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Institut National de la Santé et de la Recherche Médicale, Unité 479, Centre Hospitalier Universitaire Xavier Bichat, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2) in a phenomenon known as
the respiratory burst. O2 is the precursor of other reactive
oxygen species, which are essential for bacterial killing and amplify
inflammatory reactions (1). Neutrophil production of
O2 is dependent on the respiratory burst oxidase, or NADPH
oxidase, a multicomponent enzyme system that catalyzes NADPH-dependent
reduction of oxygen to O2 (2, 3). NADPH oxidase
is activated by a variety of agents, including fMLP, which is
representative of bacteria-derived N-formyl peptides and the
protein kinase C (PKC)3
activator PMA. These stimuli trigger biochemical cascades leading to
the phosphorylation of several proteins of the NADPH-oxidase system,
such as p47 phagocyte oxidase (p47phox), a key component in
the assembly and activation of this O2-producing machinery
(4, 5, 6). In addition to the well-documented PKC pathway,
one of these cascades involves activation of members of the
mitogen-activated protein kinase (MAPK) family. Although many authors
have implicated PKC in p47phox phosphorylation
(7, 8, 9, 10, 11), no data are available on the possible involvement
of the MAPK pathway in this process. MAPK enzymes are proline-directed serine/threonine kinases activated by phosphorylation of tyrosine and threonine residues. The MAPK superfamily is believed to be an important signaling pathway in many cell types (12, 13). Although the role of MAP kinases in proliferation and differentiation is well established, their roles and targets in terminally differentiated nonproliferating cells such as neutrophils are not known. Several recent studies have demonstrated that MAPK pathways such as extracellular signal-regulated kinases (ERK)1/2 and p38 MAPK, but not c-Jun N-terminal kinase, are activated in human neutrophils by fMLP, PMA, GM-CSF, and TNF (14, 15, 16, 17, 18, 19), but the targets of these kinases remain to be identified.
The NADPH oxidase component p47phox is phosphorylated on several serine residues, two of which are serines recognized by proline-directed kinases (20, 21). p47phox is an in vitro substrate for activated MAPK (ERK and p38 kinases), PKC, PKA, p21-activated kinase, and a phosphatidic acid-activated kinase (16, 20, 22, 23), but which of these enzymes phosphorylate p47phox in vivo, and at which site, is unknown. Several studies have shown that inhibitors of the ERK pathway and p38 MAPK inhibit the respiratory burst (14, 15, 24, 25), but the mechanisms underlying this inhibitory effect and the identity of the MAPK involved in p47phox phosphorylation in intact cells are unknown.
Myeloid cells such as neutrophils are terminally differentiated short-lived cells resistant to transfection. An alternative strategy to study the role of specific enzymes is to use cell-permeant pharmacologic inhibitors. In this study we used PD98059 and U0126, which are MEK1/2 inhibitors (26, 27), and SB203580, the p38 MAPK inhibitor (28), to analyze the role of these two MAPK pathways in p47phox phosphorylation in intact neutrophils.
We found that only the inhibitors of the ERK1/2 pathway inhibited p47phox phosphorylation in human neutrophils. In addition, we obtained evidence that the PKC and ERK1/2 pathways cooperate in p47phox phosphorylation triggered by fMLP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fMLP, PMA, and lucigenin were obtained from Sigma (St. Louis, MO); SDS-PAGE reagents were purchased from Bio-Rad (Richmond, CA); SB203580, PD98059, U0126, and GF109203X were obtained from Calbiochem (La Jolla, CA); 32Pi was obtained from NEN (Boston, MA); anti-p47phox Ab was a generous gift from Dr. B. M. Babior (The Scripps Research Institute, Division of Biochemistry, La Jolla, CA). Anti-active phosphorylated ERK1/2 Abs were purchased from New England Biolabs (Beverly, MA) and Promega (Madison, WI). Anti-ERK1 and anti-ERK2 Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human heat shock protein 27 (hsp27) was purchased from StressGen Biotechnologies (Victoria, Canada).
Neutrophil preparation
Human neutrophils were obtained from healthy volunteers by Dextran sedimentation and Ficoll-Hypaque fractionation of freshly drawn blood, as previously described (28).
