Pharmacological activation of integrin CD11b/CD18 (αMβ2, Mac-1, and CR3) shows anti-inflammatory benefits in a variety of animal models of human disease, and it is a novel therapeutic strategy. Reasoning that genetic models can provide an orthogonal and direct system for the mechanistic study of CD11b agonism, we present in this study, to our knowledge, a novel knock-in model of constitutive active CD11b in mice. We genetically targeted the Itgam gene (which codes for CD11b) to introduce a point mutation that results in the I332G substitution in the protein. The I332G mutation in CD11b promotes an active, higher-affinity conformation of the ligand-binding I/A-domain (CD11b αA-domain). In vitro, this mutation increased adhesion of knock-in neutrophils to fibrinogen and decreased neutrophil chemotaxis to a formyl–Met–Leu–Phe gradient. In vivo, CD11bI332G animals showed a reduction in recruitment of neutrophils and macrophages in a model of sterile peritonitis. This genetic activation of CD11b also protected against development of atherosclerosis in the setting of hyperlipidemia via reduction of macrophage recruitment into atherosclerotic lesions. Thus, our animal model of constitutive genetic activation of CD11b can be a useful tool for the study of integrin activation and its potential contribution to modulating leukocyte recruitment and alleviating different inflammatory diseases.

This article is featured in Top Reads, p.2325

Also known as αMβ2, Mac-1, and CR3, CD11b/CD18 is the predominant β2 integrin in polymorphonuclear leukocytes and is abundantly expressed in monocytes, macrophages, and dendritic cells (1, 2). A heterodimer of the CD11b (αM) and CD18 (β2) subunits encoded by the ITGAM and ITGB2 genes, respectively, it exists on the cell surface in two key conformational states: 1) an inactive, low-affinity, closed conformation, and 2) an active, high-affinity, open conformation (3). CD11b/CD18 is among the most versatile of all integrins, with more than 40 reported ligands (4). As a result, it plays an important role in a diversity of immunological processes, from leukocyte recruitment to the sites of tissue injury to the resolution of inflammation (57).

The affinity of CD11b/CD18 to multiple endothelial ligands (e.g., ICAM-1, fibrinogen, and endothelial protein C receptor [EPCR]) (810) has made it a promising target for the treatment of inflammatory diseases. Pharmacological blockade or genetic deletion of CD11b improved restenosis (11), cerebral and renal ischemic injury (12, 13), glomerulonephritis (14, 15), and thrombosis (16) in animal models. Based on these findings, it was initially thought that inhibition of ligand binding to CD11b/CD18 would be a potential therapeutic strategy for vascular inflammation. However, several such anti-integrin therapeutic antagonists failed to meet efficacy end points in clinical trials (17).

Similarly, large-scale, genome-wide association studies have shown that mutations that reduce activity of CD11b or render it inactive are pathogenic (1823). These studies have identified single-nucleotide polymorphisms in the human ITGAM gene that result in the production of functionally deficient CD11b/CD18 but without change in the level of surface expression (22, 24). The detected variants are associated with increased susceptibility to systemic lupus erythematosus and lupus nephritis (18, 20, 21, 25). Animal models of autoimmunity also confirmed the relationship between Mac-1 deficiency and end-organ damage (26, 27). Together, these studies suggested that integrin activation may be an alternate and highly promising therapeutic approach for these pathological conditions.

Toward that, we were among the first, to our knowledge, to develop novel small-molecule agonists of CD11b, and have shown that such agents reduce recruitment of CD11b+ cells and have therapeutic efficacy in a variety of disease models (2830). These include experimental models of vascular injury (28, 30), nephritis (28), organ allograft vasculopathy (31), liver fibrosis (32), hyperoxic lung injury (33), autoimmunity (23), and pancreatic cancer (34). Small-molecule CD11b agonists bind to an allosteric site in the αA-domain (also known as αI-domain) of CD11b, where they stabilize a more active conformation of the receptor and prime it for ligand binding (23). We and others have also shown that CD11b activation can inhibit proinflammatory TLR pathways (23, 35).

To fully understand the therapeutic potential of CD11b activation, as well as to elucidate the molecular pathways involved, alternative, orthologous approaches are needed. In this study, we present, to our knowledge, a novel knock-in (KI) mouse model expressing a constitutively active mutant CD11b. In this model, the Itgam gene was genetically targeted to introduce a mutation (I332G) that promotes a higher-affinity conformation of this integrin subunit. The ligand-binding αA-domain of CD11b contains an allosteric site, known as socket for isoleucine (SILEN) (36) or I-domain allosteric site (IDAS) (37), that modulates its conformation and binding affinity. Mutagenesis of isoleucine 332 to glycine (I332G) shifts the αA-domain into its open, ligand-competent conformation. We introduced this mutation in murine CD11b to generate CD11bI332G mice (KI mice). Our investigations show that this activating mutation decreases inflammatory cell recruitment and vascular inflammation in models of sterile peritonitis and atherosclerosis, respectively. To our knowledge, this novel experimental model can be used to gain deeper insight into mechanisms that can be further therapeutically targeted in inflammation and atherosclerosis.

The CD11bI332G targeting vector was engineered by Cyagen (Santa Clara, CA) as portrayed in Fig. 1A. Briefly, the 5′ and 3′ homology arms including exons 7–12 of the Itgam gene were amplified by PCR from bacterial artificial chromosome clones RP23-343J4 and RP23-323J3 from the C57BL/6J library. The I332G mutation (ATC to GGC) was introduced by site-directed mutagenesis into exon 9 in the 3′ homology arm. The homology arms were cloned into the targeting vector using the NotI and BsiWI (5′ arm) and XhoI and NruI (3′ arm) restriction sites. An Neo cassette flanked by two Frt sites was inserted between the homology arms. A diphtheria toxin A cassette cloned downstream of the 3′ homology arm was used for negative selection. The above cloning strategy was confirmed by PCR, restriction digestion, and sequencing.

