Rheumatology 2012;52:1018–1021
doi:10.1093/rheumatology/kes326
Advance Access publication 30 November 2012
RHEUMATOLOGY
Concise report
Hyperuricaemia elevates circulating CCL2 levels
and primes monocyte trafficking in subjects
with inter-critical gout
Rebecca Grainger1, Rene J. McLaughlin1, Andrew A. Harrison2 and
Jacquie L. Harper1
Abstract
Objective. To investigate the effect of hyperuricaemia on serum chemokine (C-C motif) ligand 2 (CCL2)
levels and blood monocytes in people with gout.
Methods. Whole blood was collected from subjects with a history of acute or chronic gout but not
currently experiencing an attack of gout, subjects with asymptomatic hyperuricaemia and healthy individuals with normouricaemia. Serum concentrations of CCL2 were measured by bead array and levels of
CD14+/CD11b+ blood monocytes determined by flow cytometry.
Results. Subjects with gout and asymptomatic hyperuricaemia had higher serum levels of CCL2 and
showed an increase in the percentage of circulating CD14+ monocytes compared with subjects with
normouricaemia.
Conclusion. Hyperuricaemia causes elevated serum CCL2 levels and increased monocyte recruitment
that may be driven by soluble uric acid-induced CCL2 production. Hyperuricaemia may initiate subclinical
priming of circulating blood monocytes for adhesion and trafficking during a gout attack.
CLINICAL
SCIENCE
Key words: hyperuricaemia, CCL2, monocytes, inflammation, gout.
Introduction
Gout is generally regarded as an articular disease, with
the manifestations related to the local inflammatory consequences of MSU crystal deposition.
Resident macrophages have been shown to be primarily responsible for initiation of inflammation and cytokine production in the early response to MSU crystals [1].
However, kinetic studies in mice have shown that monocytes are one of the initial infiltrating inflammatory cells
[1, 2] and chemokine (C-C motif) ligand 2 (CCL2) is a
key monocyte chemoattractant involved in controlling
monocyte trafficking [3, 4]. Animal models of MSU
crystal-induced inflammation have identified CCL2 as a
key local chemoattractant in synovial recruitment of
monocytes in gout [5] indicating that changes in CCL2
levels could impact significantly on monocyte recruitment
during a gout attack.
Hyperuricaemia is the primary risk factor for gout.
Soluble uric acid has been shown to induce CCL2 production in vascular smooth muscle cells [6] and hyperuricaemia
has been linked with elevated levels of serum inflammatory
markers, including CCL2 levels, in the absence of clinical
inflammation [7, 8]. Together these data indicate that hyperuricaemia may directly contribute to elevated levels of
CCL2 to facilitate monocyte release into the circulation of
individuals with hyperuricaemia and established gout even
in the absence of an inflammatory attack. In this study, we
investigated the impact of hyperuricaemia on the levels of
serum CCL2 and circulating CD14+ monocytes in subjects
with established gout but no current inflammation, and
asymptomatic hyperuricaemia.
1
Malaghan Institute of Medical Research and 2Department of
Medicine, Wellington School of Medicine and Health Sciences,
University of Otago, Wellington, New Zealand.
Materials and methods
Submitted 3 July 2012; revised version accepted 11 October 2012.
Study participants
Correspondence to: Jacquie L. Harper, Malaghan Institute of Medical
Research, PO Box 7060, Wellington 6242, New Zealand.
E-mail: [email protected]
Participants were recruited from advertisements to the
public, primary care and a rheumatology clinic and was
! The Author 2012. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
Hyperuricaemia, CCL2 and monocyte trafficking in gout
approved by the New Zealand Central Regional Ethics
Committee (CEN/06/03/018). Informed, written consent
was obtained from all participants according to the
Declaration of Helsinki. The study has been previously
described in Martin et al. [9]. Briefly, this study collected
a peripheral blood sample from subjects who had acute
gout (n = 29), chronic tophaceous gout (n = 16) (each with
no current clinical signs of inflammation) hyperuricaemia
with no history of gout (n = 15) or healthy controls (n = 31).
