Air pollution-related metals induce differential cytokine responses in bronchial epithelial cells.

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd(2+), Zn(2+) and As(3+) induced CXCL8 and IL-6 release at concentrations below 100µM, and Mn(2+) and Ni(2+) at concentrations above 200µM. In contrast, VO4(3-), Cu(2+) and Fe(2+) did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd(2+), Zn(2+) and As(3+) induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn(2+)- and As(3+)-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd(2+), Zn(2+) and As(3+) seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.

Role of size and surface area for pro-inflammatory responses to silica nanoparticles in epithelial lung cells: importance of exposure conditions.

The present study compared non-crystalline silica particles of nano (50nm)- and submicro (500nm)-size (Si50 and Si500) for the potential to induce cytokine responses in bronchial epithelial lung cells (BEAS-2B). The cell cultures were exposed to equal mass and surface area concentrations of the two particles in different exposure media; LHC-9 and DMEM:F12. The state of agglomeration was different in the two media; with marked agglomeration in LHC-9 and nearly no agglomeration in DMEM:F12. On a mass basis, Si50 was more potent than Si500 in inducing cytokine responses in both exposure media. In contrast, upon exposure to similar surface area concentrations, Si500 was more potent than Si50 in DMEM:F12. This might be due to different agglomeration/sedimentation properties of Si50 versus Si500 in the two media. However, influence of differences in particle reactivity or particle uptake cannot be excluded. The data indicated no qualitative changes in the cytokine gene-expression patterns induced by the two particles, suggesting effects through similar mechanisms. These aspects might be of importance for interpretation of in vitro studies of nanomaterials.

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene.

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 µM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 µM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

TACE/TGF-α/EGFR regulates CXCL8 in bronchial epithelial cells exposed to particulate matter components.

Airborne particulate matter (PM) may induce or exacerbate neutrophilic airway disease by triggering the release of inflammatory mediators, such as CXC chemokine ligand (CXCL)8, from the airway epithelium. It is still unclear which PM components are driving CXCL8 responses, as most candidates occur at low concentrations in the dusts. We therefore hypothesised that different PM constituents may contribute through common mechanisms to induce CXCL8. Human bronchial epithelial cells (BEAS-2B) were exposed to different PM components (Zn²âº/Fe²âº salts, 1-nitropyrene, lipopolysaccharide and diesel exhaust/mineral particles). Gene expression patterns were detected by real-time PCR array. CXCL8 responses were measured by real-time PCR and ELISA. CXCL8 regulation was assessed with a broad inhibitor panel and neutralising antibodies. Epidermal growth factor receptor (EGFR) phosphorylation was examined by immunoprecipitation and Western blotting. Component-induced gene expression was mainly linked to nuclear factor-κB, Ca²âº/protein kinase C, phospholipase C, low-density lipoprotein and mitogenic signalling. Many inhibitors attenuated CXCL8 release induced by all PM components, but to varying extents. However, EGFR inhibition strongly reduced CXCL8 release induced by all test compounds and selected compounds increased EGFR phosphorylation. Interference with transforming growth factor (TGF)-α or tumour necrosis factor-α-converting enzyme (TACE), which mediates TGF-α ectodomain shedding, also attenuated CXCL8 release. Different PM constituents induced CXCL8 partly through similar signalling pathways but the relative importance of the different pathways varied. However, TACE/TGF-α/EGFR signalling appears to be a convergent pathway regulating innate immune responses of airway epithelial cells upon exposure to multiple airborne pollutants.

Differential effects of nitro-PAHs and amino-PAHs on cytokine and chemokine responses in human bronchial epithelial BEAS-2B cells.

