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Maximal Human Neutrophil Priming for Superoxide Production and Elastase Release Requires p38 Mitogen-Activated Protein Kinase Activation
David A. Partrick, MD;
Ernest E. Moore, MD;
Patrick J. Offner, MD;
Daniel R. Meldrum, MD;
Douglas Y. Tamura, MD;
Jeffrey L. Johnson, MD;
Christopher C. Silliman, MD, PhD
Arch Surg. 2000;135:219-225.
ABSTRACT
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Hypothesis Neutrophil priming has been implicated in the development of multiple organ failure, although the precise intracellular mechanisms that regulate neutrophil priming remain unclear. Our previous work characterized platelet-activating factor (PAF) priming of human neutrophils for concordant superoxide anion (O2-) generation and elastase degranulation. The p38 mitogen-activated protein kinase (MAPK) is activated by PAF stimulation. We hypothesized that PAF-induced human neutrophil priming for O2- and elastase release is mediated via the p38 MAPK pathway.
Design Isolated neutrophils from 6 human donors were preincubated with the specific p38 MAPK inhibitor SB 203580 (1 µmol/L) or buffer (control) for 30 minutes. Cells were then primed with PAF (200 nmol/L), followed by receptor-dependent (N-formyl-methionyl-leucyl-phenylalanine, 1 µmol/L) or receptor-independent phorbol myristate acetate (PMA, 100 ng/mL) activation.
Setting Urban trauma research laboratory.
Patients Healthy volunteer donors of neutrophils.
Main Outcome Measures Maximal rate of O2- generation was measured by superoxide dismutase-inhibitable reduction of cytochrome c and elastase release by the cleavage of N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.
Results SB 203580 significantly attenuated the generation of O2- and release of elastase from neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine but not with PMA. Independent of the activator receptor status, SB 203580 almost completely blocked the exaggerated neutrophil cytotoxic response due to PAF priming.
Conclusions The p38 MAPK pathway is required for maximal PAF-induced neutrophil priming for O2- production and elastase degranulation. Therefore, the MAPK signaling cascade may offer a potential therapeutic strategy to preempt global neutrophil hyperactivity rather than attempt to nullify the end products independently.
INTRODUCTION
THE NEUTROPHIL is a pivotal cellular mediator in the pathogenesis of the adult respiratory distress syndrome (ARDS) and postinjury multiple organ failure (MOF). In the 2-event inflammatory model of organ injury,1-2 the first event is priming of the immune response. We focused on the neutrophil as a cellular surrogate of the systemic hyperinflammatory immune response. Priming of circulating neutrophils causes nonselective adherence to endothelium. The second event provokes activation of these adherent neutrophils, leading to the indiscriminate release of cytotoxic reactive oxygen metabolites and proteases into the neutrophil-endothelial cell microenvironment, which results in endothelial damage, capillary leak, and ultimately, end-stage organ dysfunction.3 In animal models, this destructive neutrophil priming via platelet-activating factor (PAF) appears to be a central process for gut ischemia and reperfusion–induced lung injury.4-5 Further clinical data have implicated PAF and similar lipid mediators in early postinjury neutrophil priming associated with the development of ARDS and MOF.6-7
In the laboratory, we have previously characterized human neutrophil priming via PAF as a concordant event resulting in the up-regulation of adhesion molecules on the cell surface, and the simultaneous generation of superoxide anion (O2-) and release of elastase after activation.8 This concordant functional response suggests a possible common priming signal transduction pathway, but intracellular signaling pathways involved in neutrophil priming have only begun to be elucidated. Intracellular PAF signal transduction appears to be mediated via tyrosine phosphorylation and activation of mitogen-activated protein kinase (MAPK) pathways.9-11 Investigators11-15 have demonstrated that p38 MAPK, 1 of 3 major MAPK families characterized, is present in human neutrophils and is activated within seconds by a variety of inflammatory stimuli. The p38 MAPK cascade has recently been shown to contribute to neutrophil priming via tumor necrosis factor  and granulocyte-macrophage colony-stimulating factor.16 Furthermore, Nick et al17 have documented the activation of p38 MAPK after exposure of neutrophils to PAF and the association of this specific kinase with a number of functional neutrophil responses.18 We investigated the role of p38 MAPK in the neutrophil priming response using a specific p38 MAPK inhibitor before PAF priming and subsequent activation of the cells. If p38 MAPK is necessary for neutrophil priming via PAF, then inhibiting p38 MAPK activity in the early postinjury period may decrease neutrophil-mediated tissue damage. Our study hypothesis is that PAF-induced human neutrophil priming for O2- generation and elastase release is mediated via the p38 MAPK pathway.
