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The Journal of Immunology, 2002, 169: 3947-3953.
Copyright © 2002 by The American Association of Immunologists

NF-{kappa}B Regulation in Human Neutrophils by Nuclear I{kappa}B{alpha}: Correlation to Apoptosis1

Susana Castro-Alcaraz, Veronika Miskolci, Bharati Kalasapudi, Dennis Davidson and Ivana Vancurova2

Division of Neonatal-Perinatal Medicine, Schneider Children’s Hospital, Long Island Jewish Medical Center-The Long Island Campus, Albert Einstein College of Medicine, and North Shore-Long Island Jewish Research Institute, New Hyde Park, NY 11040


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-{kappa}B plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-{kappa}B inhibitor, I{kappa}B{alpha}, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of I{kappa}B{alpha} in human neutrophils and to determine whether increased accumulation of I{kappa}B{alpha} in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of I{kappa}B{alpha} that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of I{kappa}B{alpha} that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of I{kappa}B{alpha} in stimulated neutrophils by blocking the I{kappa}B{alpha} nuclear export, and this is associated with inhibition of NF-{kappa}B activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-{kappa}B regulation in human neutrophils by nuclear I{kappa}B{alpha}. Our results demonstrate that the NF-{kappa}B activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of I{kappa}B{alpha} in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human neutrophils (polymorphonuclear leukocytes) have been directly implicated in the pathogenesis of inflammatory diseases, including acute respiratory distress syndrome, chronic obstructive pulmonary disease, and bronchopulmonary dysplasia (1, 2, 3, 4). Neutrophils are short-lived, terminally differentiated blood cells that constitutively undergo apoptosis, and this process is critical for the resolution of inflammation (5, 6, 7). While it has been recognized for many years that proinflammatory mediators such as TNF and LPS can modulate neutrophil survival, the responsible molecular mechanisms are still largely unknown (8, 9, 10). Recent studies have shown that both constitutive and TNF-induced apoptosis in human neutrophils is regulated by the transcription factor NF-{kappa}B (11, 12, 13).

NF-{kappa}B comprises a family of transcription factors that serve as important regulators of the genes involved in host immune and inflammatory responses, apoptosis, proliferation, and differentiation (14, 15, 16, 17, 18, 19). The most abundant and best characterized of the NF-{kappa}B dimers is the inducible p50/65 NF-{kappa}B heterodimer (20). In the classical model of NF-{kappa}B activation, NF-{kappa}B p50/65 exists in the cytoplasm of unstimulated cells in an inactive form associated with the inhibitory protein I{kappa}B{alpha} (20, 21). Following cell stimulation by extracellular stimuli, I{kappa}B{alpha} is phosphorylated through a cascade of inducible protein kinases, ubiquitinated, and selectively degraded in cytoplasm by the proteasome (21, 22). This results in unmasking of the nuclear localization sequence (NLS)3 of the NF-{kappa}B 50/65 heterodimers, which then translocate to the nucleus and stimulate transcription of inflammatory and anti-apoptotic genes. One of the first genes induced following NF-{kappa}B activation is I{kappa}B{alpha} itself, since the I{kappa}B{alpha} promoter also contains the NF-{kappa}B binding region. This newly synthesized I{kappa}B{alpha} can then enter the nucleus, remove NF-{kappa}B from gene promoters, and transport it back to the cytoplasm, representing an important feedback regulatory mechanism, also called postinduction repression (23, 24). Thus, in this classical model of NF-{kappa}B activation, NF-{kappa}B activity is regulated by cytoplasmic degradation of I{kappa}B{alpha} and nuclear translocation of NF-{kappa}B dimers, and it is terminated by nuclear entry of the newly synthesized I{kappa}B{alpha}.

Importantly, the NF-{kappa}B regulation appears to be highly tissue and cell specific (20). In human neutrophils, NF-{kappa}B was first identified by McDonald et al. (25). Interestingly, the extent of NF-{kappa}B activation in neutrophils was lower than that in peripheral blood monocytes (25). We have recently shown that resting human neutrophils, but not monocytes, contain a significant amount of I{kappa}B{alpha} in the nucleus, thus differing from most of the human cells described to date (26).

