Oxygen Radicals and Nitrite Covalent Cross-Linking of Immune

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Covalent Cross-Linking of Immune Complexes by Oxygen Radicals and Nitrite Masaaki Uesugi, Takeshi Hayashi and Hugo E. Jasin This information is current as J Immunol 1998; 161:1422-1427; ; of September 30, 2021. http://www.jimmunol.org/content/161/3/1422

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Covalent Cross-Linking of Immune Complexes by Oxygen Radicals and Nitrite1

Masaaki Uesugi,* Takeshi Hayashi,† and Hugo E. Jasin2*

We have shown that polymorphonuclear neutrophils mediate the covalent cross-linking of immune complexes (ICs) using H2O2 and myeloperoxidase (MPO). Moreover, activated superﬁcial chondrocytes produce large amounts of nitric oxide (NO), suggesting that high concentrations of these radicals may interact at the cartilage surface in rheumatoid arthritis. We describe the effects of the interaction of NO and its decay product, NO2, with H2O2 and MPO on IC cross-linking. Cross-linking was measured by resistance to the guanidine extraction of plastic-bound ICs. The combination of H2O2, MPO, and NO in the absence of O2 did not alter the magnitude of cross-linking. The addition of O2 resulted in a signiﬁcant enhancement of cross-linking (p < 0.004), suggesting that nitrite was responsible for the increase observed. Indeed, NaNO2 greatly increased H2O2-dependent cross-linking Downloaded from ؎ ؎ ؎ (control: 29.2 3.8; 1 mM NaNO2: 58.4 9.9; 10 mM: 60.4 4.2% cross-linking, p < 0.0002). Sodium azide, which is an inhibitor of MPO, completely inhibited cross-linking. These results indicated that the product of interaction of H2O2 and NO2 mediated by

MPO may be responsible for the increase in cross-linking. The generation of nitrotyrosine was demonstrated when NO2 was added ؊ to the cross-linking system. Cross-linking was also shown with an O2 -generating system and NO. Peroxynitrite alone mediated cross-linking (100 ␮M ONOO؊: 40.3 ؎ 1.9% cross-linking; p < 0.002), and the addition of MPO signiﬁcantly enhanced this effect ␮M: 57.7 ؎ 6.0%; p < 0.0002 with respect to no nitrite control). Oxygen radicals and NO are likely to interact at the cartilage 100) surface in inﬂammatory arthritis, resulting in an increase in oxidative damage within the joint cavity. The Journal of Immu- http://www.jimmunol.org/ nology, 1998, 161: 1422–1427.

ighly reactive oxygen-derived species (ROS)3 play im- tightly bound and sequestered on the pannus-free articular surface portant roles in defense mechanisms against infections (13). It was suggested that these complexes gave rise to the phe- H (1) and in tissue injury in inﬂammatory reactions (2, 3). nomenon of frustrated phagocytosis (14, 15) on the cartilage sur- One of the main reactive products generated by activated phago- face, resulting in the egestion of noxious neutrophil granular com- cytic cells is hypochlorous acid (HOCl), which is formed by the ponent such as ROS and proteases. Indeed, there is abundant

enzyme myeloperoxidase (MPO) acting on H2O2 in the presence evidence that a variety of neutrophil products are present at the by guest on September 30, 2021 of chloride ion. This oxidant is mainly responsible for the intra- cartilage surface and at the cartilage-pannus junction (16–19). cellular killing of phagocytosed bacteria (1); it also plays a role in Articular chondrocytes generate large amounts of nitric oxide tissue injury because it mediates the oxidative modification of tis- (NO) when appropriately stimulated with cytokines or bacterial sue macromolecules, the deamination and decarboxylation of pro- products (20, 21). We and others have recently shown that most of teins with the formation of aldehydes, sulfhydryl oxidation, and the oxidant gas is secreted by the chondrocytes located close to the covalent cross-linking (4–10). We have shown previously that articular surface (22, 23). Moreover, recent data indicate that the HOCl is responsible for the covalent cross-linking of proteins, human neutrophil is also able to secrete NO when appropriately Ϫ namely IgG, resulting in the generation of immune complex (IC)- stimulated (24). Chemical data suggest that NO reacts with O2 like aggregates with phlogogenic capacity (10, 11). Moreover, IgG and other reactive molecules to form stronger oxidant molecules aggregates with evidence of oxidative modification have been such as peroxynitrite (25, 26) and nitryl chloride (27). Similarly, Ϫ found in the synovial fluids of patients with rheumatoid arthritis the dismutation of O2 by superoxide dismutase and the subse- (12). Previous work in our laboratory also indicated that the ma- quent generation of H2O2 reacting with nitrite in the presence of jority of articular cartilage specimens obtained from patients with MPO may give rise to another strong oxidant, nitrogen dioxide ⅐ rheumatoid arthritis contained ICs and complement that were (NO 2) (28). Since the considerations discussed above indicate that high concentrations of ROS, neutrophil granular material such as

