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Originally published online as doi:10.1189/jlb.0103038 on October 23, 2003

Published online before print October 23, 2003
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(Journal of Leukocyte Biology. 2004;75:286-292.)
© 2004 by Society for Leukocyte Biology

Aggregation of ß2 integrins activates human neutrophils through the I{kappa}B/NF-{kappa}B pathway

Cheol Hyeon Kim1, Kyoung-Hee Lee, Choon-Taek Lee, Young Whan Kim, Sung Koo Han, Young-Soo Shim and Chul-Gyu Yoo2

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University College of Medicine; Clinical Research Institute, Seoul National University Hospital; and Lung Institute, Medical Research Center, Seoul National University, Korea

2 Correspondence: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. E-mail: cgyoo{at}snu.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are now considered central to the pathogenesis of most forms of acute lung injury. Neutrophils do not cause damage while suspended in the bloodstream; however, a release of cytotoxic agents occurs when neutrophils are adherent to endothelium, epithelium, or extracellular matrix proteins in the interstitium. Such neutrophil adherence is mediated predominantly through ß2 integrins (CD11/CD18) on its surface. This study was undertaken to investigate whether the I{kappa}B/nuclear factor (NF)-{kappa}B cascade is involved in this ß2 integrin-mediated activation of human neutrophils. ß2 Integrin Mac-1 (CD11b/CD18) aggregation was induced by antibody cross-linking of the integrins on the cell surface. ß2 Integrin aggregation induced interleukin-1ß and tumor necrosis factor-{alpha} production, which suggests the activation of neutrophils by ß2 integrin. I{kappa}B{alpha} was markedly degraded at 1 h, and NF-{kappa}B–DNA-binding activity markedly increased 2 h after ß2 integrin aggregation, which activated I{kappa}B kinase activity at 1 h. ß2 Integrin-induced cytokine production was suppressed by MG132 or SN50 pretreatment, which blocked the activation of NF-{kappa}B. These findings suggest that the activation of human neutrophils by ß2 integrin aggregation is mediated through the activation of the I{kappa}B/NF-{kappa}B pathway.

Key Words: adhesion • acute lung injury • CD11b/CD18 • IL-1ß • TNF-{alpha}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute lung injury (ALI) is a syndrome characterized by increased alveolar–capillary permeability and hypoxemia. Histologically, the hallmark of ALI is the accumulation of neutrophils in the microvasculature of the lung [1 ]. In general, the activation of neutrophils leads to the release of multiple cytotoxic products, growth factors, cytokines, and chemokines, which may enhance the inflammatory response [2 ]. Given this destructive potential, it has been proposed that neutrophils may be important in the pathogenesis of ALI [3 ]. This hypothesis has been supported in numerous in vitro and in vivo studies, and neutrophils are now considered central to the pathogenesis of most forms of ALI [2 ].

Neutrophils do not cause damage while suspended in the bloodstream; however, a release of cytotoxic agents occurs when neutrophils are adherent to endothelium or epithelium or are in contact with extracellular matrix proteins in the interstitium [4 5 6 7 ]. Thus, adhesion of neutrophil is important in inflammatory tissue injury. Neutrophil adherence to endothelial cells is mediated predominantly through interactions between ß2 integrins on the neutrophil surface and intercellular adhesion molecule 1 located on the endothelial surface. ß2 Integrins are heterodimers consisting of a common ß subunit (CD18) and an {alpha} subunit (CD11a, CD11b, CD11c, or CD11d) [8 ]. ß2 Integrins, CD11a/CD18 (lymphocyte function-associated antigen-1), CD11b/CD18 (Mac-1), CD11c/CD18 (gp150/95), and CD11d/CD18, are constitutively expressed on the surface of neutrophils [8 ]. The adhesive interactions of ß2 integrins have been reported to initiate intracellular signaling events, leading to the activation of various neutrophil functions [9 10 11 ]. ß2 Integrin-mediated signaling is known to require ß2 integrin aggregation in neutrophils, which is induced by binding to immobilized ligands but not to soluble ligands [12 ].

