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Published ahead of print on March 6, 2003, doi:10.1165/rcmb.2002-0275OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 239-244, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0275OC

Peroxynitrite Enhances Interleukin-10 Reduction in the Release of Neutrophil Chemotactic Activity

Hiroki Numanami, Dan K. Nelson, Jeffrey C. Hoyt, Jon L. Freels, Michael Habib, Jun Amano, Masayuki Haniuda, Sekiya Koyama and Richard A. Robbins

Research Service, Southern Arizona Veterans Health Care System, and Arizona Respiratory Center, University of Arizona, Tucson, Arizona; Second Department of Surgery, and First Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan

Address correspondence to: Richard A. Robbins, M.D., Chief, Research Service Line, Southern Arizona Veterans Health Care System, 3601 S. 6th Avenue, Tucson, AZ 85723. E-mail: Richard.Robbins2{at}med.va.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peroxynitrite, formed by nitric oxide and superoxide, has been shown to nitrate and reduce the function of proinflammatory proteins such as interleukin (IL)-8, monocyte chemoattractant protein-1, and eotaxin, but in contrast, to enhance the function of the anti-inflammatory cytokine IL-10 in reducing IL-1 release from blood monocytes. However, the effect of nitrated IL-10 on release of proinflammatory cytokines from lung epithelial cells is unknown. We hypothesized that peroxynitrite would enhance the capacity of human IL-10 to reduce inflammatory mediators released by epithelial cells. To test this hypothesis, recombinant human IL-10 was evaluated for its capacity to attenuate the release of neutrophil chemotactic activity and IL-8 from a human epithelial cell line in response to IL-1ß and tumor necrosis factor-{alpha}. Neutrophil chemotactic activity and IL-8 in lung epithelial culture supernatant fluids were significantly lower after culture with nitrated human IL-10 compared with non-nitrated human IL-10 controls (P < 0.05). Consistent with these results, nitrated human IL-10 attenuated IL-8 mRNA expression more than non-nitrated human IL-10 controls (P < 0.05). These data demonstrate that peroxynitrite exposed human IL-10 has enhanced anti-inflammatory activity and suggest that nitration may play a critical role in the regulation of inflammation within the lower respiratory tract.

Abbreviations: enzyme-linked immunosorbent assay, ELISA • high-power fields, HPF • interleukin, IL • neutrophil chemotactic activity, NCA • regulated upon activation, normal T cell expressed and presumably secreted, RANTES • reverse transcriptase-polymerase chain reaction, RT-PCR • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Peroxynitrite, which is formed by the reaction between two free radicals, nitric oxide and superoxide, is a nitrating agent that reacts with a variety of biomolecules including lipids, proteins, carbohydrates, and deoxyribonucleic acid (1, 2). Peroxynitrite can nitrate proteins by interacting with several amino acids, including methionine, tryptophan, cysteine, and tyrosine (3). Peroxynitrite nitrates free or protein-associated tyrosine residues to form the stable product nitrotyrosine by addition of a nitro group to the 3-position adjacent to the hydroxyl group of tyrosine (4).

Interleukin (IL)-10 is a cytokine that inhibits the production of a wide range of inflammatory and proinflammatory cytokines. For example, human IL-10 suppresses the expression of IL-2, IL-3, interferon-{gamma}, and granulocyte-macrophage colony-stimulating factor in T helper type 1 cells (5, 6); IL-4 and IL-5 in T helper type 2 cells (7); IL-8 and macrophage inflammatory protein–1 in neutrophils (8); IL-1, IL-8, and tumor necrosis factor (TNF)-{alpha} in macrophages/monocytes (9, 10); and IL-8 in human lung epithelial cells and in human vascular endothelial cells (11, 12). Hence, IL-10 is a potent anti-inflammatory cytokine that has an important role in balancing inflammatory responses (13).