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are found in diesel exhaust and air pollution particles. Along with other PAHs, many nitro-PAHs possess mutagenic and carcinogenic properties, but their effects on pro-inflammatory processes and cell death are less known. In the present study we examined the effects of 1-nitropyrene (1-NP), 3-nitrofluoranthene (3-NF) and 3-nitrobenzanthrone (3-NBA) and their corresponding amino forms, 1-AP, 3-AF and 3-ABA, in human bronchial epithelial BEAS-2B cells. The effects of the different nitro- and amino-PAHs were compared to the well-characterized PAH benzo[a]pyrene (B[a]P). Expression of 17 cytokine and chemokine genes, measured by real-time PCR, showed that 1-NP and 3-NF induced a completely different cytokine/chemokine gene expression pattern to that of their amino analogues. 1-NP/3-NF-induced responses were dominated by maximum effects on CXCL8 (IL-8) and TNF-alpha expression, while 1-AP-/3-AF-induced responses were dominated by CCL5 (RANTES) and CXCL10 (IP-10) expression. 3-NBA and 3-ABA induced only marginal cytokine/chemokine responses. However, 3-NBA exposure induced considerable DNA damage resulting in accumulation of cells in S-phase and a marked increase in apoptosis. B[a]P was the only compound to induce expression of aryl hydrocarbon receptor (AhR)-regulated genes, such as CYP1A1 and CYP1B1, but did not induce cytokine/chemokine responses in BEAS-2B cells. Importantly, nitro-PAHs and amino-PAHs induced both qualitatively and quantitatively different effects on cytokine/chemokine expression, DNA damage, cell cycle alterations and cytotoxicity. The cytokine/chemokine responses appeared to be triggered, at least partly, through mechanisms separate from the other examined endpoints. These results confirm and extend previous studies indicating that certain nitro-PAHs have a considerable pro-inflammatory potential.

Zinc- and copper-induced interleukin-6 release in primary cell cultures from rat heart.

The metals, zinc (Zn2+) and copper (Cu2+) from inhaled particulate matter may reach the systemic circulation and the cardiac tissue. In the present study, the potential of Zn2+ and Cu2+ to induce interleukin (IL)-6 responses in cardiomyocytes (CMs) and cardiac fibroblasts (CFs), in mono- and cocultures, was examined. Both metals induced IL-6 release in a concentration (20-200 microM)-dependent manner. Zn2+ appeared more potent than Cu2+ in both mono- and cocultures of CMs and CFs. In the cocultures, the basal- and metal-induced IL-6 responses were synergistically increased compared to the monocultures. Exposure to Zn2+ increased phosphorylation of the MAP-kinases, ERK1/2 and p38, in monocultures of CMs and CFs. Cu2+ induced an increased phosphorylation of p38 in both cell types and of ERK1/2 in CFs, but at higher concentrations than Zn2+. Treatment with a p38 inhibitor (SB202190) reduced the IL-6 responses to Zn2+ and Cu2+ in both cell types. Pretreatment with PD98059 to inhibit ERK1/2 was without significant effect; however, insignificant reductions was observed in the in the CFs. In conclusion, Zn2+ and Cu2+ increased IL-6 release and MAP-kinase activation in primary cardiac cells, processes known to be involved in cardiac inflammation and hypertrophy.

Cytokine and chemokine expression patterns in lung epithelial cells exposed to components characteristic of particulate air pollution.

Airborne particulate matter (PM) has a complex composition, and the relative contribution of different compounds to PM-induced effects is only partly understood. The present study compared the capability of selected components commonly found in PM, to induce pro-inflammatory responses in lung epithelial cells. Ultrafine carbon black (ufCB), ZnCl(2), FeSO(4), 1-nitropyrene (1-NP), lipopolysaccharide (LPS), and crystalline silica (positive control) were screened for effects on the expression of 84 inflammation-related genes in the bronchial epithelial cell line, BEAS-2B. A total of 22 genes were up-regulated by one or more of the tested compounds, and 5 cytokine and 11 chemokine genes were selected for further studies. After 10h exposure, silica induced significantly increased expression of CCL20, CXCL1/-3/-8/-10/-11, lymphotoxin (LT)beta and interleukin (IL)-6; ufCB induced CXCL8/-10 and -11; ZnCl(2) induced CCL11/-20/-26, CXCL1/-5/-8/-14 and tumor necrosis factor (TNF)-alpha; FeSO(4) induced a weak up-regulation of CXCL8 and TNF-alpha; LPS induced CCL20, CXCL1/-5/-8/-10/-11, LTbeta and IL-6; and 1-NP induced expression of CCL20, CXCL1/-3/-8, TNF-alpha and IL-6. Despite obvious differences, all compounds induced response-patterns that correlated relatively well with that of silica, the positive control. The predominant response appeared to be increased gene expression of neutrophil-recruiting CXC-chemokines. CXCL8 was the only gene induced by all tested PM-components, the most up-regulated on average, and also dominating the gene-expression patterns induced by coarse PM. The data show quantitative, and to a certain extent qualitative differences in cytokine/chemokine gene-expression profiles of the compounds tested. However, there were also striking similarities in the response-patterns induced by these physically/chemically widely different compounds.

