Mononuclear Cells Production by Stimulated Peripheral Blood ...

The Journal of Immunology

Enhanced Activity of Human IL-10 After Nitration in Reducing Human IL-1 Production by Stimulated Peripheral Blood Mononuclear Cells1 Jon L. Freels,* Dan K. Nelson,* Jeffrey C. Hoyt,* Michael Habib,* Hiroki Numanami,* R. Clark Lantz,† and Richard A. Robbins2* Nitric oxide and superoxide form the unstable compound, peroxynitrite, which can nitrate proteins and compromise function of proinflammatory cytokines at sites of inflammation. Reduced function of proinflammatory proteins such as IL-8, macrophage inflammatory protein-1␣, and eotaxin suggest an anti-inflammatory effect of nitration. The effects of nitration on anti-inflammatory cytokines such as IL-10 are unknown. We hypothesized that peroxynitrite would modify the function of anti-inflammatory cytokines like IL-10. To test this hypothesis, the capacity of recombinant human IL-10 to inhibit production of human IL-1␤ (IL-1) from LPS-stimulated human PBMC was evaluated. Human IL-10 was nitrated by incubation with peroxynitrite or by incubation with 3-morpholinosydnonimine, a peroxynitrite generator, for 2 h and then incubated with LPS-stimulated PBMC for 6 h, and IL-1 was measured in the culture supernatant fluids. Human IL-1 production was significantly lower in the peroxynitrite- or 3-morpholinosydnonimine-nitrated IL-10 group than in the IL-10 controls (p < 0.05, all comparisons). This finding demonstrates that although peroxynitrite inhibits proinflammatory cytokines, it may augment anti-inflammatory cytokines and further point to an important role for peroxynitrite in the regulation of inflammation. The Journal of Immunology, 2002, 169: 4568 – 4571.

H

uman IL-10 is known to be immunosuppressive by its ability to inhibit the synthesis of Th1 cell-derived cytokines, tumor necrosis factor, and IFN-␥ (1). Administration of IL-10 is currently being investigated in the prevention of acute lung injury, intestinal ischemia-reperfusion, inflammatory bowel disease, rheumatoid arthritis, psoriasis, and multiple sclerosis (2). IL-10 found on chromosome 1 (3) is an 18- to 20-kDa monomer that forms a homodimer protein consisting of 160 aa (4). IL-10 is expressed in monocytes/macrophages, CD4⫹ T cells and Th0, Th1, Th2 cell clones, CD8⫹ T cells and clones, keratinocytes, activated B cells, B lymphomas, and Burkitt lymphoma lines infected with transforming EBC strain (5). In human PBMC, IL-10 inhibits the synthesis of IL-1, IL-6, IL-8, IL-12, and TNF (6). The C-terminal end of human IL-10 contains a 9-aa peptide (Ala-TyrMet-Thr-Met-Lys-Ile-Arg-Asn) that has a tyrosine at position 153. This amino acid sequence is one of the functional domains of human IL-10 (7). Peroxynitrite compromises proinflammatory cytokine function and can be considered an anti-inflammatory agent. Peroxynitrite can form stable 3-nitrotyrosine by the addition of a nitro group to the 3-position adjacent to the hydroxyl group of the tyrosine (8). Nitration by peroxynitrite inhibits IL-8 binding to neutrophils (9), C1q binding on human IgG (10), and protein phosphorylation by

tyrosine kinase (11). Peroxynitrite is produced by the reaction between NO and superoxide (12, 13). Human IL-10 has at least one known functional domain that includes a tyrosine amino acid that may be nitrated by peroxynitrite. We hypothesized that nitration of human IL-10 would augment its anti-inflammatory effect. To test this hypothesis, human IL-10 was incubated with peroxynitrite or 3-morpholinosydnonimine (SIN-1),3 a peroxynitrite generator. The nitrated human IL-10 was then added to human PBMC and incubated, and human IL-1 production was measured.

