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Originally published online as doi:10.1189/jlb.0903422 on May 20, 2004

Published online before print May 20, 2004
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(Journal of Leukocyte Biology. 2004;76:477-483.)
© 2004 by Society for Leukocyte Biology

CP-64131, an aminobenzazepine with cytokine-like properties, stimulates human neutrophil functions through the p38-MAPK pathway

Marsha S. Anderson*,{dagger}, Cindy Knall{ddagger},1, Gail Thurman*, Don Mann§, Nancy Cusack§, Gary L. Johnson{ddagger},¶,2 and Daniel R. Ambruso*,{dagger},3

* Bonfils Blood Center, Denver, Colorado;
{ddagger} Program in Molecular Signal Transduction, Division of Basic Sciences, Department of Pediatrics, National Jewish Medical Research Center, Denver, Colorado; Departments of
Pharmacology and
{dagger} Pediatrics, University of Colorado Health Sciences Center, Denver; and
§ Central Research Division, Pfizer, Inc., Groton, Connecticut

3Correspondence: Bonfils Blood Center, 717 Yosemite Street, Denver, CO 80230. E-mail: daniel.ambruso{at}uchsc.edu


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ABSTRACT
 
CP-64131 (CP), an aminobenzazepine with cytokine-like, physiologic effects similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF, increases the number of neutrophils and stimulates marrow recovery after doxirubicin ablation. CP can also function as a neutrophil agonist, like formyl-Met-leu-Phe (fMLP). In these studies, we show that CP is unique in that it stimulates the p38-mitogen-activated protein kinase (MAPK) pathway but not extracellular signal-regulated kinase (ERK)1/2 or c-jun N-terminal kinase MAPKs in human neutrophils from peripheral blood. This is in contrast to other neutrophil agonists such as fMLP, interleukin (IL)-8, or GM-CSF, which stimulate multiple MAPK pathways. Like fMLP and IL-8, CP is capable of stimulating superoxide (O2–) production, CD11b expression, and cell polarization in human neutrophils. CP-stimulated O2– production is completely dependent on p38-MAPK activation, as determined by sensitivity to the p38-MAPK inhibitor SB203580. In contrast, SB203580 only partially inhibits expression of CD11b and has no effect on cell polarization stimulated by CP. Therefore, CP treatment of neutrophils activates p38-MAPK but has effects independent of p38-MAPK activation. In human embryonic kidney 293 cells, a human kidney epithelial cell line CP stimulates p38-MAPK and modestly activates ERK1/2. The findings define CP as a novel, small molecule, which has little cellular toxicity in vitro. CP has the ability to activate specific MAPK pathways in different cell types and should prove to be an effective agonist in combination with inhibitors to study biological responses regulated by MAPKs.

Key Words: GM-CSF • fMLP • ERK1/2


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INTRODUCTION
 
Normal neutrophil function is dependent on the ability of the cell to perform certain activities such as shape changes (polarization), expression of adhesion molecules (CD11b/CD18), and the respiratory burst [1 ]. The coordinated cytoskeletal rearrangements reflected by cell polarization are essential for cell motility. Likewise, the expression of adhesion molecules is important for tight adhesion during diapedesis. Finally, the production of superoxide anion (O2–) is critical for bactericidal activity. Genetic defects in each of these processes are associated with clinically defined diseases [1 2 3 ].

A number of signaling mechanisms, including protein phosphorylation, changes in intracellular calcium, and mitogen-activated protein kinase (MAPK) recruitment, have been proposed to regulate these processes [4 ]. MAPKs are members of a group of enzymes that form highly conserved, tiered kinase cascades that are present in virtually all cell types [5 ]. Each enzyme phosphorylates and activates the next member of the cascade. These cascades can be activated by G protein-coupled receptors and protein tyrosine kinase receptors. The MAPKs, extracellular signal-regulated kinase (ERK) and p38-MAPK, are activated in neutrophils by various agonists [6 7 8 9 10 11 ]. It is interesting that c-jun N-terminal kinase (JNK) remains quiescent in nonadherent neutrophils [8 ] but is activated in adherent neutrophils [12 ].

The correlation of individual MAPKs to the regulation of specific neutrophil functions is being established currently. In the case of O2– production, ERK and p38-MAPK have been implicated in the regulation of the oxidase [11 , 13 , 14 ]. However, whether only p38-MAPK or ERK activation is sufficient to produce O2– remains controversial. As many neutrophil agonists, such as interleukin (IL)-8 and formyl-Met-leu-Phe (fMLP), activate ERK and p38-MAPK [6 7 8 ], it is hard to determine the contribution of each individual kinase/pathway to the overall effect.

