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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Monosodium urate monohydrate (MSU) crystals have remarkable inflammatory potential. However, gouty inflammation is spontaneously self-limited, an occurrence recognized since antiquity. Gouty synovitis is driven and sustained by neutrophil influx. Importantly, macrophage phagocytosis of apoptotic (but not necrotic) neutrophils is antiinflammatory. Therefore, we tested the hypothesis that efficient clearance of apoptotic neutrophils by macrophages is one of the factors that restrains the progression of gouty inflammation. Macrophage expression of transglutaminase 2 (TG2), a multifunctional protein with reciprocally regulated transamidation and purine nucleotide–binding activities, promotes apoptotic leukocyte uptake. In this study, we tested the specific role of macrophage TG2 expression in MSU crystal–induced inflammation.

Methods

We studied MSU crystal–induced peritonitis in TG2−/− and congenic TG2+/+ mice. We also studied the effects of TG2 on apoptotic cell uptake by cultured macrophages.

Results

TG2−/− mice demonstrated more progressive neutrophilic accumulation than did TG2+/+ mice, which was associated with delayed clearance of apoptotic neutrophils during MSU crystal–induced peritonitis. We observed defective phagocytosis of apoptotic leukocytes by TG2−/− peritoneal macrophages, which was corrected by soluble extracellular TG2. Transamidation catalytic activity of TG2 was not required to mediate macrophage uptake of apoptotic leukocytes. In contrast, the TG2 nucleotide binding site residue K173 was critical for this TG2 function. TG2 bound to GDP, ADP, or ATP (but not to GTP) rescued defective apoptotic leukocyte uptake by TG2−/− macrophages.

Conclusion

Enhancement of apoptotic neutrophil uptake by macrophage-derived TG2 restrains gout-like neutrophilic peritoneal inflammation. Differential binding of TG2 by purine nucleotides may contribute to clinical variability in the extent and duration of gouty inflammation.

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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Reagents.

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. MSU crystals were prepared and verified to be free of detectable endotoxin, as previously described (4).

Mice.

TG2−/− mice, originally provided by Dr. Robert Graham (Victor Chang Research Institute, Darlinghurst, New South Wales, Australia) (27), were on a hybrid (50:50) C57BL/6J × 129 background. TG2−/− and TG2+/+ mice on this background were backcrossed onto the C57BL/6J background for an additional 5 generations prior to study, and we compared congenic TG2−/− and TG2+/+ mice in the described studies. All animal procedures were humane and institutionally approved at the peer review committee level.

MSU crystal–induced murine peritonitis.

Mice of both sexes, between 8–18 weeks of age, were injected intraperitoneally with 3 mg of MSU crystals in 1 ml of sterile phosphate buffered saline (PBS). At various times after crystal injection, mice were killed by CO2 inhalation, and their peritonea were lavaged with 4 ml of PBS containing 5 mM EDTA and 1% bovine serum albumin (BSA). The total number of cells in lavage fluid was enumerated with a hemocytometer. Differential leukocyte counting was performed on cells after sedimentation in a Cytospin 4 cytocentrifuge (ThermoShandon, Pittsburgh, PA), via modified Wright-Giemsa staining. Apoptotic neutrophils were detected by flow cytometry using TUNEL staining according to the manufacturer's instructions (Promega, Madison, WI) and Gr-1 antibody staining (BD Biosciences, San Jose, CA) for neutrophils. Levels of keratinocyte-derived chemokine (KC)/CXCL1 and TGFβ in peritoneal lavage fluids were quantified by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).

Recombinant soluble TG2.

We used characterized site-directed mutants of TG2 that selectively attenuate GTP binding (K173L) or transamidation catalytic activity (C277G) (28). Recombinant soluble His-tagged TG2 was produced by transient transfection of human HEK-293 cells and was isolated using the Probound Purification kit (Invitrogen, San Diego, CA). Nucleotide-bound soluble TG2 was prepared as previously described (28, 29). Briefly, soluble TG2 was incubated with a 10-molar excess of nucleotide in PBS containing 5 mM MgCl2 for 30 minutes at 4°C. Free nucleotide was removed by filtration through Microcone columns (Millipore, Bedford, MA). To prepare TG2 lacking bound nucleotides, soluble TG2 was incubated with 5 mM EDTA for 20 minutes at 30°C (29).