Materials and Methods PBMC Human PBMC were isolated from venous blood by Ficoll-Hypaque density centrifugation (Histopaque 1077; Sigma, St. Louis, MO). PBMC, as determined by trypan blue, consisted of ⬎98% viable monocytes. The cells were diluted in RPMI 1640 (Biofluids, Rockville, MD) containing 5% FBS, 50 U/ml penicillin, 50 ␮g/ml streptomycin to obtain a final concentration of 4 ⫻ 106 cells/ml. The PBMC were cultured in a 96-well flat-bottom culture plate (Fisher, Pittsburgh, PA). PBMC (50 ␮l), 100 ␮l of treatment material, and 20 ␮l of LPS (Escherichia coli 0127:B8 at 200 ␮g/ml) were cultured for 6 h at 37°C. Negative controls consisted of 20 ␮l of RPMI in place of LPS. The supernatant fluids from each well were transferred to a corresponding 96well round-bottom plate, covered, and stored at ⫺80°C until assayed.

Inhibition of IL-1 production by PBMCs *Research Service, Southern Arizona Veterans Health Care System, Arizona Respiratory Sciences, University of Arizona, Tucson, AZ 85723; and †Department of Cell Biology, University of Arizona, AZ 85724 Received for publication June 4, 2002. Accepted for publication August 9, 2002. 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. 1

This work was supported by a Merit Review Grant from the Veterans Administration.

Human IL-10 activity was measured by measuring human IL-1␤ (IL-1) in the supernatant fluids of cultured human PBMC by a modification of previously described methods (14). IL-1 was measured using a commercially available human IL-1␤ ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s directions. The samples for the assay were diluted 1/2.5.

Effects of IL-10 on IL-1 production Human IL-10 (IL-10) (R & D Systems) was evaluated for its ability to inhibit human IL-1 production by LPS (E. coli 0127:B8, Sigma)-stimulated

2

Address correspondence and reprint requests to Dr. Richard A. Robbins, Research Health Care Group, Southern Arizona Health Care System, 3601 South 6th Avenue, Tucson, AZ 85723. E-mail address: [email protected] Copyright © 2002 by The American Association of Immunologists, Inc.

3

Abbreviations used in this paper: SIN-1, 3-morpholinosydnonimine; IL-1, IL-1␤. 0022-1767/02/$02.00


4569

human PBMC using previously described methods (14). Human IL-10 was serially diluted 1/2 starting at a concentration of 1 ␮g/ml– 4.88 pg/ml; this became the sample material. The control groups were incubated without IL-10 in the positive control and without IL-10 and LPS in the negative control.

Effects of peroxynitrite on IL-10 inhibition of IL-1 production Peroxynitrite (Calbiochem, San Diego, CA) was evaluated for its ability to alter human IL-10-induced reduction in human IL-1 production of stimulated PBMC compared with human IL-10 alone. One milliliter of human IL-10 (2500 pg/ml) was incubated with 5 ␮l of peroxynitrite (200 mmol/ ml) for 2 h at 37°C. The nitrated human IL-10 was serial diluted from 2500 pg/ml to 4.88 pg/ml and became the sample material for the human IL-1 assay. To determine the minimal concentration of peroxynitrite on a fixed concentration of human IL-10 for maximal effect toward human IL-1 production, 1 ml of human IL-10 (500 pg/ml) was incubated with 5 ␮l of peroxynitrite (10⫺2, 10⫺3, 10⫺4, 10⫺5, 10⫺6, 10⫺7 M) for 2 h at 37°C. The nitrated human IL-10 was the sample material for the human IL-1 assay. Alteration of human IL-10 by peroxynitrite was confirmed by the detection of 3-nitrotyrosine by Western blotting using previously described methods (15).

Effects of SIN-1 on IL-10 inhibition of IL-1 production The ability of SIN-1 (Alexis, San Diego, CA), a peroxynitrite generator (16), to alter human IL-10 was evaluated. One milliliter of human IL-10 (500 pg/ml) was incubated with SIN-1 (10⫺3, 10⫺4, 10⫺6, 10⫺8M) for 2 h at 37°C before the human IL-1 assay was performed.

