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Abstract
Objectives To determine the most relevant parameters in synovial fluid (SF) during the various stages of acute gout.
Methods SFs from 38 gouty patients were analysed for white blood cell (WBC) count, percentage of polymorphonuclear cells (PMNs) and levels of interleukin 1β (IL-1β), IL-6, IL-8, tumour necrosis factorα (TNFα) and transforming growth factor β1 (TGFβ1). Patients were divided into three groups according to the length of time since onset of the attack: phase I (0–48 h), phase II (days 3–4) and phase III (days 5–7).
Results Levels of WBCs were similar in SFs from phases I and II, while phase III showed the lowest WBC count. Percentages of PMNs were raised in all SFs. None of the cytokines analysed differed between phases I and II except for TGFβ1, which was higher in phase II. IL-1β, IL-6 and TNFα were higher in group 1 than in group 3. Levels of all the cytokines assessed, with the exception of TGFβ1, were significantly lower in phase III than in phase II IL-1β, p<0.05; IL-6, p<0.01; IL-8, p<0.001; TNFα, p<0.05).TGFβ1 levels were highest in SFs from phase III.
Conclusion Cytokine levels in SFs may change depending on the different stages of acute gout, highlighting the role of TGFβ1 in the resolution of gout.
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Introduction
Several mechanisms have been suggested as underlying the inflammatory response induced by deposit of monosodium urate (MSU) crystals in articular and periarticular tissues. It has been proposed that after crystal deposition, resident phagocytic cells are activated in an attempt to clear the crystals by phagocytosis. This results in the release into the surrounding area of various inflammatory mediators, thus triggering a cascade of acute inflammatory reactions and influx of neutrophils and macrophages into the joint.1 A massive influx of leucocytes, mainly neutrophils, and high levels of cytokines such as interleukin 1β (IL-1β),2 IL-63 and tumour necrosis factor α (TNFα),4 chemokines such as IL-85 and CCL26 have been detected in the synovial fluid (SF) of patients with gout.
Additionally, in vitro, a variety of different cytokines and chemotactic factors are upregulated when various types of leucocytes are in contact with MSU crystals,3 5 7 and animal models of gout have shown that when injected into the peritoneum, the air pouch or the joint, MSU crystals induce an inflammatory response characterised by a cellular infiltrate rich in neutrophils and the production of proinflammatory cytokines as well as other inflammatory mediators.8 9
Even if left untreated, the acute inflammatory response in gout is characteristically self-limiting after 7–10 days. Various factors are thought to be involved in the mechanisms of spontaneous remission of inflammation, including the effect of plasma proteins and lipoproteins identified in SFs,10 11 the clearance of apoptotic cells,12 the induction of peroxisome proliferator-activated receptor γ,13 the upregulation of IL-10 expression7 and the monocyte–macrophage switch resulting in the production of anti-inflammatory cytokines such as transforming growth factor (TGF)β.14 High levels of TGFβ have been detected in the SFs of patients with acute gout,15 and the administration of TGFβ significantly inhibited cellular recruitment in a rat air pouch model of gout.16 More recently, it has been observed that a rapid induction of the TGFβ1 and the intracellular cytokine negative regulators CIS and SOCS3 is associated with spontaneous resolution of acute gouty arthritis.17
To shed light on the complex mechanisms involved in crystal-induced inflammation in gout, several studies have been performed, of which only a few have examined the natural course of acute gout and none of them has the characteristics of SF during the different phases of acute gout in humans. This study was performed to test SF for the levels of the most relevant parameters in gout during the various stages of acute gouty arthritis.
Materials and methods
Patients
Samples of SF were obtained from 38 patients (31 men, mean age 60.8±9.1 years, range 44–77) diagnosed with gout by urate crystal identification in SF.
All patients gave their informed consent to the study, which was approved by the local ethical committee. Only patients without anti-inflammatory treatment apart from non-steroidal anti-inflammatory drugs (NSAIDs) were enrolled in the study; in particular, none of the patients were taking colchicine or steroids or had received intra-articular injections during the 2 previous months and their hypouricaemic treatment had been stable in the past 2 months. Patients were subsequently divided into three groups according to the length of time since onset of the attack: phase I (0–48 h), phase II (days 3–4) and phase III (days 5–7). Patients' characteristics are shown in table 1.
Patient characteristics
Collection and analysis of synovial fluid
SF samples were promptly analysed after arthrocentesis by optical light microscopy. The total white blood cell (WBC) count was obtained using a standard haematological counting chamber. A differential cell count was performed by May–Grünwald–Giemsa staining. MSU crystals were identified by ordinary and polarising light microscopy. After examination, SF samples were centrifuged at 3000 g for 10 min.
Determination of cytokines and chemokines
SF concentrations of IL-1β, IL-6, IL-8, TNFα and TGFβ1 were assessed in duplicate by ELISA (eBioscience, San Diego, California, USA).
Statistical analysis
Differences in levels of parameters between independent groups were assessed by analysis of variance, followed by post hoc Dunn's test. All values are expressed as mean±SD. A p value of <0.05 is considered significant.
Results
The results of SF analysis and determination of cytokines and chemokines are shown in figure 1. The p values of the statistical analysis are summarised in table 2.