Apoptotic murine thymocytes.

Thymi were removed from mice at 4–6 weeks of age. Thymocytes were isolated by passage through 100-μm nylon cell strainers (BD Falcon, San Jose, CA). Cells were induced to undergo apoptosis by treatment with dexamethasone (1.0 μM) for 12 hours. Thymocytes were consistently ≥85% apoptotic, as evaluated by annexin V binding detected by flow cytometry.

Macrophage culture and assays of macrophage apoptotic cell tethering and uptake in vitro.

Resident mouse peritoneal macrophages were collected by lavage with ice-cold PBS containing 5 mM EDTA and 1% BSA. Bone marrow–derived macrophages were generated from mice as previously described (3). Briefly, bone marrow cells were flushed from femurs and tibias and cultured for 5–7 days in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 μg/ml streptomycin, 100 IU/ml penicillin, and 40 ng/ml of recombinant granulocyte–macrophage colony-stimulating factor (BioSource International, Camarillo, CA). THP-1 cells stably expressing antisense TG2 in pcDNA3.1neo or empty vector were a generous gift from Dr. Alexey M. Belkin (American Red Cross, Rockville, MD) (30). Macrophages were grown in RPMI 1640 supplemented with 10% FCS, 1% glutamine, 100 units/ml penicillin, 50 μg/ml streptomycin, and 300 μg/ml G418 sulfate (Calbiochem, La Jolla, CA).

For apoptotic cell tethering and uptake studies, aliquots of 0.2 × 106 cells/well were plated in 24-well plates (Costar, Cambridge, MA) in 1 ml of RPMI 1640 medium containing 10% FBS, 2 mML-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 15 ng/ml phorbol myristate acetate (PMA). After 18 hours, nonadherent cells were removed by repeated washing. One million apoptotic thymocytes were added to each well containing macrophages, for 30 minutes at 4°C for tethering or 2 hours at 37°C for phagocytosis studies. In experiments in which recombinant soluble TG2, neutralizing anti-TGFβ antibody, latency-associated peptide for TGFβ1 peptide (R&D Systems), or purified recombinant human TGFβ1 (R&D Systems) was used, the compounds were added to macrophages 30 minutes prior to the addition of apoptotic cells. Nonphagocytosed cells were removed by washing, and macrophage monolayers were fixed with 4% formalin and stained with hematoxylin and eosin. Tethering and phagocytosis were enumerated by counting the number of associated apoptotic cells per 100 macrophages/well. Results are expressed as the tethering/phagocytic index, representing the percent of macrophages that rosetted or ingested apoptotic cells multiplied by the average number of apoptotic cells associated per macrophage.

Assays of peritoneal macrophage uptake of apoptotic leukocytes in vivo.

Mice were injected intraperitoneally with 1 ml of 4% thioglycollate to generate elicited peritoneal macrophages. After 4 days, mice were injected with 2 × 107 apoptotic murine thymocytes fluorescently labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), according to the manufacturer's instructions (Molecular Probes, Eugene, OR). After 30 minutes, mice were killed, and their peritonea were lavaged with 4 ml of PBS containing 5 mM EDTA and 1% BSA. Phagocytic uptake of labeled apoptotic thymocytes was evaluated by flow cytometry, using antibody to the macrophage marker F4/80.

Assay for macrophage phagocytosis of MSU crystals.

Bone marrow–derived macrophages were treated with MSU crystals (0.5 mg/ml) in serum-free DMEM for 2 hours or 4 hours at 37°C and then washed with cold PBS containing 5 mM EDTA. The proportion of macrophages taking up MSU crystals was assessed by flow cytometry, based on increased side scatter properties (6).

Statistical analysis.

Statistical analyses were performed using one-way analysis of variance. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Altered course of MSU crystal–induced peritonitis in TG2−/− mice.

