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Originally published online as doi:10.1189/jlb.0205067 on June 7, 2005 Originally published online as doi:10.1189/jlb.0205067 on April 21, 2005

Published online before print April 21, 2005
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(Journal of Leukocyte Biology. 2005;78:210-219.)
© 2005 by Society for Leukocyte Biology

TNF-{alpha} promotes a stop signal that inhibits neutrophil polarization and migration via a p38 MAPK pathway

Mary A. Lokuta*,1 and Anna Huttenlocher*,{dagger}

* Departments of Pediatrics and
{dagger} Pharmacology, University of Wisconsin, Madison

1 Correspondence: Department of Pediatrics, University of Wisconsin, 2715 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706. E-mail: malokuta{at}facstaff.wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are a major component of the inflammatory response in patients with asthma and other inflammatory conditions. Proinflammatory cytokines, such as tumor necrosis factor {alpha} (TNF-{alpha}), are increased in the airway of patients with severe asthma and have been implicated in the recruitment of neutrophils into areas of inflammation. Here, we show that TNF-{alpha} induces a stop signal that promotes firm neutrophil adhesion and inhibits neutrophil polarization and chemotaxis to chemoattractants including interleukin-8 and C5a. TNF-{alpha} treatment of neutrophils plated on a fibrinogen-coated surface promotes firm neutrophil adhesion and the formation of vinculin-containing focal complexes. TNF-{alpha} induces a more than tenfold increase in p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase phosphorylation. Inhibition of p38 MAPK in neutrophils treated with TNF-{alpha} causes neutrophil polarization and motility. These findings suggest that TNF-{alpha} initiates a stop signal through a p38 MAPK pathway, which may promote the retention of neutrophils in inflammatory sites. Together, our data suggest that inhibition of p38 MAPK may be an attractive target to limit inflammatory responses that are mediated by TNF-{alpha}.

Key Words: chemotaxis • inflammation • asthma


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are a key component of the innate immune response and are the initial responders to infection or other inflammatory stimuli. Leukocyte recruitment to areas of inflammation is a multi-step process that involves a hierarchical response to external signals, including cytokines, chemokines, and extracellular matrix ligands [1 2 3 4 ]. The initial step in the inflammatory response is adhesion and migration of neutrophils from the microvasculature, followed by the directional migration of neutrophils into the tissues and their subsequent retention within inflammatory sites [3 , 5 , 6 ]. Each step of the process involves the integrated response to numerous inflammatory mediators and the prioritization of different directional cues. Although there has been recent progress in understanding the mechanisms that regulate neutrophil extravasation, we still have limited understanding of the mechanisms that contribute to neutrophil recruitment and retention in inflammatory sites and how these multiple directional cues are prioritized to promote an inflammatory response.

Tumor necrosis factor {alpha} (TNF-{alpha}) is a cytokine that has been implicated in neutrophil extravasation and recruitment to inflammatory sites [7 8 9 10 11 ], and its levels are increased in many inflammatory conditions including asthma [12 ]. Elevated levels of TNF-{alpha} have been observed in the bronchioalveolar lavage fluid of patients with severe exacerbations of asthma [13 ]. Further, TNF-{alpha} has been shown to be constitutively elevated in asthmatic subjects [12 , 13 ]. Studies have demonstrated the therapeutic benefit of specific therapies targeted at blocking TNF-{alpha} for the treatment of patients with chronic inflammatory conditions including rheumatoid arthritis and inflammatory bowel disease [14 15 16 17 18 ]. Additional work suggests the potential therapeutic benefits of using TNF-{alpha}-targeted therapies to treat severe asthma [19 , 20 ]. Despite its potent inflammatory effects, the role of TNF-{alpha} on leukocyte recruitment and retention during inflammation has not been well characterized. Much work has focused on the role of TNF-{alpha} in the priming of neutrophils for such functions as oxidative burst and degranulation [21 , 22 ]. More recent studies have focused on the role that TNF-{alpha} plays in neutrophil recruitment during inflammatory responses. TNF-{alpha} has been shown to up-regulate the adhesion molecules CD11b and CD15 on the surface of neutrophils via a p38 mitogen-activated protein kinase (MAPK)-dependent pathway [23 , 24 ]. T cell-derived TNF-{alpha} has been shown to be required for neutrophil recruitment during immune peritonitis via a leukotriene B4-dependent pathway [7 ]. However, the specific effect of TNF-{alpha} on the migration of neutrophils and its role in neutrophil recruitment and/or retention in tissues during inflammatory responses are still poorly understood. In fact, previous reports suggest positive and negative regulatory roles for TNF-{alpha} signaling during neutrophil motility and chemotaxis, and much work suggests that TNF-{alpha} induces inflammation indirectly via the induction of other inflammatory mediators [7 8 9 , 25 26 27 28 ].

