Mechanisms in fluoride-induced interleukin-8 synthesis in human lung epithelial cells
Introduction
Fluorides have been associated with asthmatic symptoms among workers in the aluminium industry (Søyseth and Kongerud, 1992). Exposure to low concentrations of hydrogen fluoride (HF) in a chamber has been reported to induce a weak inflammatory response in humans, with a change in cell pattern and an increase in the levels of chemoattracting cytokines in the bronchoalveolar lavage (BAL) fluid (Lund et al., 1999). Many cytokines are known to be involved in inflammatory responses (Vanhee et al., 1995, Driscoll et al., 1997) in a complex cell-to-cell network that involves epithelial cells (Adler et al., 1994, Khair et al., 1996). Interleukin-8 (IL-8) is a cytokine that primarily is involved in mobilisation of neutrophils after exposure to a large range of toxicants (Standiford et al., 1990, Kunkel et al., 1991, DeForge et al., 1993, Rosenthal et al., 1994, Lakshminarayanan et al., 1997). The important role of IL-8 in inflammatory processes was illustrated in experiments in which IL-8 antibodies partly inhibited inflammation induced by toxic agents in animals (Folkeson et al., 1995). Recently, we showed that sodium fluoride (NaF) has a strong potential to induce the synthesis of IL-8 in human epithelial lung cells in vitro (Refsnes et al., 1999a). The detailed mechanisms behind the NaF-induced IL-8 release and other cellular effects of NaF are unclear. Several reports show that NaF may exert various cellular effects both through GTP-binding-protein (G-protein)-dependent and -independent pathways (Blackmore et al., 1985, Murthy and Makhlouf, 1994, Caverzasio et al., 1998). Extensive studies in isolated plasma membranes and in various intact cell types have shown that G protein activation is mediated via formation of an AlF4−-complex, that strongly inhibits G protein-linked GTP-ases, leading to a persistent stimulation of the G proteins (Sternweiss and Gilman, 1982, Blackmore et al., 1985, English et al., 1992, Susa et al., 1997). AlF4− has been reported to induce biphasic changes in cyclic AMP (cAMP) levels subsequent to activation of Gi and Gs, and to increase the levels of diacyl glycerol, inositol-1,4,5-triphosphate and cytosolic Ca2+ subsequent to activation of Gq (Blackmore et al., 1985). Increases in the levels of these intracellular messengers are linked to activation of cAMP protein kinase (PKA), protein kinase C (PKC) and Ca2+-dependent protein kinases. Tyrosine phosphorylation has also been shown to be induced by fluorides, presumably via a G protein-linked mechanism (Caverzasio et al., 1998). NaF, without forming a complex with aluminium, is also known to inhibit tyrosine protein phosphatases (Wergedal and Lau, 1992), thereby keeping several important signal-transducing molecules in an active, phosphorylated form. NaF has also been reported to activate extracellular signal-regulated kinase (ERK) (Anderson et al., 1991, Susa et al., 1997, Wu et al., 1997) and the stress-activated MAP kinase p38 (Heidenreich et al., 1999), but it is not known to what extent this activation is mediated via G protein activation or phosphatase inhibition.
PKA-, PKC-, Ca2+- dependent protein kinase- and tyrosine kinase-linked mechanisms have been reported to be involved in IL-8 responses induced by proinflammatory cytokines and environmental stress (Zhang et al., 1992, Chabot-Fletcher et al., 1994, Kavelaars et al., 1997, Denning et al., 1998, Foreback et al., 1998, Kuhns et al., 1998, Zidovetzki et al., 1999). The role of phosphatidyl inositol-3 kinase (PI-3 kinase) in IL-8 induction has been less examined, but it has been reported that PI-3 kinase is not involved in IL-8 production in an epithelial cell line (Schulte et al., 1998). Different mitogen-activated protein kinases (MAP kinases), ERK, p38 and c-jun NH2-terminal kinase (JNK), are known to participate in the regulation of IL-8 production (Matsumoto et al., 1998, Chen et al., 1999, Hashimoto et al., 2000). The stimulation of the MAP kinase pathways may be exerted via increased phosphorylation of the kinases and/or inhibition of protein phosphatases (Lewis et al., 1998).
Our previous findings suggest the involvement of G protein activation in NaF-induced interleukin 6 (IL-6) and IL-8 responses in a human lung epithelial cell line (Refsnes et al., 1999a). In the present study the aim was to examine how activation of different G protein-linked signal-transduction systems (PKA, PKC, Ca2+-dependent protein kinases, tyrosine kinase, PI-3 kinase) and MAP kinase (ERK, p38) activation are involved in the NaF-induced IL-8 response in human epithelial lung cells (A549 cells).
Section snippets
Materials
Culture medium: Nutrient mixture F12 Ham Kaighn's modification (Cat. No N-3520), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid (HEPES), propidium iodide, forskolin, dibuturyl cAMP (dBcAMP), sodium orthovanadate, phenylmethylsulphonyl fluoride (PMSF), benzamidine, β-mercaptoethanol, dithiothreitol (DDT) were obtained from Sigma Chemical Company, St. Louis, MO. Fetal bovine serum (FBS) was from Gibco BRL, Paisley, Scotland. The antibiotics ampicillin and fungizone were from Bristol-Myers
NaF-induced IL-8 response
A549 cells were exposed to NaF (0–5 mM) for 20 h, and subsequently analysed for IL-8 release to the culture medium. A 3-fold increase was observed at 2.5 mM NaF and a maximal response was found at 3.75–5 mM, with an ∼7-fold increase (Fig. 1).
Involvement of PKC
The role of PKC in the NaF-induced IL-8 response was examined with PKC inhibitors and stimulators and by measuring PKC activity.
Discussion
Recently, we showed that NaF has a strong potential to induce the synthesis of proinflammatory cytokines (Refsnes et al., 1999a). We have now examined the involvement of different G protein-linked signal systems (PKA, PKC, Ca2+-dependent protein kinases, tyrosine kinases, PI-3 kinase) in NaF-induced IL-8 production, and also the possible involvement of MAP kinases (ERK, p38, JNK) and protein phosphatases.
Acknowledgements
We thank Tonje Skuland, Hans Jørgen Dahlman and Edel Lilleaas for excellent technical assistance.
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