In this study we characterized the mechanisms controlling the nuclear accumulation of I{kappa}B{alpha} in human neutrophils. We show for the first time that neutrophil stimulation with pro-inflammatory signals induces the degradation of I{kappa}B{alpha} that occurs in both cytoplasm and nucleus. Furthermore, we demonstrate that sustained stimulation with TNF and LPS induces resynthesis of I{kappa}B{alpha} that is translocated to the nucleus in human neutrophils, but not in monocytic cells. Our results indicate that the increased accumulation of I{kappa}B{alpha} in the nucleus of human neutrophils is associated with the inhibition of NF-{kappa}B activity, caspase-3 activation, and increased apoptosis in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

Polyclonal Abs to human I{kappa}B{alpha} (sc-371) and p21CIP1(sc-469) and mouse monoclonal anti-actin Ab (sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-SUMO-1 mAb was obtained from Zymed (San Francisco, CA), and polyclonal lactate dehydrogenase (LDH) Ab (20-LG22) was purchased from Fitzgerald Industries International (Concord, MA). HRP-conjugated anti-rabbit, anti-mouse, and anti-goat IgG secondary Abs were obtained from Amersham (Arlington Heights, IL).

Cell culture

Fresh blood was obtained from healthy adult human volunteers and collected in heparinized preservative-free tubes. Neutrophils and monocytic cells were purified under endotoxin-free conditions as described previously (26, 27). Purified cells were resuspended in RPMI 1640 supplemented with 10% low endotoxin FCS at a final concentration of 5 x 106 cells/ml and incubated at 37°C in polypropylene tubes with gentle agitation. For the inhibition experiments the cells were incubated with leptomycin B (LMB) or the vehicle solution (1/1000 volume of ethanol) for 45 min before stimulation.

Preparation of cytoplasmic and nuclear extracts

Nuclear and cytoplasmic extracts were prepared from 5 x 106 cells as described previously (26). Protein concentration was measured using Pierce Coomassie Plus protein assay kit (Pierce, Rockford, IL). Contamination of nuclear and cytoplasmic fractions by cytoplasmic and nuclear proteins, respectively, was determined by Western analysis using LDH and SUMO-1 as specific markers (26).

EMSA

The oligonucleotide used as a probe for EMSA was a 42-bp double-stranded construct (5'-TTGTTACAAGGGGACTTTCCGCTGGGGACTTTCCAGGGAGGC-3') containing two tandemly repeated NF-{kappa}B binding sites (underlined). EMSA was performed using nuclear extracts (containing 4–6 µg protein in 5–7 µl) and the above NF-{kappa}B oligonucleotide as described previously (26, 28).

Western blotting

Denatured proteins were separated on 12% denaturing polyacrylamide gels and transferred to nitrocellulose membrane as described previously (26). To confirm equal amounts of loaded proteins, the membranes were stripped and reprobed with actin Ab. The signal was developed using secondary IgG-HRP and ECL detection.

Assessment of neutrophil apoptosis

Two methods were used for the assessment of neutrophil apoptosis. For morphological assessment, neutrophils were cytocentrifuged, air-dried, stained with a commercial May-Grunwald Giemsa stain (Diff-Quick, Baxter Healthcare, Deerfield, IL), and counted using oil immersion microscopy (x100 objective). Neutrophils (minimum of 200 cells/slide) counted in randomly selected fields were scored as apoptotic and nonapoptotic based on their nuclear morphology (8, 11). The results are expressed as the mean percent apoptosis ± SEM.