To address the role of TNF-{alpha} during neutrophil migration, we examined its effect on the migration of neutrophils in the presence and absence of the inflammatory mediator interleukin-8 (IL-8) and examined the contribution of p38 MAPK signaling to this regulation. Our findings demonstrate that TNF-{alpha} promotes firm neutrophil adhesion and is inhibitory to neutrophil polarization and migration through a p38 MAPK pathway. The findings suggest that inhibition of the p38 MAPK pathway may be a useful, therapeutic target to limit TNF-{alpha}-mediated inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and cell isolation
Peripheral blood neutrophils were purified from human blood using Polymorphprep according to the manufacturer’s recommendations (Nycomed, Sheldon, UK). All donors were self-reportedly healthy, and informed consent was obtained at the time of the blood draw. The University of Wisconsin–Madison, Center for Health Sciences Human Subjects Committee approved the use of human subjects. Recombinant human TNF-{alpha} was purchased from BioSource (Camarillo, CA). IL-8, f-Met-Leu-Phe (fMLP), and fibrinogen (Fbg) were purchased from Sigma Chemical Co. (St. Louis, MO). SB203580, SB202190, SB202474, U0126, U0125, and U0124 were purchased from EMD Biosciences (San Diego, CA).

Immunofluorescent staining
Neutrophils (1.5x105) were plated on glass coverslips, which had been coated with 10 µg/mL Fbg and incubated for 30 min at 37°C, 10% CO2, in the presence of activator and/or inhibitor. Cells were then fixed with 6.6% paraformaldehyde, 0.05% glutaraldehyde, and 0.25 mg/mL saponin in phosphate-buffered saline (PBS) for 15 min as described previously [29 ]. This reaction was quenched with 0.15 M glycine in PBS for 15 min. Samples were then blocked in PBS containing 10% heat-inactivated fetal bovine serum and 0.25 mg/mL saponin at 4°C for 24–48 h. Antivinculin antibody (V-9131, Sigma Chemical Co.) was used at 1:400 in blocking buffer. Sheep anti-mouse fluorescein isothiocyanate (ICN Pharmaceuticals, Costa Mesa, CA) was used at 1:250, and rhodamine:phalloidin (Molecular Probes, Junction City, OR) was used at 1:1000 in blocking buffer. Coverslips were imaged using a 60x oil-immersion lens on a Nikon Eclipse TE300 inverted fluorescent microscope. Images were acquired with a Hamamatsu cooled charged-coupled device (CCD) video camera and captured into Metamorph v5.0 (Universal Imaging Corp., Downingtown, PA).

Video microscopy
Nontissue culture-treated dishes were coated with 2.5 µg/mL Fbg, and 1 x 106 neutrophils were plated for 30 min at 37°C, 10% CO2, in EGM-2MV (Cambrex, East Rutherford, NJ) containing TNF-{alpha} or inhibitor as noted. IL-8 or fMLP was included for the last 5 min where indicated. Dishes were then overlayed with mineral oil, placed in The Box closed system (Life Imaging Services, Reinach, Switzerland), and maintained at 37°C while imaged on an Olympus IX-70 inverted microscope (Olympus America, Melville, NY) using a 20x phase objective. Images were collected using a Coolsnap fx cooled CCD camera (Photometrics, Huntington Beach, CA) and captured into MetaView v6.2 (Universal Imaging Corp.) every 15 s for 15 min.

Chemotaxis assay
Neutrophils (1x106) were pretreated and plated as described for video microscopy. Chemoattractants were loaded into an Eppendorf femptotip, and a gradient was formed by slow release of the chemoattractant from the tip into the medium using an Eppendorf FemptoJet microinjection system with a constant back pressure of 10 hPa as described previously [30 , 31 ]. Cells were placed in The Box closed system (Life Imaging Services) and maintained at 37°C while viewed on a Nikon Eclipse TE300 inverted fluorescent microscope. Images were acquired every 15 s for 20 min with a Hamamatsu cooled CCD video camera using a 20x differential interference contrast objective and captured into Metamorph v5.0.