Although the exact amino acid sequence on human IL-10 that binds to its receptor is unknown, human IL-10 includes a tyrosine and two methionines at one of the functional domains (14). It was previously reported that the peroxynitrite enhanced the anti-inflammatory activity of human IL-10 in reducing the release of IL-1 from human peripheral blood monocytes in response to lipopolysaccharide (15). In this context, we hypothesized that nitration may regulate the anti-inflammatory activity of human IL-10 on human lung epithelial cells. In this study, human IL-10 was nitrated and its anti-inflammatory activity compared with non-nitrated IL-10 controls. Peroxynitrite enhanced the anti-inflammatory action of human IL-10, including reducing the release of neutrophil chemotactic activity (NCA), IL-8, and IL-8 mRNA expression by the human lung epithelial cell line, A549, in response to IL-1ß or TNF-{alpha}. These data suggest that nitration of human IL-10 may enhance its anti-inflammatory action within the lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Cultures
A549 cells, a pulmonary type-II epithelial cell line derived from an individual with alveolar cell carcinoma, were purchased from American Type Culture Collection (Rockville, MD). These cells retained many of the characteristics of normal type-II epithelial cells such as surfactant production, cytoplasmic multilamellar inclusion bodies, and cuboidal appearance (16). A549 cells were cultured in Ham's F-12 (GIBCO, Inc., Grand Island, NY) medium with 10% heat-inactivated fetal bovine serum. After 2–4 d in culture, the cells had reached confluence and were then used for experiments.

Nitration of IL-10 with Peroxynitrite
One milliliter of recombinant carrier-free human IL-10 (1 µg /ml; R&D Systems, Minneapolis, MN) was incubated with 5 µl of peroxynitrite (200 mM/ml; Calbiochem, San Diego, CA) for 2 h at 37°C. The nitrated IL-10 was serial diluted from 1 µg/ml to 10 ng/ml and became the sample material for the following assay. Nitration was performed just before each cell treatment. The pH after nitrated IL-10 was measured, and the change was minimal (pH = 7.41). Nitration of human IL-10 by peroxynitrite was confirmed by the detection of 3-nitrotyrosine by Western blotting and silver stain using previously described methods used for type 2 nitric oxide synthase (17). Briefly, SDS-PAGE was performed at 150 V using a 15%T resolving gel and the Laemmli tris-glycine buffer system. No reducing agent was used in sample preparation, and samples were heated to 60°C before electrophoresis to minimize reduction of 3-nitrotyrosine to 3-aminotyrosine. Western transfers were performed using PVDF membranes at 30 V for 18 h at 4°C in a tris-glycine methanol buffer. The membranes were developed using BLOTTO as the blocking agent and the appropriate primary antibody, IL-10 polyclonal antibody (R&D Systems), or 3-nitrotyosine antibody (Upstate Biotechnology, Lake Placid, NY) and secondary antibody-alkaline phosphatase conjugates (Sigma, St. Louis, MO). Silver staining of SDS-PAGE gels was performed according Morrissey (18).

IL-10, Nitrated IL-10, and Stimulants
Serum-containing media was removed from the cells by washing twice with serum-free Ham's F-12 medium, and the A549 cells were incubated Ham's F-12 medium without fetal bovine serum in the absence and presence of human recombinant IL-1ß (1 ng/ml; Sigma) or TNF-{alpha} (5 ng/ml; Sigma) at 37°C in a humidified 5% CO2 atmosphere for 48 h. These concentrations were chosen because they produced the maximal effect of the doses tested for NCA release (data not shown) without cell injury in these experimental conditions. A549 cells were pretreated with IL-10 (10 or 100 ng/ml) or nitrated IL-10 (10 or 100 ng/ml). The concentrations of human IL-10 were based on results showing that 3–5 ng/ml of human IL-10 was included in the epithelial lining fluid of healthy humans (19) and 1–100 ng/ml of human IL-10 inhibited the IL-8 production from human bronchial epithelium, human monocyte, and human neutrophils (11, 13). IL-1ß and TNF-{alpha} were tested for LPS contamination, and LPS was shown to be < 0.1 ng/ml. These cytokines did not cause A549 cell injury (no deformity of cell shape, no detachment from culture dish, and > 98% viability by trypan blue exclusion) after 48 h incubation at the highest concentration used. The culture supernatant fluid was harvested and centrifuged at 1,000 rpm for 10 min to remove cells and then frozen at -80°C until assayed. All assays were finished within 2 wk after harvesting the supernatant fluids.