MATERIALS AND METHODS
MATERIALS
L--phosphatidylcholine,  -acetyl-g-O-alkyl (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLP), cytochrome c, superoxide dismutase (SOD), N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (AAPV-pNA), N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (AAPV-CK), dextran, and Triton X-100 were purchased from Sigma-Aldrich Corp (St Louis, Mo). Ficoll-Hypaque was obtained from Pharmacia Biotech (Uppsala, Sweden). A kinetic microplate reader (THERMOmax) and software (Softmax)were purchased from Molecular Devices (Menlo Park, Calif). SB 203580 was supplied by John C. Lee, PhD, at SmithKline Beecham Pharmaceuticals (King of Prussia, Pa). SB 203580 was dissolved in dimethyl sulfoxide and diluted in normal saline containing 0.25% human serum albumin (Abbott Laboratories, North Chicago, Ill).
SAMPLE COLLECTION
After providing informed consent via a protocol approved by the University of Colorado Health Sciences Center Combined Affiliated Institutional Review Board, Denver, 6 healthy human volunteers donated venous blood through a sterile 21-gauge butterfly needle into a heparinized syringe (10 U of heparin per milliliter of blood).
NEUTROPHIL ISOLATION
Neutrophils were isolated from heparinized blood samples as described.8 Briefly, following dextran sedimentation of erythrocytes, the upper layer was removed and centrifuged at 200g for 10 minutes. The pellet was resuspended in phosphate-buffered saline and overlaid on 5 mL of Ficoll-Hypaque. This gradient was centrifuged at 400g for 30 minutes and the supernatant was discarded, leaving red blood cells and neutrophils in the bottom layer. Hypotonic lysis was performed twice at 4°C to remove contaminating red blood cells, and the neutrophils were resuspended in Krebs-Ringer phosphate with dextrose at pH 7.35 to a final concentration of 2.5x107cells/mL. The final cell population was more than 98% neutrophils by differential staining, and more than 99% viable by trypan blue exclusion.
O2- ASSAY
Generation of O2- by neutrophils was measured by SOD-inhibitable cytochrome c reduction in 96-well microplates. Isolated neutrophils were preincubated with the specific p38 MAPK inhibitor SB 203580 (1 µmol/L) or Krebs-Ringer phosphate with dextrose buffer (control) for 30 minutes in a shaking water bath at 37°C. SB 203580 (IC50=0.6 µmol/L in vitro) is a pyridinyl imidazole that binds to and blocks the activity of p38 MAPK in a highly selective manner.19-20 The concentration of SB 203580 chosen is based on previous work demonstrating that this dose effectively blocks p38 MAPK activity in neutrophils21 and inhibits neutrophil reactive oxygen metabolite production and adhesion.21-22 Neutrophil viability was again verified by trypan blue exclusion after preincubation with SB 203580, and was found to be unchanged at 99%. Of the pretreated neutrophils, 3.75x105were then added to individual wells of a microtiter plate. The PAF was added to the experimental wells to make a final concentration of 200 nmol/L, and incubation at 37°C was continued
for 5 minutes with mixing. We previously demonstrated8 that 5 minutes is the optimal incubation time for maximal priming for concordant O2- and elastase release. Control wells contained Krebs-Ringer phosphate with dextrose instead of PAF. Experimental wells contained cytochrome c (80 µmol/L), and blank wells contained SOD (15 µg/mL) to achieve a total reaction volume of 150 µL. All priming assays were completed at 37°C in duplicate with a separate SOD blank. Respiratory burst was initiated by adding either the receptor-dependent activator fMLP (1 µmol/L) or the receptor-independent activator phorbol myristate acetate (PMA, 100 ng/mL) to experimental wells. Additional wells were prepared with neutrophils that had not been preincubated with SB 203580, and these were also exposed to PAF, fMLP, PMA, PAF and fMLP, and PAF and PMA. Immediately after the addition of neutrophils, the plate was placed in a kinetic microplate reader, and absorbance was measured at 550 to 450 nm every 20 seconds for 5 minutes. Maximal rate of O2- production (Vmax) was determined by the slope of the absorbance curve over 5 points using the computer software. An extinction coefficient of 8.4x10-3 L·mol- 1·min- 1 was used as determined for the 150-µL reaction volume and the 550-nm filter in the reader. Data are recorded as superoxide Vmax (nanomoles of O2-/3.75x105 cells per minute). The PMA-induced respiratory burst confirmed neutrophil viability and oxidase functional integrity.