Nuclear factor-{kappa}B (NF-{kappa}B) is a ubiquitous transcription factor, which is normally sequestered in the cytoplasm in an inactive form by virtue of its association with a class of inhibitory proteins, I{kappa}Bs. The activation of NF-{kappa}B involves the phosphorylation of I{kappa}B by I{kappa}B kinase (IKK). The phosphorylation of I{kappa}B is followed by its degradation and the nuclear translocation of NF-{kappa}B. In the nucleus, NF-{kappa}B binds to target DNA elements and positively regulates the transcription of genes involved in immune and inflammatory responses, cell growth, and apoptosis [13 14 15 ]. Over the last decade, many studies on the basic biological characteristics of inflammation and tissue injury have implicated proinflammatory cytokine-mediated tissue injury in the pathogenesis of a wide variety of inflammatory disorders. As the majority of the genes for inflammatory mediators, such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-2, IL-6, IL-8, lymphotoxin, granulocyte macrophage-colony stimulating factor, ß-interferon, and adhesion molecules, has a {kappa}B site in the 5'-flanking region, their transcription is regulated by NF-{kappa}B activation [13 14 15 ]. Thus, the activation of the NF-{kappa}B transcriptional factor plays a central role in inflammation through its ability to induce the transcription of proinflammatory genes [13 14 15 ]. Indeed, NF-{kappa}B dysregulation is associated with various inflammatory diseases [16 17 18 ].

Neutrophil-induced tissue destruction results from the release of inflammatory mediators, which are mediated by the ß2 integrin [19 ]. In addition, most of the proinflammatory cytokines are regulated at the level of gene transcription by transcription factor NF-{kappa}B [14 , 20 ]. These findings led us to speculate that the ß2 integrin-mediated activation of neutrophils may be related to the activation of the I{kappa}B/NF-{kappa}B cascade. In human monocytes, it has been reported that ß2 integrin-mediated cytokine production results from NF-{kappa}B activation [21 , 22 ]. However, whether the activation of NF-{kappa}B is involved in the adhesion-induced activation of human neutrophils is currently unknown.

In this study, we investigated whether the I{kappa}B/NF-{kappa}B cascade is involved in the ß2 integrin-mediated activation of human neutrophils. We found that ß2 integrin Mac-1 (CD11b/CD18) aggregation induced IKK activation, I{kappa}B{alpha} degradation, NF-{kappa}B activation, and subsequent proinflammatory cytokine production. ß2 Integrin-induced cytokine production was suppressed by pretreatment with two different inhibitors of NF-{kappa}B activation, MG132 and SN50. These findings suggest that the activation of human neutrophils by ß2 integrin aggregation is mediated through the activation of the I{kappa}B/NF-{kappa}B pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of human neutrophils
Human blood was collected from healthy donors by venipuncture with a heparinized (10 units/mL) syringe. Neutrophils and monocytes (95–98% purity) were separated under endotoxin-free conditions using Ficoll-Hypaque centrifugation [23 ], and the neutrophils were subsequently purified by dextran sedimentation and hypotonic lysis of the residual erythrocytes. Cells were resuspended in HEPES buffer supplemented with 0.25% bovine serum albumin (BSA) and 0.1% glucose. The isolated samples contained >98% neutrophils by Wright staining and differential counting and demonstrated >98% trypan blue exclusion.

Reagents
Histopaque-1077 was purchased from Sigma Chemical Co. (St. Louis, MO). Mouse anti-human CD11b monoclonal antibody (mAb; BCA2) and the enzyme-linked immunosorbent assay (ELISA) kit for IL-1ß and TNF-{alpha} were supplied by R&D Systems (Minneapolis, MN). The F(ab')2 fragments of the secondary polyclonal goat anti-mouse immunoglobulin G (IgG) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-very late-activating antigen-4 (VLA-4) antibody was purchased from Chemicon International (Temecula, CA). Rabbit polyclonal anti-p65, anti-p50, anti-IKK{alpha}, antiphosphorylated extracellular signal-regulated kinase (ERK), antiphosphorylated p38 antibodies, and recombinant glutathione S-transferase (GST)–I{kappa}B{alpha} were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Dr. Stephen Haskill (Department of Microbiology and Immunology, University of North Carolina, Chapel Hill) kindly provided rabbit polyclonal antibody (No. 9) against N-terminal I{kappa}B{alpha}. Goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) and T4 polynucleotide kinase were purchased from Promega (Madison, WI). Protein-G Sepharose beads and an enhanced chemiluminescence (ECL) kit were supplied by Amersham Pharmacia Biotech (Uppsala, Sweden). Protease inhibitors were obtained from Roche (Mannheim, Germany). The proteasome inhibitor N-carbobenzoxyl-Leu-Leu-Leu-leucinal (MG132) was purchased from Peptide Institute (Osaka, Japan). [{gamma}-32P]Adenosine 5'-triphosphate (ATP) was supplied by ICN Pharmaceuticals (Costa Mesa, CA). SN50, a cell-permeable, inhibitory peptide of the nuclear translocation of NF-{kappa}B, was purchased from Biomol (Plymouth Meeting, PA).

ß2 Integrin Mac-1 (CD11b/CD18) aggregation
Neutrophils (5x106/mL) were incubated with 10 µg/mL mouse anti-human CD11b mAb in HEPES buffer, supplemented with 0.25% BSA and 0.1% glucose for 20 min at room temperature under gentle rotation. After two washes, neutrophils were suspended in HEPES buffer (5x106/mL), supplemented with 0.25% BSA and 0.1% glucose. ß2 Integrin aggregation was induced by cross-linking the primary mAb using F(ab')2 fragments of the secondary antibody at a final dilution of 1:20.

IL-1ß and TNF-{alpha} ELISA
Cell-culture supernatants of neutrophils were collected and stored at -70°C until analyzed. IL-1ß and TNF-{alpha} concentrations were quantitated using an ELISA kit according to the manufacturer’s specifications.

Western blot analysis
Stimulation of neutrophils by ß2 integrin aggregation was terminated by addition of one-third volume of 3x Laemmli buffer. All samples were immediately heated for 6 min at 100°C and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Aliquots of total cell lysates containing 30 µg total protein were resolved on 10% SDS-PAGE and transferred to nitrocellulose. The membranes were blocked with 5% skim milk–phosphate-buffered saline (PBS)/0.1% Tween 20 for 1 h before overnight incubation at room temperature with rabbit anti-I{kappa}B{alpha} antibody diluted 1:1000 in 5% skim milk–PBS/0.1% Tween 20. Membranes were washed three times in 1x PBS/0.1% Tween 20 and were incubated with HRP-conjugated secondary antibody, diluted 1:2000 in 5% skim milk–PBS/0.1% Tween 20 for 1 h. Following successive washes, membranes were developed with an ECL kit.

Electrophoretic mobility shift assays (EMSA)
The NF-{kappa}B DNA binding activity was assessed as described previously [24 ]. Briefly, the nuclear extracts were incubated for 20 min at room temperature with a radiolabeled NF-{kappa}B consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3'). In the competition experiments, a 50-fold molar excess of the unlabeled oligonucleotide was added to the binding reaction. The DNA–protein complexes were resolved on 4% nondenaturing polyacrylamide gels. The gels were dried and autoradiographed at -70°C.

IKK assay
The IKK activity was assessed using an in vitro kinase assay as described previously [25 ]. In short, the IKK complex was immunoprecipitated with anti-IKK{alpha} antibody, diluted 1:100. The immunoprecipitates were incubated at 30°C for 30 min in a kinase buffer containing 0.5 µg GST–I{kappa}B{alpha} (containing amino acids 1–317) and 10 µCi [{gamma}-32P]ATP. The kinase-reaction products were subjected to SDS-PAGE in 10% gels followed by transfer to a nitrocellulose membrane and analysis by autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Aggregation of ß2 integrins induced the production of cytokines in neutrophils
To determine whether ß2 integrin-mediated adhesive interactions are sufficient to trigger cytokine production in human neutrophils, antibody cross-linking of CD11b on the cell surface mimicked adhesion. The 18-h incubation of neutrophils in medium alone did not induce the production of IL-1ß or TNF-{alpha}. As a control group, we used treatment with mouse anti-human Ig, F(ab')2 fragments alone, and antibody to VLA-4, as the integrin VLA-4 is thought to be absent on neutrophils. Mouse anti-human Ig or anti-VLA-4 antibody did not increase the concentrations of IL-1ß and TNF-{alpha}. Treatment with F(ab')2 fragments alone induced only a slight increase in IL-1ß and TNF-{alpha} concentrations. However, the production of IL-1ß increased 120-fold by cross-linking ß2 integrins compared with the untreated cells (25±4.1 vs. 3010±137 pg/ml; Fig. 1A ). Aggregation of ß2 integrins also induced the production of TNF-{alpha} (12±1.7 vs. 1250±84 pg/ml; Fig. 1B ). These results indicate that the cross-linking ß2 integrins activate human neutrophils.



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  		Figure 1. Aggregation of ß2 integrin Mac-1 (CD11b/CD18) induces IL-1ß and TNF-{alpha} production in human neutrophils. Isolated human neutrophils (106/well) in suspension were untreated, treated with mouse anti-human Ig for 20 min, F(ab')2 fragments of the secondary antibody for 60 min, or anti-VLA-4 antibody for 20 min. To induce ß2 integrin aggregation, neutrophils were treated with 10 µg/ml mouse anti-human CD11b mAb for 20 min. After placing fresh media, cells were incubated with F(ab')2 fragments of the secondary antibody at a final dilution of 1:20 for 60 min. Culture media were removed and replaced with fresh media. Cells were then incubated in a 5% CO2 incubator at 37°C for 18 h. ELISA measured IL-1ß (A) and TNF-{alpha} (B) in the supernatant. Values are mean ± SD (n=4). 1, Untreated, freshly isolated neutrophils; 2, untreated neutrophils incubated for 18 h; 3, treated with mouse Ig; 4, treated with F(ab')2 fragments; 5, treated with anti-VLA-4 antibody; 6, integrin aggregation. *, P < 0.05, versus unstimulated control.

 
ß2 Integrin aggregation leads to the activation of the I{kappa}B/NF-{kappa}B cascade
To investigate whether the increase in cytokine production by ß2 integrin aggregation in human neutrophils is related to the I{kappa}B/NF-{kappa}B cascade, we first examined the effect of ß2 integrin aggregation on the activity of IKK, which increased 1 h after antibody cross-linking (Fig. 2 ). As the phosphorylation of I{kappa}B{alpha} by IKK is followed by its degradation, we next evaluated the time-dependent degradation of I{kappa}B{alpha}. The level of I{kappa}B{alpha} expression decreased 1 h after antibody cross-linking, which suggests the degradation of I{kappa}B{alpha}; I{kappa}B{alpha} expression returned to basal levels 2 h after antibody cross-linking (Fig. 3 ). Time-dependent NF-{kappa}B–DNA-binding activity was investigated by EMSA. NF-{kappa}B–DNA-binding activity increased 2 h after ß2 integrin aggregation. This increase in NF-{kappa}B–DNA-binding activity was abolished in the presence of an excess of unlabeled NF-{kappa}B probe ("cold probe"), suggesting the specificity of binding (Fig. 4 ). Overall, the I{kappa}B/NF-{kappa}B cascade was activated by ß2 integrin aggregation in human neutrophils. These findings suggest the possibility that the activation of human neutrophils by ß2 integrin aggregation is related to the activation of the I{kappa}B/NF-{kappa}B cascade.



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  		Figure 2. IKK activity is increased by ß2 integrin aggregation in human neutrophils. Isolated human neutrophils (106/well) in suspension were treated with 10 µg/ml mouse anti-human CD11b mAb for 20 min and were then incubated with F(ab')2 fragments of the secondary antibody at a final dilution of 1:20 at 37°C for the indicated times. IKK complex was immunoprecipitated using anti-IKK{alpha} antibody, and IKK assays were performed as described in Materials and Methods (upper panel). To ensure that equal amounts of IKK complex were immunoprecipitated, the membrane was stripped and reprobed with anti-IKK{alpha} antibody (lower panel). Results are representative of three separate experiments.

 


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  		Figure 3. ß2 integrin aggregation induces I{kappa}B{alpha} degradation in human neutrophils. Isolated human neutrophils (106/well) in suspension were treated with 10 µg/ml mouse anti-human CD11b mAb for 20 min and were then incubated with F(ab')2 fragments of the secondary antibody at a final dilution of 1:20 at 37°C for the indicated times. Equal amounts of whole-cell lysates were separated on 10% SDS-PAGE and analyzed for I{kappa}B{alpha} by Western blot analysis using rabbit polyclonal anti-I{kappa}B{alpha} antibody (upper panel). To confirm equal protein loading, the membrane was stripped and reprobed with ß-actin antibody (lower panel). Results are representative of three separate experiments.

 


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  		Figure 4. ß2 integrin aggregation activates NF-{kappa}B in human neutrophils. Isolated human neutrophils (106/well) in suspension were treated with 10 µg/ml mouse anti-human CD11b mAb for 20 min and were then incubated with F(ab')2 fragments of the secondary antibody at a final dilution of 1:20 at 37°C for the indicated times. Nuclear extracts were prepared and subjected to EMSA with a NF-{kappa}B site DNA probe as described in Materials and Methods. In a competition experiment, nuclear extracts were incubated with an unlabeled NF-{kappa}B oligonucleotide (cold probe) before the addition of the labeled NF-{kappa}B oligonucleotide probe to the binding mixture. The inducible NF-{kappa}B–DNA complex is indicated by NF-{kappa}B; ns, nonspecific band. Results are representative of three separate experiments.

 
Blockade of the I{kappa}B/NF-{kappa}B cascade suppresses ß2 integrin-mediated cytokine production
To confirm that the activation of the I{kappa}B/NF-{kappa}B cascade is a critical determining factor of ß2 integrin-mediated neutrophil activation, we examined the cytokine production by antibody cross-linking after blocking the activation of the I{kappa}B/NF-{kappa}B cascade. The activation of the I{kappa}B/NF-{kappa}B cascade was blocked by two approaches: One was blocking the degradation of I{kappa}B{alpha} by proteasome inhibitor MG132, and the other was inhibiting the nuclear translocation of NF-{kappa}B by SN50 [26 ]. I{kappa}B{alpha} degradation induced by ß2 integrin aggregation was suppressed by proteasome inhibition with MG132 (Fig. 5 ). To investigate whether MG132 or SN50 affects the survival of neutrophils, the viability of neutrophils was assessed by flow cytometry using an Annexin V–fluorescein isothiocyanate apoptosis detection kit I. Neither MG132 nor SN50 treatment affected the viability of neutrophils (untreated control: 69.14±4.8%; vehicle control: 63.1±5.2%; MG132 treatment: 57.16±5.7%; SN50 treatment: 60.46±4.9%). We also evaluated whether MG132 or SN50 globally perturbs neutrophil function. The effect of MG132 or SN50 on the lipopolysaccharide (LPS)-induced activation of mitogen-activated protein kinases, including ERK and p38, was analyzed. Pretreatment with MG132 or SN50 did not alter LPS-induced phosphorylation of ERK and p38 in human neutrophils (Fig. 6 ). In addition, LPS-induced phosphorylation of c-jun NH2-terminal kinase was not affected by MG132 or SN50 pretreatment (data not shown). ß2 Integrin-mediated IL-1ß production (2111±167 pg/ml) was suppressed by pretreatment with MG132 (381±25 pg/ml) or SN50 (169±6.4 pg/ml; Fig. 7A ). TNF-{alpha} production by antibody cross-linking (813±20 pg/ml) also decreased dramatically after pretreatment with MG132 (64±4.9 pg/ml) or SN50 (17±2.0 pg/ml; Fig. 7B ). These results indicate that the activation of the I{kappa}B/NF-{kappa}B cascade is a critical pathway to ß2 integrin-mediated neutrophil activation.



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  		Figure 5. Proteasome inhibition suppresses ß2 integrin aggregation-induced degradation of I{kappa}B{alpha} in human neutrophils. Isolated human neutrophils were incubated in the presence or absence of MG132 (200 µM) for 30 min at 37°C. Neutrophils were treated with 10 µg/ml mouse anti-human CD11b mAb and were then incubated with F(ab')2 fragments of the secondary antibody at a final dilution of 1:20 at 37°C for 60 min. Equal amounts of cellular extracts were separated on 10% SDS-PAGE. Western blot analysis for I{kappa}B{alpha} was performed using rabbit polyclonal anti-I{kappa}B{alpha} antibody (upper panel). To confirm equal protein loading, the membrane was stripped and reprobed with ß-actin antibody (lower panel). Results are representative of three separate experiments.

 


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  		Figure 6. Pretreatment with MG132 or SN50 does not disturb LPS-induced phosphorylation of ERK and p38 in human neutrophils. Isolated human neutrophils were incubated with MG132 (200 µM) or SN50 (100 µg/ml) for 50 min and were then stimulated with LPS (10 µg/ml) for 60 min at 37°C. Equal amounts of whole-cell lysates were separated on 10% SDS-PAGE and analyzed for phosphorylated ERK (p-ERK) and phosphorylated p38 (p-p38) by Western blot analysis (upper panels). To confirm equal protein loading, the membrane was stripped and reprobed with ß-actin antibody (lower panels). Results are representative of three separate experiments. 1, Vehicle control; 2, MG132; 3, SN50; 4, LPS; 5, MG132 + LPS; 6, SN50 + LPS.

 


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  		Figure 7. ß2 Integrin-induced production of IL-1ß and TNF-{alpha} is related to the I{kappa}B/NF-{kappa}B pathway. Isolated human neutrophils were incubated with MG132 (200 µM) or SN50 (100 µg/ml) for 30 min at 37°C. Neutrophils were treated with 10 µg/ml mouse anti-human CD11b mAb for 20 min. After placing fresh media, cells were incubated with F(ab')2 fragments of the secondary antibody at a final dilution of 1:20 for 60 min. Culture media were removed and replaced with fresh media. Cells were then incubated in a 5% CO2 incubator at 37°C for 18 h. ELISA measured IL-1ß (A) and TNF-{alpha} (B) in the supernatant. Values are mean ± SD (n=8). 1, Vehicle control; 2, integrin aggregation; 3, MG132 + integrin aggregation; 4, SN50 + integrin aggregation. *, P < 0.05, versus integrin aggregation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Adhesion molecules of the integrin family mediate the recruitment of neutrophils to sites of inflammation by binding to specific ligands and allowing cell–cell and cell–substrate interactions [8 ]. Integrins also transduce signals into the cells, which are thought to control adhesion-related processes, including firm attachment and spreading [27 ]. Furthermore, integrins contribute to the activation of various neutrophil functions [10 , 28 , 29 ]. Although much progress has been made in the understanding of the adhesive functions of the ß2 integrins, the intracellular events that follow their ligand interactions and allow adhesion-mediated cellular responses are still incompletely understood.

In this study, ß2 integrin-mediated adhesive interactions were mimicked by aggregation of the ß2 integrins induced by antibody cross-linking of the anti-CD11b mAb by F(ab')2 fragments of a secondary antibody. This was performed, as efficient integrin signaling requires integrin aggregation in neutrophils [19 ]. Among the ß2 integrin family members, we focused on Mac-1 (CD11b/CD18), as it seems to represent the dominant activator of cytokine gene expression in neutrophils activated by ß2 integrin aggregation [19 ].

We failed to detect I{kappa}B{alpha} in cellular extracts prepared by conventional procedures (i.e., nonionic detergent lysis or repeated freeze-thaw cycles), which is in agreement with a recent report [30 ]. This is probably a result of the release of proteolytic enzymes, which are normally stored in intracellular granules of neutrophils, resulting in the degradation of various proteins. As the strong ionic detergent SDS could inactivate proteolytic enzymes, we used SDS to prepare cellular extracts.