Injection of MSU crystals induced a rapid influx of neutrophils into the peritoneal cavities within the first 4 hours, in both TG2+/+ and TG2−/− mice (Figure 1A). Neutrophil numbers declined (by ∼25%) over the subsequent 20 hours in TG2+/+ mice peritonea, but, in contrast, peritoneal neutrophils continued to accumulate (by ∼40%) in TG2−/− mice (Figure 1A). Forty-eight hours after MSU crystal injection, the number of neutrophils present within peritonea of TG2−/− mice was approximately twice the number in TG2+/+ mice. To assess whether the increase in peritoneal cavity neutrophil numbers was mediated by delayed clearance of apoptotic cells, we first evaluated apoptotic neutrophil concentrations by flow cytometry, using the TUNEL assay and employing Gr-1 as the neutrophil surface marker.

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Figure 1. Delayed clearance of apoptotic neutrophils during monosodium urate monohydrate (MSU) crystal–induced peritonitis in transglutaminase 2 (TG2)–deficient mice. TG2+/+ and TG2−/− mice were injected intraperitoneally with 3 mg of MSU crystals. At various times thereafter, mice were killed, and their peritonea were lavaged. Lavage fluids were analyzed for A, the total neutrophil count as determined by hemocytometric enumeration and Wright-Giemsa staining, B, the number of apoptotic neutrophils as determined by TUNEL staining and flow cytometry, and levels of C, KC/CXCL1 and D, transforming growth factor β (TGFβ) as measured by enzyme-linked immunosorbent assay. Values are the mean ± SEM results for 10 mice of each genotype. ∗ = P < 0.05 by analysis of variance.

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In comparison with TG2+/+ mice, TG2−/− mice demonstrated significantly higher numbers of apoptotic neutrophils in their peritoneal cavities at 4 and 24 hours after MSU crystal injection (Figure 1B). Under these conditions, the chemokine KC/CXCL1, a neutrophil chemotaxin that mediates acute MSU crystal–induced inflammation (31), was induced at similar levels in TG2+/+ and TG2−/− mice peritonea in response to the injection of MSU crystals (Figure 1C). Furthermore, the course and extent of TGFβ induction in peritoneal fluid did not significantly differ between TG2−/− and TG2+/+ mice injected with MSU crystals (Figure 1D). These collective results suggested that the increase in peritoneal neutrophils in TG2−/− mice was driven, at least partly, by impaired clearance of apoptotic cells. Therefore, we next directly assessed apoptotic cell clearance by peritoneal macrophages in vivo.

Mice were injected intraperitoneally with fluorescently labeled apoptotic murine thymocytes as a surrogate for apoptotic neutrophils. After 30 minutes, the peritonea were lavaged, and macrophages were evaluated by flow cytometry for apoptotic thymocyte uptake. Approximately 71% of the peritoneal macrophages from TG2+/+ mice had taken up labeled apoptotic cells, but only ∼25% of the peritoneal macrophages from TG2−/− mice had engulfed apoptotic cells (Figure 2A). As observed in vivo, peritoneal macrophages from TG2−/− mice demonstrated severe impairment in phagocytosis of apoptotic thymocytes in vitro (Figure 2B). In contrast, no impairment was seen in the early association (tethering prior to phagocytosis) of apoptotic cells with the surface of macrophages from TG2−/− mice (data not shown). Mature macrophages can contribute to the resolution of gouty inflammation through the generation of antiinflammatory mediators stimulated by ingestion of MSU crystals (6, 10). However, macrophages from TG2−/− mice showed no impairment in MSU crystal uptake when compared with macrophages from TG2+/+ mice (Figure 2C), unlike the difference observed for uptake of apoptotic leukocytes.

