Interleukin-10 regulates quartz-induced pulmonary inflammation in rats

Kevin E. Driscoll1, Janet M. Carter1, Brian W. Howard1, Diana Hassenbein1, Marie Burdick2, Steven L. Kunkel2, and Robert M. Strieter2

1 The Procter & Gamble Company, Cincinnati, Ohio 45253; and 2 University of Michigan, Ann Arbor, Michigan 48109-0602

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Interleukin-10 (IL-10) can downregulate expression of several proinflammatory cytokines including chemokines. This study investigated the role of IL-10 in the acute response of the rat lung to quartz particles. Intratracheal instillation of rats with 1 mg of quartz produced an inflammatory and cytotoxic response demonstrated by increased bronchoalveolar lavage (BAL) fluid neutrophils, lactate dehydrogenase, and protein. IL-10 was detected in rat lung, but IL-10 levels were not altered by quartz. In contrast, quartz increased lung levels of the chemokine macrophage inflammatory protein-2 (MIP-2). Treatment with recombinant murine IL-10 (rmIL-10) attenuated quartz-induced pulmonary inflammation and injury. Pretreatment with anti-IL-10 antiserum enhanced inflammatory responses to quartz. Consistent with effects on quartz-induced inflammation, rmIL-10 and anti-IL-10 serum decreased and increased, respectively, lung MIP-2 mRNA and protein in response to quartz. Additionally, rmIL-10 reduced production of hydrogen peroxide, superoxide anion, and nitric oxide by BAL cells from quartz-exposed and control rats. These results demonstrate that IL-10 is expressed in rat lung and downregulates quartz-induced inflammation and cell activation. The mechanism of the anti-inflammatory action of IL-10 after quartz administration involves, at least in part, attenuation of MIP-2 expression.

macrophage inflammatory protein-2; chemokine; lung; particulate matter; hydrogen peroxide; nitric oxide

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

SILICOSIS is a chronic interstitial lung disease characterized by granulomatous inflammation and pulmonary fibrosis resulting from inhalation of various crystalline forms of silica, including quartz and cristobalite (22). The inflammatory component of silicosis appears to play a key role in the tissue injury associated with crystalline silica exposure (23). Studies (11, 13, 14) that used animal models of silicosis have demonstrated that the recruitment and activation of inflammatory cells in silica-exposed lungs result, at least in part, from the activation of alveolar macrophages and lung epithelial cells to produce proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha ), interleukin (IL)-1, and members of the chemokine cytokine family such as the neutrophil chemoattractant macrophage inflammatory protein-2 (MIP-2). Regarding the latter, lung expression of MIP-2 increases rapidly after exposure of rats to quartz, and pretreatment with neutralizing antibody to MIP-2 markedly attenuates quartz-induced pulmonary inflammation (13, 16).

The degree and persistence of inflammation in the lung and other tissues are influenced by the balance between proinflammatory factors and other processes that serve to downregulate inflammation. In this respect, a cytokine shown to exhibit an anti-inflammatory activity in a number of in vitro and in vivo models is IL-10. IL-10 was initially described as a Th2 lymphocyte-derived factor, which downregulated synthesis of interferon-gamma by Th1 lymphocytes (18). Subsequently, IL-10 was characterized as an 18-kDa protein produced by Th2 lymphocytes, macrophages, epithelial cells, and keratinocytes (27). In vitro studies have shown that IL-10 can suppress synthesis of several proinflammatory cytokines, including IL-1, TNF-alpha , IL-8, and MIP-1alpha , by monocytes, macrophages, and/or neutrophils (1, 7). Additionally, IL-10 can inhibit production of reactive oxygen and nitrogen species by macrophages (1), suppress interferon-gamma production by natural killer cells (27), stimulate monocyte production of IL-1 receptor antagonist (4), and decrease expression of major histocompatibility complex class II antigen on monocytes (7). Thus IL-10 exhibits several bioactivities in vitro consistent with downregulation of inflammatory responses. An anti-inflammatory function for IL-10 has been demonstrated in vivo in studies in which rmIL-10 was shown to decrease pulmonary inflammation in an immune complex model of rat lung injury (31) and joint inflammation in collagen-induced arthritis in rats (25). Additionally, a study (29) in humans demonstrated that rmIL-10 can prevent pulmonary granulocyte accumulation in response to a systemic endotoxin challenge. In the rodent and human studies, treatment with rmIL-10 was associated with decreased production of proinflammatory cytokines such as TNF-alpha , IL-1, and members of the chemokine family.