32P-labeling of neutrophils
Neutrophils were resuspended at a density of 108 cells/ml in phosphate-free buffer (20 mM HEPES pH 7.4, 140 mM NaCl, 5.7 mM KCl, 0.8 mM MgCl2, and 0.025% BSA), treated with diisopropylfluorophosphate (2.5 mM) for 15 min, and washed twice. Neutrophils were then loaded with 0.5 mCi/ml 32Pi for 1 h at 30°C (29) and incubated for 45 min with inhibitors or DMSO at 37°C. They were then activated with fMLP (0.1 µM) for 3 min or with PMA (500 ng/ml) for 6 min. Activation was stopped with ice-cold buffer, and cells were lysed as previously described (29). p47phox was immunoprecipitated with a specific Ab (1/200 dilution) complexed with Sepharose G beads (Pharmacia Biotech, Uppsala, Sweden). Proteins were analyzed by 10% SDS-PAGE, then blotted on a nitrocellulose membrane and detected by autoradiography.
Electrophoresis and immunoblotting
SDS-PAGE of samples and transfer to nitrocellulose were performed as previously described (28), using standard techniques (30, 31).
Two-dimensional tryptic phosphopeptide mapping
For each experiment, we used immunodetection techniques to ensure that the same quantities of p47phox were immunoprecipitated from each sample. The nitrocellulose area containing 32P-labeled p47phox was cut out and incubated for 30 min in polyvinyl pyrrolidone-40 solution for saturation, then washed with ammonium bicarbonate buffer before tryptic digestion (20, 21). Peptides were dried using a Speedvac concentrator, then washed and dried four times. Samples were resuspended in thin-layer electrophoresis buffer and electrophoresis was run at 1100 V for 25 min at 6°C. Chromatography was conducted as described elsewhere (20, 21). All the plates from the same experiment were analyzed by autoradiography for the same period of time.
Superoxide production assay
Neutrophils were resuspended in Hank’s buffer supplemented with 0.025% BSA. Cells (5 x 105) were incubated with DMSO or inhibitors for 45 min. Lucigenin (100 µM) was added and cells were stimulated with fMLP (0.1 µM) or PMA (100 ng/ml); chemiluminescence was then measured in a chemiluminometer (Autolumat LB953; Berthold, Bad-Wildbad, Germany) for 30 min at 37°C. fMLP- and PMA-induced chemiluminescence was inhibited by superoxide dismutase, indicating that the signal obtained in our conditions was mainly due to superoxide anion production.
Assay of MAPK activation
In the ERK1/2 activation assay, cell lysates were prepared as described above, analyzed by 9% SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were blocked in TBST + BSA 2.5% for 45 min, incubated overnight with anti-active phosphorylated ERK1/2 Abs (1/5000) at 4°C, washed, incubated with a secondary Ab (1/10,000) for 1 h, and detected with enhanced chemiluminescence. ERK1/2 proteins were identified with specific Abs.
For the p38 activation assay, we measured the activity of the
downstream target MAPK-activated protein kinase 2 by using hsp27 as a
specific substrate, as described by Zu et al. (19).
Briefly, neutrophils were lysed in lysis buffer (1% Triton X-100, 150
mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, and 0.2
mM PMSF), then 30 µl of the soluble fraction was added to 30 µl of
reaction buffer (40 mM HEPES pH 7.4, 20 mM MgCl2,
2 mM EGTA, 5 µM okadaic acid), 1 µg of recombinant hsp 27, and 50
µM ATP containing 1 µCi [
-32P]ATP. The
reaction mixture was incubated at 30°C for 15 min, and the reaction
was stopped by boiling in SDS-PAGE sample buffer. Phosphorylated hsp 27
was resolved by 13% SDS-PAGE and autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Phosphorylation of the cytosolic oxidase subunit
p47phox is a key event in NADPH oxidase activation. The
kinases mediating phosphorylation of p47phox in intact
neutrophils remain to be characterized. Therefore, we analyzed the
effect of the MAPK inhibitors on p47phox phosphorylation.
