The leukocyte integrin αMβ2 (CR3 or Mac-1) has both proinflammatory and immune regulatory functions. Genome-wide association studies have identified several ITGAMM subunit) single nucleotide polymorphisms that are associated with systemic lupus erythematosus. The single nucleotide polymorphism rs1143678 substitutes Pro1146 for Ser in the integrin αM cytoplasmic tail. A detailed functional characterization of this substitution is lacking. Using transfected human cell lines, reconstituted mouse bone marrow neutrophils, and bone marrow–derived macrophages (BMDMs), we showed that P1146S (PS) substitution promoted integrin αMβ2–mediated adhesion, spreading, and migration of cells on iC3b and fibrinogen. In the presence of LPS together with iC3b or fibrinogen, the expression levels of IL-6 and TNF-α in integrin αM(PS)β2 BMDMs were significantly higher than those of integrin αM(wild-type)β2 BMDMs, and they showed faster kinetics of Erk1/2 activation through the src family kinase(s)–Syk signaling pathway. Integrin αM(PS)β2 BMDMs also exhibited higher levels of active RhoA and phagocytic activity. Mechanistically, P1146S substitution in the αM cytoplasmic tail generates a noncanonical 14-3-3ζ binding site that modulates integrin αM(PS)β2 outside-in signaling.

Integrins are transmembrane heterodimers that are composed of α and β subunits, and they mediate cell–cell and cell–extracellular matrix interactions (1). The leukocyte β2 (CD18) integrin family consists of αLβ2 (CD11aCD18, LFA-1), αMβ2 (CD11bCD18, Mac-1, CR3), αXβ2 (CD11cCD18, p150,95), and αDβ2 (CD11dCD18) (2). The importance of the β2 integrins is underscored by the disease leukocyte adhesion deficiency type I in which ITGB22 subunit) gene mutations lead to defective leukocyte adhesion and migration (3). Patients with leukocyte adhesion deficiency type I are therefore prone to recurrent microbial infections that often lead to life-threatening complications.

Integrin αMβ2 is expressed in myeloid, NK, and γδ T cells (4, 5). Its ligands include iC3b, fibrinogen, ICAM-1, receptor for advanced glycation end products, myelin basic protein, LPS, and denatured proteins (2). In addition to leukocyte migration and phagocytosis, integrin αMβ2 regulates neutrophil survival, monocyte/macrophage differentiation, and immune tolerance (611). The integrin αL, αM, αX, and αD cytoplasmic tails (CTs) are divergent except for a conserved GFFKR motif (2). Integrin αM CT contains Tyr1137 and Ser1142 that are potential phosphorylation sites. Although there is limited information on the function of Tyr1137, nonconserved mutation of Ser1142 has been shown to reduce the binding of integrin αMβ2 to ICAM-1 (12). Nuclear magnetic resonance study has shown that integrin αM CT adopts a hairpin conformation that contains a membrane-proximal helix and a C-terminal loop, and Ser1142 phosphorylation did not induce any significant change to the conformation (13). Hence, phospho-Ser1142 most likely provides a docking site for yet to be identified cytosolic molecules that regulate integrin αMβ2 function.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease with a complex etiology that involves genetic predispositions and environmental factors. Genome-wide association studies have identified several susceptibility variants in the ITGAM gene (αM subunit) (1416). The nonsynonymous single nucleotide polymorphism (SNP) variant rs1143679 substitutes Arg77 for His in the integrin αM β-propeller, leading to defective ligand-binding of integrin αMβ2 (1520). It was proposed that macrophages expressing integrin αM(R77H) contribute to the onset of SLE because they are defective in clearing apoptotic bodies and they overproduce proinflammatory IL-6 (21). SNP rs1143679 together with SNPs rs1143678 and rs1143683 have been identified in some SLE patients: rs1143678 leads to P1146S substitution and rs1143683 leads to A858V substitution in the integrin αM CT and calf-1 regions, respectively (17). Neutrophils from SLE patients with rs1143678 and rs1143683 have been shown to exhibit attenuated integrin αMβ2 ligand-binding properties (22). To date, a detailed characterization of rs1143678, αM(P1146S), is lacking. In this study, we show that P1146S substitution in integrin αM CT promotes myeloid cell adhesion, spreading, migration, phagocytosis, and secretion of proinflammatory cytokines IL-6 and TNF-α. Mechanistically, P1146S substitution in the integrin αM CT generates a noncanonical 14-3-3ζ binding site. 14-3-3ζ is a cytoplasmic molecule that binds to and positively regulates β2 integrins (23, 24). Our data suggest that the aberrant association between 14-3-3ζ and integrin αM(P1146S) CT leads to the proinflammatory phenotype of myeloid cells expressing integrin αM(P1146S)β2.

K562 and 293T cell lines were obtained from the American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 and DMEM media, respectively, supplemented with 10% (v/v) heat-inactivated FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cell culture medium, FBS, and antibiotics were from HyClone (GE Healthcare Life Sciences, Logan, UT). The integrin αl-deficient Jurkat cell line JB2.7 (provided by Prof. N. Hogg, London Research Institute, London, U.K. and Prof. S.K.A. Law, School of Biological Sciences, Nanyang Technological University, Singapore) was cultured in full RPMI 1640 medium. Platinum-E retroviral packaging and L929 cell lines (provided by Prof. K.E. Karjalainen, School of Biological Sciences, Nanyang Technological University, Singapore) were cultured in full DMEM and IMDM media, respectively.

Animal studies were approved by the Institutional Animal Care and Use Committee at Nanyang Technological University, Singapore. Bone marrow cells isolated from ITGAMtm1Myd/g (integrin αM−/−) mice (The Jackson Laboratory, Bar Harbor, ME) were transduced by retroviral method with pMyc-IRES plasmid carrying the HOXB4 and Nup98 genes to generate long-term hematopoietic stem cells according to a previous study (25). In brief, Platinum-E cells were transfected with the plasmid using the calcium phosphate method. After 2 d, the culture supernatant containing the retrovirus was collected and used to transduce freshly isolated bone marrow cells. The cells were cultured and selected in IMDM medium containing 2% (v/v) FBS, 2 mM glutamine, 1.5 μg/ml puromycin supplemented with stem cell factor, and IL-6 conditioned media in a bacteriological petri dish. To generate immortalized bone marrow cells with rescued expression of integrin αM (wild-type [WT] or P1147S), immortalized bone marrow cells (5 × 106) were incubated in 5 ml of retrovirus-containing culture supernatant harvested from Platinum-E cells that were previously transfected with either empty pMyc-IRES-GFP plasmid or plasmid containing mouse integrin αM(WT) or αM(P1147S) cDNA. Polybrene (30 μg) was included in the culture mix. Cells were then harvested and sorted for GFP expression using FACS (FACSAria; BD Biosciences).

Macrophages were derived from these immortalized bone marrow cells by culturing in DMEM medium containing 10% (v/v) FBS, 2 mM glutamine, and 15% (v/v) conditioned medium of L929 cells that contained M-CSF-1. The latter was prepared by collecting the DMEM culture supernatant from L929 cells after 7 d in culture. Bone marrow–derived macrophage (BMDM) differentiation medium was changed once after 4 d in culture.

Mouse primary neutrophils were isolated by negative selection using the EasySep mouse neutrophil enrichment kit (Stemcell Technologies, Vancouver, BC, Canada). The isolated neutrophils were resuspended in appropriate medium and were used immediately in subsequent experiments.

Integrin αM−/− mice (6–8 wk of age) were subjected to two 5.5 Gy doses of gamma irradiation (each for 2 min and 4 h apart). After 24 h, transplantation was performed. Immortalized bone marrow cells that were reconstituted with integrin αM (WT, P1147S, or empty vector [EV]) were washed twice in PBS and resuspended in PBS containing 2% (v/v) FBS to a final concentration of 2.5 × 106 cells/100 μl. Two hundred microliters of cells was injected into each mouse via the retro-orbital venous route. Mice were fed with sterile water containing 1.17 g/ml neomycin for 2 wk.

Full-length human integrin αM and β2 cDNAs in expression plasmid pcDNA 3.0 were previously described (26). Human integrin αM P1146S, β2 Q746*, and β2 T758G were generated by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) and relevant primers. Full-length mouse integrin αM (WT) cDNA was purchased from Source BioScience LifeSciences (Nottingham, U.K.). To subclone the full-length mouse integrin αM (WT) cDNA into the pMyc-IRES-GFP plasmid (a gift from Prof. K.E. Karjalainen, School of Biological Sciences, Nanyang Technological University, Singapore), a silent point mutation was introduced in the αM cDNA to destroy the XhoI restriction site. The mouse integrin αM cDNA was PCR amplified using relevant primers containing NotI and XhoI restriction sites and subcloned into pMyc-IRES-GFP digested with the same enzymes. Site-directed mutagenesis was performed using relevant primers to generate mouse integrin αM P1147S in pMyc-IRES-GFP. An N-terminal hemagglutinin (HA)-tagged 14-3-3ζ (M1-S230) in pcDNA 3.1/Zeo(−) was generated by PCR subcloning using the template plasmid pGEX-4T1–14-3-3ζ obtained from Addgene (Cambridge, MA). The region Met1–Ser230 was selected based on previous studies on 14-3-3ζ in complex with CT peptides of integrins α4 (Protein Data Bank: 4HKC) and β2 (Protein Data Bank: 2V7D) (27, 28). For fluorescence resonance energy transfer (FRET) analyses, full-length human integrin αM (WT) and αM (P1146S) in pEYFP-N1 (Clontech Laboratories, Mountain View, CA) (29) were generated by PCR subcloning of the respective cDNAs into the plasmids using the KpnI and AgeI restriction sites. The construct therefore carries the integrin αM cDNA sequence with a C-terminal EYFP sequence. 14-3-3ζ (M1-S230) in pECFP-C1 was generated by PCR subcloning using the KpnI and BamHI restriction sites. The construct therefore contains an N-terminal ECFP sequence followed by the 14-3-3ζ sequence. Escherichia coli expression plasmid pET14b containing 14-3-3ζ (M1-S230) was generated by Shanghai ShineGene Molecular Biotech (Shanghai, China).

