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Oxidized ₂-Macroglobulin (₂M) Differentially Regulates Receptor Binding by Cytokines/Growth Factors: Implications for Tissue Injury and Repair Mechanisms in Inflammation¹

Sean M. Wu^*, Dhavalkumar D. Patel and Salvatore V. Pizzo^2,*

Departments of ^* Pathology and Medicine, Division of Rheumatology, Duke University Medical Center, Durham, NC 27710

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
₂M binds specifically to TNF-, IL-1ß, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), ß-nerve growth factor (ß-NGF), platelet-derived growth factor (PDGF), and TGF-ß. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of ₂M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized ₂M exhibits increased binding to TNF-, IL-2, and IL-6 and decreased binding to ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2. Hypochlorite oxidation of methylamine-treated ₂M (₂M*), an analogue of the proteinase/₂M complex, also results in decreased binding to bFGF, ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2. Concomitantly, we observed decreased ability to inhibit TGF-ß binding and regulation of cells by oxidized ₂M and ₂M*. We then isolated ₂M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-ß compared with ₂M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, ß-NGF, PDGF, and TGF-ß from binding to ₂M.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 720-kDa homotetrameric broad-spectrum proteinase inhibitor ₂M is found in the plasma at high (micromolar) concentrations (for a review, see 1 . Interaction between ₂M and proteinases in plasma and extracellular fluids involves a unique trapping mechanism by which the proteinase is incorporated covalently into the ₂M molecule via a unique ß-cysteinyl--glutamyl thioester bond. Following scission of the thioester bond, the conformation of ₂M changes to a more compacted structure, exposing its receptor recognition sites for binding to both the low density lipoprotein receptor-related protein (LRP)³ (2, 3) and the ₂M signaling receptor (4, 5). This conformational change can be generated chemically by reacting ₂M with small primary amine nucleophiles such as methylamine, forming an ₂M/methylamine complex (hereafter designated ₂M*) that behaves in many ways identically with a proteinase/₂M complex (6).

Although ₂M has traditionally been viewed as a plasma and inflammatory fluid proteinase scavenger, evidence has accumulated in recent years suggesting that in vivo ₂M can bind to cytokines and growth factors such as TNF-, IL-1ß, IL-2, IL-6, bFGF, ß-NGF, PDGF, and TGF-ß (for a review, see Refs. 7 and 8). Binding to ₂M abolishes the ability of most cytokines/growth factors to regulate cell functions while enhancing the ability of a few others (9, 10, 11, 12, 13, 14, 15, 16). Cytokines bind to ₂M with affinities that vary from micromolar K_d values for early inflammatory mediators such as TNF-, to nanomolar K_d values for late inflammatory mediators such as bFGF, ß-NGF, PDGF, and TGF-ß (10, 17, 18, 19, 20). It has been shown that very little free TGF-ß and PDGF are found in the circulation, since 85 to 90% of them are bound to ₂M (10, 13, 14, 21). Yet, to date no mechanism has been demonstrated in vivo that inhibits the binding of growth factors to ₂M, raising the question of how growth factors are able to function when ₂M is present in high concentrations in the plasma and inflammatory fluids. Given that bFGF, ß-NGF, PDGF, and TGF-ß have been implicated in tissue injury repair mechanisms such as angiogenesis, fibroblast proliferation, smooth muscle cell proliferation, collagen deposition, and neuronal regeneration (22, 23, 24, 25), it appears that a mechanism must exist that inhibits the binding of ₂M to these growth factors and/or allows these growth factors to be released from ₂M to regulate cell functions.

We and others are investigating the role of oxidants in abolishing the ability of ₂M to inhibit proteinases. It is well known that reactive oxygen species such as superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorite play an important role during acute and chronic inflammation (26, 27, 28, 29). In addition to neutralizing bacteria, these neutrophil-derived oxidants accelerate tissue destruction by acting either directly on cells, causing apoptosis and tissue necrosis, or indirectly by altering the proteinase-proteinase inhibitor balance (30). Hypochlorite, produced by the neutrophil H₂O₂-myeloperoxidase-Cl^- system, but not H₂O₂ or hydroxyl radical from metal-catalyzed oxidation, can abolish the ability of ₂M to inhibit proteinases at low micromolar concentrations (31, 32). The biologic concentration of hypochlorite during inflammation can be as high as millimolar (30). Reactions of hypochlorite with ₂M occur predominantly at methionine and tryptophan residues, although we have recently shown that lysine is a susceptible target of oxidation as well (32, 33). Hypochlorite oxidation of ₂M results in fragmentation of ₂M tetramers into dimers, whereas the effect of oxidation on ₂M* is currently unknown.

Since neutrophil-derived oxidants are presumed to be released concomitant with cytokines/growth factors, and increased concentrations of ₂M in tissue fluids have been demonstrated in a number of inflammatory diseases such as rheumatoid arthritis (RA) (34), pulmonary emphysema (35), pneumonia (36), and periodontitis (37), we hypothesize that hypochlorite oxidation may serve as a physiologically relevant mechanism that regulates the binding of cytokines and growth factors to ₂M.

In this study, we found that hypochlorite oxidation decreases the binding of ₂M and ₂M* to tissue repair growth factors such as ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2. On the other hand, we found that hypochlorite oxidation enhances the binding of ₂M, but not that of ₂M*, to acute phase cytokines such as TNF-, IL-2, and IL-6. Additional experiments using ₂M purified from human RA synovial fluid (RASF) indicate that this protein is significantly oxidized and that its binding to TGF-ß is decreased. Given these findings, we propose that ₂M oxidation is a switch mechanism that reverses the cytokine/growth factor binding profile of ₂M, thus facilitating the transition from the early phase of inflammation, when tissue injury and destruction predominate, to the late phase, when tissue repair and remodeling are required.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

¹²⁵I-labeled Bolten Hunter reagent and [methyl-³H]thymidine were purchased from New England Nuclear Life Science Products (Boston, MA). RPMI 1640, DMEM, L-glutamine, penicillin/streptomycin, HBSS, and FBS were purchased from Life Technologies (Gaithersburg, MD). HEPES, sodium hypochlorite, L-methionine, L-glycine, 5,5'-dithio-bis(2-nitrobenzoic acid), and EDTA were purchased from Sigma (St. Louis, MO). All other reagents were of the highest quality commercially available.