32P-loaded neutrophils were incubated for 45 min
with PD98059 (50 µM), an inhibitor of MEK1/2 (the upstream activator
of ERK1/2) or with SB203580 (10 µM), an inhibitor of p38 MAPK, then
stimulated with fMLP (10-7 M) or PMA (0.5
µg/ml). p47phox was immunoprecipitated with a specific Ab
and then analyzed by SDS-PAGE, Western blot, and autoradiography. The
results (Fig. 1
A,
32P-p47phox) showed that PD98059,
unlike SB203580, clearly inhibited p47phox phosphorylation
triggered by the chemotactic peptide fMLP (51.2 ± 6.9% of the
control, n = 5, p < 0.05 and
101.7 ± 0.3% of the control, n = 5, for PD98059
and SB203580, respectively, as determined by radioactivity counting).
However, the same concentration of PD98059 only weakly inhibited the
phosphorylation of p47phox when the latter was induced by
PMA, a direct PKC activator (Fig. 1B). Specific detection of
p47phox with an Ab (Fig. 1, p47phox) showed
that the same amount of p47phox was immunoprecipitated from
each sample. Fig. 2, A and
B, clearly shows that in the conditions used above, PD98059
inhibited the phosphorylation of ERK1/2 as measured with a
phosphospecific Ab; SB203580 inhibited the phosphorylation of hsp27, a
known substrate of MAPK-activated kinase 2 (a downstream target of p38
MAPK). In keeping with the literature, these concentrations of PD98059
and SB203580 inhibited superoxide production in fMLP-stimulated cells
(data not shown). Taken together, these results suggest that although
both ERK1/2 and p38 MAPK are involved in superoxide production, only
the ERK1/2 pathway participates in the phosphorylation of
p47phox.
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The results obtained above by the use of PD98059 strongly suggest
the involvement of ERK1/2 in fMLP-induced p47phox
phosphorylation. Because chemical compounds may affect cellular
functions unrelated to their intended targets, we sought to confirm our
observations by using a chemically unrelated inhibitor of MEK1/2,
namely U0126 (27). As shown in Fig. 3, the phosphorylation of
p47phox was effectively inhibited by incubation of
neutrophils for 30 min with 10 µM U0126 (61.8 ± 6.6% of the
control, n = 6, p < 0.01). Like
PD98059, U0126 also inhibited superoxide production of fMLP-activated
neutrophils (53.2 ± 5, 32.8 ± 5.3, and 21.4 ± 4.8%
of the control for 10, 25, and 50 µM U0126, respectively;
n = 3, p < 0.01). Under these
conditions U0126 completely blocks ERK1/2 activation (data not
shown).
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To link the inhibitory effect of PD98059 on p47phox
phosphorylation and ERK1/2 activation, we performed parallel
concentration-effect studies of these two processes. As shown in Fig. 4, the inhibitory effect of PD98059 on
fMLP-induced p47phox phosphorylation (Fig. 4A) was concentration dependent, running parallel to the
inhibitory effect on MAPK activation (Fig. 4B). This
inhibitory effect reached a plateau at a concentration of 50 µM
(48.8 ± 6.8% of the control, n = 6,
p < 0.01, determined by densitometric analysis). In
contrast, PD98059 weakly inhibited PMA-induced p47phox
phosphorylation (Fig. 4C) (20.2 ± 7.8% inhibition,
n = 5, p < 0.01) at concentrations
that completely inhibit ERK1/2 activation (Fig. 4D). These
results suggest that ERK1/2, directly or indirectly, play a major role
in p47phox phosphorylation in fMLP-activated neutrophils.
However, in PMA-activated neutrophils, although ERK1/2 was activated,
it appeared to have a minimal role in p47phox
phosphorylation.
