoyl-DL-arginine-naphthylamide HCl and diazoic dye Fast Corinth V in phosphate buffer pH 6.0 in the presence of EDTA and cysteine. A significant activity of cathepsin B was shown in lining mesothelial cells, in macrophages of the submesothelial infiltrations, as well as in fibroblasts prominent in the deep areas of rheumatoid synovial membranes. In the cell pellets of synovial fluids the highest activity of cathepsin B was found in the macrophages and polymorphonuclear leukocytes, accompanied by a variable activity in lymphocytes. The considerable activity of cathepsin B, an enzyme with degradative action upon collagen and proteoglycans, in the main cellular populations of rheumatoid synovial membranes and fluids, suggests its involvement in the genesis and maintenance of rheumatoid lesions.     

Early rheumatoid arthritis is characterized by a distinct and transient synovial fluid cytokine profile of T cell and stromal cell origin

Pathological processes involved in the initiation of rheumatoid synovitis remain unclear. We undertook the present study to identify immune and stromal processes that are present soon after the clinical onset of rheumatoid arthritis (RA) by assessing a panel of T cell, macrophage, and stromal cell related cytokines and chemokines in the synovial fluid of patients with early synovitis. Synovial fluid was aspirated from inflamed joints of patients with inflammatory arthritis of duration 3 months or less, whose outcomes were subsequently determined by follow up. For comparison, synovial fluid was aspirated from patients with acute crystal arthritis, established RA and osteoarthritis. Rheumatoid factor activity was blocked in the synovial fluid samples, and a panel of 23 cytokines and chemokines measured using a multiplex based system. Patients with early inflammatory arthritis who subsequently developed RA had a distinct but transient synovial fluid cytokine profile. The levels of a range of T cell, macrophage and stromal cell related cytokines (e.g. IL-2, IL-4, IL-13, IL-17, IL-15, basic fibroblast growth factor and epidermal growth factor) were significantly elevated in these patients within 3 months after symptom onset, as compared with early arthritis patients who did not develop RA. In addition, this profile was no longer present in established RA. In contrast, patients with non-rheumatoid persistent synovitis exhibited elevated levels of interferon-γ at initiation. Early synovitis destined to develop into RA is thus characterized by a distinct and transient synovial fluid cytokine profile. The cytokines present in the early rheumatoid lesion suggest that this response is likely to influence the microenvironment required for persistent RA.     

IL-6/IFN-beta 2 in synovial effusions of patients with rheumatoid arthritis and other arthritides. Identification of several isoforms and studies of cellular sources

We have looked for IL-6, a cytokine that has immunomodulating and inflammation-associated activities, in joint exudates (fluid and mononuclear cells) from patients with rheumatoid arthritis and other arthritides using both biologic and biochemical assays. IL-6 was assessed by its ability to stimulate alpha 1-antichymotrypsin secretion from the human hepatoma cell line Hep3B clone 2, an activity which is blocked by an antiserum to Escherichia coli derived IL-6, and by the growth of the IL-6-dependent murine hybridoma 7TD1 cell line. IL-6 isoforms in synovial fluid were characterized by immunoaffinity chromatography followed by Western blotting. The presence of IL-1 in synovial fluids and its production by synovial fluid mononuclear cells was monitored by Western blotting and indirect immunofluorescence with polyclonal anti-IL-1 beta antisera. In an analysis of 30 effusions from 27 rheumatoid patients with acutely inflamed joints, abundant quantities of IL-6 (greater than 2 ng/ml) were detected in 23 by the alpha 1-antichymotrypsin bioassay. Several rheumatoid synovial fluids also had elevated IL-6 levels in the 7TD1 bioassay. Seven of nine nonrheumatoid effusions also contained high levels of IL-6 (greater than 2 ng/ml). No IL-1 (less than 0.25 ng/ml) could be detected by Western blotting in 10 rheumatoid effusions even though eight of these contained high levels of IL-6. The IL-6 activity could be neutralized with a rabbit antiserum to rIL-6. Multiple IL-6 isoforms (25, 30, 45 kDa) were present in two rheumatoid and one traumatic effusion studied. Fresh mononuclear cells isolated from various synovial effusions did not appear to make IL-6 constitutively, as no IL-6 could be detected in the media of cells cultured for 12 to 18 h after isolation. Similarly, there was no constitutive production of IL-1 by these cells. However, synovial fluid mononuclear cells could be induced to secrete both IL-6 and IL-1 after stimulation with LPS. The LPS-responsive cells were monocytes and not lymphocytes or dendritic cells. These findings suggest that IL-6 is involved in inflammatory joint disease. However, the primary cells synthesizing it may be located in the synovial lining instead of the joint exudate.     