ELASTASE ASSAY
Neutrophil elastase release was measured by the cleavage of the specific elastase substrate AAPV-pNA. A total of 6.25x105 neutrophils were preincubated with the specific p38 MAPK inhibitor SB 203580 (1 µmol/L) or Krebs-Ringer phosphate with dextrose buffer (control) for 30 minutes at 37°C. Triton X-100 (0.1% final concentration) was added to an additional sample of neutrophils at the same concentration for quantification of the total neutrophil elastase content. Following SB 203580 preincubation, neutrophils were primed with 200-nmol/L PAF for 5 minutes, and activated with either the receptor-dependent activator fMLP (1 µmol/L) or the receptor-independent activator PMA (100 ng/mL) for an additional 10 minutes. Microcentrifuge tubes were also prepared with neutrophils that had not been preincubated with SB 203580 and were similarly exposed to PAF, fMLP, PMA, PAF and fMLP, or PAF and PMA as controls. Samples were then centrifuged at 400g for 5 minutes, and the cell-free supernatant was added to individual wells in the microplate. All wells also contained AAPV-pNA (0.33 mmol/L) diluted in HEPES (33.3 mmol/L) and sodium chloride (0.17 mol/L) buffer, and blank wells contained AAPV-CK (0.17 mmol/L) to achieve a total reaction volume of 150 µL. All priming experiments were completed at 37°C in duplicate, with a separate AAPV-CK blank. The plate was incubated for 60 minutes at 37°C, and absorbance was measured at 405 nm. An extinction coefficient of 8.8x103 cm·mol-1·L-1 was used to calculate the units of elastase released. This number was then divided by the total neutrophil elastase content (Triton X-100 sample) to yield the percentage of total neutrophil elastase.
STATISTICAL ANALYSIS
Data were compared by an analysis of variance using the Scheffe F procedure for post hoc comparisons. Data are reported as mean±SEM. P<.05 was considered statistically significant. All groups contained 6 separate human donors.
RESULTS
RECEPTOR-DEPENDENT NEUTROPHIL ACTIVATION
Used as a cellular surrogate of the systemic immune response, neutrophil priming and activation correlated with the 2-event model in vivo. In vitro, our investigations2, 8 into the neutrophil priming and activation sequence used PAF as a primer and fMLP as an activating stimulus. Human fMLP receptor is an example of a G protein–linked chemotactic receptor23 documented to activate the p38 MAPK signaling pathway.13-14,17 This well-characterized stimulant (a component of bacterial cell walls) was therefore used to investigate neutrophil receptor-dependent activation of O2- generation (Figure 1). The fMLP activation alone stimulated neutrophil O2- generation (0.9±0.3 nmol of O2-/3.75x105 cells per minute) above basal levels (0.03±0.02 nmol of O2-/3.75x105 cells per minute). This fMLP-stimulated O2- response was significantly inhibited by the specific p38 MAP kinase inhibitor SB 203580 (0.4±0.1 nmol of O2-/3.75x105 cells per minute). In addition, as demonstrated in previous work8 from our laboratory, PAF priming resulted in significantly increased production of O2- (7.9±0.6 nmol of O2-/3.75x105 cells per minute) compared with fMLP stimulation alone. This PAF O2- priming response was similarly inhibited by SB 203580 (4.5±0.5 nmol of O2-/3.75x105 cells per minute) but not completely blocked to basal levels.
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Figure 1. Superoxide anion (O2-) production from isolated neutrophils in response to platelet-activating factor (PAF)–induced priming and receptor-dependent activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Data are expressed as mean±SEM from 6 donors. Superoxide Vmax equals nanomoles of O2-/3.75x105 cells per minute. Asterisk indicates P=.002 compared with control value. Dagger indicates P=.001 compared with fMLP control value.