For measurement of caspase-3 activity, we used a colorimetric assay kit from R&D Systems (Minneapolis, MN). Briefly, cells (2.5 x 106) were lysed in 63 µl lysis buffer (R&D Systems) on ice (15 min), and whole cell lysates were centrifuged (14,000 x g for 10 min). Fifty microliters of whole cell lysates (containing ~50 µg protein) were then diluted to 100 µl with reaction buffer (R&D Systems) and incubated with 5 µl Asp Glu Val Asp-p-nitroanaline substrate (R&D Systems) for 2 h at 37°C. Absorbance was determined at 410 nm. Blanks in the absence of cell lysate were conducted to determine background absorbance.

Data analysis

The data presented here represent a minimum of three experiments and, where appropriate, are expressed as the mean ± SEM. Data were analyzed using an InStat software package (GraphPad, San Diego, CA). Statistical significance was evaluated by Mann-Whitney test, and p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Newly synthesized I{kappa}B{alpha} accumulates in the nucleus of TNF-stimulated neutrophils, but not monocytic cells

First we analyzed whether I{kappa}B{alpha} induced by postinduction repression translocates to the nucleus in TNF-stimulated human neutrophils and monocytic cells. Cells were stimulated with TNF for 0, 15, 60, and 120 min, and cytoplasmic and nuclear extracts were prepared and analyzed by Western blotting using I{kappa}B{alpha} Ab. The purity of cytoplasmic and nuclear fractions was monitored using LDH and SUMO-1 as specific markers, respectively (26). In neutrophils, TNF stimulation leads to rapid (15-min) depletion of I{kappa}B{alpha} in both cytoplasmic and nuclear fractions (Fig. 1Go), and the newly synthesized I{kappa}B{alpha} induced after prolonged neutrophil stimulation with TNF (60 and 120 min) translocates without delay to the nucleus.



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  		FIGURE 1. I{kappa}B{alpha} induced after sustained stimulation with TNF accumulates in the nucleus of human neutrophils, but not monocytic cells. Western analysis was performed using I{kappa}B{alpha} polyclonal Ab of cytoplasmic and nuclear extracts prepared from neutrophils and monocytic cells stimulated with TNF (10 ng/ml) for 0, 15, 60, and 120 min with and without prior incubation with CHX (100 µg/ml, 15 min). To confirm equal protein loading, the membranes were stripped and reprobed with actin Ab. The presence of cytoplasmic proteins in nuclear fraction of neutrophils was evaluated by reprobing the membrane with LDH Ab. Nuclear contamination in the cytoplasmic fraction was assessed using SUMO-1-specific Ab.

 
In monocytic cells, no I{kappa}B{alpha} was detected in the nuclear fraction at any time point (Fig. 1Go), indicating that in these cells neither basal nor newly synthesized I{kappa}B{alpha} can accumulate in the nucleus. However, since in monocytic cells the kinetics of I{kappa}B{alpha} degradation in the cytoplasm were slower than those in the neutrophils, the following experiments were performed to distinguish between basal and newly synthesized I{kappa}B{alpha}. Both cell types were stimulated with TNF as described above in the absence and the presence of prior pretreatment with cycloheximide (CHX), an inhibitor of protein synthesis. This CHX experiment confirmed that while in neutrophils I{kappa}B{alpha} is degraded almost completely within 15 min after TNF stimulation, in monocytic cells a significant amount of basal I{kappa}B{alpha} is present in the cytoplasm even after 2-h stimulation with TNF, due to its slower degradation.

Neutrophil stimulation with TNF induces nuclear shuttling of I{kappa}B{alpha}

To further characterize the nuclear transport of I{kappa}B{alpha} in the neutrophils, we used leptomycin B (LMB), a specific inhibitor of nuclear export that interferes with the interaction between nuclear export sequences (NES), and CRM1, a NES receptor, belonging to the {beta}-importin family (29, 30). As shown in Fig. 2GoA, blocking the nuclear export with LMB resulted in substantially increased nuclear accumulation of I{kappa}B{alpha} in TNF-stimulated (2 h) neutrophils (lanes 7 and 8), while the effect on the nuclear accumulation of I{kappa}B{alpha} in unstimulated cells was much less pronounced (lanes 3 and 4). In the TNF-stimulated neutrophils two different scenarios could account for the LMB-induced nuclear accumulation of I{kappa}B{alpha}. In the first model the newly synthesized I{kappa}B{alpha} shuttles between nucleus and cytoplasm of TNF-stimulated neutrophils, and blocking the nuclear export results in I{kappa}B{alpha} accumulation in the nucleus. Alternatively, this I{kappa}B{alpha} protein accumulated in the nucleus of TNF-stimulated neutrophils in the presence of LMB could be the original, basal I{kappa}B{alpha}, escaping TNF-induced degradation, provided that this degradation occurs only in the cytoplasm.