Proteins

Human native ₂M was purified from plasma according to a previously described protocol (2). ₂M was at least 90% active against proteinases as determined by thioester titration using the 5,5'-dithio-bis(2-nitrobenzoic acid) assay (38). ₂M* was prepared as previously described (39). Both ₂M and ₂M* were free of endotoxin as determined by the Limulus amebocyte lysate test purchased from the Associates of Cap Cod (Falmouth, MA) performed according to the manufacturer’s suggested protocol (n = 2). Recombinant carrier-free human TGF-ß1, TGF-ß2, PDGF-BB, IL-6, ß-NGF, and bFGF were purchased from R&D Systems (Minneapolis, MN). Recombinant carrier-free human TNF-, IL-1ß, and IL-2 were purchased from Genzyme Diagnostics (Cambridge, MA). All ¹²⁵I-labeled cytokines/growth factors were either labeled with [¹²⁵I]Bolten Hunter reagent according to the manufacturer’s recommended protocol or purchased from New England Nuclear Life Science Products. The specific radioactivity of the labeled ligands ranged from 500 Ci/mmol ([¹²⁵I]TNF-) to 4000 Ci/mmol ([¹²⁵I]TGF-ß1). Differences in specific radioactivity between commercially purchased proteins and our own preparations were estimated to be <20% (n = 4). Both labeled and unlabeled proteins were reconstituted with 0.1% BSA in PBS, pH 7.4, and stored at -20°C. ¹²⁵I-labeled cytokines/growth factors were used within 2 wk of labeling, and unlabeled proteins were stored at -20°C and used within 3 mo.

Oxidation of ₂M and ₂M*

Oxidation of ₂M and ₂M* was performed as previously described with minor modifications (33). In brief, ₂M and ₂M* were incubated with sodium hypochlorite (0–100 µM) for 15 min at 37°C in PBS, pH 7.4. The sodium hypochlorite concentration was determined spectrophotometrically at 292.5 nm with an extinction coefficient of = 206 M^-1 cm^-1 at pH 7.4 (40). At the end of the incubation, 200 µM L-methionine was added to the mixture to quench residual oxidants.

¹²⁵I-labeled cytokine/growth factor binding to oxidized ₂M and oxidized ₂M*

¹²⁵I-labeled cytokines/growth factors (0.3–0.5 ng) were added to ₂M and ₂M* that were oxidized with 0 to 100 µM of hypochlorite and incubated for 2 h at 37°C. Following incubation, the mixture was loaded onto either native pore-limit gels or a nonreducing SDS gels to separate bound from unbound ¹²⁵I-labeled ligands. Following electrophoresis, gels were stained with Coomassie brilliant blue to verify equal loading of ₂M into each lane. Gels were subsequently dried and exposed to a PhosphorImager (Molecular Dynamics Sunnyvale, CA) plate for 16 h before the plate was developed and the bands were quantified. To eliminate the possible overexposure of the PhosphorImager plate, we performed control experiments to verify that the relative intensity of the bands on the PhosphorImager plate corresponded to the radioactivity detected by gamma counting of each band (n = 4). Nonspecific binding was determined by the binding of [¹²⁵I]ligand in the presence of a 1000-fold excess of unlabeled ligand. Specific noncovalent binding of [¹²⁵I]cytokines/growth factors to ₂M was determined by subtracting radioactivity associated with ₂M in nonreducing SDS gels from radioactivity associated with ₂M in native pore-limit gels. Nonspecific binding was approximately 0 to 30% and varied with each growth factor. At least three independent binding assays for each cytokine/growth factor and oxidized ₂M or oxidized ₂M* pairs were performed.

Protein gel electrophoresis

Native pore-limit gel electrophoresis was performed as described previously (2). In brief, 5 to 15% gradient polyacrylamide gels in 8.9 mM Tris, 8.9 mM boric acid, and 0.2 mM EDTA, pH 8.8, were made immediately before use. ₂M and oxidized ₂M alone or incubated with ¹²⁵I-labeled cytokines/growth factors (25 µl) in nonreducing, nondenaturing sample buffer were added to each lane and run for 3 h at 150 V. Subsequently, gels were stained with Coomassie brilliant blue and destained for 4 h in methanol/acetic acid. To ensure that ₂M-bound cytokines/growth factors did not dissociate during the destaining procedure, identical gels were autoradiographed without staining/destaining in some experiments. No difference was detected between gels that were stained and gels that were not stained. Nonreducing SDS-gel electrophoresis was performed as previously described (33).

Cell surface binding assay

CCL64 mink lung epithelial cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in 150-cm² flasks in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 25 mM HEPES, and 15 ml of 10,000 U/ml penicillin/streptomycin. Cell surface receptor ligand binding assays were performed as previously described (39). Cells were seeded into 24-well plates at 500,000 cells/well and allowed to adhere and grow until confluence (1–2 days) in a 5% CO₂ humidified incubator at 37°C. [¹²⁵I]TGF-ß1 (0.5 ng) was then added into each well in the presence or the absence of the indicated concentrations of ₂M or oxidized ₂M in HBSS containing 5% BSA and 25 mM HEPES, pH 7.4, and incubated at 4°C for 16 h. In some experiments, receptor-associated protein (RAP; 6.65 µM), which inhibits binding of all ligands to LRP (41), was also added to determine the amount of oxidized ₂M/[¹²⁵I]TGF-ß1 complex binding to the cell surface via the scavenger receptor LRP. Following incubation, unbound ligand was washed twice with ice-cold HBSS buffer and solubilized with 0.5 M NaOH/0.1% SDS for 4 h at 25°C before gamma counting using CliniGamma 1272 from LKB-Wallac (Turku, Finland). Total binding was determined by measuring the binding of [¹²⁵I]TGF-ß1 in the absence of ₂M. Nonspecific binding was determined by measuring the binding in the presence of a 1000-fold molar excess of unlabeled TGF-ß1 and range from 20 to 30% of total binding.