Effects of H2O2 on IL-10 inhibition of IL-1 production To contrast with peroxynitrite and to examine the effects of oxidation of cysteine residues (17), the effects of H2O2 (10⫺5–10⫺7 M) on human IL-10 were also evaluated similar to peroxynitrite.

Effects of DTT on IL-10 inhibition of IL-1 production To evaluate the possibility of thiol formation as a cause of the enhanced human IL-10 activity (17), DTT (10⫺5 M) was added to nitrated human IL-10 and evaluated for its ability to reverse the enhanced nitrated IL-10 activity.

Effect of desferrioxamine on peroxynitrite-induced enhancement of IL-10 activity The capacity of the reducing agent, desferrioxamine, to attenuate the effect of peroxynitrite on human IL-10 was assessed (9). Desferrioxamine (50 ␮M; Sigma) and peroxynitrite (10⫺4 M) were added to human IL-10 (500 pg/ml) and incubated for 2 h at 37°C before evaluating for its capacity to inhibit human IL-1 release from PBMC.

Statistics The differences between groups were tested using one-way ANOVA. In all cases, a p value of ⬍0.05 was considered significant. Data in figures are expressed as mean ⫾ SEM.

FIGURE 1. Enhancement of IL-10 activity by peroxynitrite. Human IL-10 was incubated with peroxynitrite (10⫺4 M, nitrated IL-10) or medium alone for 2 h at 37°C. Horizontal axis, IL-10 concentration; vertical axis, IL-10 activity expressed as the percent inhibition of human IL-1 release. n ⫽ 4 for each concentration.

Peroxynitrite added to PBS and incubated for 2 h at 37°C before addition to human IL-10 resulted in no significant increase in human IL-1 inhibition ( p ⬎ 0.05; data not shown). Peroxynitrite (10⫺3–10⫺7) incubated with human IL-10 (500 pg/ml) produced a dose-dependent enhancement of human IL-10 inhibition of human IL-1 production (Fig. 2). Peroxynitrite concentration of 10⫺4 resulted in the production of human IL-1 equal to the negative control. The positive control had human IL-1 production equal to the peroxynitrite concentration of 10⫺7. Human IL-10 exposed to peroxynitrite resulted in the detection of 3-NT by Western blotting (data not shown). Effects of SIN-1 on IL-10 inhibition of IL-1 production SIN-1 (10⫺3–10⫺8) incubated with human IL-10 (500 pg/ml) produced a dose-dependent enhancement of IL-10 inhibition of human IL-1 production (Fig. 3). A SIN-1 concentration of 10⫺3 had human IL-1 production similar to that of negative control. The positive control had human IL-1 production equal to the SIN-1 concentration of 10⫺8. The 50% maximal inhibition was ⬃6.4 ⫻ 10⫺5 M. Effect of hydrogen peroxide on peroxynitrite-induced enhancement of IL-10 activity Exposure of human IL-10 to hydrogen peroxide did not significantly alter IL-10 activity (IL-10 alone 30.7 ⫾ 2.0% inhibition; H2O2 10⫺5 M 28.6 ⫾ 13.6% inhibition; H2O2 10⫺6 M 29.7 ⫾ 4.9% inhibition; H2O2 10⫺7 M 35.2 ⫾ 4.9% inhibition).

Results Effects of IL-10 and nitrated IL-10 on IL-1 production Incubation of human PBMC with human IL-10 demonstrated a dose-dependent inhibition of human IL-1 production. (Fig. 1) Human IL-10 at a concentration of 1 ␮g/ml had the maximal inhibition of human IL-1 production, whereas a human IL-10 concentration of 4.88 pg/ml had insignificant inhibition and was comparable with the positive control. In contrast, human IL-10 exposed to peroxynitrite was significantly more potent in inhibiting human IL-1 production. The curve of human IL-1 inhibition is shifted to the left in the peroxynitrite-exposed human IL-10 group compared with human IL-10 alone. Fifty percent of maximal inhibition for peroxynitrite-exposed human IL-10 was ⬃300 pg/ml. In comparison, the 50% of maximal inhibition of human IL-10 not exposed to peroxynitrite was ⬃4116 pg/ml, suggesting that nitrated IL-10 is ⬃14-fold more potent at 50% of maximal inhibition.