The Relative Composition of the Inflammatory Infiltrate as an Additional Tool for Synovial Tissue Classification

Objectives

Traditionally, differences in absolute numbers of cells expressing a certain marker (e.g., positive staining cells per mm²) have been used in immunohistological synovial tissue classification. We have begun to evaluate the relative composition of the inflammatory infiltrates, i.e. percentages of inflammatory cell types in inflammatory infiltrates, as an alternate classification tool that may potentially improve tissue diagnostics, subgrouping in clinical trials, and understanding of pathogenesis of inflammatory and noninflammatory arthropathies.

Methods

Synovial tissue specimens (normal synovium, n=15; orthopedic arthropathies, n=6; osteoarthritis, n=26; early undifferentiated arthritis, n=10; rheumatoid arthritis, n=26; chronic septic arthritis, n=11) were stained for CD15, CD68, CD3, CD20, and CD38. Densities of cells expressing a given marker were determined in the superficial subintima. Binary and multicategory receiver operating characteristic (ROC) analysis and naïve Bayes classifier were used to compare the abilities of (1) the absolute densities of cells expressing a given marker (absolute method) with (2) the percentages of these cells in the inflammatory cell population (relative method) to differentiate among the six tissue classes.

Results

The inflammatory infiltrates in normal synovium and the orthopedic arthropathies consisted almost exclusively of CD68+ and CD3+ cells. Notable fractions of CD20+ and CD38+ cells appeared in a subset of osteoarthritis samples, and increased further in early, rheumatoid and chronic septic arthritis. ROC analyses and naïve Bayes classifier ranked the absolute method above the relative method in terms of overall discriminatory ability. The relative method became slightly superior when the samples were also stratified according to the total number of inflammatory cells/mm².

Conclusions

This exploratory investigation featuring a variety of joint disorders revealed that measuring the relative proportions of inflammatory cell types may aid in synovial tissue classification if the samples are also stratified according to the intensity of inflammation.     

In vitro responses to a 65-kilodalton mycobacterial protein by synovial T cells from inflammatory arthritis patients

Bacterial Ag, especially those of mycobacteria, have been implicated in the pathogenesis of experimental inflammatory arthritis in rodents, while in man, reactive arthritis has a clear temporal relationship to infection with particular bacteria. To investigate the role of immune responses to bacterial Ag in inflammatory arthritis, we have examined the proliferative responses of paired synovial fluid and PBMC when stimulated with 1) suspensions of irradiated or heat-killed bacteria associated with reactive arthritis (ReA), 2) purified protein derivative, 3) a recombinant 65-kDa heat shock protein of Mycobacterium leprae. The 65-kDa Ag was stimulatory to synovial fluid mononuclear cells, but not PBMC, from patients with different arthropathies, including most of those with ReA, but also some with rheumatoid arthritis. Furthermore, the magnitude of these responses correlated more closely with responses to ReA-associated bacteria (such as Salmonella), than with responses to the mycobacterial Ag represented in purified protein derivative. These results suggest that the 65-kDa molecule, which is common to a wide range of bacteria, may be an important immunogen for the T cell-mediated immune responses within the joint in different clinically defined inflammatory arthropathies.     