Cell proliferation assays

For TGF-ß studies, we used CCL64 cells that were cultured as described above, except that 2% FBS was used during the experiment to minimize the interference by bovine macroglobulins to the assay. Cell proliferation assays were performed according to a standard protocol (14). In brief, 1 day before experimentation, cells were trypsinized and plated into 96-well tissue culture plates from Costar (Cambridge, MA) at 5000 cells/well and incubated overnight. On the day of the experiment, TGF-ß at the indicated concentrations was added alone or in the presence of 1 mg/ml ₂M, oxidized ₂M, ₂M*, or oxidized ₂M* and incubated for 16 h at 37°C in a 5% humidified CO₂ incubator. Following incubation [methyl-³H]thymidine (0.5 µCi) was added to each well, and the plates were incubated for an additional 5 h. Cells were then trypsinized and harvested using a Skatron (Sterling, VA) cell harvester, and the cell-associated radioactivity was counted in a MINAXIß liquid scintillation counter from Packard Instruments (Downers Grove, IL). As controls, CCL64 cells were incubated with ₂M, ₂M*, oxidized ₂M, and oxidized ₂M* in the absence of TGF-ß and harvested in parallel with the experimental wells.

For bFGF assays, fetal bovine heart endothelial cells obtained from American Type Culture Collection were cultured in 75-cm² flasks in DMEM supplemented with 10% FBS, 50 ng/ml bFGF, 2 mM L-glutamine, 25 mM HEPES, and 15 ml of 10,000 U/ml penicillin/streptomycin until 80% confluent and then transferred to bFGF-deficient medium for 48 h to reach quiescence. One day before experimentation, quiescent cells were trypsinized from flasks, plated into 96-well plates at 2000 cells/well in bFGF-deficient medium, and allowed to adhere. On the day of experimentation, cell medium was replaced with ₂M (either oxidized or nonoxidized) alone (1 mg/ml) or ₂M and various concentrations of bFGF-containing medium and allowed to incubate for 48 h at 37°C. Following incubation, cells were pulsed with [methyl-³H]thymidine (0.5 µCi) for an additional 5 h and then harvested for scintillation counting.

For TNF- assays, murine fibrosarcoma (WEHI 13VAR) cells that are highly sensitive to TNF--induced cell death were used. These cells were cultured under the same conditions as CCL64 cells described above. Assays for TNF--induced cell death was performed as described previously (42) with modifications. Cells were cultured in 75-cm² flasks and plated into 96-well plates 2 days before experimentation at 10,000 cells/well. On the day of experimentation, ₂M alone (1 mg/ml) or ₂M and various concentrations of TNF- were added to each well in the presence of 10 µg/ml of cycloheximide and allowed to incubate for 24 h at 37°C. Following incubation, cell viability was measured using Celltiter 96 (Promega, Madison, WI) according to the manufacturer’s suggested protocol and verified by cell counting.

Collection of synovial fluids from patients with RA and osteoarthritis (OA)

Synovial fluids from six patients fulfilling the American College of Rheumatology’s revised criteria for the classification of RA (43) and five patients fulfilling criteria for OA were obtained from the Rheumatology Clinic of Duke University Medical Center (Durham, NC). Informed consent was obtained in each case for the use of these fluids. Synovial fluids were aspirated as a standard procedure to drain inflamed joint effusions. Fluids were anticoagulated with 5 mM EDTA or 10 U/ml heparin and frozen immediately at -70°C. Before analysis synovial fluids were thawed and treated with a mixture of proteinase inhibitors to give a final concentration of 2 mM PMSF, 2 mM 3,4-dichloroisocoumarin, 5 mM 1,10-phenanthroline, and 2 µM E-64. Cell debris was removed by centrifugation.

Affinity purification of ₂M from synovial fluid

Polyclonal Abs against human ₂M were made in New Zealand White rabbits and isolated using ₂M-Sepharose prepared by coupling ₂M to cyanogen bromide-activated Sepharose purchased from Pharmacia (Uppsala, Sweden). Purified Ab was then coupled to cyanogen bromide-activated Sepharose according to the manufacturer’s suggested protocol and incubated for 2 h with RA and OA synovial fluid prepared as described above and subsequently diluted threefold with PBS, pH 7.4. Following incubation, ₂M bound to anti-₂M IgG-Sepharose was eluted with 0.1 M Tris/0.5 M NaCl, pH 10.8, and immediately readjusted to pH 7.4. As controls, ₂M from healthy human donor plasma (n = 6) was isolated using the same procedure as that used for synovial fluid ₂M. The ₂M protein concentration was determined both spectrophotometrically using A₂₈₀ (1%; 1 cm) = 8.93 (44) and with bicinchoninic acid protein assay (Pierce, Rockford, IL), and the purity of the protein was verified by gel electrophoresis and Western blotting.

Protein carbonyl content determination

Measurement of the extent of oxidation in synovial fluid ₂M compared with plasma ₂M was performed using 2,4-dinitrophenylhydrazine (DNPH) derivatization of protein carbonyls as previously described (45) with modifications. One hundred micrograms of protein in 800 µl of PBS was added to 200 µl of 10 mM DNPH in 2 M HCl and incubated at 25°C for 1 h. Following incubation, proteins were precipitated with 150 µl 70% TCA and placed on ice for 10 min. Proteins were then centrifuged at 800 x g for 10 min. Protein pellets were washed with ethyl acetate/ethanol (1/1, v/v), and the centrifugation/washing process was repeated two more times before final solubilization in 6 M guanidine-HCl, pH 7.4. Each protein sample was then scanned from 200 to 500 nm using a Beckman DU-640 spectrophotometer (Arlington Heights, IL), and the quantity of protein carbonyl was calculated using an extinction coefficient for dinitropheynlhydrazone of 22,000 M^-1 cm^-1 (45). To control for the possible loss of proteins during washing steps, all spectrophotometric readings were adjusted to an identical A₂₈₀. Background absorption was determined by experiments performed in the absence of DNPH, and this value was used as the blank for each reading.

