Rheumatoid Arthritis Neutrophils Research Articles

Rheumatoid peripheral blood phagocytes are primed for activation but have impaired Fc-mediated generation of reactive oxygen species

Significant levels of circulating immune complexes (ICs) containing rheumatoid factors and immunoglobulin G in peripheral blood are a characteristic feature of rheumatoid arthritis (RA). ICs interact through Fcγ receptors (FcγR) to activate phagocytes in numerous inflammatory processes. The high concentration of neutrophils in synovial fluid during active phases of the disease, together with their destructive capacity, pose important questions as to their role in the pathogenesis of RA. Functional defects in RA or control peripheral blood neutrophil FcγRs were examined with a specific FcγR-mediated reactive oxygen species (ROS) assay. Heterologous cross-linking of FcγRIIa and FcγRIIIb on neutrophils resulted in a significantly decreased production of ROS by RA cells compared with controls matched for age and sex. However, expression and homologous ligation of receptors did not differ between these groups. These data suggest that neutrophil priming does occur before emigration into the joint and that blood neutrophils from patients with RA have a functional impairment in cooperative FcγR-mediated ROS generation. This may account for the increased susceptibility to bacterial infection that arises in patients with severe disease.

Secretion of oncostatin M by neutrophils in rheumatoid arthritis

Neutrophils are known to express and release a large number of proinflammatory cytokines when they are stimulated by inflammatory stimuli. The objective of this study was to determine whether neutrophils express oncostatin M (OSM), a member of the interleukin-6 family of cytokines that has been implicated in the pathogenesis of inflammatory joint disease. Neutrophils were isolated from the blood of healthy volunteer donors and from the blood and synovial fluid of patients with rheumatoid arthritis (RA). OSM levels were measured in cell extracts and in culture supernatants by Western blotting. Total RNA was isolated from control and granulocyte-macrophage colony-stimulating factor (GM-CSF)-treated neutrophils, and OSM messenger RNA levels were quantified by hybridization of a radiolabeled probe. GM-CSF stimulated a rapid and transient expression and release of OSM from blood neutrophils, which was more rapid than the expression and release from blood monocytes. A 28-kd protein was identified in cell extracts, but an additional 25-kd isoform was detected in culture supernatants. Synovial fluid neutrophils could not be stimulated to express OSM, but this cytokine was detected in cell-free supernatants at various levels. Blood neutrophils can be stimulated to express and rapidly release large quantities of OSM. We propose that this important cytokine is released from neutrophils as they infiltrate rheumatoid joints and, thus, contribute to the complex cytokine network that characterizes RA.

Synovial fluid neutrophils transcribe and express class II major histocompatibility complex molecules in rheumatoid arthritis.

To investigate a potential interaction between neutrophils and T cells in rheumatoid arthritis (RA), by defining the optimal conditions for induction of class II major histocompatibility complex (MHC) expression on peripheral blood neutrophils in vitro and investigating the capacity for neutrophils to express class II MHC molecules in RA. Surface expression of class II MHC and costimulatory molecules by peripheral blood and synovial fluid (SF) neutrophils obtained from healthy controls and patients with RA was measured by flow cytometry and fluorescence microscopy. Intracellular class II MHC protein and messenger RNA (mRNA) were detected by Western blotting and Northern blotting, respectively. Freshly isolated peripheral blood neutrophils from controls did not express surface class II MHC; expression was induced by culture with appropriate cytokines. Freshly isolated peripheral blood neutrophils from patients with RA expressed mRNA, but there was no surface expression of class II MHC. Freshly isolated SF neutrophils from patients with RA contained high levels of class II MHC mRNA, did not express surface class II MHC, but did have large intracellular amounts of this protein as detected by Western blotting. After culture for 20 hours in vitro, SF neutrophils from RA patients expressed large amounts of surface class II MHC but very low levels of costimulatory molecules CD80 and CD86. Fluorescence microscopy localized surface class II MHC to discrete areas on the neutrophil. Class II MHC-expressing neutrophils stimulated T cell proliferation. Peripheral blood neutrophils from patients with RA but not healthy controls express class II MHC mRNA. SF neutrophils in RA synthesize and express large amounts of class II MHC but not costimulatory molecules. This might underlie a novel interaction with T cells that is important in terms of disease pathology.

