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(Circulation Research. 2001;89:859.)
© 2001 American Heart Association, Inc.

Molecular Medicine

Leukocyte Integrin Mac-1 Recruits Toll/Interleukin-1 Receptor Superfamily Signaling Intermediates to Modulate NF-B Activity

Can Shi, Xiaobin Zhang, Zhiping Chen, Martyn K. Robinson, Daniel I. Simon

From the Cardiovascular Division (C.S., X.Z., Z.C., D.I.S.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; and Celltech, Inc (M.K.R.), Slough, England.

Correspondence to Daniel I. Simon, MD, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Tower 3, Boston, MA 02115. E-mail dsimon{at}rics.bwh.harvard.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—— The leukocyte integrin Mac-1 (Mß2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-B activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1–clustered compared with –nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor–associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family–like signaling pathway. Mac-1–dependent activation of NF-B was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-ß–activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family–like cascade to modulate NF-B activity. This represents a new pathway for integrin-dependent modulation of gene expression.

Key Words: leukocytes • integrins • signal transduction • gene expression

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Receptors of the integrin family mediate adhesion of cells to extracellular matrices as well as intercellular interactions that are central to inflammation, immunity, hemostasis, and tumor metastasis.¹ These adhesive interactions transduce "outside-in" signals that control complex cell functions, such as proliferation, differentiation, and survival, and require the regulation of gene expression.² The cytoplasmic domains of integrin and ß subunits do not have intrinsic catalytic activity, indicating that interaction with other transducing molecules is crucial for integrin-mediated signaling. Ligation of ß1- and ß3-integrins has been shown to activate numerous signal transduction pathways, including Ras,³ nonreceptor tyrosine kinases (eg, Src,⁴ Fyn⁵), focal adhesion kinase,⁶ mitogen-activated protein (MAP) kinases,⁷ c-Jun N-terminal kinase (JNK),⁸ and phosphatidylinositol 3-kinase.⁹ The formation of focal adhesion complexes containing both cytoskeletal and catalytic signaling proteins is capable of directly altering gene transcription.²

Neutrophil and monocyte recruitment in acute inflammation is mediated in part by the ß2-integrin family of receptors, LFA-1 (Lß2, CD11a/CD18), Mac-1 (Mß2, CD11b/CD18), and p150,95 (Xß2, CD11c/CD18).¹⁰ Engagement of ß2-integrins by a broad repertoire of ligands generates outside-in signals leading to inflammatory cell activation. In the case of Mac-1, this activation induces the expression of genes encoding for cytokines (eg, IL-1ß^11,12 and TNF-¹³) and tissue factor.¹⁴ Although leukocyte integrins have been implicated in modulating activator protein-1 (AP-1) and nuclear factor-B (NF-B) activity,¹⁵ the signal transduction pathways involved are incompletely defined.

We have recently shown that monoclonal antibody (mAb) blockade¹⁶ or absence¹⁷ of Mac-1 modulates the biological response to vascular injury, reducing vessel wall inflammation and neointimal thickening after experimental angioplasty. This led us to hypothesize that clustering and activation of Mac-1 may initiate a novel gene program that promotes vascular inflammation. Our goal was to identify genes regulated by Mac-1 and to characterize this signaling pathway. Gene transcription was analyzed using cDNA arrays from Mac-1–clustered compared with –nonclustered monocytic THP-1 cells. We found that Mac-1 clustering induced the expression of genes encoding for cytokines, chemokines, transcription factors, signaling intermediates, and numerous expression sequence tags. Increased expression of the signal transducer TRAF6¹⁸ in Mac-1–clustered cells led us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family–like signaling pathway.^18–20 Here, we report that Mac-1 physically associates with IRAK1 to induce NF-B activity through TRAF6 and TAK1. This represents a new pathway for integrin-dependent modulation of gene expression.