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  		FIGURE 2. Neutrophil stimulation with TNF induces I{kappa}B{alpha} nuclear shuttling. A, Western analysis of cytoplasmic and nuclear extracts prepared from unstimulated and TNF-stimulated (10 ng/ml, 2 h) neutrophils preincubated with LMB (20 nM, 45 min) or vehicle solution (1/1000 volume of ethanol). B, Western analysis of cytoplasmic and nuclear extracts prepared from TNF-stimulated (10 ng/ml, 2 h) neutrophils preincubated with LMB (20 nM, 45 min) with and without CHX (100 µg/ml).

 
To differentiate between these two models, neutrophils were stimulated with TNF for 2 h with and without prior incubation with LMB and CHX (Fig. 2GoB). As expected, no I{kappa}B{alpha} was detected in the cytoplasmic fraction in neutrophils stimulated 2 h with TNF in the presence of CHX, independently of LMB treatment (Fig. 2GoB, lanes 3 and 4). In the nucleus, the TNF-induced resynthesis of I{kappa}B{alpha} that is increased by blocking the nuclear export with LMB (lanes 6 and 7) is prevented by pretreatment with CHX (lanes 6 and 8). These results imply that the I{kappa}B{alpha} protein accumulating in the nucleus of stimulated neutrophils treated with LMB is the newly synthesized I{kappa}B{alpha}, induced by postinduction repression, and that neutrophil stimulation with pro-inflammatory signals induces continuous nuclear-cytoplasmic shuttling of I{kappa}B{alpha}. Surprisingly, no I{kappa}B{alpha} was detected in the nucleus of TNF-stimulated neutrophils in the presence of CHX and LMB (lane 9). The effect of CHX alone on I{kappa}B{alpha} expression and stability was minimal (lanes 5 and 10) and was comparable to that of TNF (2 h) alone (lanes 1 and 6). Together, these results suggested that the TNF-induced degradation of I{kappa}B{alpha} might occur also in the nucleus.

Neutrophil stimulation induces I{kappa}B{alpha} degradation that occurs in the cytoplasm as well as in the nucleus

In Fig. 1Go we have shown that neutrophil stimulation with TNF results in rapid depletion of I{kappa}B{alpha} in both cytoplasm and nucleus. However, it was not clear whether I{kappa}B{alpha} is degraded independently in both compartments or whether it is degraded exclusively in the cytoplasm after TNF induces its nuclear shuttling. To distinguish between these two models, neutrophils were stimulated 15 min with TNF with and without prior incubation with LMB and/or CHX (Fig. 3Go). As expected, in the cytoplasm 15-min stimulation with TNF led to degradation of I{kappa}B{alpha} (lanes 1 and 3), independently of LMB or CHX (lanes 4–6). In the nucleus, inhibition of nuclear export with LMB resulted in a partial, TNF-induced (15-min) degradation of I{kappa}B{alpha} (lanes 2 and 4), independent of new protein synthesis (lane 6). This result together with the data shown in Fig. 2GoB (lane 9) indicated that I{kappa}B{alpha} can be degraded in the nucleus as well.



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  		FIGURE 3. Nuclear and cytoplasmic degradation of I{kappa}B{alpha} in human neutrophils. Western analysis of cytoplasmic and nuclear extracts prepared from neutrophils that were either unstimulated or stimulated with TNF (10 ng/ml) for 15 min with and without prior incubation with LMB (20 nM, 45 min) and CHX (100 µg/ml, added at the same time as LMB).