Expression of vascular endothelial growth factor by synovial fluid neutrophils in rheumatoid arthritis (RA).

Most of the leucocytes infiltrating rheumatoid synovial fluid (SF) are neutrophils capable of producing a variety of inflammatory mediators known to contribute significantly to the disease process during active RA. In the present study, we investigated the contribution made by SF neutrophils to the elevated levels of vascular endothelial growth factor (VEGF) seen in rheumatoid SF. Rheumatoid SF neutrophils were found to contain significantly larger amounts of both VEGF protein and its mRNA than peripheral blood neutrophils from either RA patients or healthy controls. Levels of cell-associated VEGF were well correlated with free VEGF in SF, which was significantly higher than in SF from osteoarthritis patients. Levels of SF neutrophil-associated VEGF also correlated with RA disease activity and cell surface integrin expression. Thus, SF neutrophil-associated VEGF may be considered an indicator of both local and systemic inflammation of RA, contributing to the neovascularization seen during RA synovitis.

Oxidative stress in rheumatoid arthritis leukocytes: suppression by rutin and other antioxidants and chelators

The enhanced production of superoxide ion and peroxynitrite by bloodstream neutrophils and of superoxide ion by monocytes from rheumatoid arthritis (RA) patients was registered. It was suggested that NADPH oxidase together with NO synthase were the major sources of superoxide ion in RA neutrophils, while in RA monocytes superoxide ion was produced by NADPH oxidase and mitochondria. Among the different free radical inhibitors studied (antioxidant enzymes, SOD and catalase; free radical scavengers, bioflavonoid rutin and mannitol; and the iron chelator desferrioxamine), SOD and rutin were the most efficient suppressors of oxygen radical overproduction by RA neutrophils, while mannitol and desferrioxamine were inactive. Thus, in contrast to Fanconi anemia (FA) leukocytes (Korkina LG et al., J Leukocyte Biol 1992;52:357–62), iron-catalyzed hydroxyl radical formation was unimportant in RA leukocytes, which mainly produced superoxide ion. Natural non-toxic bioflavonoid rutin (vitamin P) inhibited oxygen radical overproduction in both RA and FA in an equally efficient manner and therefore may be considered as a useful supporting pharmaceutical agent for the treatment of “free radical” pathologies.

Seeing the wood for the trees: the forgotten role of neutrophils in rheumatoid arthritis

Seeing the wood for the trees: the forgotten role of neutrophils in rheumatoid arthritis

Immunolocalization of bradykinin receptors on human synovial tissue

Kinins have been implicated in the pathogenesis of experimental and clinical inflammatory arthritis. Previous studies have reported increased amounts of plasma and tissue kallikreins in synovial fluid, raised kinin levels and an upregulation of kinin B2 receptors on synovial fluid neutrophils in rheumatoid arthritis. Bradykinin binding sites have been identified on human synovial cells by autoradiographic localization and Scatchard analysis. This study was undertaken to localize immunohistochemically kinin B1 and B2 receptors on human synovial tissue. Synovial tissue was obtained at the time of joint replacement surgery or arthroscopic synovectomy in six patients (two RA, two OA and two with avascular necrosis). Tissue sections were immunolabelled for kinin B1 and B2 receptors and viewed by light and confocal microscopy. No immunolabelling of the kinin receptors was observed in the method controls. In all patients labelling for kinin B2 receptors was observed in the synovial lining cells, fibroblasts and endothelial lining cells of blood vessels. There was no immunolabelling for kinin B1 receptors in all samples. These findings further support a role for the B2 receptors in joint diseases. There did not appear to be an induction of the kinin B1 receptor in human synovial tissue obtained from patients with chronic arthritis. However, further studies are required to assess the role of B1 receptors in active joint inflammation.