After 48 h, the concentrations of IL-10 were evaluated in some culture supernatant fluids (n = 5, cells cultured in media alone 324 ± 13 pg/ml, cells cultured with TNF-{alpha} 5 ng/ml 369 ± 38 pg/ml) using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems).

Effects of IL-10 and Nitrated IL-10 on Neutrophil Chemotactic Activity by A549 Supernatant Fluids
Polymorphonuclear leukocytes were purified from heparinized normal human blood by the method of Böyum (20). The resulting cell pellet consisted of > 96% neutrophils and > 98% viable cells as determined by trypan blue and erythrosin exclusion. The cells were suspended in Hanks' balanced salt solution (GIBCO, Inc., Grand Island, NY) containing 2% bovine serum albumin (Sigma) at pH 7.42 to give a final concentration of 3.0 x 106 cells/ml. This suspension was used for the neutrophil chemotaxis assay.

The chemotaxis assay was performed by a 48-well microchemotaxis chamber (NeuroProbe Inc., Cabin John, MD), as has been previously described (21). Briefly, 25 µl of the harvested supernatant fluids were placed into the lower wells, and a 10-µm-thick polyvinylpyrrolidone-free polycarbonate filter (Nucleopore, Pleasanton, CA), with a pore size of 3 µm, was placed over the bottom wells. The silicon gasket and upper pieces of the chamber were applied, and 50 µl of the cell suspension was placed into the upper wells above the filter. The chambers were incubated in humidified air in 5% CO2 at 37°C for 30 min. After incubation, the chamber was disassembled and nonmigrated cells were wiped away from the filter. The filter was then immersed in methanol for 5 min, stained with Diff-Quik (American Scientific Product, McGraw Park, IL), and mounted on a glass slide. Cells that completely migrated through the filter were counted by using light microscopy in ten random high power fields (HPF) per well.

Measurement of IL-8
The concentrations of IL-8 were measured in the cell culture supernatant fluids using commercially available ELISA kits (R&D Systems) according to the manufacturer's instructions in duplicate. The frozen supernatant fluids were thawed just before being assayed and were diluted 100-fold before measurement of IL-8 by ELISA. The minimum concentration detected by the ELISA was 10 pg/ml. Intra-assay precision of this ELISA kit is 4.56 ± 0.15%, and interassay precision is 6.70 ± 1.45%.

Evaluation of IL-8 mRNA Expression
IL-8 mRNA was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). A549 cells were incubated with the cytokines for 12 h, and total cellular RNA was extracted from adherent cells using a modification of the methods of Chomczynski and Sacchi (22). The RNA was reverse transcribed using a commercially available kit (Promega, Madison, WI). One microgram of the reverse transcribed DNA was then mixed with Ready-to-Go PCR Beads (Pharmacia, Piscataway, NJ) and the front and back primers, using a commercially available primer pairs (R&D Systems), added at 0.3 µM final concentration. PCR was performed in a Perkin-Elmer model 480 thermal cycler using 94°C for 2 min and 24 cycles consisting of 94°C for 45 s, primer annealing at 55°C for 45 s, primer extension at 72°C for 45 s, followed by 72°C for an additional 7 min. ß-Actin was used as a "housekeeping gene" with PCR. The DNA was subjected to agarose gel and the intensity of the bands quantified by densitometry. The results were expressed as the ratio of intensity to the ß-actin.

