Innate Immunity in Triggering and Resolution of Acute

Gouty Inflammation David M. Rose, DVM, PhD, and Ru Liu-Bryan, PhD

Corresponding authorRu Liu-Bryan, PhD VA Medical Center, Department of Medicine, University of California, 3350 La Jolla Village Drive, 111K, San Diego, CA 92161, USA. E-mail: rliu@vapop.ucsd.edu

Current Rheumatology Reports 2006, 8:209–214Current Science Inc. ISSN 1523-3774Copyright © 2006 by Current Science Inc.

Gout has long been recognized as a disease of recurrent bouts of acute inflammation that undergo self-resolution. This inflammation is triggered by the body’s response to monoso-dium urate (MSU) crystals. In this paper, we focus on recent studies that describe how interactions of MSU crystals with the components of the innate immune system trigger acute gouty inflammation as well as mechanisms that are involved in the resolution of this inflammation. Specifically, we describe how toll-like receptors mediate the uptake of MSU crystals involved in the initiation and resolution of gouty inflammation. We also describe recent findings on the role of apoptotic clearance in the resolution of gouty inflammation. In addition, how therapies used to treat gout act on the innate immune system to inhibit MSU crystal-induced inflammation and promote the resolution of inflammation is discussed.

IntroductionIn gout, the deposition of monosodium urate (MSU) crystals in articular joints and bursal tissues can be either non-symptomatic or associated with the pathogenesis of acute, episodic, self-limiting joint inflammation. Over time, acute intermittent gouty inflammation can progress to chronic synovitis that may lead to bone and cartilage destruction [1]. This paper outlines the recent advances in the understanding of innate immunity in triggering and resolution of MSU crystal-induced inflammation.

Triggering of Acute Gouty InflammationThe pathologic hallmark of acute gout is neutrophil influx into the synovium and joint fluid (See Fig. 1) [1]. Because neutrophils are absent in the normal joint, the

interaction of MSU crystals with resident cells in the joint (principally synovial lining cells) is believed to be the pri-mary factor for acute neutrophil ingress and paroxysms of gouty inflammation episodes [1]. Neutrophil ingress into gouty joint appears to be driven synergistically by IL-1 and tumor necrosis factor alpha (TNFα) induced by MSU crystals, which in turn stimulate endothelium adhesion molecule expression, such as E-selectin [2], and by criti-cal chemotactic activities of CXCL8 and closely related chemokine ligands of CXCR2 [3,4]. Uptake of the MSU crystals by neutrophils entering the joint drives release of mediators, including calgranulins S100A8 and S100A9, which are quite abundant in the neutrophil cytosol, and the neutrophil activation by crystals and by soluble mediators promotes further amplification of the acute gouty inflammation [5].

MSU crystal-induced cell activationInert MSU crystals have a highly negatively charged, reactive surface that is known to avidly bind many plasma proteins [6]. Although such MSU crystals binding interactions are nonspecific through hydrogen and electrostatic bonds, MSU does associate preferentially with selected proteins in complex mixtures, as illustrated by apoB and complement protein binding in crystals exposed to human plasma [7].

Monosodium urate crystals are shown to activate cells through phagocytosis that can be greatly enhanced by opsonization by IgG or complement components [8], or through direct interactions with cell surface receptors, such as leukocyte integrin CD11b/CD18 (CR3) and the Fc receptor CD16 [9]. The interaction of MSU crystals with cells leads to rapid activation of a variety of stress kinase signaling pathways typically used by ligands recognized in a “cognate” fashion, such as immunoglobulin, with exam-ples of the signaling players including phosphatidylinositol 3-kinase (PI3K), Src, Syk, Pyk2, and MAP kinases [10]. One important outcome of the MSU crystal stimula-tion of cells is the induction of expression of NF-κB and AP-1 transcription factor-dependent genes including a broad array of inflammatory mediators, such as cyclooxy-genase 2, TNFα, IL-1 and IL-6, and the CXCR2-binding chemokines CXCL8 (IL-8) and CXCL1 (GROα) [2,11,12].