Whereas several of the factors exerting a proinflammatory action after exposure to quartz and other pneumotoxic particles have been identified, little is known about factors that may attenuate inflammatory responses to particle exposure. Because there is evidence from in vitro and in vivo studies that IL-10 can regulate cytokines that contribute to particle-induced inflammation, we hypothesized that this cytokine may influence the response of the lung to quartz. To evaluate this possibility, we characterized inflammation and chemokine expression in rats treated with either anti-IL-10 antibody or rmIL-10. Our results indicate that IL-10 acts as a constitutive anti-inflammatory factor in the rat lung and that its effect on quartz-induced inflammation is mediated, in part, by attenuation of MIP-2 gene expression.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Treatment with anti-IL-10 antiserum, rmIL-10, and quartz. Specific pathogen-free male Fischer 344 (F-344) rats (Charles River Breeding Laboratories, Kingston, NY), 12-14 wk old and ~200 g in body mass, were pretreated by intraperitoneal injection with 1 ml of saline, nonimmune rabbit serum, or a rabbit anti-murine IL-10 serum. The development of specific anti-IL-10 antisera was reported previously (32); a 1:1,000 dilution of the anti-IL-10 antiserum neutralizes 30 ng of IL-10 protein. Two hours after pretreatment, rats were intratracheally instilled with saline or a saline suspension of 1 mg of quartz at a dose volume of 1 ml/kg body wt as described previously in detail (14). The quartz particles (Minusil 5; Pennsylvania Sand & Glass, Pittsburgh, PA) had a median diameter of 0.9 ± 1.8 (geometric SD) µm and a surface area of 4.5 m2/g and were heated for 2 h at 200°C for sterilization and inactivation of any endotoxin present before use. Three days after quartz exposure, the animals were killed by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and exsanguinated via the abdominal aorta. The left lung lobe was removed and frozen in liquid nitrogen for analysis of MIP-2 mRNA and protein. Bronchoalveolar lavage (BAL) was performed on the right lung lobe six times with 5 ml of Ca- and Mg-free phosphate-buffered saline (PBS; pH 7.2) as previously described (14). The BAL fluid was centrifuged (300 g), and the acellular supernatant from the first two washes were pooled and analyzed for total protein and lactate dehydrogenase (LDH) as indicators of lung injury. Total protein was determined with the Bio-Rad method (3), and LDH was assayed on a Hitachi 705 autoanalyzer with commercially available kits (Boehringer Mannheim). BAL fluid cells were quantified by hemocytometer counting, and cell viability was determined by exclusion of trypan blue dye. Cell differentials were performed on cytocentrifuge preparations that were fixed in methanol and stained with Diff-Quik (Sigma, St. Louis, MO).

To determine the effect of rmIL-10 protein on the acute lung response to quartz, we intratracheally instilled specific pathogen-free male F-344 rats with 1 mg of quartz as described above with and without 50 µg of rmIL-10 (Pepro Tech, Rocky Hill, NJ) added to the instillation materials. Because of the short 2-h half-life of rmIL-10 (26), responses were characterized 24 h after exposure. The left lung lobe was removed and frozen in liquid nitrogen for analysis of MIP-2 mRNA and protein or IL-10 protein. BAL was performed on the right lung lobe six times with 5 ml of Ca- and Mg-free PBS (pH 7.2). The cellular and acellular components of the BAL fluid were analyzed as described above.