Statistical Analysis
Data was analyzed by one-way ANOVA with Fisher's protected least significant difference (Fisher's PLSD). In all cases, a P value less than 0.05 were considered significant. Data in figures were expressed as means ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nitration of IL-10 with Peroxynitrite
Western blotting of recombinant IL-10 and nitrated IL-10 with an IL-10 polyclonal antibody revealed both at the appropriate molecular weight (Figure 1B). Corresponding to this band was a clearly identifiable band that marked for 3-nitrotyrosine antibody (Figure 1A, lane 2). Silver staining indicated only the band of appropriate molecular weight of IL-10 (Figure 1C, lanes 2 and 3). No peroxynitrite-induced cleavage of IL-10 was identified.



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  		Figure 1. (A) Western transfer of nitrated IL-10 developed with 3-nitrotyrosine antibody. Lane 1: molecular weight standards (pyruvate kinase from chicken muscle 58 kD, lactic dehydrogenase from rabbit muscle 36.5 kD, triosephosphate isomerase from rabbit muscle 26.6 kD); lane 2: nitrated IL-10 (1 µg, 18.6 kD); lane 3: human serum albumin (0.5 µg, 66.5 kD). (B) Western transfer of nonreduced native and nitrated IL-10 developed with polyclonal IL-10 antibody. Lane 1: molecular weight standards; lane 2: native IL-10 (1.1 µg); lane 3: nitrated IL-10 (1.1 µg). (C) Silver stained electrophoresis gel of native and nitrated IL-10. Lane 1: molecular weight standards; lane 2: native IL-10 (0.7 µg); lane 3: nitrated IL-10 (0.7 µg).

 
NCA
Both human IL-10 and nitrated IL-10 inhibited NCA from A549 stimulated with IL-1ß and TNF-{alpha} (Figure 2). Nitrated IL-10 reduced NCA from the stimulated A549 more than non-nitrated IL-10 (IL-1ß with 10 ng/ml nitrated IL-10 45.7 ± 4.2 Cell/HPF; IL-1ß with 10 ng/ml non-nitrated IL-10 68.4 ± 6.9 Cell/HPF; TNF-{alpha} with 10 ng/ml nitrated IL-10 47.6 ± 8.6 Cell/HPF; TNF-{alpha} with 10 ng/ml non-nitrated IL-10 66.8 ± 7.6 Cell/HPF; Figure 2, n = 6). Human IL-10 or nitrated IL-10 alone had no effect on baseline release of NCA under control conditions (P > 0.05, Figure 2, n = 6).



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  		Figure 2. Inhibition of neutrophil chemotactic activity (NCA) from A549 cells stimulated with IL-1ß and TNF-{alpha} by human IL-10 and nitrated IL-10. NCA is on the vertical axis and experimental groups are on the horizontal axis. Values are expressed as mean ± SD. n = 6 each condition. Human IL-10 or nitrated IL-10 alone had no effect on baseline release of NCA under control conditions (P > 0.05). ***P < 0.001 compared with A549 culture supernatant fluids with IL-1ß or TNF-{alpha}. #P < 0.05 between supernatant fluids from cells cultured with human IL-10 and nitrated IL-10. ##P < 0.01 between supernatant fluids from cells cultured with human IL-10 and nitrated IL-10. ###P < 0.001 between supernatant fluids from cells cultured with human IL-10 and nitrated IL-10.

 
Effects of Human IL-10 and Nitrated IL-10 on IL-8 Production from A549
A549 spontaneously released IL-8, but IL-1ß or TNF-{alpha} increased the release of IL-8 from A549 (Figure 3, n = 4). The release of IL-8 was inhibited by both human IL-10 and nitrated IL-10. Nitrated IL-10 was significantly more effective in reducing IL-8 release than non-nitrated IL-10 (IL–1ß with 10 ng/ml nitrated IL-10 1,589.13 ± 33.09 pg/ml; IL–1ß with 10 ng/ml non-nitrated IL-10 1,834.99 ± 71.11 pg/ml; TNF-{alpha} with 10 ng/ml nitrated IL-10 2,037.93 ± 258.73pg/ml; TNF-{alpha} with 10 ng/ml non-nitrated IL-10 2,119.97 ± 21.97 pg/ml; Figure 3, n = 4). Human IL-10 and nitrated IL-10 had no stimulating effect on release of IL-8 from nonstimulated A549 (P > 0.05, Figure 3, n = 4).



