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This Article Full Text (PDF) All Versions of this Article: 16/1/129 01-0817fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by ALVARADO-KRISTENSSON, M. Articles by ANDERSSON, T. Search for Related Content PubMed PubMed Citation Articles by ALVARADO-KRISTENSSON, M. Articles by ANDERSSON, T.

p38 Mitogen-activated protein kinase and phosphatidylinositol 3-kinase activities have opposite effects on human neutrophil apoptosis ¹

Experimental Pathology, Department of Laboratory Medicine, Lund University, U-MAS, S-205 02 Malmö, Sweden

³Correspondence: Experimental Pathology, Lund University, U-MAS, Ent. 78, Fl. 3, SE-205 02 Malmö, Sweden. E-mail: tommy.andersson{at}exppat.mas.lu.se

SPECIFIC AIMS

Neutrophil apoptosis is essential for resolution of inflammatory reactions, but there is only limited information regarding the involvement of protein phosphorylation events in the regulation of human neutrophil apoptosis. Therefore, our specific objective was to investigate the role of two apoptosis/survival-associated protein kinases—p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-K)—in the regulation of Fas-mediated neutrophil apoptosis.

PRINCIPAL FINDINGS

1. Activity of p38 MAPK during neutrophil apoptosis
Western blot analysis with a specific antibody (Ab) recognizing the active form of p38 MAPK revealed constitutive activity of the enzyme in cells isolated by two different methods and immediately analyzed. Such an activity has previously been reported in human neutrophils and freshly isolated thymocytes. Here we made the novel finding that this activity was transiently lost but subsequently regained during spontaneous and Fas-induced apoptosis. This transient loss of p38 MAPK activity was independent of the absence or presence of fetal calf serum in the cell culture medium.

To further confirm our observation of a transient inhibition of p38 MAPK activity during neutrophil apoptosis, we analyzed its activity by an in vitro kinase assay in which the p38 MAPK-induced phosphorylated form of activating transcription factor 2 (ATF2)_19–96 is detected by Western blot. The results were confirmed by this approach, which again revealed a transient and early inhibition of p38 MAPK activity (Fig. 1 , representative blot and filled circles in the diagram). Even more interesting was the observation that during the transient inhibition of p38 MAPK activity (at 60 min) there is a statistically significant (P<0.05) increase in caspase-3 activity (Fig. 1 , open triangles).

View larger version (24K): [in this window] [in a new window]   Figure 1. Correlation between p38 MAPK activity and DEVDase activity during anti-Fas Ab exposure. In all experiments, incubations were terminated by lysis of the cells and samples of incubated cells were taken for immunoprecipitation of p38 MAPK or determination of DEVDase (caspase-3) activity. The activity of p38 MAPK in the immunoprecipitates was determined by incubation with ATP and GST-ATF2 and subsequent Western blot analysis of phosphorylated ATF2 (n=7); DEVDase activity was determined by incubation with DEVD-AMC and analyzed as percent of free AMC (n=4).

Addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), a well-known survival factor for neutrophils, counteracted the transient inhibition of p38 MAPK activity. In parallel control experiments, we ascertained that the addition of GM-CSF reduced the number of condensed nuclei by more than half. These results suggest that the transient inhibition of p38 MAPK participates in the regulation of apoptosis of isolated human neutrophils.

2. Role of p38 MAPK in regulation of neutrophil apoptosis
A p38 MAPK inhibitor, SB203580, significantly increased the spontaneous and anti-Fas-induced percentage of phosphatidylserine (PS) -exposing cells. Due to the controversy regarding the effect of p38 MAPK on neutrophil apoptosis, we examined the effect of SB203580 on nuclear condensation and caspase activity. We found that SB203580 increased the percentage of condensed nuclei and the IETDase (primarily caspase-8) and DEVDase (primarily caspase-3) activities during both spontaneous and anti-Fas-induced apoptosis.

To further explore the possibility of p38 MAPK being a survival signal in neutrophils, we tested the activity of established survival signals present in other cell types. We found no activation of p21ras or p42/p44 MAPK in neutrophils undergoing spontaneous or anti-Fas Ab-induced apoptosis. The lack of p21ras activation does not exclude a role for Akt in neutrophil apoptosis since there are alternative ways by which Akt can be activated. In most cells, Akt functions as a survival signal. We could show by Western blot analysis that there was neither an initial nor late activation of Akt during either spontaneous or anti-Fas-mediated apoptosis. These observations suggest the presence of other survival mechanisms in these cells, and p38 MAPK activity is consequently proposed as such a signal.