Data analysis

In studies of [¹²⁵I]cytokine/growth factor binding to oxidized ₂M and oxidized ₂M*, the K_d values were determined by least squares curve fitting using the SYSTAT program (version 5.04, Systat, Evanston, IL). We chose to determine the K_d values using this method because it gave more consistent data (r² >0.95 for all calculations) than those derived from Scatchard plots. The K_d values determined from Scatchard analysis, however, were within the SE. In cell proliferation assays the EC₅₀ was also determined using SYSTAT. The growth factor effects (percentages) on cell proliferation was determined as was previously described (46): % effect = EC₅₀ (control)/EC₅₀ (treatment) x 100, where EC₅₀ (control) represents half-maximum growth factor effects in the absence of ₂M, and EC₅₀ (treatment) represents half-maximum growth factor effects in the presence of ₂M or oxidized ₂M.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Structural disruption of oxidized ₂M

The effects of various physiologically relevant concentrations of hypochlorite on the structures of ₂M and ₂M* were determined. As shown in Figure 1, ₂M oxidation at >50 µM hypochlorite concentrations resulted in significantly faster electrophoretic migration. The faster migratory position corresponded to ₂M dimers as previously described (32) and not ₂M*, which migrated only slightly faster (by 2 mm) than ₂M tetramers in the same gel system (data not shown). This effect was absent with oxidation of ₂M*, which remained as intact tetramers at the concentrations shown. A slight decrease in staining by Coomassie brilliant blue was evident in 100 µM hypochlorite-oxidized ₂M and ₂M*. Decreased staining of chlorinated ₂M has been previously reported (31, 33). Additional verifications using Western blotting analysis and autoradiography of radiolabeled proteins showed equivalent protein quantity in all lanes (data not shown).

View larger version (50K): [in this window] [in a new window]   FIGURE 1. Structural disruption of oxidized 2M. 2M (top gel) and 2M* (bottom gel) were oxidized using the indicated concentrations of hypochlorite for 15 min at 37°C. Residual oxidants were quenched with 200 µM L-methionine. Samples (5 µg each) were then loaded onto a native pore-limit gel and electrophoresed for 3 h followed by Coomassie brilliant blue staining. Tetrameric 2M is denoted by T, and dimeric 2M is denoted by D.

Binding of ¹²⁵I-labeled cytokines/growth factors to oxidized ₂M

To determine whether oxidation affects the binding of ₂M to cytokines/growth factors, we performed in vitro binding experiments using ¹²⁵I-labeled TNF-, IL-2, IL-6, bFGF, ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2. These labeled ligands were incubated for 2 h with 20 µl of 0.25 mg/ml ₂M oxidized at the indicated concentrations. Unbound ligands were separated by native pore-limit electrophoresis. Figure 2 shows the ₂M-associated radioactivity for each labeled cytokine/growth factor binding assay with ₂M. As can be seen, the affinity of ₂M for TNF-, IL-2, IL-6, and bFGF increases with oxidation. It appears that the increase in cytokine/growth factor binding is not dependent on the tetramer to dimer transition, since some ligands, such as IL-2 and IL-6, show increased binding to ₂M even while it is still in the tetrameric state. In contrast, the binding of oxidized ₂M to ß-NGF, TGF-ß1, TGF-ß2, and, to a lesser extent, PDGF-BB decreases with increasing oxidant concentrations. The decrease in binding is more dramatic for TGF-ß2 than ß1, possibly reflecting the higher affinity of ₂M binding to TGF-ß2 than TGF-ß1 (17). It is important to point out that the specific radioactivity is different for each cytokine, and therefore the binding intensities cannot be compared between cytokines. Since methionine was added to quench residual oxidants, control experiments were performed to determine whether methionine alone or methionine sulfoxide, the product of the reaction between methionine and hypochlorite, can affect the binding of cytokine/growth factors to ₂M. ¹²⁵I-labeled ligands were incubated with ₂M in the presence or the absence of methionine or methionine/hypochlorite mixture. In three independent experiments with [¹²⁵I]TGF-ß1, PDGF-BB, and TNF-, there was no difference in the amount of cytokine/growth factor bound to ₂M (data not shown).

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Binding of 125I-labeled cytokines/growth factors to oxidized 2M. 125I-labeled TNF-, IL-2, IL-6, bFGF, ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2 were added to 2M oxidized with the indicated concentrations of hypochlorite. The samples were then incubated for 2 h at 37°C followed by native pore-limit gel electrophoresis. Gels were then stained, dried, and developed by autoradiography. T represents the position of 2M tetramers, and D represents the position of 2M dimers. Each gel is the representative binding data for three or more sets of independent experiments for each cytokine/growth factor and 2M pairs performed in triplicate.

Binding of ¹²⁵I-labeled cytokines/growth factors to oxidized ₂M*

Given that oxidation appears to alter cytokine/growth factor binding to ₂M and that ₂M-proteinase complexes may represent a significant portion of the ₂M in inflammatory fluids (30, 47, 48), we performed ₂M* binding experiments using the same ¹²⁵I-labeled ligands as those in Figure 2. Figure 3 shows the changes in ₂M*-associated ¹²⁵I-labeled cytokine/growth factor binding with increasing oxidant concentrations. In contrast to binding to oxidized ₂M, all the labeled ligands showed either a decrease in affinity for oxidized ₂M* or no effect.

View larger version (41K): [in this window] [in a new window]   FIGURE 3. Binding of 125I-labeled cytokines/growth factors to oxidized 2M*. 125I-labeled TNF-, IL-2, IL-6, bFGF, ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2 were added to 2M* oxidized with the indicated concentrations of hypochlorite. Samples were then electrophoresed and developed as described in Figure 2. T represents the position of 2M* tetramers. Each gel shows the representative binding data for three sets of independent experiments for each cytokine/growth factor and 2M* pairs performed in triplicate.

