Roger C. Bucknall

Neutrophil function in inflammation and inflammatory diseases

In inflammatory conditions such as RA, the neutrophil has tended to be dismissed as a short-lived, terminally differentiated, irrelevant bystander cell. However, this is clearly not the case. A better understanding of the complex heterogeneous pathways and processes that constitute RA, in parallel with a more sophisticated knowledge of neutrophil biology has identified many potential roles for these cells in the persistence of inflammation and progression of joint damage, which should not be underestimated. Not only are neutrophils found in high numbers within the rheumatoid joint, both in synovial tissue and in joint fluid, they have a huge potential to directly inflict damage to tissue, bone and cartilage via the secretion of proteases and toxic oxygen metabolites, as well as driving inflammation through antigen presentation and secretion of cytokines, chemokines, prostaglandins and leucotrienes. Drugs already used to treat RA down-regulate many neutrophil functions, including migration to the joint, degranulation and production of inflammatory mediators, and these cells should be considered as important targets for the development of new therapies in the future.

Neutrophils isolated from the synovial fluid of patients with rheumatoid arthritis: priming and activation in vivo.

The oxidative metabolism of neutrophils isolated from the bloodstream and synovial fluid of 16 patients with rheumatoid arthritis was compared by measuring the ability of neutrophils to generate luminol dependent chemiluminescence and to secrete O2-. Measurements of receptor mediated--that is, N-formyl-methionyl-leucyl-phenylalanine stimulated--activation or receptor and second message independent--that is phorbol myristate acetate stimulated--activation showed that synovial fluid neutrophils had biochemical characteristics to suggest that they had been either up-regulated (primed) or down-regulated (activated) in vivo. These conclusions were confirmed by comparison of these responses with the changes in oxidative metabolism observed during in vitro priming and activation of control neutrophils: synovial fluid neutrophils possessed lower levels of myeloperoxidase than paired bloodstream cells, and unlike bloodstream cells could not be primed in vitro. These data thus suggest that synovial fluid neutrophils have been exposed to both priming and activating agents within rheumatoid joints.

Neutrophil apoptosis in rheumatoid arthritis is regulated by local oxygen tensions within joints

Neutrophils are normally short‐lived cells and die by apoptosis, but when recruited into tissues, their apoptosis is delayed, and they survive for much longer time periods. In inflammatory diseases, such as rheumatoid arthritis (RA), this delayed apoptosis may lead to increased tissue damage and a failure of the inflammation to resolve. However, there are conflicting reports in the literature as to whether neutrophil apoptosis is delayed or accelerated in rheumatoid joints. In this report, we show that neutrophils isolated from the ynovial fluid (SF) of patients with RA show accelerated rates of apoptosis when incubated ex vivo and that SF, despite containing a variety of antiapoptotic cytokines, is proapoptotic. Paradoxically, levels of the key neutrophil survival protein Mcl‐1 are elevated in freshly isolated SF neutrophils compared with matched peripheral blood samples from the same patients, indicating that delayed neutrophil apoptosis has been signaled in vivo as the cells enter the joints. However, when SF was added to neutrophils and incubated under hypoxia (1% O2), conditions known to exist in vivo within joints, the SF was antiapoptotic. These data reveal that the rheumatoid synovial joint contains a complex mixture of pro‐ and antiapoptotic factors and that the low, local oxygen tensions that exist within these joints can exert profound effects on neutrophil survival. These experiments also highlight the importance of performing in vitro experiments under laboratory conditions that closely mimic those that occur in vivo; otherwise, misleading conclusions may be drawn.

Neutrophils from the synovial fluid of patients with rheumatoid arthritis express the high affinity immunoglobulin G receptor, FcγRI (CD64): role of immune complexes and cytokines in induction of receptor expression