IL-1beta as a determinant in silica-induced cytokine responses in monocyte-endothelial cell co-cultures.

Alveolar macrophages and endothelial cells are both involved in lung inflammation and remodeling of lung alveolar structures. In the present study, monocytes (precursors for macrophages) were exposed to crystalline silica and examined for pro- and anti-inflammatory cytokine responses in non-contact co-cultures with endothelial cells. The time courses for silica-induced release of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-8 both from co-cultures and monocyte mono-cultures showed an early peak at 5-10 h, almost no response at 20 h, and a strong increase at 43 h. At 43 h, co-cultures also showed strongly increased IL-6 levels. Steady-state levels of mRNA roughly exhibited the same pattern of early up-regulation and reduced levels at 20 h. Compared with monocyte mono-cultures, silica induced a strong release of IL-1beta, IL-6, and IL-8, but not of TNF-alpha, after 43 h in co-cultures, whereas at 5 and 10 h a significant difference was only observed for the silica-induced IL-8 response. An antagonist to the IL-1 receptor strongly reduced IL-6 and IL-8 levels, whereas antibodies to TNF-alpha increased the levels of IL-1beta and IL-8. Thus, IL-1beta is suggested to be an important triggering factor that determines the silica-induced release of several of the other cytokines in this co-culture system.

Differential binding of cytokines to environmentally relevant particles: a possible source for misinterpretation of in vitro results?

Inflammation is considered as a key event in adverse health effects associated with exposure to ambient particulate matter. The inflammatory potential of particles is often compared using in vitro cell systems, where the particle-induced release of pro-inflammatory cytokines is measured. A major concern in these assays is the potential of particles to bind cytokines, which may lead to an underestimation of the inflammatory potential. We therefore investigated the cytokine binding to a selection of particle samples, including particles collected from outdoor sources (wood combustion, traffic) and particles commonly used to model environmental sources (ultrafine carbon black, diesel, quartz), for a range of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IL-8). Furthermore, the influence of serum proteins and particle- and cytokine concentrations on the cytokine binding was studied. Cytokines primarily bound to carbonaceous particles (up to 85%), not to mineral particles. Furthermore, depending on the type of cytokine, the cytokine binding could be reduced partly or completely by adding serum proteins to the cell growth medium or particle suspensions. Based on these observations we recommend either to adjust culturing and exposure conditions to prevent cytokine binding, or to adjust the measured cytokine release by application of correction factors obtained from cytokine binding experiments.

Importance of size and composition of particles for effects on cells in vitro.