Serum collection
Peripheral blood was collected into additive free tubes.
Serum was separated by centrifugation (1900 g, 10 min)
and stored at 70 C for subsequent analysis.
Monocyte purification
Peripheral blood was collected in K3EDTA tubes and peripheral blood mononuclear cells (PBMCs) were separated
out by sedimentation using Polymorphprep (Invitrogen,
Auckland, New Zealand) as per manufacturer’s instructions. Any contaminating red blood cells were removed
with lysis buffer (Invitrogen) and the PBMCs were
washed twice with PBS (Invitrogen). CD14+ monocytes
were purified from PBMCs using positive immunomagnetic selection with MACS CD14 microbeads (Miltenyi
Biotec, Pharmaco, New Zealand) as per manufacturer’s
instructions. Monocyte (CD45+/CD14+) purity >94% was
determined by flow cytometric analysis (FITC-Mouse antihuman-CD45, PE-CD11b, PE-Cy7-CD14; ebiosciences,
San Diego, CA, USA).
Elevated serum CCL2 correlates with elevated
serum uric acid
Analysis of serum CCL2 levels from the four study groups
showed higher concentrations of CCL2 in subjects with
acute and chronic gout and asymptomatic hyperuricaemia compared with healthy controls (Fig. 1A). Due to the
presence of elevated serum CCL2 levels in subjects with
asymptomatic hyperuricaemia, serum CCL2 was stratified
based on the presence of hyperuricaemia (50.42 mM/
7.1 mg/dl). As shown in Fig. 1B there was an increase in
serum CCL2 in subjects with hyperuricaemia.
Elevated serum CCL2 correlates with increased
circulating CD14+ monocytes and up-regulation of
adhesion molecule CD11b
CCL2 is considered to be the primary chemoattractant for
monocyte/macrophages. Analysis of monocyte composition in total white blood cells revealed an increase in circulating CD14+ blood monocytes associated with serum
CCL2 levels (Fig. 1C) that was independent of gouty disease (Fig. 1D).
Elevated levels of CCL2 have also been linked to
up-regulation of the adhesion molecule CD11b on monocyte/macrophages [10]. As shown in Fig. 1E, there was a
positive correlation between serum CCL2 levels and
monocyte CD11b expression levels. When CD11b was
analysed by subject group we saw a significant increase
in CD11b expression on monocytes from acute gout subjects and a trend towards elevated CD11b on CD14+
monocytes from individuals with asymptomatic hyperuricaemia and chronic gout (Fig. 1F).
Serum analysis
Serum was analysed for CCL2 using a Luminex multiplex
bead array (Invitrogen), as per the manufacturer’s instructions. Serum uric acid levels were measured in a commercial laboratory (Aotea Pathology, Wellington, New
Zealand).
Statistical analysis
Data were analysed using Prism (Version 5.0d; GraphPad,
La Jolla, CA, USA). Exploratory data analyses confirmed
that continuous variables were not normally distributed so
serum concentrations of cytokines were assessed using
non-parametric tests (Kruskal–Wallis test with Dunn’s
post test, Mann–Whitney and Spearman’s r). For all statistical tests a P < 0.05 was considered statistically
significant.
Results
Study participants
Full demographic data have been previously reported [9].
The mean (95% CI) serum uric acid levels of the four subject groups were control 0.35 mM (0.33, 0.36 mM), asymptomatic hyperuricaemic 0.45 mM (0.44, 0.47 mM), acute
gout 0.46 mM (0.44, 0.48 mM) and chronic gout 0.48 mM
(0.45, 0.51 mM). Uric acid levels represent 5.9 mg/dl,
7.6 mg/dl, 7.7 mg/dl and 8.1 mg/dl, respectively.
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Discussion
Here we show that serum CCL2 levels positively correlate
with serum uric acid in both individuals with gout and with
asymptomatic hyperuricaemia. Soluble uric acid has been
shown to induce CCL2 production in vascular smooth
muscle cells [6, 11] and may represent one source of
CCL2 in subjects with hyperuricaemia.