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p47phox is phosphorylated on several serine residues,
which can be phosphorylated in vitro by PKC, PKA, and MAPKs (ERK1/2 and
p38). The peptides containing these serines can be analyzed by tryptic
phosphopeptide mapping. In vitro, MAPK phosphorylates only one peptide
but PKC phosphorylates several other peptides. The possibility that
PD98059 or SB203580 might inhibit the phosphorylation of one or more
individual sites in p47phox was studied by using
two-dimensional peptide mapping. Fig. 5
shows that when neutrophils were stimulated with PMA, phosphorylation
of p47phox occurred on several peptides; some major
peptides were strongly phosphorylated and a few minor peptides were
less phosphorylated. PD98059 inhibited the phosphorylation of three
minor peptides (dotted lines) and minimally affected the
phosphorylation of other major peptides. However, when fMLP was the
stimulus (Fig. 6), the phosphopeptides
were located in the same position on the map but were much less
phosphorylated, except for one peptide. PD98059 strongly inhibited the
phosphorylation of all the major peptides (dotted lines), including
those phosphorylated in PMA-stimulated conditions. As mentioned in
Materials and Methods, in each experiment we ensured by
immunodetection techniques that the same quantities of
p47phox were immunoprecipitated from each sample. In
addition, all the plates from the same experiment were analyzed by
autoradiography for the same period of time. These results suggest
that, in fMLP-stimulated cells, ERK1/2 plays an important role in
p47phox phosphorylation by regulating the phosphorylation
of all the major sites. Unlike PD98059, SB203580 had no effect on
PMA-induced phosphorylation, but had a moderate stimulatory effect on
fMLP-induced phosphorylation when analyzed by two-dimensional
phosphopeptide mapping (Figs. 5 and 6).
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The chemotactic peptide fMLP is known to activate several PKC
isoforms that participate in the phosphorylation of
p47phox. We postulated that the PKC system and ERK1/2
pathway could converge to phosphorylate p47phox. Therefore,
we analyzed the effect of PKC and MAPK inhibitors alone, and in
combination, on the phosphorylation of p47phox. Fig. 7A shows that when PD98059 (50
µM) and GF109203X (5 µM) were added alone, they partially inhibited
fMLP-induced p47phox phosphorylation (59.3 ± 7.4 and
67.2 ± 1.2% of the control, respectively), and that their
combination resulted in a clearly additive inhibitory effect
(lanes 3 and 4 compared with lane
5) (35.5 ± 2.2% of the control). In keeping with the above
results, PMA-induced p47phox phosphorylation was more
sensitive to GF109203X (Fig. 7C), with only a weak
additional effect of the MEK inhibitor PD98059 (24.4 ± 14.4,
108.5 ± 17.7, and 21.3 ± 1.9% of the control for
GF109203X, PD98059, and GF109203X + PD98059, respectively). The MAPK
activation blots clearly show that GF109203X does not inhibit ERK1/2
activation in fMLP-activated cells (Fig. 7B) while
inhibiting this process when it was induced with PMA, thus explaining
the strong effect of GF109203X on PMA-induced p47phox
phosphorylation (Fig. 7D). The two-dimensional peptide map
in these conditions showed that all of the sites were inhibited (data
not shown). Taken together, these results suggest that after PMA
stimulation, PKC plays the major role in p47phox
phosphorylation, whereas in more physiological conditions (fMLP
stimulation), ERK1/2 and PKC participate together, directly or
indirectly, in the phosphorylation of p47phox.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under physiological conditions, represented here by fMLP stimulation of human neutrophils, several kinase pathways (PKC, p21-activated kinase, MAPK) are activated via the serpentine receptor. However, the kinases involved in p47phox phosphorylation have not yet been identified, although it has been reported that PKC (7, 8, 9, 10, 11) and some PKC isoforms may be involved in the process (32, 33, 34). Here we provide clear evidence that ERK1/2 are implicated in the phosphorylation cascade of p47phox in intact cells. The data also suggest cooperation between PKC and ERK1/2 in this process. It is noteworthy that p47phox is phosphorylated on multiple serine residues that could be targeted by different kinases, thus explaining the tight control of NADPH oxidase.
