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Ozone in ambient air may cause various effects on human health, including decreased lung function, asthma exacerbation, and even premature mortality. These effects have been evidenced using various clinical indicators that, although sensitive, do not specifically evaluate the O(3)-increased lung epithelium permeability. In the present study, we assessed the acute effects of ambient O(3) on the pulmonary epithelium by a new approach relying on the assay in serum of the lung-specific Clara cell protein (CC16 or CC10). We applied this test to cyclists who exercised for 2 hr during episodes of photochemical smog and found that O(3) induces an early leakage of lung Clara cell protein. The protein levels increased significantly into the serum from exposure levels as low as 0.060-0.084 ppm. Our findings, confirmed in mice exposed to the current U.S. National Ambient Air Quality Standards for O(3) (0.08 ppm for 8 hr) indicate that above the present natural background levels, there is almost no safety margin for the effects of ambient O(3) on airway permeability. The assay of CC16 in the serum represents a new sensitive noninvasive test allowing the detection of early effects of ambient O(3) on the lung epithelial barrier.

Clara cell secretory protein (CC16, CC10, or CCSP), the major secretory protein of the Clara cell, presents several biologic properties, suggesting that it may play a protective role against intrapulmonary inflammatory processes. The aim of the present study was to investigate the changes of CC16 concentrations in the lung, bronchoalveolar lavage fluid (BALF), and serum of rats with acute lung injury induced by lipopolysaccharide (LPS). These changes were compared with Clara cell density, CC16 mRNA level in the lung and classic indices of inflammation in BALF. Injected at doses of 10, 100, or 200 microgram/100 g body weight, LPS induced an acute lung inflammation as estimated by an increased influx of cells and albumin in the BALF. This inflammatory response was associated with a marked reduction of CC16 concentrations in BALF and lung homogenate as well as of the CC16 mRNA levels in the lung. At the highest dose of LPS, the CC16-positive cell density in the bronchiolar epithelium was also decreased. In serum, by contrast, the concentration of CC16 was elevated as a consequence of increased airway permeability. Pretreating rats intraperitoneally with dexamethasone (2 mg/kg) significantly lowered the leukocyte influx and attenuated the albumin increase in BALF. Dexamethasone, however, failed to prevent the increased airway permeability to CC16, suggesting that during inflammation different mechanisms regulate the leakage of proteins across the alveolocapillary barrier depending on the direction of passage and/or the size of the protein. Our results show a marked decrease of the secretion and synthesis of CC16 during LPS-induced acute lung inflammation.

Using two-dimensional electrophoresis, we have recently identified in human bronchoalveolar lavage fluid a novel protein, termed B166, with a molecular mass of 17 kDa. Here, we report the cloning of human and rat cDNAs encoding B166, which has been renamed AOEB166 for antioxidant enzyme B166. Indeed, the deduced amino acid sequence reveals that AOEB166 represents a new mammalian subfamily of AhpC/TSA peroxiredoxin antioxidant enzymes. Human AOEB166 shares 63% similarity with Escherichia coli AhpC22 alkyl hydroperoxide reductase and 66% similarity with a recently identified Saccharomyces cerevisiae alkyl hydroperoxide reductase/thioredoxin peroxidase. Moreover, recombinant AOEB166 expressed in E. coli exhibits a peroxidase activity, and an antioxidant activity comparable with that of catalase was demonstrated with the glutamine synthetase protection assay against dithiothreitol/Fe3+/O(2) oxidation. The analysis of AOEB166 mRNA distribution in 30 different human tissues and in 10 cell lines shows that the gene is widely expressed in the body. Of interest, the analysis of N- and C-terminal domains of both human and rat AOEB166 reveals amino acid sequences presenting features of mitochondrial and peroxisomal targeting sequences. Furthermore, human AOEB166 expressed as a fusion protein with GFP in HepG2 cell line is sorted to these organelles. Finally, acute inflammation induced in rat lung by lipopolysaccharide is associated with an increase of AOEB166 mRNA levels in lung, suggesting a protective role for AOEB166 in oxidative and inflammatory processes.