Determination of the oxidation-induced changes in binding K_d values of ₂M and ₂M* to cytokines/growth factors

Of the eight cytokines/growth factors studied, the most significant changes in binding were observed with TNF-, PDGF-BB, ß-NGF, and TGF-ß. To further quantify the oxidation-induced changes in the binding affinity of ₂M for these cytokines/growth factors, we performed concentration-dependent binding assays. Figure 4A shows the results of [¹²⁵I]TNF- binding to oxidized ₂M and oxidized ₂M*. Figure 4B shows the results of [¹²⁵I]PDGF-BB binding. Similar experiments were performed for [¹²⁵I]TGF-ß1 (Fig. 4C) and for [¹²⁵I]ß-NGF (Fig. 4D). A summary of the binding K_d values for these experiments is presented in Table I. As shown in the table, the affinity of ₂M for binding to TNF- increased by 5.3-fold with oxidation. The affinities for TGF-ß1 and ß-NGF decreased by 3.1- and 5.9-fold, respectively. With oxidized ₂M*, an 8.5- and 13.1-fold decreases in affinity were observed for binding to PDGF-BB and TGF-ß1, respectively. These binding K_d values are in close agreement with the results of previous studies using a combined protein cross-linking/electrophoresis assay (17) despite the report that 10 to 20% dissociation of radiolabeled ligand from ₂M is possible during 2-h gel electrophoresis in the absence of cross-linking. We have attempted the combined protein cross-linking/electrophoresis method for our studies but have found a large decrease in the cross-linking efficiency for oxidized proteins compared with nonoxidized proteins, possibly because the lysine residues that are necessary for cross-linking using bis-(sulfosuccinimidyl) suberate have been modified by oxidation (33). Since the binding K_d values between our studies and the previous studies are similar, we assume that the amount of radioligand dissociation during gel electrophoresis does not significantly alter the measurement of K_d values.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Determination of the oxidation-induced changes in binding Kd values of 2M and 2M* to cytokines/growth factors. [125I]TNF- (A), [125I]PDGF-BB (B), [125I]TGF-ß1 (C), or [125I]ß-NGF (D) was incubated with the indicated concentrations of 2M (open circle), oxidized 2M (closed circle), 2M* (open square), and oxidized 2M* (closed square) for 2 h at 37°C followed by native pore-limit gel electrophoresis and denaturing SDS-PAGE. Gels were then stained, dried, and developed by autoradiography. Radioactivity associated with each 2M band was quantified on the PhosphorImager and represented by arbitrary units (AU). Specific noncovalent binding and lines that represent the best least square fit to the data are plotted in the figure. The data represent the mean of three independent experiments performed in triplicate.

View this table: [in this window] [in a new window]   Table I. (Kd) Values for 125I-cytokine/growth factor binding to 2M, 2M*, oxidized 2M, and oxidized 2M*1

Inhibition of [¹²⁵I]TGF-ß1 binding to cell surface receptors by oxidized ₂M and oxidized ₂M*

The binding of cytokines/growth factors to cell surface receptors decreases significantly in the presence of ₂M or ₂M* (10, 13, 14). This effect is directly related to the affinities of ₂M and ₂M* for binding to cytokines/growth factors. Given that oxidation alters the binding affinities of ₂M and ₂M* to these growth factors, we postulated that this may result in altered growth factor binding to cells. We chose [¹²⁵I]TGF-ß as the model ligand for these binding experiments because TGF-ß has high affinities for binding to ₂M and ₂M*, and these interactions may play significant roles in inflammation in vivo (7, 8). Figure 5A shows the results of [¹²⁵I]TGF-ß1 binding to CCL64 cells in the presence of ₂M or oxidized ₂M. As shown in Figure 5, a significant decrease in [¹²⁵I]TGF-ß1 binding was observed in the presence of ₂M (IC₅₀ = 130 nM). This effect was reduced by 4.1-fold in the presence of oxidized ₂M (IC₅₀ = 530 nM). Similar results were obtained with ₂M* and oxidized ₂M*, where the IC₅₀ increased from approximately 10 to 90 nM (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   FIGURE 5. Inhibition of [125I]TGF-ß1 binding to cell surface receptors by oxidized 2M and oxidized 2M*. A, CCL64 cells were grown in 24-well plates until confluent and incubated with [125I]TGF-ß1 (5 ng/well) and the indicated concentrations of 2M (open circle) or 100 µM hypochlorite-oxidized 2M (closed circle) at 4°C for 16 h. Cells were then washed twice with ice-cold buffer and solubilized before gamma counting. Total binding is defined as binding of [125I]TGF-ß1 in the absence of 2M or oxidized 2M (11,000 cpm/well). B, Experiments identical to those in A were performed, except that 2M* (open square) or oxidized 2M* (closed square) was added. Data represent the mean ± SEM of three independent experiments performed in triplicate.

The binding of TGF-ß1 to cell surface receptors is higher in the presence of oxidized ₂M compared with nonoxidized protein (Fig. 5A). This is most likely due to the decreased binding affinity between ₂M and TGF-ß as a result of oxidation. There is another explanation, however, that must be considered. We have recently shown that while unmodified ₂M does not bind to LRP, oxidation results in the exposure of its receptor recognition sites for binding to LRP (33). It is possible that the increase in TGF-ß binding to the cell surface in the presence of oxidized ₂M is the result of the formation of TGF-ß-oxidized ₂M/LRP complexes on the cell surface in addition to TGF-ß/TGF-ß receptor complexes. This would be consistent with the hypothesis that receptor-recognized forms of ₂M may serve as a vehicle that carries growth factors to the cell surface for delivery to growth factor receptors (9, 16). To investigate whether LRP may be involved in binding to oxidized ₂M/TGF-ß complexes, we incubated CCL64 cells with oxidized ₂M and [¹²⁵I]TGF-ß1 in the presence or the absence of a 50-fold molar excess of RAP, which competes for the binding of all ligands to LRP (41) (Fig. 6). As shown in Figure 6, RAP does not significantly alter the cell surface binding of [¹²⁵I]TGF-ß1 in the presence of oxidized ₂M. RAP alone also has no effect on [¹²⁵I]TGF-ß1 binding.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Determination of the role of LRP in regulating [125I]TGF-ß1 binding to cells. CCL64 cells were grown in 24-well plates until confluent and incubated at 4°C for 16 h with [125I]TGF-ß1 (0.5 ng/well) alone or in combination with RAP (6.6 µM), 2M (133 nM), 100 µM hypochlorite-oxidized 2M (133 nM), or unlabeled TGF-ß1 (0.5 µg) as indicated. Following incubation, cells were washed twice with ice-cold buffer and solubilized before gamma counting. Total binding represents the binding of [125I]TGF-ß1 alone (11,000 cpm/well). Data represent the mean ± SEM of three independent experiments performed in triplicate.