Measurement of hydrogen peroxide, superoxide anion, and nitric oxide. To characterize inflammatory cell production of superoxide anion and hydrogen peroxide, cells obtained by BAL were suspended in phenol red-free Earle's balanced salt solution (GIBCO BRL, Gaithersburg, MD) and seeded into 96-well tissue culture plates (Corning, Corning, NY) at 1 × 105 cells/well. Superoxide anion levels were determined on the basis of the oxidation of cytochrome c as described by Pick and Mizel (30). Briefly, 100 µl of 50 µM cytochrome c (Sigma) diluted in phenol red-free Earle's balanced salt solution were added to the BAL cell cultures, and at 15-min intervals over a 2-h period, the absorbance of each culture well was determined at 550 nm with the use of an EL311 Biotek microplate reader (Biotek Instruments, Winooski, VT). Control wells consisted of BAL cells with cytochrome c and 100 U of superoxide dismutase (Sigma). Superoxide anion concentrations were calculated with the extinction coefficient for reduced and oxidized cytochrome c (30). Hydrogen peroxide production was determined by the horseradish peroxidase-dependent conversion of phenol red by hydrogen peroxide into a compound with absorbance at 600 nm as described by Ding et al. (8). Briefly, 100 µl of phenol red solution (in mM: 140 NaCl, 10 KH2PO4, 5.5 dextrose, and 0.56 phenol red; Sigma) containing 6 U/ml of type VI horseradish peroxidase (Sigma) were added to cell cultures. Cells were incubated for 2 h at 37°C, followed by addition of 10 µl of 1 N NaOH. Absorbance at 600 nm was determined with an EL311 Biotek microplate reader. Hydrogen peroxide concentrations were calculated from a standard curve. To assess nitric oxide release, BAL cells were suspended in RPMI 1640 medium and seeded at 1 × 105 cells/well in 96-well plates. Cells were incubated overnight at 37°C in 5% CO2, after which 100 µl of cell supernatant were removed, and the cells were added to 100 µl of Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% phosphoric acid] and incubated in the dark for 10 min at room temperature (8). Absorbance was measured at 550 nm with an EL311 Biotek microplate reader, and concentrations were determined with a standard curve of sodium nitrite.

RT-PCR analysis of lung MIP-2 mRNA. MIP-2 mRNA transcript levels were assessed by PCR amplification of the MIP-2 cDNA as described by Driscoll et al. (13). Briefly, RNA was extracted as described by Chomczynski and Sacchi (6) from the left lung lobe of 2 animals/treatment group. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was evaluated as an internal control. RNA from the lungs of an untreated F-344 rat and from an F-344 rat instilled with 10 µg of lipopolysaccharide and killed 6 h after exposure was analyzed concurrently with unknown lung RNA samples as negative and positive controls for chemokine mRNA expression. The primers, designed from the published sequences for MIP-2 (12) and GAPDH (19), were as follows: MIP-2, 5'-GGCACATCAGGTACGATCCAG-3' and 5'-ACCCTGCCAAGGGTTGACTTC-3'; and GAPDH, 5'-CAGGATGCATTGCTGACAATC-3' and 5'-GGTCGGTGTGAACGGATTTG-3'. PCR reactions were overlaid with mineral oil. Amplification was carried out through 22-30 cycles of denaturation at 94°C for 1 min, oligo annealing at 55°C for 1 min, and extension at 72°C for 2 min. Reactions were electrophoresed in 1.5% agarose gels containing ethidium bromide in Tris-acetate-EDTA buffer to visualize the MIP-2 and GAPDH PCR products. Confirmation that the PCR products obtained with the primer sequences were MIP-2 or GAPDH was obtained by Southern analysis of the PCR products and probing with oligonucleotide probes complementary to mRNA sequences internal to the PCR primer sequences used (data not shown).