FIGURE 2. Dose-responsive enhancement of IL-10 activity by peroxynitrite. Human IL-10 (500 pg/ml) was incubated with peroxynitrite for 2 h at 37°C. Horizontal axis, Peroxynitrite concentration; vertical axis, IL-10 activity expressed as the percent inhibition of human IL-1 release. n ⫽ 6 for each condition. ⴱ, p ⬍ 0.05 compared with 0 (medium alone).

4570

FIGURE 3. Dose-responsive enhancement of IL-10 activity by the peroxynitrite generator SIN-1. SIN-1 was incubated with human IL-10 (500 pg/ml) for 2 h at 37°C. Horizontal axis, SIN-1 concentration; vertical axis, IL-10 activity expressed as the percent inhibition of human IL-1 release. n ⫽ 6 for each condition.

Effect of DTT on peroxynitrite-induced enhancement of IL-10 activity In separate experiments, exposure of human IL-10 to peroxynitrite resulted in significant enhanced IL-10 activity (IL-10 500 pg/ml 28.7 ⫾ 2.0% inhibition; nitrated IL-10 59.3 ⫾ 2.1% inhibition). Addition of DTT to the nitrated IL-10 resulted in no significant change compared with nitrated IL-10 alone (56.0 ⫾ 4.3% inhibition). Effect of desferrioxamine on peroxynitrite-induced enhancement of IL-10 activity Addition of the reducing agent, desferrioxamine, before incubation with peroxynitrite attenuated the enhancing effects of peroxynitrite on human IL-1 release from PBMC (Fig. 4, p ⬍ 0.01). Desferrioxamine did not alter human IL-1 release when added directly to the human PBMC IL-1 assay (data not shown).

Discussion This study shows that human IL-10 when incubated with peroxynitrite has enhanced activity to inhibit human IL-1 production of stimulated PBMC. Incubation of peroxynitrite with human IL-1␤ standard before the ELISA did not alter the results of the standard curve. SIN-1, a peroxynitrite generator, induced a significant con-

FIGURE 4. Attenuation of peroxynitrite-induced enhancement of IL-10 activity by desferrioxamine. Human IL-10 (500 pg/ml) was incubated with peroxynitrite (OONO⫺, 10⫺4) with and without desferrioxamine (50 ␮M) for 2 h at 37°C. n ⫽ 4 for each condition. ⴱ, p ⬍ 0.05 compared with IL-10 alone; ⴱⴱ, p ⬍ 0.05 compared with IL-10 plus OONO⫺.