Regulation of cytokines/growth factor functions by oxidized ₂M and oxidized ₂M*

The biologic activities of various cytokines/growth factors are decreased in the presence of ₂M and ₂M* (13, 14). To determine whether the altered binding interaction between oxidized ₂M and ₂M* with these cytokines/growth factors can translate into altered cytokine/growth factor bioactivity, we performed in vitro bioassays to test the activities of these cytokines/growth factors in the presence of oxidized and nonoxidized ₂M and ₂M*. Figure 7A shows that in the presence of oxidized ₂M, TNF- activity is inhibited by 66% compared with nonoxidized ₂M, which had no effect. In the presence of ₂M*, however, no significant difference in inhibition was observed between oxidized and nonoxidized protein, consistent with the in vitro binding data. Figure 7B shows that the biologic activity of TGF-ß is significantly decreased in the presence of ₂M (62%) and ₂M* (82%). In the presence of oxidized proteins, however, this inhibition is abolished. Figure 7C shows the effects of ₂M and oxidized ₂M on bFGF bioactivity. Interestingly, there is no difference in inhibition between ₂M and oxidized ₂M despite the apparent increase in oxidized ₂M binding to bFGF in vitro (Fig. 2). For ₂M*, however, a significant decrease in inhibition of bFGF bioactivity is observed with the oxidized protein compared with the nonoxidized protein.

View larger version (19K): [in this window] [in a new window]   FIGURE 7. Regulation of TNF-, TGF-ß, and bFGF functions by oxidized 2M and oxidized 2M*. A, WEHI 13VAR cells were incubated with various concentrations of TNF- in the absence or the presence of 1.0 mg/ml 2M (or 2M*; closed bar) or 1.0 mg/ml 100 µM hypochlorite oxidized 2M (or 2M*; open bar). Following incubation, cell viability was measured using Celltiter 96. TNF- activity in the absence of 2M is defined as 100% activity (EC50 = 0.03 ng/ml). Changes in TNF- activity were calculated as described in Materials and Methods. B, TGF-ß assays were performed as described in A, except that CCL64 cells were used, and cell proliferation was determined using [methyl-3H]thymidine incorporation. TGF-ß activity in the absence of 2M is defined as 100% TGF-ß activity (EC50 = 8 pM). C, bFGF assays were performed as described in B, except that fetal bovine heart endothelial cells were used. The bFGF activity in the absence of 2M is defined as 100% bFGF activity (EC50 = 0.6 ng/ml). Data represent the mean ± SEM of three independent experiments performed in quadruplicate. *, Statistically significant difference (p < 0.05) in cytokine/growth factor activity between oxidized 2M (or 2M*) and nonoxidized 2M (or 2M*) using two-tailed Student’s t test.

Characterization of ₂M from RA and OA synovial fluid

Given that oxidation regulates ₂M-cytokine/growth factor binding in vitro, we investigated the in vivo relevance of this process by asking whether oxidized ₂M is present in the tissue fluids of patients with acute inflammatory diseases such as RA. This hypothesis seems likely given that oxidized proteins have been demonstrated in RASF (49), and increased levels of ₂M are present in this fluid during inflammation (34, 50). ₂M was isolated from the knee joint synovial fluid of six patients with active RA and five patients with OA by affinity chromatography using rabbit anti-human ₂M polyclonal Ab, and the results were verified by Western blotting. As controls, ₂M was also isolated from the plasma of six healthy volunteers by the same method. To determine whether the rheumatoid synovial fluid ₂M is oxidized, we performed DNPH derivatization, which measures the carbonyl content of proteins due to oxidation. Figure 8A shows the mean protein carbonyl content of plasma ₂M, plasma ₂M oxidized in vitro with 100 µM hypochlorite, RASF ₂M, and osteoarthritis synovial fluid (OASF) ₂M. As shown in Figure 8, the level of protein carbonyl was approximately sevenfold higher for RASF ₂M compared with plasma ₂M (p < 0.005). This level is comparable to the level of protein carbonyl generated by oxidizing plasma ₂M in vitro with 100 µM hypochlorite.

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Characterization of RA and OA synovial fluid 2M. A, Plasma 2M (1), plasma 2M oxidized with 100 µM hypochlorite (2), RASF 2M (3), and OASF 2M (4) were treated with DNPH, and the quantity of protein carbonyl was measured as described in Materials and Methods. B, [125I]TGF-ß1 (0.5 ng) was incubated with plasma 2M (1), plasma 2M* (2), plasma 2M oxidized with 100 µM hypochlorite (3), RASF 2M (4), and OASF 2M (5) for 2 h at 37°C. Protein was then electrophoresed on a native pore-limit gel and developed by autoradiography as described in Materials and Methods. Data shown represent the mean ± SEM from three independent experiments for all patient samples. *, Two-tailed Student’s t test comparing RASF 2M with plasma 2M (p = 0.001) and OASF 2M (p = 0.02). **, Two-tailed Student’s t test comparing RASF 2M with plasma 2M (p = 0.02) and OA synovial fluid 2M (p = 0.04).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have identified a physiologic oxidant that can potentially regulate the interaction between ₂M and inflammatory cytokines/growth factors in vivo. The reaction between ₂M and hypochlorite, a powerful oxidant released by the neutrophil H₂O₂-myeloperoxidase-Cl^- system, results in fragmentation of the ₂M tetramer into dimers with an enhanced binding capacity toward acute inflammatory cytokines such as TNF-, IL-2, and IL-6. Binding of ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2 to oxidized ₂M, on the other hand, is significantly decreased. We investigated the potential biologic relevance of this change by demonstrating that the ability of ₂M and ₂M* to inhibit the bioactivity of TGF-ß and bFGF is abolished by oxidation. To determine whether ₂M in inflammatory tissue is oxidized in vivo, we isolated ₂M from the synovial fluid of patients with active RA and found that this protein is significantly more oxidized than plasma and OASF ₂M. The binding of TGF-ß to this protein is decreased as well. Taken together, it appears that oxidation may play an important role in regulating inflammatory cytokine/growth factor functions by altering the affinity of extracellular binding proteins such as ₂M for these signaling molecules.