This study also showed that elevated serum CCL2 was
associated with an increase in both circulating CD14+
monocytes and monocyte CD11b expression. CD14+
monocytes are one of the first leucocytes to migrate to
inflammatory sites during a gout attack. The observed increase in circulating blood monocytes associated with
elevated serum uric acid and CCL2 levels in the absence
of active inflammation may serve to provide a pool of
monocytes primed for rapid trafficking in response to inflammatory insults. Monocyte migration is mediated by
the up-regulation of adhesion molecules such as CD11b,
which binds intercellular adhesion molecule 1 (ICAM-1) on
endothelial cells to facilitate cell adhesion. CCL2 has been
shown previously to increase CD11b expression [10].
Consistent with this, CD11b was also up-regulated on
monocytes in this study. Interestingly, the most significant
increase in CD11b expression was observed on monocytes from acute gout subjects and was not directly
related to uric acid levels. Increased CD11b expression
1019
Rebecca Grainger et al.
FIG. 1 Hyperuricaemia elevates serum CCL2 and monocyte trafficking.
(A) Serum CCL2 levels for healthy controls (HC), subjects with asymptomatic hyperuricaemica (HUA), acute gout (AG) and
chronic gout (CG). (B) Serum CCL2 levels in subjects with normouricaemia (40.41 mM/6.9 mg/dl) and hyperuricaemia
(50.42 mM/7.1 mg/dl). (C) Percentage of CD14+ cells vs serum CCL2. (D) Percentage of CD14+ cells vs subject group.
(E) Mean fluorescent intensity (MFI) of monocyte CD11b vs serum CCL2. (F) MFI of monocyte CD11b vs subject group.
*P < 0.05, ***P < 0.001. Statistical analyses were by (A, D, F) Kruskal–Wallis test with Dunn’s post-test, (B) Mann–Whitney
test and (C, E) Spearman’s correlation.
on monocytes in acute gout subjects could indicate
heightened responsiveness of these cells to migration signals triggered by local inflammatory stimuli such as MSU
crystals. Up-regulation of CD11b would facilitate monocyte adhesion to the vascular wall and therefore the process of diapedesis. In this way, up-regulation of CD11b
could accelerate monocyte migration to sites of acute
MSU crystal deposition leading to rapid inflammation
such as that observed during an acute attack of gout.
Our findings may also have relevance to the elevated
cardiovascular risk observed in gout subjects [12–14].
Hyperuricaemia, elevated serum CCL2 levels and
increased circulating leucocyte numbers are commonly
associated with higher risk of cardiovascular disease
[15, 16]. The migration of monocytes to inflamed sites
on the arterial wall and their subsequent transformation
into foam cells plays a pivotal role in plaque formation in
1020
atherosclerosis [17, 18]. The presence of higher numbers
of circulating monocytes in subjects with hyperuricaemia
and gout could be a contributing factor in the higher risk of
cardiovascular disease in these individuals [19] where
increased expression of CD11b on monocytes could act
to facilitate monocyte adhesion and migration into the
vascular endothelium.
In conclusion, we show elevated levels of serum CCL2
in subjects with hyperuricaemia, with and without gout,
resulting in increased CD14+ monocytes in the circulation.
These findings indicate an important role for serum uric
acid in the modulation of monocyte trafficking and function and may be of particular importance in acute gout
attacks. Future prospective studies examining these inflammatory processes in the context of gout, hyperuricaemia and the potential relationship with atherosclerosis will
be necessary to confirm and expand the current findings.
www.rheumatology.oxfordjournals.org
Hyperuricaemia, CCL2 and monocyte trafficking in gout
.
Rheumatology key message
8 Ruggiero C, Cherubini A, Ble A et al. Uric acid and inflammatory markers. Eur Heart J 2006;27:1174–81.
Hyperuricaemia elevates serum CCL2 production
and monocyte trafficking in patients with gout.
9 Martin WJ, Grainger R, Harrison A, Harper JL. Differences
in MSU-induced superoxide responses by neutrophils
from gout subjects compared to healthy controls and a
role for environmental inflammatory cytokines and hyperuricemia in neutrophil function and survival. J Rheumatol
2010;37:1228–35.
Funding: This work was supported by Arthritis New
Zealand (grant: R184); Health Research Council of New
Zealand (grant: 06024); Henry Cotton Trust; and the
Wellington Medical Research Foundation (2011/203).
Disclosure statement: The authors have declared no
conflicts of interest.
References
1 Martin WJ, Walton M, Harper J. Resident macrophages
initiating and driving inflammation in a monosodium urate
monohydrate crystal-induced murine peritoneal model of
acute gout. Arthritis Rheum 2009;60:281–9.
2 Schiltz C, Liote F, Prudhommeaux F et al. Monosodium
urate monohydrate crystal-induced inflammation in vivo:
quantitative histomorphometric analysis of cellular events.
Arthritis Rheum 2002;46:1643–50.
3 Rollins BJ. Monocyte chemoattractant protein 1: a potential regulator of monocyte recruitment in inflammatory
disease. Mol Med Today 1996;2:198–204.
4 Serbina NV, Pamer EG. Monocyte emigration from bone
marrow during bacterial infection requires signals
mediated by chemokine receptor CCR2. Nat Immunol
2006;7:311–7.
5 Matsukawa A, Miyazaki S, Maeda T et al. Production and
regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced
arthritis in rabbits: roles of tumor necrosis factor alpha,
interleukin-1, and interleukin-8. Lab Invest 1998;78:
973–85.
6 Kanellis J, Watanabe S, Li JH et al. Uric acid stimulates
monocyte chemoattractant protein-1 production in vascular smooth muscle cells via mitogen-activated protein
kinase and cyclooxygenase-2. Hypertension 2003;41:
1287–93.
7 Wasilewska A, Tenderenda E, Taranta-Janusz K,
Tobolczyk J, Stypulkowska J. Markers of systemic inflammation in children with hyperuricemia. Acta Paediatr
2012;101:497–500.
www.rheumatology.oxfordjournals.org
10 Jiang Y, Beller DI, Frendl G, Graves DT. Monocyte
chemoattractant protein-1 regulates adhesion molecule
expression and cytokine production in human monocytes.
J Immunol 1992;148:2423–8.
11 Kang DH, Han L, Ouyang X et al. Uric acid causes vascular
smooth muscle cell proliferation by entering cells via a
functional urate transporter. Am J Nephrol 2005;25:
425–33.
12 Krishnan E, Baker JF, Furst DE, Schumacher HR. Gout
and the risk of acute myocardial infarction. Arthritis Rheum
2006;54:2688–96.
13 Choi HK, Curhan G. Independent impact of gout on mortality and risk for coronary heart disease. Circulation 2007;
116:894–900.
14 Cukurova S, Pamuk ON, Unlu E, Pamuk GE, Cakir N.
Subclinical atherosclerosis in gouty arthritis patients: a
comparative study. Rheumatol Int 2012;32:1769–73.
15 Kocaman SA, Sahinarslan A, Cemri M, Timurkaynak T,
Boyaci B, Cengel A. Independent relationship of serum
uric acid levels with leukocytes and coronary atherosclerotic burden. Nutr Metab Cardiovasc Dis 2009;19:
729–35.
16 Coll B, Alonso-Villaverde C, Joven J. Monocyte chemoattractant protein-1 and atherosclerosis: is there room
for an additional biomarker? Clin Chim Acta 2007;383:
21–9.
17 Nasir K, Guallar E, Navas-Acien A, Criqui MH, Lima JA.
Relationship of monocyte count and peripheral arterial
disease: results from the National Health and Nutrition
Examination Survey 1999–2002. Arterioscler Thromb Vasc
Biol 2005;25:1966–71.
18 Gerszten RE, Tager AM. The monocyte in atherosclerosis—should I stay or should I go now? N Engl J Med
2012;366:1734–6.
19 Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nat Rev Cardiol 2010;7:
77–86.
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