TRIzol reagent (Invitrogen) was used to extract total RNA from cells. RNA was air dried and resuspended in RNAse-free water. Quantitative RT-PCR (qRT-PCR) was performed using the Ambion Cells-to-CT kit (Life Technologies, Carlsbad, CA) on a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA). The cycling parameters were 95°C for 10 min, 95°C for 30 s, 58°C for 1 min, and repeated from step 2 for 39 times, followed by melt-curve analysis from 58°C to 95°C with a 0.5°C increment every 10 s. The specificity of the primers was verified by analyzing the melt-curve data and by performing agarose gel electrophoresis of amplified products from standard RT-PCR. Mouse IL-6 primers were: forward, 5′-GCCAGAGTCCTTCAGAGAGA-3′, reverse, 5′-TCTTGGTCCTTAGCCACTCC-3′. Mouse TNF-α primers were: forward, 5′-CTCCCAGGTTCTCTTCAAGG-3′, reverse, 5′-TGGAAGACTCCTCCCAGGTA-3′. Mouse GAPDH primers were: forward, 5′-GGTGAAGGTCGGTGTGAACG-3′, reverse, 5′-CTCGCTCCTGGAAGATGGTG-3′.

Cells (2 × 106) were transfected with relevant plasmids (8 μg each) by electroporation on a Microporator MP-100 according to the manufacturer’s instructions (Invitrogen) (30). Electroporated cells were resuspended in 2 ml of culture medium containing 20% (v/v) heat-inactivated FBS and maintained in culture for 24 h before being used in subsequent experiments.

Transfected JB2.7 and K562 cells were analyzed by flow cytometry as previously described (31). Cells were incubated in PBS containing 20 μg/ml primary Ab (mAb LPM19c) for 30 min on ice. Cells were washed in PBS and incubated in PBS containing FITC-conjugated sheep anti-mouse F(ab′)2 secondary Ab (1:400 dilution) (Sigma-Aldrich) for 30 min on ice. Stained cells were washed in PBS and analyzed on a FACSCalibur, FACS LSR II, or FACS LSRFortessa X-20 flow cytometer (BD Biosciences). Data were analyzed and presented using FlowJo software (Tree Star, Ashland, OR). Neutrophils and BMDMs were analyzed by flow cytometry using the following Abs: allophycocyanin/Cy7 anti-mouse/human CD11b, allophycocyanin/Cy7 rat IgG2b κ isotype control (BioLegend, San Diego, CA), PE anti-mouse Ly6G/Ly6C (Gr-1), PE anti-mouse F4/80, and PE anti-rat IgG2b κ isotype control (eBioscience, San Diego, CA).

An uncoated μ-slide I0.4 Luer flow chamber (ibidi, Munich, Germany) was coated with 7.5 μg/ml iC3b (Complement Technology, Tyler, TX) in PBS at 4°C overnight (32). The solution was removed and replaced with 0.2% (w/v) polyvinylpyrrolidone (10,000) in PBS at room temperature (RT) for 1 h to block nonspecific binding sites. Bone marrow–isolated neutrophils (5 × 105) were incubated in 1 ml of HBSS (1 mM CaCl2, 1 mM MgCl2, 5% [v/v] heat-inactivated FBS, 10 mM HEPES [pH 7.4]) without or with 200 nM phorbol dibutyrate (PdBu) for 5 min at 37°C. Cells were then infused into the chamber at a constant shear stress of 0.4 dyne/cm2 with an automated syringe pump (Harvard Apparatus, Holliston, MA). The experiment was performed at 37°C in a custom-built microscope-stage incubator equipped with a ×10 objective lens and a CCD camera. The number of adherent cells in four different fields (dimension 1 mm versus 1 mm) was recorded. The average number of adherent cells in four fields was calculated. Data were analyzed and presented based on the following: number of adherent cells with PdBu stimulation − number of adherent cells without stimulation.

Similar experiments were performed using transfected JB2.7 and K562 cells except that 6 × 105 cells were used and the applied shear stresses were 0.6 and 0.5 dyne/cm2, respectively. Data for JB2.7 cells were analyzed and presented based on the following: number of adherent cells with PdBu − number of adherent cells without PdBu. In the experiment using transfected K562 cells, the β2 integrin activating Ab KIM185 (10 μg/ml) was included (33).

In the experiment involving HA–14-3-3ζ overexpression in K562 cells, cells were transfected with β2 T758G and αM(WT)enhanced yellow fluorescent protein (eYFP) or αM(PS)eYFP and HA–14-3-3ζ or empty plasmid by electroporation. Cells were pretreated without or with 200 nM PdBu for 5 min at 37°C followed by shear flow analyses on iC3b-coated flow chamber as described above. LPM19c (10 μg/ml) was included to demonstrate ligand-binding specificity.

BMDMs (1 × 105) were resuspended in 200 μl of culture medium and dispensed into one well of the PolySorp microtiter plate (Nalge Nunc, Rochester, NY) that was precoated overnight with 80 μg/ml fibrinogen (Sigma-Aldrich) in sodium bicarbonate buffer (pH 9). Cells were incubated under culture conditions for 15 and 30 min. Four wells were used for each time point. Untreated cells were used for the 0 min time point. At each time point, cells were harvested and lysed in lysis buffer containing protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitors (Nacalai Tesque, Kyoto, Japan). Total cell lysate was used in subsequent immunoblotting assays. To examine the activation of MAPKs in BMDMs on iC3b, the same procedure was used except that cells were dispensed into wells of standard cell culture microtiter plates that were preincubated with 1:1 (v/v) mixture of mouse serum and HBSS buffer for 2 h at 37°C. This method is based on serum iC3b deposition on cell culture plastic as previously described (34, 35). For experimental conditions that required integrin activation, 100 ng/ml LPS (Sigma-Aldrich) or 1 mM MnCl2 were used. In the experiments involving Src family kinase (SFK) inhibition, cells were pretreated with 20 μM PP2 or the inactive analog PP3 for 2 h under culture conditions. Syk inhibition in cells was performed by pretreating cells with 20 μM piceatannol or DMSO carrier for 1 h under culture conditions. Inhibitors previously mentioned were purchased from Abcam (Cambridge, U.K.).

Cells were lysed in lysis buffer (1% [v/v] Igepal, 150 mM NaCl, and 50 mM Tris [pH 7.5]) containing protease inhibitors (Roche) and phosphatase inhibitors (Nacalai Tesque). Total protein concentration was determined using the bicinchoninic acid assay kit (Thermo Scientific). Proteins were resolved by SDS-PAGE under reducing conditions and electrotransferred onto polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories). For total protein immunoblotting, PVDF membrane was incubated in TBST blocking buffer containing 5% (w/v) nonfat milk for 30 min at RT. The membrane was incubated in TBST buffer containing relevant primary Abs at 4°C overnight. The following primary Abs were used: rabbit anti-MAPK1/2 (Erk1/2) (Millipore, Billerica, MA), rabbit anti-p38 MAPK (Cell Signaling Technology, Danvers, MA), rabbit anti-HA (Delta Biolabs, Gilroy, CA), and rabbit anti–14-3-3ζ (Abcam). To detect the phosphorylated proteins, PVDF membrane was incubated in TBST blocking buffer containing 5% (w/v) BSA for 30 min at RT. The following Abs were used: mouse anti-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) and rabbit anti–phospho-p38 MAPK (Thr180/Tyr182) (both from Cell Signaling Technology). The membrane was washed three times in TBST buffer followed by incubation in TBST buffer containing appropriate HRP-conjugated secondary Abs for 1 h at RT. Protein bands were detected by enhanced chemiluminescence using autoradiography or on a ChemiDoc Touch Gel and Western blot imaging System (Bio-Rad Laboratories). To determine the level of phosphorylated Erk1/2 or p38, the membrane was immunoblotted for the phosphorylated protein and the same membrane was stripped and reimmunoblotted for total protein.

Acceptor-photobleaching FRET analysis was performed as previously described but with modifications (29). K562 cells were transfected by electroporation with plasmids β2 Q746* and αM(WT)eYFP or αM(PS)eYFP and enhanced cyan fluorescent protein (eCFP)–14-3-3ζ or pECFP-C1 EV (10 μg each). Transfected cells were treated without or with 200 nM PdBu for 5 min at 37°C and centrifuged onto poly-l-lysine slides by cytospinning. FRET was performed on a Zeiss LSM 510 confocal microscope (Carl Zeiss, Thormwood, NY) using the following laser lines and emission filters: eCFP (excitation wavelength at 458 nm and band-pass filter 470–500 nm); eYFP (excitation wavelength at 514 nm and low-pass filter 539 nm). eYFP was photobleached between the fifth and sixth time points by scanning the entire cell 20 times using the 514 argon laser line that was set at the maximum intensity. The eCFP signal of the cell membrane was acquired before and after the photobleach. FRET efficiency was calculated using the equation: FRET efficiency = [(I6I5)/I6] × 100, where I is the eCFP intensity at the nth time point. Control samples that were not subjected to eYFP photobleaching (unbleached) were also analyzed.

Macrophage adhesion and spreading on iC3b and fibrinogen were determined by the electric cell–substrate impedance sensing (ECIS) method (36). The wells of a 16-well E-plate (ACEA Biosciences, San Diego, CA) were incubated with 4 mg/ml dithiobis(succinimidyl propionate) (Pierce; Thermo Fisher Scientific, Rockford, IL) in DMSO (Thermo Scientific) for 30 min at RT. The wells were washed twice in molecular biology–grade water and incubated with 80 μg/ml mouse fibrinogen (Haematologic Technologies, Essex Junction, VT) in PBS for 3 h at RT. Thereafter, the wells were washed twice with PBS. Each well was filled with 100 μl of culture medium and the background signal was measured on a real-time cell electronic system (ACEA Biosciences). Macrophages (1 × 105) were seeded into each well, without or with 100 ng/ml LPS. The cell index, which corresponds to cell adhesion/spreading, was determined during a period of 100 min. Each datum point represents the SD of technical triplicates. Data points were plotted as a function of time at 10-min intervals. For macrophage adhesion/spreading on iC3b, each dithiobis(succinimidyl propionate)–treated well was coated with 7.5 μg/ml iC3b in PBS for 3 h at RT.

Transwell migration assays were performed using the Corning Costar Transwell permeable supports (Thermo Fisher Scientific, Waltham, MA). The Transwell insert (8 μM) was coated with 80 μg/ml fibrinogen in PBS overnight at 4°C. The insert was washed with PBS and placed into a well (24-well format) filled with 500 μl of BMDM culture medium containing 100 ng/ml mouse SDF-1α (PeproTech, Rocky Hill, NJ). Cells were incubated in serum- and M-CSF-1–free culture medium for 6 h under culture conditions. Cells (8 × 104) were resuspended in 300 μl of serum- and M-CSF-1–free culture medium without or with 100 ng/ml LPS and were placed in the upper chamber followed by incubation under culture conditions for 16 h. A cotton swab was used to remove cells that did not migrate through the upper chamber insert. Cells underneath the upper chamber insert were fixed in 4% (w/v) paraformaldehyde in PBS for 10 min at RT. Cells were permeabilized with cytoskeletal buffer containing 0.25% (v/v) Triton X-100 for 2 min at RT followed by staining in crystal violet solution (0.5% w/v) for 20 min at RT. Stained cells were extensively washed in PBS. The dye was extracted using 0.25% (v/v) Triton X-100 in double distilled H2O and absorbance at 595 nm was measured on a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories).