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What is the principal mechanism by which the inert MSU crystals initially transduce signaling at the cell mem-brane to produce a cascade of cell activation? The answer to this question has remained elusive. Recent studies implicate the important role of innate immune recogni-tion of naked MSU crystals in gouty inflammation.

Innate immune recognition of the naked MSU crystal surface as a pathogen-associated molecular pattern Innate immunity provides a first line of defense against infection through primitive responses that are non-spe-cific and broad in spectrum [13]. Unlike adaptive immune responses, stereotypic innate immune “early induced” responses do not directly induce immunologic memory or lasting protective immunity, consistent with the nature of recurrent acute gouty attack. Thus, innate rather than adap-tive immunity is critical in acute gouty inflammation.

In a series of recent studies, we have implicated innate immune inflammatory responses to the naked MSU crys-tal surface in the pathogenesis of acute gout. Previous studies have shown that MSU crystals activate the classic and alternative complement pathways in vitro, leading to elaboration of C5a, which acts as a leukocyte chemoat-tractant through C5 cleavage catalyzed by the MSU crystal surface [14,15]. Using the C6-deficient rabbit, we revealed a substantial role of the local assembly of the C5b-C9 membrane attack complex (MAC) in acute inflammation in experimental gouty arthritis [16]. Specifically, C6 defi-ciency was associated with less inflammatory response to MSU crystals in vivo. Moreover, we recently demonstrated that MSU crystals activate cells of mesenchymal origin in a manner that requires canonical signal transduction through toll-like receptor 2 (TLR2), a pattern recognition receptor that plays a central role in innate immune recog-nition of components of numerous microbial pathogens, including gram-positive bacteria and lipopeptides [17]. In addition, we determined recognition of naked MSU crystals by TLR2 and TLR4 to be critical for the capacities of inert MSU crystals to induce macrophage phagocytosis of crystals and expression of pro-inflammatory cytokines in vitro (demonstrated under serum-free conditions) and acute inflammatory responses in vivo [18••]. In this con-text, the intracellular adaptor protein for both TLR2 and TLR4, myeloid differentiation factor 88 (MyD88), plays a key role in transducing TLR2 and TLR4 responses to MSU crystals. Furthermore, we recently also found that CD14, another pattern recognition receptor that has been shown to functionally interact with TLR2 and TLR4 [19,20], directly binds naked MSU crystals in vitro and mediates MSU crystal-induced expression of pro-inflammatory cyto-kines in vitro (demonstrated under serum-free conditions) and acute inflammatory responses in vivo [21]. These findings suggest that MSU crystals may act as a “danger” motif analogous to pathogen-associated molecular pattern (PAMP) and that innate immune recognition of naked

MSU crystals by CD14 and specific TLRs is a determinant of the inflammatory potential of MSU crystals.

Recognition of ligands by TLRs facilitates TLR dimer-ization, which triggers activation of signaling pathways that eventually leads to activation of NF-κB. In this con-text, TLR2 forms heterodimers with TLR1 and TLR6, but TLR4 forms homodimers [13]. The roles in recognizing MSU crystals of distinct TLR2 heterodimers and TLR4 accessory molecule MD-2 are not known yet. In addition, how CD14 cooperates with TLR2 and TLR4 in mediating MSU crystal-induced inflammation remains to be stud-ied. Furthermore, since MSU crystals nonspecifically bind many plasma proteins and also engage integrins such as CD11b/CD18, it is not surprising that MSU crystals have the potential to nonspecifically engage CD14 and TLR complexes with other proteins that synergistically recognize PAMPs. Activation of CD11b/CD18 by certain bacterial products has been shown to mediate through a pathway involving CD14 and TLR2 in leukocytes [22]. Thus, MSU crystals have the potential to engage the signaling complexes including CD14, TLR2, TLR4, and CD11b/CD18 that modulate responsiveness to MSU crystals in leukocytes.