	Materials and Methods

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Biological Reagents and Expression Vectors
Anti-CD11b mAbs included LPM19c (DAKO) and LM2/1 (Dr Lloyd Klickstein, Brigham and Women’s Hospital). The stimulating CD18 mAb KIM127²¹ was a gift of Dr Martyn Robinson (Celltech). Anti–human IRAK1 antibodies were from Transduction Laboratories and Santa Cruz Biotechnology, and polyclonal antibodies including anti-human CD11b, MyD88, TRAF6, and TAK1 were purchased from Santa Cruz Biotechnology. Transforming growth factor-ß1 (TGF-ß1) was from Collaborative Research, and 1,25-(OH)₂ vitamin D₃ was from Calbiochem. Wild-type MyD88 and dominant negative [MyDC(155-296)] expression constructs were provided by Dr Ruslan Medzhitov (Yale University School of Medicine).²² Wild-type TRAF6 and dominant negative [TRAF6(289-522)] human expression constructs were provided by Dr David Goeddel (Tularik).¹⁸ Wild-type TAK1 and TAK1 binding protein (TAB1), as well as a kinase-defective dominant negative, TAK1(K63W), expression vectors were provided by Dr Jun Ninomiya-Tsuji (Nagoya University, Nagoya, Japan).²⁰

Cell Lines
THP-1 monocytic (American Type Culture Collection) and 293 cells transfected with human CD11b and CD18 (Mac-1 293) were provided by Dr Li Zhang (American Red Cross, Holland Laboratory, Rockville, MD)²³ and were maintained as previously described. ²⁴

Adhesion Assays
Unstimulated and cytokine-treated [1 ng/mL TGF-ß1 and 50 nmol/L 1,25-(OH)₂ vitamin D₃] THP-1 cells were used in adhesion experiments to immobilize fibrinogen, as reported.²⁴ Briefly, tissue culture dishes and 6- to 96-well plates were coated overnight with 10 µg/mL human fibrinogen depleted of plasminogen, von Willebrand factor, and fibronectin (Enzyme Research Laboratory) and then blocked with 0.2% gelatin or coated with gelatin alone. Fibrinogen used was pretreated with END-X B15 Endotoxin Removal Resin (Associates of Cape Cod, Inc) and gelatin was endotoxin-free. Adhesion was induced with the ß2-stimulating KIM 127 mAb (5 µg/mL) in the presence and absence of 10 µg/mL LPM19c, 5 mmol/L EDTA, polymyxin B (10 µg/mL), recombinant human IL-1ß (10 ng/mL, R&D Systems), or recombinant human IL-1R antagonist (50 ng/mL, R&D Systems) at 37°C for 0 to 8 hours. Plates were washed with phosphate-buffered saline (3 to 5 times), and adhesion quantified by measuring the fluorescence of BCECF AM (Molecular Probes)-loaded cells using a Cytofluor II fluorescence multi-well microplate reader (PerSeptive Biosystems). Endogenous IL-1ß production under these adhesion conditions was measured using a commercially available ELISA kit (Pierce Endogen).

Reporter Assays
THP-1 cells (10⁷) were transfected using DEAE/Dextran (Pharmacia) with indicated amounts of expression plasmids, pNF-B-Luc (Stratagene), and pSV-ß-Gal reporter plasmids (Promega). The amount of total DNA was kept constant by adding empty pcDNA3.1 plasmid DNA. After 72 hours, transfected cells were then used in adhesion assays in 6-well plates, as described above, and Mac-1–clustered and –nonclustered cells lysed for measurement of luciferase activity using reagents from Promega. ß-Gal activity was analyzed with High Sensitivity ß-Gal Assay Kit (Stratagene). Luciferase activities were determined and normalized on the basis of cell number using BCECF AM and ß-galactosidase activity. Each experiment was repeated 3 to 5 times with qualitatively similar results.