3. Effects of the antagonistic ZB4 anti-Fas Ab on p38 MAPK activity and apoptosis
We found that preincubation with the ZB4 Ab had no effect on the transient inhibition of p38 MAPK activity during spontaneous and Fas-induced apoptosis. However, it impaired the Fas-induced increase in p38 MAPK activity after 4 h. In accordance with previous results, the Ab inhibited anti-Fas-induced apoptosis, but had no effect on spontaneous apoptosis. These data support the idea that spontaneous neutrophil apoptosis occurs independent of Fas.

4. Effects of caspase-3 and caspase-8 inhibition on p38 MAPK activity and neutrophil apoptosis
To further explore the interaction between p38 MAPK and caspases, we tested the effects of cell-permeable caspase inhibitors on the activity of p38 MAPK during Fas-induced neutrophil apoptosis. Both IETD-fmk and DEVD-fmk counteracted the transient inactivation of p38 MAPK during Fas-induced apoptosis. The relevance of using these inhibitors was documented in parallel experiments in which both IETD-fmk and DEVD-fmk partially protected neutrophils from anti-Fas-induced apoptosis.

5. Activation of PI 3-K during neutrophil apoptosis
Even though Akt is not activated during Fas-induced apoptosis, it does not exclude activation and a role for PI 3-K in the regulation of neutrophil apoptosis, since Akt is only one of its downstream targets. Indeed, we found a significant and rapid increase in PI 3-K activity, peaking around 30–60 min, during Fas-induced apoptosis (Fig. 2 A) but only a very low, if any, activity during spontaneous apoptosis (Fig. 2A ). The activation of PI 3-K was accompanied by a subsequent translocation of p85 PI 3-K to a membrane fraction where the enzyme remained even after its activity had declined (Fig. 2B ). Note that the peak in PI 3-K activity occurred at the same time the activity of p38 MAPK was transiently impaired (Fig. 1 ; for comparison, indicated as a dashed line in Fig. 2A ). The opposite behavior of PI 3-K and p38 MAPK activities was matched by their effects on neutrophil apoptosis (Fig. 2C, D ). We found that two different inhibitors of PI 3-K (wortmannin and LY294002) significantly inhibited Fas-mediated PS exposure and chromatin condensation. We investigated a possible interdependence between PI 3-K and p38 MAPK signaling. Neither wortmannin nor LY294002 had any effect on transient inhibition of p38 MAPK activity during Fas-induced apoptosis (Fig. 2E ). In reversed experiments, we found no statistically significant effects of SB203580 on Fas-induced PI 3-K activity (Fig. 2F ).

View larger version (37K): [in this window] [in a new window]   Figure 2. PI 3-K activity and effect of pharmacological inhibition of PI 3-K activity on anti-Fas Ab-induced apoptosis. A, B) Cells were incubated in the absence or presence of anti-Fas Ab. A) The incubations were terminated by lysis and immunoprecipitation of PI 3-K. The activity of PI 3-K in each precipitate was then immediately analyzed. The panel shows the mean values of the accumulated PI 3-K activity in the two situations. Open circles represent untreated cells and filled circles represent anti-Fas Ab treated cells. The broken line depicts the activity of p38 MAPK and is taken from Fig. 1 to enable a direct comparison of the two signals. B) Incubations were terminated by placing the cells on ice, after which the membrane proteins were analyzed by Western blot with an antibody against the p85 subunit of PI 3-K. The blots are representative of 6 separate experiments. C, D) Results obtained from cells incubated in the absence or presence of wortmannin (50 nM) or LY294002 (1 µM) for 30 min on ice and then at 37°C in the presence of anti-Fas Ab. C) Aliquots of cells were stained after 16 h with annexin V-FITC and analyzed by flow cytometry (n=9). D) Neutrophils were treated as in panel C but stained after 8 h with acridine orange to assess nuclear morphology. The results are presented as the percentage of cells with apoptotic (condensed) chromatin relative to that found in untreated cells (n=5). E) Representative Western blots of samples obtained from cells treated as in panels C, D. All blots were probed with an antibody against the active form of p38 MAPK (P-p38), then stripped and reanalyzed with an antibody against total p38 MAPK (p38). F) Results from cells incubated in the absence or presence 20 µM SB203580 for 30 min on ice and then at 37°C in the presence of the anti-Fas Ab. PI 3-K activity was determined after 0, 60, or 120 min as outlined in panel A (n=6). The SB203580 inhibitor had no statistically significant effects.