PEROXYNITRITE INCREASES IL-10 ACTIVITY centration-dependent enhancement of human IL-10 ability to inhibit human IL-1 production similar to that of peroxynitrite. The addition of desferrioxamine, a reducing agent, to peroxynitrite inhibited the enhanced activity of nitrated human IL-10. Previous investigations have suggested that peroxynitrite can interact with several proteins, hormones, and neurotransmitters. In general, most studies have suggested inactivation as a consequence of interaction with peroxynitrite (18). Consistent with these results, the proinflammatory cytokines IL-8, macrophage chemotactic protein-1␣, RANTES, and eotaxin all reduced chemotactic activity when exposed to peroxynitrite or SIN-1 (9, 19 –21). In contrast, this study shows an enhanced activity of the anti-inflammatory cytokine, IL-10. However, consistent with previous studies, this investigation supports the concept that peroxynitrite exhibits a predominantly anti-inflammatory effect. Coincubation of human IL-10 with the iron chelator, desferrioxamine, in iron-free conditions inhibited peroxynitrite-induced enhancement of human IL-10 activity. However, desferrioxamine is also a peroxynitrite scavenger independent of iron chelation (22). Peroxynitrite nitrates tyrosine residues (13). A tyrosine residue at aa 153 in the presumptive functional domain is a potential target site for the interaction of IL-10 and peroxynitrite (7). These results are consistent with tyrosine nitration by peroxynitrite as a mechanism for enhancement of IL-10 activity. However, peroxynitrite may potentially affect protein function by other mechanisms including nitration of methionine (23). The presumed active site of human IL-10 contains two methionines (7). Also nitration of tryptophan (24) or formation of S-nitrosothiol groups on cysteines (25) can occur with peroxynitrite. Although these residues are not at the presumed functional domain, it is possible that interaction of peroxynitrite with these amino acids might result in a conformational change altering protein function. The mechanism of enhanced activity induced by nitration is unknown. The experiments with DTT suggest that thiol formation is not sufficient to explain the increase in activity. This is consistent with the lack of cysteines at the presumed active site (7). If formation of 3-nitrotyrosine on IL-10 were the mechanism of enhancement, other mechanisms of nitration such as nitrite and myeloperoxidase would presumably do the same (26). IL-10 exerts its anti-inflammatory activity by interaction with its cell surface receptor (27). Experiments were performed to determine whether the mechanism of enhancement IL-10 activity exposed to peroxynitrite was an augmentation in cell surface binding to the IL-10 receptor. However, the human IL-10 ELISA used for these studies (R&D Systems) did not completely recognize nitrated IL-10 and the results were inconclusive. Other potential mechanisms including reduction in catabolism of IL-10 by steric hindrance or other mechanisms are also possible. IL-10 has been proposed as a potential therapy for a variety of inflammatory disorders including acute lung injury, intestinal ischemia-reperfusion, inflammatory bowel disease, rheumatoid arthritis, psoriasis, and multiple sclerosis (2). This study suggests that the potency of IL-10 can be increased after being exposed to peroxynitrite. This study examines specifically the effects of human IL-10 on stimulated human PBMC production of human IL-1 in vitro. Studies would need to be undertaken to find whether this augmented activity is also found in vivo. This study demonstrates enhanced activity of IL-10 with nitration, suggesting a mechanism to attenuate inflammation at inflammatory sites where peroxynitrite and protein nitration is expected. Furthermore, these studies may have therapeutic implications if IL-10 is demonstrated to have therapeutic value in human disease.


References 1. Vieira, P., R. de Waal-Malefyt, M. N. Dang, K. E. Johnson, R. Kastelein, D. F. Fiorentino, J. E. deVries, M. G. Roncarolo, T. R. Mosmann, and K. W. Moore. 1991. Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: homology to Epstein-Barr virus open reading frame BCRFI. Proc. Natl. Acad. Sci. USA 88:1172. 2. Opal, S. M., and V. A. DePalo. 2000. Anti-inflammatory cytokines. Chest 117: 1162. 3. Kim, J. M., C. I. Brannan, N. G. Copeland, N. A. Jenkins, T. A. Khan, and K. W. Moore. 1992. Structure of the mouse IL-10 gene and chromosomal localization of the mouse and human genes. J. Immunol. 148:3618. 4. Keystone, E., J. Wherry, and P. Grint. 1998. IL-10 as a therapeutic strategy in the treatment of rheumatoid arthritis. Rheum. Dis. Clin. North Am. 24:629. 5. Moore, K. W., A. O’Garra, R. de Waal Malefyt, P. Vieira, and T. R. Mosmann. 1993. Interleukin-10. Annu. Rev. Immunol. 11:165. 6. Tan, J. C., S. Braun, H. Rong, R. DiGiacomo, E. Dolphin, S. Baldwin, S. K. Narula, P. J. Zavodny, and C. C. Chou. 1995. Characterization of recombinant extracellular domain of human interleukin-10 receptor. J. Biol. Chem. 270:12906. 7. Gesser, B., H. Leffers, T. Jinquan, C. Vestergaard, N. Kirstein, S. Sindet-Pedersen, S. L. Jensen, K. Thestrup-Pedersen, and C. G. Larsen. 1997. Identification of functional domains on human interleukin 10. Proc. Natl. Acad. Sci. USA 94:14620. 8. Radi, R., J. S. Beckman, K. M. Bush, and B. A. Freeman. 1991. Peroxynitrite oxidation of sulfhydryls: the cytotoxic potential of superoxide and nitric oxide. J. Biol. Chem. 266:4244. 9. Sato, E., K. L. Simpson, M. B. Grisham, S. Koyama, and R. A. Robbins. 2000. Reactive nitrogen and oxygen species attenuate interleukin-8-induced neutrophil chemotactic activity in vitro. J. Biol. Chem. 275:10826. 10. McCall, M. N., and S. B. Easterbrook-Smith. 1989. Comparison of the role of tyrosine residues in human IgG and rabbit IgG in binding of complement subcomponent C1q. Biochem. J. 257:845. 11. Gow, A. J., D. Duran, S. Malcolm, and H. Ischiropoulos. 1996. Effects of peroxynitrite-induced protein modifications on tyrosine phosphorylation and degradation. FEBS Lett. 385:63. 12. Beckman, J. S., T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman. 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87:1620.

