The present study was undertaken to address the mechanisms of NF-κB activation in lung epithelium by respirable quartz particles. In a previous study, we could already show that quartz exposure leads to an increased nuclear staining of the NF-κB subunit RelA in type II epithelial cells as well as alveolar macrophages . Now, we have provided further in vivo evidence for the actual activation of the classical NF-κB pathway in both cell types, the pathway involving phosphorylation of IκBα at serines 32/36, tagging this inhibitor protein for degradation. In our in vivo model, the observed activation of NF-κB in rat lung tissue was accompanied by increased mRNA expression of iNOS, which has been shown to be involved in inflammation [14, 17] and to be induced on the mRNA level by silica exposure in rat lung . iNOS is known to be regulated at least partly by NF-κB [31–34]. A classic pro-inflammatory gene induced by NF-κB is COX-2, known to occupy an important position in the regulation of pulmonary inflammation . Although the role of COX-2 in inflammation is complex and the concept of a general pro-inflammatory role has been disproved , in the acute stage of pulmonary disease COX-2 expression is considered to promote inflammation . COX-2 is known to be mainly regulated on the level of transcription  and the gene has been shown to be induced in lung epithelial type II cells . In the current study, COX-2 was not significantly induced in vivo following quartz exposure on the mRNA level. In contrast, we observed a significant increase of HO-1 mRNA expression in quartz exposed rat lungs. HO-1 induction is considered as a sensitive oxidative stress marker  and has been shown to be increased in serum of silicotic patients as well as in lung tissue of silica-exposed mice [39, 40]. Our findings provide further support for the well-known role of oxidative stress in silica-elicited pathogenesis [15, 16].
Our parallel in vitro approach focused on the investigation of the role of macrophages in NF-κB activation in lung epithelial cells. Therefore, we compared epithelial NF-κB activation in response to treatment with cell-free supernatants from quartz-treated macrophages to the direct treatment with quartz particles. Evaluation of NF-κB pathway activation on multiple levels is necessary, as there might be different mechanisms involved. While cytokines like TNFα and IL-1β induce the canonical pathway characterized by IκBα Ser32/36-phosphorylation and degradation, quartz particles have also been shown to activate NF-κB in macrophages via tyrosine phosphorylation of IκBα . In the present study, we observed that supernatants of quartz-treated NR8383 alveolar macrophages caused Ser32/36 phosphorylation and degradation of IκBα, the main NF-κB inhibitor protein, in epithelial cells. These supernatants were also found to cause nuclear translocation and DNA binding of the RelA NF-κB subunit, part of the most common RelA-p50 heterodimer that is generally known as NF-κB. Only RelA contains a transactivation domain and thus induces transcription of inflammatory genes , while p50 homodimers are known to inhibit transcription by competing for and blocking available DNA docking sites . In association with NF-κB pathway activation, supernatants from the quartz-treated macrophages also caused a massive increase in the number of iNOS gene transcripts in the RLE rat lung epithelial cells. In sharp contrast to these findings, direct treatment with quartz only induced a marginal reduction of IκBα levels after the longest treatment. Apart from reflecting active degradation, the observed IκBα decline may also be a result of an inhibited de novo synthesis . In association with this, quartz itself was also found to be a rather weak inducer of iNOS, when compared to supernatants of the quartz-treated macrophages. However, direct treatment with quartz resulted in a more than 100-fold upregulation of COX-2 mRNA in the RLE cells, an effect that was much stronger than that observed with the macrophage supernatants. An NF-κB-independent induction of COX-2 by silica appears to contrast with previous findings by Choi and colleagues . However, in their study fibroblasts were used, and cell type-specific differences in COX-2 regulation are a possibility. Although COX-2 expression is considered to be regulated through NF-κB signalling, other transcription factors have been implicated as well, both in general [46, 47] and specifically after silica treatment . For instance, in lung epithelial cells, it has been shown that zinc-induced COX-2 induction is not reduced by NF-κB inhibition .