 
To further analyze the nuclear degradation of I{kappa}B{alpha}, neutrophils were stimulated with TNF in the presence of CHX and LMB, and nuclear I{kappa}B{alpha} degradation was measured at 0–60 min. As shown in Fig. 4Go, blocking the nuclear I{kappa}B{alpha} export with LMB did not prevent time-dependent degradation of I{kappa}B{alpha} in the nucleus. The lower panel shows analysis of another nuclear protein, p21CIP1 (28), under these conditions, demonstrating the specificity of TNF-induced nuclear degradation for I{kappa}B{alpha}. Together, these results demonstrate that in stimulated human neutrophils, I{kappa}B{alpha} is degraded in response to stimulation with proinflammatory signals in the nucleus as well as in the cytoplasm.



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  		FIGURE 4. Time course of TNF-induced I{kappa}B{alpha} degradation in the nucleus of human neutrophils. Western analysis of nuclear extracts prepared from neutrophils incubated with LMB (20 nM, 45 min) and CHX (100 µg/ml, added at the same time as LMB) and stimulated with TNF for 0–60 min. To confirm specificity for I{kappa}B{alpha} degradation as well as equal protein loading, the membrane was stripped and reprobed with p21CIP1 polyclonal Ab.

 
LMB induces nuclear accumulation of I{kappa}B{alpha} in stimulated neutrophils, but not monocytic cells, and this is associated with NF-{kappa}B inhibition and increased apoptosis

Since apoptosis in neutrophils as well as other cell types has been shown to be mediated by the inhibition of NF-{kappa}B (11, 31), we investigated whether LMB-induced increased nuclear accumulation of I{kappa}B{alpha} in stimulated neutrophils is associated with NF-{kappa}B inhibition and increased apoptosis.

Although in our initial studies of the regulation of nuclear accumulation of I{kappa}B{alpha} we used TNF for neutrophil stimulation, to investigate the role of nuclear I{kappa}B{alpha} in the induction of apoptosis we used LPS. Like TNF, LPS evokes activation of NF-{kappa}B in human neutrophils (25, 26, 32). However, unlike TNF, which inhibits apoptosis after 20-h incubation, while it accelerates neutrophil apoptosis in a subpopulation of cells at earlier (2–8 h) times (33), LPS promotes neutrophil survival (34, 35).

Similarly as in TNF-stimulated neutrophils, in neutrophils stimulated with LPS, LMB significantly increased the nuclear accumulation of I{kappa}B{alpha} (Fig. 5Go), again demonstrating that the newly synthesized I{kappa}B{alpha} induced by sustained neutrophil stimulation with pro-inflammatory signals continuously shuttles between nucleus and cytoplasm. The LMB-induced increased nuclear accumulation of I{kappa}B{alpha} was accompanied by inhibition of NF-{kappa}B, measured as the extent of NF-{kappa}B DNA binding in neutrophils stimulated with LPS as well as TNF (Fig. 6Go). This correlates well with the previously reported in vitro inhibition of NF-{kappa}B DNA binding by exogenously added purified recombinant I{kappa}B{alpha} protein (36).



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  		FIGURE 5. LMB induces nuclear accumulation of I{kappa}B{alpha} in LPS-stimulated neutrophils. Western analysis of cytoplasmic and nuclear extracts prepared from neutrophils stimulated with LPS (1 µg/ml) for 0, 0.5, 1, 2, and 3 h, with and without LMB (20 nM, 45-min preincubation).

 


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  		FIGURE 6. LMB-induced nuclear accumulation of I{kappa}B{alpha} in stimulated neutrophils results in the inhibition of NF-{kappa}B DNA binding. A, EMSA of NF-{kappa}B DNA binding in nuclear extracts prepared from LPS-stimulated (1 h, 1 µg/ml) neutrophils incubated in the presence (45 min, 20 nM) and the absence of LMB. B, EMSA of NF-{kappa}B DNA binding in nuclear extracts prepared from neutrophils stimulated with TNF (1 h, 10 ng/ml) and incubated with LMB as described above.