Since CD14, TLR2, and TLR4 are expressed in synovial lining cells [22,23,24], MSU crystals may activate syno-vial lining cells through direct engagement with these pattern recognition receptors. In addition, these recep-tors, which are also expressed in neutrophils [25,26], may be involved in mediating activation of neutrophils recruited in the joint to ampify synovitis once the acute gouty inflammatory process has been initiated.

Synovial MSU crystals deposited in microscopic tophi have been observed to be tightly packed in a core contained by a protein-rich wall that includes fibrinogen [27]. As such, our recent findings on recognition of naked MSU crystals by pattern recognition receptors, and the known association of acute gouty attacks with rapid rises and falls in serum urate, suggest that gouty inflammation is triggered by de novo formation of uncoated MSU crys-tals in the joint or release from synovial tophi of MSU crystals liberated from coating proteins by factors includ-ing partial crystal dissolution.

Very recently, the intracellular NALP3/cryopyrin inflammasome has been implicated as playing a pivotal role in acute gouty inflammation [28••]. The inflamma-some is formed by members of the NALP protein family, such as NALP1, NALP2, or NALP3. Upon activation, the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) connects the NALPs with cas-pase-1, which cleavages the precursor pro-IL-1β (p35) to the active mature IL-1β protein (p17) [29]. NALP3/cryopyrin is mainly expressed in leukocytes [29]. MSU crystal-induced IL-1β activation is impaired in macrophages from mice deficient in NALP3/cryopyrin, ASC, or caspase-1 in vitro [28••]. In addition, impaired neutrophil influx is found in an in vivo peritonitis model in inflammasome-deficient

Innate Immunity in Triggering and Resolution of Acute Gouty Inflammation Rose and Liu-Bryan 211

mice in response to MSU crystals [28••]. Like TLRs, NALPs also contain a leucine-rich repeat (LRR) domain. Muramyl dipeptide (MDP), a degradation product of peptidoglycan, activates a NALP3/cryopyrin inflammasome probably through the LRR domain [30], suggesting a fundamental role of intracellular innate immune recognition of PAMPs by NALPs. The mechanism by which endocytosed MSU crystals are sensed by the NALP3/cryopyrin inflamma-some is currently unknown. Whether MSU crystals are directly recognized by NALP3/cryopyrin or through inter-mediary protein(s) is not clear.

Taken as a whole, innate immune recognition of MSU crystals on the cell surface by CD14, TLR2, and TLR4 and intracellulalry by NALP3/cryopyrin, promotes MSU crystal-induced inflammation.

Resolution of Gouty InflammationThe acute gouty inflammatory response is typically self-limiting within 7 to 10 days. Increasingly, a bet-ter understanding of the “self-limiting” nature of acute gouty inflammation is emerging. The resolution of gouty inflammation follows the course of resolution of acute inflammation in general; namely, [31] safe disposal of the initiating cause of the inflammation (in the case of gout, removal and/or neutralization of MSU crystals), [32] clearance of apoptotic cells and cel-lular debris, and [33•] a switch in the milieu of soluble mediators from pro-inflammatory to anti-inflamma-tory (Fig. 1). An increased understanding of how each of these processes contributes to the resolution of gout will lead to a better understanding of why some forms of gout are chronic and will also identify potential therapeutic targets whose manipulation may speed in the recovery of gouty attacks.

Disposal and neutralization of MSU crystalsThe direct removal of free MSU crystals and/or coat-ing crystals to mask their pro-inflammatory properties contributes to the resolution of gouty inflammation. However, this is not the “fin de l’histoire” in the resolu-tion of gout as it is well established that MSU crystals can be found in asymptomatic joints [31]. Macrophages can avidly take up MSU crystals. This uptake has been shown to be dependent on the state of differentiation of the macrophages [32,33•]. Monocytes/macrophages are heterogeneous in form and function [34]. Work by Yagnik et al. [32] using murine monocyte/macrophage cell lines in various states of differentiation has shown that more mature macrophages take up greater amounts of MSU crystals. This trend has also been observed in primary macrophages isolated from humans [33•]. Thus, although more immature macrophages found at the site of early gout may contribute to the initiation of inflam-mation, macrophages present later contribute to the resolution through the clearance of MSU crystals.

