SM inhalation causes acute airway inflammation and tissue injury, presumably due to the premature, sudden and massive release of destructive enzymes and mediators of inflammation [3
]. Currently there are no effective antidotes for SM-induced inflammation and injury. Macrolide antibiotics have been reported to have immunomodulatory effects and to be effective in treatment of chronic airway inflammation through actions other than their bactericidal activity [17
]. In this study, we evaluated the potential of macrolide antibiotics as antidotes for SM-induced injury and inflammation using roxithromycin as a representative macrolide antibiotic for the tests.
Airway inflammation and injury by SM is a complex phenomenon that involves airway epithelial cells, cilia cells, goblet cells and smooth muscle cells interacting with one another under the control of a network of cytokines and other mediators. SAE and BTE cells are considered the first line of defense against inhaled chemicals as they are in direct contact with these chemicals and participate in the pathogenesis of airway injury through the release of oxidants and inflammatory cytokines. It is therefore important to understand the interaction between these cells and the chemical insult, as well as their response to an insult/therapeutic treatment combination such as SM/roxithromycin.
The effect of roxithromycin on SM-induced cell injury was first tested by the MTS cell viability assay and the Calcein AM/EthD-1 fluorescence staining viability assay. The MTS assay showed a concentration-dependent cytotoxicity induced by SM in SAE and BTE cells. For both cell types, 100 μM roxithromycin alleviated the SM cytotoxicity. Calcein AM/EthD-1 fluorescence staining confirmed these results. Together, these results demonstrated the protective effect of roxithromycin against SM-induced injury.
Proinflammatory cytokines play a major role in both acute and chronic inflammatory processes, including those produced by SM. However, these cytokines, particularly when produced in excess, can be pathogenic. Previous studies demonstrated that in vivo
damage to the skin by SM results in an immunological response defined by increased gene expression of the inflammatory cytokines [6
]. In this study we examined the expression of four major inflammatory cytokines, IL-1β, IL-6, IL-8, and TNF. The basal expression levels of the four cytokines were all very low in normal SAE and BTE cells, although IL-6 and IL-8 accumulated continuously in the culture medium from unstimulated cells grown in cell culture. However, exposure of SAE and BTE cells to SM in vitro
resulted in a rapid increase in the mRNA levels in the cell and the release of the cytokine protein into culture supernatants for all of the four cytokines. Both the mRNA expression and protein release increased several fold in SM-exposed cells relative to unexposed cells for IL-1β and TNF, and increased 10- to 20-fold in the cases of IL-6 and IL-8. Although the final supernatant concentrations for IL-1β and TNF remained low (<100 pg/ml), the concentrations of IL-6 and IL-8, on the other hand, reached very high levels (10 ng/ml – 30 ng/ml). These results suggest that these two inflammatory mediators may play a major role in the mediation of the inflammatory and immune responses initiated by SM inhalation.
In light of the potential role of IL-6 and IL-8 in inflamed airway epithelium, an understanding of the regulation of their production may provide valuable information for treatment of SM exposure. A significant increase in IL-8 release was also reported in human epidermal keratinocytes following SM exposure and has been proposed as a biomarker for SM-induced inflammation [21
]. It is also intriguing to note that IL-6 was reported as an anti-inflammatory cytokine in human plasma samples [22
] and in human monocytes [23
]. Therefore, it is reasonable to speculate that IL-6 is a pleiotropic cytokine whose anti- or pro-inflammatory properties depend on the cell type from which it is produced and to which it is targeted.
iNOS has been suggested to be an important biomarker of inflammation, as its overexpression leads to excessive production of NO, which contributes to the pathophysiology of inflammation and the resultant tissue damage [10
]. NO is involved in several types of acute and chronic inflammation [24
]. Overproduction of NO by type II NOS or iNOS is associated with the development of airway inflammation [14
]. However, the basal level expression of iNOS was low in SAE and BTE cells and therefore was difficult to detect by immunocytochemistry using conventional fluorophores (Texas Red, fluorescein, etc
.). To improve signal stability and quantitation, an optically stable, new class fluorophore for immunocytochemical detection was employed in this study. Detection of iNOS was based on fluorescence from streptavidin-linked inorganic semiconductor nanocrystals of cadmium selenide [(CdSe)ZnS]. As fluorescence of nanomaterialfluorophores was significantly brighter and more photostable than organic fluorophores such as Texas Red and fluorescein [25
], we were able to detect iNOS in both unexposed and SM-exposed SAE and BTE cells.
The reduced overexpression of proinflammatory cytokines and iNOS by roxithromycin in SM-exposed SAE and BTE cells is in agreement with previous in vivo
and in vitro
studies (for a complete review see [27
]) and further supports the contention that macrolides can inhibit the production of proinflammatory mediators and cytokines. This suppressive effect of roxithromycin may not be accounted for by its antimicrobial properties but rather by its anti-inflammatory actions, as evidenced by the fact that other antibiotics, such as amoxicillin, cefaclor, penicillin, and cephalosporin, had no such effects even at high concentration (data not shown). Thus the immunomodulatory effects of antibiotics appear to be specific to macrolides. Similar findings were made by Kohri et al
The protective effect of roxithromycin on SM cytotoxicity could be explained, at least in part, by its ability to inhibit the overexpression of pro-inflammatory cytokines and mediators. As mentioned previously, Qabar et al
] reported that suppressed expression of the proinflammatory cytokines IL-8 and IL-6 in human epidermal keratinocytes, mediated by overexpressing the anti-inflammatory cytokine IL-10, led to increased viability of SM-treated cells. Also, 1-alpha, 25-dihydroxyvitamin D3 enhanced cell proliferation in human skin cells stimulated with SM by suppressing expression of the inflammatory mediators IL-6 and IL-8 [29
]. On the other hand, Stone et al
] reported that inflammatory cytokines, such as TNF-α and IL-1β enhanced SM toxicity on murine macrophages. The protective effect of roxithromycin seen in this study, an increase in cell viability by ~2.5 and ~3.0 times for SAE and BTE cells respectively, is higher than that of 1-alpha, 25-dihydroxyvitamin D3 on HEK cells, an increase of ~1.5 times in cell proliferation [29
]; although the results are not directly comparable as the cells used in the two studies are different.
The subcellular mechanism of the anti-inflammatory effect of macrolides remains unknown. However, it is well known that the expression of several genes involved in the immune and inflammatory response (e.g., iNOS, TNF, IL-1, IL-6, IL-8) are regulated at the transcriptional level by nuclear factor-κB (NF-κB) (for a recent review, see [31
]). Thus, it is conceivable that macrolide antibiotics may act as anti-inflammatory agents by preventing the activation of NF-κB. Ichiyama et al
] demonstrated that clarithromycin, another macrolide widely used clinically, inhibits NF-κB-dependent reporter gene expression in transfected pulmonary epithelial cells, providing direct evidence in support of specific effects of macrolides on NF-κB activation. However, it is now held that the molecular mechanism(s) by which macrolides inhibit pro-inflammatory cytokine responses in the respiratory epithelium varies depending upon the macrolide used, the activating stimulus, and the cell type examined [27
]. Thus, further studies are required to determine whether NF-κB is the target molecule for roxithromycin in the signal transduction pathway in SM-exposed SAE and BTE cells.