Clara cell protein (CC16) is a 16-17-kDa protein secreted by Clara cells in the bronchial lining fluid of the lung from which it passively diffuses into serum before being eliminated by the kidneys. The concentration of CC16 in serum has recently been proposed as a peripheral marker of the integrity of Clara cells and/or of the bronchoalveolar/blood barrier. To evaluate the sensitivity of this new lung marker to acute epithelial damage induced by ozone (O(3)), CC16 was determined in the serum of rats after a single 3-h exposure to 0.3, 0.6, or 1 ppm O(3). The urinary excretion of the protein was also studied in rats repeatedly exposed to 1 ppm O(3), 3 h/day, for up to 10 days. The concentrations of CC16 in the lung or trachea homogenates, the lung CC16 mRNA levels, and classical markers of lung injury in bronchoalveolar lavage fluid (BALF) were also determined. O(3) produced a transient increase of CC16 concentration in serum that reached values on average 13 times above normal 2 h after exposure to 1 ppm O(3). The intravascular leakage of CC16 was dose-dependent and correlated with the extent of lung injury as assessed by the levels of total protein, LDH, and inflammatory cells in BALF. This effect was most likely responsible for the concomitant marked reduction of CC16 concentrations in BALF and lung homogenate, since the CC16 mRNA levels in the lungs were unchanged and the absolute amounts of CC16 leaking into serum or lost from the respiratory tract were similar. These changes were paralleled by an elevation of the urinary excretion of CC16 resulting from an overloading of the tubular reabsorption process. These results demonstrate that the assay of CC16 in serum and even in urine represents a new noninvasive test to detect the increased lung epithelial permeability induced by O(3)., (Copyright 1999 Academic Press.)

The 16 kDa Clara cell protein (CC16), an abundant component of airway secretions, has recently been proposed in humans as a pulmonary marker measurable not only in bronchoalveolar lavage fluid (BALF) but also in serum. The aim of the present study was to investigate the changes and determinants of CC16 concentrations in these fluids in normal rats and rats with lung injury. Female Sprague-Dawley rats were given a single i.p. injection of arachis oil (n=20) or chemicals in arachis oil (n=10) that mainly damage Clara cells (4-ipomeanol (IPO) 8 mg x kg(-1) and methylcyclopentadienyl manganese tricarbonyl (MMT) 5 mg x kg(-1)) or endothelial cells (alpha-naphthylthiourea (ANTU) 5 mg x kg(-1)). CC16 concentration (mean+/-sD in microg x L(-1)), measured by a sensitive latex immunoassay, was significantly reduced in BALF of all treated groups (IPO 380+/-100; MMT 730+/-200; ANTU 1,070+/-200; controls 1,700+/-470). The same pattern of decrease was observed in the labelling of Clara cells with an anti-CC16 antiserum as well as in the CC16 messenger ribonucleic acid levels assessed by Northern enzyme-linked immunosorbent assay. In serum, by contrast, CC16 was significantly increased in all treated groups (IPO 31+/-7; MMT 22+/-12; ANTU 52+/-24; controls 15+/-6). This rise of CC16 in serum was associated with an elevation of albumin in BALF which is an index of increased bronchoalveolar/blood barrier permeability. In conclusion, lung injury induces a decrease of the 16 kDa Clara cell protein in bronchoalveolar lavage fluid owing to a reduced production by damaged Clara cells, and an increase in serum protein levels resulting from its enhanced leakage across the bronchoalveolar/blood barrier. This study provides new insights into the understanding of the changes of lung secretory proteins in bronchoalveolar lavage fluid and serum.

Glutathione (GSH) is an essential antioxidant tripeptide that protects mammalian cells against oxidants and xenobiotics. Patients with fibrotic lung disorders have very low levels of GSH in their alveolar epithelial lining fluid (ELF), whereas transforming growth factor (TGF)-beta is overexpressed in their alveolar epithelial cells. We observed that TGF-beta1 increased susceptibility of the human alveolar epithelial cell line A549 to H2O2-mediated cytotoxicity (P < 0.05), decreased the activities of the antioxidant enzymes glutathione reductase and catalase by 31%, and markedly decreased GSH content in A549 cells (P < 0.01). GSH depletion was associated with an equivalent decrease in the activity of the rate-limiting enzyme in GSH synthesis, gamma-glutamylcysteine synthetase (gamma-GCS) (P < 0.01). Western blot analysis confirmed that the loss of gamma-GCS activity was associated with a marked decrease in gamma-GCS heavy subunit (gamma-GCShs) protein. TGF-beta1 suppressed the steady-state level of messenger RNA (mRNA) for the gamma-GCShs gene, with a maximal effect at 24 h. The half-life of gamma-GCShs mRNA was not affected by TGF-beta1, but transcription of the gene was downregulated as determined with nuclear run-on assays. Our findings indicate for the first time that TGF-beta1 is a potent inhibitor of GSH synthesis in human lung epithelial cells, and that the inhibition is mediated, at least in part, by a transcriptional effect on the gene encoding gamma-GCShs. Regulation of gamma-GCShs gene expression by TGF-beta1 is likely to play an important role in lower respiratory tract GSH homeostasis, and may represent a novel target for therapeutic efforts in lung fibrosis.