Analysis of lung tissue for IL-10 and MIP-2 proteins. IL-10 and MIP-2 proteins were determined in lung tissue homogenates as described by Standiford et al. (32). Briefly, homogenates were prepared by suspending the left lung lobe in a lysis buffer (PBS containing 2 mM phenylmethylsulfonyl fluoride and 1 mg/ml each of aprotinin, antipain, leupeptin, and pepstatin) with the use of a Polytron (Brinkmann Instruments, Westbury, NY). The homogenates were centrifuged at 2,000 g for 10 min, and supernatants were filtered through a 0.2-µm Millipore filter. The level of immunoreactive IL-10 protein was determined by double-ligand ELISA as described in detail elsewhere (32). Immunoreactive MIP-2 protein was determined with a commercially available MIP-2 ELISA (Biosource, Camarillo, CA). The MIP-2 antisera did not cross-react with the structurally related chemokines cytokine-induced neutrophil chemoattractant, MIP-1alpha , MIP-1beta , and IL-8.

Statistical analysis. Data were analyzed by ANOVA, with group differences determined by the Newman-Keuls test (38).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Expression of anti-inflammatory (IL-10) and proinflammatory (MIP-2) cytokines during quartz-induced inflammation. Lung IL-10 levels were determined in rat lungs 6 h, 1 day, and 3 days after intratracheal instillation of saline or 1 mg of quartz. Rat lung tissue contained levels of IL-10 protein that ranged from 480 to 690 pg/ml lung homogenate. Quartz exposure had no effect on rat lung IL-10 concentrations (Fig. 1). Because a previous study (13) indicated that the chemokine MIP-2 contributes to quartz-induced neutrophil recruitment in rats, we characterized lung MIP-2 levels in quartz-exposed and control rat lungs. As shown in Fig. 1, lung MIP-2 protein increased significantly after quartz exposure. A 12-fold increase in MIP-2 was observed 6 h after exposure, with the levels of MIP-2 remaining elevated approximately twofold over those in control lungs 3 days after exposure.


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Fig. 1.   Interleukin (IL)-10 protein (A) and macrophage inflammatory protein (MIP)-2 (B) in rat lung homogenates after quartz exposure. Cytokine protein concentrations in homogenates of left lung lobe are shown 6, 24, and 72 h after intratracheal instillation exposure of rats (n = 6) to saline or saline suspension of quartz particles. Values are means ± SD. * Significant difference from respective saline control group, P < 0.05.

Treatment with anti-IL-10 augments quartz-induced inflammation. Exposure of rats to quartz produced pulmonary inflammation and tissue injury as indicated by significant increases in BAL fluid neutrophils, LDH, and protein (Figs. 2 and 3). Pretreatment of rats with an antiserum to IL-10 increased the severity of the lung response to quartz as demonstrated by significantly greater increases in BAL fluid neutrophils and LDH (Figs. 2 and 3). Saline or a nonimmune serum pretreatment did not alter BAL fluid parameters after instillation of saline or quartz.


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Fig. 2.   Number of macrophages (A) and neutrophils (B) in bronchoalveolar lavage (BAL) fluid from rats exposed to quartz and pretreated with neutralizing anti-IL-10 antiserum. Rats (n = 4) were pretreated with intraperitoneal injection of saline, nonimmune serum, or anti-IL-10 serum 2 h before intratracheal instillation of saline (-) or a saline suspension of quartz particles (+). Responses were examined 3 days after quartz exposure. Values are means ± SD. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and quartz-exposed saline- and nonimmune serum-pretreated groups.


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Fig. 3.   BAL fluid lactate dehydrogenase (LDH; A) and total protein (B) from rats exposed to quartz and pretreated with neutralizing anti-IL-10 antiserum. Rats (n = 4) were treated with intraperitoneal injection of saline, nonimmune serum, or anti-IL-10 serum 2 h before intratracheal instillation with saline or saline suspension of quartz particles. Amount of LDH and protein was determined 3 days after quartz exposure as an indicator of lung injury and edema. Values are means ± SD. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and quartz-exposed saline- and nonimmune serum-pretreated groups.