Two-dimensional phosphopeptide mapping analysis of p47phox isolated from EBV-transformed B lymphocytes and human neutrophils shows that the same peptides are phosphorylated in both cells. The use of site-directed mutagenesis of p47phox in B lymphocytes revealed that serines 303–370 are phosphorylated and that each peptide on the map contains one or several phosphorylated serines (21). The results reported here show that in the case of fMLP, PD98059 clearly inhibits the peptides containing serines 303 + 304, 315, 320, and 345 + 348. ERK1/2 phosphorylate p47phox in vitro on only one peptide containing serines located in the MAPK recognition sequence, i.e., 345 + 348 (16, 21). As the other p47phox serines have not been identified as ERK substrates (21), the observation that PD98059 inhibits all the major phosphopeptides in fMLP-stimulated cells suggests that ERK1/2 regulates non-MAPK sites by an unknown mechanism. One possibility is that p47phox phosphorylation by ERK1/2 induces conformational changes that make the other sites more accessible to their protein kinases. Indeed, it was recently shown that p47phox phosphorylation in vitro induces conformational changes of the protein (35, 36). The other possibility is that ERK1/2 phosphorylate p47phox on their specific serines and that, in addition, they may be upstream regulators of other kinases such as MAPK-activated protein kinase which, in turn, could phosphorylate p47phox. A hierarchical organization of p47phox phosphorylation has previously been suggested (37, 38, 39). In addition, we recently demonstrated that an early phosphorylation step took place under conditions of priming by proinflammatory cytokines such as GM-CSF (40). Whether or not MAPK are involved in this process is currently under investigation.
The results obtained here also confirm that PKCs play a predominant role in p47phox phosphorylation in PMA-activated cells, and suggest that PKCs could simultaneously activate the ERK1/2 pathway, which might also contribute to p47phox phosphorylation. However, the role of ERK1/2 MAPK seems to be minor in PMA activation conditions, as PD98059 only slightly inhibited p47phox phosphorylation and the respiratory burst (data not shown). This is in keeping with previous results showing that mutation of p47phox at Ser345 and Ser348 (sites within MAPK recognition sequence) moderately decreases the respiratory burst in transfected B lymphoblasts stimulated with PMA (41).
Unlike PD98059, the p38 inhibitor SB203580 did not decrease
p47phox phosphorylation induced by PMA or fMLP, while
nonetheless inhibiting the respiratory burst (Refs. 14, 19
and results not shown). This result is in agreement with a recent
publication (42) that shows that SB203580 was without
effect on p47phox phosphorylation. p38 MAPK could affect
other events necessary for NADPH activation such as the phosphorylation
of other oxidase components or proteins involved in NADPH oxidase
activation, such as cytoskeleton reorganization or phospholipase
A2 activity. It has been reported that MAPK-activated
kinase phosphorylates hsp27 (43) and lymphocyte-specific
protein 1 (44), which are both involved in actin
polymerization (44, 45). Fig. 8 illustrates a summary of the different
pathways possibly activated by fMLP or PMA leading to
p47phox phosphorylation and NADPH oxidase activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jamel El-Benna, Institut National de la Santé et de la Recherche Médicale, Unité 479, Center Hospitalier Universitaire Xavier Bichat, 16 rue Henri Huchard, 75877 Paris Cedex 18, France.
3 Abbreviations used in this paper: PKC, protein kinase C; MAPK, mitogen-activated protein kinase; p47phox, p47 phagocyte oxidase; ERK, extracellular signal-regulated protein kinase; hsp27, heat shock protein 27.
Received for publication January 28, 2000. Accepted for publication August 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and GM-CSF. J. Leukocyte Biol. 64:537.[Abstract]
or FMLP stimulation. J. Immunol. 160:1982.