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Similar data were obtained with measured neutrophil elastase release using the receptor-dependent activator fMLP (Figure 2). The fMLP activation alone stimulated elastase release (17.9%±1.2% total neutrophil elastase) above basal levels (9.4%±0.9%). This fMLP-stimulated elastase response was significantly inhibited by SB 203580 (12.3%±1.2% total neutrophil elastase). When the cells were initially primed with PAF, the resultant elastase release was significantly increased (42.5%±3.9% total neutrophil elastase). This PAF elastase priming response was also inhibited by SB 203580 (23.9%±3.1%) but, again, not completely blocked to basal levels. These data demonstrate that p38 MAPK partially mediates PAF priming and fMLP activation of neutrophils for both O2- production and elastase release. However, since fMLP activates p38 MAPK, one cannot discriminate the effect of inhibiting p38 MAPK on PAF priming from its effect on fMLP activation.
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Figure 2. Elastase release from isolated neutrophils in response to platelet-activating factor (PAF)–induced priming and receptor-dependent activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Data are expressed as mean±SEM from 6 donors. Elastase release equals percentage of total neutrophil elastase content (Triton X-100 cell lysis). Asterisk indicates P=.005 compared with control value. Dagger indicates P=.003 compared with fMLP control value.
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RECEPTOR-INDEPENDENT NEUTROPHIL ACTIVATION
Recognizing that fMLP activates p38 MAPK, independent of PAF,13-14,17 the receptor-independent activator PMA was used to determine whether p38 MAPK mediates PAF priming of the neutrophil functional response (Figure 3). The PMA activation alone stimulated neutrophil O2- generation (2.8±0.7 nmol of O2-/3.75x105 cells per minute) above basal levels (0.03±0.02 nmol of O2-/3.75x105 cells per minute). However, in contrast to fMLP receptor-dependent activation, the O2- response stimulated by PMA receptor-independent activation was not significantly inhibited by the specific p38 MAPK inhibitor SB 203580 (2.3±0.5 nmol of O2-/3.75x105 cells per minute). The PAF priming followed by PMA activation resulted in significantly increased production of O2- (5.4±0.9 nmol of O2-/3.75x105 cells per minute) compared with PMA stimulation alone. This PAF priming of the respiratory burst was inhibited by SB 203580 (2.4±0.4 nmol of O2-/3.75x105 cells per minute), decreasing it to the same level produced with PMA stimulation alone. Therefore, PMA receptor-independent activation discriminates the effect of p38 MAPK inhibition on PAF priming from activation.
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Figure 3. Superoxide anion (O2-) production from isolated neutrophils in response to platelet-activating factor (PAF)–induced priming and receptor-independent activation with phorbol myristate acetate (PMA). Data are expressed as mean±SEM from 6 donors. Superoxide Vmax equals nanomoles of O2-/3.75x105 cells per minute. Asterisk indicates P=.01 compared with control value. Dagger indicates P=.03 compared with PMA control value.
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Similar data were obtained with measured neutrophil elastase release using PMA activation (Figure 4). The PMA activation alone stimulated elastase release (29.4%±2.4% total neutrophil elastase) above basal levels (9.4%±0.9%). This PMA-stimulated elastase response was not significantly inhibited by SB 203580 (25.9%±4.6% total neutrophil elastase). When the cells were initially primed with PAF, the resultant elastase release in response to PMA was significantly increased (43.6%±3.9% total neutrophil elastase). This PAF elastase priming response was also nearly completely inhibited by SB 203580 (28.3%±4.7% total neutrophil elastase) down to the same level released with PMA stimulation alone. These data, using receptor-independent activation, demonstrate that p38 MAPK is required for maximal PAF priming of neutrophils for both O2- production and elastase release.
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Figure 4. Elastase release from isolated neutrophils in response to platelet-activating factor (PAF)–induced priming and receptor-independent activation with phorbol myristate acetate (PMA). Data are expressed as mean±SEM from 6 donors. Elastase release equals percentage of total neutrophil elastase content (Triton X-100 cell lysis). Asterisk indicates P=.004 compared with control value. Dagger indicates P=.01 compared with PMA control value.