Cell counts and concentrations of cytokines and chemokines in synovial fluids (SFs). (A) Number of white blood cells (WBC). (B) Percentage of polymorphonuclear cells (PMNs). SF concentrations of (C) (closed rectangle) interleukin (IL)-1β and (open rectangle) tumour necrosis factor α (TNFα), (D) (closed circle) IL-8 and (open circle) IL-6, (E) (closed triangle) transforming growth factor-β1 (TGFβ1). Values are expressed as the mean±SD. #phase I versus phase II, #p<0.05; *phase I versus phase III, ***p<0.001; +phase II versus phase III, +p<0.05, ++p<0.01, +++p<0.001. The p values of the statistical analysis are summarised in table 2. Phase I (0–48 h), phase II (days 3–4) and phase III (days 5–7).
p Values of statistical analysis of cell counts and concentrations of cytokines and chemokines in SFs
Levels of WBC were similar in SFs from phase I and phase II patients (29.7±10.6 103/mm3, 29.8±3.1 103/mm3), while phase III patients showed the lowest WBC count (8.4±6.0 103/mm3, p<0.001 vs groups 1 and 2). All SFs contained raised percentages of polymorphonuclear cells (PMNs), no difference being seen between the three groups (80.9±9.2%, 81.5±7.2%, 73.4±7.0%). Levels of all the proinflammatory cytokines tested were high in phase I and II. Although the concentrations of IL-1β, IL-6 and TNFα indicated a tendency towards a decrease in phase II (40.73±10.68 pg/ml, 983.64±156.41 pg/ml and 71.27±15.25, respectively) as compared with phase I (63.9±31.8 pg/ml, 1226.2±424.2 pg/ml and 85.2±18.8 pg/ml, respectively), the differences did not reach statistical significance (p>0.05). IL-1β, TNFα and especially IL-6 were markedly decreased in phase III (23.5±9.1 pg/ml, p<0.001 vs group 1 and p<0.05 vs group 2; 43.9±11.7 pg/ml, p<0.001 vs group 1 and p<0.05 vs group 2; 570.0±113.1 pg/ml, p<0.001 vs groups 1 and 2, respectively). In contrast, concentrations of IL-8 were found to be increased in SFs from phase II patients (840.91±145.56 pg/ml) as compared with SFs from phase I patients (690.62±328.97 pg/ml), but again the difference was not statistically significant. As shown in table 2, in phase III patients, IL-8 was present at significantly lower concentrations than in phase II patients (441.36±72.46 pg/ml, p<0.001). The levels of all cytokines assessed, with the exception of TGFβ1, were significantly lower in phase III than in phase II. Moreover, TGFβ1 was the only cytokine measured that showed differences between phase I and phase II, TGFβ1 levels being significantly higher in group 2 (116.7±11.4 ng/ml, p<0.05). The level of TGFβ1 was highest in SFs from phase III patients (173.±13.8 ng/ml).
Discussion
This is the first study demonstrating that in human SFs levels of proinflammatory and anti-inflammatory cytokines may change depending on the stages of acute gout. Studies like this have been rarely performed owing to the difficulty in finding untreated gouty patients with joint effusions in different phases of the disease.
In our study SFs from 38 patients with gout were divided into three groups according to the length of time since onset of the acute attack. This subdivision was established arbitrarily on the basis of our knowledge of the pathophysiology of the inflammatory process and mainly on the typical clinical evolution of gout.
After the division into groups, we observed that the cell counts did not differ significantly between the first and the second phase, showing that the severity of inflammation decreased only after day 4. This observation was borne out by the analysis of pro- and anti-inflammatory cytokines/chemokines. Although IL-1β, IL-6 and TNFα levels tended to decrease between phases I and II, there were no significant differences between the two groups.
IL-8 is known to be the principal CXC chemokine involved in neutrophil migration in response to MSU crystals.5 Although IL-8 decreased in phase III, its high levels could explain the high percentage of PMNs also seen in this phase. Therefore, our results suggest that IL-8 might be the main chemotactic factor causing neutrophil infiltration in the late phase of acute gouty arthritis.
A significant reduction in most of the parameters considered was seen in phase III in comparison with phases I and II; however, the loss of the ability to secrete proinflammatory cytokines in response to MSU crystals was accompanied by a gain in the ability to release TGFβ1.
TGFβ1 is thought to be the prime mediator in the active resolution of gouty inflammation.18 Raised levels of TGFβ1 have been reported in SFs of patients with gout and pseudogout, suggesting that TGFβ1 may be involved in inflammatory quiescence during intercritical phases.15 19 20 Both in vitro and in vivo studies have demonstrated that TGFβ1 acts as a potent anti-inflammatory cytokine in MSU crystal-induced inflammation.14 16
Our observation that TGFβ1 increased in SF before a decrease of inflammatory parameters, strongly supports an integral role for TGFβ1 as a key factor in inducing auto-remission of inflammation in acute gout attacks. In the early stage of the disease, a high level of TGFβ1 may play the role of a chemotactic factor, but at a later stage, acts as an immunosuppressor.
Our study has a few limitations, such as the arbitrary definition of the acute gout phases and, most of all, the differences in the disease duration, which at times is difficult to determine with accuracy. However, it will contribute to a better definition of the individual role of each molecule involved in gouty arthritis and to the design of new therapeutic strategies for its management.
References
Footnotes
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Competing interests None.
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Patient consent Obtained.
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Ethics approval Obtained.
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Provenance and peer review Not commissioned; externally peer reviewed.