A primary goal of current research on particle-induced health effects is to reveal the critical characteristics that determine their biological effects. Experimental studies have shown that smaller particles induce stronger biological effects than larger particles of similar composition, due to their larger surface area to mass ratio. However, correlation for variations in surface area could not account for variation in biological reactivity among particles of differential composition. Hence, the importance of size and surface area does not override the importance of particle composition. Moreover, different particle characteristics appear to be involved in different biological effects in vitro. Our studies show that mineral particle-induced apoptosis mostly seems to depend on particle size, whereas composition and surface reactivity appeared to be most important for the proinflammatory potential of the particles. The ability of the particles to generate reactive oxygen species in vitro was not correlated with either inflammatory markers or apoptosis, suggesting that other mechanisms are at play. A single, specific component of the mineral particles, explaining the differences in response, has not been identified. In European-wide studies such as the Respiratory Allergy and Inflammation due to Air Pollution (RAIAP) study, particles have been sampled in different locations to study season- and site-dependent variations in responses particles, such as markers of inflammatory and allergic reactions in cells and animals. The data indicate that coarse particles can induce at least as strong inflammatory responses as fine particles. The allergic responses tended to be more associated with the organic fraction (PAH) of particles, whereas the inflammatory reactions seemed to be more associated with metals and endotoxin. Overall, coarse PM was found to have an inflammatory potential similar to fine PM on an equal mass basis. Even though one has to take into account different concentrations in ambient air as well as differences in respiratory system deposition of the size fractions, the potential of coarse particles to induce pulmonary effects should not be neglected.

Particulate matter properties and health effects: consistency of epidemiological and toxicological studies.

Identifying the ambient particulate matter (PM) fractions or constituents, critically involved in eliciting adverse health effects, is crucial to the implementation of more cost-efficient abatement strategies to improve air quality. This review focuses on the importance of different particle properties for PM-induced effects, and whether there is consistency in the results from epidemiological and experimental studies. An evident problem for such comparisons is that epidemiological and experimental data on the effects of specific components of ambient PM are limited. Despite this, some conclusions can be drawn. With respect to the importance of the PM size-fractions, experimental and epidemiological studies are somewhat conflicting, but there seems to be a certain consistency in that the coarse fraction (PM10-2.5) has an effect that should not be neglected. Better exposure characterization may improve the consistency between the results from experimental and epidemiological studies, in particular for ultrafine particles. Experimental data indicate that surface area is an important metric, but composition may play an even greater role in eliciting effects. The consistency between epidemiological and experimental findings for specific PM-components appears most convincing for metals, which seem to be important for the development of both pulmonary and cardiovascular disease. Metals may also be involved in PM-induced allergic sensitization, but the epidemiological evidence for this is scarce. Soluble organic compounds appear to be implicated in PM-induced allergy and cancer, but the data from epidemiological studies are insufficient for any conclusions. The present review suggests that there may be a need for improvements in research designs. In particular, there is a need for better exposure assessments in epidemiological investigations, whereas experimental data would benefit from an improved comparability of studies. Combined experimental and epidemiological investigations may also help answer some of the unresolved issues.

Mineral particles of varying composition induce differential chemokine release from epithelial lung cells: importance of physico-chemical characteristics.

Presently, little is known about the potential health effects of mineral particles other than asbestos and quartz. In this study, a human epithelial lung cell line (A549), primary human small airway epithelial cells (SAECs) and primary rat type 2 (T2) cells were exposed to stone quarry particles of two size fractions (<10 and <2.5 microm) from nine different rock samples. The ability to induce the release of chemokines from lung cells was investigated and compared with the particles' mineral and element composition and the amount of soluble elements. The stone particles induced the release of only low levels of interleukin (IL)-8 from A549 cells. In contrast, some of the other particles induced the release of high levels of macrophage inflammatory protein (MIP)-2 from T2 cells, and high levels of IL-8 from SAECs. Differences in particle surface area could account for differences in activity between the <10 and <2.5 microm fractions of six out of the nine rock samples. For two samples the <2.5 microm fraction was most active and for one sample the <10 microm fraction was most active. Content of the mineral plagioclase displayed a strong, negative correlation with the potential to induce MIP-2, whereas the mineral pyroxene was positively correlated with MIP-2 induction. However, neither plagioclase nor pyroxene content was sufficient to explain differences in bioactivity between the particles. No statistically significant correlation was found between the amounts of total or soluble elements and MIP-2 release. In conclusion, the results suggest that mineral particles with a high content of plagioclase have a low potential to induce a pro-inflammatory response. However, a particular mineral or element responsible for eliciting strong increases in chemokine release could not be identified. Thus, at present it appears that analysing mineral and element content is insufficient to predict stone particle bioactivity, and that biological testing is a necessity.

Release of inflammatory cytokines, cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size fractions.