Interferon (IFN)-gamma-induced inhibition of type II epithelial cell thymidine incorporation (45% decrease vs. control) was restored by cocultures with mitogen-activated peripheral blood mononuclear cells (PBMC) and conditioned media (CM) from mitogen-activated PBMC. Successive exposure of type II cells to IFN-gamma and interleukin (IL)-2 produced similar alterations in thymidine incorporation. Given that IL-2 is a powerful pleiotropic cytokine produced by lymphoid and myeloid cells, the presence of IL-2 receptors (IL-2R) was assessed in primary cultures of rat type II pneumocytes (TIIP) and the nontransformed alveolar type II epithelial cell line L2. The presence of IL-2R membrane protein on rat type II cells was established by immunodetection assays. The expression of all three murine IL-2R alpha-, beta-, and gamma-chain RNA transcripts in primary TIIP cultures and L2 cells was highlighted by reverse transcriptase-polymerase chain reaction analysis. Overall, these experiments demonstrate, for the first time, that type II epithelial cells can express functional IL-2R, confirming TIIP as a potential "partner" in the lung immune system. Consequently, it can be speculated that TIIP are new cellular targets for lymphokines using (IL-2R) gamma-chain-bearing receptors in lung distal air-spaces.

After acute lung injury, altered bronchioloalveolar epithelia must be repaired quickly in order to restore lung function. During reepithelialization, type II cells initially appear to migrate and spread over a remodeled matrix; then a secondary proliferative phase occurs. It was hypothesized that 1) type II cells can develop locomotion in vitro that is modulated by growth factors, proinflammatory cytokines, and substrate adhesion molecules and 2) migration and proliferation of type II cells can occur as distinctive processes. Chemotaxis assays were elaborated using short term cultures of rat type II pneumocytes. Epidermal growth factor (EGF), transforming growth factor-alpha, laminin, fibronectin were found to be the main attractants for type II cells with respective increases of approximately 8.5-, 10.5-, 8-, and 7-fold in cell migration (P<0.05 vs. control). Laminin induced gradient-dependent and random cell migration. Addition of laminin with EGF had a synergistic effect in promoting cell migration (approximately 30-fold increase over control, P<0.05). Interferon-gamma and interleukin-6 inhibited EGF-induced type II cell migration, whereas tumor necrosis factor-alpha and interleukin-1beta acted as primers for type II cell migration (approximately 1.5-fold increase over control, P<0.05. Type II cells did not need to be in a proliferative phase in order to exhibit motility. New insights regarding the regulatory processes for type II cell migration are especially relevant in our understanding of early events occurring during epithelial repair after acute lung injury.

Ozone (O3) is one of the major irritant oxidant gases in photochemical smog. In the present study, the in vitro effect of low concentrations of O3 (0.1 to 1 ppm) was evaluated on cell viability and cytokine secretion by alveolar macrophages (AM) from guinea pigs and healthy subjects. Cell injury was estimated immediately after O3 exposure by evaluation of ATP cell content (measured by bioluminescence) and lactic dehydrogenase (LDH) release in the culture medium. No cytotoxic effect was found: the ATP cell content of both guinea pig AM and human AM did not significantly change after O3 exposure and similarly the LDH release in the culture medium was unchanged. AM-derived cytokines (tumor necrosis factor alpha [TNF alpha], interleukin-1 beta [IL-1 beta], interleukin-6 [IL-6], and interleukin-8 [IL-8]) were evaluated in AM supernatants. O3 exposure was associated with a significant increase in cytokine secretion, with a peak value at 0.4 ppm O3. The exposure of the guinea pig AM to 0.4 ppm O3 for 60 min increased the IL-6 activity by 252 +/- 60% and TNF activity by 202 +/- 35%. The increase in monokine production by the human AM was 443 +/- 208% for TNF alpha, 484 +/- 171% for IL-1 beta, 383 +/- 147% for IL-6, and 226 +/- 45% for IL-8 after a 60-min exposure to 0.4 ppm O3. Lowest O3 concentrations (0.1 and 0.2 ppm) only increased TNF alpha secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Clara cell protein (CC-16, also designated CC-10) is synthesized by the bronchiolar epithelium and has been suggested as an inhibitor of phospholipase A2 (PLA2) activity. Therefore, CC-16 is a candidate for controlling inflammatory events in the lung. Because CC-16 amounts and function may be altered in fibrosing lung diseases in which bronchiolar injury has been reported, it was measured in alveolar fluids and sera. Secretory PLA2 activity in alveolar fluids and the influence of CC-16 on platelet-derived growth factor-induced human fibroblast chemotaxis and cytosolic PLA2 activity were also explored. CC-16 content was decreased in alveolar fluids from idiopathic pulmonary fibrosis (IPF: 1.3 +/- 0.1 mg/L) and bleomycin lung (1.1 +/- 0.2 versus 2.1 +/- 0.2 mg/L in controls, p < 0.05), whereas there was a three- to ninefold increase in secretory PLA2 activity (p < 0.05 versus controls). CC-16 inhibited fibroblast chemotaxis in a dose-dependent manner (90% inhibition at 30 micrograms/ml CC-16). This inhibition was reversed by reducing CC-16. CC-16 was also able to lower fibroblastic cytosolic PLA2 activity by 50% in vitro. In summary, CC-16 is able to inhibit fibroblast chemotaxis in vitro by mechanisms that may be related to a blockage of cytosolic PLA2 activity. It can be postulated that CC-16 deficiency may contribute to fibroblast burden activity in fibrosing lung diseases.

