Reduced glutathione as a physiological co-activator in the activation of peptidylarginine deiminase

Background Citrullination catalysed by peptidylarginine deiminases (PADs) plays an important pathogenic role in anti-citrullinated protein antibody (ACPA)-positive rheumatoid arthritis (RA) and, possibly, several other inflammatory diseases. Non-physiological reducing agents such as dithiothreitol (DTT) are normally added to the reaction buffer when determining PAD activity in vitro. We investigated the ability of reduced glutathione (GSH), the most abundant intracellular small-molecule thiol in vivo, to activate PADs. Methods Activity of recombinant human (rh) PAD2 and PAD4, PADs contained in synovial fluid (SF) samples from RA patients and PADs released from phorbol 12-myristate 13-acetate (PMA)-stimulated cells was measured using an in-house PAD activity assay detecting citrullination of fibrinogen. Results No activity of rhPAD2, rhPAD4 or PADs within SF was observed without addition of an exogenous reducing agent. Activity of both recombinant and SF PAD was observed in the presence of 1 mM DTT or 10–15 mM GSH. Following stimulation with PMA, human isolated leucocytes, but not mononuclear cells, released enzymatically active PAD, the activity of which was abolished upon pre-incubation of the cells with the glutathione reductase inhibitor 2-AAPA. No PAD activity was observed in the corresponding supernatants, but addition of exogenous GSH restored activity. Conclusions Catalytic activity of PAD requires reducing conditions. GSH meets this requirement at concentrations comparable with those found within cells. Active PAD, reduced by GSH, is released from PMA-stimulated granulocytes, but becomes inactivated in the extracellular space.

Most studies on PAD activity and functional studies of citrullinated proteins have been based on in vitro citrullination using the reducing agent dithiothreitol (DTT) and exogenously added calcium. The calcium dependency of PADs is well described: upon binding of calcium, Cys645 in PAD4 (Cys647 in PAD2) translocates into a position in the catalytic site, where nucleophilic attack on guanidinium groups of target arginines takes place [10,11]. The PAD requirement for calcium is met in the extracellular space [12] and, under certain circumstances, also intracellularly [13]. The dependency of PADs on reducing agents is less well understood. Reduction of the active site thiol Cys645/647 is likely to precede attack on the guanidinum carbon of arginine. Thus, the redox balance might be an additional regulator for PAD's catalytic activity. While DTT is a nonphysiological synthetic molecule, reduced glutathione (GSH) is a physiological reducing agent that may facilitate PAD activity in vivo. GSH is a linear tripeptide of L-glutamine, L-cysteine and glycine, and contains a sulfhydryl (SH) group on the cysteinyl portion, accounting for its strong electron-donating character. GSH is the most abundant intracellular small-molecule thiol, and is essential for maintaining the thiol status of various molecules. GSH has many biological roles, including protection against reactive oxygen and nitrogen species (ROS/NOS), which are reduced by two GSH molecules forming oxidized glutathione (GSSG) in the process [14]. GSH has been demonstrated in cytosol and in organelles of virtually all cells of the body at the low millimolar range [15], whereas extracellular levels are two to three orders of magnitude lower [16,17].
Owing to involvement in many cellular functions, it is not surprising that dysregulation of GSH has been associated with various diseases [14]. The glutathione reductase, which converts GSSG to GSH, has been found upregulated in synovial fluid (SF) from RA patients [18], and blood from RA patients contains higher levels of GSH, as well as higher GSH:GSSG ratios, than blood from healthy controls [19].
We hypothesize that, in addition to calcium, reducing agents are required for PADs to become enzymatically active, and that GSH is the major physiological reducing agent in this respect.
Using an assay for citrullination of matrix-bound fibrinogen, we here analyse the impact of GSH on recombinant human PADs (rhPADs), on PADs contained in SF and on PADs released from stimulated human leucocytes.