To further investigate the mechanism of NF-κB activation in the lung epithelial cells, we evaluated the roles of the most likely relevant candidates, i.e. the pro-inflammatory cytokines TNFα and IL-1β, as well as the involvement of ROS. TNFα and IL-1β are considered important factors in the development of silicosis [12–14] and are well-known inducers of NF-κB in various cell types [8–11]. Both cytokines were produced by the NR8383 cells upon quartz treatment, and the concentrations as measured in the supernatants of macrophage treated with quartz for various periods matched well with the ability of these supernatants to activate NF-κB. Remarkably however, inhibition of TNFα or IL-1β with their respective neutralizing antibodies did not influence NF-κB-inducing potential of the supernatants from quartz-treated NR8383 cells. Even when both cytokines were neutralized simultaneously in the supernatants from the quartz-treated macrophages, epithelial NF-κB activation could not be inhibited. Taken together, these findings suggest that neither TNFα nor IL-1β plays a crucial role in quartz-induced NF-κB activation in lung epithelial cells, and that other factors are at least involved or possibly even more important. This is in accordance with previous investigations in our laboratory, which indicated that in vivo NF-κB activation by quartz may be, at least in part, TNFα-independent .
To address the potential role of ROS in the macrophage-mediated NF-κB activation in RLE cells the effects of H2O2, BSO and NAC were evaluated. An H2O2 concentration of 50 μM was used, as it was shown to induce a strong increase in NF-κB DNA binding activity in Jurkat cells , while in addition, in our lab it proved to be one of the highest concentrations applicable without causing significant cytotoxicity in RLE cells (data not shown). H2O2 is well known to possess NF-κB activating properties, albeit in a cell specific-manner [19, 50]. H2O2 is formed upon the dismutation of superoxide anions which are generated in large amounts by activated macrophages during inflammation. Although the generation of ROS from DQ12-treated NR8383 macrophages was shown in this study in a concentration and time dependent manner, H2O2 treatment did not lead to any notable IκBα degradation in the RLE cells. This observation might be due to the fact that H2O2 has been found to activate NF-κB through a non-classical pathway in some cell types, involving tyrosine phosphorylation of IκBα and direct activation of RelA . However, nuclear extracts from H2O2 treated RLE cells failed to increase DNA binding activity of RelA in our hands (data not shown). In line with these observations, we also did not observe any effect of the macrophage supernatants on NF-κB pathway activation in RLE cells upon modification of the intracellular glutathione content with BSO or NAC. In alveolar epithelial cells, glutathione is considered to be the most important intracellular antioxidant . Lower levels of intracellular glutathione have been associated with enhanced NF-κB activation . In Jurkat cells, increasing intracellular glutathione levels using NAC has been shown to block H2O2-induced NF-kB activation . There is also evidence for the interplay between ROS and cytokines in NF-κB induction . In our hands however, depleting the intracellular glutathione concentration to 5% of its normal level using BSO or augmenting it to 133% using NAC did not influence NF-κB activation in macrophage supernatant-treated or H2O2-treated RLE cells. It remains to be investigated whether in our experiments the protective role of glutathione is taken over by other intracellular thiols or thioredoxin .
Our findings suggest that oxidative stress is not a key mechanism in the activation of NF-κB in lung epithelial cells by quartz. This is evidenced by the inability of H2O2 to directly activate NF-κB, as well as the finding that modulation of the intracellular antioxidant level did not influence classical NF-κB pathway activation. As ROS are highly reactive and extremely short-lived, stronger proof for this concept might be obtained from future in vitro co-culture experiments where macrophages and type II epithelial cells are cultured simultaneously, so that macrophages are allowed to adhere to lung epithelial cells, providing an oxidative stress setting more similar to the in vivo situation. In a mixed co-culture of primary rat macrophages and primary epithelial type II cells however, no significant induction of several proinflammatory genes, including iNOS, was found after silica treatment . The discrepancy between these investigations and the current study might be due to the different experimental set-up. Although primary cells are of higher biological relevance, the isolation of these cells might induce stress-related genes in the cells, which could make it harder to observe differences between treated and control cells. Another possibility is that direct contact of macrophages and epithelial cells diminishes iNOS induction by quartz treatment, possibly due to a lower activation of macrophages by surfactant production, which was shown in a follow-up to the former study .