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  		Figure 3. Inhibition of the IL-8 release from A549 cells by human IL-10 and human nitrated IL-10. Both inhibited IL-8 production from A549 cells in response to IL-1ß (A) and TNF-{alpha} (B), but nitrated IL-10 was significantly stronger than non-nitrated IL-10. The concentration of IL-8 is on the vertical axis and experimental groups are on the horizontal axis. Values are expressed as mean ± SD. n = 4 each condition. Human IL-10 and nitrated IL-10 had no effect on IL-8 release from nonstimulated A549 (P > 0.05). **P < 0.01 compared with supernatant fluids from cells cultured with IL-1ß or TNF-{alpha}. ***P < 0.001 compared with supernatant fluids from cells with IL-1ß or TNF-{alpha}. #P < 0.05 between cell culture supernatant fluids with human IL-10 and nitrated IL-10. ##P < 0.01 between cell culture supernatant fluids with human IL-10 and nitrated IL-10. ###P < 0.001 between cell culture supernatant fluids of human IL-10 and nitrated IL-10.

 
Effects of Human IL-10 and Nitrated IL-10 on mRNA Expression from A549
Semiquantitative RT-PCR was performed to evaluate the effect of human IL-10 and nitrated IL-10 on IL-8 mRNA expression in A549. IL-1ß–induced IL-8 mRNA expressions were suppressed by pretreatment with human IL-10 and nitrated IL-10 (Figure 4, n = 3). IL-8 mRNA was significantly more suppressed by nitrated IL-10 in comparison to normal human IL-10 (IL-1ß with 10 ng/ml nitrated IL-10 1.034 ± 0.028; IL–1ß with 10 ng/ml non-nitrated IL-10 1.142 ± 0.031).



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  		Figure 4. Semiquantitative RT-PCR was performed to evaluate the effect of human IL-10 and nitrated IL-10 on IL-8 mRNA expression in A549. IL-1ß–induced IL-8 mRNA expressions were slightly but significantly suppressed by pretreatment with human IL-10 and nitrated IL-10. IL-8 mRNA was suppressed more by nitrated IL-10 compared with non-nitrated IL-10. Human IL-10 and nitrated IL-10 had no stimulating effect on IL-8 mRNA from nonstimulated A549 (P > 0.05). Lanes 1, 2, and 3 indicated that cells were cultured with medium. Cells were cultured with 1 ng/ml of IL-1ß (lanes 4, 5, and 6), with 10 ng/ml of human IL-10 (lanes 7, 8, and 9) or with 10 ng/ml of nitrated IL-10 (lanes 10, 11, and 12). Cells were cultured only with 10 ng/ml human IL-10 (lane 13) or 10 ng /ml nitrated IL-10 (lane 14). The density of IL-8/ß-actin are on the vertical axis and experimental groups are on the horizontal axis. Values are expressed as mean ± SD. n = 3 each condition. *P < 0.05 compared with supernatant fluids from cells cultured with IL-1ß. **P < 0.01 compared with supernatant fluids from cells cultured with IL-1ß. #P < 0.05 between supernatant fluids from cells cultured with human IL-10 and nitrated IL-10.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we demonstrated that human IL-10 inhibited the release of NCA, IL-8, and IL-8 mRNA expression in a human lung epithelial cell line stimulated with IL-1ß or TNF-{alpha}. In addition, human IL-10 exposed to peroxynitrite suppressed these inflammatory mediators more than non-nitrated human IL-10 controls. These data suggest that peroxynitrite may enhance anti-inflammatory capacity of human IL-10 on human lung epithelial cells in response to the proinflammatory cytokines.

