Elsevier

Clinical Immunology

Volume 142, Issue 3, March 2012, Pages 362-372
Clinical Immunology

Alternative activation in systemic juvenile idiopathic arthritis monocytes

https://doi.org/10.1016/j.clim.2011.12.008Get rights and content

Abstract

Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14++CD16 and CD14+CD16+ monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1β after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.

Highlights

► Monocytes are implicated in SJIA pathogenesis based on clinical and mechanistic data. ► We characterized the activation phenotype of SJIA monocytes during flare and quiescence. ► SJIA monocytes, especially during flare, show a mixed M1/M2-like activation phenotype. ► During quiescence SJIA monocytes tend to exhibit a M2-like phenotype. ► Circulating monocytes may play a pro- but also an anti-inflammatory role in SJIA.

Introduction

Systemic juvenile idiopathic arthritis (SJIA) is a rheumatic condition characterized by remitting fever, transient rash, and relapsing arthritis. Although SJIA has been considered an autoimmune disease, the paucity of specific autoantibodies or pre-disposing major histocompatibility complex (MHC) alleles suggest an auto-inflammatory nature, in contrast to the non-systemic subtypes of JIA [1], [2]. Consistent with this, SJIA clinical features include thrombocytosis, granulocytosis, and the up-regulation of acute-phase proteins [3]. The fact that macrophage activation syndrome (MAS) and amyloidosis are complications of SJIA further supports the involvement of the innate immune system and monocyte/macrophages in particular [4].

Mechanistic studies of SJIA also point to activation of the monocyte lineage. Transcriptome analyses of PBMC reflect likely stimulation of innate immune pathways in monocytes at flare [5], [6], [7], [8]. Pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, IL-18, and TNFα, are elevated in the serum and/or synovial fluid of SJIA patients with active disease [9], [10]; activated monocytes are potential sources of these cytokines. Importantly, biologic therapies targeting IL-1β and IL-6 ameliorate disease, at least in subsets of patients [6], [11], [12], [13], [14]. High serum concentrations of calcium-binding proteins S100A8, S100A9, and S100A12 correlate with disease activity; these proteins also indicate the activation of monocytes and/or granulocytes [3]. Thus, numerous lines of evidence suggest that activation of monocytes may play an important role in SJIA pathophysiology.

Monocytes can be induced into specific activation phenotypes, depending on the microenvironment. Activation by IFN-γ and LPS results in “classical” or M1 monocytes/macrophages and is strongly linked with T helper 1 (Th1) polarization, whereas activation by type 2 cytokines IL-4 and IL-13 results in “alternative” or M2 monocyte/macrophages, which are associated with Th2 polarization [15]. M1 and M2 phenotypes likely represent the extremes of a continuum of monocyte activation states. M1 polarization confers a pro-inflammatory phenotype, associated with elevated production of IL-1β, TNFα, and IL-6 [16] and enhanced killing of intracellular pathogens [17]. M2 polarization is associated with regulatory and inhibitory functions, counterbalancing proinflammatory mechanisms, and M2 monocytes provide a niche for chronic infection by some pathogens, such as parasites and certain bacteria [17], [18]. Although much remains to be clarified regarding the alternative activation of monocytes/macrophages [19], the M1/M2 paradigm already has been used to characterize monocyte/macrophage activation in situations other than Th1/Th2 dominance. Other M2-associated conditions include endotoxin tolerance [20], obesity and insulin resistance, atherogenesis and tumor-associated macrophages [21]. Notably, cells with a mixed M1/M2 phenotype, such as monocytic myeloid-derived suppressor cells (MDSC) have been described in cancer, infectious diseases, and autoimmune diseases, and are potent inhibitors of immune responses, both adaptive as well as innate responses [22], [23].