CD11bI332G KI mice were generated by Cyagen. Briefly, the targeting vector was linearized with NotI and electroporated into C57BL/6 embryonic stem (ES) cells. Ninety-three G418-resistant clones were screened by PCR, of which 23 positive clones were sent to sequencing. Fourteen ES clones were positive by sequencing, six of which were expanded for Southern blot confirmation using KpnI and EcoRI digestion, followed by hybridization with a probe complementary to the Neo cassette (Supplemental Fig. 1). Clones 1B6 and 1C9 were selected for blastocyst injection and chimera production. Chimeras were bred with C57BL/6 females to test germline transmission. Three pups from clone 1B6 and three from clone 1C9 were identified as positive germline F1 founders by PCR and sequencing. F1 founders were crossed with homozygous ROSA26::FLPe KI mice (38) (stock no. 003946; The Jackson Laboratory, Bar Harbor, ME) at the University of Miami to excise the Neo cassette. Mac-1I332G heterozygotes and negative for the Neo insertion were then backcrossed with C57BL/6 mice to remove the ROSA26::FLPe allele. Once achieved, mice were further backcrossed into the C57BL/6 background for six to eight generations. Primers 5′-AAGGAATATCTTTTGCTGGACGTACG-3′ and 5′-CAGAAGGCAGCTTAGAACAGGGC-3′ were used to screen for the excision of Neo, whereas 5′-CACTGATATTGTAAGTAGTTTGC-3′ and 5′-CTAGTGCGAAGTAGTGATCAGG-3′ were used to detect the ROSA26::FLPe allele. The Mac-1I332G colony is maintained as heterozygotes, from which CD11bI332G homozygotes and CD11b wild-type (CD11bWT) littermates are obtained for experiments. Genotyping of the Itgam wild-type (WT) and I332G alleles is performed with primers 5′-ATTTAGCTTTGGCTCCTTGGCAAC-3′ and 5′-TGGAGCAAGTCAGACCCAAATGTC-3′, followed by enzymatic digestion with BglII. This strategy results in 107- and 162-bp bands for the WT allele and the undigested 269-bp band for the I332G variant (Fig. 1B). All animal procedures were performed according to the National Institutes of Health guidelines (Guide for the Care and Use of Laboratory Animals) and approved by the respective Institutional Animal Care and Use Committees at the University of Miami Miller School of Medicine and Rush University Medical Center.

Peritonitis was induced in 14–16-wk-old CD11bWT and CD11bI332G mice of both sexes by injecting 3 ml of a sterile 3% Brewer’s thioglycollate broth (Sigma-Aldrich, St. Louis, MO). To analyze cell recruitment, leukocyte cell suspensions were obtained by lavage of the peritoneal cavity with 10 ml of PBS at 4 and 48 h after injection. Cells were collected by centrifugation for 10 min at 250 × g and washed twice with PBS before analysis.

Hypercholesterolemia was induced in 12-wk-old CD11bWT and CD11bI332G mice of both sexes after administration of a single tail-vein injection of the recombinant rAAV8-D377Y-mPCSK9 virus (University of North Carolina at Chapel Hill Vector Core, Chapel Hill, NC) (2.0 × 1010 copies per mouse) according to the published protocol (39). The exogenous expression of PCSK9 enhances internalization and degradation of hepatic low-density lipoprotein receptors. One week after the virus injection, mice started feeding on a cocoa butter Teklad research custom diet (catalog TD.88051; Envigo, Somerset, NJ) for 12 wk ad libitum. Cholesterol levels were measured 1 wk after diet initiation and at the end of the 12 wk diet using cholesterol test strips by PTS Panels (Indianapolis, IN). After completion of the diet, nonfasting blood was submitted to the University of Miami Division of Comparative Pathology for complete blood count and blood chemistry, and whole aortas were submitted to Eehscience (Pickerington, OH) for independent determination of atherosclerotic burden by Oil Red O staining. Serial cross-sections of the brachiocephalic trunk were obtained for plaque characterization. Lesion size and macrophage infiltration were quantified at three positions along the brachiocephalic artery (∼550, ∼450, and ∼350 μm from the aortic arch).

Peritoneal neutrophils were isolated 4 h after sterile 3% Brewer’s thioglycollate injection. Cell purity was assessed by flow cytometry. Cell adhesion assays were performed as previously described (40) and used immobilized fibrinogen as Mac-1 ligand. Briefly, 384-well microplates (Corning, Corning, NY) were coated with 15 μg/ml fibrinogen in PBS containing 1 mM of each Ca2+ and Mg2+ by incubating overnight at 4°C. The nonspecific sites were blocked with 1% gelatin in TBS, followed by washing three times with TBS. Cells were suspended in TBS containing 1 mM of each Ca2+ and Mg2+ (TBS2+ buffer) or in TBS containing 1 mM Mn2+ in the presence or absence of an anti-CD11b blocking Ab (clone M1/70, 10 μg/ml). Cells were incubated in the ligand-coated wells (3000 cells per well) for 10 min at 37°C. The assay plates were then gently inverted and kept in the inverted position for 30 min at room temperature to dislodge nonadherent cells. The remaining adherent cells were fixed using 4% formaldehyde and quantified by automated imaging microscopy as previously described (41). Assays were performed in triplicate wells and at least three independent times.

Peritoneal neutrophils were obtained as above. Neutrophil chemotaxis on planar surfaces was performed using 48-well Zigmond chambers (Neuro Probe, Gaithersburg, MD) as previously described (40). Briefly, cells were preincubated for 10 min on fibrinogen-coated (25 μg/ml) glass coverslips in a humidified chamber with RPMI 1640 supplemented with 1% FBS. The coverslips were then placed on top of Zigmond chambers, and fMLF (Sigma-Aldrich) gradient was created by placing the assay buffer (RPMI 1640 with 1% FBS) in one well of the chamber and 10 μM fMLF in assay buffer in the other well. The migration of neutrophils toward fMLF was recorded at 5–30-s intervals for a period of 25 min using a Nikon Eclipse 90i microscope, as previously described (40, 42). Images were acquired using a Nikon DS camera with a PLAN APO ×20 differential interference contrast microscopy objective and the Nikon Imaging software. Analysis of neutrophil migration was performed with the motile population that had moved more than 10 μm using the ImageJ software (National Institutes of Health, Bethesda, MD) with manual cell tracking using the chemotaxis and migration tool plugins (Ibidi) for ImageJ. Data from 30 neutrophils per mouse strain were quantified in at least three independent experiments.