Electrophoretic Mobility-Shift Assay
Nuclear extracts were prepared as described²⁵ and protein concentration determined and verified by test SDS-PAGE. Nuclear protein (10 µg) was incubated with 0.5 ng ³²P-end-labeled NF-B consensus oligonucleotide (Santa Cruz Biotechnology) in binding buffer, as described.²⁵ Anti-p65 supershift mAb was from Santa Cruz Biotechnology. DNA-protein complexes were then resolved by 5% PAGE gel in buffer containing 45 mmol/L Tris, pH 8.3, 45 mmol/L boric acid, and 1 mmol/L EDTA. Dried gels were subjected to autoradiography. Nuclear extracts were prepared from at least two independent experiments.

cDNA Arrays and Northern Analysis
One microgram of polyA-mRNA extracted from Mac-1–clustered and –nonclustered THP-1 cells was ³²P-labeled through synthesis of first strand cDNA directed by SuperScript II RNase H^- reverse transcriptase (Life Technologies). GeneFilter GF200 (Research Genetics) containing 5184 noncontrol cDNA spots were then successively hybridized with labeled probes according to manufacturer’s protocol. Hybridized filters were then analyzed using a PhosphorImager.

Northern analysis was used to confirm cDNA array data. THP-1 cell mRNA was prepared from total RNA, subjected to formaldehyde gel electrophoresis, and transferred to Duralon-UV membrane (Stratagene). Filters were then hybridized with ³²P-labeled TRAF6 cDNA probe (Research Genetics) in QuickHyb solution (Stratagene).

Immunoprecipitation and Immunoblotting
Forty eight hours after transfection, Mac-1 293 (10⁷) or THP-1 (10⁸) cells were harvested from 6-well plates or dishes, washed once in PBS, and lysed for 1 hour on ice in 0.5 to 1.0 mL lysis buffer (150 mmol/L NaCl, 15 mmol/L Hepes containing 1% Triton X-100, 1 mmol/L PMSF, and Calbiochem protease inhibitor cocktail set III). Cellular debris was removed by centrifugation at 10 000g for 15 minutes. An aliquot of lysate was removed to verify the presence of the transfected proteins in the lysate before immunoprecipitation. The remaining lysates were precleared with two changes of Protein A/G-sepharose during an overnight incubation at 4°C. Precleared lysates were incubated with 4 µg of purified antibody (LM2/1 anti-CD11b mAb) or nonimmune IgG for 6 hours at 4°C. Immune complexes were precipitated with 25 µL of Protein A/G-sepharose during overnight incubation at 4°C. Sepharose beads were washed extensively in lysis buffer. Samples were then eluted from the beads by boiling in SDS sample buffer, run on 8% reducing SDS-PAGE, and transferred to nitrocellulose. The membranes were then blotted with indicated antibodies, and the bands visualized with HRP-conjugated secondary antibody followed by the enhanced chemiluminescence Western blotting detection system (NEN Life Science Products).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To identify molecules involved in transducing integrin-dependent signals, we used cDNA array analysis comparing gene expression in Mac-1–clustered and –nonclustered monocytic THP-1 cells. Mac-1 was clustered by inducing adhesion of THP-1 cells to immobilized fibrinogen. We have previously shown that stimulation of THP-1 cells (which constitutively express Mac-1) with TGF-ß1 and 1,25-(OH)₂ vitamin D₃ increases Mac-1 surface expression 2.0-fold.²⁶ When cytokine-treated THP-1 cells were stimulated with KIM 127, a mAb to CD18 which induces a change in the conformation of CD18 and promotes LFA-1–, Mac-1–, and p150,95-dependent adhesion,²¹ they adhered robustly to wells coated with fibrinogen and blocked with gelatin, but not to wells coated with gelatin alone (Figure 1A). Adhesion of the cells to fibrinogen was inhibited by LPM19c, a mAb that binds to the I-domain of the M subunit of Mac-1 (CD11b) and blocks ligand binding,²⁷ indicating that, under these experimental conditions, THP-1 cell adhesion to fibrinogen is Mac-1–dependent.