Recently, a compound was developed that has unique biochemical and biological properties. CP-64131 (CP) is a low molecular weight aminobenzazepine whose cytokine-like effects are similar to granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage (GM)-CSF in animal models [15 , 16 ]. It increases total white blood cell number, expands the neutrophil population, and stimulates marrow recovery after doxirubicin ablation. CP also induces tyrosine phosphorylation of p38-MAPK in human neutrophils [15 ]. As a result of these preliminary studies, we set out to define the effects of this compound on human neutrophils. Specifically, we studied the effects of CP on MAPK activation, the respiratory burst, up-regulation of CD11b, and neutrophil polarization.


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MATERIALS AND METHODS
 
Preparation of neutrophils
Endotoxin-free reagents and plastics were used in all isolations. Human neutrophils used in the kinase assays and polarization assay were isolated from peripheral blood of healthy donors obtained by venipuncture using dextran sedimentation followed by Percoll gradient centrifugation as described previously [7 ]. For the kinase assays, neutrophils were suspended in Krebs-Ringer phosphate buffer containing dextrose (KRPD), containing 0.25% human serum albumin (HSA), 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 5 µg/ml leupeptin at 2 x 107 cell/ml, and were incubated for 30 min at 37°C before the various kinase assays. Human neutrophils used in the O2– and CD11b assays were isolated from peripheral blood of healthy donors obtained by venipuncture using dextran sedimentation, Ficoll-Hypaque gradient centrifugation, and hypotonic lysis of red blood cells, as described previously [17 ]. For O2– assays, neutrophils were suspended in KRPD at 2.5 x 107 cells/ml and used within 60 min of isolation.

Human embryonic kidney (HEK)293 cells
HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum as described previously [18 ].

ERK assay in neutrophils
ERK was assayed as described previously [7 ]. Neutrophils (3x107) were stimulated with buffer only, CP (4 µM), tumor necrosis factor {alpha} (TNF-{alpha}; 10 ng/ml), fMLP (25 nM), or phorbol 12-myristate 13-acetate (PMA; 200 ng/ml) for the indicated times. ERK was purified by immunoprecipitation, and its activity was measured in an in vitro kinase assay using myelin basic protein as substrate. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), substrate-incorporated, radioactive phosphate was quantified by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).

ERK assay in HEK293 cells
Human epithelial-derived HEK293 cells were grown to 90% confluence and serum-starved in DMEM containing 0.1% bovine serum albumin overnight before stimulation. Cells were stimulated with CP (4 µM) or epidermal growth factor (EGF; 30 ng/ml) for the indicated times. Cell lysates were normalized for protein content, and the assay was then performed as described above.

p38-MAPK assay in neutrophils
The p38-MAPK assay was performed as described previously [11 ]. Briefly, neutrophils (4x107) were stimulated with CP, fMLP, or TNF-{alpha} at the concentrations noted above for the indicated times. p38-MAPK was purified by immunoprecipitation, and its activity was measured in an in vitro kinase assay using activating transcription factor-21–100 as substrate. Following SDS-PAGE, substrate-incorporated, radioactive phosphate was quantified by PhosphorImager analysis.

p38-MAPK assay in HEK293 cells
HEK293 cells were prepared as described for the ERK assay. Cells were stimulated with fMLP, TNF-{alpha}, or UV irradiation for the indicated times. The assay was performed as described above.

JNK assay
The JNK assay was performed as described previously [19 ]. Briefly, HEK293 cells were prepared as described for the ERK assay. The cells were stimulated with CP, TNF-{alpha}, or UV irradiation for the indicated times. Following stimulation, JNK was purified using glutathione S-transferase c-jun beads, and JNK activity was measured using an in vitro kinase reaction with c-jun as substrate. Following SDS-PAGE, substrate-incorporated, radioactive phosphate was quantified by PhosphorImager analysis.

Respiratory burst assay
Polymorphonuclear neutrophils (PMNs; 2.5x106/ml) were incubated with buffer or CP (4 µM) for 15 min at 37°C. Then buffer, PMA (200 ng/ml), or fMLP (1 µM) was added, and the rate of O2– production was assessed. The maximal initial rate (over 5 min) of O2– production was measured using a standard superoxide dismutase (SOD)-inhibited cytochrome c reduction assay as described previously [2 , 17 ].