Several studies have shown that particles of smaller size may be more potent than larger to induce inflammatory and toxic responses in cultured lung cells. However, the relative importance of different size fractions of ambient PM to induce such effects is still not known. In this study, we investigated the potency of different size fractions of urban ambient air particles to induce release of inflammatory cytokines in the human alveolar cell line A549 and primary rat type 2 cells. A mineral-rich ambient air PM10 sample collected in a road tunnel (road PM10) was also included. The coarse fraction of the urban ambient air particles demonstrated a similar or higher potency to induce release of the proinflammatory cytokines IL-8/MIP-2 and IL-6 compared to the fine and ultrafine fractions. The coarse fraction was also the most toxic in both cell systems. In contrast to the A549 cells, no induction of cytokine release was induced by the ultrafine particles in the primary type 2 cells. The mineral-rich road PM10 may be equally or more potent than the various size fractions of the ambient air particles to induce cytokines in both cell types. In conclusion, the coarse fraction of ambient particles may be at least as potent by mass as smaller fractions to induce inflammatory and toxic effects in lung cells.

Mechanisms involved in the induction of apoptosis by T-2 and HT-2 toxins in HL-60 human promyelocytic leukemia cells.

T-2 and HT-2 toxins belong to a group of mycotoxins that are widely encountered as natural contaminants known to elicit toxic responses in hematopoietic cells. In the present study, HL-60 cells were used to characterize the apoptotic effects of T-2 and a major metabolite, HT-2, and to examine the mechanisms involved. Apoptotic cells were identified microscopically by chromatin condensation and nuclear fragmentation, by flow cytometric analysis, and by DNA gel electrophoresis. T-2 and HT-2 induced concentration-dependent apoptosis after 24 h in HL-60 cells, starting at concentrations of 3.1 and 6.25 ng/ml respectively. An increased number of apoptotic cells could be observed 4-6 h after exposure to 12.5 ng/ml of toxin. Little cytotoxicity (plasma membrane damage) was observed even after exposure to concentrations of toxins (25-50 ng/ml) inducing apoptosis in 60-100% of the cells. The apoptotic process was almost completely blocked in the presence of the general caspase inhibitor zVAD.fmk. In contrast, no or only minor effects were observed with the more specific caspase inhibitors DEVD.CHO, IETD.fmk, and DEVD.fmk. As judged by Western blotting, the levels of several procaspases (-3, -7, -8, -9, but not -12) were reduced 3-6 h after exposure to toxin. Substantial increases in the presumed active form(s) of caspase-8 and -9 were observed. Furthermore, poly(ADP-ribose) polymerase (PARP) was already markedly cleaved 3 h after toxin treatment, indicative of active caspase-3 and -7. No or only minor changes in Bcl-2, Bcl-XL and Bax levels were observed. BAPTA-AM and ZnCl2 blocked the degradation of procaspases, the fragmentation of PARP, and the induction of apoptosis. In summary, both T-2 and HT-2 induced apoptosis, with T-2 being somewhat more potent than HT-2. The divalent calcium concentration, [Ca2+], appears to be involved in the activation of several caspases, resulting in DNA fragmentation, chromosomal condensation, and nuclear fragmentation.

Cadmium-induced apoptosis of primary epithelial lung cells: involvement of Bax and p53, but not of oxidative stress.

The lung is a target organ for cadmium (Cd) toxicity. Apoptosis induced by cadmium acetate (CdAc) was studied in alveolar type 2 cells and Clara cells isolated from rat lung. Relatively low concentrations of CdAc (1-10 micromol/L) induced apoptosis after exposure for 20 h. Type 2 cells were more sensitive than Clara cells to Cd-induced apoptosis and loss of cell viability. On exposure to 10 micromol/L CdAc, the levels of the apoptosis-modulating proteins p53 and Bax were increased at 2 h and 5-12 h, respectively. The expression of p53 preceded the expression of Bax and the apoptotic process. The exposure to 10 micromol/L CdAc did not significantly increase the formation of cellular reactive oxygen species (ROS). However, after exposure to a high concentration of CdAc (100 micromol/L), a 30% increase of the ROS level was observed. No significant nitric oxide production was measured following CdAc exposure. Catalase, superoxide dismutase, dimethyl sulfoxide, or tetramethylthiourea did not protect against Cd-induced apoptosis. In conclusion, the results show that Clara cells and type 2 cells are sensitive to Cd-induced apoptosis. Increased levels of p53 and Bax are suggested to be involved in the apoptosis. The apoptosis did not appear to be mediated by oxidative pathways.

