In this study, we demonstrate that TNFα- and LPS-stimulated neutrophils exhibit a previously unrecognized NFκB activation that lasts up to 12 h. In contrast to the early activation of NFκB that is regulated by the nuclear levels of IκBα (24
), this delayed NFκB activity in 9 h stimulated neutrophils is increased despite the presence of IκBα in the nucleus. Based on this and previous studies from our laboratory (25
), we propose a new model of NFκB regulation during persistent neutrophil stimulation with inflammatory signals. In the resting neutrophils, IκBα is localized in the nuclear matrix that also contains NFκB p50 and p65 subunits, and by binding to p65 NFκB it inhibits NFκB activity. After neutrophil stimulation with proinflammatory signals, both the nuclear and cytoplasmic IκBα are degraded within 30 min, thus releasing p65 NFκB from the inhibitory complex to bind to NFκB-dependent promoters (25
). Our results indicate that during persistent neutrophil stimulation, IκBα is again synthesized and translocates to the nucleus (). This is supported by our previous study demonstrating that cycloheximide treatment prevents the reappearance of both nuclear and cytoplasmic IκBα after 2-h neutrophil stimulation with LPS or TNFα (25
). Thus, it seems that the newly synthesized nuclear IκBα that associates with p65 NFκB in the nuclear matrix (), is no longer sufficient to inhibit the persistent NFκB activity. The reduced ability of the nuclear IκBα to inhibit NFκB DNA binding at 9 h after stimulation suggests that the newly synthesized nuclear IκBα may be posttranslationally modified, and its binding to p65 NFκB does not inhibit, or may even stimulate, the NFκB DNA binding. Alternatively, the concentration of the newly synthesized IκBα in the nucleus might be lower than the concentrations of NFκB proteins. In addition, it cannot be completely ruled out at present that even though IκB
is not degraded after neutrophil stimulation, it can still regulate the transcriptional activity of NFκB. Studies are in progress to discriminate between these models, and to identify the exact mechanisms by which the nuclear IκB proteins regulate the NFκB-dependent transcription in stimulated human neutrophils.
The nuclear matrix is a specialized proteinaceous nuclear structure that serves as a scaffold for chromatin loops. Studies have suggested that the nuclear matrix is tightly associated with transcriptionally active, but not inactive, DNA (45
). To our knowledge, this study provides the first demonstration that IκBα associates with the nuclear matrix. Our results are consistent with the morphological study of Trubiani et al. (47
) showing that in stimulated epithelial cells, the subnuclear localization of NFκB p50 and p65 proteins is the nuclear matrix. We suggest that the nuclear localization of IκBα and attachment to nuclear matrix represent one of the underlying mechanisms for the decreased activation of NFκB in human neutrophils (34
) and for the increased apoptosis of these cells.
The subunit composition of NFκB complexes activated in LPS- and TNFα-stimulated neutrophils at 9 h () is the same as the NFκB subunit composition at 30 min (36
): p50/50 homodimers and p50/65 heterodimers. In addition, Western blotting ( and ) as well as immunofluorescence microscopy () revealed that the p50 and p65 NFκB nuclear protein levels are not substantially changed during neutrophil stimulation with TNFα or LPS, indicating that the persistent NFκB activity in stimulated human neutrophils is not regulated primarily by the nuclear translocation of NFκB subunits. Thus, when assessing NFκB activity during neutrophil-mediated inflammatory disorders, it is important to analyze the extent of NFκB activity by EMSA, and not only the nuclear levels or translocation of NFκB subunits.
There is an urgent need for early detection and safer and more specific therapies that minimize tissue injury for ALI, sepsis, and BPD. Neutrophil apoptosis, regulated by NFκB, plays a critical role in the resolution of inflammation associated with these disorders (28
). Although studies have shown that NFκB is activated in patients with ALI, sepsis, and BPD (4
), the stage of NFκB activation in these patients is not known. It seems plausible that it is the persistent NFκB activity, and not the early NFκB activation induced by neutrophil stimulation for 30 min, that is associated with the tissue injury in these inflammatory disorders. Whereas TNFα, one of the first mediators of sepsis (50
), is released early during neutrophil stimulation, IL-8 is released during later time points (). We hypothesize that different genes may be activated by NFκB during different times of neutrophil stimulation. In this scenario, the TNFα transcription would be expected to be induced by the early NFκB activation in LPS-stimulated neutrophils, and the IL-8 transcription during the second wave. It might be possible that the second wave of NFκB activation in LPS-stimulated neutrophils is caused by the release of TNFα or other NFκB-regulated mediator. However, this does not seem very likely, because the second wave of NFκB activation in LPS-stimulated neutrophils is not associated with the degradation of cytoplasmic or nuclear IκBα (). A detailed analysis by chromatin immunoprecipitation of the nuclear IκB and/or NFκB complexes that are recruited to NFκB promoters during neutrophil stimulation will be essential to identify the specific NFκB-regulated proinflammatory and anti-apoptotic genes synthesized during different stages of neutrophil activation.
Previous studies have shown that the early activation of NFκB is regulated by PKCδ, IκB kinase, and p38 MAP kinase and by PP1/PP2A phosphatases (24
); however, the mechanisms that regulate the persistent NFκB activity during continuous neutrophil stimulation are unknown. In addition, inhibitors of NFκB, such as dexamethasone or cur-cumin, inhibit production of NFκB-dependent proinflammatory cytokines and induce neutrophil apoptosis (52
). It will therefore be important to identify the upstream signaling mechanisms that regulate the persistent NFκB activity and to determine whether this delayed NFκB activity in continuously stimulated human neutrophils can be inhibited by antiinflammatory or proapoptotic drugs like dexamethasone and curcumin.
Previous work from our laboratory has demonstrated that there are significant differences in the NFκB regulation between human neutrophils and other cells, such as monocytic cells (24
). The early stage of NFκB activation in human neutrophils is regulated predominantly by the nuclear levels of IκBα (24
). This study shows that NFκB is persistently activated in human neutrophils stimulated with LPS or TNFα, but this persistent NFκB activation is independent of the nuclear levels of IκBα. A better understanding of the mechanisms regulating the initiation and persistence of NFκB activation in human neutrophils may provide new information for the development of early detection and safer therapies for BPD, ALI, sepsis, and other neutrophil-mediated inflammatory disorders.