View larger version (22K): [in this window] [in a new window]   Figure 1. Clustering of Mac-1 induces activation of NF-B and expression of TRAF6. A, Clustering of Mac-1. Cytokine-treated THP-1 cells were added to fibrinogen-coated and gelatin-blocked wells. Adhesion was promoted by the addition of the anti-CD18 stimulating mAb KIM 127 (5 µg/mL) in the presence and absence of anti-CD11b mAb LPM19c (10 µg/mL) or EDTA (5 mmol/L). After washing, adhesion was quantified by measuring the fluorescence of BCECF AM–loaded THP-1 cells. Triplicate determination (mean±SD) representative of 3 to 5 separate experiments. B, EMSA demonstrating Mac-1–dependent NF-B binding activity. THP-1 cell adhesion to fibrinogen was stimulated by KIM127 in the absence or presence of EDTA or LPM19c. Nuclear extracts from these THP-1 cells were analyzed by EMSA with labeled NF-B oligonucleotide probes. Some samples were preincubated with 20- and 200-fold excess unlabeled NF-B oligonucleotide, anti-p65 mAb, or nonimmune IgG. C, Clusterting of Mac-1 induces expression of TRAF6. Northern blots containing 1 µg/lane of mRNA isolated from Mac-1–clustered and –nonclustered THP-1 cells were probed with 32P-labeled TRAF6 cDNA. RNA size is indicated in kilobases.

We then asked whether clustering of Mac-1 induced NF-B transcriptional activity. Nuclear extracts from THP-1 cells were prepared and used in electrophorectic mobility-shift assays (EMSA) to test the ability of NF-B complexes to bind to a labeled NF-B probe. Clustering of Mac-1 resulted in increased binding of the NF-B probe that was supershifted with anti-p65 mAb, but not nonimmune IgG (Figure 1B). Formation of the protein-DNA complex was inhibited by a 20- to 200-fold excess of unlabeled NF-B oligonucleotide. Integrin- and, in particular, Mac-1–dependence was established by the finding that EDTA and the anti-CD11b mAb LPM19c, which blocks fibrinogen binding to Mac-1,²⁷ impaired protein-DNA binding.

Mac-1–dependent gene expression was examined using a GeneFilter cDNA array and labeled cDNA probes prepared from adherent (ie, Mac-1–clustered) THP-1 cells and from cells treated identically except that adhesion was inhibited by LPM19c (ie, Mac-1–nonclustered). After hybridization, array analysis indicated Mac-1–dependent expression of a cDNA clone corresponding to TNF receptor associated factor 6 (TRAF6), a signaling intermediate in the Toll/IL-1 receptor family cascade¹⁸ (data not shown). Induction of TRAF6 gene expression by Mac-1 was confirmed by Northern analysis using labeled TRAF6 cDNA (Figure 1C).