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Figure 2. Impaired phagocytosis of apoptotic cells by transglutaminase 2 (TG2)–deficient macrophages. A, Flow cytometry dot plot of F4/80+ peritoneal macrophages with associated 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)–positive apoptotic thymocytes. Apoptotic murine thymocytes labeled with CFSE were injected intraperitoneally in TG2+/+ and TG2−/− mice 4 days after thioglycollate treatment. Thirty minutes after apoptotic thymocyte injection, mice were killed, and the peritonea were lavaged. Note the reduced association of apoptotic cells with TG2−/− macrophages. B, In vitro phagocytosis of apoptotic thymocytes by peritoneal macrophages from TG2+/+ and TG2−/− mice. Leukocyte phagocytosis assays were performed as described in Materials and Methods. The phagocytic index represents the percent of macrophages that ingested apoptotic cells multiplied by the average number of apoptotic thymocytes taken up per macrophage. C, Monosodium urate monohydrate (MSU) crystal uptake. Bone marrow–derived macrophages from TG2+/+ and TG2−/− mice were treated with MSU crystals (0.5 mg/ml) for 2 hours or 4 hours at 37°C. MSU crystal uptake was analyzed by flow cytometry based on increased side scatter properties of cells. The proportion of macrophages taking up crystal is shown. D, Phagocytosis of apoptotic thymocytes by THP-1 cells stably transfected with antisense TG2 or vector control. Results are representative of 4 separate experiments performed in duplicate. Values are the mean ± SEM. ∗ = P < 0.05 by analysis of variance.

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To rule out the possibility that the defect in apoptotic cell phagocytosis arose as an artifact of genetic manipulation of the mice or as a compensatory response to TG2 knockout in the germline, we examined phagocytosis of apoptotic leukocytes by THP-1 cells stably expressing TG2 in an antisense orientation (32), which we verified to have ∼80% reduction in TG2 expression, by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/Western blot analysis (data not shown). PMA-differentiated THP-1 cells with TG2 knockdown had ∼75% impairment in apoptotic thymocyte phagocytosis relative to THP-1 cells transfected with empty vector (Figure 2D).

Critical role of TGFβ activation and TG2 nucleotide binding but not transamidation catalytic activity in phagocytosis of apoptotic cells by macrophages.

Despite lacking signal peptide, TG2 is released into the extracellular environment and can bind the cell surface (26, 33). TG2 possesses reciprocally regulated transamidation and purine nucleotide–binding activities, the latter of which is linked to GTPase/ATPase catalytic activities (26, 34, 35). TG2 transamidation activity is dependent on binding of Ca++ and is inhibited by guanine nucleotide binding, whereas TG2 GTPase/ATPase catalytic activity is attenuated in the Ca++-bound state (34, 35). TG2 activities also depend on subcellular localization (26). For example, on the cell surface, TG2 functions as an integrin coreceptor for fibronectin (32), and extracellular TG2 regulates cell adhesion and migration independently of enzymatic TG2 functions (30, 32). We observed that recombinant exogenous TG2 rescued the defect in apoptotic cell phagocytosis of TG2−/− macrophages in a dose-dependent manner (Figure 3A). This effect was not shared by heat-denatured soluble TG2 (Figure 3B). These results pointed to an extracellular locus of TG2 action in the engulfment of apoptotic cell by macrophages.

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Figure 3. Rescue of the apoptotic leukocyte engulfment defect in macrophages from transglutaminase 2 (TG2)–deficient macrophages by recombinant soluble TG2 but not by the TG2 guanine binding-site mutant TG2(K173L). Peritoneal macrophages from TG2+/+ and TG2−/− mice were treated with the indicated concentrations of soluble TG2 before (A) or after (B) heat denaturation or with recombinant soluble TG2 (0.5 μg/ml) harboring selective mutations that disrupt transamidation activity (C277G) (C) or guanine nucleotide binding (K173L) (D). Thirty minutes after the addition of soluble protein, macrophages were evaluated for phagocytosis of apoptotic thymocytes, as described in Materials and Methods. Values are the mean ± SEM of 4 separate experiments performed in duplicate.

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TGFβ is released by activated macrophages, and TG2 transamidation catalytic activity promotes maturation of the latent TGFβ protein complex and stimulates activation of TGFβ (24, 25). We confirmed that function-blocking antibody to TGFβ inhibited phagocytosis of apoptotic cells by TG2+/+ peritoneal macrophages (22), and a fragment of the TGFβ1-associated latency-associated peptide for TGFβ1 peptide that effectively neutralizes active TGFβ inhibited apoptotic cell engulfment in TG2+/+ macrophages (data not shown). Moreover, exogenous active TGFβ1 rescued the defect in apoptotic cell engulfment in TG2−/− macrophages (data not shown). Unexpectedly, however, a soluble TG2 point mutant (C277G) with attenuated TG catalytic activity but intact nucleotide-binding activity retained the capacity to rescue the defect in apoptotic cell phagocytosis in TG2−/− peritoneal macrophages (Figure 3C). Furthermore, a competitive substrate inhibitor of TG catalytic activity, monodansyl cadaverine, failed to inhibit apoptotic cell phagocytosis in peritoneal macrophages from TG2+/+ mice (data not shown).