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COMMENT
Investigation into the pathogenesis of MOF has implicated the neutrophil as a key cellular mediator.24-25 Our laboratory and clinical work2 focused on the mechanisms responsible for early postinjury neutrophil hyperactivity. These studies and others have further elucidated the neutrophil priming-activation sequence that forms the framework for the 2-event model of postinjury MOF. Animal studies4-5 have invoked PAF-induced neutrophil priming as a pivotal event for gut ischemia and reperfusion–induced lung injury. Our clinical studies6-7 also suggest that PAF and similar inflammatory lipid mediators contribute to postinjury neutrophil priming and the development of MOF. Further in vitro studies8 on neutrophil functional response demonstrate that PAF primes these cells concordantly for superoxide production and elastase release. Enhanced protease degranulation may be even more important than reactive oxygen metabolites for neutrophil-mediated tissue injury.26 Although traditional therapy has concentrated on nullifying cellular end products (antioxidants or antiproteases), these therapies have largely been unsuccessful. We therefore investigated more proximal aspects in this cascade of events. Specifically, our efforts focused on signal transduction pathways involved in the concordant neutrophil priming response to determine whether a common intracellular signaling pathway existed. Elucidating the cellular signaling that determines the neutrophil primed state and its functional response may allow the development of strategies targeted to modify the systemic inflammatory response. Intervention at the level of signal transduction may prove to be more effective clinically than manipulation of neutrophil priming end products.
The MAPKs are a family of serine and threonine protein kinases regulated by both tyrosine and threonine phosphorylation,27-28 and have been implicated in a broad variety of cellular functions.29 This group of enzymes is recruited in response to various cellular stresses, including heat stress, osmotic stress, lipopolysaccharide, cytokine stimulation, and bacterial phagocytosis.11-12,28, 30-31 Previous investigators using multiple cell types have characterized 3 basic MAPK pathways that function as parallel signal processors: extracellular signal-related kinase 1/2 (ERK 1/2 or p42/44 MAPK), c-Jun amino-terminal kinase and stress-activated protein kinase (JNK/SAPK), and p38 MAPK.32 Cross-talk between these parallel pathways is crucial to the coordinated responses of cells, resulting in an integration of multiple effector mechanisms.15, 33
Recent work has demonstrated that at least 2 of these MAPK pathways are involved in neutrophil cytotoxicity. ERK 1/2 were the first cloned and well-characterized mammalian MAPKs, and are generally associated with mitogenesis and cellular differentiation.33 Previous work22 in our laboratory revealed that blockade of ERK 1/2 had no effect on O2- production, and actually increased elastase degranulation from fMLP-activated neutrophils. The p38 MAPK is the second MAPK pathway delineated in neutrophils and is activated by many agonists, including tumor necrosis factor , lipopolysaccharide, and granulocyte-macrophage colony-stimulating factor.11-12,14-15 Returning to our neutrophil priming and activation paradigm, we and others previously demonstrated13-14,22 that neutrophil activation with fMLP results in p38 MAPK activation. Nick et al17 have documented the activation of p38 MAPK after exposure of neutrophils to PAF and its association with a number of functional neutrophil responses. However, the concordant neutrophil functional response associated with the neutrophil primed state, and its associated signaling cascade, has not been clarified.
We began our investigations into the neutrophil priming signaling cascade using the well-characterized, receptor-dependent chemoattractant fMLP as the activating stimulus. The O2- and elastase functional responses to fMLP activation are decreased about 44% and 69%, respectively, by specific inhibition of p38 MAPK. This inhibition may indicate that, although fMLP primarily functions via the p38 MAPK pathway, other signaling events are also required, and further downstream kinases may be activated to bypass the selective blockade caused by SB 203580. These results agree with other investigators14, 17 who have documented that fMLP-induced neutrophil O2- production and migration, adherence, and interleukin 8 production can be at least partially inhibited by selective p38 MAPK inhibition (SB 203580 or SK&F 86002). The PAF priming followed by fMLP activation was also inhibited by SB 203580 but, again, other signaling cascades were able to partially circumvent the inhibition. Furthermore, we could not delineate the selective effects of p38 MAPK inhibition on PAF priming vs the effects on fMLP activation.