FluoSpheres carboxylate-modified microspheres (1.0 μm, crimson fluorescent, excitation/emission of 625/645) (Invitrogen) were incubated in HBSS buffer containing 20% (v/v) mouse serum at 4°C overnight. Beads were washed and resuspended in HBSS before use. BMDMs (5 × 105) were preincubated in 500 μl of culture medium for 30 min at 4°C or 37°C. Cells were mixed with the beads at a cell/bead ratio of 1:100 in the presence or absence of LPS (100 ng/ml) for 30 min at 4°C or 37°C. Nonphagocytosed beads were removed by washing cells in 500 μl of PBS containing 4 mM EDTA and 1% (w/v) BSA. Cells were subjected to flow cytometry analysis. Expression index (EI) was calculated based on percentage gated positive × geometric mean. Phagocytic index was calculated and plotted based on EI at 37°C − EI at 4°C.

Production of cytokines in cells was detected using the GolgiPlug kit (BD Biosciences). BMDMs were incubated in serum- and M-CSF-1–free medium (1 ml) containing 1 μg of GolgiPlug (BD Biosciences) for 2 h under culture conditions. Cells (5 × 105) were resuspended in 200 μl of full medium containing 0.2 μg of GolgiPlug without or with 100 ng/ml LPS and plated onto iC3b-coated culture dish (35) for 6 h under culture conditions. Cells were harvested and stained with either allophycocyanin-conjugated anti-mouse TNF-α or anti-mouse IL-6 Ab (eBioscience) according to the manufacturer’s instructions followed by flow cytometry analyses. Fold expression was calculated based on EI and the expression level in cells bearing integrin αM(WT)β2 in the absence of LPS was normalized to 1.0.

In brief, E. coli BL21(DE3) cells were transformed with pET14b–6xHis–14-3-3ζ (Met1–Ser230). Transformed cells were grown at 37°C in Luria–Bertani medium until it reached an OD600 reading of 0.6–0.7. Isopropyl β-d-thiogalactoside was added to the culture to an end concentration of 0.5 mM followed by incubation for 10–12 h at 18°C. Cells were pelleted by centrifugation and lysed by sonication in 50 mM sodium phosphate buffer (pH 8) containing 300 mM NaCl followed by affinity purification on a Ni-NTA column (GE Healthcare, Amersham, U.K.). Recombinant protein was further purified by size exclusion chromatography in 20 mM sodium phosphate buffer (pH 6) containing 50 mM NaCl using a Hiload Superdex 75 16/26 GL preparative column (flow rate of 0.3 ml/min) on a ÄKTAFPLC UPC-900 system (GE Healthcare).

N-terminal biotin-labeled peptides of full-length integrin αM (WT) and αM (pS1142) tails were synthesized by ChinaPeptides (Shanghai, China). N-terminal biotin-labeled peptides of full-length αM (PS) and αM (P1146pS) tails were synthesized by GL Biochem (Shanghai, China). Pull-down assays were performed by mixing 60 μl of streptavidin agarose beads (Sigma-Aldrich) with or without (for uncoated) 20 μg of peptide and 3 μg of purified His6–14-3-3ζ (M1-S230) in 25 mM Tris (pH 7.4) containing 150 mM NaCl, 0.1% (w/v) BSA, 0.02% (v/v) Tween 20, and 0.5 mM TCEP. The mixture was rotated for 30 min at RT followed by washing in the same buffer. Proteins were eluted by boiling the beads in SDS sample loading buffer containing 40 mM DTT followed by SDS-PAGE and immunoblotting to detect 14-3-3ζ.

E. coli BL21(DE3) cells were transformed with GST–rhotekin binding domain (RBD) pGEX-2T plasmid (Addgene) or pGEX-KG empty plasmid. Cells in 500 ml of Luria–Bertani medium with an OD600 reading of 0.6 were induced to express the GST-RBD or GST by adding isopropyl β-d-thiogalactoside to a final concentration of 0.2 mM followed by incubation for 6 h at 30°C. Cells were pelleted by centrifugation and lysed by sonication in lysis buffer (100 mM NaCl, 0.5 mM PMSF, 0.5 mM TCEP, and 50 mM Tris-HCl [pH 7.4]) containing protease inhibitors. GST-RBD or GST was affinity purified on a glutathione-Sepharose column. An active RhoA pull-down assay was performed according to the protocol of the RhoA G-LISA activation assay kit (Cytoskeleton, Denver, CO) but with modifications. Glutathione-Sepharose bead suspension (15 μl) was washed once in 50 mM Tris-HCl (pH 7.5) buffer containing 2% (v/v) Igepal, 500 mM NaCl, and 10 mM MgCl2. Beads and 45 μg of either GST-RBD or GST were incubated in the same buffer for 1 h at 4°C with rotation. At the end of the incubation, beads were washed in the same buffer. BMDMs were serum and M-CSF-1 starved for 6 h. Thereafter, 1 × 105 cells were resuspended in 200 μl of full culture medium containing 100 ng/ml LPS and seeded into one well of the PolySorp microtiter plate that was precoated with 80 μg/ml mouse fibrinogen. For each time point, cells from 12 wells were used. Cell lysate containing 60 μg of total proteins as determined by bicinchoninic acid assay was used for each pull-down assay.

Statistical analyses were performed using the Prism software (GraphPad Software, La Jolla, CA). A Student t test (two-tailed and unequal variance) was used unless indicated otherwise. A p value <0.05 was considered significant.

The nonsynonymous SNP (rs1143678) leads to P1146S substitution in human integrin αM CT (Fig. 1A). Human and mouse integrin αM CTs have high sequence identity. The residue corresponding to Pro1146 of human integrin αM CT is Pro1147 in mouse integrin αM CT. P1146S (human) and P1147S (mouse) substitutions are henceforth referred to as PS. To examine the effects of PS substitution on integrin αMβ2 function, the human T cell line JB2.7 that does not express the α subunits of the β2 integrins was transfected to express either human αM(WT) or αM(PS) (Fig. 1B). Under shear flow and in the presence of PdBu, which induces integrin activation, the number of integrin αM(PS)β2 cells that adhered to iC3b was significantly higher than that of integrin αM(WT)β2 cells. To determine whether PS substitution promotes integrin αMβ2 inside-out or outside-in signaling, similar experiments were performed using transfected human chronic myelogenous leukemia K562 cells without treatment or treated with the β2 integrin–activating mAb KIM185 that bypasses inside-out signaling (33) (Fig. 1C). In the presence of mAb KIM185, the number of integrin αM(PS)β2 cells that adhered to iC3b was markedly higher than that of integrin αM(WT)β2 cells. These data suggest that PS substitution promotes integrin αMβ2 outside-in signaling.

FIGURE 1.

ITGAM SNP rs1143678 that substitutes Pro1146 for Ser in human integrin αM CT promotes cell adhesion. (A) Amino acid sequences of human and mouse integrin αM CTs. SNP rs1143678 is indicated. Pro1146 is conserved in humans and mice. (B) Expression levels of αM(WT)β2 and αM(P1146S)β2 integrins in transfected human JB2.7 cells were analyzed by flow cytometry using the integrin αM–specific mAb LPM19c (upper panel). Cells (6 × 105 per sample) were treated with PdBu (200 nM) and perfused into iC3b-coated (7.5 μg/ml) parallel flow chamber at a shear stress of 0.6 dyne/cm2 (lower panel). Data were analyzed based on: number of adherent cells with PdBu − number of adherent cells without PdBu. Data points are mean ± SD of three independent experiments. (C) Expression levels of αM(WT)β2 and αM(P1146S)β2 integrins in transfected K562 cells were analyzed by flow cytometry using the mAb LPM19c (upper panel). Adhesion of cells to iC3b in the absence or presence of β2-activating mAb KIM185 (33) (10 μg/ml) (lower panel) under a shear stress of 0.5 dyne/cm2 was examined. Cells (6 × 105) were used in each condition. Data points are mean ± SD of two independent experiments. (D) Neutrophils isolated from WT mice and integrin αM−/− mice bone marrows were analyzed by flow cytometry (upper panel). The following Abs were used: allophycocyanin/Cy7 anti-CD11b and its isotype control (BioLegend), and PE anti-mouse Ly6G/Ly6C (Gr1) and its isotype control (eBioscience). Adhesion of PdBu-treated neutrophils (5 × 105 per sample) to iC3b under a shear stress of 0.4 dyne/cm2 was examined (lower panel). Data points are mean ± SD (n = 3 mice per group). (E) Neutrophils isolated from the bone marrows of integrin αM−/− mice that were transplanted with reconstituted bone marrow cells [EV, αM (WT) or αM (P1147S)] were analyzed by flow cytometry (upper panel). Adhesion of PdBu-treated neutrophils to iC3b under a shear stress of 0.4 dyne/cm2 was examined (lower panel). Data points are mean ± SD. N values indicate number of mice. Flow cytometry data were analyzed using FlowJo software (Tree Star). A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

FIGURE 1.

ITGAM SNP rs1143678 that substitutes Pro1146 for Ser in human integrin αM CT promotes cell adhesion. (A) Amino acid sequences of human and mouse integrin αM CTs. SNP rs1143678 is indicated. Pro1146 is conserved in humans and mice. (B) Expression levels of αM(WT)β2 and αM(P1146S)β2 integrins in transfected human JB2.7 cells were analyzed by flow cytometry using the integrin αM–specific mAb LPM19c (upper panel). Cells (6 × 105 per sample) were treated with PdBu (200 nM) and perfused into iC3b-coated (7.5 μg/ml) parallel flow chamber at a shear stress of 0.6 dyne/cm2 (lower panel). Data were analyzed based on: number of adherent cells with PdBu − number of adherent cells without PdBu. Data points are mean ± SD of three independent experiments. (C) Expression levels of αM(WT)β2 and αM(P1146S)β2 integrins in transfected K562 cells were analyzed by flow cytometry using the mAb LPM19c (upper panel). Adhesion of cells to iC3b in the absence or presence of β2-activating mAb KIM185 (33) (10 μg/ml) (lower panel) under a shear stress of 0.5 dyne/cm2 was examined. Cells (6 × 105) were used in each condition. Data points are mean ± SD of two independent experiments. (D) Neutrophils isolated from WT mice and integrin αM−/− mice bone marrows were analyzed by flow cytometry (upper panel). The following Abs were used: allophycocyanin/Cy7 anti-CD11b and its isotype control (BioLegend), and PE anti-mouse Ly6G/Ly6C (Gr1) and its isotype control (eBioscience). Adhesion of PdBu-treated neutrophils (5 × 105 per sample) to iC3b under a shear stress of 0.4 dyne/cm2 was examined (lower panel). Data points are mean ± SD (n = 3 mice per group). (E) Neutrophils isolated from the bone marrows of integrin αM−/− mice that were transplanted with reconstituted bone marrow cells [EV, αM (WT) or αM (P1147S)] were analyzed by flow cytometry (upper panel). Adhesion of PdBu-treated neutrophils to iC3b under a shear stress of 0.4 dyne/cm2 was examined (lower panel). Data points are mean ± SD. N values indicate number of mice. Flow cytometry data were analyzed using FlowJo software (Tree Star). A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

Close modal

The effects of PS substitution on integrin αMβ2 function were further verified using mouse myeloid cells. Adhesion of integrin αM−/− neutrophils to iC3b in the presence of PdBu was significantly less than that of WT neutrophils (Fig. 1D). We next isolated bone marrow cells from integrin αM−/− mice and performed retrovirus-mediated transduction of these cells with the Nup-98-HOXB4 expression plasmid. This procedure has been reported to enable self-renewal of bone marrow hematopoietic stem cells in vitro but they retained their capacity to differentiate in vitro and in vivo (25). These cells were then transduced with pMyc-IRES-GFP EV, pMyc-mouse αM(WT)-IRES-GFP or pMyc-mouse αM(PS)-IRES-GFP. GFP+ cells were isolated by FACS and then transplanted into irradiated integrin αM−/− mice. Bone marrow neutrophils were isolated from these mice 7 wk later. Integrin αM(WT) and αM(PS) expression levels were analyzed by flow cytometry (Fig. 1E). In line with previous data, the number of neutrophils expressing integrin αM(PS)β2 that adhered to iC3b under shear flow was significantly higher than that of integrin αM(WT)β2 and EV neutrophils (Fig. 1E).

Considering the low percentage (∼21%) of integrin αM+ neutrophils that could be isolated from the transplanted mice, we decided to perform the rest of the study using F4/80+ macrophages that were obtained from in vitro differentiation of integrin αM(WT) and αM(PS) bone marrow cells (37, 38) (Fig. 2A). Integrin αM(PS)β2 BMDMs showed relatively higher levels of basal and LPS-activated adhesion and spreading on ligands than those of integrin αM(WT)β2 and EV cells as determined by the ECIS method (Fig. 2B). LPS was included because it activates macrophages via TLR4 signaling (39, 40). These data are in line with the increased adhesive properties of mouse neutrophils and human cell lines expressing integrin αM(PS)β2. Furthermore, the migration of LPS-stimulated integrin αM(PS)β2 BMDMs on fibrinogen was significantly faster than integrin αM(WT)β2 and EV cells in Transwell assays (Fig. 2C).

FIGURE 2.

Integrin αM(P1147S)β2 BMDMs exhibited enhanced adhesion, spreading, and migration on ligands. (A) Flow cytometry analysis of BMDMs transduced with EV, αM (WT), or αM (P1147S). P1 → P2 → P3 indicates that the cells in P3 were derived from the gated population P2, and P2 was a subpopulation of P1. The Abs used were allophycocyanin/Cy7 anti-CD11b and PE anti-mouse F4/80 and their respective isotype controls. (B) ECIS measurements of BMDMs (1 × 105 cells per condition) spreading on fibrinogen (80 μg/ml)– or iC3b (7.5 μg/ml)–coated wells without or with LPS (100 ng/ml) treatment. Data points are mean ± SD of technical triplicates. Data shown are from one representative experiment of three independent experiments performed. (C) BMDM migration assays were performed in 24 Transwells for 16 h under culture conditions. The upper chamber insert (8 μM) was coated with fibrinogen (80 μg/ml). Serum-starved BMDMs (8 × 104 cells per condition) in serum-free medium were placed into the upper chamber whereas the bottom chamber was filled with complete medium containing mouse SDF-1α (100 ng/ml). BMDMs that migrated through the pores of the insert were fixed and stained in crystal violet solution followed by dye extraction and OD595 measurement. Absorbance values were normalized with that of integrin αM(WT)β2 cells in the presence of LPS assigned as 1.0. Data points are mean ± SD of three independent experiments. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

FIGURE 2.

Integrin αM(P1147S)β2 BMDMs exhibited enhanced adhesion, spreading, and migration on ligands. (A) Flow cytometry analysis of BMDMs transduced with EV, αM (WT), or αM (P1147S). P1 → P2 → P3 indicates that the cells in P3 were derived from the gated population P2, and P2 was a subpopulation of P1. The Abs used were allophycocyanin/Cy7 anti-CD11b and PE anti-mouse F4/80 and their respective isotype controls. (B) ECIS measurements of BMDMs (1 × 105 cells per condition) spreading on fibrinogen (80 μg/ml)– or iC3b (7.5 μg/ml)–coated wells without or with LPS (100 ng/ml) treatment. Data points are mean ± SD of technical triplicates. Data shown are from one representative experiment of three independent experiments performed. (C) BMDM migration assays were performed in 24 Transwells for 16 h under culture conditions. The upper chamber insert (8 μM) was coated with fibrinogen (80 μg/ml). Serum-starved BMDMs (8 × 104 cells per condition) in serum-free medium were placed into the upper chamber whereas the bottom chamber was filled with complete medium containing mouse SDF-1α (100 ng/ml). BMDMs that migrated through the pores of the insert were fixed and stained in crystal violet solution followed by dye extraction and OD595 measurement. Absorbance values were normalized with that of integrin αM(WT)β2 cells in the presence of LPS assigned as 1.0. Data points are mean ± SD of three independent experiments. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

Close modal

Integrin αMβ2–mediated activation of Erk1/2 has been reported (41). Therefore, we asked whether the kinetics of Erk1/2 activation induced by integrin αM(PS)β2 and αM(WT)β2 are different. BMDMs expressing these integrins were seeded into empty culture wells in the presence of LPS- or ligand-coated culture wells in the absence of LPS with different incubation times. Cells were collected and lysed followed by immunoblotting to determine the levels of activated Erk1/2 and p38 MAPK (42) (Fig. 3A). There was no significant difference in the activation kinetics of Erk1/2 or p38 MAPK between the two groups of cells under all three conditions. We next examined cells that were seeded into ligand-coated culture wells in the presence of LPS or Mn2+, which activates integrin αMβ2 (32, 43). Under these conditions, faster kinetics of Erk1/2 activation was detected in integrin αM(PS)β2 BMDMs as compared with those in integrin αM(WT)β2 BMDMs (Fig. 3B). There was, however, no significant difference in the kinetics of p38 activation, suggesting signaling specificity that involves integrin αMβ2 and Erk1/2. Taken together, these data suggest that the overt activation of Erk1/2 mediated by integrin αM(PS)β2 requires both integrin activation and ligand engagement. SFKs and Syk are downstream effectors of integrin αMβ2 signaling (2). The Syk inhibitor piceatannol and the pan-SFK inhibitor PP2 but not its inactive analog PP3 effectively reduced the level of Erk1/2 activation in both integrin αM(PS)β2 and αM(WT)β2 BMDMs (Supplemental Fig. 1).

FIGURE 3.

Integrin αM(P1147S)β2 BMDMs exhibited faster kinetics of Erk1/2 activation compared with integrin αM(WT)β2 BMDMs in the presence of LPS together with ligand. (A) BMDMs transduced with αM (WT) or αM (P1147S) were treated with either LPS (100 ng/ml) or ligand (80 μg/ml fibrinogen-coated PolySorp microtiter wells or iC3b-coated culture dish as described previously) (34, 35). At different time points, cells were harvested and lysed followed by immunoblotting to detect activated Erk1/2 and p38. The following Abs were used: rabbit anti-Erk1/2 (total), mouse anti-pErk1/2 (Thr202/Tyr204), rabbit anti-p38 (total), and rabbit anti-p-p38 (Thr180/Tyr182). There was no marked difference in the kinetics of Erk1/2 or p38 activation between cells expressing αM(P1147S)β2 and αM(WT)β2 integrins. (B) Similar to (A) except that cells were treated with ligand together with LPS or MnCl2 (1 mM). The levels of Erk1/2 activation at the 15 min time point in integrin αM(P1147S)β2 BMDMs were significantly higher than those of integrin αM(WT)β2 BMDMs. There was no major difference in the kinetics of p38 activation between these cells. Immunoblot images shown are representative of three independent experiments. In all experiments, the membranes used to detect pErk1/2 and p-p38 were stripped and reblotted to detect total Erk1/2 and p38, respectively. Densitometry analyses were performed using ImageJ. Numbers below protein bands represent the mean (±SEM) fold differences of phospho-protein/total protein relative to that of WT sample at time point 0 (normalized to 1.0) from three independent experiments. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05. ns, not significant.

FIGURE 3.

Integrin αM(P1147S)β2 BMDMs exhibited faster kinetics of Erk1/2 activation compared with integrin αM(WT)β2 BMDMs in the presence of LPS together with ligand. (A) BMDMs transduced with αM (WT) or αM (P1147S) were treated with either LPS (100 ng/ml) or ligand (80 μg/ml fibrinogen-coated PolySorp microtiter wells or iC3b-coated culture dish as described previously) (34, 35). At different time points, cells were harvested and lysed followed by immunoblotting to detect activated Erk1/2 and p38. The following Abs were used: rabbit anti-Erk1/2 (total), mouse anti-pErk1/2 (Thr202/Tyr204), rabbit anti-p38 (total), and rabbit anti-p-p38 (Thr180/Tyr182). There was no marked difference in the kinetics of Erk1/2 or p38 activation between cells expressing αM(P1147S)β2 and αM(WT)β2 integrins. (B) Similar to (A) except that cells were treated with ligand together with LPS or MnCl2 (1 mM). The levels of Erk1/2 activation at the 15 min time point in integrin αM(P1147S)β2 BMDMs were significantly higher than those of integrin αM(WT)β2 BMDMs. There was no major difference in the kinetics of p38 activation between these cells. Immunoblot images shown are representative of three independent experiments. In all experiments, the membranes used to detect pErk1/2 and p-p38 were stripped and reblotted to detect total Erk1/2 and p38, respectively. Densitometry analyses were performed using ImageJ. Numbers below protein bands represent the mean (±SEM) fold differences of phospho-protein/total protein relative to that of WT sample at time point 0 (normalized to 1.0) from three independent experiments. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05. ns, not significant.