The aim of the current study was to assess the in vitro effects of nickel hydroxy carbonate (NiHC) at noncytotoxic concentrations on the production of cytokines such as tumour necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in alveolar macrophages (AMs). The effect of NiHC was evaluated in both unstimulated AMs and cells activated by lipopolysaccharide (LPS). Cytotoxicity was related to lactate dehydrogenase release and ATP cell content. The results confirm that NiHC at concentrations of 0.125, 1.25 and 3.125 micrograms NiHC 10(-6) cells was not cytotoxic. The NiHC exposure of unstimulated AMs significantly increased the release of TNF-alpha at all concentrations and that of IL-6 at 1.25 micrograms NiHC 10(-6) cells. LPS addition significantly increased the secretion of both cytokines. However, NiHC did not cause a significant increase in the release of TNF-alpha and IL-6 in LPS-stimulated cells. In conclusion, the ability of NiHC to activate AMs and to release increased amounts of pro-inflammatory mediators may be responsible, at least partly, for inflammation and pneumotoxicity associated with nickel exposure.

The aim of this investigation was to assess the cytotoxic effect of nickel hydroxy carbonate (NiHC) on guinea pig alveolar macrophages (AMs) by studying ultrastructural modifications and by determining beta-glucuronidase (BG) and lactate dehydrogenase (LDH) activities, as well as cellular ATP content. The ultrastructural studies revealed phagocytosis of NiHC particles and a general vacuolisation of the cells, especially at high concentrations. X-ray microprobe analyses of these particles demonstrated the presence of Ni, P and Ca which suggests the formation of Ni-P-Ca complexes. In exposed cells, a biphasic change in intracellular ATP concentrations was observed which could indicate 'activation' of AMs at low concentrations and inhibition of energy generation at higher concentrations. As for enzymatic activities, a dose-dependent increase in LDH release was observed except at low doses which increased ATP. There was a good correlation between ATP decrease and LDH release, consistent with a dose-dependent cytotoxic effect of NiHC. However beta-glucuronidase activity remained unchanged at all NiHC concentrations. It has been concluded that NiHC undergoes an intracellular, biological transformation to form Ni-P-Ca. Further investigations are needed to determine the precise nature and importance of these complexes.

The metabolic effects of different concentrations of nickel hydroxycarbonate (NiHC) on guinea pig alveolar macrophages (GPAMs) were investigated. Exposure to high concentrations of NiHC (6.25 and 12.5 micrograms 10(-6) cells) led to cell vacuolization. Morphological changes were associated with a dramatic reduction in the steady-state level of cellular adenosine triphosphate (ATP), i.e. ATP levels were reduced by 35% (P less than 0.001) and 53% (P less than 0.01), respectively. Low concentrations of NiHC (0.0625 and 0.125 microgram 10(-6) cells) did not induce morphological changes but increased cellular ATP content by 19% (P less than 0.01) and 12% (P less than 0.05), respectively. Effects of NiHC (0.125 and 6.25 micrograms 10(-6) cells) on cell oxidative metabolism were studied. The chemiluminescence was significantly increased (P less than 0.001) by the lower but not the higher concentration. A slight inhibition of total superoxide dismutase (P less than 0.05) and a decrease of catalase activity were demonstrated (P less than 0.05) for the high dose, while the low dose decreased the levels of reduced and total glutathione. In conclusion, the effects of NiHC on alveolar macrophages are characterized by an overproduction of free radicals for low concentrations and the depletion of cellular reserve energy, particularly ATP, for high concentrations.