Neutrophil expression of CD11b
CD11b expression was determined as described previously [20 ]. Briefly, neutrophils (1x106) were incubated with dimethyl sulfoxide (DMSO) or SB203580 (10 µM) for 30 min at 37°C before stimulation. Neutrophils (1x106/ml) were then incubated with buffer alone, fMLP (25 nM), or CP (4 µM) for the indicated times and stained with phycoerythrin-labeled mouse anti-human CD11b antibody (Becton Dickinson, San Jose, CA), and fluorescence staining was quantified by flow cytometry.

Polarization assay
Neutrophils (3x106) were pretreated with DMSO or SB203580 (10 µM) for 30 min at 37°C before stimulation. Neutrophils were stimulated with buffer alone, CP (4 µM), or fMLP (25 nM) and were spotted immediately onto coverslips (precoated with 5% fetal calf serum for 2 h and washed in KRPD/HSA). Samples were incubated for 30 min at 37°C in a humidified CO2 incubator. Samples were fixed and permeabilized for 10 min at room temperature with 3.7% formalin containing 0.1 mg/ml lysophosphatidylcholine. The coverslips were gently washed once with phosphate-buffered saline (PBS), drained, spotted with PBS containing rhodamine-phalloidin (165 nM, Molecular Probes, Eugene, OR) and Hoechst 33342 (10 µg/ml), and incubated for 30 min at room temperature in the dark. The coverslips were washed with PBS, mounted in glycerol containing 100 mM Tris (pH 8), sealed with nail polish, and stored at –20°C until examined. The slides were imaged with a 100x objective using the Everest digital imaging microscopy system with SlideBook software (Intelligent Imaging Innovations, Denver, CO).

Time-lapse video microscopy
Neutrophils (3x106) were placed in a heated dish/stage apparatus (Bioptics, Beaver Falls, PA) and stimulated with buffer alone or CP (4 µM). Images were collected at 15 s intervals with a 40x water objective on the Everest system using SlideBook.

Statistical analysis
Statistical analysis of assays was completed using JMP statistical software. Paired and unpaired t-tests were used as appropriate.


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RESULTS
 
CP stimulation of MAPKs in human neutrophils
As CP was previously reported to have G-CSF/GM-CSF-like properties [15 , 16 ], the ability of CP to activate p38-MAPK, ERK1/ERK2, or JNK in human neutrophils was investigated. The magnitude of CP-induced p38-MAPK activation was equivalent to that stimulated by fMLP (Fig. 1A ). It is interesting that the time-course of activation was similar to that reported for TNF-{alpha} [9 ] with a maximal response at 10–15 min (Fig. 1B) . In contrast to GM-CSF [21 ], CP did not activate ERK (Fig. 1C and 1D) . Similarly, CP did not activate JNK (data not shown). We previously reported the absence of a JNK response in human neutrophils stimulated by various agonists [8 , 9 ]. Therefore, CP appears to be a selective activator of the p38-MAPK pathway in human neutrophils.



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  		Figure 1. CP stimulation of MAPKs in human neutrophils. (A) p38 activity in human PMNs stimulated with CP (4 µM), fMLP (25 nM), and TNF-{alpha} (10 ng/ml). Results are expressed as mean ± SEM, n = 8. (B) Time curve for CP-induced p38 activation of human PMNs, which were incubated with 4 µM CP for indicated times, and p38 activity was measured. Bars represent mean ± SEM for three to five experiments. (C) ERK activity of human PMNs incubated with various stimuli. PMNs were incubated with 4 µM CP (15 min), 25 nM fMLP (3 min), or buffer (15 min), and ERK activity was measured as in Materials and Methods. Bars represent mean ± SEM for three separate experiments performed in triplicate. (D) Time curve for ERK activity in human PMNs. Cells were incubated for the indicated times with the stimulus, and ERK activity assay was completed. Data are represented as mean ± SEM for four separate experiments. Asterisks denote statistical differences from resting, basal, or O-time activity: *, P < 0.05; **, P < 0.005; ***, P < 0.0005; unpaired t-test.