Transwell assay
Neutrophil migration by Transwell was determined using standard methods [32 ]. In brief, 3 µm pore Transwell filters (Costar, Corning, NY) were coated with 2.5 µg/mL Fbg, and 4 x 105 neutrophils were treated for 30 min in EGM-2MV containing treatments as noted. EGM-2MV alone or EGM-2MV containing 1.25 nM IL-8, with or without TNF-{alpha} as noted, was placed in the bottom chamber; 4 x 105 neutrophils were placed in the top chamber, with or without TNF-{alpha} as noted; and samples were incubated at 37°C, 10% CO2, for 3 h. EDTA was then added to the bottom chamber, and the plate was incubated at 4°C for 10 min. The top chambers were then removed, and the neutrophils present in the bottom chamber were counted. The number of neutrophils that migrated across the filter was determined and expressed relative to control.

Adhesion assay
Neutrophils were incubated in EGM-2MV containing 1.93 µM of the cell-permeant fluorescent indicator calcein-acetoxymethyl ester (Molecular Probes) for 15 min at 37°C and 10% CO2. Cells were then washed with Dulbecco’s PBS (DPBS) without (w/o) Ca2+/Mg2+ (Mediatech, Herndon, VA). Neutrophils were brought to 1 x 106 cells/mL in DPBS w/o Ca2+/Mg2+ for the standard curve or brought to 1 x 106 cells/mL in EGM-2MV and treated accordingly. Neutrophils (1x106) were then allowed to adhere for 30 min on 2.5 µg/mL Fbg-coated, black-sided, clear-bottom 96-well plates (Greiner, Kremsmuenster, Upper Austria). During the last 15 min, IL-8 or fMLP was added to the appropriate wells. Unbound cells were then removed by washing and vigorous shaking. Fluorescence excitation/emission at 485/535 nm was determined using a TECAN SPECTRAFluor Plus fluorometer. Samples were run in quadruplicate. A standard curve was included on each plate, and linear regression was performed with Magellan v2.50 software to determine the number of neutrophils adhered in each well.

Western blot analysis of p38 and extracellular signal-regulated kinase (ERK) MAPK activity
Neutrophils (1x107) were stimulated with IL-8, TNF-{alpha}, or fMLP for 5 min in EGM-2MV in suspension. Neutrophils were then lysed in 50 mM HEPES, pH 7.6, 75 mM NaCl, 0.25% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 µM NaF, 5 mM MgCl2 with protease-inhibitor cocktail (P-8340, Sigma Chemical Co.), phosphatase-inhibitor cocktail (P-5725, Sigma Chemical Co.), 2 mM phenylmethylsulfonyl fluoride, 100 µM sodium orthovanadate, 900 µM benzamidine, and 1 mM phenantroline. Samples were subjected to three alternating freeze/thaw cycles, and the debris was pelleted. Protein concentration was determined using the Pierce (Rockford, IL) bicinchoninic acid assay, and 40 µg protein per lane was loaded onto a 4–15% Tris:HCl ReadyGel (Bio-Rad, Hercules, CA). Samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose using standard methods. Anti-p38 MAPK and antiphospho-p38 MAPK antibodies (Biosource) were used at 1:1000 to detect total and active p38 MAPK. Anti-ERK1/2 was used at 1:1000 (Biosource). Antiphospho-ERK1/2 (Santa Cruz Biotechnology, CA) was used at 1:200. Detection was performed using Alexa-Fluor® 680 goat anti-mouse immunoglobulin G (IgG; Molecular Probes) and IRDyeTM 800 CW goat anti-rabbit IgG (Rockland Chemicals, Gilbertsville, PA) antibodies on an Odyssey infrared-imaging system (LI-COR Biosciences, Lincoln, NE). Quantification was determined using the Odyssey infrared-imaging system software, Version 1.2.15.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} induces firm neutrophil adhesion but not polarization
Previous studies have demonstrated that TNF-{alpha} is critical for neutrophil recruitment to inflamed tissues in vivo [7 , 9 ]. The chemoattractant IL-8 has also been implicated in the neutrophil-mediated inflammatory response in many conditions, including asthma [25 ]. To determine the mechanism of TNF-{alpha} and IL-8 proinflammatory effects, we examined neutrophil morphology and adhesion on a Fbg-coated surface in response to TNF-{alpha} or IL-8. In agreement with previously published reports [33 ], treatment with TNF-{alpha} induced dose-dependent neutrophil spreading (Fig. 1A ) and firm adhesion (Fig. 2 ). TNF-{alpha} failed to induce neutrophil polarization or the localization of actin to a leading pseudopod and instead, promoted the formation of vinculin-containing focal complexes that were not polarized (Fig. 1B) . In contrast, IL-8 induced a poor-spreading response, weak adhesion with few or no vinculin-containing focal contacts, and a highly polarized morphology with actin localization to the leading edge.