Inflammation and cellular oxidant production after quartz is attenuated by rmIL-10. Treatment of rats with rmIL-10 attenuated quartz-induced lung inflammation and tissue injury (Figs. 4 and 5). rmIL-10 significantly reduced the increases in BAL fluid neutrophil numbers after quartz exposure, although the number of inflammatory cells remained elevated over saline-treated control animals. Levels of BAL fluid LDH and protein were significantly less in animals treated with rmIL-10 and quartz than in animals treated with quartz alone.


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Fig. 4.   Effect of recombinant murine IL-10 (rmIL-10) on number of macrophages (A) and neutrophils (B) in BAL fluid 1 day after quartz exposure. Rats (n = 3) underwent intratracheal instillation of saline or a saline suspension of quartz particles with or without rmIL-10. Values are means ± SD. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and rmIL-10-treated groups.


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Fig. 5.   Effect of rmIL-10 on BAL fluid LDH (A) and total protein (B) after quartz exposure. Rats (n = 3) underwent intratracheal instillation of saline or a saline suspension of quartz particles with or without rmIL-10. LDH and protein levels were determined 1 day after exposure as indicators of lung injury and edema. Values are means ± SD. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and rmIL-10-treated groups.

A previous study (5) demonstrated that quartz in vitro and in vivo activates lung inflammatory cells to produce reactive oxygen species, and this is thought to contribute to the tissue injury associated with quartz exposure. As shown in Fig. 6, instillation of quartz resulted in a significant increase in the production of hydrogen peroxide, superoxide anion, and nitric oxide by inflammatory cells obtained by BAL. rmIL-10 treatment reduced basal production of hydrogen peroxide and nitric oxide by BAL cells from control rats. Coadministration of rmIL-10 with quartz significantly reduced production of reactive oxygen and nitrogen species by BAL cells.


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Fig. 6.   Effect of rmIL-10 on quartz activation of inflammatory cell hydrogen peroxide (H2O2; A), nitric oxide (nitrate; B), and superoxide anion (O-2·; C) production. Rats (n = 3) underwent intratracheal instillation of saline or a saline suspension of quartz particles with or without rmIL-10. BAL was performed 1 day after exposure, and BAL cells were cultured to determine release of reactive oxygen and nitrogen species. Values are means ± SD. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and rmIL-10-treated groups; ddager  saline control group.

IL-10 regulates quartz-induced expression of MIP-2. To investigate whether IL-10 might influence quartz-induced inflammation by influencing MIP-2 expression, we determined the effect of anti-IL-10 antiserum or rmIL-10 on lung MIP-2 mRNA and protein. Treatment of rats with quartz increased MIP-2 mRNA, with the increase in message appearing greater for rats pretreated with anti-IL-10 antiserum (Fig. 7A). Minimal or no MIP-2 message was detected in rats treated with saline or nonimmune serum. Administration of rmIL-10 attenuated quartz-induced MIP-2 mRNA (Fig. 8A). Consistent with the effects on gene expression, quartz increased MIP-2 protein in BAL fluid, whereas pretreatment with antiserum against IL-10 resulted in an increase in BAL fluid MIP-2 protein that was significantly greater than that seen with quartz alone (Fig. 7B). In contrast, rmIL-10 inhibited the quartz-induced increases in BAL fluid MIP-2 protein (Fig. 8B).


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Fig. 7.   Effect of anti-IL-10 antiserum treatment on quartz-induced MIP-2 mRNA and MIP-2 protein in rat lung homogenates. Rats underwent intraperitoneal injection of saline, nonimmune serum, or anti-IL-10 serum 2 h before intratracheal instillation with saline or a saline suspension of quartz particles. Responses were characterized 3 days after exposure. A: RT-PCR products for MIP-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs amplified from rat lung RNA; n = 2 rats/treatment. B: MIP-2 protein in homogenates of left lung lobe. Values are means ± SD; n = 3 rats. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and quartz-exposed saline- and nonimmune serum-pretreated groups.