CP stimulation of MAPKs in HEK293 cells
To determine whether selective activation of p38-MAPK by CP was restricted to neutrophils, human HEK293 cells were stimulated with CP, EGF, TNF, UV, or buffer alone, and p38-MAPK, ERK, and JNK activities were measured. Similar to neutrophils, p38-MAPK was activated in CP-stimulated HEK293 cells to levels comparable with those seen with TNF-{alpha} (Fig. 2A ). In contrast to neutrophils, CP stimulated ERK activity in HEK293 cells, albeit modestly relative to that observed with EGF (Fig. 2B) . This activity was maximal at 5 min and returned to baseline levels within 30 min (Fig. 2D) . Like neutrophils, no JNK activation was observed in HEK293 cells stimulated with CP, although TNF-{alpha} and UV irradiation stimulated strong JNK activation (Fig. 2C and 2E) . These data suggest that CP, although preferentially activating p38-MAPK, can activate ERK in some cell types.



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  		Figure 2. CP stimulation of MAPKs in HEK293 cells. (A) p38 activity. HEK293 cells were incubated with 4 µM CP (15 min) and 10 ng/ml TNF-{alpha} (10 min), and activity assays were completed as in Materials and Methods. The data are presented as mean ± SEM for three separate experiments. (B) ERK activity. HEK293 cells were stimulated with 4 µM CP (15 min) or 30 ng/ml EGF (10 min), and the ERK activity was completed as described in Materials and Methods. Results are represented as mean ± SEM for three separate experiments. (C) JNK activity. HEK293 cells were incubated with stimuli in A, and JNK activity was measured as described in Materials and Methods. (D) ERK activity time-course. HEK293 cells were stimulated with 4 µM CP (•) or 30 ng/ml EGF ({blacksquare}) for the indicated times, and the ERK activity was completed as described in Materials and Methods. Results are represented as mean ± SEM for three separate experiments. (E) JNK activity time-course. HEK293 cells were incubated with 4 µM CP (•) and 10 ng/ml TNF-{alpha} ({blacksquare}) for the indicated times, and JNK activity was measured as described in Materials and Methods. Results are represented as mean ± SEM for three separate experiments.

CP stimulates the production of O2– in neutrophils
Because of its biological effects in animals [15 , 16 ] and activity on signaling pathways noted above, we investigated the ability of CP to stimulate or enhance the neutrophil respiratory burst. CP itself activated the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase with the rate of O2– production approximately sixfold higher than buffer-treated cells (P<0.0005; Fig. 3A ). CP and fMLP synergized to give a nearly 11-fold stimulation of O2– from neutrophils compared with fMLP alone (P<0.0005). O2–in neutrophils stimulated with CP plus fMLP was 12.47 ± 1.22 nmol/min (mean±SEM) versus 1.15 ± 0.66 nmol/min (mean±SEM) in fMLP only-treated cells (Fig. 3A) . CP plus PMA also synergized to stimulate O2– production but not to the same magnitude as fMLP (Fig. 3A) . The effects of CP alone on the oxidase were time-dependent. Figure 3B presents SOD-inhibitable cytochrome c reduction as MOD units over time after addition to neutrophils in the assay mix. Cytochrome c reduction progresses at a linear rate after 10 min. Enhancement of the response to fMLP or PMA was optimal after a 10- to 15-min preincubation with CP (data not shown). In addition, O2– production by CP alone was not associated with any changes in cytosolic calcium (data not shown).



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  		Figure 3. Effects of CP on O2– production by PMNs. (A) Effect of CP on the respiratory burst. PMNs were preincubated for 15 min with buffer or CP (4 µM), then buffer, PMA (200 ng/ml), or fMLP (1 µM) was added, and the rate of O2– production was assessed. Numbers are mean ± SEM for three separate experiments. ***, P < 0.0005. (B) Mean optical density (MOD) at 550 nM over time representing SOD-inhibitable cytochrome c reduction over time after addition of CP (4 µM) to neutrophils. Data are representative of five separate experiments. After 10 min, a linear rate of cytochrome c reduction was seen. (C) Inhibition of CP induced O2– production. PMNs were pretreated with buffer or SB203580 at various concentrations, 4 µM CP was added and incubated for 10 min, and O2– production was assessed. Numbers represent mean ± SEM for three separate experiments. (D) Inhibition of CP induced enhancement of the fMLP response by SB203580. Cells were preincubated with buffer or various concentrations of SB203580, 4 µM CP was added for 10 min, fMLP (1 µM) was then added, and O2– production assessed. Numbers represent mean ± SEM for three separate experiments. Asterisks denote statistical difference from resting activity: *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