Primary macrophages were collected by peritoneal lavage from 14- to 16-wk-old CD11bWT and CD11bI332G mice of both sexes, 4 d after injecting 3 ml of sterile 3% Brewer’s thioglycollate broth (Sigma-Aldrich). Cells were adherence purified for 1 h, followed by a wash with PBS to remove nonadherent cells. Adherent cells were suspended in DMEM supplemented with 10% FBS and counted. One million cells per well were plated in 12-well plates and allowed to settle down for 2 d before treatment. Cells were stimulated with 50 ng/ml of LPS from Escherichia coli K12 (InvivoGen, San Diego, CA), and supernatants were collected at baseline and at 2, 4, and 8 h after treatment. Levels of secreted IL-1β, IL-6, and TNF-α were quantified by ELISA following the manufacturer’s protocols (R&D Systems, Minneapolis, MN).

Three brachiocephalic artery cross-sections per animal, corresponding to positions ∼550, ∼450, and ∼350 μm from the aortic arch were costained with anti–galectin-3 (Mac2) and smooth muscle actin (SMA) Abs to identify macrophages and smooth muscle cells, respectively. Briefly, Ag retrieval was performed in 10 mM sodium citrate, 0.05% Tween 20 (pH 6) solution for 30 min in 95°C water, followed by treatment with 3% hydrogen peroxide and TNB blocking solution (no. FP1020; PerkinElmer, Waltham, MA). Then, slides were incubated with rat anti-mouse Mac2 Ab (1:50, no. 125402; BioLegend, San Diego, CA) and mouse anti-human SMA Ab (1:200, no. M0851; Dako) overnight at 4οC. The next day, the slides were incubated with Alexa Fluor 546 goat anti-rat Ab (1:1000, no. A11081; Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 488 goat anti-mouse Ab (1:1000, no. A11029; Thermo Fisher Scientific) for 45 min. Sections were counter stained with 300 nM DAPI solution (no. D1306; Thermo Fisher Scientific) in PBS for 3 min and mounted in DABCO antifading polyvinyl alcohol mounting medium (no. 10981; Sigma-Aldrich). Sections were examined in an Olympus Ix71 inverted microscope and photographed using the Olympus cellSens Standard software. Image analysis was performed with ImageJ (National Institutes of Health).

All Abs and reagents were purchased from BioLegend except when indicated otherwise. Quantification of integrin subunit expression used the Abs CD11a PE (no. 101107), CD11b PerCP (no. 101230), CD11c BV650 (no. 564079; BD Biosciences), CD18 APC (no. 562828; BD Biosciences), CD115 BV605 (catalog no. 135577), and Gr-1 PE/Dazzle 594 (no. 108452). Briefly, cells were washed with PBS twice and counted, and Fc receptors were blocked with anti-mouse CD16/32 Ab for 10 min at 4°C. Cells were washed with PBS, and True-Stain Monocyte Blocker was added before labeling with eBioscience Fixable Viability Dye eFluor 660 (Thermo Fisher Scientific) for 30 min. Cells were washed with FACS buffer with 0.1% sodium azide (flow buffer), and True-Stain Monocyte Blocker was added again, followed by 30-min incubation with the remaining labeling Abs. Excess Abs were washed, and cells were fixed with flow cytometry buffer supplemented with 1% paraformaldehyde prior to flow cytometric analyses. Flow cytometry data were read using a BD LSRFortessa High Throughput Sampler analyzer (BD Biosciences) and processed with the FlowJo software (Ashland, OR).

Statistical analyses were performed in GraphPad Prism 5 (San Diego, CA). Normally distributed values are presented as mean ± SEM and compared using a two-tailed Student t test or one-way ANOVA. Nonnormally distributed data are presented as median ± interquartile range and compared using the Mann–Whitney U test. A p value <0.05 was considered significant.

Isoleucine 332 in CD11b, corresponding to residue 316 after processing the N-terminal signal peptide, has been identified as a key residue in stabilizing the inactive low-affinity conformation of the CD11b/CD18 integrin receptor (36). The regulatory role of this residue relies on its hydrophobic interactions with the SILEN (Fig. 1C). The I332G substitution weakens these interactions and favors the active, high-affinity, open conformation of the CD11b αA-domain (36). However, consequences of such activation of CD11b in animals are currently not fully understood. Therefore, to investigate the role of active CD11b in vivo, we generated mice harboring the I332G-activating CD11b mutation following the strategy delineated in Fig. 1. Briefly, a genetic construct was designed to replace the WT exon 9 in the Itgam gene with the mutant exon 9 downstream of an Frt-flanked Neo cassette (Fig. 1A). The construct was inserted by homologous recombination in ES cells, followed by antibiotic selection, PCR screening, and Southern blot confirmation (Supplemental Fig. 1). After blastocyst injection and chimera production, heterozygous founders were crossed with ROSA26::FLPe mice to generate Neo-deleted CD11bI332G KI animals (KI animals), and backcrossed successively into the C57BL/6 background. Fig. 1B presents the genotyping analysis of CD11bI332G KI and littermate WT mice.

FIGURE 1.