Phosphorylation of I{kappa}B{alpha} by activated IKK leads to its degradation, which is followed by NF-{kappa}B activation. Thus, the signaling sequence of I{kappa}B/NF-{kappa}B cascade is IKK activation, I{kappa}B{alpha} degradation, the nuclear translocation of NF-{kappa}B, and transcriptional induction of cytokine genes. In this study, a perfect temporal correlation was found between I{kappa}B/NF-{kappa}B signaling and cytokine production. Activation of IKK and I{kappa}B{alpha} degradation was induced 1 h after ß2 integrin aggregation, increase in NF-{kappa}B–DNA-binding activity at 2 h, and IL-1ß and TNF-{alpha} production at 18 h. These findings suggest that the I{kappa}B/NF-{kappa}B cascade may mediate cytokine production by ß2 integrin aggregation. To confirm this further, we examined the effect of blocking NF-{kappa}B activation on IL-1ß and TNF-{alpha} production induced by ß2 integrin aggregation. NF-{kappa}B activation was blocked by two different methods: One was inhibiting I{kappa}B{alpha} degradation, and the other was blocking NF-{kappa}B activation directly. As I{kappa}B{alpha} degradation is mediated through the proteasome pathway, proteasome inhibitor MG132 was used to inhibit I{kappa}B{alpha} degradation. We found that I{kappa}B{alpha} degradation by ß2 integrin aggregation was completely suppressed by pretreatment with MG132 in neutrophils. IL-1ß and TNF-{alpha} production by ß2 integrin aggregation was markedly suppressed by MG132 pretreatment. However, as various other proteins—cyclins, cyclin-dependent kinase inhibitors, and transcription factors, such as myc, p53, c-fos, and c-jun—are also degraded by the proteasome [31 , 32 ], the suppression of ß2 integrin-mediated cytokine production by MG132 pretreatment may be a result of the effects of one or more of these proteins. Thus, we next blocked NF-{kappa}B activation directly using SN50, which is a cell-permeable peptide that inhibits the nuclear translocation of NF-{kappa}B [26 ] and analyzed its effect on cytokine production. SN50 suppressed IL-1ß and TNF-{alpha} production by ß2 integrin aggregation. These findings indicate that neutrophil activation by ß2 integrin aggregation is mediated through the activation of the I{kappa}B/NF-{kappa}B pathway. This result is in accordance with that of Rezzonico et al. [22 ], who found that engagement of ß2 integrins provided activation signals leading to I{kappa}B{alpha} degradation, subsequent nuclear translocation of NF-{kappa}B, and chemokine production in human monocytes. Although they did not investigate the effect of ß2 integrin aggregation on IKK activity, we extended the experiments to IKK assays, showing that ß2 integrin aggregation activated IKK. Taken together, these findings suggest that ß2 integrin-mediated signaling is similar in neutrophils and monocytes.

Little is known about upstream regulator(s) of the I{kappa}B/NF-{kappa}B pathway in ß2 integrin-mediated activation of human neutrophils. In macrophages, ß1 integrin engagement led to the translocation of the adaptor protein c-Cbl to the cell membrane [33 ]. This adhesion-induced redistribution as well as the tyrosine phosphorylation of c-Cbl were found to critically depend on src kinases, forming a complex with c-Cbl, which was also found to form a complex with phosphatidylinositol 3-kinase (PI-3K) upon engagement of ß1 and ß3 integrins, resulting in the activation of PI-3K/Akt in macrophages [34 ]. In a recent study, the most prominent protein that became tyrosine-phosphorylated upon ß2 integrin aggregation was identified as c-Cbl in human neutrophils [35 ], and the redistribution and activation of the src-family tyrosine kinases to the cytoskeletal fraction were observed in human neutrophils upon adhesion to immobilized fibrinogen or incubation on surface-bound anti-ß2 subunit antibodies [36 , 37 ]. These findings implicate various tyrosine kinases in integrin signaling in neutrophils and macrophages. Tyrosine kinases can activate PI-3K. The PI-3K/Akt pathway has been shown to participate in signaling pathways that lead to NF-{kappa}B activation and subsequent production of proinflammatory cytokines in multiple cell types, including murine neutrophils exposed to endotoxin [38 39 40 41 ]. Thus, the upstream regulators of ß2 integrin-mediated activation of the I{kappa}B/NF-{kappa}B pathway may be tyrosine kinases and the PI-3K/Akt pathway in neutrophils. However, direct evidence for this has not been reported.

Adhesion is important in neutrophil activation, and activated neutrophils are now considered central to the pathogenesis of acute lung injury. In addition, NF-{kappa}B-dependent, proinflammatory cytokine production has been suggested as an important pathogenetic mechanism of acute lung injury. This was supported by findings that NF-{kappa}B activation correlated with prognosis and with mortality as a result of sepsis or acute respiratory distress syndrome [42 43 44 45 ]. In the present study, we demonstrated that the activation of human neutrophils by ß2 integrin aggregation is mediated through the activation of the I{kappa}B/NF-{kappa}B pathway. Taken together, integrin-mediated activation of NF-{kappa}B in neutrophils may be related to the pathogenesis of acute lung injury, and this could be a good therapeutic target for new, anti-inflammatory treatment.


   ACKNOWLEDGEMENTS
 
This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (02-PJ1-PG3-20706-0009).


   FOOTNOTES
 
1 Current address: Department of Internal Medicine, Korea Cancer Center Hospital, Seoul 139-706, Korea. Back

Received January 23, 2003; revised September 19, 2003; accepted September 22, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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