In previous studies, we have demonstrated that modification of the surface of DQ12 quartz particles, e.g. using the polymer PVNO, leads to a marked inhibition of inflammation and toxicity in rat lungs after instillation [21, 24, 57]. In the present study, we found marked effects of particle surface modification in our in vitro model: When cells were treated with supernatant from PVNO-quartz-treated macrophages, IκBα phosphorylation and degradation was abolished, compared to the effect of supernatants obtained from macrophages that were treated with the non-coated quartz. Also, the induction of the two main genes induced by macrophage supernatants in our system, iNOS and COX-2, was found to be abrogated. A similar inhibition was found for direct quartz treatment, as HO-1 and COX-2 induction was almost brought back to the control level. Our current findings with the PVNO-modified particles point towards a specific effect of quartz particles, and suggest that particles of lower toxicity or reactivity may not trigger either the macrophage-driven or direct particulate effects on the epithelial cells. Currently we are investigating this by evaluating a set of particle types with different size and surface properties. Altogether our study indicates the existence of a differentially mediated pro-inflammatory activation of lung epithelial cells in quartz-exposed lung, which in both cases appears to be driven by the particle surface properties: Firstly, quartz particles can induce cellular oxidative stress leading to an NF-κB-independent increase in expression of HO-1 and COX-2. We hypothesise that the transcription factor nuclear factor E2-related factor 2 (nrf-2), which was elegantly shown to be the main transcription factor regulating HO-1 expression , is at least in part involved in these observed direct quartz effects. Secondly, stable mediators released from quartz-treated macrophages strongly induce the classical NF-κB pathway as well as iNOS. Remarkably, the involvement of the most likely candidates for this indirect, macrophage-mediated activation, i.e. TNFα, IL-1β and H2O2 could be ruled out. In further experiments we could also exclude potential contributions of macrophage-derived NO as well as of proteases, which both have been shown to be involved in activation of the NF-κB pathway [59, 60]. In line with observations by others who have used primary macrophages , quartz did not elicit any detectable NO production from quartz-treated NR8383 cells, measured using the Griess assay, in contrast to lipopolysaccharide (LPS, 0.1 or 1 g/l) (data not shown). The effect of proteases could be ruled out by the addition of protease inhibitors to the supernatants of quartz-treated NR8383 cells (data not shown). Among various potential candidates, one could also consider prostaglandins (PG), which are known to be released from macrophages. However, there is considerable experimental data suggesting that many of the arachidonic acid metabolites rather act as NF-κB inhibitors and thereby provide a negative feedback loop for COX-2 expression [62, 63]. Indeed, the well investigated metabolite PGE2, which is considered as an acute pro-inflammatory mediator, has been shown to increase the transactivation potential of the RelA subunit of NF-κB in intestinal epithelial cell lines. However, this effect was demonstrated to occur in the absence of IκBα degradation and nuclear accumulation of NF-κB . Thus, macrophage-derived prostaglandins including PGE2, unlikely explain for the observed macrophage-mediated phosphorylation and degradation of IκBα and subsequent translocation of RelA in the RLE cells. Although the responsible mechanism is yet to be identified, our data indicate that for therapeutic or possible preventive approaches, an intervention at the macrophage product level might not be feasible, as targeting of the main individual mediators suspected to be involved (TNFα, IL-1β) was unsuccessful. Additionally, in such intervention direct particle effects on epithelial cells (e.g. COX-2 induction) would persist. Instead, a more promising strategy should focus on upstream processes, e.g. by targeting the initial activation of macrophages by the quartz particles. Preferentially, one should aim at blocking the secretion of mediators from macrophages without interfering with their phagocytic properties that are essential for lung homeostasis and are known to provide the hallmark of particle clearance out of the alveoli via the lymph nodes. Recent in vitro investigations in our laboratory  indicate that this may possibly be achieved by targeting Fc II-receptor signalling pathway in the macrophages.