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Fig. 8.   Effect of rmIL-10 on quartz-induced lung MIP-2 gene expression and MIP-2 protein in rat lung homogenates. Rats underwent intratracheal instillation of saline or a saline suspension of quartz particles with or without rmIL-10. Responses were characterized 1 day after exposure. A: RT-PCR products for MIP-2 and GAPDH mRNA amplified from rat lung RNA; n = 2 rats/treatment. B: MIP-2 protein concentrations in homogenates of left lung. Values are means ± SD; n = 3 rats. Significant difference (P < 0.05) from: * respective saline-instilled control group; dagger  respective saline-instilled control group and quartz-exposed saline- and nonimmune serum-pretreated groups.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Inhalation of quartz can result in lung injury and fibrosis secondary to the inflammatory response elicited by this mineral dust (20, 21). Several studies (11, 13, 14) have shown that members of the chemokine cytokine family, including MIP-2, contribute to quartz-induced inflammatory cell recruitment. In addition to proinflammatory chemokines, cytokines such as IL-10, IL-4, and transforming growth factor-beta , exist with the potential to exert an anti-inflammatory effect on the response of the lung to inhaled particles (27, 28, 34). Regarding IL-10, this cytokine was reported to attenuate pulmonary inflammation elicited by immune complexes or endotoxin (28, 29, 31). Here we show that IL-10 can downregulate the rat lung inflammatory and cell activation response to quartz by mechanisms that involve suppression of quartz-induced expression of the chemokine MIP-2. These observations support a key role for IL-10 and MIP-2 as anti- and proinflammatory cytokines regulating inflammation within the lower respiratory tract during quartz-induced lung injury.

The rat lung contained basal levels of IL-10 that were not altered by quartz exposure. In contrast, quartz markedly increased lung MIP-2 protein levels. This apparent quartz-induced imbalance between pro- and anti-inflammatory cytokines in the rat lung is likely a factor in the marked pulmonary inflammatory response elicited by this mineral dust. That IL-10 can downregulate the response of the lungs to quartz was demonstrated by administration of exogenous IL-10, which attenuated quartz-induced pulmonary inflammation, cytotoxicity, and inflammatory cell oxidant production. That endogenous IL-10 exerts an anti-inflammatory action toward quartz responses was shown by the greater than threefold increase in quartz-induced inflammation in rats passively immunized with a neutralizing anti-IL-10 antiserum. Apparently, however, the levels of IL-10 were insufficient to fully compensate for the potent proinflammatory action of quartz. In this respect, our observations suggest that variations in basal lung IL-10 levels can influence susceptibility to inhaled materials. In this respect, cystic fibrosis patients have been reported to have lower levels of IL-10 in the respiratory tract fluids than normal subjects (2). Similar to silica responses in rats treated with anti-IL-10 antiserum, the depressed IL-10 levels in the lungs of cystic fibrosis patients may result in enhanced local inflammatory responses to inhaled pathogens.

In contrast to our observations with quartz, increases in tissue IL-10 have been described during the course of inflammatory responses elicited by immunogenic stimuli. For example, in a murine model of pneumococcal pneumonia, IL-10 levels increased in the lung over a period of 3 days after infection (33), and in mice exposed systemically to endotoxin, plasma IL-10 was observed to increase 2 and 6 h after exposure (32). Also, in collagen-induced arthritis in the rat, IL-10 in joint tissue increased by 44 days after the initial immunization (25). In the present study, the absence of any apparent increase in IL-10 in quartz-exposed rat lungs may reflect an inherent difference between quartz and the eliciting agents used in the other studies. In this respect, it is tempting to speculate that the lack of IL-10 induction after quartz may be a factor that contributes to the progressive nature of quartz-induced lung disease (15, 22).