Cells, serum and SF
Blood samples were obtained from healthy donors attending the Blood Bank at Copenhagen University Hospital, Rigshospitalet, Denmark. All donors were anonymous to the investigators.
One hour after collection, serum was isolated from venous blood drawn into 10 ml dry Vacutainer tubes (BD Bioscience, Brøndby, Denmark) by centrifugation at 400 × g for 10 min at 20°C. Pooled serum from blood group AB-positive donors, henceforward referred to as "AB serum", was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were isolated from venous blood drawn into 10 ml lithium-heparin tubes (BD Bioscience). Blood leucocytes were isolated after lysis of erythrocytes in heparinized blood by incubation with ammonium chloride (In Vitro As, Fredensborg, Denmark) for 7 min. Mononuclear cells (MNCs) were isolated by gradient centrifugation of heparinized blood using LymphoPrep™ (Axis-Shield, Oslo, Norway). Before use, both cell preparations were washed twice in RPMI 1640, 25 mM Hepes containing 0.42 mM calcium nitrate, L-glutamine and gentamicin (In Vitro As).
SF samples from nine ACPA-positive RA patients, fulfilling the American College of Rheumatology 1987 diagnostic criteria [20], were obtained from Dr Ladislav Senolt, Charles University in Prague, Czech Republic. The study was approved by the Ethics Committee of the Institute of Rheumatology and written informed consents were obtained from all patients prior to initiation of the study. Samples were centrifuged at 1900 × g for 10 min to remove cells and were stored at -80°C prior to analysis.

Assay for cell-mediated PAD activity
Isolated leucocytes or MNCs were added to microtitre wells coated with fibrinogen, washed and blocked as already described, and incubated for 180 min under agitation at RT. Purified cells were diluted 1:1 with RPMI 1640 to a final concentration of 5 % AB serum and, when relevant, 15 nM phorbol 12-myristate 13-acetate (PMA) and 50 μM 2-AAPA (which were incubated for 20 min with cells prior to stimulation). Cells were removed by washing four times in PBS and 0.05 % Tween-20, and plates were developed as already described.

Results
Reduction of PADs is a prerequisite for enzyme activity PAD activity was determined in microtitre plates containing immobilized human fibrinogen, using an antibody specifically reactive with citrullinated fibrinogen. In the presence of DTT (1 mM in Tris-HCl buffer containing 10 mM CaCl 2 ) as a reducing agent, we observed ample citrullination of human fibrinogen by rhPAD2 and/or rhPAD4 (Fig. 1a). No citrullination was observed when a pool of SF from four RA patients was used, although we have previously shown that SF contained PAD2 and 1.5-2.5 mM calcium [12,23]. The addition of rhPAD2 and rhPAD4 to SF did not result in detectable fibrinogen citrullination, except when DTT was also present. A similar pattern was observed for a pool of sera from five healthy donors.

GSH-mediated reduction can activate PADs
To investigate whether GSH could substitute for DTT as a reducing agent, we examined the enzymatic activity of rhPAD2 and rhPAD4 over a range of GSH concentrations and at a fixed CaCl 2 concentration of 10 mM (Fig. 1b). Catalytic activity required GSH concentrations above 1 mM, reached a peak around 10-15 mM and declined with further increasing GSH concentrations. At GSH concentrations above 25 mM, no PAD activity was observed. In contrast, the activity of both rhPAD isoforms reached a plateau at DTT concentrations between 1 mM and 50 mM (data not shown). As shown previously with DTT as a reducing agent [12], PAD2 showed higher efficacy than PAD4 in the employed assay (Fig. 1b).

Calcium is required for GSH-mediated PAD activity
As expected, the catalytic activity of both rhPAD2 and rhPAD4 was calcium dependent, irrespective of whether GSH or DTT was used as a reducing agent (Fig. 2). Higher calcium concentrations were required to obtain

PAD activity in SF is dependent on reducing conditions
To examine the PAD-reducing capacity of GSH in a physiologically relevant setting, we examined the catalytic activity of native PAD enzymes contained in a pool of SF from five RA patients in the presence and absence of GSH. ELISA verified that the pool contained PAD, the concentration of PAD2 being 18 ng/ml. As shown in Fig. 3, pure SF as well as SF supplemented with 10 mM CaCl 2 failed to exhibit catalytic activity above that observed in negative controls containing the calcium chelator EDTA, which blocks PAD activity. However, when SF was supplemented with 10 mM GSH in the presence or absence of exogenously added CaCl 2 , substantial citrullination was observed.