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2 generation but not degranulation or adherence in differentiated HL60 cells. J. Biol. Chem. 273:27292.This article has been cited by other articles:
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C. Dewas, P. M.-C. Dang, M.-A. Gougerot-Pocidalo, and J. El-Benna TNF-{alpha} Induces Phosphorylation of p47phox in Human Neutrophils: Partial Phosphorylation of p47phox Is a Common Event of Priming of Human Neutrophils by TNF-{alpha} and Granulocyte-Macrophage Colony-Stimulating Factor J. Immunol., October 15, 2003; 171(8): 4392 - 4398. [Abstract] [Full Text] [PDF]
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N. Moghaddami, M. Costabile, P. K. Grover, H. P. A. Jersmann, Z. H. Huang, C. S. T. Hii, and A. Ferrante Unique Effect of Arachidonic Acid on Human Neutrophil TNF Receptor Expression: Up-Regulation Involving Protein Kinase C, Extracellular Signal-Regulated Kinase, and Phospholipase A2 J. Immunol., September 1, 2003; 171(5): 2616 - 2624. [Abstract] [Full Text] [PDF]
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 M. Schafer, C. Schafer, N. Ewald, H.M. Piper, and Th. Noll Role of Redox Signaling in the Autonomous Proliferative Response of Endothelial Cells to Hypoxia Circ. Res., May 16, 2003; 92(9): 1010 - 1015. [Abstract] [Full Text] [PDF]
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Q. Chen, D. W. Powell, M. J. Rane, S. Singh, W. Butt, J. B. Klein, and K. R. McLeish Akt Phosphorylates p47phox and Mediates Respiratory Burst Activity in Human Neutrophils J. Immunol., May 15, 2003; 170(10): 5302 - 5308. [Abstract] [Full Text] [PDF]
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L. K. Parvathenani, S. Tertyshnikova, C. R. Greco, S. B. Roberts, B. Robertson, and R. Posmantur P2X7 Mediates Superoxide Production in Primary Microglia and Is Up-regulated in a Transgenic Mouse Model of Alzheimer's Disease J. Biol. Chem., April 4, 2003; 278(15): 13309 - 13317. [Abstract] [Full Text] [PDF]
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 V. M. Lee, P. A. Quinn, S. C. Jennings, and L. L. Ng NADPH Oxidase Activity in Preeclampsia With Immortalized Lymphoblasts Used as Models Hypertension, April 1, 2003; 41(4): 925 - 931. [Abstract] [Full Text] [PDF]
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G. M. Fuhler, A. L. Drayer, and E. Vellenga Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia Blood, February 1, 2003; 101(3): 1172 - 1180. [Abstract] [Full Text] [PDF]
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A. C. MacKinnon, A. Buckley, E. R. Chilvers, A. G. Rossi, C. Haslett, and T. Sethi Sphingosine Kinase: A Point of Convergence in the Action of Diverse Neutrophil Priming Agents J. Immunol., December 1, 2002; 169(11): 6394 - 6400. [Abstract] [Full Text] [PDF]
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M. S. Huber-Lang, E. M. Younkin, J. V. Sarma, S. R. McGuire, K. T. Lu, R. F. Guo, V. A. Padgaonkar, J. T. Curnutte, R. Erickson, and P. A. Ward Complement-Induced Impairment of Innate Immunity During Sepsis J. Immunol., September 15, 2002; 169(6): 3223 - 3231. [Abstract] [Full Text] [PDF]
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 S. Du, B. McLaughlin, S. Pal, and E. Aizenman In Vitro Neurotoxicity of Methylisothiazolinone, a Commonly Used Industrial and Household Biocide, Proceeds via a Zinc and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase-Dependent Pathway J. Neurosci., September 1, 2002; 22(17): 7408 - 7416. [Abstract] [Full Text] [PDF]
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C. M. Rosenberger and B. B. Finlay Macrophages Inhibit Salmonella Typhimurium Replication through MEK/ERK Kinase and Phagocyte NADPH Oxidase Activities J. Biol. Chem., May 17, 2002; 277(21): 18753 - 18762. [Abstract] [Full Text] [PDF]
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C. Pilette, Y. Ouadrhiri, J. Van Snick, J.-C. Renauld, P. Staquet, J.-P. Vaerman, and Y. Sibille IL-9 Inhibits Oxidative Burst and TNF-{alpha} Release in Lipopolysaccharide-Stimulated Human Monocytes Through TGF-{beta} J. Immunol., April 15, 2002; 168(8): 4103 - 4111. [Abstract] [Full Text] [PDF]
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M. O. Price, L. C. McPhail, J. D. Lambeth, C.-H. Han, U. G. Knaus, and M. C. Dinauer Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system Blood, April 15, 2002; 99(8): 2653 - 2661. [Abstract] [Full Text] [PDF]
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P. J. Mansfield, V. Hinkovska-Galcheva, S. S. Carey, J. A. Shayman, and L. A. Boxer Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D Blood, February 15, 2002; 99(4): 1434 - 1441. [Abstract] [Full Text] [PDF]
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 M. J. Alcorn, J. L. Booth, K. M. Coggeshall, and J. P. Metcalf Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2 J. Virol., July 15, 2001; 75(14): 6450 - 6459. [Abstract] [Full Text]
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