The molecular mechanisms responsible for effective clearance of MSU crystals involve numerous cell surface receptors capable of engaging crystals. The most promi-nent MSU recognition receptors include the integrins, Fc-receptors, and pattern recognition receptors [9,18••]. The receptors used to engage MSU crystals may affect not only crystal uptake but also how the macrophage responds to crystals (discussed below). For example, we have recently demonstrated that CD14, expressed highly on immature macrophages, is not required for MSU crystal uptake but is important in triggering pro-inflam-matory mediator release after crystal engagement [18••]. In contrast, TLR2 and TLR4 directly participate in mac-rophage phagocytosis of MSU crystals and also mediate MSU crystal-induced TGFβ expression [18••].

The materials coating naked MSU crystals affect how the crystals engage macrophages and the subsequent response triggered by the crystals. Given the physical-chemical nature of MSU crystals, they can non-covalently bind a number of biologic molecules, including immu-noglobulins (Igs), complement factors, extracellular matrix molecules, and lipoproteins [6,14,35]. Crystals coated with Igs, fibronectin, and complement fragments are known to trigger pro-inflammatory mediator release [9,16]. On the other hand, crystals coated with lipopro-tein containing apolipoproteins B and E are relatively inert with respect to neutrophil activation [36]. Conse-quently, a dynamic change in MSU crystal coating during the course of a gouty attack may influence the extent and duration of the inflammation.

Clearance of apoptotic cells and the resolution of gouty inflammationThe efficient and rapid clearance of apoptotic neutrophils by macrophages and non-professional phagocytic cells contrib-utes to the resolution of inflammation. Indeed, in several animal models of inflammation, defects in apoptotic cell clearance lead to over exuberant and chronic inflammation as well as predisposing to the development of autoimmunity [37,38]. Clearance of apoptotic neutrophils contributes to resolution of inflammation in two major ways. First, the removal of spent neutrophils prior to secondary necrosis prevents the release of injurious intracellular molecules contained within the neutrophil, which can exacerbate the inflammatory response [37]. Second, the phagocytosis of apoptotic cells triggers the release of anti-inflammatory mediators such as TGFβ, IL-10, and prostaglandins [39]. How macrophages recognize and engulf apoptotic cells has been extensively reviewed elsewhere [37].

Direct evidence for the role of apoptotic cell clearance in the resolution of gouty inflammation has been lacking. However, in other models of inflammation, the recognition and clearance of apoptotic cells results in accelerated resolu-tion of inflammation [40]. Thus, it is intriguing to speculate that apoptotic neutrophil clearance by macrophages may contribute to the resolution of gouty inflammation.

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Anti-inflammatory mediators and the resolution of gouty inflammationOver the course of gouty inflammation there is a switch in mediator profile from pro-inflammatory, present early in inflammation, to anti-inflammatory, which arises later in the response. These anti-inflammatory media-tors such as TGFβ, IL-10, PGD2, and 15deoxy-PGJ2 (ligand of PPARγ) are thought to play an important role in orchestrating the resolution of gouty inflamma-tion [41–45]. Macrophages are a major source of these anti-inflammatory mediators, whose release is triggered by the uptake of MSU crystals as well as apoptotic cells [39,41]. Additional evidence for the role of these media-tors in the resolution phase comes from animal studies in which exogenous introduction of these mediators dampens MSU crystal-induced inflammation [42–45].