After binding to the cell surface IL-1 receptors, IL-1 triggers a signaling cascade that results in activation of NF-B, a ubiquitously expressed transcription factor that controls the expression of many immune- and inflammatory-response genes.²⁸ Several adapter proteins are involved in events associated with this cytokine-induced effect including IL-1 receptor accessory protein,²⁹ MyD88,^22,30 IRAK1,¹⁹ TRAF6,¹⁸ and a succession of kinase enzymes, including TAK1,²⁰ NF-B–inducing kinase (NIK),³¹ and IB kinases.³² To determine if TRAF6 directly regulated Mac-1–dependent NF-B transcriptional activity, we transiently cotransfected THP-1 cells with an NF-B luciferase reporter gene in the presence of wild-type or a dominant negative TRAF6 construct, TRAF6(289-522).¹⁸ These TRAF6 constructs have been used previously to characterize IL-1 signaling in 293 cells: overexpression of wild-type TRAF6 activated, and dominant negative TRAF6(289-522) inhibited, NF-B activation signaled by IL-1 but not by TNF.¹⁸ Transfected THP-1 cells were stimulated with KIM127 to induce adhesion to fibrinogen and adherent cells were then assayed for luciferase activity. Adhesion of transfected cells to fibrinogen resulted in a 23-fold increase in the relative activation of the NF-B reporter compared with cells stimulated with KIM127 but exposed to wells coated with gelatin alone (ie, Mac-1–nonclustered, vector alone) (Figure 2A). Therefore, in accordance with our EMSA findings (Figure 1B), this increase in NF-B activity is dependent on the clustering of Mac-1. NF-B activity after Mac-1 clustering was potentiated by wild-type TRAF6, and attenuated dose-dependently by dominant negative TRAF6 (percent maximal inhibition=76±9%, n=3), compared with vector alone (Figure 2A); in contrast, these TRAF6 constructs did not affect NF-B activity in Mac-1–nonclustered THP-1 cells. Taken together, these observations indicate that TRAF6 is involved in the regulation of Mac-1–dependent NF-B transcriptional activity.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of wild-type and dominant negative TRAF6, TAK1, and MyD88 on Mac-1–dependent NF-B activation in THP-1 cells. A, TRAF6 and Mac-1–dependent NF-B activation in THP-1 cells. THP-1 cells (2–4x107) were transfected with 5 µg pNF-luc reporter gene plasmid, 5 µg pRSV-ß-gal, and expression plasmids for 40 µg pcDNA3.1 (vector), 40 µg wild-type TRAF6, and different amounts of dominant negative TRAF6(289-522) (10, 25, and 40 µg) supplemented with pcDNA3.1 to achieve 40 µg total DNA. Luciferase activities in Mac-1–clustered (+) and –nonclustered (-) cells were determined, normalized on the basis of cell number and ß-galactosidase activity, and plotted as fold-activation compared with nonclustered, vector alone treatment. B, TAK1 and Mac-1–dependent NF-B activation in THP-1 cells. Identical transfection procedure outlined above except for use of expression plasmids for 40 µg pcDNA3.1, wild-type TAK1/TAB1 (20 µg of each DNA combined), and different amounts of dominant negative TAK1(K63W) (10, 25, and 40 µg) supplemented with pcDNA3.1 to achieve 40 µg total DNA. C, MyD88 and Mac-1–dependent NF-B activation in THP-1 cells. Identical transfection procedure outlined above except for use of expression plasmids for 40 µg pcDNA3.1, 40 µg wild-type MyD88, and different amounts of dominant negative MyDC(155-296) (10, 25, and 40 µg) supplemented with pcDNA3.1 to achieve 40 µg total DNA. Nonclustered cells were also treated with 10 ng/mL IL-1ß. Triplicate determination (mean±SD) representative of 2 to 4 independent experiments.

TAK1 is a MAP kinase kinase kinase immediately downstream of TRAF6.²⁰ THP-1 cell transfection experiments were also performed using wild-type and dominant negative TAK1 constructs cotransfected with NF-B luciferase reporter. Because TAK1 activity requires the presence of its binding protein TAB1, transfection of wild-type TAK1 was accompanied by cotransfection with TAB1.²⁰ Similar to the upstream signaling intermediate TRAF6, wild-type TAK1 potentiated and dominant negative TAK1 attenuated (percent maximal inhibition=63±8%, n=3) NF-B activity after Mac-1 clustering (Figure 2B). TAK1 constructs had no effect on NF-B activity in Mac-1–nonclustered cells.

Next, we examined the role of the upstream signaling intermediate MyD88 in Mac-1–dependent NF-kB activity by transfecting cells with wild-type or a dominant negative MyD88 construct, MyDC(155-296). These Myd88 constructs have been used previously to characterize IL-1 signaling in 293 cells.²² NF-B activity after Mac-1 clustering in THP-1 cells was largely unaffected by these MyD88 constructs (Figure 2C). Importantly, overexpression of wild-type MyD88 potentiated, and dominant negative MyDC(155-296) attenuated, NF-B activity stimulated by IL-1ß in nonclustered THP-1 cells (Figure 2C). Taken together, these observations indicate that TRAF6 and TAK1, but unlikely MyD88, are involved in the regulation of Mac-1–dependent NF-B transcriptional activity.

To eliminate the possibility that NF-B reporter activity was mediated by endogenously produced IL-1,¹³ we examined the effects of a recombinant IL-1 receptor antagonist (IL-1RA) on NF-B activity. The concentration of IL-1ß under identical conditions of Mac-1 clustering was determined (4.2±2.9 pg/mL; mean±SD, n=15) and cells were incubated with 50 ng/mL IL-1RA, resulting in a 10⁴-fold excess concentration that is sufficient to block the biological response of IL-1ß. Neither stimulation of Mac-1–dependent NF-B activity by wild-type TRAF6 or TAK1 nor attenuation by dominant negative TRAF6 or TAK1 was affected by addition of IL-1RA (data not shown), indicating that binding of IL-1 to THP-1 cell IL-1 receptors was not responsible for NF-B activation under these experimental conditions. Furthermore, unlike IL-1 signaling, which is modulated by MyD88 (Figure 2C and Medzhitov et al²²), Mac-1–dependent NF-B activity was largely unaffected by wild-type or dominant negative MyD88, suggesting that Mac-1 signaling cannot be explained solely by paracrine IL-1 effects and is distinct from IL-1 signaling.