Enzymatic activity of PAD released from stimulated leucocytes
To examine whether PAD released from cells is catalytically active, we cultivated blood leucocyte cells in microwells coated with fibrinogen to allow citrullination in situ (Fig. 4a). No catalytic activity was observed in wells containing unstimulated cells, but stimulation of the leucocytes with PMA resulted in substantial citrullination of fibrinogen (Fig. 4a, grey columns). As expected, this signal could be inhibited by EDTA. Addition of exogenous rhPAD2 did not further increase the catalytic activity in the wells, which was tested in experiments involving leucocytes from 10 different donors (Fig. 4b), indicating that the requirements for PAD activity were not met in the medium. Addition of DTT enhanced activity of the released PAD and rendered the exogenously added rhPAD2 active (Fig. 4b). Taken together, these data suggest that stimulation of leucocytes by PMA leads to the release of PAD, which is temporarily enzymatically active and loses its activity in the oxidative environment.
In contrast with total leucocytes, purified MNCs did not show PAD activity upon stimulation (Fig. 4c), suggesting that granulocytes were the main providers of catalytically active PAD (Fig. 4a). In agreement with the finding that exogenous rhPAD2 did not enhance the catalytic activity in wells containing PMA-stimulated cells, rhPAD2 added to supernatants from the unstimulated or PMA-stimulated cells was not enzymatically active unless supplemented with GSH (data not shown). The highest activity was observed at GSH concentrations around 20-25 mM, and no activity was observed at a concentration of 50 mM (data not shown).
To determine whether PADs released from stimulated leucocytes (Fig. 4a) had gained catalytic activity through reduction by endogenous GSH, we stimulated leucocytes with PMA in the presence of a selective GRI (2-AAPA) [24] (Fig. 5a). Inclusion of the GRI abrogated enzymatic  activity signals to those observed in the presence of EDTA. Importantly, GRI did not prevent PAD from being released. Thus, inclusion of GRI diminished neither the PAD2 concentration (Fig. 5b) nor the PAD activity (Fig. 5c) of the supernatants. No activity was observed in supernatants without exogenously added GSH (data not shown).