Peripheral blood neutrophil leukotriene B4 release and migration in rheumatoid arthritis.

The present study was designed to compare peripheral blood neutrophil migration and leukotriene (LT) release between patients with rheumatoid arthritis (RA) and healthy controls and to correlate the neutrophil functions with clinical disease activity. Nineteen patients with moderately active RA and 19 age and sex matched healthy volunteers participated in this study. Isolated peripheral blood neutrophils from RA patients released equal amounts of LTB4 but their random migration was enhanced as compared with neutrophils from healthy controls. LTB4 release in whole blood was significantly lower in samples from RA patients than in those from the healthy volunteers (13.5 +/- 1.4 and 19.1 +/- 1.4 ng/10(6) neutrophils respectively; P < 0.001). LTB4 release from isolated RA neutrophils correlated with the levels of C-reactive protein, duration of morning stiffness and Ritchie articular swelling index. Concentrations of hyaluronate, cyclic AMP and 13, 14-dihydro-15-keto prostaglandin were not different between patients with RA and healthy volunteers. Neither was there any difference in TXB2 production by platelets during blood clotting. In conclusion, peripheral blood neutrophils of RA patients seem to be primed and/or activated as their random migration is enhanced as compared with those of healthy volunteers. In RA, LTB4 release from peripheral blood neutrophils seems to reflect the clinical activity of the disease. However, RA neutrophils released smaller (in whole blood) or equal (isolated cells) amount of LTB4 as compared with the respective controls. These contradictory findings suggest that LTB4 release from peripheral blood neutrophils has no major role in the regulation of disease activity in rheumatoid arthritis.

Tumor necrosis factor priming of peripheral blood neutrophils from rheumatoid arthritis patients.

Recently it was shown that tumor necrosis factor-alpha (TNF) receptors on neutrophils may be down-regulated after stimulation with proinflammatory mediators. Since in rheumatoid arthritis neutrophils are likely to encounter these mediators in the circulation, we tested the hypothesis that rheumatoid arthritis neutrophil TNF receptors are down-regulated. Peripheral blood neutrophils from patients with rheumatoid arthritis and healthy subjects were compared with respect to their TNF binding activity and ability to be primed by TNF. There were no differences between rheumatoid arthritis and control neutrophils in receptor-mediated TNF binding, superoxide release in response to agonist, and TNF priming of this respiratory burst or in the ability to degrade cartilage in vitro and TNF priming for increased cartilage damage. It is evident that rheumatoid arthritis blood neutrophils retain the ability to bind TNF and can be primed by TNF for increased oxygen radical production and augmented cartilage damage. These findings further implicate the role of neutrophils in the pathogenesis of arthritis.

Relationship between interleukin-8 and neutrophil adhesion molecules in rheumatoid arthritis

To evaluate the role of interleukin-8 (IL-8) on the activation of neutrophils in rheumatoid arthritis (RA), we measured IL-8 and the adhesion molecules, L-selectin (CD62L), CD1 1b and CD18, on neutrophils in paired peripheral blood and synovial fluid of RA patients. Synovial fluid IL-8 levels were significantly increased compared to peripheral blood. L-selectin was split off and CD1 1b and CD 18 were upregulated on neutrophils in the synovial fluid. A positive correlation occurred between the IL-8 concentration and CD18 or CD1 1b densities on neutrophils in the synovial fluid (r = 0.75, P < 0.005 and r = 0.60, P < 0.05, respectively). Peripheral blood neutrophils of the patients were desensitised with IL-8 in vivo, as shown by the significantly lower L-selectin shedding after in vitro IL-8 stimulation: 1.6 times decrease for patients vs 3.2 for controls (P < 0.05). In conclusion, these results add further evidence for the role of IL-8 in the activation of neutrophils in RA.

Neutrophil trafficking into inflamed joints in patients with rheumatoid arthritis, and the effects of methylprednisolone.