To eliminate the possibility that NF-B reporter activity was mediated by contaminant endotoxin,³³ we also examined the effects of polymyxin B on NF-B activity. Mac-1–dependent NF-B activity assessed by EMSA and reporter assays was unaffected by addition of polymyxin B (data not shown), indicating that endotoxin contamination was not responsible for NF-B activation under these experimental conditions. Finally, the binding of nuclear NF-B complexes to NF-B probe was inhibited by the addition of reagents that solely blocked Mac-1 adhesion to fibrinogen (eg, EDTA or LPM19c) (Figure 1B), ruling out further an endotoxin effect.

In IL-1 receptor signaling, the association of IRAK1 with the IL-1 receptor, IL-1 receptor accessory protein, and the adapter protein MyD88 is accompanied by the recruitment and extensive phosphorylation of IRAK1.¹⁹ A marker of IRAK1 phosphorylation is the altered migration of IRAK by immunoblot analysis with nonphosphorylated forms of IRAK1 appearing at 80kDa and phosphorylated forms up to 100kDa. Therefore, we examined the status of IRAK1 using immunoblot analysis after clustering Mac-1. Mac-1–dependent adhesion to fibrinogen led to a significant change in the pattern of IRAK1-immunoreactive bands with the rapid appearance of 100kDa forms of IRAK1 (Figure 3A). Cells in which Mac-1 was not clustered contained predominantly lower molecular weight forms of IRAK1. Thus, clustering of Mac-1 leads to similar changes in IRAK1, as previously observed for IL-1 receptor signaling.

View larger version (23K): [in this window] [in a new window]   Figure 3. Physical association of Mac-1 and IRAK1. A, Effect of Mac-1 clustering on IRAK1. THP-1 cells were treated with KIM127 to stimulate adhesion to fibrinogen. At indicated times, adherent cells were lysed in situ with SDS-PAGE–reduced sample buffer and then immunoblotted sequentially with anti-IRAK and anti-tubulin mAbs. B, Physical association of Mac-1 and IRAK1 in THP-1 cells. Cytokine-treated THP-1 cells (1x108) were treated with 1 mmol/L MnCl2 to activate Mac-143 and promote adhesion to fibrinogen. Adherent cell lysates were prepared and immunoprecipitation performed with anti-CD11b mAb LM2/1 or nonimmune IgG. The immunoprecipitates were blotted with polyclonal anti-IRAK1, stripped, and re-blotted for CD11b to verify immunoprecipitation of CD11b. C, Physical association of Mac-1 and IRAK1 in Mac-1–transfected 293 cells. Mac-1 293 (2x107 to 4x107) cells were transfected with an expression plasmid for wild-type IRAK1. After 48 hours, cell lysates were prepared and immunoprecipitation performed with anti-CD11b mAb LM2/1 or nonimmune IgG. The immunoprecipitates were blotted with polyclonal anti-IRAK1, stripped, and re-blotted for CD11b to verify immunoprecipitation of CD11b. Lysates of the IRAK1-transfected Mac-1 293 cells were immunoblotted with anti-IRAK1 to monitor expression of IRAK1.

Finally, we explored whether there might be a physical basis for the observed functional linkage between Mac-1 and signaling intermediates of the IL-1 receptor pathway. We observed that Mac-1–dependent adhesion to fibrinogen was followed rapidly by the recruitment of IRAK1 into the Triton X-100 soluble fraction (data not shown) that is known to contain Mac-1²⁷; no significant changes were noted in the recruitment of TRAF6 or TAK1 (data not shown). Lysates from THP-1 cells adherent to fibrinogen were prepared and then subjected to coimmunoprecipitation using anti-CD11b mAb. The coimmunoprecipitated products were serially immunoblotted using anti-IRAK1 and anti-CD11b antibodies. As indicated in Figure 3B, IRAK1 coprecipitated with CD11b, but not with nonimmune IgG.