*Division of Rheumatology and Clinical Immunology, University of Arkansas for MPO, and NO may interact at the articular surface in inflammatory Medical Sciences and John L. McClellan Veterans Administration Center, Little arthritis, the present studies were conducted to study the effects of Rock, AR 72205; and †Department of Orthopedic Surgery, Yokohama City Univer- NO and its decay products on the protein cross-linking activity of sity School of Medicine, Yokohama, Japan neutrophil-derived ROS. Received for publication August 18 1997. Accepted for publication March 30, 1998. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods Reagents 1 This work was supported by U.S. Public Health Service Grant AR-16209. Ј 2 Address correspondence and reprint requests to Dr. Hugo E. Jasin, UAMS-Mail Slot Sodium hypochlorite and 5,5 -dithiobis(2-nitrobenzoic acid) were pur- 509, 4301 W. Markham, Little Rock, AR 72205. chased from Sigma (St. Louis, MO). Rabbit anti-BSA Ab was purified as described previously (10, 29). Purified human MPO was a gift from Dr. 3 Abbreviations used in this paper: ROS, reactive oxygen-derived species; IC, immune complex; GO, glucose oxidase; MPO: myeloperoxidase; XO: xanthine oxidase; Isaac Ginsburg (Hebrew University, Hadassah School of Dental Medicine, PMN, polymorphonuclear neutrophil; NO, nitric oxide; TNB, 5-thio-2-nitrobenzoic Jerusalem, Israel) (10). NO gas (10% concentration) was purchased from acid; PBS-RS, PBS containing 10% heat-inactivated rabbit serum; SNP, sodium ni- Matheson (Montgomeryville, PA). HOCl was obtained by vacuum distil- troprusside. lation of a 5% solution of sodium hypochlorite (pH 7.5) (30). The resulting

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 1423 product was stored at Ϫ80°C. Concentrations were determined by spectropho- ⑀ ϭ Ϫ1 Ϫ1 tometric analysis ( 235 100 M cm ) (31) (Beckman Instruments, Ful- lerton, CA). Mouse monoclonal anti-nitrotyrosine IgG was purchased from Upstate Biotechnology (Waltham, MA). Affinity-purified goat anti-mouse Ig was purchased from Biosource International (Camarillo, CA). Preparation of NO-saturated HBSS HBSS was saturated with NO gas as described by Harry et al. (32) with modifications. A total of 10 ml of HBSS without phenol red was bubbled for 15 min with nitrogen gas and subsequently with 10% NO gas followed by air for 15 min in studies in which the presence of O2 was required. NO-HBSS was prepared freshly before use. NO2 concentrations were determined by the Griess reaction (33). Briefly, 100 ␮l of diluted NO-saturated HBSS or standard solution was mixed with 50 ␮l of 0.1% naphtha- lene diamine dihydrochloride and 50 ␮l of 1% sulfanilamide, 5% phosphoric acid. Standards were prepared with 10 to 250 ␮M sodium nitrite. After a 5-min incubation, the OD at 540 nm was measured with a microplate reader (400ATC, SLT Labs, Triangle Park, NC). Synthesis of 5-thio-2-nitrobenzoic acid (TNB) FIGURE 1. Effect of NO gas in air-saturated HBSS on the H2O2-MPO-

The synthesis of TNB was conducted according to Ching et al. (34). A total mediated covalent cross-linking of ICs. Plastic-bound BSA/anti-BSA com- Downloaded from of 20 mM of sodium borohydride was added to a 1 mM solution of 5,5Ј- plexes were incubated for 1 h with 22 mU/ml of MPO and 0.1 U/ml GO Ϯ dithiobis(2-nitrobenzoic acid) in 50 mM potassium phosphate buffer (pH NO gas. *p Ͻ 0.0002; n ϭ 4. 6.6) plus 5 mM EDTA. The solution was incubated at 37°C for 30 min. The ⑀ ϭ concentrations were determined by spectrophotometric analysis ( 412 13,600 MϪ1 cmϪ1) (35). Colorimetric assay for H2O2 Synthesis of peroxynitrite Hydrogen peroxide was measured as described by Pick and Keisari (37). The method is based on the colorimetric measurement of oxidized phenol http://www.jimmunol.org/ Peroxynitrite was synthesized from sodium nitrite and hydrogen peroxide red. Concentrations were calculated from a standard curve using serial according to Rakesh and Victor (36). Two syringes containing 6 ml of 2 M dilutions of an H2O2 solution. cold NaNO2 and 6.6 ml of 30% cold H2O2 plus 5% HNO3 were used to mix the two solutions rapidly before neutralization with 6 ml of 4.2 M NaOH Purification of blood leukocytes solution that had been stirred and kept in an ice bath. Residual H2O2 was removed by passing the solution through a column of granular MnO2 that PMNs were purified from 15 ml of heparinized blood from healthy donors had been rinsed with 0.5 M NaOH and kept at 4°C. The concentration of by isopicnic centrifugation using Polymorphoprep (Accurate Chemical and ⑀ ϭ peroxynitrite was determined spectrophotometrically ( 302 1670 Scientific, Westbury, NY). PMNs were washed twice with large amounts MϪ1 cmϪ1). of HBSS without phenol red; contaminating E were lysed with 0.2% NaCl solution. After further washing with HBSS, the cell suspension was ad- Cross-linking of plastic-bound ICs justed to 106 cells/ml. As a rule, cell viability was Ͼ99% as determined by by guest on September 30, 2021 the trypan blue dye exclusion test. We have described previously the methodology for the generation of cross- linked ICs on plastic plates (10). Round-bottom, flexible, polyvinyl chlo- Detection of nitrotyrosine ride 96-well microtiter plates (Dynatech Laboratories, Chantilly, VA) were coated with 50 ␮l BSA 20 ␮g/ml in saline solution. After a 2-h incubation, The nitrosation of the tyrosine residues of BSA mediated by H2O2, MPO, the wells were washed twice with 100 ␮l of PBS and blocked twice with and nitrite was detected by radioimmunoassay as described by Crow et al. 100 ␮l of PBS containing 10% heat-inactivated rabbit serum (PBS-RS) for with some modifications (38). We coated 96-well polyvinyl chloride plates 10 min. ICs were generated by2hofincubation with 50 ␮l of a 1:100 with 50 ␮l BSA solution, 20 ␮g/ml for2hatroom temperature. The coated dilution of affinity-purified 125I-labeled rabbit-anti-BSA Ab in PBS-RS at wells were incubated with 44 mU/ml of MPO, 0.1 U/ml of GO, and 0.1 to room temperature. The cross-linking of ICs was achieved by incubation 10 mM NaNO2 in chloride-free buffer (0.02 M KH2PO4,0.08MNa2HPO4, with MPO, glucose oxidase (GO), and glucose as a source of hydrogen 10 mg/dl MgSO4, and 100 mg/dl glucose (pH 7.4)) for1hat37°C in the peroxide. The wells contained 22 mU/ml of MPO, 0.1 U/ml of GO with or presence of increasing concentrations of sodium nitrite or sodium nitrate as without NaNO2, or 0.01 to 1 mM of NO-saturated HBSS in a total volume control. Additional controls consisted of wells in which MPO was omitted. of 50 ␮l. Additional experiments were conducted with 10 ␮M HOCl, The wells were then washed twice with chloride-free phosphate buffer, MPO, and GO or with 0.1 and 1.0 mU/ml xanthine oxidase (XO) and 1.5 twice with PBS, and twice with PBS and 0.05% Tween 20 for 10 min. mM hypoxanthine with or without 2.0 mM sodium nitroprusside (SNP). Next, the wells were incubated with 50 ␮l of a 1:500 dilution of anti- After incubation with MPO, HOCl, or XO at 37°C for 1 h, the reaction was nitrotyrosine Ab in PBS and 0.05% Tween 20 for2hatroom temperature. terminated by removing the HBSS and washing twice with 100 ␮l of PBS- After washing twice with PBS and blocking twice with PBS and 0.05% RS. Sextuplicate wells were divided into two groups. The first group of Tween 20, the wells were incubated with 50 ␮l of a 1:100 dilution of triplicate wells was washed twice with 100 ␮l of 0.1 N HCl containing 10% 125I-labeled goat anti-mouse Ig in PBS and 0.05% Tween 20 for 2 h. Sub- rabbit serum and twice with 100 ␮l of 4 M guanidine solution (4 M gua- sequently, the wells were washed twice and counted with an autogamma nidine, 0.1 M EDTA, 0.1 M NaCl, 0.05 M Tris, and 0.01 M sodium azide) scintillation spectrometer. for 10 min each to remove noncovalently bound IgG. The other group of wells was washed four times with PBS-RS for 10 min. Controls consisted Statistics of wells incubated without MPO. Plastic-bound radioactivity was measured The number of experiments (n) refers to the number of separate experi- in a Cobra-2 autogamma scintillation spectrometer (Packard Instrument ments, where the results for each experiment were the mean value of trip- Company, Meriden, CT). The quantification of the cross-linking of ICs was licate wells. The results were analyzed by ANOVA. calculated as the percent radioactivity bound ϭ cpm/well washed with HCl and 4 M guanidine/cpm/well washed with PBS ϫ 100; net percent bound ϭ percent bound with MPO Ϫ percent bound without MPO. Results

Colorimetric assay for oxidative activity Incubating ICs with GO-glucose as a source of H2O2 and also with MPO resulted in covalent cross-linking of high magnitude as The oxidative activity of MPO or polymorphonuclear neutrophils (PMNs) shown previously (Fig. 1) (10). Incubating ICs with GO, MPO, was measured as described by Ching et al. (34). The wells containing 250 mU/ml of MPO, 0.01 U/ml of GO, or 5 ϫ 105 PMN/ml, and 70 ␮Mof and NO-saturated HBSS in the presence of O2, however, almost doubled cross-linking at an initial concentration of 1 mM (no NO: TNB in HBSS with or without 0.1 to 10 mM NaNO2 were incubated for 1 h at 37°C. The OD was measured at 412 nm. 31.7 Ϯ 5.2%; 0.01 mM NO: 25.1 Ϯ 5.2%; 0.1 mM NO: 43.6 Ϯ 1424 PROTEIN CROSS-LINKING BY NITRITE

FIGURE 2. Effect of sodium nitrite on the H2O2-MPO-mediated cross- FIGURE 4. Effect of sodium nitrite on the chloride-free H2O2-MPO- linking of ICs. For experimental details, see Figure 1. *p Ͻ 0.008; **p Ͻ Downloaded from mediated cross-linking of ICs. *p Ͻ 0.01; **p Ͻ 0.0001; n ϭ 3. 0.006; n ϭ 4.

Ϯ Ͻ 4.0; 1 mM NO: 67.3 5.9%; p 0.0002) (Fig. 1). In the absence was not required, and its cross-linking capacity was inhibited by of O2, no increase in covalent cross-linking was detected (data not nitrite. shown). The next series of experiments was designed to investigate the http://www.jimmunol.org/ The above results suggested that the oxidative product of NO, effects of nitrite on MPO activity. The activity of MPO, as mea- NO2, was responsible for the increase in IC cross-linking. Indeed, sured by the oxidation of TNB, was significantly enhanced by the H2O2-dependent cross-linking of plastic-bound ICs in the pres- nitrite in a dose-dependent manner (Fig. 6). Similarly, the addition ence of MPO was significantly increased by 1 and 10 mM NaNO2 of increasing concentrations of nitrite to stimulated PMNs resulted Ϯ Ϯ Ͻ (no NO2: 29.2 3.8%; 1 mM NaNO2: 58.4 9.9%; p 0.008; in a significant increase in oxidative activity as measured with Ϯ 10 mM NaNO2: 60.4 4.2%) (Fig. 2). Sodium nitrate in concen- TNB as a substrate (Fig. 7). To rule out the possibility that the trations as high as 10 mM did not affect cross-linking (data not nitrite effects observed may have been due to increased H2O2 pro- shown). Moreover, when NaNO2 was added to an optimal dilution duction by GO, the enzyme activity was measured in the presence of purified HOCl, the salt inhibited protein cross-linking in a dose- or absence of sodium nitrite. As shown in Figure 8, 1 mM of by guest on September 30, 2021 dependent fashion (Fig. 3). As previously shown (10), H2O2 and sodium nitrite did not affect the production of H2O2;10mMof MPO in the absence of chloride resulted in insignificant cross- sodium nitrite partially reduced its production in the face of a linking. However, the absence of chloride did not affect nitrite- concomitant increase in protein cross-linking, as shown previously dependent cross-linking (Fig. 4). These findings reinforced the (Fig. 2). suggestion that nitrite was able to mediate this phenomenon in the Since the nitrosation of tyrosine can be generated by the product presence of H2O2 and MPO. This conclusion was supported by the of H2O2 and nitrite (28), experiments were conducted to examine observation that sodium azide, which is an inhibitor of MPO, also this possibility. Plastic-bound BSA was incubated with the H2O2- inhibited nitrite-dependent cross-linking (Fig. 5). The aggregate of generating system in the presence or absence of nitrite. As shown these results indicated that a nitrite-mediated enhancement of protein cross-linking required the presence of MPO and H2O2; HOCl

FIGURE 5. Effect of sodium azide on the H2O2-MPO-mediated cross- FIGURE 3. Effect of sodium nitrite on the HOCl-mediated cross-link- linking of ICs. Control: no sodium nitrite or azide (100%); Open bars: with ing of ICs. Plastic-bound ICs were incubated with 10 ␮M freshly distilled sodium azide; stippled bars: no sodium azide. *p Ͻ 0.04 with respect to an HOCl Ϯ sodium nitrite. *p Ͻ 0.02; **p Ͻ 0.004; n ϭ 3. absence of azide control; n ϭ 3. The Journal of Immunology 1425

FIGURE 6. Effect of sodium nitrite on MPO activity as measured by FIGURE 8. Effect of sodium nitrite on H O production by GO. *p Ͻ TNB oxidation. *p Ͻ 0.001; n ϭ 3. 2 2 0.0001; n ϭ 3. Downloaded from in Figure 9, easily detectable nitrosation was seen after a 1-h in- Ϫ Ϯ Ͻ cubation with 0.1 mM nitrite, reaching a maximal increase in ni- ONOO : 40.3 1.9% cross-linking; p 0.002) and the addition ␮ Ϫ Ϯ trotyrosine at a nitrite concentration of 1 mM. No nitrosation was of MPO signiﬁcantly enhanced this effect (1.0 M ONOO : 6.0 ␮ Ϫ Ϯ ␮ Ϫ Ϯ detected with nitrate or when MPO was omitted; this observation 5.1%; 10 M ONOO : 7.1 5.5%; 100 M ONOO : 55.7 ϭ

6.0%; p 0.05 vs no MPO) (Fig. 10). http://www.jimmunol.org/ supports our previous findings, which indicated that H2O2 and nitrite were necessary and sufficient for the generation of nitrotyrosine and protein cross-linking. Discussion The aggregate of these results suggested that the product of There is considerable evidence that in inflammatory foci such as interaction between NO2 and H2O2 that was catalyzed by MPO the involved joint in rheumatoid arthritis, ROS and NO-derived may have been responsible for the increase in IC cross-linking reactive products play important pathogenic roles in tissue damage Ϫ observed. Since activated PMNs generate O2 , an alternative path- (39). Thus, evidence of oxidative damage of rheumatoid synovial way may involve the product of the interaction of this radical with fluid proteins has been reported by us (12) and others (40); in NO, namely peroxynitrite. The next series of experiments were addition, increased concentrations of nitrite (41) and the presence by guest on September 30, 2021 designed to explore this possibility. Adding increasing concentra- of nitrotyrosine (42) in synovial fluids indicate that NO-derived Ϫ tions of NaNO2 to the O2 generated by XO and hypoxanthine did products also play a significant role. There is also abundant evi- not result in any detectable cross-linking or nitrotyrosine genera- dence that a variety of neutrophil components are present at the tion (data not shown). However, significant protein cross-linking cartilage surface and at the cartilage-pannus junction (16-19). We was obtained (O.1 mU/ml XO: 4.1 Ϯ 0.9%, p Ͻ 0.02; 1.0 mU/ml have previously shown that one of the major oxidative products of XO: 5.2 Ϯ 1.4%, p Ͻ 0.05, crosslinking over control without SNP) PMNs, namely HOCl, is responsible for the covalent cross-linking Ϫ when a NO donor (SNP, 1 mM) was added to the O2 -generating of ICs (10). Moreover, we have also obtained evidence that su- system, suggesting that the resulting peroxynitrite was responsible perficial articular chondrocytes generate large amounts of NO for these results. Indeed, increasing concentrations of freshly syn- when appropriately stimulated with cytokines or bacterial products thesized peroxynitrite alone mediated cross-linking (1.0 ␮M ONOOϪ: 2.2 Ϯ 3.7%; 10 ␮M ONOOϪ: 0.7 Ϯ 3.7%; 100 ␮M

FIGURE 9. Effect of sodium nitrite on the H2O2-MPO-mediated generation of nitrotyrosine. Plastic-bound BSA was incubated with increasing FIGURE 7. Effect of sodium nitrite on the oxidative activity of PMNs concentrations of sodium nitrite in the presence or absence of MPO. Open as measured by TNB oxidation. *p Ͻ 0.003; n ϭ 4. bars: no MPO; stippled bars: with MPO. *p Ͻ 0.0001; n ϭ 3. 1426 PROTEIN CROSS-LINKING BY NITRITE

form ONOOϪ, which is a well-known nitrosating and cross-link- Ϫ ing species (38, 39). Moreover, O2 generated by inflammatory cells would readily dismutate to H2O2 in the presence of the superoxide dismutase found in inflammatory cells and rheumatoid synovial fluids (46, 47). This oxidant may mediate the same effects in conjunction with nitrite and MPO as shown in these studies. To complicate things even further, tyrosine chlorination and nitrosation may also occur as a result of the interaction of HOCl and NO, giving rise to nitryl chloride or similar chloride- and NO-derived free radicals (27, 48). Finally, ONOOϪ can decay spontaneously to form hydroxyl radical and nitrogen dioxide (25), another molecule which is thought to mediate tyrosine nitrosation (28). The presence of the inhibitors of these reactions (chloride, as shown here, or bicarbonate (49) in body fluids) introduces yet another complicat- ing factor. At the present time, it is not possible to determine which or how many of these pathways may be operative in inflammation. The relatively large concentrations of nitrite that are required to FIGURE 10. Effect of peroxynitrite on the cross-linking of ICs. Open mediate protein cross-linking in our experiments raise questions as ϭ ϭ Downloaded from bars: no MPO; stippled bars: with MPO. *p 0.05 vs no MPO; n 4. to the biologic significance of the results, since the nitrite concentrations measured in inflammatory fluids are lower by at least two orders of magnitude. Several factors may help explain this require- (23). Recent data by other investigators indicate that the human ment. The nitrite “accumulation” measured in biologic fluids prob- neutrophil is also able to secrete NO when appropriately stimu- ably represents a gross underestimation, taking into account its Ϫ lated (24). Chemical data suggest that NO reacts with O2 and rapid equilibration with the vascular compartment, the removal of other reactive molecules to form strong oxidant molecules such as nitrite by locally produced HOCl (27), and the real concentration http://www.jimmunol.org/ Ϫ peroxynitrite (25, 26) and nitryl chloride (27). Alternatively, O2 - probably achieved within the inflammatory cell phagosomes in derived H2O2 reacting with nitrite also gives rise to another strong which H2O2, MPO, and NO synthase are known to coexist. More- ⅐ oxidant, probably NO2 (28). However, NO has been shown to have over, higher nitrite concentrations were required to prevent protein both deleterious and protective effects in pathologic states. The fragmentation mediated by strong oxidative attack, because we considerations discussed above suggest that high concentrations of used limiting amounts of MPO and GO in our experiments. ROS, PMN granular products, and NO may interact at the articular Regardless of the relative importance of each of the reactions surface in inflammatory arthritis; this finding prompted the present outlined above, one of the possible corollaries of the results pre-

studies dealing with the effects of NO and its decay products on the sented stems from our previous observations that both in the su- by guest on September 30, 2021 oxidizing activity of PMN-derived ROS. perficial layers of cartilage obtained from an experimental model The results obtained indicated that NO gas under anaerobic con- of rheumatoid arthritis (50) and in superficial cartilage from pa- ditions did not interfere with HOCl-mediated protein cross-linking. tients with this disease (13), we found large amounts of ICs tightly However, NO significantly enhanced cross-linking in the presence bound to the tissue, so that even exposure to high molar guanidine of O2, suggesting that the oxidation end-product of NO, namely solutions was not sufficient to extract them. Extraction could only nitrite, may have been responsible for this enhancement. Addi- be achieved with a dissolution of the tissue using bacterial colla- tional experiments clearly indicated that NO2 and not NO3 was genase (51), suggesting that the ICs were covalently bound to the able to increase MPO-mediated cross-linking in the absence of underlying cartilage macromolecules. Ongoing studies in our lab- chloride ion. Indeed, NO2 was shown to partially quench HOCl- oratory may help clarify some of the unresolved issues discussed mediated protein cross-linking, suggesting that cross-linking was above. solely the result of the interaction of NO2 with MPO and H2O2. The enhanced oxidative capacity of MPO in the presence of NO2 was also evident when an alternative substrate (TNB) was used, References and, more pertinent to biology, when PMNs were the source of 1. Babior, B. M. 1978. Oxygen-dependent microbial killing by phagocytes. N. Engl. ROS. These findings are also in accordance to previous work J. Med. 298:659. 2. Miller, R. A., and B. E. Britigan. 1995. The formation and biologic significance showing that MPO reacts directly both with NO2 (28, 43) and Ϫ of phagocyte-derived oxidants. J. Invest. Med. 43:39. ONOO (44). 3. Klebanoff, S. J. 1982. Oxygen-dependent mechanisms of phagocytes. In Ad- Finally, the covalent cross-linking of ICs was demonstrated with vances in Host Defense Mechanisms, Vol. 1. J. I. Gallin and A. S. Fauci, eds. Ϫ Raven Press, New York, p. 111. a source of O2 and a NO donor, and in addition with a low Ϫ 4. Clark, R. A. 1983. Extracellular effects of the myeloperoxidase-hydrogen perox- concentration of freshly synthesized ONOO that was further ide-halide system. In Advances in Inflammation Research, Vol. 5. enhanced by the addition of MPO. The modest increase in cross- G. Weissman ed. Raven Press, New York, p. 107. linking observed when MPO was added suggests that ONOOϪ 5. Sizer, I. W. 1953. Oxidation of proteins by tyrosinase and peroxidase. In Ad- vances in Enzymology and Related Subjects of Biochemistry, Vol. 14. F. F. Nord, was able to mediate this reaction by itself, since most of the cross- ed. Wiley Interscience Publishers, Inc., New York, p. 129. linking observed took place with the oxidant alone. 6. Strauss, R. R., B. B. Paul, A. A. Jacobs, and A. J. Sbarra. 1971. Role of the Ϫ The chemical pathways resulting in the modification of macro- phagocyte in host-parasite interactions: Myeloperoxidase-H2O2-Cl -mediated al- molecules as found in inflammatory foci in vivo are complicated dehyde formation and its relationship to antimicrobial activity. Infect. Immun. 3:595. and have not been completely clarified. The t1/2 of NO is probably 7. Zgliczynski, J. M., W. Stelmaszynska, W. Ostrowski, J. Nashalski, and J. Sznajd. very short in inflammatory fluids (45), reacting with various sub- 1968. Myeloperoxidase of human leukaemic leucocytes: oxidation of amino acids in the presence of hydrogen peroxide. Eur. J. Biochem. 4:540. strates and decaying to its oxidized byproduct, NO2, which has 8. Selvaraj, R., B. B. Paul, R. R. Strauss, A. A. Jacobs, and A. J. Sbarra. 1974. been found to be increased in inflammatory synovial fluids (41). Ϫ Ϫ Oxidative peptide cleavage and decarboxylation by the MPO-H2O2-Cl antimi- Since NO is generated intracellularly, it may react with O2 to crobial system. Infect. Immun. 9:255. The Journal of Immunology 1427

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