Close modal

qRT-PCR was performed to examine the mRNA expression levels of IL-6 and TNF-α in the BMDMs. In the presence of ligand and LPS, the expression levels of IL-6 and TNF-α transcripts were higher in integrin αM(PS)β2 BMDMs than in integrin αM(WT)β2 BMDMs (Fig. 4A). These findings were verified by performing intracellular staining of IL-6 and TNF-α using the GolgiPlug kit and then analyzed by flow cytometry (Fig. 4B). We next performed a bead-based phagocytosis assay to examine the phagocytic capacity of these cells (Fig. 4C). The level of serum-opsonized bead engulfment in integrin αM(PS)β2 BMDMs was markedly higher than that of integrin αM(WT)β2 BMDMs. RhoA is involved in integrin αMβ2–mediated phagocytosis of complement-opsonized particles (44). To determine whether these cells have different levels of active RhoA, we performed a RhoA pull-down assay (Fig. 4D). The level of active RhoA was significantly higher in integrin αM(PS)β2 BMDMs than in integrin αM(WT)β2 BMDMs at the 15 min time point. Taken together, these data suggest that PS substitution in integrin αM CT causes BMDMs to become proinflammatory and to exhibit enhanced phagocytosis capacity.

FIGURE 4.

Increased phagocytosis capacity and production of IL-6 and TNF-α in integrin αM(P1147S)β2 BMDMs. (A) BMDMs transduced with EV, αM(WT) or αM(P1147S) were treated with LPS (100 ng/ml) together with fibrinogen (top panel) or iC3b (bottom panel) and incubated for 6 h under culture conditions. Cells were harvested to isolate total RNA followed by qRT-PCR analysis of IL-6 and TNF-α gene expression. Data are presented as normalized fold expression with that of integrin αM(WT)β2 cells in the presence of LPS assigned the value 1.0. Data points are mean ± SD of two independent experiments for the EV BMDMs and three independent experiments for the integrin αM(WT) and αM(P1147S) BMDMs. (B) BMDMs were plated on iC3b-coated culture dish (34, 35) without or with LPS and incubated for 6 h under culture conditions. Intracellular cytokines were stained using the GolgiPlug kit and relevant Abs and analyzed by flow cytometry. Data are from at least two independent experiments and presented as normalized fold expression with that of integrin αM(WT)β2 without LPS assigned the value 1.0. (C) BMDMs were incubated with mouse serum-coated FluoSpheres (1.0 μm) (excitation/emission 625/645) at a cell/bead ratio of 1:100 with or without LPS (100 ng/ml) for 30 min in culture medium and analyzed by flow cytometry as described in 2Materials and Methods. Data points are mean ± SD of at least two independent experiments. (D) BMDMs were treated with LPS together with fibrinogen. At different time points, cells were harvested and lysed followed by active RhoA pull-down assay using either GST or GST-RBD beads. Immunoblotting was performed to detect bead-bound active RhoA. Densitometry analyses were performed using ImageJ software. Numbers below protein bands represent the mean (±SEM) fold differences of active RhoA relative to those of the EV BMDM GST-RBD pull-down sample at time point 0 (normalized to 1.0) from three independent experiments. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

FIGURE 4.

Increased phagocytosis capacity and production of IL-6 and TNF-α in integrin αM(P1147S)β2 BMDMs. (A) BMDMs transduced with EV, αM(WT) or αM(P1147S) were treated with LPS (100 ng/ml) together with fibrinogen (top panel) or iC3b (bottom panel) and incubated for 6 h under culture conditions. Cells were harvested to isolate total RNA followed by qRT-PCR analysis of IL-6 and TNF-α gene expression. Data are presented as normalized fold expression with that of integrin αM(WT)β2 cells in the presence of LPS assigned the value 1.0. Data points are mean ± SD of two independent experiments for the EV BMDMs and three independent experiments for the integrin αM(WT) and αM(P1147S) BMDMs. (B) BMDMs were plated on iC3b-coated culture dish (34, 35) without or with LPS and incubated for 6 h under culture conditions. Intracellular cytokines were stained using the GolgiPlug kit and relevant Abs and analyzed by flow cytometry. Data are from at least two independent experiments and presented as normalized fold expression with that of integrin αM(WT)β2 without LPS assigned the value 1.0. (C) BMDMs were incubated with mouse serum-coated FluoSpheres (1.0 μm) (excitation/emission 625/645) at a cell/bead ratio of 1:100 with or without LPS (100 ng/ml) for 30 min in culture medium and analyzed by flow cytometry as described in 2Materials and Methods. Data points are mean ± SD of at least two independent experiments. (D) BMDMs were treated with LPS together with fibrinogen. At different time points, cells were harvested and lysed followed by active RhoA pull-down assay using either GST or GST-RBD beads. Immunoblotting was performed to detect bead-bound active RhoA. Densitometry analyses were performed using ImageJ software. Numbers below protein bands represent the mean (±SEM) fold differences of active RhoA relative to those of the EV BMDM GST-RBD pull-down sample at time point 0 (normalized to 1.0) from three independent experiments. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

Close modal

We next examined whether PS substitution in integrin αM CT could lead to aberrant interaction with cytosolic protein, thereby modulating the functions of integrin αMβ2. The 14-3-3 family of proteins, which forms homo- or heterodimers, binds phospho-Ser/Thr–containing sequences, and they regulate diverse cellular processes (45). 14-3-3ζ interacts with the phospho-Thr triplet of the integrin β2 CT and it positively regulates β2 integrin–ligand binding (23, 24). This interaction could induce integrin clustering based on structure analysis of a 14-3-3ζ homodimer that was in complex with two integrin β2 CT peptides (28). We hypothesized that PS substitution in integrin αM CT could generate an aberrant phospho-Ser site that is recognized by 14-3-3ζ. To this end, we expressed and purified recombinant 14-3-3ζ(M1-S230) as previously described (27, 28). A pull-down assay was performed using the recombinant 14-3-3ζ, streptavidin-coated agarose beads, and the following biotin-tagged full-length integrin αM CT peptides: WT, pS1142, P1146S, and P1146pS (Fig. 5A). The peptide pS1142 was included in the assay because phosphorylation of Ser1142 in integrin αM(WT) CT has been shown to positively regulate the ligand-binding function of integrin αMβ2 (12) (Fig. 1A). 14-3-3ζ associated with P1146S and P1146pS but not with WT and pS1142 peptides. These data suggest that PS substitution in integrin αM CT generates a 14-3-3ζ binding site, and the interaction apparently does not require the phosphorylation of Ser1146.

FIGURE 5.

14-3-3ζ interacts with integrin αM(P1146S) CT. (A) Pull-down assay was performed using recombinant purified 14-3-3ζ and Sepharose beads conjugated with different integrin αM CT peptides. Immunblotting was performed to detect bead-bound 14-3-3ζ. Densitometry analyses were performed using ImageJ. Numbers below the blot represent mean (±SEM) fold intensity differences relative to those of αM(P1146S) (normalized to 1.0) from three independent experiments. (B) K562 cells were transfected with expression plasmids by electroporation. Transfected cells were subjected to YFP-photobleaching FRET analysis (29, 30, 43). Percentage FRET efficiency was calculated and data were plotted. Data shown are collated from at least three independent experiments with each symbol depicting one cell. (C) Similar to (B) except that cells were treated with PdBu (200 nM) before FRET analysis. (D) FRET analysis was performed using K562 cells transfected with the plasmids indicated to demonstrate that substituting Thr758 for Gly in integrin β2 CT effectively disrupts its binding to 14-3-3ζ. To mimic Thr758 phosphorylation that is required for 14-3-3ζ binding (28), the β2 T758E mutant was included as a positive control. (E) Functional assay to examine the interaction between 14-3-3ζ and integrin αM (P1146S). K562 cells were transfected with the expression plasmids β2 (T758G) and αM (WT) or αM (P1146S) together with HA–14-3-3ζ as indicated. β2 (T758G) was used instead of β2 (WT) to eliminate 14-3-3ζ–β2 CT binding that could complicate the analysis. Cells were subjected to flow assay on iC3b under a shear stress of 0.5 dyne/cm2. PdBu (200 nM) or/and LPM19c mAb (20 μg/ml) was included where indicated. Data points are mean ± SD of three independent experiments. The expression of HA–14-3-3ζ in cells was verified by immunoblotting using the anti-HA Ab. GAPDH serves as loading control. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

FIGURE 5.

14-3-3ζ interacts with integrin αM(P1146S) CT. (A) Pull-down assay was performed using recombinant purified 14-3-3ζ and Sepharose beads conjugated with different integrin αM CT peptides. Immunblotting was performed to detect bead-bound 14-3-3ζ. Densitometry analyses were performed using ImageJ. Numbers below the blot represent mean (±SEM) fold intensity differences relative to those of αM(P1146S) (normalized to 1.0) from three independent experiments. (B) K562 cells were transfected with expression plasmids by electroporation. Transfected cells were subjected to YFP-photobleaching FRET analysis (29, 30, 43). Percentage FRET efficiency was calculated and data were plotted. Data shown are collated from at least three independent experiments with each symbol depicting one cell. (C) Similar to (B) except that cells were treated with PdBu (200 nM) before FRET analysis. (D) FRET analysis was performed using K562 cells transfected with the plasmids indicated to demonstrate that substituting Thr758 for Gly in integrin β2 CT effectively disrupts its binding to 14-3-3ζ. To mimic Thr758 phosphorylation that is required for 14-3-3ζ binding (28), the β2 T758E mutant was included as a positive control. (E) Functional assay to examine the interaction between 14-3-3ζ and integrin αM (P1146S). K562 cells were transfected with the expression plasmids β2 (T758G) and αM (WT) or αM (P1146S) together with HA–14-3-3ζ as indicated. β2 (T758G) was used instead of β2 (WT) to eliminate 14-3-3ζ–β2 CT binding that could complicate the analysis. Cells were subjected to flow assay on iC3b under a shear stress of 0.5 dyne/cm2. PdBu (200 nM) or/and LPM19c mAb (20 μg/ml) was included where indicated. Data points are mean ± SD of three independent experiments. The expression of HA–14-3-3ζ in cells was verified by immunoblotting using the anti-HA Ab. GAPDH serves as loading control. A Student t test (two-tailed and unequal variance) was used for statistical analyses. *p < 0.05.