To probe further this physical association between Mac-1 and IRAK1, we turned to 293 cells stably transfected with human Mac-1 (Mac-1 293 cells). We have previously exploited the higher level of Mac-1 expression in Mac-1 293 compared with THP-1 cells to investigate physical interactions between Mac-1 and membrane-associated proteins using immunoprecipitation techniques.³⁴ The expression of Mac-1 in stably transfected 293 cells was evaluated using flow cytometry and confirmed greater (23-fold) CD11b expression in Mac-1 293 compared with THP-1 cells (data not shown). 293 cells over-expressing Mac-1 were cotransfected with wild-type MyD88, IRAK1, TRAF6, or TAK1 and then subjected to coimmunoprecipitation using the CD11b-specific mAb LM2/1. The coimmunoprecipitated products were serially immunoblotted using anti-MyD88, anti-IRAK1, anti-TRAF6, and anti-TAK1 antibodies. As indicated in Figure 3C, IRAK1 coprecipitated with CD11b, but not with nonimmune IgG. In contrast, we were unable to detect comparable association of MyD88, TRAF6, or TAK1 with CD11b.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have shown that Mac-1 recruits a Toll/IL-1 receptor superfamily–like cascade, physically interacting with IRAK1 to induce NF-B activity through TRAF6 and TAK1. This represents a new pathway for integrin-dependent modulation of gene expression (Figure 4). Evidence that NF-B activation depends on outside-in signaling generated by Mac-1 includes the following: (1) NF-B DNA binding was induced by Mac-1–dependent adhesion to fibrinogen and abolished by a mAb that blocks fibrinogen binding to Mac-1; and (2) NF-B activity was modulated by TRAF6 and TAK1 constructs only if Mac-1 was clustered.

View larger version (37K): [in this window] [in a new window]   Figure 4. Schematic diagram of proposed model for Mac-1–dependent NF-B activation. Clustering of Mac-1 leads to association of Mac-1 with the serine-threonine kinase IRAK1 and activation of NF-B that is relayed through TRAF6 and TAK1 (dashed box). The involvement of additional kinases, including NIK and Iß kinase, is neither supported nor refuted by our data, but rather is inferred from a representative model of NF-B signaling proposed by Ninomiya-Tsuji et al. 20

After binding to the cell-surface IL-1 receptor, IL-1 triggers a cascade of signaling events, including activation of JNK and NF-B, which upregulates the expression of inflammatory-related genes in the nucleus.³⁵ The first signaling event is the ligand-induced complex formation of IL-1 receptor and IL-1 receptor accessory protein.²⁹ The adapter protein MyD88 is next recruited to this complex, which in turn enables the association of IRAK1. ^22,30 IRAK1 gets highly phosphorylated, leaves the receptor complex, and interacts with TRAF6.¹⁸ The IRAK1-TRAF6 interaction triggers kinase cascades that lead to the activation of the IB kinases and c-Jun N-terminal kinase,²⁰ which phosphorylate IB and c-Jun, respectively. Although there are many similarities among Toll/IL-1 receptor and Mac-1 signaling, important differences are discernable. Toll and IL-1 receptors use the adapter protein MyD88 immediately upstream of IRAK1.²² In contrast, NF-kB activity stimulated by Mac-1 appears to be likely MyD88-independent. We were unable to identify MyD88 in association with Mac-1 or the Mac-1-IRAK1 complex, even after overexpression of MyD88 in Mac-1 293 cells, and observed no significant effect of MyD88 constructs on Mac-1–dependent NF-B activity. We propose that the interaction of IRAK1 with Mac-1 may involve an unidentified adapter protein (Figure 4). Identification of this adapter protein, the importance of the kinase activity of IRAK1 in NF-B activation by Mac-1, and the undefined role of downstream signaling intermediates, such as NIK and IB kinases, are the focus of ongoing studies.