Reactive oxygen species produced by activated neutrophils and macrophages have been recognized as a hallmark of inflammation (26, 27, 30). Interactions between oxidants and proteins lead to modification of amino acid residues such as cysteine, methionine, histidine, tryptophan, tyrosine, and lysine and increase the carbonyl content and negative surface charge of proteins (26, 27). A large body of evidence has supported the role of oxidants in disease pathogenesis. Oxidation of low density lipoprotein leads to foam cell formation and atherosclerosis, while cigarette smoking generates inhaled oxidants that induce emphysema (28, 51). Despite a long standing interest in the role of oxidants in disease, only recently has there been in-depth investigation of the molecular mechanisms involved in oxidant-mediated tissue injury. Oxidants can interact directly with cells, causing activation of endogenous oxidative stress-mediated signaling pathways involving extracellular-receptor mediated kinase, c-jun N-terminal-activated kinase/stress-activated protein kinase, and/or NF-ß, resulting in either cell proliferation or apoptosis depending on the cell type (52, 53, 54). Activation of the NF-ß pathway induces the production of TNF-, IL-1ß, IL-2, and IL-6 (54), which can further activate the inflammatory cascade. Interaction of oxidants with proteinase inhibitors such as ₁-proteinase inhibitor, secretory leukocyte proteinase inhibitor, and ₂M destroys the inhibitory activity of these antiproteinases while enhancing the proteolytic activity of latent collagenase (30). This may contribute to tissue destruction in adult respiratory distress syndrome, RA, pulmonary emphysema, and glomerulonephritis. On the other hand, oxidants may also be involved in tissue repair mechanisms by stimulating cell proliferation and collagen deposition either alone or in conjunction with growth factors such as PDGF, TGF-ß, bFGF, and ß-NGF (54).

Evidence has accumulated in recent years that ₂M, in addition to its ability to inhibit proteinases, can bind to various cytokines and growth factors with high affinity. Binding of ₂M to these molecules generally results in neutralization of their activities on various different cell types (6, 7). It has been hypothesized that the physiologic role of ₂M in binding to these growth factors is to down-regulate the effects of these extremely potent growth factors by inhibiting their interactions with cell surface receptors and by internalizing bound growth factors via LRP. However, the available data suggest that a mechanism must exist that allows growth factors to be released from ₂M during inflammation. It seems paradoxical that during inflammation when the concentration of ₂M in tissue fluid increases dramatically due to increased vascular permeability and local synthesis, the concentration of growth factors increases as well. In addition, ₂M is present in inflammatory fluids at micromolar concentrations, whereas the affinity of ₂M for growth factors is on the order of nanomolar (10, 17, 18, 19, 20). At this concentration most, if not all, growth factors should be bound to ₂M, yet a large increase in growth factor level and activity has been detected in inflammatory fluids (24, 55, 56). Moreover, despite the finding that TGF-ß and PDGF are both predominantly bound to ₂M in the plasma, to our knowledge no data has demonstrated the isolation of TGF-ß or PDGF-BB/₂M complexes from inflammatory lesions. We hypothesize that hypochlorite oxidation, which selectively and potently inactivates ₂M inhibition of proteinases in vitro, might serve as a mechanism that abolishes the binding of growth factors to ₂M in vivo.

Our initial survey of the binding of eight different cytokines and growth factors to oxidized ₂M reveals an interesting and unexpected finding. The binding of acute inflammatory mediators such as TNF-, IL-2, and IL-6 to ₂M appears to increase significantly with oxidation. In the absence of oxidation, these cytokines all bind to ₂M with micromolar affinity; however, with oxidation, the affinity increases approximately fivefold to nanomolar affinity. The binding K_d value observed between oxidized ₂M and TNF- (340 nM) is similar to the binding K_d values of soluble growth factor receptors to growth factors (57), highlighting the potential importance of this interaction.

The mechanism responsible for this increase is unknown. Binding of cytokines/growth factors to ₂M involves a number of mechanisms including, but not limited to, noncovalent binding, trapping within the ₂M cage, covalent incorporation via the glutamine side chain of the thioester, or disulfide cross-linking (7). Interesting differences in cytokine binding to ₂M and various ₂M/proteinase complexes have been demonstrated (9, 18, 58, 59). Our cytokine binding assay measures only noncovalent association with ₂M and oxidized ₂M. It is possible that changes in other modes of binding can influence the overall effects observed in comparing ₂M and oxidized ₂M. We did not, however, observe any difference in covalent association with oxidized ₂M relative to ₂M, and it appears unlikely that noncovalent trapping could be responsible for this increase given that oxidation fragments ₂M tetramers into dimers. Since the treatment of ₂M with methylamine or plasmin results in increased binding to TNF- (9), it is possible that oxidation can induce a similar structural change; however, neither earlier studies of hypochlorite oxidation of ₂M (31, 32) nor our own studies (33) have demonstrated cleavage of the thioester bond and the major conformational change that resembles ₂M* in oxidized ₂M. Mechanisms that may be responsible for the increased TNF-, IL-2, and IL-6 binding to oxidized ₂M include increased electrostatic interaction, exposure of a previously buried cytokine binding site, increased affinity to an existing cytokine binding site, and increased access to this site. Given that very little is currently known about the exact mechanism of noncovalent cytokine/growth factor binding to ₂M, additional studies will be necessary to determine which of these mechanisms is likely to play a role.