CP stimulation of O2– in neutrophils requires p38-MAPK activity
As selective activation of p38-MAPK by CP (as opposed to ERK or JNK) was seen, we investigated p38-MAPK regulation of the production of O2– in neutrophils stimulated with CP. In three separate experiments, the specific p38-MAPK inhibitor SB203580 [22 ] completely inhibited CP-induced activation of the oxidase (Fig. 3C) . SB203580 also completely inhibited the production of O2– by CP plus fMLP (Fig. 3D) . In contrast, the MAPK and ERK (MEK) inhibitor PD98059 [23 ], which blocks ERK activation, had no effect on the CP or CP plus fMLP-induced O2– production (data not shown). The effect seen here with SB203580 is similar to that recently reported by Dang et al. [24 ] for fMLP- or PMA-stimulated neutrophils. These data taken together suggest that CP, fMLP, and PMA regulate O2– production through p38-MAPK. It is interesting that our data suggest that the activation of p38-MAPK by CP alone, CP/fMLP, or CP/PMA is sufficient to activate the oxidase without activation of ERK. This is in contrast to the observation of Dang and co-workers [24 ] in which they reported the need for ERK and p38-MAPK phosphorylation of p67Phox.

CP-induced expression of CD11b requires p38-MAPK activity
As CP activated the NADPH oxidase system, we evaluated whether CP could increase expression of CD11b, another critical neutrophil function, and whether this was dependent on p38-MAPK activation. Like O2– production, CP stimulated an approximately fivefold increase in CD11b expression compared with control (P<0.005; Fig. 4 ). However, unlike CP-stimulated O2– production, CP-stimulated up-regulation of CD11b was significantly but only partially inhibited by the p38-MAPK inhibitor SB203580 (P<0.05; Fig. 4 ). Similar results were observed for fMLP. These data suggest that CP, although activating p38-MAPK, can also activate other signal transduction molecules that lay up-stream of or parallel to p38-MAPK.



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  		Figure 4. CP induced expression of CD11b. PMNs were preincubated for 30 min with buffer or SB203580 (10 µg/ml). Expression of PMN CD11b was measured after a 15-min incubation with 4 µM CP or 25 nM fMLP. Results are represented as mean ± SEM of mean channel fluorescence for four separate experiments.

CP-induced neutrophil polarization does not require p38-MAPK activity
As many neutrophil stimuli trigger not only O2– production and CD11b up-regulation but also morphological changes, we determined the ability of CP to induce neutrophil polarization. CP induced the polarization of primary human neutrophils including the formation of lamellipodia and filipodia, as demonstrated by F-actin staining (Fig. 5A ) and time-lapse video microscopy (Fig. 5B) . It is interesting that the induction of neutrophil polarization was not sensitive to the p38-MAPK inhibitor SB203580 (Fig. 5A) , suggesting that CP is acting on a signal-transduction molecule that can regulate p38-MAPK and cell morphology.



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  		Figure 5. Neutrophil polarization stimulated by CP. (A) Neutrophils were pretreated with DMSO or SB203580 (SB; 10 µM) for 30 min at 37°C before stimulation with buffer or CP (4 µM) for 30 min at 37°C. Actin was visualized with rhodamine-phalloidin (red), and nuclei were visualized with Hoechst 33342 (blue). Digital images were collected at 0.3 µm intervals along the z-axis of the neutrophil using a 100x objective on an Everest imaging system with SlideBook software, and deconvolution was performed. Images shown are three-dimensional reconstructions and are representative of three independent experiments performed with neutrophils isolated from three different donors. (B) Neutrophils were placed in a heated dish/stage apparatus and stimulated with CP (4 µM). Images were collected at 15 s intervals using a 40x water objective. Interval numbers are shown in the upper right-hand corner with the same neutrophil in all images designated by the arrow.


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DISCUSSION
 
In the current study, we have demonstrated the ability of CP, an aminobenzazepine with cytokine-like effects [15 , 16 ], to activate specific MAPKs in human neutrophils and HEK293 cells. CP selectively activated p38-MAPK in human neutrophils. This is in contrast to other previously characterized neutrophil agonists, such as fMLP or IL-8, which activate p38-MAPK and ERK [25 ]. It is interesting that CP was also able to activate p38-MAPK in HEK293 cells, which are of human epithelial origin [26 ]. However, CP activation of MAPKs was not restricted to p38-MAPK in HEK293 cells, as CP was able to modestly activate ERK in these cells.