Rat MIP-2 is a member of the C-X-C chemokine family and is structurally and functionally related to the human GRO chemokines (12). MIP-2 is a potent neutrophil chemoattractant and a mitogen for epithelial cells, and it plays a significant role in mediating the rat lung inflammatory response to quartz as well as to other inhaled agents (e.g., asbestos, ozone, and endotoxin; Refs. 9, 10, 12, 13, 17, 33, 37). Consistent with previous observations (10, 13) and a key role of MIP-2 in quartz-induced inflammation (13), the present study demonstrates that this chemokine increases greater than sixfold shortly after quartz exposure and remains elevated throughout a 3-day postexposure period. Importantly, rmIL-10 markedly attenuated quartz-induced increases in both lung MIP-2 mRNA and protein, whereas passive immunization with anti-IL-10 antiserum increased lung MIP-2 protein levels above those seen with quartz alone. These results indicate downregulation of MIP-2 is responsible, at least in part, for the effects of IL-10 on quartz-induced inflammation. At present, the mechanism(s) by which IL-10 inhibits quartz activation of MIP-2 is uncertain. However, it is noteworthy that IL-10 can inhibit nuclear translocation of a transcription factor known as nuclear factor-kappa B (35) that regulates activation of MIP-2 gene transcription (36).

In addition to modulating neutrophilic inflammation, treatment with rmIL-10 or anti-IL-10 serum decreased or increased lung injury, respectively, after quartz exposure. Also, rmIL-10 significantly attenuated production of reactive oxygen and nitrogen species by inflammatory cells after quartz exposure. Whereas the mechanism of rmIL-10-mediated decreases in hydrogen peroxide, superoxide anion, and nitric oxide was not investigated, previous studies (1, 21) have demonstrated that IL-10 can attenuate production of hydrogen peroxide and nitric oxide by macrophages. Additionally, MIP-2 can stimulate an oxidative burst by rat neutrophils (20), and the ability of rmIL-10 to attenuate expression of this chemokine may contribute to decreased oxidant generation in quartz-exposed animals. The concurrence among quartz-induced inflammation, oxidant production, and lung injury further supports a key role for inflammatory cells in the tissue damage resulting from quartz exposure. This observation is consistent with that of Henderson et al. (23), who demonstrated that neutrophil depletion in rats results in a significant attenuation in lung injury from subsequent quartz exposure. Collectively, these findings indicate that a significant component of the tissue damage occurring after quartz exposure occurs secondary to MIP-2 expression and the recruitment and activation of inflammatory cells.

In summary, the present study demonstrates that treatment with rmIL-10 attenuates quartz-induced pulmonary inflammation, tissue injury, and production of reactive oxygen and nitrogen species in rat lungs, whereas passive immunization with anti-IL-10 serum enhances the inflammatory response to quartz. The modulation of quartz-induced responses by IL-10 was associated with decreases in MIP-2 mRNA and protein, indicating the anti-inflammatory action of IL-10 was mediated, at least in part, by a downregulation of MIP-2 gene expression. Basal levels of IL-10 were detected in the rat lung; however, IL-10 did not increase after quartz exposure. These observations suggest that IL-10 acts as a constitutive anti-inflammatory factor serving to attenuate the inflammatory response to quartz. The absence of any detectable induction of IL-10 in response to quartz may be a factor contributing to the progressive nature of quartz-induced lung disease in rodents and humans.

    ACKNOWLEDGEMENTS

These studies were partially supported by National Heart, Lung, and Blood Institute Grant P50-HL-56402 (to S. L. Kunkel and R. M. Strieter).

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: K. E. Driscoll, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd., Mason, OH 45040-9462.

Received 9 February 1998; accepted in final form 10 June 1998.

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Abstract
Introduction
Methods
Results
Discussion
References

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