To investigate the trafficking of circulating blood neutrophils and synovial fluid neutrophils in rheumatoid arthritis (RA) patients and the influence of a 1,000-mg intravenous pulse of methylprednisolone succinate (MP). Neutrophils were isolated from the circulation and from the knee synovial compartments of subjects with RA. Circulating neutrophils were labeled with technetium-99 hexametazime (99mTc-HMPAO) and reinjected intravenously. Synovial fluid neutrophils were labeled from indium-111 oxine and reinjected into the knee from which they were isolated. Gamma camera images were obtained at intervals up to 24 hours post MP. Each patient had a baseline study (no MP) and a study in which MP was administered either 4 hours before (2 patients), 10 minutes before (1 patient), or 30 minutes to 1.5 hours after (6 patients) injection of the radiolabeled neutrophils. Subsequent analysis allowed quantitation of the neutrophil uptake into and clearance from the knee as a function of time. Nine patients who had not received glucocorticoids in the previous 3 months were studied. MP significantly decreased neutrophil ingress in 13 of the 16 knees studied (almost total inhibition in 5 knees), and this occurred within 1.5 hours of MP administration in all except 1 knee. At 24 hours after MP administration, there was a significant increase in visual analog scale (VAS) scores for well-being and significant decreases in scores on the modified Health Assessment Questionnaire (P<0.05), tender joints (P<0.005), VAS for pain (p<0.005), and generalized stiffness (P<0.005), as well as a decrease in the C-reactive protein level (P<0.05). MP had no effect on neutrophil egress (2 patients). Two additional patients who were receiving oral glucocorticoids were studied. One of them was clinically unresponsive to oral prednisolone, and MP had no effect on neutrophil ingress. The other patient showed no neutrophil ingress during the baseline study. This was confirmed by the presence of a noninflammatory synovial fluid at arthrocentesis. Neutrophil ingress into and egress from inflamed joints can be accurately monitored using radiolabeled neutrophils and quantitative gamma camera imaging. MP rapidly and substantially decreases neutrophil ingress into inflamed joints. In contrast, MP has no effect on neutrophil egress from the joint.

Differences in oxidative response of subpopulations of neutrophils from healthy subjects and patients with rheumatoid arthritis.

To determine whether blood neutrophils from healthy individuals and blood and synovial fluid neutrophils from patients with rheumatoid arthritis (RA) responded differently to priming agonists and stimuli of the oxidative burst and, if so, whether this was a property of a subpopulation of neutrophils. Continuous flow electrophoresis was used to separate neutrophils into subpopulations based upon quantitative differences in net negative surface charge. The generation of superoxide anion (O2-) was used as a measure of oxidative activity using 10(-7) mol/l N-formyl-methionylleucyl-phenylalanine (FMLP) as the stimulating agonist and 10(-8) mol/l platelet activating factor (PAF) as the priming agent. The production of O2- by blood and synovial fluid neutrophils from RA patients in response to FMLP was greater than that observed with control blood neutrophils (p < 0.001). Priming of normal blood neutrophils with PAF increased their FMLP induced oxidative burst (p < 0.001), but PAF treatment had no effect on rheumatoid neutrophils. Neutrophils from synovial fluid of RA patients were less electronegative than paired blood samples and exposure of blood neutrophils to FMLP but not PAF reduced their surface charge. Continuous flow electrophoresis isolated three neutrophil subpopulations: cells of least surface electronegativity were ascribed to pool P1 and cells of greatest surface electro-negativity to P3. Normal blood neutrophils from P3, but not P1, showed increased oxidative activity after PAF priming (twofold increase; p < 0.01), whereas the responsiveness of rheumatoid blood and synovial fluid neutrophils from P1 and P3 was not modified by PAF treatment under the same conditions. It is suggested that most of the circulating neutrophils in RA are already in a state of readiness to generate O2- upon activation by an inflammatory stimulus. This is in contrast to normal blood neutrophils, which have both responsive and non-responsive subpopulations with respect to priming agonists.

Interleukin-1 expression by neutrophils in rheumatoid arthritis.