Mechanisms in fluoride-induced interleukin-8 synthesis in human lung epithelial cells.

Sodium fluoride (NaF) has previously been reported to induce a strong IL-8 response in human epithelial lung cells (A549) via mechanisms that seem to involve the activation of G proteins. In the present study the signal pathways downstream of the G proteins have been examined. NaF induced a weak, but sustained increase in PKC activity. In contrast, the PKC activator TPA induced a relatively strong, but transient effect and augmented the NaF-induced PKC activity. TPA induced a marked IL-8 response compared to NaF. PDB, another PKC activator, was less effective, but augmented the IL-8 response to NaF. Pretreatment with TPA for 20 h, or the PKC inhibitor GF109203X for 1 h, abolished the basal and NaF-induced PKC activities and partially prevented the NaF-induced IL-8 response. Inhibition of the MAP kinase p38 by SB202190 partially reduced the IL-8 response to NaF, whereas a reduction in ERK activity by PD98059 led to an increased response. The NaF-induced IL-8 response was weakly augmented by the PKA stimulator forskolin and the G(i) inhibitor pertussis toxin. The PKA inhibitor H89 seemed to reduce the NaF-induced IL-8 response, but the measured effect was not statistically significant. BAPTA-AM, KN93 and W7, that inhibit Ca(2+)-linked effects, did not affect the IL-8 response. Furthermore, the tyrosine kinase inhibitor genestein, the PI-3 kinase inhibitor wortmannin and phosphatase inhibition were without effects. In conclusion, the data suggest that NaF-induced increase of IL-8 in A549 cells involved PKC- and p38-linked pathways, whereas an ERK-dependent pathway counteracted the response. Tyrosine kinases, Ca(2+)-linked pathways, PI-3 kinase, PKA and phosphatase inhibition seem to play no or minor roles in the fluoride-induced IL-8 response.

Persistent versus transient map kinase (ERK) activation in the proliferation of lung epithelial type 2 cells.

Type 2 pneumocytes are progenitor cells of alveolor epithelium and important for reepithelialization following lung injury. This study examined the role of persistent versus transient mitogen-activated protein (MAP) kinase (extracellular signal-regulated kinase; ERK) in type 2 cell proliferation. Three different types of agents; epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate (TPA), and fetal bovine serum (FBS) induced different patterns of ERK activation. FBS induced a strong and persistent MAP kinase response, whereas the effect of EGF was transient with a strong activation at 5 minutes and only a slight stimulation at 4 hours. The TPA response was more prolonged than the EGF response, but not by far as strong and persistent as the FBS response. Activation by EGF and TPA and the early response induced by FBS were strongly reduced by the MEK inhibitor PD98059. The sustained FBS-induced ERK activation was inhibited by approximately 50%. The total number of cell, the percentage of cells in S and G2/M phase of the cell cycle and the incorporation of 3H-thymidine into DNA were strongly increased in response to FBS, whereas EGF and TPA were without effect. The proliferation was reduced by approximately 50% after pretreatment with PD98059. The results indicate that a persistent ERK activation of a critical size leads to type 2 cell proliferation, and that the proliferative response may also depend on a MEK-independent ERK activation.

Fluoride-induced apoptosis in epithelial lung cells involves activation of MAP kinases p38 and possibly JNK.