In contrast to the acute inflammatory mediators, our studies show that the binding of growth factors, such as ß-NGF, PDGF-BB, TGF-ß1, and TGF-ß2, to oxidized ₂M is significantly decreased. The affinity of PDGF-BB decreased approximately 9-fold, while the affinity of TGF-ß2 decreased 13-fold. The mechanism of binding by these growth factors to ₂M and ₂M* is primarily noncovalent, and cross-competition between PDGF-BB and TGF-ß1 has been demonstrated (60), suggesting that these growth factors may bind to the same site on ₂M. Oxidation can alter the overall structure of ₂M in such a way as to destroy the three-dimensional conformation required for growth factor binding. This hypothesis is consistent with our recent finding that oxidized ₂M is partially unfolded (33). It is also possible that oxidation selectively modifies a site on ₂M that is involved in binding to growth factors. Supporting this hypothesis is a recent study demonstrating that a polypeptide corresponding to the N-terminal sequence of ₂M binds to TGF-ß (61). Identification of the amino acid residues in this sequence susceptible to oxidation may shed light on the nature of TGF-ß binding to ₂M.

The selective regulation of ₂M cytokine/growth factor binding by oxidation offers an intriguing interpretation for the potential physiologic significance of this interaction. During acute inflammation, activated neutrophils release proteinases and oxidants as part of the endogenous defense mechanisms against foreign organisms. As a result, susceptible extracellular proteins such as ₁-proteinase inhibitor and ₂M may also be oxidized (30). Although the effects of oxidation on cytokines/growth factors have not been reported, nor have oxidized cytokines/growth factors been isolated from inflammatory fluids, the possibility exists that oxidation may directly alter cytokine/growth factor functions. The finding that oxidized ₂M binds with greater affinity to acute phase cytokines such as TNF-, IL-2, and IL-6 suggests that oxidized ₂M may play an anti-inflammatory role by inhibiting the progression of the proinflammatory cascade induced by these molecules. In this regard, the decreased binding of oxidized ₂M and ₂M* to TGF-ß, PDGF-BB, and ß-NGF, all of which have been considered as inflammatory cytokines/growth factors, argues against the anti-inflammatory role of oxidized ₂M. Inflammation, however, is complex process involving multiple different phases (62). Growth factors such as ß-NGF, bFGF, PDGF-BB, and TGF-ß have generally been considered mediators of tissue repair processes including neurite out-growth, angiogenesis, fibroblast proliferation, and collagen deposition. It is possible that oxidation of ₂M may play a different role at different stages of the inflammatory process. In this regard, it is worth mentioning that ₂M oxidation, which results in greater in vitro binding affinity to bFGF, has no differential effect on bFGF stimulation of endothelial cell proliferation (Fig. 7C). Oxidation of ₂M*, however, results in decreased binding affinity to bFGF as well as decreased ability to inhibit the biologic activity of bFGF. Taken together, we offer the hypothesis that oxidation may facilitate the transition from tissue damage to repair during inflammation by enhancing the ability of ₂M to bind to proinflammatory molecules while decreasing its ability to bind to tissue repair molecules. This would suggest that in the absence of oxidation (as in patients with chronic granulomatous disease) chronic and persistent inflammation may occur due to poor transition from acute inflammation to resolution. It should be pointed out that this hypothesis may apply only to the inflammatory process once neutrophil activation has occurred, since chemokines, which play important roles in leukocyte chemotaxis and transmigration into inflammatory sites, were not investigated in this study, and HOCl is known to be released by neutrophils only after they have become activated. Additional in vivo studies will be necessary to determine whether this hypothesis is correct. We herein offer some additional evidence that suggests our hypothesis is possible.

Using RA as a model of an inflammatory disease with a prominent tissue repair phase, we isolated synovial fluid ₂M from the knee joints of six patients with this disease. RASF ₂M from these patients was sevenfold more oxidized than control plasma ₂M and threefold more oxidized than ₂M isolated from OASF, which is generally considered a noninflammatory fluid. The binding of RASF ₂M to TGF-ß1 was significantly decreased as well. Protein oxidation has been demonstrated in patients with RA, adult respiratory disease syndrome, atherosclerosis, bronchitis, and a number of other inflammatory diseases (49, 63, 64, 65). Oxidation of ₂M has been suggested to be the mechanism involved in enhanced tissue degradation in RA (66). To date, however, no study has isolated and characterized oxidized ₂M in disease tissues and fluids. Our studies with RASF ₂M are the first to demonstrate the presence of oxidized ₂M in human disease tissue. That RASF ₂M, moreover, has significantly decreased ability to bind to TGF-ß1 suggests that oxidation may play a major role in regulating the functions of extracellular cytokine/growth factor binding proteins.

	Acknowledgments

 
We thank Drs. George Cianciolo and Maureane Hoffman for their comments on the manuscript, and Marie Thomas for her assistance with preparation of the manuscript.

	Footnotes

 
¹ This work was supported in part by National Heart Lung and Blood Institute (Grant HL-24066, in part by NIAMS (National Institute of Arthritis and Musculoskeletal and Skin Diseases) Grant AR39162 (to D.D.P.), and in part by Medical Scientist Training Program GM-07171 (to S.M.W.) and a predoctoral research fellowship from the American Heart Association, North Carolina affiliate (to S.M.W.).

² Address correspondence and reprint requests to Dr. Salvatore V. Pizzo, Department of Pathology, Duke University Medical Center, Durham, NC 27710. E-mail address:

³ Abbreviations used in this paper: LRP, low density lipoprotein receptor-related protein; ₂M*, ₂-macroglobulin-methylamine; bFGF, basic fibroblast growth factor; ß-NGF, ß-nerve growth factor; PDGF-BB, platelet-derived growth factor BB homodimer; RA, rheumatoid arthritis; RASF, rheumatoid arthritis synovial fluid; RAP, receptor-associated protein; OA, osteoarthritis; DNPH, 2,4-dinitrophenylhydrazine; OASF, osteoarthritis synovial fluid.

⁴ In this paper, the abbreviation ₂M will be used to represent all ₂M with the native conformation, and ₂M* will be used to represent ₂M-proteinase as well as ₂M-methylamine complexes.

Received for publication January 15, 1998. Accepted for publication June 17, 1998.

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