 
Caspase-3 activation has been widely demonstrated to be one of the early events in cells undergoing apoptosis, including neutrophils (37, 38). Therefore, we determined whether the LMB-induced increased accumulation of I{kappa}B{alpha} in the nucleus of human neutrophils is associated with caspase-3 activation. As shown in Fig. 7GoA, neutrophil preincubation with LMB increased caspase-3 activity in neutrophils stimulated with LPS, and this was most prominent after 3-h incubation with LPS (p < 0.01; n = 6). To confirm the above results by an independent method, we determined apoptosis in LMB-treated neutrophils by morphological evaluation (8, 11). Consistent with caspase-3 activity, LMB accelerated the induction of apoptosis in LPS-stimulated neutrophils (Fig. 7GoB), and as with caspase-3 activity, this was most significant after 3-h stimulation with LPS (p < 0.01; n = 5).



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  		FIGURE 7. LMB-induced nuclear accumulation of I{kappa}B{alpha} in LPS-stimulated neutrophils is associated with caspase-3 activation and apoptosis. A, Colorimetric determination of caspase-3 activity in whole cell extracts prepared from neutrophils (2 x 106) stimulated with LPS (1 µg/ml) for 0, 1, 2, and 3 h, with and without LMB (20 nM, 45-min preincubation). Statistical significance was evaluated using the Mann-Whitney test. B, Morphological assessment of apoptotic neutrophils stimulated with LPS in the presence and the absence of LMB as described above. Statistical significance was evaluated using the Mann-Whitney test.

 
To further support the role of nuclear I{kappa}B{alpha} in the inhibition of NF-{kappa}B activity and the induction of apoptosis in human neutrophils and to exclude the possibility that LMB-mediated inhibition of NF-{kappa}B and induction of neutrophil apoptosis were due to the cytotoxic effect of LMB, we evaluated the effect of LMB on I{kappa}B{alpha} nuclear-cytoplasmic shuttling, NF-{kappa}B activation, and caspase-3 activity in human monocytic cells. As shown in Fig. 8GoA, LMB did not induce nuclear translocation of I{kappa}B{alpha} in LPS-stimulated monocytic cells, confirming that in these cells I{kappa}B{alpha} is not imported to the nucleus and remains localized in the cytoplasm. Importantly, in stimulated monocytic cells, LMB did not inhibit DNA binding of the inducible NF-{kappa}B 50/65 heterodimer (Fig. 8GoB). Furthermore, LMB treatment of LPS-stimulated monocytic cells did not significantly induce caspase-3 activity (p > 0.3; n = 6; Fig. 8GoC).



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  		FIGURE 8. LMB does not cause nuclear translocation of I{kappa}B{alpha}, NF-{kappa}B inhibition, and caspase-3 activation in LPS-stimulated human monocytic cells. A, Western analysis of cytoplasmic and nuclear extracts prepared from monocytic cells stimulated with LPS (1 µg/ml) for 0, 0.5, 1, 2, and 3 h, with and without LMB (20 nM, 45-min preincubation). B, EMSA of NF-{kappa}B DNA binding in nuclear extracts prepared from LPS-stimulated (1 h, 1 µg/ml) monocytic cells incubated in the presence (45 min, 20 nM) and the absence of LMB. C, Colorimetric determination of caspase-3 activity in whole cell extracts prepared from monocytic cells (2 x 106) stimulated with LPS (1 µg/ml) for 0, 1, 2, and 3 h, with and without LMB (20 nM, 45-min preincubation). Statistical significance was evaluated using the Mann-Whitney test.