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  		Figure 1. Effect of TNF-{alpha} on neutrophil morphology. (A) Neutrophils were plated for 30 min on 2.5 µg/mL Fbg in EGM-2MV at 37°C and 10% CO2, and activators were added as noted. Neutrophils were (a) unstimulated, treated with TNF-{alpha} at (b) 250 ng/mL, (c) 25 ng/mL, (d) 2.5 ng/mL, or (e) 0.25 ng/mL, (f) treated with 100 nM fMLP, or treated with IL-8 at (g) 125 nM, (h) 12.5 nM, (i) 1.25 nM, or (j) 0.125 nM. Morphology was assessed, and all samples were subsequently examined by time-lapse videomicroscopy as described in Materials and Methods. Shown are representative frames of at least four replicate experiments. Original bar = 50 µm. (B) Neutrophils were plated on glass coverslips coated with 10 µg/mL Fbg, treated with 25 ng/mL TNF-{alpha}, 12.5 nM IL-8, or 100 nM fMLP, and incubated at 37°C, 10% CO2, for 30 min. Samples were then processed as described in Materials and Methods and stained for vinculin or for actin with phalloidin. Shown are representative cells from three replicate experiments. Original bar = 10 µm.

 


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  		Figure 2. Neutrophil adhesion is regulated by TNF-{alpha}, IL-8, and fMLP. Neutrophils were loaded as described in Materials and Methods and were plated on 2.5 µg/mL Fbg in black-sided, 96-well plates. Cells were unstimulated (un) or treated with the indicated concentrations of TNF-{alpha}, IL-8, or fMLP. Shown are the sample means ± SEM for four replicate experiments. *, Statistical significance as compared with unstimulated, as determined by the two-tailed Student’s t-test (P≤0.05).

 
Previous studies have demonstrated that high ligand density and increased cell-substratum adhesiveness may inhibit cell polarization [34 ]. To determine if all chemoattractants that mediate strong adhesion are also inhibitory to neutrophil polarization, we examined the effects of the end target, bacterially derived chemoattractant fMLP on neutrophil polarization and adhesion. Similar to TNF-{alpha}, fMLP induced firm neutrophil adhesion to Fbg-coated surfaces (Fig. 2) ; however, fMLP induced a highly polarized morphology with actin reorientation to the leading edge (Fig. 1) . Together, these findings indicate that different inflammatory mediators have distinct effects on cellular morphology and polarization that are independent of their effects on cellular adhesiveness.

TNF-{alpha} inhibits neutrophil chemotaxis to IL-8
Previous studies have suggested a promigratory and inhibitory role for TNF-{alpha} during neutrophil migration [7 , 26 ]. To assess whether TNF-{alpha} promotes neutrophil migration, time-lapse videomicroscopy was performed, and the migration speeds of neutrophils treated with TNF-{alpha} or IL-8 were determined. TNF-{alpha} does not promote a significant increase in random migration of neutrophils at most concentrations examined (Fig. 3A ). However, at low concentrations (≤0.025 ng/mL), there is a modest increase in neutrophil chemokinesis (data not shown). Together, the data suggest that TNF-{alpha} may promote neutrophil chemokinesis at low concentrations, but there is a dose-dependent inhibition of chemokinesis at most concentrations of TNF-{alpha}.



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  		Figure 3. Effects of TNF-{alpha} on neutrophil migration. (A) Neutrophils were treated as in Figure 1A and were unstimulated (un) or stimulated with 250 ng/mL TNF-{alpha}, 25 ng/mL TNF-{alpha}, 2.5 ng/mL TNF-{alpha}, 0.25 ng/mL TNF-{alpha}, 125 nM IL-8, 12.5 nM IL-8, 1.25 nM IL-8, or 0.125 nM IL-8. Chemokinetic motility was examined, and migration rates were determined using time-lapse videomicroscopy, as described in Materials and Methods. Shown are the means of the samples ± SEM for three replicate experiments. *, Statistical significance as compared with unstimulated, as determined by the two-tailed Student’s t-test (P≤0.05). (B) Neutrophils were added to the top well of a 3-µm pore Transwell. TNF-{alpha} (250 ng/ml or 25 ng/ml) was added to the top and/or bottom chambers, as noted, and incubated in the absence (–) or presence of 12.5 nM IL-8, placed in the bottom of the Transwell. Samples were incubated for 3 h, 37°C, 10% CO2. Samples were then processed as described in Materials and Methods and expressed relative to the unstimulated control. Shown are the means of the samples ± SEM for five replicate experiments. #, Statistical significance as compared with unstimulated, as determined by the two-tailed Student’s t-test (P≤0.05). *, Statistical significance as compared with IL-8 in the absence of any TNF-{alpha}, as determined by the two-tailed Student’s t-test (P≤0.05). (C) Neutrophils were untreated or pretreated with 25 ng/mL TNF-{alpha} for 30 min 37°C, 10% CO2, on 2.5 µg/mL Fbg. IL-8 was loaded into a Femtotip, and a chemotactic gradient was formed. Neutrophil chemotaxis was assessed by time-lapse videomicroscopy as described in Materials and Methods. Original bar = 50 µm.