Exposure to fluorides can induce inflammatory reactions, cell cycle arrest, and apoptosis in different experimental systems. Fluorides are known G-protein activators, but less is known about fluoride effects downstream of G-protein activation. The aim of this study was to elucidate whether the induction of apoptosis by fluorides and inhibition of proliferation is mediated by MAP kinases in primary rat lung, alveolar type 2 cells and the human epithelial lung cell line A549. Sodium fluoride (NaF) induced apoptosis in both cell types but at different concentrations, with the primary cells being more sensitive to NAF: Proliferation of the type 2 cells and A549 cells was inhibited in the presence of NAF: NaF induced a prolonged activation of MAP kinase ERK. NaF also activated p38 and JNK in A549 cells for several hours (maximally 6-fold and 3-fold increase, respectively). Inhibition of ERK with the MEK1,2 inhibitor PD98059 increased apoptosis 2-fold, whereas the inhibitor of p38, SB202190, decreased the level of apoptotic cells by approximately 40%. SB202190 also inhibited apoptosis by almost 40% when ERK activity was reduced in the presence of PD98059. Neither PD98059 nor SB202190 did affect the NaF-induced inhibition of proliferation. These observations indicate that activation of MAP kinases p38 and possibly JNK are involved in NaF-induced apoptosis of epithelial lung cells, whereas ERK activation seems to counteract apoptosis in epithelial lung cells. In contrast, activation of ERK and p38 are not involved in NaF-induced inhibition of cell proliferation.

Cell-specific expression of CCAAT/enhancer-binding protein delta (C/EBP delta) in epithelial lung cells.

CCAAT/enhancer-binding proteins (C/EBP) constitute a family of transcription factors that are involved in regulation of proliferation and differentiation in several cell types. In epithelial lung cells the C/EBP alpha isoform seems to play a role in the regulation of surfactant proteins (SP) and Clara cell specific protein (CCSP), whereas the roles of C/EBP beta and C/EBP delta are unclear. We have examined the protein levels of C/EBP delta in bronchiolar Clara cells and alveolar type 2 cells, and its relation to the expression of lung specific proteins and cell proliferation. The protein expression of C/EBP delta was high in freshly isolated Clara cells compared to type 2 cells. In both cell types C/EBP delta levels increased during culture. Alterations of the levels of C/EBP delta did not correspond with the proliferation levels of Clara cells, but seemed to correspond in type 2 cells. Clara cell secretory protein (CCSP) was highly expressed in freshly isolated Clara cells, in contrast to type 2 cells. SP-D and CYP2B1 were expressed at somewhat higher levels in Clara cells than in type 2 cells, whereas SP-A exhibited highest expression in type 2 cells. During culture the levels of all these lung proteins were strongly reduced. However, compared to with serum we found an increase in CCSP in Clara cell cultures without serum, and this correlated with an increase in C/EBP delta. Overall our in vitro data suggest that C/EBP delta alone is not related to the maintenance of proteins involved in differentiation.

Fluoride-induced interleukin-6 and interleukin-8 synthesis in human epithelial lung cells.

Exposure to fluorides has been associated with asthmatic symptoms among workers in the aluminium industry. In a recent experimental study hydrogen fluoride (HF) was found to induce a weak inflammatory response in humans. In the present study the potential of sodium fluoride (NaF) and HF to induce cytokine response was examined and how these responses are modulated by Al3+ in a human epithelial lung cell line (A549). Dose-response experiments showed a maximal release of IL-6 and IL-8 at a concentration of 5 mM NaF 24 h after addition. The responses to HF were of a similar magnitude as for NaF. Time-course experiments showed a NaF-induced IL-6 response at 5 h, whereas an IL-8 response was observed after 10 h. Cycloheximide treatment completely abolished the NaF-induced cytokine responses. A marked increase in the mRNA level for IL-6 was observed already 2 h after exposure to 5 mM NaF, and presumably is a prerequisite for the subsequent increase of IL-6. The fluoride-induced effects on IL-6 and IL-8 release were strongly reduced by pretreatment with deferoxamine (an Al3+-chelator), and enhanced by addition of Al3+. This indicates that an AlF4-- complex, a known activator of GTP-binding proteins, is involved in fluoride-induced IL-6 and IL-8 responses in A549 cells.