Elsevier

Toxicology Letters

Volume 193, Issue 3, 1 April 2010, Pages 252-260
Toxicology Letters

Cadmium-induced inflammatory responses in cells relevant for lung toxicity: Expression and release of cytokines in fibroblasts, epithelial cells and macrophages

https://doi.org/10.1016/j.toxlet.2010.01.015Get rights and content

Abstract

Inhalation is an important route of cadmium (Cd) exposure, and the lung is considered to be one of the main target organs of Cd toxicity. Pulmonary inflammation seems to be involved in development of many lung diseases. In the present study we show that Cd2+ at fairly low concentrations affects gene expression of several different cytokines/chemokines in human M1 fibroblasts. The chemokines CXCL2, CXCL3, IL-8/CXCL8 and CCL26, the pro-inflammatory cytokine IL-6 and the receptor IL-1RL1 were expressed at high levels after exposure to 7 μM Cd2+ for 7 h. The expression of some important cytokines was further studied in two different primary cell cultures from rat lungs. Cd2+ induced cytokine responses at low concentrations (3–6 μM) and early time-points both in type 2 epithelial cell-enriched cultures and alveolar macrophages. However, the two primary lung cells displayed different patterns of cytokine release. Cd2+ induced an increased release of IL-6 and MIP-2/CXCL2 from the epithelial cells and MIP-2, IL-1β and TNF-α from alveolar macrophages. In conclusion, the marked up-regulation of different cytokines in these cell types, that are important in development of lung injury and disease, suggests that inflammation may contribute in Cd-induced lung damage.

Introduction

Cadmium (Cd) is frequently used in manufacture of batteries, plastics, and fertilizers and is a widespread industrial pollutant that may represent a health hazard. Burning of fossil fuels is also an important source for release of Cd to the atmosphere. In addition, cigarette smoke is regarded as a major source of human exposure (Jarup and Akesson, 2009). Inhalation is an important route of Cd exposure, and the lung is considered to be one of the main target organs of Cd-induced toxicity. Inhalation of Cd has been reported to be associated with lung tumors, pulmonary fibrosis and emphysema (Oberdorster, 1992). Inflammation is a common denominator in development and exacerbation of these and other lung diseases. Inflammatory processes involve the production and release of a range of signalling molecules such as cytokines, leukotrienes/prostaglandins, and adhesion molecules, which operate in a complex network between epithelial cells, fibroblasts and immune cells such as macrophages, neutrophils, eosinophils and T-cells (Davies and Holgate, 2002, Mills et al., 1999, Thannickal et al., 2004). Inflammatory responses normally resolve with transient tissue remodeling and repair. However, progression to a state of chronic inflammation may lead to permanent tissue damage through the continuous release of endogenous oxidants and toxic proteases by activated immune cells.

Few studies have focused on the involvement of inflammation in the Cd-induced lung toxicity. However, inhalation of cadmium oxide fume has been reported to result in pulmonary inflammatory processes in rats and mice, with influx of neutrophils and proliferation of alveolar macrophages and epithelial lung cells (McKenna et al., 1997). Inflammatory phenomena have also been suggested as the most likely events responsible for induction of lipid peroxidation in lung tissue following acute exposure of rats to Cd salts (Manca et al., 1994). Moreover, Cd-induced hepatotoxicity has been related to inflammation (Rikans and Yamano, 2000). Cd is also an activator of transcription factors, such as the AP-1 and NF-κB, which are known to be involved in the synthesis of various inflammatory mediators (Hart et al., 1999, Hyun et al., 2007, Liu et al., 2002, Souza et al., 2004, Thevenod, 2009).

Inflammatory responses induced by Cd or other toxic agents involve cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-8/CXCL8 and IL-6 (Horiguchi et al., 1993, Hyun et al., 2007, Kayama et al., 1995a, Kayama et al., 1995b). Responses mediated by TNF-α in both liver and lung include inflammatory cell infiltration, oxygen radical generation and fibrogenesis. TNF-α, along with IL-1β, is important in the onset of inflammatory processes that regulate expression of other cytokines and chemokines (Barksby et al., 2007, Grivennikov et al., 2006, Shimabukuro et al., 2003). IL-8 in humans and the functional analogue macrophage inflammatory protein (MIP)-2/CXCL2 in rats have been associated with pulmonary edema, mobilization and activation of neutrophils, as well as bronchoconstriction (Fox et al., 2005, Mills et al., 1999). Another important pro-inflammatory cytokine is IL-6, known to promote induction of acute phase proteins. This pleiotropic cytokine may also be involved in regulation of the transition from acute to chronic inflammation and stimulation of T- and B-lymphocytes (Gabay, 2006). Increased plasma activity of IL-6 and TNF were observed following Cd administration in rats, and this systemic inflammatory response was suggested to be involved in the pulmonary toxicity of Cd (Kataranovski et al., 1998).

To enhance knowledge of Cd-induced lung toxicity, we have now studied changes in cytokine gene expression and release in fibroblasts, epithelial cells and macrophages. These are all important cell types in the development of lung injury (Chung, 2005, Davies and Holgate, 2002, Thannickal et al., 2004) and are therefore useful models for studying cytokine responses related to inflammation. Cd-responsive genes in M1 fibroblasts were screened using a microarray analysis, with focus on gene expression of various cytokines and their receptors. To confirm that changes of cytokine expression affect the protein release of some crucial cytokines, the release of IL-8, IL-6, IL-1β and TNF-α from the M1 fibroblasts was investigated. Effects of Cd in fibroblasts were furthermore compared with effects in primary epithelial cells and macrophages from rat lungs.

Section snippets

Chemicals

Collagen (type III), insulin, hydrocortisone, transferrin, epidermal growth factor (EGF), protease (type 1: crude), DNase I (type III), propidium iodide (PI), Hoechst 33258 [2′-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole] and Hoechst 33342 [2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole] were obtained from Sigma–Aldrich, St. Louis, MO, USA; cadmium acetate [CdAc, Cd(C2H3O2)2·2H2O] and cadmium chloride from E. Merck, Darmstadt, Germany.

cDNA microarray analysis on various cytokines in Cd-exposed M1 human fibroblasts

To characterize the cytokine/chemokine responses induced by Cd2+, the M1 human fibroblast cell line was exposed to 7 μM Cd2+. The cells were screened for Cd-inducible genes by microarray hybridization. Several stress response genes, well-known to be up-regulated by Cd in different cellular systems (Andrew et al., 2003, Koizumi and Yamada, 2003, Li et al., 2008, Yamada et al., 2009), also showed an enhanced response in our cell line (data not shown). In addition, Cd induced a range of cytokines,

Discussion

Due to the importance of the lung as a target organ of Cd toxicity, and the role of pulmonary inflammation for development of many lung diseases, the potential effects of Cd on cytokine expression and release are of great interest to investigate. In the present study we show that Cd2+ at fairly low concentrations affects gene expression of several different cytokines/chemokines in human M1 fibroblasts. Among them the chemokines IL-8/CXCL8 and the pro-inflammatory cytokine IL-6 were expressed at

Conflict of interest

No conflict of interest.

Funding

The microarray studies were funded by the “Tordis og Fritz C Riebers Legat, Bergen” and the Research Council of Norway, Functional Genomics (FUGE).

Acknowledgements

The expert assistance of Edel M. Lilleaas, Tonje S. Skuland and Hans J. Dahlmann is very much appreciated.

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