The molecular targets for CP are not currently defined. One set of targets might be at the level of the MAPK kinases (MKK3 or MKK6), which directly activate p38-MAPK but do not activate other MAPKs [5 ]. In HEK293 cells, there would have to be some activation of MKK1 or -2 that allowed the modest activation of the ERK pathway. An alternate point for CP stimulation of the p38-MAPK pathway would be at the level of the low molecular weight GTPases Rac and/or Cdc42, which regulate p38-MAPK activation [5 ]. GTPases seem a more likely target than MKKs, as CP stimulates neutrophil polarization, a Rac/Cdc42-regulated response, independent of p38-MAPK activation. Recently, fMLP was shown to activate Rac2 and Cdc42 in neutrophils [27 ]. Although Rac1 and Cdc42, in other cell types, can regulate JNK activation and associate with MEK kinase 1 (MEKK1), which can activate the ERK pathway, activation of JNK [8 ] and MEKK1 (C. Knall, unpublished observation) is not detected in nonadherent human neutrophils. Therefore, activation of Rac and/or Cdc42 by CP would effectively result in a selective activation of p38-MAPK in human neutrophils.

The selective action of CP on MAPK signaling and neutrophil function suggests that CP, in combination with p38-MAPK inhibitors, may be a valuable tool for investigating the regulation of biological responses downstream of and parallel to p38-MAPK. The ability of specific MAPKs to regulate neutrophil functions such as O2– production, CD11b expression, and neutrophil polarization has remained unclear. For example, ERK and p38-MAPK activities have been implicated in the regulation of O2– production [11 , 13 , 14 ]. Our data now demonstrate that activation of p38-MAPK, as opposed to any other MAPKs, is necessary and sufficient to regulate the production of O2– in neutrophils. p38-MAPK was previously shown to phosphorylate p47-phox, a critical component of the oxidase [14 ]. Also, p38-MAPK is required for cytosolic phospholipase A2 (cPLA2) activation in neutrophils stimulated by TNF-{alpha} [28 ], and arachidonic acid, the product of cPLA2 activity, is required for the activation of the oxidase [29 ]. Therefore, p38-MAPK can regulate the oxidase through two distinct mechanisms.

In contrast to the regulation of O2– production, the expression of CD11b by CP or fMLP was only partially (50%) blocked by inhibiting p38-MAPK activation. This suggests that there are two distinct pathways, one p38-MAPK-dependent and the other p38-MAPK independent, which can regulate the increased expression of CD11b on the neutrophil cell surface. We have recently determined that functional Rac2 is not required for the expression of CD11b on neutrophils stimulated by fMLP or platelet-activating factor [2 ]. This observation together with our current data indicate that the CP- and fMLP-activated, p38-MAPK-dependent, pathway-stimulating, increased CD11b expression is not regulated by Rac2.

As discussed above, Cdc42 can also regulate the activation of p38-MAPK [5 ] and the oxidase [30 ]. Therefore, CP may be activating the p38-MAPK pathway through Cdc42 itself or through one of its effector molecules such as p21-activated kinase (PAK), which can regulate p38-MAPK and the oxidase [31 ] (Fig. 6 ). Our observation that CP induces neutrophil polarization and significant filipodia, which are regulated by Cdc42, supports the hypothesis that CP is exerting its effects at least in part through Cdc42 or a Cdc42 effector molecule. Furthermore, the fact that CP is able to induce polarization and filipodia formation in the presence of a p38-MAPK inhibitor supports the idea that CP targets are upstream of p38-MAPK. CP will be an invaluable small molecule agonist to study cell-shape change and MAPK regulation.



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  		Figure 6. Proposed model for CP regulation of neutrophil function. CP is an agonist at the level of Cdc42/PAK, resulting in p38-MAPK-independent regulation of filipodia formation but p38-MAPK-dependent regulation of O2– production and CD11b expression.


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ACKNOWLEDGEMENTS
 
This work was supported in part by The Children’s Hospital Research Institute (M. S. A.), National Institutes of Health HL09640 (C. K.) and GM30324 (G. L. J.), the Bonfils Blood Center (D. R. A.), the Stacy Marie True Memorial Trust, and the Central Research Division, Pfizer, Inc. C. K. was The Helen Wohlberg and Herman Lambert Fellow in Cancer Biology. M. S. A. and C. K. contributed equally to this work.


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FOOTNOTES
 
1 Current address: Immunology Program, Lovelace Respiratory Research Institute, Albuquerque, NM 87108. Back

2 Current address: Department of Pharmacology, CB#7365, 1108 Mary Ellen Jones Building, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7365. Back

Received September 11, 2003; revised April 8, 2004; accepted April 13, 2004.


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