 
To determine whether TNF-{alpha} modulates the directional migration to IL-8, Transwell assay and live videomicroscopy were performed with neutrophils exposed to a gradient of IL-8. It is interesting that our data indicate that TNF-{alpha} is inhibitory to neutrophil chemotaxis toward IL-8 (Fig. 3C) and C5a (data not shown). Furthermore, the presence of TNF-{alpha} inhibited neutrophil chemotaxis to IL-8 when present in the upper or lower portion of the Transwell chamber (Fig. 3B) . This suggests that TNF-{alpha} does not have significant chemotactic properties but is instead inhibitory to neutrophil chemotaxis. In addition, in an under-agarose chemotactic assay, TNF-{alpha} failed to induce neutrophil chemotaxis, and IL-8, C5a, and fMLP induced significant neutrophil migration in the direction of the well containing the inflammatory mediator (data not shown). Taken together, the data suggest that TNF-{alpha} is inhibitory to neutrophil chemotaxis and may act to promote neutrophil retention within inflammatory sites.

TNF-{alpha} induces p38 MAPK more than ERK-MAPK phosphorylation
Previous work has demonstrated a key role for p38 MAPK during neutrophil chemotaxis to specific inflammatory mediators, including to the end-target chemoattractant, fMLP [25 , 35 , 36 ]. Additional work has shown that although p38 MAPK inhibitors can decrease neutrophil migration to IL-8, neutrophil chemotaxis to IL-8 relies more heavily on the phosphatidylinositol-3 kinase pathway [35 ]. In agreement with previously published work [21 ], we find that various concentrations of TNF-{alpha} induced more than a tenfold increase in p38 MAPK phosphorylation, and IL-8 stimulation of primary neutrophils induced only a three- to fourfold increase in p38 MAPK activity (Fig. 4A and 4B ). In contrast, the ERK-MAPK pathway was more markedly increased by neutrophils treated with different concentrations of IL-8 (nine- to 12-fold) than by TNF-{alpha} (two- to fourfold). However, 100 nM fMLP induced enhanced phosphorylation of p38 MAPK and ERK-MAPK (ten- and 11-fold, respectively). Together, these findings demonstrate the differential regulation of the p38 MAPK and ERK-MAPK pathways in primary neutrophils by different inflammatory mediators.



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  		Figure 4. Regulation of p38 MAPK and ERK1/2-MAPK by TNF-{alpha}. (A) Neutrophils were incubated in suspension in EGM-2MV in the absence (un) or presence of activators at the indicated concentrations for 5 min at 37°C, 10% CO2. Cells were then spun and lysed, and samples were examined by Western blot as described in Materials and Methods. Shown is a representative of four replicate experiments with simultaneous detection of phospho-p38 MAPK and total p38 MAPK. The overlay shows the phospho-p38 MAPK in green and total p38 MAPK in red with areas of overlay indicated in yellow. (B) The Western blot replicates were quantified, and the sample means are shown ± SEM. *, Statistical significance as compared with unstimulated, as determined by the two-tailed Student’s t-test (P≤0.05). (C) Neutrophils were treated as in A. Shown is a representative of four replicate experiments with simultaneous detection of phospho-ERK1/2 and total ERK1/2. The overlay shows the phospho-ERK1/2 in red and total ERK1/2 in green with areas of overlap indicated in yellow. (D) The Western blot replicates were quantified, and the sample means are shown ± SEM. *, Statistical significance as compared with unstimulated, as determined by the two-tailed Student’s t-test (P≤0.05).

 
p38 MAPK is required for TNF-{alpha} effects on neutrophil adhesion, polarization, and migration
To examine the mechanism of TNF-{alpha} effects on neutrophil polarization and migration, we characterized the effects of cell-permeable p38 MAPK and ERK-MAPK inhibitors on the morphology, adhesion, and migration of neutrophils treated with TNF-{alpha}. We found that inhibition of p38 MAPK using SB202190 or SB203580 reduced neutrophil adhesion induced by TNF-{alpha} to 37.1% ± 6.7 or 44.8% ± 2.6, respectively, as compared with that induced by TNF-{alpha} alone (Fig. 5 ). This reduced adhesion was not seen with the negative-control compounds, SB202474 (99.2%±7.5) or U0124 (97.0%±6.6), nor was it seen upon inhibition of the upstream regulator of ERK1/2 activity, MAPK kinase (MEK)1/2, with U0125 (81.4%±13.5) or U0126 (90.3%±5.8). These findings suggest that p38 MAPK is required for the firm adhesion mediated by TNF-{alpha}.