Generation of CD11bI332G KI mice. (A) Schematic of the targeting strategy. From top to bottom, the diagrams indicate the genomic structure of the WT allele, the targeting vector, and the targeted KI allele. Confirmatory Southern blots used a probe complementary to the Neo cassette and DNA digested with EcoRI or KpnI (see Supplemental Fig. 1). (B) Genotyping of the WT and KI alleles in mice by PCR, followed by BglII digestion. The I332G mutation disrupts a BglII recognition site in exon 9 of the Itgam gene, generating a 269-bp band in KI homozygotes and 107- and 162-bp bands for the WT allele. (C) Partial amino acid alignment of the human, WT mouse, and mutant (I332G) mouse CD11b subunits of integrin CD11b/CD18. The mutant position is highlighted in black. The boxed sequence delineates the CD11b A-domain, with asterisks indicating amino acid residues that form a hydrophobic coordination socket (known as SILEN) for WT Ile332. Amino acid numbering is based on the full-length proteins in GenBank accession numbers NP_001139280.1 (https://www.ncbi.nlm.nih.gov/protein/NP_001139280.1) and NP_001076429.1 (https://www.ncbi.nlm.nih.gov/protein/NP_001076429.1) for human and mouse, respectively.

FIGURE 1.

Generation of CD11bI332G KI mice. (A) Schematic of the targeting strategy. From top to bottom, the diagrams indicate the genomic structure of the WT allele, the targeting vector, and the targeted KI allele. Confirmatory Southern blots used a probe complementary to the Neo cassette and DNA digested with EcoRI or KpnI (see Supplemental Fig. 1). (B) Genotyping of the WT and KI alleles in mice by PCR, followed by BglII digestion. The I332G mutation disrupts a BglII recognition site in exon 9 of the Itgam gene, generating a 269-bp band in KI homozygotes and 107- and 162-bp bands for the WT allele. (C) Partial amino acid alignment of the human, WT mouse, and mutant (I332G) mouse CD11b subunits of integrin CD11b/CD18. The mutant position is highlighted in black. The boxed sequence delineates the CD11b A-domain, with asterisks indicating amino acid residues that form a hydrophobic coordination socket (known as SILEN) for WT Ile332. Amino acid numbering is based on the full-length proteins in GenBank accession numbers NP_001139280.1 (https://www.ncbi.nlm.nih.gov/protein/NP_001139280.1) and NP_001076429.1 (https://www.ncbi.nlm.nih.gov/protein/NP_001076429.1) for human and mouse, respectively.

Close modal

CD11bI332G KI mice are normal in size and in reproductive and social behavior. Their complete blood count at 16 wk showed no significant differences from littermate WT mice (Table I). Surface expression of the integrin subunits CD11b and CD18, and of other α subunits (CD11a and CD11c) from the β2 integrin family, in peripheral blood neutrophils and monocytes was also similar between KI and WT animals (Supplemental Fig. 2).

Table I.
Complete blood count analysis of peripheral blood collected from 16-wk-old WT and CD11bI332G KI mice
WT (n = 8)CD11bI332G KI (n = 8)p Value
Complete Blood Count    
 WBC (1 × 103/μl) 3.2 ± 1.5 2.2 ± 0.5 0.09 
 RBC (1 × 106/μl) 7.8 ± 0.5 7.4 ± 0.6 0.11 
 Hemoglobin (g/dl) 10.9 ± 0.5 10.5 ± 0.5 0.16 
 Hematocrit (%) 37.3 ± 2.3 35.3 ± 2.5 0.12 
 MCV (fl) 47.5 ± 1.2 47.9 ± 1.6 0.61 
 MCH (pg) 14.0 (13.3–14.0) 14.0 (14.0–14.8) 0.50 
 MCHC (%) 29.5 ± 1.6 30.0 ± 0.8 0.44 
 Segs (1 × 103/μl) 0.2 (0.1–0.3) 0.2 (0.1–0.2) 0.24 
 Bands (1 × 103/μl) N.D. N.D. — 
 Lymphocytes (1 × 103/μl) 2.4 ± 1.0 1.7 ± 0.4 0.09 
 Monocytes (1 × 103/μl) 0.4 ± 0.3 0.3 ± 0.1 0.09 
 Eosinophils (1 × 103/μl) 0.03 (0.0–0.04) 0.02 (0.0–0.03) 0.41 
 Basophils (1 × 103/μl) N.D. N.D. — 
 NRBC N.D. N.D. — 
WT (n = 8)CD11bI332G KI (n = 8)p Value
Complete Blood Count    
 WBC (1 × 103/μl) 3.2 ± 1.5 2.2 ± 0.5 0.09 
 RBC (1 × 106/μl) 7.8 ± 0.5 7.4 ± 0.6 0.11 
 Hemoglobin (g/dl) 10.9 ± 0.5 10.5 ± 0.5 0.16 
 Hematocrit (%) 37.3 ± 2.3 35.3 ± 2.5 0.12 
 MCV (fl) 47.5 ± 1.2 47.9 ± 1.6 0.61 
 MCH (pg) 14.0 (13.3–14.0) 14.0 (14.0–14.8) 0.50 
 MCHC (%) 29.5 ± 1.6 30.0 ± 0.8 0.44 
 Segs (1 × 103/μl) 0.2 (0.1–0.3) 0.2 (0.1–0.2) 0.24 
 Bands (1 × 103/μl) N.D. N.D. — 
 Lymphocytes (1 × 103/μl) 2.4 ± 1.0 1.7 ± 0.4 0.09 
 Monocytes (1 × 103/μl) 0.4 ± 0.3 0.3 ± 0.1 0.09 
 Eosinophils (1 × 103/μl) 0.03 (0.0–0.04) 0.02 (0.0–0.03) 0.41 
 Basophils (1 × 103/μl) N.D. N.D. — 
 NRBC N.D. N.D. — 

Values are presented as mean ± SD or median (interquartile range). Dashes indicated p values could not be calculated. MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; N.D., not detected; NRBC, nucleated RBC; Seg, segmented neutrophils.