Anti-inflammatory mediators contribute to the resolution of gouty inflammation through several mechanisms. TGFβ released by MSU crystal-stimu-lated macrophages suppresses endothelial E selectin

expression, which in turn can lessen further neutrophil recruitment [41]. PGD2 promotes neutrophil apoptosis and in conjunction with other lipid mediators can stimulate macrophage uptake of apoptotic cells [46]. Furthermore, in addition to its role in suppressing leukocyte activation, TGFβ also promotes fibroblast dif-ferentiation and fibrosis, which can contribute to wound repair and stabilization of MSU tophi.

Pharmacologic intervention in the innate immune system during gouty inflammation Many of the therapeutic “gold-standards” in gout treatment may act, at least in part, by modulating the innate immune system. Nonsteroidal anti-inflammatory drugs (NSAIDs), both nonspecific cyclooxygenase (COX)-1 and COX-2 and more selective COX-2 inhibitors, are a first line therapy for acute gout [1]. No doubt these compounds act to inhibit MSU crystal induced pro-inflammatory lipid mediator release, but they may also act in the generation of anti-inflammatory lip-ids such as resolvins and protectins, which actively promote

Figure 1. Scheme for the initiation and resolution of monosodium urate (MSU) crystal-induced gouty inflammation. During the acute phase (left panel), MSU crystals interact with pattern recognition receptors (PRRs) expressed on synoviocytes within the joint, resulting in the release of pro-inflammatory cytokines and chemokines. This results in the recruitment of circulating leukocytes and the additional release of inflammatory mediators. During the resolution phase of gouty inflammation (right panel), MSU crystals are disposed of through macrophage engulfment or neutralized by coating. In addition, the clearance of apoptotic cells as well as crystal triggers the generation of anti-inflammatory mediators. TGFβ⎯transforming growth factor β.

Innate Immunity in Triggering and Resolution of Acute Gouty Inflammation Rose and Liu-Bryan 213

the resolution of inflammation [47]. NSAIDs may promote the accumulation of early precursors of these anti-inflam-matory lipids, which can be processed through enzymatic and non-enzymatic mechanisms. Synthetic corticosteroids or induction of endogenous glucocorticoids with adrenocor-ticotropic hormone (ACTH) are also used in the treatment of gout [1]. These compounds have profound anti-inflam-matory activity, which is probably mediated by multiple mechanisms. Given the evidence for the involvement of TLRs in MSU crystal-induced inflammation, recent studies demonstrating antagonism of TLR-triggered expression of inflammatory response genes by glucocorticoids may be one major mechanism mediating the beneficial effect of these compounds in gout [48]. Yet another mechanism may be through glucocorticoid’s capacity to stimulate macrophage phagocytosis of apoptotic neutrophils [49]. Colchicine is another gout treatment that has broad anti-inflammatory effects. Central to colchicine’s mode of action is inhibi-tion of microtubule assembly in cells. Recently, colchicines have been shown to inhibit MSU crystal-induced NALP3/cryopyrin inflammasome activation and IL-1β generation [28••]. It is speculated that colchicines may potentially pre-vent MSU crystal endocytosis and/or presentation of MSU crystal to the NALP3/cryopyrin inflammasome complex.

ConclusionsThere is abundant evidence that the innate immune system is critical for the initiation and resolution of MSU crystal-mediated gouty inflammation. MSU crys-tals, coated and potentially uncoated, are recognized by many of the same receptors of the innate immune system used to engage pathogens, including TLRs, integrins, and Fc-receptors. Recognition through these receptors leads to pro-inflammatory mediator genera-tion and recruitment of leukocytes. In acute gout, this pro-inflammatory phase is followed-up by self-limiting resolution. This resolution is mediated by safe disposal of MSU crystals, clearance of apoptotic leukocytes and cellular debris, and a switch in mediator profile from pro-inflammatory to anti-inflammatory. Furthermore, many of the therapeutics used in the treatment of gout may have their primary action in modulation of the innate immune system to inhibit initiation of gouty inflammation or speed its resolution.

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