We therefore used the receptor-independent activator PMA to determine if p38 MAPK mediates PAF priming of the neutrophil functional response, independent of its effects on fMLP activation. As reported by others12, 14 and documented in our experiments, PMA-induced O2- production and elastase release were not inhibited by SB 203580. Therefore, PMA does not rely on p38 MAPK signal transduction for either the resulting neutrophil respiratory burst or the degranulation response. Indeed, PMA has been shown to activate the Raf kinases, MEK-1 and MEK-2, p42/44 ERK MAPKs, and p38 MAPK and, therefore, has many available downstream kinases to bypass any selective blockade of p38 MAPK.12 The PAF priming followed by activation with PMA resulted in a significantly increased neutrophil functional response measured for both O2- production and elastase release. This PAF priming was completely inhibited by SB 203580. Therefore, PAF appears to have a much more limited set of downstream kinases to activate compared with either fMLP or PMA. Figure 5 schematically represents these proposed intracellular neutrophil signal transduction cascades with multiple areas of cross-talk and intercommunication.
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Figure 5. Mitogen-activated protein kinase (MAPK) pathways in neutrophils in response to platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and phorbol myristate acetate (PMA) that have been associated with activation of cytotoxic mechanisms. Specific pathways within the shaded box remain to be fully elucidated, and there are multiple areas of cross-talk and intercommunication. O2-indicates superoxide anion; PKC, protein kinase C; and ERK, extracellular signal-related kinase.
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Collectively, these data demonstrate that maximal PAF-induced neutrophil priming for O2- production and elastase degranulation requires p38 MAPK signal transduction. Therefore, p38 MAPK appears to serve a proinflammatory role in the neutrophil. Further work34 in pulmonary endothelial cells also demonstrated a proinflammatory role for p38 MAPK, since inhibition of this signaling pathway results in decreased adhesion molecule expression. Therefore, inhibition of p38 MAPK activity may offer a potential therapeutic target to reduce neutrophil-mediated hyperinflammation and, ultimately, postinjury MOF and ARDS by altering both the neutrophil and endothelial cell response during systemic inflammatory response syndrome. We recognize that this is a complex system, and that MAPK inhibition may affect other immune cells such as T-cells and monocytes.35-38 However, these effects may add to the anti-inflammatory effects demonstrated on the neutrophil. In addition, 4 distinct isoforms of p38 MAPK have been identified in mammalian cells (the original p38, as well as p38, p38 , and p38 ).18, 39 Only the p38 and p38 MAPK isoforms have been detected in neutrophils.18, 38 Further elaboration of these intricate and complicated signal transduction pathways involved in the potentially destructive neutrophil-endothelial cell interaction may generate more specific inhibitors and afford the possibility to selectively modulate the inflammatory response.
| Statement of Clinical Relevance
Investigation into the pathophysiology of MOF has implicated the neutrophil as a key cellular mediator. We previously characterized PAF priming of human neutrophils as a concordant event resulting in adhesive neutrophils with maximal cytotoxic potential. Whereas traditional therapy has concentrated on extracellular events such as nullifying cellular end products, intervention at the level of signal transduction may prove to be more effective clinically. The concordant neutrophil functional response suggests a possible common priming signal transduction pathway, but precise intracellular signaling pathways involved in neutrophil priming remain unclear. Our data demonstrate that maximal PAF-induced neutrophil priming for O2- production and elastase degranulation requires p38 MAPK signal transduction. The p38 MAPK thus appears to serve a proinflammatory role in the neutrophil, and inhibition of p38 MAPK activity may offer a potential therapeutic strategy to reduce global neutrophil-mediated hyperinflammation and ultimately, postinjury ARDS and MOF. Further elucidation of these complicated signal transduction pathways involved in the potentially destructive neutrophil-endothelial cell interaction may generate more specific inhibitors and afford the possibility to selectively modulate the inflammatory response.
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AUTHOR INFORMATION
This study was supported in part by grants P50GM49222 and T32GM08315 and Clinical Associate Physician Award M01-RR00069 from the General Clinical Research Centers Program, National Institutes of Health, Bethesda, Md.
Presented at the 19th Annual Meeting of the Surgical Infection Society, Seattle, Wash, April 29, 1999.
Corresponding author: Ernest E. Moore, MD, Department of Surgery, Denver Health Medical Center, 777 Bannock St, Denver, CO 80204 (e-mail: emoore{at}dhha.org).
From the Department of Surgery, Denver Health Medical Center (Drs Partrick, Moore, Offner, Meldrum, Tamura, and Johnson), and Bonfils Blood Center (Dr Silliman), University of Colorado Health Sciences Center, Denver.
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