Neutrophils isolated from the synovial fluid of 16/24 patients with rheumatoid arthritis expressed FcγRI (CD64), the high‐affinity receptor for monomeric immunoglobulin G (IgG), on their cell surface. Receptor expression ranged from 17% to 168% of the level of expression obtained after incubation of control blood neutrophils with 100 U/ml interferon‐γ (IFN‐γ) for 24 hr in vitro. Similarly, mRNA for FcγRI was detected in synovial fluid neutrophils from 12/15 patients and transcript levels ranged from 5% to 200% of the values obtained after treatment of blood neutrophils with IFN‐γ for 4 hr in vitro. No surface expression nor mRNA were detected in freshly isolated blood neutrophils from either patients or from healthy controls. Addition of cell‐free synovial fluid to control blood neutrophils induced both mRNA and surface expression of FcγRI to levels that were comparable to those achieved after addition of IFN‐γ. Neither soluble nor insoluble immune complexes appeared to be involved in induction of FcγRI expression in spite of the ability of these complexes to induce protein biosynthesis. Synovial fluid‐induced expression of FcγRI was partially blocked by incubation with neutralizing IFN‐γ antibodies, whilst neutralizing interleukin (IL)‐6 antibodies had little effect. Levels of IFN‐γ measured within these synovial fluids ranged from 0 to 2·7 U/ml, well within the range known to induce neutrophil FcγRI expression. These data thus indicate that gene expression in synovial fluid neutrophils is selectively activated as the cells enter the diseased joint. Furthermore, these data indicate that induced expression of FcγRI may alter the ability of infiltrating neutrophils to respond to IgG‐containing immune complexes present in these joints.

Insoluble and soluble immune complexes activate neutrophils by distinct activation mechanisms: changes in functional responses induced by priming with cytokines

Background: Rheumatoid synovial fluid contains both soluble and insoluble immune complexes that can activate infiltrating immune cells such as neutrophils. Objectives: To determine if these different complexes activate neutrophils through similar or different receptor signalling pathways. In particular, to determine the circumstances which result in the secretion of tissue damaging reactive oxygen metabolites and granule enzymes. Methods: Blood neutrophils were incubated with synthetic soluble and insoluble immune complexes and the ability to generate reactive oxidants tested by luminescence or spectrophotometric assays that distinguished between intracellular and extracellular production. Degranulation of myeloperoxidase and lactoferrin was determined by western blotting. The roles of FcγRII (CD32) and FcγRIIIb (CD16) were determined by incubation with Fab/F(ab`)2 fragments before activation. The effect of cytokine priming was determined by incubation with GM-CSF. Results: Insoluble immune complexes activated unprimed neutrophils, but most of the oxidants produced were intracellular. This activation required FcγRIIIb, but not FcγRII function. Soluble complexes failed to activate unprimed neutrophils but generated a rapid and extensive secretion of reactive oxygen metabolites when the cells were primed with granulocyte-macrophage colony stimulating factor (GM-CSF). This activity required both FcγRII and FcγRIIIb function. Insoluble immune complexes activated the release of granule enzymes from primed or unprimed neutrophils, but the kinetics of release did not parallel those of secretion of reactive oxygen metabolites. Only primed neutrophils released enzymes in response to soluble complexes. Conclusions: Soluble and insoluble immune complexes activate neutrophils by separate receptor signalling pathways. Profound changes in neutrophil responsiveness to these complexes occur after cytokine priming.

Fcγ receptors in autoimmune diseases

Fcγ‐receptors (Fcγ‐R) recognise the Fc portion of IgG and thus form a link between humoral and cellular immunity. These receptors are expressed by a variety of immune cells, and they function in the binding of immune complexes or IgG‐opsonised particles, such as microbial pathogens. The are three major types of Fcγ‐R, namely Fcγ‐RI (CD64), Fcγ‐RII (CD32) and Fcγ‐RIII (CD16), and these differ in their ability to bind IgG and complexes. There are many isoforms of these receptors and a number of recently identified polymorphisms in their structure. This review describes the structure and function of these Fcγ‐Rs, and highlights how gene deficiencies and polymorphisms may contribute to the pathology of human diseases.

Analysis of SF and plasma cytokines provides insights into the mechanisms of inflammatory arthritis and may predict response to therapy

OBJECTIVES Biologic drugs have revolutionized the care of RA, but are expensive and not universally effective. To further understand the inflammatory mechanisms underlying RA and identify potential biomarkers predicting response to therapy, we measured multiple cytokine concentrations in SF of patients with inflammatory arthritides (IAs) and, in a subset of patients with RA, correlated this with response to TNF-α inhibition. METHODS SF from 42 RA patients and 19 non-RA IA patients were analysed for 12 cytokines using a multiplex cytokine assay. Cytokines were also measured in the plasma of 16 RA patients before and following treatment with anti-TNF-α. Data were analysed using Mann-Whitney U-test, Spearmans rank correlation and cluster analysis with the Kruskal-Wallis test with Dunns post-test analysis. RESULTS RA SF contained significantly elevated levels of IL-1β, IL-1ra, IL-2, IL-4, IL-8, IL-10, IL-17, IFN-γ, G-CSF, GM-CSF and TNF-α compared with other IA SF. RA patients who did not respond to anti-TNF therapy had elevated IL-6 in their SF pre-therapy (P < 0.05), whereas responders had elevated IL-2 and G-CSF (P < 0.05). Plasma cytokine concentrations were not significantly modulated by TNF inhibitors, with the exception of IL-6, which decreased after 12 weeks (P < 0.05). CONCLUSIONS Cytokine profiles in RA SF vary with treatment and response to therapy. Cytokine concentrations are significantly lower in plasma than in SF and relatively unchanged by TNF inhibitor therapy. Concentrations of IL-6, IL-2 and G-CSF in SF may predict response to TNF inhibitors.