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  		Figure 5. Requirement of p38 MAPK for TNF-{alpha}-induced adhesion. Neutrophils were loaded as described in Materials and Methods and plated on 2.5 µg/mL Fbg in black-sided, 96-well plates. Cells were unstimulated (un) or treated with 25 ng/mL TNF-{alpha} in the presence or absence of the indicated inhibitors. SB203580 was used at 5 µM, SB202190 was used at 10 µM, SB202474 was used at 5 µM, U0124 was used at 50 µM, U0125 was used at 25 µM, and U0126 was used at 50 µM. Shown are the sample means ± SEM for five replicate experiments. *, Statistical significance as compared with TNF-{alpha} treatment alone as determined by the two-tailed Student’s t-test (P≤0.05).

 
To determine whether p38 MAPK also mediates the dose-dependent, inhibitory effects of TNF-{alpha} on migration, we assessed the effects of p38 MAPK inhibitors on neutrophil chemokinesis in the presence of TNF-{alpha} alone. We examined neutrophil migration by time-lapse videomicroscopy and found that the inhibition of p38 MAPK with SB203580 or SB202190 reduced neutrophil spreading and enhanced neutrophil polarization and neutrophil migration rates by approximately twofold (Fig. 6 ). This effect was not seen with the negative-control compound SB202474 or with the MEK1/2 inhibitors U0125 and U0126 (Fig. 6 and data not shown). In accordance with work by Heit et al. [35 ], neutrophils that have been stimulated with IL-8 after pretreatment with SB203580 or SB202190 showed migration rates that were unchanged as compared with IL-8 alone. Together, these findings suggest that p38 MAPK mediates the stop signal that inhibits neutrophil migration and polarization by TNF-{alpha} stimulation. Further, the findings suggest that the inhibitory effects of TNF-{alpha} on neutrophil migration are mediated by p38 MAPK.



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  		Figure 6. The role of p38 MAPK on neutrophil morphology and migration induced by TNF-{alpha}. (A) Neutrophils were plated in the presence or absence of activator and/or inhibitor and imaged as in Figure 1A . Cells were treated with 25 ng/mL TNF-{alpha} alone or in the presence of 5 µM SB203580, 10 µM SB202190, 5 µM SB202474, or 50 µM U0126. Likewise, neutrophils were treated with 12.5 nM IL-8 alone or in the presence of 5 µM SB203580, 10 µM SB202190, 5 µM SB202474, or 50 µM U0126. All samples were subsequently examined by time-lapse videomicroscopy. Shown are representative frames of at least four replicate experiments. Original bar = 50 µm. (B) The path of each cell was analyzed as a function of time, as described previously [31 ] and the mean speeds shown ± SEM. *, Statistical significance as compared with TNF-{alpha} treated alone, as determined by the two-tailed Student’s t-test (P≤0.05).

 
To further characterize the role of p38 MAPK signaling in neutrophil polarization in response to TNF-{alpha}, immunofluorescent staining of neutrophils treated with TNF-{alpha} in the presence and absence of p38 MAPK inhibitors was performed. Neutrophils treated with TNF-{alpha} alone exhibited peripheral actin and large focal contacts containing vinculin, as shown in Figure 1 and as previously reported [33 ]. However, upon the inhibition of p38 MAPK by SB203580 or SB202190, the focal contacts were lost, and actin was localized toward the leading edge of the neutrophil (Fig. 7 ), a morphology that is typical of migrating neutrophils. The negative-control compound SB202474 and the MEK inhibitor U0126 had no effect on the polarization of TNF-{alpha}-treated neutrophils. Together, our data suggest that TNF-{alpha} is inhibitory to neutrophil polarization and migration through a p38 MAPK pathway.



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  		Figure 7. p38 MAPK regulates TNF-{alpha} effects on neutrophil polarization. Neutrophils were plated on coverslips coated with 10 µg/mL Fbg and processed as in Figure 1B . Samples were treated with TNF-{alpha} alone or in the presence of 5 µM SB203580, 10 µM SB202190, 5 µM SB202474, or 50 µM U0126. Shown are representative cells of three replicate experiments. Original bar = 10 µm.