To determine if neutrophils from blood and synovial fluid of patients with rheumatoid arthritis and other joint arthropathies express interleukin-1 beta mRNA. RNA was isolated from neutrophils from patient and control blood, and synovial fluid of patients, probed in northern blots, and quantified by densitometry. It was also isolated and analysed from control blood neutrophils after incubation in vitro with granulocyte macrophage colony stimulating factor (GM-CSF). Neutrophils from the synovial fluid of patients with rheumatoid arthritis contained low levels of mRNA for interleukin-1 beta--between 0.1 and 2% of those observed during stimulation of control neutrophils with GM-CSF for one hour. Higher levels (4-40% of the maximal GM-CSF values) were observed in blood neutrophils from patients with rheumatoid arthritis. Neutrophils contribute to the cytokine network in rheumatoid arthritis. In some circumstances, activation of transcription may occur within the circulation of these patients.

Growth-related gene product alpha. A chemotactic cytokine for neutrophils in rheumatoid arthritis.

Leukocyte recruitment is critical in the inflammation seen in rheumatoid arthritis (RA). To determine whether the chemokine growth-related gene product alpha (gro alpha) plays a role in this process, we examined synovial tissue (ST), synovial fluid (SF), and plasma samples from 102 patients with arthritis. RA SF contained more antigenic gro alpha (mean 5.3 +/- 1.9 ng/ml) than did SFs from either osteoarthritis (OA) or other forms of arthritis (mean 0.1 ng/ml) (p < 0.05). RA plasma contained more gro alpha (mean 4.3 +/- 1.8 ng/ml) than normal plasma (mean 0.1 ng/ml) (p < 0.05). RA ST fibroblasts (1.2 x 10(5)/cells/mI RPMI 1640/24 h) produced antigenic gro alpha (mean 0.2 +/- 0.1 ng/ml), and this production was increased significantly upon incubation with TNF-alpha (mean 1.3 +/- 0.3 ng/ml) or IL-1 beta (mean 2.3 +/- 0.6 ng/ml) (p < 0.05). Cells from RA SF also produced gro alpha: neutrophils (PMNs) (10(7) cells/mI/24 h) produced 3.7 +/- 0.7 ng/ml. RA SF mononuclear cells produced gro alpha, particularly upon incubation with LPS or PHA. Immunoreactive ST gro alpha was found in greater numbers of RA compared with either OA or normal lining cells, as well as in RA compared with OA subsynovial macrophages (p < 0.05). IL-8 accounted for a mean of 36% of the RA SF chemotactic activity for PMNs, while epithelial neutrophil-activating peptide-78 accounted for 34%, and gro alpha for 28%, of this activity. Combined neutralization of all three chemokines in RA SFs resulted in a mean decrease of 50% of the chemotactic activity for PMNs present in the RA SFs. These results indicate that gro alpha plays an important role in the ingress of PMNs into the RA joint.

Production of PAF-acether by synovial fluid neutrophils in rheumatoid arthritis.

PAF-acether (PAF) is a pro-inflammatory phospholipid molecule potentially involved in the pathogenesis of arthritis. PAF and related metabolites have been isolated in the synovial fluid from patients with arthritis. The aim of this study was to determine fluid and blood in patients with rheumatoid arthritis. Blood neutrophils from normal donors were also studied for their capacity to form PAF. Neutrophils were stimulated with the calcium ionophore A23187 (2 microM) for 1 to 60 min. PAF released in the medium and PAF associated to cells were measured. In synovial fluid neutrophils. PAF production began as soon as 1 min of stimulation (16.1 +/- 6.3 pmol per 1 x 10(6) cells) and reached a maximum at 20 min: 29.2 +/- 2.8 pmol per 1 x 10(6) cells (mean +/- SEM, n = 5). The amount of PAF released in the supernatant increased with the length of stimulation, similar amounts of PAF were produced by blood neutrophils isolated from the joint had a lower capacity to produce PAF than blood neutrophils from the same patients. The present results demonstrate the synthesis and release of PAF by synovial fluid neutrophils. They suggest that neutrophils may be source of PAF locally present in the joint. Newly synthesized PAF could participate in the amplification of the local inflammatory reaction.

