Interleukin-10 regulates quartz-induced pulmonary inflammation in rats.

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.

Crocidolite activates NF-kappa B and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants.

Nuclear factor kappa B (NF-kappa B) is a transcription factor that regulates expression of several genes coding for inflammatory and immunoregulatory proteins including the neutrophil chemotactic cytokine MIP-2. In previous studies we found that crocidolite asbestos activates the nuclear translocation of NF-kappa B as well as MIP-2 gene expression in rat alveolar type II cells. Here we report that both crocidolite-induced NF-kappa B activation of MIP-2 gene expression can be attenuated by the antioxidant tetramethylthiourea, suggesting the dependence of these responses on oxidative stress. Crocidolite exposure of RLE-TN cells also increased production of H2O2, a response that was inhibited by the mitochondrial respiratory chain inhibitor thenoyltrifluoroacetone (TTFA). TTFA treatment of RLE-6TN cells also inhibited crocidolite-induced nuclear translocation of NF-kappa B and MIP-2 gene expression. These results indicate crocidolite exposure of rat alveolar type II cells results in increased production of mitochondrial-derived hydrogen peroxide and that mitochondrial-derived oxidants contribute to crocidolite activation of NF-kappa B and increases in MIP-2 gene expression.

Modulation of mitochondrial gene expression in pulmonary epithelial cells exposed to oxidants.

Oxidants are important in the regulation of signal transduction and gene expression. Multiple classes of genes are transcriptionally activated by oxidants and are implicated in different phenotypic responses. In the present study, we performed differential mRNA display to elucidate genes that are induced or repressed after exposure of rat lung epithelial (RLE) cells to H2O2 or crocidolite asbestos, a pathogenic mineral that generates oxidants. After 8 or 24 hr of exposure, RNA was extracted, reverse transcribed, and amplified by polymerase chain reaction with degenerate primers to visualize alterations in gene expression. The seven clones obtained were sequenced and encoded the mitochondrial genes, NADH dehydrogenase subunits ND5 and ND6, and 16S ribosomal RNA. Evaluation of their expression by Northern blot analysis revealed increased expression of 16S rRNA after 1 or 2 hr of exposure to H2O2. At later time periods (4 and 24 hr), mRNA levels of 16S rRNA and NADH dehydrogenase were decreased in H2O2-treated RLE cells when compared to sham controls. Crocidolite asbestos caused increases in 16S rRNA levels after 8 hr of exposure, whereas after 24 hr of exposure to asbestos, 16S rRNA levels were decreased in comparison to sham controls. In addition to these oxidants, the nitric oxide generator spermine NONOate caused similar decreases in NADH dehydrogenase mRNA levels after 4 hr of exposure. The present data and previous studies demonstrated that all oxidants examined resulted in apoptosis in RLE cells during the time frame where alterations of mitochondrial gene expression were observed. As the mitochondrion is a major organelle that controls apoptosis, alterations in expression of mitochondrial genes may be involved in the regulation of apoptosis.

Cytokines and particle-induced inflammatory cell recruitment.

The inflammatory response is a key component of host defense. However, excessive or persistent inflammation can contribute to the pathogenesis of disease. Inflammation is regulated, in part, by cytokines, which are small, typically glycosylated proteins that interact with membrane receptors to regulate cellular processes such as proliferation, differentiation, and secretion. During the past 10 years studies in humans and experimental animals have demonstrated that a cytokine called tumor necrosis factor alpha (TNF-alpha) plays a key role in the initiation of inflammatory responses in the lung and other tissues, including inflammation resulting from inhalation of noxious particles. There is now compelling evidence that one of the pathways by which inhaled particles stimulate the recruitment and subsequent activation of inflammatory cells is through the activation of lung macrophages to release TNF-alpha. TNF-alpha then acts via paracrine and autocrine pathways to stimulate cells to release other cytokines known as chemokines, which are directly chemotactic to leukocytes and other cells that participate in inflammatory and wound healing responses. In addition to a TNF-alpha-mediated pathway, there is growing evidence that some particles such as quartz and crocidolite can directly activate lung epithelial cells to release chemokines such as macrophage inflammatory protein-2, cytokine-induced neutrophil chemoattractant, and interleukin-8. A direct stimulatory effect of particles on lung epithelium may represent an additional or alternate pathway by which inhaled particles may elicit inflammation in the lung. Recent studies have suggested that oxidative stress may be a component of the mechanism by which particles activate cytokine production in cells such as macrophages and epithelial cells. The contribution of oxidative stress to particle-induced cytokine gene expression appears to be mediated, at least in part, through activation of the transcription factor nuclear factor kappa B.

Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells.