 
The fact that in monocytic cells, in the absence of nuclear translocation of I{kappa}B{alpha}, LMB does not significantly affect NF-{kappa}B and caspase-3 activities suggests that the LMB-mediated inhibition of NF-{kappa}B and increased apoptosis in the neutrophils are caused by the increased amount of I{kappa}B{alpha} in the nucleus, rather than by LMB itself. Together, these results indicate that the increased I{kappa}B{alpha} in the nucleus of human neutrophils inhibits NF-{kappa}B DNA binding activity and suggest that this may represent one of the underlying mechanisms for the increased rate of neutrophil apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that NF-{kappa}B activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells. The current model of NF-{kappa}B regulation in human neutrophils by nuclear I{kappa}B{alpha} is presented in Fig. 9Go. First, we demonstrate that sustained stimulation with proinflammatory signals induces resynthesis of I{kappa}B{alpha} that is translocated to the nucleus in neutrophils, but not monocytic cells. Second, we show, for the first time, that neutrophil stimulation results in rapid I{kappa}B{alpha} degradation that occurs in both cytoplasm and nucleus. Third, our results indicate that the increased nuclear accumulation of I{kappa}B{alpha} in human neutrophils is associated with the inhibition of NF-{kappa}B activity and the induction of apoptosis in these cells.



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  		FIGURE 9. Current model of NF-{kappa}B regulation in human neutrophils by nuclear I{kappa}B{alpha}.

 
I{kappa}B{alpha} protein contains both NLS activity (24, 39) and NESs (40, 41, 42, 43). Recent studies have shown that although in unstimulated epithelial cells, I{kappa}B{alpha} is localized almost exclusively in the cytoplasm, blocking the CRM1-specific export with LMB induces nuclear localization of I{kappa}B{alpha}, indicating nuclear-cytoplasmic trafficking of basal I{kappa}B{alpha} in unstimulated epithelial cells (40, 41). Moreover, these studies implied that cytoplasmic localization of I{kappa}B{alpha} in epithelial as well as other human cells is established by a potent CRM1-dependent nuclear export process that is dominant over the relatively weaker NLS-dependent nuclear import (41). In contrast, in human neutrophils blocking the CRM1-dependent export did not substantially increase nuclear accumulation of basal I{kappa}B{alpha} in unstimulated cells (Fig. 2GoA). This implies that in unstimulated neutrophils, I{kappa}B{alpha} nuclear import is dominant over its export, inhibition of which has little effect on I{kappa}B{alpha} nuclear accumulation.

The lack of significant nuclear trafficking of I{kappa}B{alpha} in resting neutrophils contrasts with its continuous shuttling in neutrophils stimulated with proinflammatory signals. Since blocking the I{kappa}B{alpha} nuclear export in stimulated neutrophils results in I{kappa}B{alpha} nuclear accumulation (Figs. 2Go and 5Go) and inhibition of NF-{kappa}B activity (Fig. 6Go), mechanisms controlling I{kappa}B{alpha} nuclear trafficking in human neutrophils may represent a new potential target for the regulation of NF-{kappa}B-driven gene expression in these cells. We and others (44, 45) have previously demonstrated that the rate of nuclear transport and the extent of nuclear accumulation of nuclear proteins are regulated by phosphorylation. In this context, I{kappa}B{alpha} has been shown to be phosphorylated, in addition to the I{kappa}B kinase (IKK) that phosphorylates it at serine residues 32 and 36 (46, 47), by casein kinase II (48), protein kinase C (49), tyrosine kinase (50), and DNA-dependent protein kinase (51). However, only phosphorylation by IKK leads to the signal-induced proteolytic degradation of I{kappa}B{alpha}. Phosphorylations by casein kinase II, DNA-dependent protein kinase, and tyrosine kinase were not demonstrated to result in I{kappa}B{alpha} degradation, albeit they regulate NF-{kappa}B activity. Therefore, it seems plausible that one of the mechanisms by which they control NF-{kappa}B activation is by regulating the nuclear trafficking of I{kappa}B{alpha}. Studies are currently in progress to determine the phosphorylation sites and corresponding protein kinases involved in the regulation of nuclear transport of I{kappa}B{alpha} in resting and stimulated human neutrophils.