 
Neutrophils stimulated with TNF-{alpha} and IL-8 develop a polarized migratory morphology
To determine if TNF-{alpha} is inhibitory to IL-8-mediated migration under all conditions, we examined the effects of TNF-{alpha} treatment on the random motility induced by IL-8 in a uniform concentration of chemoattractant. It is interesting that TNF-{alpha} was not inhibitory to neutrophil polarization or migration in a uniform concentration of IL-8. Figure 8 shows that when neutrophils are treated with TNF-{alpha} and IL-8, they develop a polarized morphology similar to that seen with IL-8 alone (Fig. 8A) , and their migration rates are increased relative to TNF-{alpha} alone (Fig. 8B ; 184.40±6.75 vs. 87.15±26.99). However, the directional migration in a gradient of chemoattractant is reduced (Fig. 3) , suggesting that TNF-{alpha} stimulation perturbs directional sensing in a gradient of IL-8 but does not affect the random motility induced in uniform concentrations of IL-8. Together, our findings demonstrate that TNF-{alpha} is inhibitory to neutrophil chemotaxis to IL-8 but not chemokinesis induced by IL-8, demonstrating a complicated cross-talk between different inflammatory mediators in the regulation of neutrophil migration.



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  		Figure 8. Effects of TNF-{alpha} and IL-8 on neutrophil chemokinesis and signaling. (A) Neutrophils were treated as in Figure 1A , and the morphology was examined as described. Neutrophils were stimulated with (a) 25 ng/mL TNF-{alpha}, (b) 12.5 nM IL-8, or (c) 25 ng/mL TNF-{alpha} + 12.5 nM IL-8. (B) Neutrophils were examined by time-lapse videomicroscopy and were unstimulated (un) or stimulated with 25 ng/mL TNF-{alpha}, 12.5 nM IL-8, or 25 ng/mL TNF-{alpha} + 12.5 nM IL-8. Migration rates were determined as described in Materials and Methods. Shown are the means of the samples ± SEM for three replicate experiments. *, Statistical significance as compared with TNF-{alpha} treatment alone, as determined by the two-tailed Student’s t-test (P≤0.05). Neutrophils were incubated in EGM-2MV in the absence (un) or presence of 25 ng/mL TNF, 12.5 nM IL-8, or both for 5 min at 37°C, 10% CO2. Cells were then spun and lysed, and samples were examined by Western blot as described. Shown is a representative of four replicate experiments with simultaneous detection of phospho-p38 MAPK and total p38 MAPK (C) or simultaneous detection of phospho-ERK1/2 and total ERK1/2 (D) as in Figure 4 . The Western blot replicates were quantified, and the sample means are shown ± SEM *, Statistical significance as compared with unstimulated, as determined by the two-tailed Student’s t-test (P≤0.05).

 
To determine if TNF-{alpha} modifies the signaling induced by IL-8, neutrophils were treated with TNF-{alpha} and IL-8. We found that p38 MAPK and ERK are significantly up-regulated (10.7±2.8 and 11.1±5.0, respectively). This up-regulation of activities does not appear to be synergistic, as the levels of p38 MAPK activation are similar to those with TNF-{alpha} alone (11.1±1.8), whereas the levels of ERK activation are comparable with those with IL-8 alone (8.8±1.3). Further, the levels of p38 and ERK-MAPK phosphorylation, after stimulation with the combination of TNF-{alpha} plus IL-8, are comparable with that induced by fMLP, which also induces a polarized and migratory phenotype (10.4±2.9 and 11.6±3.1, respectively). Together, the findings suggest that TNF-{alpha} modifies the signaling induced by IL-8 and its capacity to induce directional migration but does not affect its ability to induce chemokinesis in a uniform concentration of attractant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
TNF-{alpha} is a cytokine that plays an important role in chronic inflammation in many disease states including rheumatic diseases and asthma. TNF-{alpha}-targeted therapies are highly effective for the treatment of chronic inflammatory conditions [12 , 16 , 18 , 37 ]. Despite its importance, there is still limited understanding of the mechanism of TNF-{alpha} effects on neutrophil recruitment and retention during inflammation. We now provide evidence that TNF-{alpha} is inhibitory to neutrophil motility and chemotaxis by promoting firm adhesion and limiting neutrophil polarization. These findings suggest that TNF-{alpha} may promote inflammatory responses by mediating neutrophil retention within tissues, rather than providing a directional cue. We also show that TNF-{alpha} inhibitory effects on neutrophil polarization and migration are mediated by a p38 MAPK-dependent pathway. Inhibition of p38 MAPK increased neutrophil random motility and polarization in the presence of TNF-{alpha}. Together, these findings suggest that inhibition of p38 MAPK may be a powerful approach to limit inflammatory responses that are mediated by TNF-{alpha}.