Interleukin-18 induces serum amyloid A (SAA) protein production from rheumatoid synovial fibroblasts

Interleukin-18 (IL-18) is a novel proinflammatory cytokine that was recently found in synovial fluids and synovial tissues from patients with rheumatoid arthritis (RA). To investigate the role of IL-18 in rheumatoid synovitis, the levels of IL-18 and serum amyloid A (SAA) were measured in synovial fluids from 24 patients with rheumatoid arthritis (RA) and 13 patients with osteoarthritis (OA). The levels of IL-18 and SAA in the synovial fluids were elevated in RA patients. In contrast, the levels of IL-18 in synovial fluids from OA patients were significantly lower compared to those of RA patients. SAA was not detected in synovial fluids from OA patients. The expression of SAA mRNA in rheumatoid synovial cells was also examined. SAA4 mRNA, which was constitutively expressed by rheumatoid synovial cells, was not affected by IL-18 stimulation. Although acute phase SAA (A-SAA, SAA1 + 2) mRNA was not detected in unstimulated synovial cells, its expression was induced by IL-18 stimulation. By immunoblot, we demonstrated that IL-18 induced the SAA protein synthesis from rheumatoid synovial cells in a dose-dependent manner. These results indicate a novel role for IL-18 in rheumatoid inflammation through the synovial SAA production.     

Transforming growth factor-beta production by synovial tissues from rheumatoid patients and streptococcal cell wall arthritic rats. Studies on secretion by synovial fibroblast-like cells and immunohistologic localization

The growth of synovial fibroblast-like cells from patients with rheumatoid arthritis and rats with streptococcal cell wall (SCW)-induced arthritis in vitro under anchorage-independent conditions is inhibited by transforming growth factor-beta (TGF-beta). Because this growth factor is present in rheumatoid synovial fluids, we studied whether this cytokine might be secreted by cells in rheumatoid synovial tissue. We show that synovial tissues from patients with rheumatoid arthritis and osteoarthritis, and rats with SCW-induced arthritis, contain TGF-beta-1 mRNA. TGF-beta, predominantly type 1, was spontaneously secreted in vitro by synovial tissue explants and synovial fibroblast-like cells. In addition, TGF-beta could be detected immunohistochemically in cells throughout rheumatoid and SCW-induced arthritic rat synovial tissues. Finally, exogenous TGF-beta induced collagen and inhibited collagenase mRNA levels by cultured synoviocytes. These data support an autocrine role for TGF-beta in the regulation of synoviocytes in rheumatoid arthritis and, in light of its demonstrated effects on the immune system, suggest that TGF-beta might also have important paracrine effects on infiltrating inflammatory cells.     

Differential synthesis of 5-lipoxygenase in peripheral blood and synovial fluid neutrophils in rheumatoid arthritis

In order to investigate 5-lipoxygenase enzyme regulation in neutrophils during an inflammatory reaction, we studied 5-lipoxygenase mRNA levels, as well as de novo enzyme synthesis, in resting and activated neutrophils isolated from normal individuals and patients with rheumatoid arthritis. The approach used was to analyze these activities in resting peripheral blood neutrophils of normal individuals on the one hand and in peripheral blood and matched synovial fluid neutrophils isolated from patients with rheumatoid arthritis on the other hand. Our first observation was that resting peripheral blood neutrophils of either normal individuals or patients show detectable levels of 5-lipoxygenase mRNA and are able to synthesize the enzyme de novo. Our second observation was that inflammatory activated neutrophils from synovial fluid reveal lower 5-lipoxygenase mRNA levels and enzyme synthesis than do the patient-matched peripheral blood cells. This is in spite of the fact that, for other proteins, synovial fluid neutrophils are equally or more active than their peripheral blood counterparts. We conclude that peripheral blood neutrophils are capable of synthesizing the enzyme, thus ensuring the turnover of the protein. Furthermore, complex regulatory mechanisms appear to take place in response to inflammation as it occurs in synovial fluids of patients with rheumatoid arthritis, leading to decreased mRNA levels and enzyme synthesis. Possible mechanisms of regulation are discussed and are presently under investigation.     

Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease

Background

Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man.

Methods/Principal Findings

We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (^99mTc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4×10⁻³ (0.95−5.1×10⁻³) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion.

Conclusions/Significance

The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.