Next, we treated peritoneal macrophages from TG2−/− mice with the TG2 GTP binding-site mutant K173L, which lacks guanine nucleotide binding and GTPase activities but has preserved TG catalytic activity (28). The K173L TG2 mutant failed to rescue the TG2−/− macrophage defect in apoptotic cell uptake (Figure 3D). When recombinant soluble TG2 that was stripped of nucleotide-binding capacity by EDTA treatment was added to TG2−/− peritoneal macrophages, it too was unable to correct the defect in phagocytosis (Figure 4A). In contrast, recombinant TG2 pretreated with ADP or ATP, or nonhydrolyzable ATPγS or GDPβS, corrected the TG2−/− macrophage defect in apoptotic leukocyte uptake (Figure 4B).

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Figure 4. Differential effect of transglutaminase 2 (TG2) nucleotide binding on macrophage phagocytosis of apoptotic cells. Peritoneal macrophages from TG2+/+ and TG2−/− mice were treated with recombinant soluble (Sol.) TG2 (0.5 μg/ml) loaded with different nucleotides prior to evaluation of apoptotic cell phagocytosis, as described in Materials and Methods. A, Macrophages were treated with wild-type soluble TG2, TG2 treated with EDTA to strip bound nucleotides, or mutant TG2 unable to bind guanine nucleotides (TG2[K173L]). B, Recombinant soluble TG2 was specifically loaded with ADP or GTP or with nonhydrolyzable nucleotides (GTPγS, ATPγS, or GDPγS), as indicated. C, Peritoneal macrophages from TG2−/− mice were treated with recombinant soluble wild-type TG2 or TG2 harboring a selective mutation that disrupts transamidation activity (C277G) (0.5 μg/ml) with or without the addition of neutralizing anti–transforming growth factor β (anti-TGFβ) antibody (10 μg/ml) 30 minutes prior to evaluation of apoptotic cell phagocytosis. Upper horizontal line represents the mean phagocytic index for untreated TG2+/+ macrophages, while the lower horizontal line represents the mean phagocytic index for untreated TG2−/− macrophages. Values are the mean ± SEM of 4 separate experiments performed in duplicate. ∗ = P < 0.05.

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TG2 in the GTPγS-bound conformational state has attenuated transamidation and GTPase activities but remains capable of activating cells by integrin-dependent signaling (28). We observed that GTP-bound TG2 and GTPγS-bound TG2 were unable to rescue the TG2−/− macrophage uptake defect for apoptotic leukocytes (Figure 4B). Furthermore, GTP-bound TG2 and GTPγS-bound TG2 inhibited phagocytosis of apoptotic cells by TG2+/+ macrophages (Figure 4B). Finally, function-blocking anti-TGFβ antibody blocked rescue of the apoptotic leukocyte uptake defect by not only soluble wild-type TG2 but also the soluble C277G TG2 mutant lacking transamidation activity (Figure 4C). These results pointed to a mechanistic model for TG2 in antiinflammatory apoptotic leukocyte uptake by the macrophage, which is presented schematically in Figure 5.

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Figure 5. Model showing the role of transglutaminase 2 (TG2) in phagocytosis of apoptotic cells by macrophages. In this model, which is supported by the results of the current study, TG2 is externalized and rests on the cell surface in a GDP/ADP- or ATP-bound conformation. Upon apoptotic cell tethering to the macrophage surface by 1 or more recognition receptors, active transforming growth factor β (TGFβ) is liberated from latent TGFβ in a TG2-dependent manner. Active TGFβ provides signals that stimulate phagocytosis of the tethered apoptotic cell, and macrophage ingestion of the apoptotic leukocyte is antiinflammatory.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Denise L. Cecil and Peter Scott (VA Medical Center/University of California, San Diego) for their assistance with several of the experiments.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES