In acute gouty arthritis, monosodium urate monohydrate (MSU) crystal deposits in joints trigger inflammation; this process is largely dependent on neutrophil recruitment and proinflammatory mediator generation by resident cells in the joint and by recruited phagocytes (1, 2). The capacity of MSU crystals to engage plasma membrane and intracellular innate immune pattern–recognition receptors (Toll-like receptor 2 [TLR-2] and TLR-4, and the NALP3 inflammasome, respectively) modulates crystal inflammatory potential (3–5), as do changes in plasma proteins and complement components that directly interact with MSU crystals (6–8). Despite the remarkable capacity of MSU crystals to induce multiple mediators of inflammation (1, 8), self-limitation of the acute gouty attack, typically culminating in spontaneous resolution within 1–2 weeks, has been recognized for more than 2 millenia (8).
Natural mechanisms limiting acute gouty inflammation include antiinflammatory disposal of MSU crystals through phagocytosis by mature macrophages, associated with generation of antiinflammatory transforming growth factor β (TGFβ) in vitro and in vivo (6, 9, 10). For example, cells isolated from resolving MSU crystal–induced skin blisters elaborate increased levels of TGFβ (9). MSU crystals also induce the release of other antiinflammatory mediators such as prostaglandin D2 (PGD2) (11), and MSU crystals up-regulate the level of the inflammation-limiting receptor peroxisome proliferator–activated receptor γ in mononuclear phagocytes (12).
In certain forms of acute inflammation, the uptake of apoptotic neutrophils by macrophages has been implicated in spontaneous resolution (13–15). Physiologic disposal of the intact apoptotic neutrophil corpse by the macrophage suppresses the release of cytotoxic granule contents from necrotic neutrophils that can promote tissue damage (16). Furthermore, up-regulated expression of antiinflammatory mediators including TGFβ, interleukin-10, and PGE2 and down-regulated expression of proinflammatory cytokines including tumor necrosis factor α (TNFα) are among the functionally significant changes stimulated in macrophages by phagocytosis of the apoptotic (but not the necrotic) neutrophil corpse (17, 18).
Phagocytosis of apoptotic cells by macrophages is mediated by changes on the apoptotic cell surface recognized by macrophage plasma membrane proteins, including scavenger receptors and integrins (19). Alterations on apoptotic cells that are functionally involved in recognition by the macrophage include exposure of phosphatidylserine (19). Certain soluble factors provide a “bridge” linking the apoptotic cell with the macrophage (20). The first events in the effective removal process appear to involve tethering of the apoptotic cell to the macrophage surface, followed by a “tickling” event that signals the macrophage to engulf the apoptotic cell and release antiinflammatory mediators (21).
Recently, the multifunctional molecule transglutaminase 2 (TG2) has been elucidated to be a major mediator of apoptotic thymocyte uptake by cultured macrophages (22). Impaired apoptotic cell engulfment by macrophages from TG2−/− mice is associated with dysregulated proinflammatory cytokine production in vitro and in vivo and prolonged acute lead nitrate–induced hepatic inflammation (23). Furthermore, splenomegaly, autoantibodies, and immune complex glomerulonephritis develop in aging TG2−/− mice in vivo, which is consistent with autoimmunity (22).
In the current study, we tested the hypothesis that TG2 modulates acute, neutrophil-driven, self-limiting gout-like inflammation in murine peritoneum by regulating apoptotic neutrophil clearance. We used neutrophil accumulation at the MSU crystal–induced inflammation locus as a standard benchmark (1, 8). Significantly, provision of exogenous active TGFβ corrects the apoptotic leukocyte uptake defect in TG2−/− macrophages in vitro, and TG2 catalyzes transamidation that promotes the maturation of TGFβ from a latent to an active protein (24, 25). Reciprocal to TG2 transamidation activity in the calcium-bound state is the capacity of TG2 to bind purine nucleotides and thereby exert conformation-dependent effects on cell differentiation as well as signaling effects via GTPase and ATPase activities (26). Therefore, we also investigated TG2 structure–function requirements for macrophage clearance of apoptotic leukocytes.
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The capacity of MSU crystals to induce a plethora of mediators of inflammation is clearly subject to natural restraining mechanisms that limit inflammation and tissue damage in gouty arthritis. Here, we observed that TG2, in large part by functioning to promote antiinflammatory apoptotic neutrophil engulfment in vivo, limited acute MSU crystal–induced inflammation, using neutrophil accumulation as a standard benchmark (1, 8). Specifically, delayed clearance of apoptotic neutrophils was demonstrated in TG2−/− mice during MSU crystal–induced peritonitis. In contrast, TG2 clearly was not required for the initiation phase of MSU crystal–induced neutrophilic inflammation in the peritoneum, which included a burst in KC/CXCL1 expression and rapid influx of neutrophils during the first 4 hours after MSU crystal injection.
In the last 24 hours of MSU crystal–induced peritonitis, TG2−/− mice had significantly more neutrophils present in the peritonea. Although most of these neutrophils were apoptotic, many nonapoptotic neutrophils were also present. This may have been the result of delayed apoptosis of the TG2−/− neutrophils or additional recruitment of neutrophils. We favor the latter explanation, because the presence of apoptotic neutrophils that are not rapidly cleared can lead to secondary necrosis and cell lysis, releasing proinflammatory mediators (16). In addition, although engulfment of apoptotic cells triggers antiinflammatory mediator release from macrophages, engulfment of late apoptotic or necrotic cells triggers proinflammatory mediator release (36). The mechanisms involved in both scenarios may have contributed to further neutrophil recruitment. However, TG2 did not appear to limit gouty inflammation by promoting MSU crystal clearance, because macrophages from TG2−/− mice had no defect in MSU crystal uptake in vitro.