To investigate mechanisms underlying development of lung adenomas and carcinomas in rats exposed to poorly soluble particles the relationships between particle exposure, inflammation and mutagenesis in rat alveolar type II cells were characterized. Rats were exposed to saline or saline suspensions of 10 and 100 mg/kg of alpha-quartz, carbon black or titanium dioxide by intratracheal instillation. Fifteen months after exposure, bronchoalveolar lavage (BAL) cells were characterized as to number and type and lung histopathology performed. The alveolar type II cells were isolated and cultured in 6 thioguanine (6TG) containing media to select for mutation in the hprt gene. The potential contribution of lung inflammatory cells to in vivo mutagenic responses, were evaluated by co-culturing BAL cells with the rat alveolar epithelial cell line, RLE-6TN for 24 h and the RLE-6TN cells selected for 6TG resistance. Neutrophilic inflammation was detected in all rats exposed to 10 and 100 mg/kg of alpha-quartz and carbon black and 100 mg/kg titanium dioxide; epithelial hyperplasia was observed in rats exposed to 10 and 100 mg/kg of alpha-quartz and 100 mg/kg carbon black. Hprt mutation frequency was increased in alveolar type II cells from rats exposed to 10 and 100 mg/kg of alpha-quartz, 100 mg/kg carbon black and 100 mg/kg titanium dioxide. In vitro exposure of RLE-6TN cells to BAL cells from rats treated with 10 and 100 mg/kg of alpha-quartz or 100 mg/kg carbon black increased hprt mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RLE-6TN cells, however, the mutagenic activity appeared greatest for neutrophils. Addition of catalase to BAL cell:RLE-6TN co-cultures inhibited the increase in hprt mutation frequency. These studies demonstrate exposure of rats to doses of particles producing significant neutrophilic inflammation is associated with increased mutation in rat alveolar type II cells. The ability of particle-elicited macrophages and neutrophils to exert a mutagenic effect on epithelial cells in vitro supports a role for these inflammatory cells in the in vivo mutagenic effects of particle exposure. The inhibition of BAL cell-induced mutations by catalase implies a role for cell-derived oxidants in this response.

Pulmonary inflammatory, chemokine, and mutagenic responses in rats after subchronic inhalation of carbon black.

Chronic inhalation of carbon black can produce carcinomas in rat lungs. At present the mechanisms underlying the rat lung tumor response to carbon black are unknown, although a significant role for inflammation and cell proliferation has been postulated. To investigate the processes which may contribute to development of rat lung tumors after carbon black exposure, we characterized the effects of subchronic inhalation of carbon black by rats on mutagenesis in alveolar epithelial cells, pulmonary inflammation, inflammatory cytokine/growth factor expression, and lung histopathology. Briefly, rats were exposed for 6 hr/day, 5 days/week for up to 13 weeks to 1.1, 7.1, and 52.8 mg/m3 carbon black and the effects on the lung were characterized after 6.5 and 13 weeks of exposure and 3 and 8 months of recovery. Endpoints characterized after carbon black exposure included mutation in the hprt gene of alveolar epithelial cells, changes in bronchoalveolar lavage fluid markers of lung injury and inflammation, expression of mRNA for the chemokines, MIP-2 and MCP-1, and lung histopathology. Lung burdens of carbon black were also determined. After 13 weeks of exposure to 1.1, 7.1, and 52.8 mg/m3 carbon black, lung burdens were 354, 1826, and 7861 micrograms carbon black, respectively. The lung clearance of carbon black appeared impaired after exposure to 7.1 and 52.8 mg/m3 carbon black, with the effects being more pronounced at the higher exposure level. Subchronic inhalation of 1.1 mg/m3 carbon black did not elicit any detectable adverse lung effects. A significant increase in hprt mutation frequency in alveolar epithelial cells was detected immediately after 13 weeks of exposure to 7.1 and 52.8 mg/m3 carbon black as well as after 3- and 8-month recovery periods for the group exposed to 52.8 mg/m3. No increase in hprt mutation frequency was observed for epithelial cells obtained from rats exposed to 1.1 mg/m3 carbon black. The observation that genotoxic effects (i.e., mutations) on alveolar epithelial cells occurred only after carbon black exposures which resulted in significant inflammation and epithelial hyperplasia supports the hypothesis that inflammatory cell-derives oxidants and increased cell proliferation play a role in the pathogenesis of rat lung tumors in response to carbon black.

TNF alpha and increased chemokine expression in rat lung after particle exposure.

Macrophage inflammatory protein 2 (MIP-2) and CINC (Cytokine-Induced-Neutrophil-Chemoattractant) are members of the chemokine family of inflammatory and immunoregulatory cytokines. MIP-2 and CINC exhibit potent neutrophil chemotactic activity and are thought to be key mediators of inflammatory cell recruitment in response to tissue injury and infection. In the present studies, we examined the potential involvement of MIP-2 and CINC in particle-elicited inflammation in the rat lung and the role of TNF alpha in particle-induced chemokine expression. Acute intratracheal instillation exposure of F344 rats to alpha quartz or titanium dioxide was shown to markedly increase steady-state levels of MIP-2 and CINC mRNA in lung tissue; a response which was associated with a significant increase in neutrophils in the bronchoalveolar lavage fluid. Additional studies demonstrated that acute inhalation of crocidolite fibers by rats also induced increased MIP-2 and CINC expression. Since previous studies had demonstrated that TNF alpha stimulates MIP-2 and CINC expression in vitro and that particle exposure induces TNF alpha production in rat lung we examined the role of TNF alpha in alpha quartz-induced MIP-2 gene expression. We demonstrated that passive immunization of mice against TNF alpha markedly attenuated the increased lung MIP-2 mRNA seen in response to alpha quartz inhalation. Collectively, these findings suggest that the chemokines MIP-2 and CINC play a role in neutrophil recruitment to the rat lung after particle exposure and indicate that particle-induced expression of these chemokines is mediated, at least in part, by production of TNF alpha.

Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein 2.

Short-term exposure of humans and animals to ozone results in increased lung neutrophils; however, the mechanisms underlying this response are not completely understood. We examined the potential involvement of the neutrophil chemotactic factor, macrophage inflammatory protein 2 (MIP-2), in ozone-induced inflammation. Exposure-response relationships for ozone and MIP-2 expression were characterized by exposing C57B1/6 mice to 0.1-2 ppm ozone for 3 hr and determining lung levels of MIP-2 mRNA 6 hr after exposure. Temporal relationships between ozone and MIP-2 were determined by exposing mice (2 ppm ozone x 3 hr) and characterizing MIP-2 mRNA expression 0, 2, 6, and 24 hr after exposure. Neutrophils in lung lavage fluid were determined in both exposure-response and time course studies. Ozone concentrations > or = 1.0 ppm increased MIP-2 mRNA and this increase corresponded with recruitment of neutrophils. MIP-2 mRNA was increased immediately after ozone exposure and decreased to control levels by 24 hr. To examine the role of direct oxidant effects in ozone-induced MIP-2 expression, alveolar macrophages were exposed in vitro for 4 hr to 10(-10)-10(-5) M hydrogen peroxide and MIP-2 expression was characterized. MIP-2 mRNA levels in lung macrophages were increased by > or = 10(-9) M hydrogen peroxide. In summary, our findings suggest the chemotactic protein MIP-2 may be responsible, at least in part, for ozone-induced increases in lung neutrophils and indicate that direct exposure of alveolar macrophages to an oxidant is sufficient to induce MIP-2 expression.

Macrophage inflammatory proteins 1 and 2: expression by rat alveolar macrophages, fibroblasts, and epithelial cells and in rat lung after mineral dust exposure.

Macrophage inflammatory proteins 1 alpha and 2 (MIP-1 alpha, MIP-2) are members of a growing family of cytokines thought to play a role in host defense. MIP-1 alpha and MIP-2 were previously identified in the mouse and shown to stimulate inflammatory cell recruitment. To better understand the potential role of MIP-1 alpha and MIP-2 in lung defense, we investigated the ability of rat lung cells to express mRNA for and/or secrete MIP-1 alpha and MIP-2 proteins in vitro and characterized expression of these cytokines in rat lung after in vivo exposure to silica (SiO2) or titanium dioxide (TiO2). In response to lipopolysaccharide, rat alveolar macrophages expressed increased levels of MIP-1 alpha and MIP-2 mRNA and secreted proteins (identified by N-terminal sequencing) homologous to mouse MIP-1 alpha and MIP-2. Rat alveolar macrophage MIP-1 alpha and MIP-2 mRNA expression was also increased by tumor necrosis factor-alpha (TNF) and adherence to plastic. Studies with a rat fibroblast and epithelial cell line demonstrated that MIP-2, but not MIP-1 alpha, expression can be detected in these cells after stimulation with TNF. Intratracheal instillation studies with SiO2 and TiO2 showed that inflammatory doses of these dusts increase MIP-1 alpha and MIP-2 mRNA expression in whole lung and that increased gene expression preceded the accumulation of inflammatory cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Discordant segregation of Na+,K(+)-adenosine triphosphatase alleles and essential hypertension.

OBJECTIVES: To determine whether the alpha 2 and or beta 1 isoforms of the Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) are involved in the pathogenesis of essential hypertension. DESIGN: Segregation analysis of polymorphic DNA markers was used to test the involvement of Na+,K(+)-ATPase in essential hypertension. PARTICIPANTS: Children with persistent hypertension having one parent with essential hypertension were included in the study. Criteria for persistent hypertension were blood pressure readings with systolic and/or diastolic levels exceeding the 95th percentile based upon age and sex. The diagnosis of hypertension for adults, including parents and older siblings, was confirmed using criteria recommended in the 1988 report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. RESULTS: In three essential hypertensive families consisting of 18 members including 11 hypertensives, several obligate recombinants between the Na+,K(+)-ATPase alpha 2 isoform marker and the hypertension phenotype were observed. Similarly, in one hypertension family consisting of four members, obligate recombinants between the beta 1 isoform marker and the disease were observed. CONCLUSIONS: The discordant segregation of the alpha 2 and beta 1 isoform markers and essential hypertension suggests that neither the Na+,K(+)-ATPase alpha 2 nor beta 1 isoform genes play a primary role in the pathogenesis of hypertension in the families studied.