Integrin-extracellular matrix interactions initiate a variety of signaling events including the induction of calcium transients, changes in cAMP levels, redistribution of phosphatidylinositol 3-kinase, activation of the Na⁺/H⁺ antiporter, and increases in tyrosine phosphorylation.⁹ Integrin-mediated cell adhesion regulates gene expression through the activation of transcription factors. For ß1- and ß3-integrins, there is increasing evidence that gene expression is mediated through integrin-linked kinase, an ankyrin repeat-containing serine-threonine protein kinase that is capable of interacting directly with the cytoplasmic domain of ß1- and ß3- integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions.³⁶ Integrin-linked kinase modulates gene expression by phosphorylating protein kinase B and glycogen synthase kinase-3, thereby promoting ß-catenin translocation to the nucleus and complex formation with the lymphoid enhancer binding factor (LEF-1) transcription factor.

Although leukocyte ß2-integrins have been implicated in modulating AP-1 and NF-B activity,¹⁵ the signal transduction pathways involved have remained incompletely defined. Engagement of Mac-1 and p150,95 by antibodies or soluble ligands has been shown to induce IL-1ß production in human monocytes through MAP kinase–dependent pathways.¹² A direct interaction between signaling intermediates and ß2-integrins has remained elusive until the recent demonstration that the cytoplasmic tail of LFA-1 interacts with the transcriptional coactivator JAB-1 and modulates AP-1 activity by regulating JAB-1 nuclear localization.³⁷ Our observation that Mac-1 associates with IRAK1 and modulates NF-B activity in a cascade involving TRAF6 and TAK1 provides a new pathway for integrin-dependent modulation of gene expression.

Integrin-mediated signaling requires integrin cross-linking, cellular adhesion, and cytoskeletal rearrangement.³⁸ We initiated Mac-1 signaling and NF-B activation in our study by using immobilized fibrinogen to cluster Mac-1. However, because Mac-1 binds a broad repertoire of ligands,³⁹ including, among others, C3bi, intercellular adhesion molecule-1 (ICAM-1), factor X, glycoprotein Ib, and heparin, it is likely that these ligands could also support activation signaling through ligation of Mac-1.

Distinct cell-associated receptors on leukocytes, such as CD14 and homologues of Drosophila Toll, allow the direct recognition of pathogen-associated molecules and trigger natural or innate immune response by inducing the production of inflammatory cytokines and costimulatory molecules that signal subsequently to activate adaptive immunity. Several components of the IL-1 pathway are also used by Toll receptors, including TLR-2 and TLR-4.⁴⁰ because Mac-1 can bind, in a complement-independent fashion, a number of protozoal, bacterial, and fungal components as well as LPS-binding protein (reviewed in Cuzzola et al⁴¹), our observation that Mac-1 is also capable of recruiting Toll/IL-1 receptor signaling intermediates may have significant implications for a role of Mac-1 in innate immunity.

The intensity of inflammation is determined by both the extent of leukocyte recruitment and the proinflammatory actions of infiltrating leukocytes. Leukocyte Mac-1 regulates important leukocyte functions including adhesion, migration, proteolysis, phagocytosis, and oxidative burst.³⁹ Targeting leukocyte receptors like Mac-1 interrupts the adhesive and migratory capability of leukocytes and reduces tissue injury in models of inflammation. NF-B regulates genes encoding for cytokines (TNF-, interleukins, and granulocyte-macrophage colony-stimulating factor), chemokines (macrophage chemotactic protein 1, macrophage inflammatory protein 1, IL-8, eotaxin), adhesion molecules (ICAM-1, vascular cell adhesion molecule-1), and enzymes (inducible nitric oxide synthase, 5-lipoxygenase, cyclooxygenase-2),⁴² thereby amplifying and perpetuating the inflammatory response. Mac-1–mediated activation of NF-B is therefore an obvious target for a new type of antiinflammatory treatment.

	Acknowledgments

 
This research was supported by National Institutes of Health Grants R01 HL57506 and R01 DK55656 (to D.I.S.).

Received May 22, 2001; revision received September 4, 2001; accepted September 13, 2001.

	References

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