Originally, I{kappa}B{alpha} has been thought to be phosphorylated by the IKK complex, ubiquitinated and degraded by the proteasome exclusively in the cytoplasm (52). However, recent studies suggested that a functional ubiquitin-proteasome system might be operative within the nucleus as well. In this study we have established that in human neutrophils, nuclear export is not required for the stimulus-induced degradation of I{kappa}B{alpha}. This is consistent with recent studies using epithelial cells, demonstrating ubiquitin-dependent nuclear degradation of I{kappa}B{alpha} and MyoD, another transcription factor (53, 54). In addition, a recent study by Birbach et al. (55) demonstrated continuous nuclear-cytoplasmic shuttling of signaling kinases upstream of I{kappa}B{alpha}, I{kappa}B kinase, and NF-{kappa}B-inducing kinase. Although the specific role and importance of nuclear protein degradation are not known at present, it is clear that the ubiquitin-mediated protein degradation is regulated by compartmentalization within the cell. In this respect it is interesting to point out that while in neutrophils, I{kappa}B{alpha} is degraded in the nucleus as well as in the cytoplasm, in monocytic cells I{kappa}B{alpha} is degraded exclusively in the cytoplasm (Fig. 1Go). Thus, the nuclear degradation of I{kappa}B{alpha} may represent a new additional level of NF-{kappa}B regulation in response to neutrophil stimulation with pro-inflammatory signals.

Our results show that LMB causes nuclear entrapment of I{kappa}B{alpha} in stimulated human neutrophils, but not in monocytic cells (Figs. 5Go and 8GoA), confirming that in the monocytic cells, I{kappa}B{alpha} is not imported to the nucleus. The LMB-induced increased nuclear accumulation of I{kappa}B{alpha} in the neutrophils was accompanied by NF-{kappa}B inhibition (Fig. 6Go) and increased caspase-3 activation and apoptosis (Fig. 7Go). Since LMB was shown to decrease cell viability after long term exposure (2–8 days) in BCR-ABL tyrosine kinase-transformed cells (56), it cannot be completely excluded that the LMB-induced NF-{kappa}B inhibition and induction of neutrophil apoptosis are caused by a more general effect that is unrelated to the increased amount of I{kappa}B{alpha} in the nucleus. However, this seems unlikely, because in monocytic cells, where I{kappa}B{alpha} remains exclusively cytoplasmic regardless of LMB treatment, LMB did not significantly affect NF-{kappa}B and caspase-3 activities. Furthermore, recent studies showed that in human prostate cancer cells and lymphocytes, short term LMB treatment (<=24 h) was not sufficient to induce apoptotic changes (57, 58).

In conclusion, in this study we present a new model of NF-{kappa}B regulation in human neutrophils by nuclear I{kappa}B{alpha}. Our results indicate that the increased accumulation of I{kappa}B{alpha} in the nucleus of human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells. Identification of the key molecular mechanisms regulating nuclear accumulation of I{kappa}B{alpha} in neutrophils will not only expand our understanding of NF-{kappa}B regulation in these cells, but might also provide a new class of drug targets to selectively modify neutrophil survival and pro-inflammatory gene expression.


   Acknowledgments
 
We thank Dr. A. Vancura for helpful comments and critical reading of the manuscript, and Dr. M. Yoshida (Department of Biotechnology, University of Tokyo, Tokyo, Japan) for generous gift of leptomycin B.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant HD39643 and a North Shore-Long Island Jewish Health System Faculty Research Award (to I.V.). Back

2 Address correspondence and reprint requests to Dr. Ivana Vancurova, Long Island Jewish Medical Center, Research Building B-49, 270-05 76th Avenue, New Hyde Park, New York 11040. E-mail address: vancurov{at}lij.edu Back

3 Abbreviations used in this paper: NLS, nuclear localization sequence; CHX, cycloheximide; IKK, I{kappa}B kinase; LDH, lactate dehydrogenase; LMB, leptomycin B; NES, nuclear export signal. Back

Received for publication March 19, 2002. Accepted for publication July 29, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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