Close modal

To verify the interaction between integrin αM(PS) CT and 14-3-3ζ, we performed acceptor-photobleaching FRET on transfected cells. K562 cells were transfected with different combinations of the following expression plasmids: β2 Q746*, αM(WT)eYFP, αM(PS)eYFP, eCFP–14-3-3ζ, and eCFP EV (Fig. 5B). β2 Q746* is a truncated integrin β2 mutant in which a stop codon was introduced after Gln746, thereby removing the 14-3-3ζ binding site from the β2 CT. Hence, any FRET signal detected is a result of the interaction between 14-3-3ζ and αM but not β2 integrin CT. Without PdBu treatment, the basal FRET level of cells expressing αM(WT), β2 Q746*, and 14-3-3ζ was not significantly different from that of cells expressing αM(PS), β2 Q746*, and 14-3-3ζ (Fig. 5B). Basal FRET levels in these two groups of cells were higher than those of control groups (cells expressing the eCFP EV), which could be attributed to the method’s high sensitivity. In the presence of PdBu, the FRET level of cells expressing αM(PS), β2 Q746*, and 14-3-3ζ was significantly higher than that of cells expressing αM(WT), β2 Q746*, and 14-3-3ζ (Fig. 5C), suggesting that the interaction between integrin αM(PS) CT and 14-3-3ζ in cells requires integrin activation. These data are in line with our earlier observations that cellular stimulation triggers overt responses from cells expressing integrin αM(PS)β2.

To verify the dependency of the interaction between integrin αM(PS) CT and 14-3-3ζ on cellular stimulation, we performed additional functional assays. In order not to disrupt interactions between other cytosolic proteins and integrin β2 CT, which are required for the function of integrin αMβ2, we generated an expression plasmid containing full-length integrin β2 with a point mutation T758G. We showed by FRET assay that this mutant interacted minimally with 14-3-3ζ (Fig. 5D). We next transfected K562 cells with HA–14-3-3ζ and integrin αM(WT)β2(T758G) or αM(PS)β2(T758G) and examined the binding of these cells to iC3b under shear flow (Fig. 5E). In the absence of PdBu stimulation, there was no significant difference between the two groups of cells despite them overexpressing HA–14-3-3ζ. When treated with PdBu, a significantly higher number of cells expressing integrin αM(PS)β2(T758G) and HA–14-3-3ζ adhered to iC3b compared with cells expressing integrin αM(WT)β2(T758G) and HA–14-3-3ζ. Ligand-binding specificity was demonstrated by including the function-blocking Ab LPM19c.

The ITGAM SNP rs1143678 leads to P1146S substitution in the integrin αM CT. In this study, we showed that PS substitution promotes integrin αMβ2-mediated cell adhesion, spreading, migration, and phagocytosis. The effects of P1146S substitution on integrin αMβ2–mediated functions were most pronounced when cells were stimulated with LPS or PdBu and in the presence of ligands, suggesting that PS substitution primarily modulates integrin αMβ2 outside-in signaling. This is also evident from the shear flow iC3b-binding data of transfected K562 cells treated with β2 integrin–activating mAb KIM185, which bypassed inside-out integrin αMβ2 activation.

The clustering of integrin αMβ2 activates Erk1/2 through the Src/Syk pathway (10). We showed that integrin αM(PS)β2 also signals through this pathway. However, BMDMs expressing integrin αM(PS)β2 that were treated with LPS together with iC3b or fibrinogen exhibited faster kinetics of Erk1/2 activation and produced higher levels of IL-6 and TNF-α when compared with integrin αM(WT)β2 BMDMs. These findings corroborate well with the overproduction of IL-6 and TNF-α detected in SLE patients (46, 47). Apart from genetic factors, environmental factors can contribute to the onset of SLE (48). TLRs are involved in the etiology of SLE (49). TLR-4 recognizes Gram-negative bacteria LPS (50). Overt signaling from TLR-4 has been shown to break immune tolerance, leading to lupus-like renal disease in mice (51). When lupus-prone C57BL/6lpr/lpr mice were crossed with TLR-4 −/− mice, their progenies produced lower levels of autoantibodies and they were less susceptible to renal injuries, suggesting that TLR-4 is involved in the onset of SLE (52). It is therefore physiologically relevant to treat BMDMs with LPS to examine the effects of PS substitution on integrin αMβ2 function. In addition to LPS, heat shock protein 60 and high mobility group box 1 protein released from damaged cells interact with TLR-4 (53, 54). Hence microbial infections and tissue injuries may exacerbate the inflammatory responses in individuals carrying the ITGAM SNP rs1143678 allele.

Mechanistically, PS substitution in integrin αM CT generates an aberrant 14-3-3ζ binding site. 14-3-3ζ positively regulates β2 integrin–mediated functions by interacting with a phospho-Thr triplet motif in the β2 CT (23, 24). Although 14-3-3 proteins bind phospho-Ser– or phospho-Thr–containing motifs in their binding partners, non–phospho-Ser/Thr ligands have been identified (5558). Our data suggest that the interaction between 14-3-3ζ and integrin αM(PS) CT is not dependent on the phosphorylation of Ser1146. Structural study has shown that the nonphosphorylated exo-S peptide interacts with a flexible binding groove in 14-3-3β (59). Whether integrin αM(PS) CT interacts with the binding groove or another site in 14-3-3ζ remains to be investigated.

Hetero- or homodimeric 14-3-3 proteins have been identified (60, 61). Structural study demonstrated that a 14-3-3ζ homodimer can associate with two integrin β2 CT peptides, suggesting a role of 14-3-3ζ in promoting the formation of integrin cluster (28). We therefore proposed a model in which the PS substitution in integrin αM CT could lead to overt outside-in signaling and inflammatory response (Fig. 6). In the absence of cellular stimulation by LPS or phorbol ester, the binding of 14-3-3ζ to the PS site is not favorable because the CTs of integrin αM(PS)β2 are in a clasped conformation, and the site may be occluded by negative regulators that directly interact with the integrin CTs. This is supported by FRET data that showed a significant increase in the interaction between 14-3-3ζ and integrin αM(PS) CT compared with integrin αM(WT) CT only under conditions that promote integrin activation.

FIGURE 6.

A model illustrating the possible mechanisms by which P1146S substitution in integrin αM CT leads to overt integrin αMβ2–mediated responses. (A and B) P1146S substitution in integrin αM CT generates a noncanonical 14-3-3ζ binding site. However, it does not induce significant aberrant effects on an inactive integrin. The clasped conformation of the integrin αM and β2 CTs and/or the presence of negative regulators that bind to the CTs could prevent 14-3-3ζ from binding to this site. (C) Upon integrin activation by LPS or phorbol ester, the negative regulator dissociates from the integrin CTs and the α and β CTs separate from each other, which permits 14-3-3ζ to bind the αM(P1146S) CT. (D) A 14-3-3ζ homodimer could then bind to two integrin αM(P1146S) CTs, leading to aberrant integrin clustering and SFK–Syk–Erk1/2 signaling. 14-3-3ζ that is bound to integrin αM(P1146S) CT could also interact with cytosolic molecules involved in other signaling pathways. In this model, P1146S substitution affects primarily integrin αMβ2 outside-in signaling.

FIGURE 6.

A model illustrating the possible mechanisms by which P1146S substitution in integrin αM CT leads to overt integrin αMβ2–mediated responses. (A and B) P1146S substitution in integrin αM CT generates a noncanonical 14-3-3ζ binding site. However, it does not induce significant aberrant effects on an inactive integrin. The clasped conformation of the integrin αM and β2 CTs and/or the presence of negative regulators that bind to the CTs could prevent 14-3-3ζ from binding to this site. (C) Upon integrin activation by LPS or phorbol ester, the negative regulator dissociates from the integrin CTs and the α and β CTs separate from each other, which permits 14-3-3ζ to bind the αM(P1146S) CT. (D) A 14-3-3ζ homodimer could then bind to two integrin αM(P1146S) CTs, leading to aberrant integrin clustering and SFK–Syk–Erk1/2 signaling. 14-3-3ζ that is bound to integrin αM(P1146S) CT could also interact with cytosolic molecules involved in other signaling pathways. In this model, P1146S substitution affects primarily integrin αMβ2 outside-in signaling.

Close modal

When cells are stimulated by LPS or phorbol ester, this leads to integrin activation through the dissociation of the negative regulator from the integrin αM(PS)β2 CTs and the separation of the integrin αM(PS) CT from the β2 CT, thereby permitting 14-3-3ζ to bind to the PS site. A 14-3-3ζ homodimer can simultaneously bind to two integrin αM(PS) CTs and induce integrin clustering, which in turn triggers downstream cellular responses. Because 14-3-3 proteins interact with a large number of cytosolic signaling molecules, the aberrant interaction between 14-3-3ζ and integrin αM(PS) CT could activate signaling pathways other than the SFK–Syk–Erk1/2 pathway.

An interesting point of discussion refers to ITGAM SNP rs1143679 that leads to R77H substitution in the integrin αM β-propeller. It has been shown to disrupt the formation of catch bonds between integrin αM and its ligand, which impairs the ligand-binding function of integrin αMβ2 (1820). However, there are different observations reported in terms of the phagocytic capacity and cytokine production of cells bearing rs1143679 (62). Integrin αMβ2 is involved in immune tolerance (63). It was therefore proposed that rs1143679 contributes to the onset of SLE because it reduces macrophage clearance of apoptotic bodies and it leads to deregulated production of inflammatory cytokines (21). Our findings that ITGAM SNP rs1143678 did not abrogate the ligand-binding function of integrin αMβ2 and that it is proinflammatory may appear contrary to the effects of ITGAM SNP rs1143679. However, studies have shown that integrin αMβ2 can serve both pro- and anti-inflammatory functions that are dependent on cell type and modulated by its cross-talk with membrane and cytosolic molecules, including CD14, FcγRIIA, FcγRIIIB, uPAR, and TLRs (62). Zhou et al. (22) reported that neutrophils from donors having all three allelic variants ITGAM SNP rs1143678 (P1146S), ITGAM SNP rs1143679 (R77H), and ITGAM SNP rs1143683 (A858V substitution in the calf-1 domain of integrin αM) were defective in complement-opsonized phagocytosis and adhesion to ICAM-1. It is likely that R77H is dominant over P1146S because R77H directly abrogates integrin αM–ligand interaction. This would have blunted the effects of P1146S that are dependent on integrin activation and ligand engagement. In the same study (22), phagocytosis and adhesion defects were also observed in neutrophils from donors having both ITGAM SNP rs1143678 (P1146S) and ITGAM SNP rs1143683 (A858V). A detailed functional characterization of A858V substitution is needed to explain the phenotype presented.