CONCLUSION

During our investigation of the role of p38 MAPK in neutrophil apoptosis, we made, by two independent methods, the novel observation of an early and significant decrease in the constitutive activity of this kinase. We also found that activation of caspase-3 (DEVDase activity) was initiated during reduced p38 MAPK activity and that the survival signaling cytokine GM-CSF counteracted the transient inhibition of p38 MAPK in isolated neutrophils. These findings suggest that p38 MAPK contributes to neutrophil survival and that the early Fas-independent drop in p38 MAPK activity during spontaneous and Fas-induced apoptosis constitutes a regulatory step in the initiation of neutrophil apoptosis. To further explore the possibility that p38 MAPK contributes to survival of human neutrophils, we repressed p38 MAPK activity by exposing the cells to SB203580, an inhibitor of p38 MAPK. We found that pharmacological inhibition of p38 MAPK activity augmented both spontaneous and Fas-mediated apoptosis, revealed by analyzing activation of caspase-8 (IETDase activity) and caspase-3 (DEVDase activity), increased chromatin condensation, and increased exposure of PS on the surface of the cells. We noted no activation of p21ras, p42/p44 MAPK, or Akt. This suggests that neutrophils have other survival mechanism, and we propose that activation of p38 MAPK is one such mechanism. This conclusion agrees well with recent findings in granulocytes from Fas-deficient mice. These findings are supported indirectly by functional studies of neutrophils showing that activation of p38 MAPK is also triggered by LPS and GM-CSF, which are known to counteract or delay apoptosis in these leukocytes.

In line with our conclusion that the transient inhibition of p38 MAPK activity favors neutrophil apoptosis, we noted a rapid Fas-induced activation of PI 3-K that peaked while p38 MAPK activity was low and most likely is responsible for the augmentation of neutrophil apoptosis that was elicited by Fas. Such an apoptosis-promoting effect of PI 3-K has been reported in other cell systems and is strengthened by our data showing the absence of Akt activation (a well-known PI 3-K-induced survival signal) during neutrophil apoptosis. Furthermore, the finding of low p38 MAPK activity concurrent with a peak in PI 3-K activity and the recent report that p38 MAPK in human neutrophils functions as PDK2, an enzyme necessary for PI 3-K-induced activation of Akt, readily explain why we did not observe activation of Akt despite a Fas-induced stimulation of PI 3-K. Consequently, we conclude that a rapid and transient inhibition of a p38 MAPK survival signal and simultaneous augmentation of a proapoptotic PI 3-K signal contribute to Fas-induced apoptosis in isolated neutrophils. By comparison, the more modest spontaneous apoptotic response is characterized by a transient decrease in p38 MAPK activity but the lack of a significant PI 3-K signal. These conclusions agree with our observation that the anti-Fas antagonist ZB4 antibody had no effect on inhibition of p38 MAPK activity.

We conclude that both spontaneous and Fas-induced apoptosis are favored by the transient decrease in the p38 MAPK survival signal, whereas the Fas-induced transient increase in PI 3-K activity is a proapoptotic signal in isolated neutrophils.

View larger version (14K): [in this window] [in a new window]   Figure 3. Schematic diagram describing intracellular signaling events in human neutrophils that participate in the regulation of anti-Fas-induced apoptosis in these cells. This overview is based on the present findings as well as those previously published by other investigators and does not exclude participation of other signaling pathways or complementary signals within the presented diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0817fje; to cite this article, use FASEB J. (November 29, 2001) 10.1096/fj.01-0817fje

² Present address: Division of Medical Microbiology, Faculty of Health Science, SE-581 85 Linköping, Sweden.

This Article Full Text (PDF) All Versions of this Article: 16/1/129 01-0817fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by ALVARADO-KRISTENSSON, M. Articles by ANDERSSON, T. Search for Related Content PubMed PubMed Citation Articles by ALVARADO-KRISTENSSON, M. Articles by ANDERSSON, T.