The functional effect of the I332G mutation was tested in peritoneal neutrophils using adhesion and chemotaxis assays (Figs. 24). In agreement with the activating role of this mutation (36), neutrophils from CD11bI332G mice showed increased static adhesion to fibrinogen than those from WT controls under physiological conditions (1 mM Ca2+ and 1 mM Mg2+; Fig. 2). The enhanced adhesion of KI neutrophils was CD11b dependent and could be neutralized with an anti-CD11b blocking Ab (M1/70). Both WT and CD11bI332G neutrophils reached similar levels of adhesion in the presence of the activating cation Mn2+ (Fig. 2) (43).

FIGURE 2.

Adhesion of neutrophils from CD11b WT and KI (I332G) mice. Adhesion of peritoneal neutrophils to fibrinogen in the presence of physiological divalent cations (1 mM Ca2+ and 1 mM Mg2+), the activating cation Mn2+ (1 mM), and the anti-CD11b blocking Ab clone M1/70. Bars represent the mean ± SEM, n = 4 per group in three independent experiments. N.S., not significant.

FIGURE 2.

Adhesion of neutrophils from CD11b WT and KI (I332G) mice. Adhesion of peritoneal neutrophils to fibrinogen in the presence of physiological divalent cations (1 mM Ca2+ and 1 mM Mg2+), the activating cation Mn2+ (1 mM), and the anti-CD11b blocking Ab clone M1/70. Bars represent the mean ± SEM, n = 4 per group in three independent experiments. N.S., not significant.

Close modal
FIGURE 4.

Time-lapse video and photography of chemotaxing neutrophils from CD11b WT and KI (I332G) mice. (A) Time-lapse videos of representative peritoneal neutrophils during chemotaxis toward an fMLF gradient. (B) Microphotographs of representative chemotaxing neutrophils from CD11bWT and CD11bI332G mice in response to an fMLF stimulus.

FIGURE 4.

Time-lapse video and photography of chemotaxing neutrophils from CD11b WT and KI (I332G) mice. (A) Time-lapse videos of representative peritoneal neutrophils during chemotaxis toward an fMLF gradient. (B) Microphotographs of representative chemotaxing neutrophils from CD11bWT and CD11bI332G mice in response to an fMLF stimulus.

Close modal

Firmer adhesion of neutrophils is expected to result in impaired chemotaxis in response to an fMLF gradient (28), and indeed, we observed that KI neutrophils showed significantly reduced chemotaxis compared with WT cells (Figs. 3, 4A). As with chemotaxis of neutrophils upon pharmacologic CD11b activation (28), the chemotaxing KI neutrophils showed prevalence of elongated, trailing uropods compared with those from WT animals (Fig. 4B). These results suggest increased adhesion and reduced detachment from the substrate as the reasons for their impaired motility.

FIGURE 3.

Chemotaxis of neutrophils from CD11b WT and KI (I332G) mice. (A) Spider plots delineating the chemotaxis of peritoneal neutrophils in response to an fMLF gradient. (BD) Quantification of Euclidean distance (B), accumulated distance (C), and cell velocity (D) in 30 migration tracks from CD11bWT and CD11bI332G neutrophils as shown in (A). Bars represent the mean ± SEM, n = 30 per group in three independent experiments.

FIGURE 3.

Chemotaxis of neutrophils from CD11b WT and KI (I332G) mice. (A) Spider plots delineating the chemotaxis of peritoneal neutrophils in response to an fMLF gradient. (BD) Quantification of Euclidean distance (B), accumulated distance (C), and cell velocity (D) in 30 migration tracks from CD11bWT and CD11bI332G neutrophils as shown in (A). Bars represent the mean ± SEM, n = 30 per group in three independent experiments.

Close modal

The CD11b/CD18 integrin plays a fundamental role in the recruitment of neutrophils and monocytes to sites of inflammation (5, 44, 45). Therefore, we tested the effects of the CD11bI332G mutation in inflammatory cell recruitment in vivo in the setting of thioglycollate-induced peritonitis. The number of infiltrated neutrophils and macrophages in the peritoneal cavity was measured at two time points after thioglycollate injection (Fig. 5A, 5B). Neutrophil counts were significantly lower in KI versus WT mice 4 h after injection but equivalent at 48 h in both animal groups (Fig. 5A). Similarly, there was a significant decrease in peritoneal macrophage infiltration in CD11bI332G mice compared with WT animals at the 48-h time point (Fig. 5B). These results indicate a reduction and/or delay in leukocyte recruitment in KI mice in the setting of acute sterile inflammation.

FIGURE 5.

Inflammatory cell recruitment and cytokine secretion in thioglycollate-induced peritonitis. (A and B) Cell counts of neutrophils (A) and macrophages (B) in the peritoneum of CD11bWT and CD11bI332G mice by flow cytometry at 4 and 48 h after sterile thioglycollate injection. Neutrophils were selected as Gr-1hi, whereas macrophages were identified as F4/80+. Bars represent the mean ± SEM, n = 7–9 mice per group. (CE) Secretion of inflammatory cytokines by peritoneal macrophages isolated at 4 d after thioglycollate injection. Cells were count normalized and stimulated with LPS for increasing periods of time in vitro. Bars represent the mean ± SEM, n = 4 mice per group in three independent experiments. N.S., not significant.

FIGURE 5.

Inflammatory cell recruitment and cytokine secretion in thioglycollate-induced peritonitis. (A and B) Cell counts of neutrophils (A) and macrophages (B) in the peritoneum of CD11bWT and CD11bI332G mice by flow cytometry at 4 and 48 h after sterile thioglycollate injection. Neutrophils were selected as Gr-1hi, whereas macrophages were identified as F4/80+. Bars represent the mean ± SEM, n = 7–9 mice per group. (CE) Secretion of inflammatory cytokines by peritoneal macrophages isolated at 4 d after thioglycollate injection. Cells were count normalized and stimulated with LPS for increasing periods of time in vitro. Bars represent the mean ± SEM, n = 4 mice per group in three independent experiments. N.S., not significant.

Close modal

Primary macrophages were obtained from the peritoneal cavity of CD11bI332G and WT animals at 4 d after thioglycollate-induced peritonitis. Count-normalized cells were plated and cultured for 2 d in the absence of CD11b-specific ligands. After this pretreatment period, cells were stimulated with LPS, and cytokine secretion was quantified at three time points (Fig. 5C–E). CD11bI332G cells showed significantly lower IL-1β and TNF-α secretion starting at 2 h after treatment (Fig. 5C, 5E). Significantly reduced IL-6 levels were also observed starting at 4 h poststimulation (Fig. 5D). These results parallel published reports with pharmacological CD11b-activating agents (23, 46) and are further supported by the opposite pattern in CD11b knockout (KO) animals (47).

CD11b is a key mediator of vascular inflammation and atherosclerosis by regulating leukocyte adhesion and extravasation across the endothelium via its ligands ICAM-1, lipoprotein(a), and others (5, 7, 48). We previously showed that pharmacologic activation of CD11b reduces influx of macrophages after arterial injury (28). To investigate whether activation of CD11b is also atheroprotective, we used KI animals in a model of atherosclerosis. Hyperlipidemia was induced in cohorts of CD11bI332G and WT mice via injection of the recombinant rAAV8-D377Y-mPCSK9 virus, followed by 12 wk of high-fat diet, a recently established model of atherosclerosis (39). Both experimental groups had overt hypercholesterolemia at the end point (Supplemental Fig. 3A, 3B). However, CD11bI332G mice showed significantly lower atherosclerotic burden than WT animals, particularly in the thoracic and abdominal aorta (Fig. 6). These results were confirmed in cross-sections of the brachiocephalic artery (Fig. 7). Lesion size (as percentage of total wall area) was significantly smaller in CD11bI332G mice compared with their WT counterparts (Fig. 7B). In addition, the number of infiltrated macrophages per section was significantly lower in the former (Fig. 7C). This suggests that CD11b activation has a protective role in atherosclerosis in great part because of a reduction of macrophage recruitment to atherosclerotic lesions.

FIGURE 6.

Atherosclerosis development in the aorta of CD11b WT and KI (I332G) mice. (A) Heatmap and percentage of plaque burden by area in whole aortas from CD11bWT (n = 10) and CD11bI332G hyperlipidemic mice (n = 13). (BD) Quantification of plaque burden in the aortic arch (B), the thoracic aorta (C), and the abdominal aorta (D) of hyperlipidemic mice. Error bars represent the median and interquartile range, n = 10–13 per group. N.S., not significant.

FIGURE 6.

Atherosclerosis development in the aorta of CD11b WT and KI (I332G) mice. (A) Heatmap and percentage of plaque burden by area in whole aortas from CD11bWT (n = 10) and CD11bI332G hyperlipidemic mice (n = 13). (BD) Quantification of plaque burden in the aortic arch (B), the thoracic aorta (C), and the abdominal aorta (D) of hyperlipidemic mice. Error bars represent the median and interquartile range, n = 10–13 per group. N.S., not significant.

Close modal
FIGURE 7.

Atherosclerosis development in the brachiocephalic trunk of CD11b WT and KI (I332G) mice. (A) Representative cross-sections of advanced atherosclerotic lesions in the brachiocephalic artery of CD11bWT and CD11bI332G hyperlipidemic mice. Galectin-3 (Mac2)+-positive macrophages/foam cells are stained in yellow, SMA+ cells are in green, and nuclei are counter stained in blue (DAPI). Scale bar, 100 μm. (B and C) Quantification of lesion size as percentage of total wall area (B) and Mac2+ macrophage counts per section (C) in brachiocephalic artery sections of CD11bWT and CD11bI332G mice. Values represent the average of three positions in the artery (at ∼550, ∼450, and ∼350 μm from the aortic arch). Error bars represent the median and interquartile range, n = 7 per group.

FIGURE 7.

Atherosclerosis development in the brachiocephalic trunk of CD11b WT and KI (I332G) mice. (A) Representative cross-sections of advanced atherosclerotic lesions in the brachiocephalic artery of CD11bWT and CD11bI332G hyperlipidemic mice. Galectin-3 (Mac2)+-positive macrophages/foam cells are stained in yellow, SMA+ cells are in green, and nuclei are counter stained in blue (DAPI). Scale bar, 100 μm. (B and C) Quantification of lesion size as percentage of total wall area (B) and Mac2+ macrophage counts per section (C) in brachiocephalic artery sections of CD11bWT and CD11bI332G mice. Values represent the average of three positions in the artery (at ∼550, ∼450, and ∼350 μm from the aortic arch). Error bars represent the median and interquartile range, n = 7 per group.

Close modal

Integrin CD11b/CD18 is essential for a diversity of functions in immune cells (2, 4). In addition to regulating cell adhesion, migration, and phagocytosis, CD11b/CD18 modulates pro- and anti-inflammatory signaling (23, 4951). Our recent studies show that CD11b acts as a negative regulator of proinflammatory TLR signaling and of B cell autoreactivity (23) and that pharmacologic activation of CD11b reduces inflammation and injury (2831, 33). However, our current knowledge of CD11b functions and downstream signaling pathways is largely based on pharmacological agents and CD11b KO models, whereas a genetic activation model was missing. In this study, to our knowledge, we present a novel genetic model of constitutive CD11b activation to study the role of active integrin CD11b/CD18 in inflammatory injury in vivo.

Our results show that global, constitutive CD11bI332G KI animals developed reduced inflammatory injury in two different models of inflammation. In vitro studies with KI, neutrophils showed impaired chemotaxis and pronounced uropod elongation in response to an fMLF gradient. Directional motility of neutrophils requires a tightly coordinated and self-organizing cell polarity. Redistribution of β2 integrins and regulation of their ligand-binding affinity along the cell body is one of the mechanisms by which neutrophils regulate polarized adhesion (52). Ultimately, after initial adhesion and cell elongation, the trailing edge of the cell (uropod) needs to be able to detach and retract to promote forward protrusion of the leading edge (pseudopod) in the direction of the chemoattracting agent (52, 53). In migrating neutrophils, CD11b/CD18 redistributes to the uropod, where it stabilizes microtubules and regulates myosin L chain–mediated uropod contractility (5457). The morphology of chemotaxing neutrophils from CD11bI332G KI mice is indicative of impaired uropod detachment as a result of CD11b/CD18 integrin activation. In vivo, both CD11b+ neutrophils and monocytes from CD11bI332G mice showed a significant reduction in inflammatory cell recruitment in the setting of peritonitis. These data confirm our previous findings that CD11b activation can efficiently regulate neutrophil and monocyte infiltration (28, 29).

Finally, using a model of chronic vascular inflammation, we show that genetic activation of CD11b results in a significant decrease in aortic and brachiocephalic plaque burden in hyperlipidemic KI mice compared with WT controls. Our results suggest that a reduction in macrophage infiltration is one of the mechanisms behind CD11b-mediated atheroprotection. Nonetheless, future studies will help determine whether other mechanisms contribute to this effect, including lower macrophage retention or proliferation in plaques, and/or a regulatory role in macrophage activation and polarization. Previous studies have associated CD11b/CD18 surface upregulation and activation in macrophages with increased macrophage egression from atherosclerotic plaques (58) and the peritoneum (6). Others have proposed that active CD11b suppresses the alternative activation of macrophages and decreases foam cell formation in vitro through the downregulation of lipid peroxidation mechanisms and scavenger receptors (49, 59). However, a clear understanding of these mechanisms is lacking because of the use on nonselective integrin activating agents and the absence of a murine model of CD11b activation in which to test these findings in vivo. Similarly, CD11b KO mice have provided conflicting data (60, 61), likely because of compensatory effects by other integrins, among other factors. Despite the previously reported atheroprotective role of CD11b in females, but not males (61), we observed a similar trend of decreased plaque burden in both sexes. Unfortunately, our study was not adequately powered for gender effects. Therefore, this is a question that deserves further attention. Future investigations on the role of activated CD11b on controlling atherogenesis and/or enhancing atherosclerosis regression are warranted.

In conclusion, our work presents, to our knowledge, a novel murine model that constitutively expresses an active variant of the CD11b/CD18 integrin. Given the recent interest in integrin activation as a potential therapeutic approach, we expect that this model will not only provide a unique tool for future studies on the effects of CD11b activation in a variety of disease settings but will also be extremely useful in fully elucidating underlying mechanisms of action that can support therapeutic drug development.

This work was supported by National Heart, Lung, and Blood Institute/National Institutes of Health Grants R01-HL125672 (to R.I.V.-P. and O.C.V.) and K08-HL151747 (to L.M.), National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health Grants R01-DK121227 (to R.I.V.-P.) and R01-DK107984 and R01-DK084195 (to V.G.), and American Heart Association Predoctoral Fellowship 18PRE34030314 (to Z.M.Z.). This project was also supported in part by Bears Care, the Department of Internal Medicine at Rush University Medical Center, and the National Cancer Institute/National Institutes of Health Grant R01-CA244938 (to V.G.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

CD11bWT

CD11b wild-type

ES

embryonic stem

KI

knock-in

KO

knockout

SILEN

socket for isoleucine

SMA

smooth muscle actin

WT

wild-type.