Discussion
The physiological agent(s) responsible for reducing PADs to their physiologically active state have not been identified. In general, PAD activity studies in vitro have used DTT or a related compound not present in nature as a reducing agent. Thus, enzymatic activity of PADs contained in SF [12,23], in cell lysates [25] or released by cells [26,27] has generally not been examined under physiological conditions, because DTT has been included in the experiments. We hypothesized that GSH is a natural reducing agent required for PADs to be enzymatically active.
Indeed, enzymatic activity of rhPAD2 and rhPAD4 was observed at GSH concentrations corresponding to those Fig. 4 Release of catalytically active peptidylarginine deiminase (PAD) from PMA-stimulated granulocytes. a Microtitre plates coated with human fibrinogen were incubated for 3 h at RT with blood leucocytes resuspended in RPMI medium containing 5 % normal human serum with or without 25 mM EDTA. Cells were stimulated with PMA (15 nM) and subsequent citrullination of fibrinogen was measured using mAb 20B2 as the detecting antibody. Average and upper range of duplicate optical density (OD) measurements shown for three donors. b Enzymatic activity of PMA-stimulated leucocytes from 10 individual donors was examined in a similar manner in the presence and absence of exogenous rhPAD2 (300 ng/ml) and/or 1 mM dithiothreitol (DTT). Shown are averages of duplicate measurements after subtraction of background values obtained in the presence of EDTA. c Using the same procedure, mononuclear cells (MNCs) from donor 1 in a were assessed. Average and upper range of duplicate OD measurements are shown. PMA phorbol 12-myristate 13-acetate  [15]. To our knowledge, intracellular GSH levels exceeding 15 mM have not been observed in vivo, and therefore the diminished PAD activity observed at high concentrations of exogenously added GSH in this study may not be physiologically relevant.
We did not observe any PAD activity in pooled SF from RA patients without addition of GSH or DTT. Spengler et al. [27] recently reported weak PAD activity in pure, freshly obtained SF from untreated RA patients, which was higher than in SF from OA patients, albeit 100-fold lower than the activity in the presence of DTTcontaining citrullination buffer. The differences between their observations and ours may rely on their use of a different assay for protein citrullination and, possibly, usage of freshly isolated SFs. The low or absent PAD activity in SF observed here and by others [27] suggests that an essential factor was missing for PADs to function optimally. Our finding that addition of DTT or GSH to SF strongly enhanced PAD activity indicates that a reducing agent(s) is this essential factor.
Circulating proteins produced outside joints (e.g. fibrinogen produced in the liver) are present in a citrullinated form in SF from RA patients [28,29], suggesting that extracellular citrullination occurs within the joints, where the calcium concentration is sufficiently high for PADs to be active. Our finding that PMAstimulated leucocytes cultured in microtitre wells were capable of citrullinating fibrinogen coated in wells suggests that the leucocytes either released PAD in reduced form or co-released substance(s) capable of reducing PAD.
Evidence for GSH being critical for reduction of PAD came from the finding that citrullination was abrogated by addition of the highly specific GRI 2-AAPA [24] to cells before incubation in the wells. Extracellular leucocyte PAD most probably originates from granulocytes, because PMA-stimulated MNCs devoid of granulocytes were not capable of citrullinating fibrinogen. The presence of PAD2 and of PAD activity (after addition of GSH) in supernatants from the cultures exposed to 2-AAPA suggested that 2-AAPA did not inhibit PAD release. However, we cannot rule out that 2-AAPA has effects on PAD activity other than that caused by enhancement of the GSSG:GSH ratio. The enzymatic activity of the supernatants was low compared with the activity observed in the presence of cells, suggesting that citrullination of fibrinogen took place in the close vicinity of the cells, where high local concentrations of reduced PADs could be obtained. It is likely that PADs are rapidly oxidized, and thereby inactivated, upon release from granulocytes.
With respect to citrullination of intracellular proteins, the PAD requirement for GSH is clearly met intracellularly [15]. Indeed, intracellularly located citrullinated proteins have been observed in the synovium of RA patients, indicating that calcium concentrations high enough for PADs to be active can be reached intracellularly under certain circumstances [13]. Accordingly, PAD enzymes are known to modulate gene expression [30][31][32]. It is noteworthy that GSH can also be recruited to the nucleus (e.g. during the early phase of cell proliferation [33]) and is also involved in gene expression [34,35].
A limitation of this study is that we cannot distinguish between PAD2 and PAD4 activity. Granulocytes are known to release both isoforms upon PMA stimulation [26,27]. We have shown previously that~150-fold more rhPAD4 than rhPAD2 is required to citrullinate the epitope recognized by the detecting antibody in the assay used in this study [12], and thus it can be speculated that PAD2 is primarily responsible for the citrullination observed. We cannot exclude an influence of other physiological reducing agents on PAD activation in vivo. Indeed, thioredoxin has been reported to be 5fold increased in SF of RA patients compared with OA patients [36]. However, the complete abrogation of cell-mediated citrullination by blockade of glutathione reductase suggests that GSH is predominantly responsible for reduction of PAD under normal physiological conditions.

Conclusion
This study shows for the first time that the physiological reducing agent GSH, at concentrations similar to those present in cytosol, is capable of reducing PADs to a degree that suffices for citrullination to occur. We found that the thiol status of PADs is as important as binding of calcium for PAD's catalytic activity. Competing interests DD and CHN are inventors for two patents concerning therapeutic use of monoclonal antibodies against PADs. MEB, MAS and GJMP have no competing interests related to the work described in this article.
Authors' contributions DD designed the study, carried out experiments and drafted the manuscript. MEB assisted with study design, experiments and helped to revise the manuscript critically. MAS assisted with experiments and helped to revise the manuscript critically. GJMP expressed, purified and tested recombinant proteins used for the experiments and helped to revise the manuscript critically. CHN designed the study and drafted the manuscript. All authors read and approved the final manuscript.