Despite the pro-inflammatory nature of the SJIA cytokine environment, some markers associated with an M2 profile are highly expressed in SJIA. These include IL-1Ra [24], IL-10 [7], MS4A4A [8], surface [25] and soluble CD163 [26] and serum heme oxygenase-1 [27], [28]. These results suggest that monocytes may play both pro- and anti-inflammatory roles in SJIA.

Human monocytes also can be divided into two major subsets, based on cell surface expression of CD14 and CD16 [29], [30]. The CD14++CD16 subset (CD14+ subset, hereafter) is predominant (~ 85% of monocytes) in the absence of infection. This subset expresses higher levels of molecules with potential antimicrobial function [31] and of receptors involved in endocytosis [32]. The CD14+CD16+ subset (CD16+ subset) is expanded in a range of inflammatory conditions, including Crohn's disease, rheumatoid arthritis, asthma, sarcoidosis [33], Kawasaki disease [34] and hemophagocytic syndrome [35]. This subset is associated with production of pro-inflammatory cytokines and has elevated Fc-mediated phagocytosis [33]. The CD14+ and CD16+ subsets appear to have a common myeloid progenitor [32], and may even have a precursor (CD14+)/product (CD16+) relationship [30]. However, the circulating subsets express a distinct profile of adhesion molecules, chemokine receptors and cell activation markers, with CD16+ expressing more macrophage- and dendritic cell-related markers [32].

Previous work from our group demonstrated expansion of the monocyte lineage in active SJIA [36]. Both CD14+ and CD16+ subsets contribute to this increase and proportions observed in normal individuals are maintained. Further, both at flare and quiescence, we observed increased expression of the CD14 and CD16 surface markers in their respective subsets, a sign of monocyte activation [36]. In addition, increased CD14 expression may be linked to increased SJIA monocyte resistance to apoptosis [37], another phenotype we have described [38].

To further explore the role of monocytes in SJIA pathogenesis, we sought to determine the activation profile of circulating CD14+ and CD16+ monocyte subsets in SJIA at different disease stages. We analyzed gene expression, surface phenotype and cytokine production. Our results suggest a novel activation state of circulating SJIA monocytes that may shed light on the role that these cells play in disease.

Section snippets

Subject population and clinical data collection

The Institutional Review Board of Stanford University approved this study. SJIA patients were followed at the Pediatric Rheumatology Clinic at Lucile Packard Children's Hospital, and were enrolled after consent. All SJIA study subjects met the International League of Associations for Rheumatology criteria for JIA [39]. Comprehensive clinical information was collected at each SJIA patient visit [36]. Clinical status was assigned according to our scoring system to grade severity of systemic

Activation phenotype of SJIA PBMC by gene expression

In a microarray screen of paired flare/quiescence PBMC samples from 14 SJIA subjects, we, like others [5], [6], [7], [8], found that gene expression patterns correlate with disease activity (Fig. 1A). In the “flare signature,” we observed mRNAs linked to M1 and M2 phenotypes [15], [20], [47], [48], [49], indicating that the PBMCs either included a mixture of M1 and M2 cells or included cells with a mixed M1/M2 phenotype. Follow-up analysis by RT-qPCR showed that, for ~ 40% of M1- and

Discussion

Multiple lines of evidence implicate monocytes in SJIA pathogenesis. However, the specific role of monocytes in SJIA is still unknown, and relatively few studies have examined monocyte phenotype in depth. Using the framework of M1 and M2 phenotypes, we analyzed the activation state of SJIA monocyte subsets during flare and quiescence. Our analysis highlights a mixed M1/M2-type activation program, especially during disease flare. Our results showing expression of M2-related genes and surface

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

We would like to thank the patients, their families and the medical staff of Pediatric Rheumatology and Pediatric Endocrinology Clinics at Lucile Packard Hospital for Children. We would also like to thank Stanley Cohen for advice, and Sidthi Sanjanwala and Kevin Phillips for help with cytokine experiments. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Arthritis and Musculoskeletal and Skin Diseases or

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