In conclusion, we showed that the SLE-associated nonsynonymous ITGAM SNP rs1143678 promotes integrin αMβ2 proinflammatory functions. Mechanistically, P1146S substitution in the integrin αM CT generates a noncanonical 14-3-3ζ binding site that leads to aberrant integrin αMβ2 outside-in signaling. ITGAM SNP rs1143678 could therefore exacerbate inflammatory responses and tissue injuries in SLE patients that are exposed to not only self-antigens but also a milieu of environmental factors, including microbial pathogens.

We are very grateful to K.E. Karjalainen and S.C. Loh (School of Biological Sciences, Nanyang Technological University, Singapore) for providing advice and assistance for the generation and transplantation of the immortalized bone marrow cells. We thank Z.H. Xue (School of Biological Sciences, Nanyang Technological University, Singapore) for advice on the Syk-Erk signaling study. We thank L.G. Ng and laboratory members (Singapore Immunology Network [SIgN0], A*STAR, Singapore) for helpful advice.

This work was supported by Ministry of Education, Singapore Grant MOE2010-T2-2-014 (to S.-M.T.) and Nanyang Technological University, Singapore Grant M4081320 (to S.-M.T.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

BMDM

bone marrow–derived macrophage

CT

cytoplasmic tail

eCFP

enhanced cyan fluorescent protein

ECIS

electric cell–substrate impedance sensing

EI

expression index

EV

empty vector

eYFP

enhanced yellow fluorescent protein

FRET

fluorescence resonance energy transfer

HA

hemagglutinin

PdBu

phorbol dibutyrate

PVDF

polyvinylidene difluoride

qRT-PCR

quantitative RT-PCR

RBD

rhotekin binding domain

RT

room temperature

SFK

Src family kinase

SLE

systemic lupus erythematosus

SNP

single nucleotide polymorphism

WT

wild-type.