We investigated the effect of human IL-10 and nitrated IL-10 on the lung epithelial cell line because epithelial cells are the first lung cells to contact noxious or infectious agents. Moreover, these cells play a role in regulating the lung inflammatory environments by producing mediators, including IL-8, in response to a variety of proinflammatory stimuli (23, 24). Hence, lung epithelial cells are ideally situated to regulate the defense mechanisms for lung inflammation. In vitro, it was reported that lung epithelial cells produced IL-10 in response to variety of stimuli (19, 25). Although the anti-inflammatory effects of IL-10 on monocytes, macrophages, and lymphocytes have been examined, fewer studies have investigated the effects of IL-10 on lung epithelial cells.

Previous work has demonstrated that the neutrophil chemotactic activity released by lung epithelial cells, including A549 cells, is heterogeneous, consisting of lipid and nonlipid chemotactic factors (26). At 48 h, blocking experiments with anti–IL-8 antibodies suggest that IL-8 may account for as much as 70% of the neutrophil chemotactic activity released by IL-1ß–stimulated A549 cells (26). The present study did not evaluate A549 cells for IL-10 receptors, but IL-10 receptor 1 expression has been observed on many nonhemopoietic cells (27). IL-10 receptor 2 gene is expressed by many human cells and tissues (28). Consistent with our data, Gudmundsson and Hunninghake (29) reported that IL-10 reduces cytokine secretion form A549 cells implying the presence of IL-10 receptors.

It has been suggested that peroxynitrite played an important role in inflammation, fibrosis, and immunity of the human airways (30). Peroxynitrite can react readily with phenolic and aromatic compounds such as tyrosine to form nitrated and dimerized products such as 3-nitrotyrosine (31). Nitration has been proposed to be major regulator of protein function (32). Recent studies showed that peroxynitrite reduced function of the inflammatory cytokines including IL-8, monocyte chemoattractant protein-1, and RANTES (33, 34). In this study, we investigated the role of peroxynitrite on human IL-10–induced anti-inflammatory activities. These studies revealed that IL-8 production, NCA, and IL-8 mRNA expression were inhibited significantly more by nitrated IL-10 than by non-nitrated human IL-10 control. These data suggested that peroxynitrite might enhance human IL-10–induced anti-inflammatory actions, whereas peroxynitrite usually inhibited the protein functions by nitration.

The mechanisms by which peroxynitrite enhances human IL-10 function are still unclear. Human IL-10 is an 18.6-kD homodimeric protein with subunits having a length of 160 amino acids. The carboxyl-terminal end of human IL-10 contains a nine–amino acid sequence including tyrosine and methionine (Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn), which has been shown to be a key functional site of human IL-10 involved in the inhibition of cytokine production (14). Nitration at the functional domain of human IL-10 may enhance the interaction between human IL-10 and its cell surface receptor. Although we attempted binding studies, the IL-10 ELISA used for these studies (R&D Systems) did not completely recognize nitrated IL-10, and the results were inconclusive. Alternatively, nitration may induce a conformational change in IL-10. A conformational change may protect proteins from enzymatic degradation and inactivation including phosphorylation, attenuate the uptake of proteins by serum/interstitial fluid factors, or promote the hydrophobic active state of proteins that may be inserted directly into plasma and intracellular organelle of activated cells, thereby modulating signal transduction of inflammatory mediators.

Neutrophil locomotion into the alveolar space from the bloodstream plays an important role in host defenses during lung injury, inflammation, and subsequent tissue remodeling (35). In agreement with this concept, it might have therapeutic merit that human IL-10 inhibits excessive neutrophil recruitment from the bloodstream by chemotactic factors, such as IL-8. The present study revealed that lung epithelial cells had the potential for contributing to airway inflammation by releasing NCA and suggested that human IL-10 and nitrated IL-10 might reduce lung inflammation by modulating the responsiveness of the epithelium.

In summary, we found that human IL-10 co-incubated with peroxynitrite inhibited NCA significantly stronger than normal human IL-10 in vitro. This data suggest that peroxynitrite might enhance anti-inflammatory activity of human IL-10 and play an important role in regulating neutrophil migration during inflammation.


   Acknowledgments
 
This study was supported by a Merit Review grant from the Veterans' Administration.

Received in original form November 26, 2002

Received in final form February 25, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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