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.

Effects of fasting on disease activity, neutrophil function, fatty acid composition, and leukotriene biosynthesis in patients with rheumatoid arthritis.

Fourteen patients with rheumatoid arthritis (RA) were studied before, during, and after a 1-week total fast. Disease activity decreased, as did the neutrophil release of lysozyme induced by the ionophore A23187. The ability of zymosan-activated RA patient serum to aggregate control neutrophils was reduced, together with serum concentrations of C3. The relative contents of arachidonic acid and eicosapentaenoic acid were increased in serum, platelets, and neutrophils, whereas levels of linoleic acid and linolenic acid were unchanged. Fasting also reduced the release of leukotriene B4 from neutrophils. We thus conclude that a reduced ability to generate cytotaxins, reduced release of enzyme, and reduced leukotriene formation from RA neutrophils, together with an altered fatty acid composition of membrane phospholipids, may be mechanisms for the decrease of inflammatory symptoms that results from fasting.

Neutrophil responsiveness to chemoattractant tripeptide in rheumatoid arthritis.

Neutrophils isolated from medication-free rheumatoid arthritis (RA) patients were assayed for responsiveness to the bacterial chemoattractant tripeptide formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe). Rheumatoid arthritis neutrophil preparations contained significantly lower percentages of rapidly migrating cells. This relative hyporesponsiveness of RA neutrophils was related to impaired sensing of chemotactic gradients. Rheumatoid neutrophil abnormalities in sensing of and responding to chemotactic gradients were not associated with resting or f-Met-Leu-Phe-induced changes in arachidonic acid metabolism.

Enhanced capacity for release of leucotriene B4 by neutrophils in rheumatoid arthritis.

The calcium dependent metabolism of endogenous arachidonic acid (AA) was investigated in 17 patients with rheumatoid arthritis during treatment with dextropropoxyphene alone and in 25 healthy volunteers. Incorporation of [1-14C]AA into intracellular phospholipids of purified neutrophils was achieved by incubation until steady state before activation with ionophore A23187. Analysis of extracellular metabolites was performed by extraction, thin layer chromatography, autoradiography, and laser densitometry. The patients showed a twofold increase in the total capacity for oxidation of AA. Release of leucotriene B4 (LTB4) and its omega oxidation products, 20-OH LTB4 and 20-COOH LTB4, was 29%, range 11-48%, in patients compared with 8%, range 4-12%, in healthy volunteers. Total amounts of radioactivity released and the specific activity of LTB4, as assessed by high pressure liquid chromatography, were equal in experimental and control groups. The demonstrated increased capacity for metabolism of AA to the major proinflammatory metabolite, LTB4, via the 5-lipoxygenase pathway may contribute to perpetuation of inflammation and to tissue destruction in rheumatoid arthritis.

Nitroblue tetrazolium dye reduction by peripheral leukocytes from rheumatoid arthritis and systemic lupus erythematosus patients measured by a histochemical and spectrophotometric method.

Abstract Phagocytic activity of peripheral leukocytes in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients was measured by in vitro reduction of nitroblue tetrazolium (NBT) dye employing histochemical and spectrophotometric methods. Neutrophils from rheumatoid patients reduced dye normally. Resting SLE neutrophils had lower mean NBT dye reduction than normal control or rheumatoid neutrophils. This decrease in NBT reduction was statistically significant. SLE patients treated with suppressive doses of prednisone (> 10 mg. per day) as well as untreated patients demonstrated this abnormality. SLE neutrophils stimulated by in vitro phagocytosis of latex particles demonstrate an incremental increase in NBT dye reduction comparable to normal control and RA neutrophils. Infected SLE patients, both untreated and steroid-treated cases, failed to demonstrate the anticipated increase in NBT dye reduction. This study presents data indicating that the histochemical and spectrophotometric procedure for measuring NBT dye reduction can be modified to yield comparable results. The current findings support other investigations that have shown serum and/or cell-mediated defects in phagocytosis can be detected in leukocytes obtained from SLE patients.