1
Hynes
R. O.
2002
.
Integrins: bidirectional, allosteric signaling machines.
Cell
110
:
673
687
.
2
Tan
S. M.
2012
.
The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties.
Biosci. Rep.
32
:
241
269
.
3
van de Vijver
E.
,
Maddalena
A.
,
Sanal
Ö.
,
Holland
S. M.
,
Uzel
G.
,
Madkaikar
M.
,
de Boer
M.
,
van Leeuwen
K.
,
Köker
M. Y.
,
Parvaneh
N.
, et al
.
2012
.
Hematologically important mutations: leukocyte adhesion deficiency (first update).
Blood Cells Mol. Dis.
48
:
53
61
.
4
Larson
R. S.
,
Springer
T. A.
.
1990
.
Structure and function of leukocyte integrins.
Immunol. Rev.
114
:
181
217
.
5
Graff
J. C.
,
Jutila
M. A.
.
2007
.
Differential regulation of CD11b on γδ T cells and monocytes in response to unripe apple polyphenols.
J. Leukoc. Biol.
82
:
603
607
.
6
Frommhold
D.
,
Kamphues
A.
,
Hepper
I.
,
Pruenster
M.
,
Lukic
I. K.
,
Socher
I.
,
Zablotskaya
V.
,
Buschmann
K.
,
Lange-Sperandio
B.
,
Schymeinsky
J.
, et al
.
2010
.
RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo.
Blood
116
:
841
849
.
7
Arnaout
M. A.
,
Todd
R. F.
 III
,
Dana
N.
,
Melamed
J.
,
Schlossman
S. F.
,
Colten
H. R.
.
1983
.
Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol).
J. Clin. Invest.
72
:
171
179
.
8
Ding
Z. M.
,
Babensee
J. E.
,
Simon
S. I.
,
Lu
H.
,
Perrard
J. L.
,
Bullard
D. C.
,
Dai
X. Y.
,
Bromley
S. K.
,
Dustin
M. L.
,
Entman
M. L.
, et al
.
1999
.
Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration.
J. Immunol.
163
:
5029
5038
.
9
Walzog
B.
,
Jeblonski
F.
,
Zakrzewicz
A.
,
Gaehtgens
P.
.
1997
.
Beta2 integrins (CD11/CD18) promote apoptosis of human neutrophils.
FASEB J.
11
:
1177
1186
.
10
Xue
Z. H.
,
Zhao
C. Q.
,
Chua
G. L.
,
Tan
S. W.
,
Tang
X. Y.
,
Wong
S. C.
,
Tan
S. M.
.
2010
.
Integrin αMβ2 clustering triggers phosphorylation and activation of protein kinase Cδ that regulates transcription factor Foxp1 expression in monocytes.
J. Immunol.
184
:
3697
3709
.
11
Han
C.
,
Jin
J.
,
Xu
S.
,
Liu
H.
,
Li
N.
,
Cao
X.
.
2010
.
Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b.
Nat. Immunol.
11
:
734
742
.
12
Fagerholm
S. C.
,
Varis
M.
,
Stefanidakis
M.
,
Hilden
T. J.
,
Gahmberg
C. G.
.
2006
.
α-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation.
Blood
108
:
3379
3386
.
13
Chua
G. L.
,
Tang
X. Y.
,
Amalraj
M.
,
Tan
S. M.
,
Bhattacharjya
S.
.
2011
.
Structures and interaction analyses of integrin αMβ2 cytoplasmic tails.
J. Biol. Chem.
286
:
43842
43854
.
14
Harley
J. B.
,
Alarcón-Riquelme
M. E.
,
Criswell
L. A.
,
Jacob
C. O.
,
Kimberly
R. P.
,
Moser
K. L.
,
Tsao
B. P.
,
Vyse
T. J.
,
Langefeld
C. D.
, et al
The International Consortium for Systemic Lupus Erythematosus Genetics (SLEGEN)
.
2008
.
Genome-wide association scan in women with systemic lupus erythematosus identifies susceptibility variants in ITGAM, PXK, KIAA1542 and other loci.
Nat. Genet.
40
:
204
210
.
15
Nath
S. K.
,
Han
S.
,
Kim-Howard
X.
,
Kelly
J. A.
,
Viswanathan
P.
,
Gilkeson
G. S.
,
Chen
W.
,
Zhu
C.
,
McEver
R. P.
,
Kimberly
R. P.
, et al
.
2008
.
A nonsynonymous functional variant in integrin-αM (encoded by ITGAM) is associated with systemic lupus erythematosus.
Nat. Genet.
40
:
152
154
.
16
Yang
W.
,
Zhao
M.
,
Hirankarn
N.
,
Lau
C. S.
,
Mok
C. C.
,
Chan
T. M.
,
Wong
R. W.
,
Lee
K. W.
,
Mok
M. Y.
,
Wong
S. N.
, et al
.
2009
.
ITGAM is associated with disease susceptibility and renal nephritis of systemic lupus erythematosus in Hong Kong Chinese and Thai.
Hum. Mol. Genet.
18
:
2063
2070
.
17
Han
S.
,
Kim-Howard
X.
,
Deshmukh
H.
,
Kamatani
Y.
,
Viswanathan
P.
,
Guthridge
J. M.
,
Thomas
K.
,
Kaufman
K. M.
,
Ojwang
J.
,
Rojas-Villarraga
A.
, et al
.
2009
.
Evaluation of imputation-based association in and around the integrin-α-M (ITGAM) gene and replication of robust association between a non-synonymous functional variant within ITGAM and systemic lupus erythematosus (SLE).
Hum. Mol. Genet.
18
:
1171
1180
.
18
MacPherson
M.
,
Lek
H. S.
,
Prescott
A.
,
Fagerholm
S. C.
.
2011
.
A systemic lupus erythematosus-associated R77H substitution in the CD11b chain of the Mac-1 integrin compromises leukocyte adhesion and phagocytosis.
J. Biol. Chem.
286
:
17303
17310
.
19
Rhodes
B.
,
Fürnrohr
B. G.
,
Roberts
A. L.
,
Tzircotis
G.
,
Schett
G.
,
Spector
T. D.
,
Vyse
T. J.
.
2012
.
The rs1143679 (R77H) lupus associated variant of ITGAM (CD11b) impairs complement receptor 3 mediated functions in human monocytes.
Ann. Rheum. Dis.
71
:
2028
2034
.
20
Rosetti
F.
,
Chen
Y.
,
Sen
M.
,
Thayer
E.
,
Azcutia
V.
,
Herter
J. M.
,
Luscinskas
F. W.
,
Cullere
X.
,
Zhu
C.
,
Mayadas
T. N.
.
2015
.
A lupus-associated Mac-1 variant has defects in integrin allostery and interaction with ligands under force.
Cell Rep.
10
:
1655
1664
.
21
Fagerholm
S. C.
,
MacPherson
M.
,
James
M. J.
,
Sevier-Guy
C.
,
Lau
C. S.
.
2013
.
The CD11b-integrin (ITGAM) and systemic lupus erythematosus.
Lupus
22
:
657
663
.
22
Zhou
Y.
,
Wu
J.
,
Kucik
D. F.
,
White
N. B.
,
Redden
D. T.
,
Szalai
A. J.
,
Bullard
D. C.
,
Edberg
J. C.
.
2013
.
Multiple lupus-associated ITGAM variants alter Mac-1 functions on neutrophils.
Arthritis Rheum.
65
:
2907
2916
.
23
Fagerholm
S.
,
Morrice
N.
,
Gahmberg
C. G.
,
Cohen
P.
.
2002
.
Phosphorylation of the cytoplasmic domain of the integrin CD18 chain by protein kinase C isoforms in leukocytes.
J. Biol. Chem.
277
:
1728
1738
.
24
Nurmi
S. M.
,
Gahmberg
C. G.
,
Fagerholm
S. C.
.
2006
.
14-3-3 proteins bind both filamin and αLβ2 integrin in activated T cells.
Ann. N. Y. Acad. Sci.
1090
:
318
325
.
25
Ruedl
C.
,
Khameneh
H. J.
,
Karjalainen
K.
.
2008
.
Manipulation of immune system via immortal bone marrow stem cells.
Int. Immunol.
20
:
1211
1218
.
26
Tan
S. M.
,
Hyland
R. H.
,
Al-Shamkhani
A.
,
Douglass
W. A.
,
Shaw
J. M.
,
Law
S. K.
.
2000
.
Effect of integrin β2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function.
J. Immunol.
165
:
2574
2581
.
27
Bonet
R.
,
Vakonakis
I.
,
Campbell
I. D.
.
2013
.
Characterization of 14-3-3-ζ interactions with integrin tails.
J. Mol. Biol.
425
:
3060
3072
.
28
Takala
H.
,
Nurminen
E.
,
Nurmi
S. M.
,
Aatonen
M.
,
Strandin
T.
,
Takatalo
M.
,
Kiema
T.
,
Gahmberg
C. G.
,
Ylänne
J.
,
Fagerholm
S. C.
.
2008
.
β2 Integrin phosphorylation on Thr758 acts as a molecular switch to regulate 14-3-3 and filamin binding.
Blood
112
:
1853
1862
.
29
Vararattanavech
A.
,
Tang
M. L.
,
Li
H. Y.
,
Wong
C. H.
,
Law
S. K.
,
Torres
J.
,
Tan
S. M.
.
2008
.
Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin.
Biochem. J.
410
:
495
502
.
30
Tang
X. Y.
,
Li
Y. F.
,
Tan
S. M.
.
2008
.
Intercellular adhesion molecule-3 binding of integrin αLβ2 requires both extension and opening of the integrin headpiece.
J. Immunol.
180
:
4793
4804
.
31
Tan
S. M.
,
Robinson
M. K.
,
Drbal
K.
,
van Kooyk
Y.
,
Shaw
J. M.
,
Law
S. K.
.
2001
.
The N-terminal region and the mid-region complex of the integrin β2 subunit.
J. Biol. Chem.
276
:
36370
36376
.
32
Xue
Z. H.
,
Feng
C.
,
Liu
W. L.
,
Tan
S. M.
.
2013
.
A role of kindlin-3 in integrin αMβ2 outside-in signaling and the Syk-Vav1-Rac1/Cdc42 signaling axis.
PLoS One
8
:
e56911
.
33
Robinson
M. K.
,
Andrew
D.
,
Rosen
H.
,
Brown
D.
,
Ortlepp
S.
,
Stephens
P.
,
Butcher
E. C.
.
1992
.
Antibody against the Leu-CAM beta-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events.
J. Immunol.
148
:
1080
1085
.
34
Andersson
J.
,
Ekdahl
K. N.
,
Larsson
R.
,
Nilsson
U. R.
,
Nilsson
B.
.
2002
.
C3 adsorbed to a polymer surface can form an initiating alternative pathway convertase.
J. Immunol.
168
:
5786
5791
.
35
Hirahashi
J.
,
Mekala
D.
,
Van Ziffle
J.
,
Xiao
L.
,
Saffaripour
S.
,
Wagner
D. D.
,
Shapiro
S. D.
,
Lowell
C.
,
Mayadas
T. N.
.
2006
.
Mac-1 signaling via Src-family and Syk kinases results in elastase-dependent thrombohemorrhagic vasculopathy.
Immunity
25
:
271
283
.
36
Feng
C.
,
Li
Y. F.
,
Yau
Y. H.
,
Lee
H. S.
,
Tang
X. Y.
,
Xue
Z. H.
,
Zhou
Y. C.
,
Lim
W. M.
,
Cornvik
T. C.
,
Ruedl
C.
, et al
.
2012
.
Kindlin-3 mediates integrin αLβ2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1).
J. Biol. Chem.
287
:
10714
10726
.
37
Trouplin
V.
,
Boucherit
N.
,
Gorvel
L.
,
Conti
F.
,
Mottola
G.
,
Ghigo
E.
.
2013
.
Bone marrow-derived macrophage production.
J. Vis. Exp.
81
:
e50966
. Available at: https://www.jove.com/video/50966/bone-marrow-derived-macrophage-production.
38
Austyn
J. M.
,
Gordon
S.
.
1981
.
F4/80, a monoclonal antibody directed specifically against the mouse macrophage.
Eur. J. Immunol.
11
:
805
815
.
39
Chow
J. C.
,
Young
D. W.
,
Golenbock
D. T.
,
Christ
W. J.
,
Gusovsky
F.
.
1999
.
Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction.
J. Biol. Chem.
274
:
10689
10692
.
40
Mosser, D. M., and X. Zhang. 2008. Activation of murine macrophages. Curr. Protoc. Immunol. Chapter 14: Unit 14.12. doi:10.1002/0471142735.im1402s83
.
41
Rezzonico
R.
,
Chicheportiche
R.
,
Imbert
V.
,
Dayer
J. M.
.
2000
.
Engagement of CD11b and CD11c β2 integrin by antibodies or soluble CD23 induces IL-1β production on primary human monocytes through mitogen-activated protein kinase-dependent pathways.
Blood
95
:
3868
3877
.
42
Cargnello
M.
,
Roux
P. P.
.
2011
.
Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases.
Microbiol. Mol. Biol. Rev.
75
:
50
83
.
43
Tang
M. L.
,
Vararattanavech
A.
,
Tan
S. M.
.
2008
.
Urokinase-type plasminogen activator receptor induces conformational changes in the integrin αMβ2 headpiece and reorientation of its transmembrane domains.
J. Biol. Chem.
283
:
25392
25403
.
44
Caron
E.
,
Hall
A.
.
1998
.
Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.
Science
282
:
1717
1721
.
45
Fu
H.
,
Subramanian
R. R.
,
Masters
S. C.
.
2000
.
14-3-3 proteins: structure, function, and regulation.
Annu. Rev. Pharmacol. Toxicol.
40
:
617
647
.
46
Mihara
M.
,
Nishimoto
N.
,
Ohsugi
Y.
.
2005
.
The therapy of autoimmune diseases by anti-interleukin-6 receptor antibody.
Expert Opin. Biol. Ther.
5
:
683
690
.
47
Weckerle
C. E.
,
Mangale
D.
,
Franek
B. S.
,
Kelly
J. A.
,
Kumabe
M.
,
James
J. A.
,
Moser
K. L.
,
Harley
J. B.
,
Niewold
T. B.
.
2012
.
Large-scale analysis of tumor necrosis factor α levels in systemic lupus erythematosus.
Arthritis Rheum.
64
:
2947
2952
.
48
Tsokos
G. C.
2011
.
Systemic lupus erythematosus.
N. Engl. J. Med.
365
:
2110
2121
.
49
Marshak-Rothstein
A.
2006
.
Toll-like receptors in systemic autoimmune disease.
Nat. Rev. Immunol.
6
:
823
835
.
50
Vaure
C.
,
Liu
Y.
.
2014
.
A comparative review of Toll-like receptor 4 expression and functionality in different animal species.
Front. Immunol.
5
:
316
.
51
Liu
B.
,
Yang
Y.
,
Dai
J.
,
Medzhitov
R.
,
Freudenberg
M. A.
,
Zhang
P. L.
,
Li
Z.
.
2006
.
TLR4 up-regulation at protein or gene level is pathogenic for lupus-like autoimmune disease.
J. Immunol.
177
:
6880
6888
.
52
Lartigue
A.
,
Colliou
N.
,
Calbo
S.
,
François
A.
,
Jacquot
S.
,
Arnoult
C.
,
Tron
F.
,
Gilbert
D.
,
Musette
P.
.
2009
.
Critical role of TLR2 and TLR4 in autoantibody production and glomerulonephritis in lpr mutation-induced mouse lupus.
J. Immunol.
183
:
6207
6216
.
53
Vabulas
R. M.
,
Ahmad-Nejad
P.
,
da Costa
C.
,
Miethke
T.
,
Kirschning
C. J.
,
Häcker
H.
,
Wagner
H.
.
2001
.
Endocytosed HSP60s use Toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells.
J. Biol. Chem.
276
:
31332
31339
.
54
Park
J. S.
,
Svetkauskaite
D.
,
He
Q.
,
Kim
J. Y.
,
Strassheim
D.
,
Ishizaka
A.
,
Abraham
E.
.
2004
.
Involvement of Toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 protein.
J. Biol. Chem.
279
:
7370
7377
.
55
Wang
B.
,
Yang
H.
,
Liu
Y. C.
,
Jelinek
T.
,
Zhang
L.
,
Ruoslahti
E.
,
Fu
H.
.
1999
.
Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display.
Biochemistry
38
:
12499
12504
.
56
Petosa
C.
,
Masters
S. C.
,
Bankston
L. A.
,
Pohl
J.
,
Wang
B.
,
Fu
H.
,
Liddington
R. C.
.
1998
.
14-3-3ζ binds a phosphorylated Raf peptide and an unphosphorylated peptide via its conserved amphipathic groove.
J. Biol. Chem.
273
:
16305
16310
.
57
Ottmann
C.
,
Yasmin
L.
,
Weyand
M.
,
Veesenmeyer
J. L.
,
Diaz
M. H.
,
Palmer
R. H.
,
Francis
M. S.
,
Hauser
A. R.
,
Wittinghofer
A.
,
Hallberg
B.
.
2007
.
Phosphorylation-independent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.
EMBO J.
26
:
902
913
.
58
Zhai
J.
,
Lin
H.
,
Shamim
M.
,
Schlaepfer
W. W.
,
Cañete-Soler
R.
.
2001
.
Identification of a novel interaction of 14-3-3 with p190RhoGEF.
J. Biol. Chem.
276
:
41318
41324
.
59
Yang
X.
,
Lee
W. H.
,
Sobott
F.
,
Papagrigoriou
E.
,
Robinson
C. V.
,
Grossmann
J. G.
,
Sundström
M.
,
Doyle
D. A.
,
Elkins
J. M.
.
2006
.
Structural basis for protein–protein interactions in the 14-3-3 protein family.
Proc. Natl. Acad. Sci. USA
103
:
17237
17242
.
60
Jones
D. H.
,
Ley
S.
,
Aitken
A.
.
1995
.
Isoforms of 14-3-3 protein can form homo- and heterodimers in vivo and in vitro: implications for function as adapter proteins.
FEBS Lett.
368
:
55
58
.
61
Chaudhri
M.
,
Scarabel
M.
,
Aitken
A.
.
2003
.
Mammalian and yeast 14-3-3 isoforms form distinct patterns of dimers in vivo.
Biochem. Biophys. Res. Commun.
300
:
679
685
.
62
Rosetti
F.
,
Mayadas
T. N.
.
2016
.
The many faces of Mac-1 in autoimmune disease.
Immunol. Rev.
269
:
175
193
.
63
Ehirchiou
D.
,
Xiong
Y.
,
Xu
G.
,
Chen
W.
,
Shi
Y.
,
Zhang
L.
.
2007
.
CD11b facilitates the development of peripheral tolerance by suppressing Th17 differentiation.
J. Exp. Med.
204
:
1519
1524
.

The authors have no financial conflicts of interest.

Supplementary data