This study identified externalization of TG2 as being central to promoting the capacity of TGFβ activation to trigger apoptotic cell engulfment by macrophages. TG2-catalyzed transamidation promotes the maturation of several polypeptides, including not only TGFβ (24, 25) but also phospholipase A2 (37). An unexpected finding of this study was that the capacity of recombinant exogenous TG2 to facilitate TGFβ promotion of apoptotic cell ingestion did not require the transamidation activity of TG2. Moreover, induction of active TGFβ was essential for the capacity of transamidation catalytic–site dead TG2 to promote apoptotic leukocyte uptake by macrophages. However, we do not exclude the potential for TG2 also to promote spontaneous resolution of acute neutrophilic inflammation in vivo, partly through other TG2 functions dependent on transamidation activity, such as modulation of extracellular matrix remodeling (26).
In this study, differential adenine and guanine nucleotide binding to extracellular TG2 was observed to critically regulate the capacity of TG2 to enhance phagocytosis of apoptotic leukocytes by macrophages. TG2 is constitutively latent in the cytosol in a GDP-bound state, and guanine nucleotide binding to TG2 is clearly essential for not only TG2 GTPase/ATPase signaling activities but also placement of TG2 in conformation states that can facilitate physical interactions with several α integrin cytosolic tails and drive integrin-mediated effects on p38 mitogen-activated protein kinase signaling and cell differentiation (28). In this context, TG2-induced clustering of cell-surface integrins alters the actin cytoskeleton via RhoA activation, and this is independent of both transamidation and GTP/ATPase activity of TG2 (38). However, such an effect is unlikely to be the mechanism responsible for TG2-mediated engulfment of apoptotic cells, because RhoA activation has been demonstrated to antagonize apoptotic cell engulfment (39).
Because external TG2 enhanced phagocytosis of apoptotic cells by macrophages in a manner dependent on activation of TGFβ but not the transamidation catalytic activity of TG2, we speculate that TG2 could function as part of a membrane complex bringing together TGFβ and other factors that modulate TGFβ activation. Intriguingly, there was no difference between TG2−/− and TG2+/+ mice in the levels of active TGFβ in the peritoneal lavage fluid during the course of MSU crystal–induced peritonitis. Nevertheless, TGFβ appears to be a major mediator driving the resolution of gouty inflammation (1, 9, 40). We have not yet determined whether TG2 directly alters the capacity of TGFβ to associate with cells and/or extracellular matrix. However, the capacity of exogenous active TGFβ to rescue the apoptotic leukocyte uptake defect in TG2-deficient macrophages argues that TG2 does function primarily to mediate signaling by activated TGFβ. Our results collectively suggest that the transamidation-independent effects of TG2 on apoptotic leukocyte uptake appear more likely to be mediated by cooperativity of TG2 in recognition events on the macrophage cell surface that drive uptake of the already tethered apoptotic leukocyte (Figure 5). It will be of interest to determine whether apoptotic leukocyte ingestion is mediated by alternative nonenzymatic TG2 functions such as fibronectin binding (26, 35) and not solely by the effects of differential purine nucleotide binding by TG2 (34, 41, 42).
The accumulation of extracellular purine nucleotides, including ATP, via active secretion by injured cells or from cell necrosis provides “danger signals” indicative of tissue stress (43, 44). For example, free ATP at high concentrations can stimulate inflammation in part by promoting NALP3 inflammasome activation (45). However, ATP and other purine nucleotides, and their derivatives such as adenosine, also can initiate antiinflammatory processes (43, 44). Suppression of TNFα production through purinergic receptor signaling is one example (46). Our results suggest additional mechanisms by which extracellular nucleotides may modify inflammation through the regulation of TG2-mediated clearance of apoptotic cells by macrophages. It is noteworthy that guanine nucleotide binding to TG2 stabilizes TG2 by reducing sensitivity of TG2 to proteolytic degradation (34). Moreover, our results suggest that the exchange of specific purine nucleotides on extracellular TG2, or TG2 GTPase activity hydrolyzing GTP to GDP, can markedly alter the inflammation-regulatory activity of TG2.
Limitations of this study include the artificiality of acute injection of free synthetic MSU crystals as a model (2) and the lack of direct assessment of the pathology of inflammation occurring at the serosal surface in the murine MSU crystal–induced peritonitis model. Consequently, we did not evaluate the role of TG2 in the resolution of acute tissue edema or in the process of extracellular matrix remodeling in inflamed tissues. A substantial limitation of this study is a species difference in the expression of the enzyme uricase, which catalyzes uric acid degradation and thereby drives MSU crystal dissolution. Mice express functional uricase, whereas the enzyme has been mutationally silenced in humans (47). We speculate that the ultimate resolution of MSU crystal–induced inflammation in both TG2−/− and TG2+/+ mice was reflective of urate crystal lysis that would not have been as accelerated in humans. We do not know whether the concomitant presence of uricase and MSU crystals also regulated the clearance of apoptotic cells by hydrogen peroxide generation or potential effects of oxidative stress modulating TGFβ binding and signaling by macrophages.
We conclude that insufficient TG2 expression and externalization, and altered regulation of TG2 function by differential purine nucleotide binding, could contribute to enhanced neutrophil-driven inflammation in acute and chronic gout. Hyperuricemia in some patients with gout is driven by increased purine nucleotide turnover (48), and it is possible that such a state impacts on the course of inflammation in gout partly by regulation of TG2 function. It also will be of interest to discern whether differences in TG2 expression account for clinical variability in gouty inflammation. Finally, our results suggest that local delivery of specific forms of nucleotide-bound TG2 could provide a novel approach to limiting neutrophil-driven inflammation in refractory gouty arthritis.