4571 13. Ischiropoulos, H., L. Zhu, J. Chen, M. Tsai, J. C. Martin, C. D. Smith, and J. S. Beckman. 1992. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch. Biochem. Biophys. 298:431. 14. Wang, P., P. Wu, M. I. Siegel, R. W. Egan, and M. M. Billah. 1994. IL-10 inhibits transcription of cytokine genes in human peripheral blood mononuclear cells. J. Immunol. 153:811. 15. Robinson, V. K., E. Sato, D. K. Nelson, S. L. Camhi, R. A. Robbins, and J. C. Hoyt. 2001. Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro. Free Radical Biol. Med. 30:986. 16. Holm, P., H. Kankaanranta, T. Metsa-Ketela, and E. Moilanen. 1998. Radical releasing properties of nitric oxide donors GEA 3162, SIN-1 and S-nitroso-Nacetylpenicillamine. Eur. J. Pharmacol. 346:97. 17. Wong, P. S., J. P. Eiserich, S. Reddy, C. L. Lopez, C. E. Cross, and A. van der Vliet. 2001. Inactivation of glutathione S-transferases by nitric oxidederived oxidants: exploring a role for tyrosine nitration. Arch. Biochem. Biophys. 394:216. 18. Nakaki, T., and T. Fujii. 1999. Nitration modifying function of proteins, hormones and neurotransmitters. Jpn. J. Pharmacol. 79:125. 19. Sato, E., K. Simpson, M. Grisham, S. Koyama, and R. Robbins. 1999. Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte chemotactic activity in vitro. Am. J. Physiol. 277:L543. 20. Sato, E., K. L. Simpson, M. B. Grisham, S. Koyama, and R. A. Robbins. 1999. Effects of reactive oxygen and nitrogen metabolites on RANTES- and IL-5 induced eosinophil chemotactic activity in vitro. Am. J. Pathol. 155:591. 21. Sato, E., K. Simpson, M. Grisham, S. Koyama, and R. Robbins. 2000. Effects of reactive oxygen and nitrogen metabolites on eotaxin induced eosinophil chemotactic activityin vitro. Am. J. Respir. Cell Mol. Biol. 22:61. 22. Denicola, A., J. M. Souza, R. M. Gatti, O. Augusto, and R. Radi. 1995. Desferrioxamine inhibition of the hydroxyl radical-like reactivity of peroxynitrite: role of the hydroxamic groups. Free Radical Biol. Med. 19:11. 23. Vogt, W. 1995. Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radical Biol. Med. 18:93. 24. Alvarez, B., H. Rubbo, M. Kirk, S. Barnes, B. A. Freeman, and R. Radi. 1996. Peroxynitrite-dependent tryptophan nitration. Chem. Res. Toxicol. 9:390. 25. Gow, A. J., D. G. Buerk, and H. Ischiropoulos. 1997. A novel reaction mechanism for the formation of S-nitrosothiol in vivo. J. Biol. Chem. 272:2841. 26. Eiserich, J. P., M. Hristova, C. E. Cross, A. D. Jones, B. A. Freeman, B. Halliwell, and A. van der Vliet. 1998. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391:393. 27. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19:683.