There is limited understanding of the mechanisms that promote neutrophil retention within inflammatory sites and the contribution of these mechanisms to chronic inflammation. There is evidence that elevated levels of TNF-{alpha} in different tissues, including the synovium and airway, correlate with neutrophil numbers [12 ], suggesting that TNF-{alpha} may contribute to neutrophil recruitment or retention. Previous studies have suggested a key role for TNF-{alpha} during neutrophil transmigration across cell barriers [38 , 39 ], likely by affecting intercellular adhesion or by affecting the expression of adhesion molecules or the production of other inflammatory mediators such as IL-8. However, there has been much controversy in the literature about the effects of TNF-{alpha} on neutrophil motility and chemotaxis. Our findings suggest that TNF-{alpha} does not promote neutrophil random motility, except possibly at low concentrations. Likewise, there is no evidence for any chemotactic role for TNF-{alpha}, suggesting that it may not have a direct chemotactic effect on neutrophils. In fact, the presence of TNF-{alpha} inhibits neutrophil chemotaxis to other inflammatory mediators, thereby potentially promoting neutrophil retention in tissues that have the highest concentrations of TNF-{alpha}. It is interesting that there is complex cross-talk between TNF-{alpha} and IL-8 effects on neutrophil motility, with TNF-{alpha} inhibiting IL-8-mediated chemotaxis but not chemokinesis.

During an inflammatory response, leukocytes must be able to sense and respond to a complex milieu of signals generated at the site of infection or injury, including divergent inflammatory mediators, i.e., TNF-{alpha} and IL-8. The complex nature of the inflammatory environment within leukocyte-recruiting tissues requires that these cells be able to prioritize the signals. Previous work has shown that during neutrophil recruitment to inflammatory sites, neutrophils are able to first migrate toward one chemoattractant source and subsequently, to a different chemoattractant source in series [40 , 41 ]. Therefore, in responding to the multiplicity of signaling mediators, the leukocyte must consolidate the signals such that each signal is converted into further biochemical events, which result in a subsequent biological response, or that the signal be dissipated within the signaling networks [42 ]. It has been shown that neutrophils will preferentially migrate toward end-target molecules and ignore more general, endogenous, chemoattractant gradients, likely through receptor desensitization [40 , 43 44 45 46 ]. However, this desensitization does not occur in all classes of inflammatory mediators. Chemokines tend not to suppress cellular responses to other attractants and are present in abundance during inflammatory insults. Instead, when the neutrophil is exposed to an environment in which multiple, cell-derived, inflammatory mediators are present, the cells must process and prioritize these signaling pathways simultaneously [6 , 47 , 48 ]. Our evidence suggests that depending on the nature of the signals (directional vs. random), there may be opposite effects on the migratory response, supporting a complicated cross-talk between these inflammatory mediators.

We provide evidence that p38 MAPK is critical for the inhibitory effects of TNF-{alpha} on neutrophil polarization and migration. These findings are in accordance with a recent paper that demonstrates that TNF-{alpha} is inhibitory to fibroblast filopodia formation through a p38 MAPK pathway [49 ]. It is interesting that their findings suggest that TNF-{alpha} initially promotes filopodial formation through activation of cdc42 but subsequently, activates p53 through a p38 MAPK-dependent pathway, thereby inhibiting filopodia formation. Similar to our findings, they show that inhibition of p38 MAPK promotes filopodia formation in the presence of TNF-{alpha} in murine embryonic fibroblasts [49 ]. Our data support this hypothesis and suggest that p38 MAPK signaling may in fact be inhibitory to motility by regulating cell polarization.

In summary, we have found that TNF-{alpha} inhibits neutrophil migration and chemotaxis by promoting firm adhesion and inhibiting cell polarization. The nature of the signals provided to neutrophils can yield divergent effects, as demonstrated by the inhibitory effects of TNF-{alpha} on neutrophil chemotaxis but not chemokinesis in response to IL-8. We also show that p38 MAPK, which is required for migration to end-target chemoattractants, such as fMLP [35 ], is inhibitory to neutrophil polarization and migration in the presence of TNF-{alpha}. We hypothesize that TNF-{alpha}-mediated effects on neutrophil adhesion and migration are key mechanisms that promotes neutrophil retention within inflammatory sites via a p38 MAPK pathway. These findings suggest that the anti-inflammatory effects of p38 MAPK inhibitors may be mediated in part by anti-TNF-{alpha} effects and may provide a powerful target to control chronic inflammation.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institute of Health Grant #1PO1AI50500-01. The authors thank Brian Kayon for his invaluable assistance in cell tracking. We also thank Kate Cooper for her critical review of this manuscript.

Received February 2, 2005; revised March 18, 2005; accepted March 25, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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