A comparison of MR and CT in suspected sacroiliitis

OBJECTIVE A prospective study to compare the MR and CT images of patients with suspected sacroiliitis and to establish the optimal MR sequences to demonstrate the changes of sacroiliitis was conducted. MATERIALS AND METHODS Thirty-nine patients and nine controls were imaged in the axial plane, with SE T1-, T2-weighted fast spin echo (T2), T1 with fat suppression (T1WFS), and fast short tau inversion recovery (fast STIR) sequences on a 1.5 T system. The sacroiliac joints of all patients were imaged with CT. The images were evaluated by two independent radiologists. Following the blinded reading, direct comparison of T1 and T1WFS, T2, and fast STIR of the CT positive group was made to determine the optimal MR sequences. RESULTS The sensitivity and specificity of MR images for the detection of cortical erosions and subchondral sclerosis when compared to CT images were 100 and 94.3%, respectively; interobserver variation was low (k = 0.80). T1WFS and fast STIR images were superior to T1 and T2 images, respectively, in demonstrating the changes of sacroiliitis. CONCLUSION MRI (T1WFS and fast STIR) can replace CT in cases with a strong clinical suspicion of sacroiliitis and equivocal or normal plain radiographs.

Activation of the neutrophil myeloperoxidase-H2O2 system by synovial fluid isolated from patients with rheumatoid arthritis.

Synovial fluid isolated from 16 patients with rheumatoid arthritis activated luminol dependent chemiluminescence in bloodstream neutrophils, and the maximal activity stimulated varied over a 50-fold range. In contrast, these same fluids only activated a much lower range (two- to threefold) of maximal rates of lucigenin dependent chemiluminescence and cytochrome c reduction, two assays which only measure oxidant secretion which is independent of myeloperoxidase. Over 95% of the luminol dependent chemiluminescence activated by all samples was inhibited by azide (indicating its dependence upon myeloperoxidase), but anti-(myeloperoxidase) IgG (which specifically inhibits only the extracellular activity of this enzyme) only inhibited the response stimulated by some samples: those fluids which activated the highest luminol dependent chemiluminescence also stimulated the greatest activity of an extracellular myeloperoxidase-H2O2 system. A clear correlation was shown to exist between the activity of myeloperoxidase already present in the fluids (after its secretion from neutrophils in situ within the rheumatoid joint) and the ability of the fluid to activate luminol dependent chemiluminescence. It is concluded, therefore, that all synovial fluid samples tested possess almost equivalent levels of a factor(s) which activated O2-/H2O2 secretion and that the variations in the measured activity of the extracellular myeloperoxidase-H2O2 system are dependent upon the level of degranulation which had occurred within the joint.

Receptor expression in synovial fluid neutrophils from patients with rheumatoid arthritis.

OBJECTIVES--The aim of this study was to determine if neutrophils isolated from the blood and synovial fluid of patients with rheumatoid arthritis had patterns of receptor expression resembling those of blood neutrophils from controls which had been activated and primed in vitro. METHODS--Fluorescence activated cell sorting was used to measure receptor expression in paired blood and synovial fluid neutrophils from patients and in control neutrophils exposed to phorbol myristate acetate and granulocyte-macrophage colony stimulating factor. RESULTS--There was no significant difference in the patterns of receptor expression in blood neutrophils from patients and healthy controls, but neutrophils in the synovial fluid had been primed and activated within the joint. About 50% of rheumatoid synovial fluid neutrophil samples expressed Fc gamma RI, a high affinity receptor for monomeric IgG, which is only expressed in neutrophils exposed to cytokines. CONCLUSIONS--Synovial fluid neutrophils are activated and primed within the inflamed joint and hence their ability to respond to activating factors such as immune complexes will be modulated. As the expression of Fc gamma RI requires active biosynthesis, this work indicates that selective gene activation occurs when neutrophils are recruited into rheumatoid joints.