1
Arnaout
,
M. A.
1990
.
Structure and function of the leukocyte adhesion molecules CD11/CD18.
Blood
75
:
1037
1050
.
2
Schittenhelm
,
L.
,
C. M.
Hilkens
,
V. L.
Morrison
.
2017
.
β2 integrins as regulators of dendritic cell, monocyte, and macrophage function.
Front. Immunol.
8
:
1866
.
3
Nishida
,
N.
,
C.
Xie
,
M.
Shimaoka
,
Y.
Cheng
,
T.
Walz
,
T. A.
Springer
.
2006
.
Activation of leukocyte beta2 integrins by conversion from bent to extended conformations.
Immunity
25
:
583
594
.
4
Podolnikova
,
N. P.
,
A. V.
Podolnikov
,
T. A.
Haas
,
V. K.
Lishko
,
T. P.
Ugarova
.
2015
.
Ligand recognition specificity of leukocyte integrin αMβ2 (Mac-1, CD11b/CD18) and its functional consequences.
Biochemistry
54
:
1408
1420
.
5
Dunne
,
J. L.
,
R. G.
Collins
,
A. L.
Beaudet
,
C. M.
Ballantyne
,
K.
Ley
.
2003
.
Mac-1, but not LFA-1, uses intercellular adhesion molecule-1 to mediate slow leukocyte rolling in TNF-alpha-induced inflammation.
J. Immunol.
171
:
6105
6111
.
6
Cao
,
C.
,
D. A.
Lawrence
,
D. K.
Strickland
,
L.
Zhang
.
2005
.
A specific role of integrin Mac-1 in accelerated macrophage efflux to the lymphatics.
Blood
106
:
3234
3241
.
7
Kourtzelis
,
I.
,
I.
Mitroulis
,
J.
von Renesse
,
G.
Hajishengallis
,
T.
Chavakis
.
2017
.
From leukocyte recruitment to resolution of inflammation: the cardinal role of integrins.
J. Leukoc. Biol.
102
:
677
683
.
8
Diamond
,
M. S.
,
D. E.
Staunton
,
S. D.
Marlin
,
T. A.
Springer
.
1991
.
Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation.
Cell
65
:
961
971
.
9
Altieri
,
D. C.
,
F. R.
Agbanyo
,
J.
Plescia
,
M. H.
Ginsberg
,
T. S.
Edgington
,
E. F.
Plow
.
1990
.
A unique recognition site mediates the interaction of fibrinogen with the leukocyte integrin Mac-1 (CD11b/CD18).
J. Biol. Chem.
265
:
12119
12122
.
10
Fink
,
K.
,
H. J.
Busch
,
N.
Bourgeois
,
M.
Schwarz
,
D.
Wolf
,
A.
Zirlik
,
K.
Peter
,
C.
Bode
,
C.
von Zur Muhlen
.
2013
.
Mac-1 directly binds to the endothelial protein C-receptor: a link between the protein C anticoagulant pathway and inflammation?
PLoS One
8
: e53103.
11
Rogers
,
C.
,
E. R.
Edelman
,
D. I.
Simon
.
1998
.
A mAb to the beta2-leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits.
Proc. Natl. Acad. Sci. USA
95
:
10134
10139
.
12
Soriano
,
S. G.
,
A.
Coxon
,
Y. F.
Wang
,
M. P.
Frosch
,
S. A.
Lipton
,
P. R.
Hickey
,
T. N.
Mayadas
.
1999
.
Mice deficient in Mac-1 (CD11b/CD18) are less susceptible to cerebral ischemia/reperfusion injury.
Stroke
30
:
134
139
.
13
Dehnadi
,
A.
,
A.
Benedict Cosimi
,
R.
Neal Smith
,
X.
Li
,
J. L.
Alonso
,
T. K.
Means
,
M. A.
Arnaout
.
2017
.
Prophylactic orthosteric inhibition of leukocyte integrin CD11b/CD18 prevents long-term fibrotic kidney failure in cynomolgus monkeys.
Nat. Commun.
8
:
13899
.
14
Hirahashi
,
J.
,
K.
Hishikawa
,
S.
Kaname
,
N.
Tsuboi
,
Y.
Wang
,
D. I.
Simon
,
G.
Stavrakis
,
T.
Shimosawa
,
L.
Xiao
,
Y.
Nagahama
, et al
.
2009
.
Mac-1 (CD11b/CD18) links inflammation and thrombosis after glomerular injury.
Circulation
120
:
1255
1265
.
15
Tang
,
T.
,
A.
Rosenkranz
,
K. J.
Assmann
,
M. J.
Goodman
,
J. C.
Gutierrez-Ramos
,
M. C.
Carroll
,
R. S.
Cotran
,
T. N.
Mayadas
.
1997
.
A role for Mac-1 (CDIIb/CD18) in immune complex-stimulated neutrophil function in vivo: Mac-1 deficiency abrogates sustained Fcgamma receptor-dependent neutrophil adhesion and complement-dependent proteinuria in acute glomerulonephritis.
J. Exp. Med.
186
:
1853
1863
.
16
Wang
,
Y.
,
H.
Gao
,
C.
Shi
,
P. W.
Erhardt
,
A.
Pavlovsky
,
D. A.
Soloviev
,
K.
Bledzka
,
V.
Ustinov
,
L.
Zhu
,
J.
Qin
, et al
.
2017
.
Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα. [Published erratum appears in 2017 Nat. Commun. 8: 16124.]
Nat. Commun.
8
:
15559
.
17
Krams
,
M.
,
K. R.
Lees
,
W.
Hacke
,
A. P.
Grieve
,
J.-M.
Orgogozo
,
G. A.
Ford
;
ASTIN Study Investigators
.
2003
.
Acute stroke therapy by inhibition of neutrophils (ASTIN): an adaptive dose-response study of UK-279,276 in acute ischemic stroke.
Stroke
34
:
2543
2548
.
18
Nath
,
S. K.
,
S.
Han
,
X.
Kim-Howard
,
J. A.
Kelly
,
P.
Viswanathan
,
G. S.
Gilkeson
,
W.
Chen
,
C.
Zhu
,
R. P.
McEver
,
R. P.
Kimberly
, et al
.
2008
.
A nonsynonymous functional variant in integrin-alpha(M) (encoded by ITGAM) is associated with systemic lupus erythematosus.
Nat. Genet.
40
:
152
154
.
19
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
.
20
Han
,
S.
,
X.
Kim-Howard
,
H.
Deshmukh
,
Y.
Kamatani
,
P.
Viswanathan
,
J. M.
Guthridge
,
K.
Thomas
,
K. M.
Kaufman
,
J.
Ojwang
,
A.
Rojas-Villarraga
, et al
.
2009
.
Evaluation of imputation-based association in and around the integrin-alpha-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
.
21
Yang
,
W.
,
M.
Zhao
,
N.
Hirankarn
,
C. S.
Lau
,
C. C.
Mok
,
T. M.
Chan
,
R. W. S.
Wong
,
K. W.
Lee
,
M. Y.
Mok
,
S. N.
Wong
, 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
.
22
Zhou
,
Y.
,
J.
Wu
,
D. F.
Kucik
,
N. B.
White
,
D. T.
Redden
,
A. J.
Szalai
,
D. C.
Bullard
,
J. C.
Edberg
.
2013
.
Multiple lupus-associated ITGAM variants alter Mac-1 functions on neutrophils.
Arthritis Rheum.
65
:
2907
2916
.
23
Faridi
,
M. H.
,
S. Q.
Khan
,
W.
Zhao
,
H. W.
Lee
,
M. M.
Altintas
,
K.
Zhang
,
V.
Kumar
,
A. R.
Armstrong
,
C.
Carmona-Rivera
,
J. M.
Dorschner
, et al
.
2017
.
CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus.
J. Clin. Invest.
127
:
1271
1283
.
24
Rosetti
,
F.
,
Y.
Chen
,
M.
Sen
,
E.
Thayer
,
V.
Azcutia
,
J. M.
Herter
,
F. W.
Luscinskas
,
X.
Cullere
,
C.
Zhu
,
T. N.
Mayadas
.
2015
.
A lupus-associated mac-1 variant has defects in integrin allostery and interaction with ligands under force.
Cell Rep.
10
:
1655
1664
.
25
Lee
,
Y. H.
,
S. C.
Bae
.
2015
.
Erratum to: Association between the functional ITGAM rs1143679 G/A polymorphism and systemic lupus erythematosus/lupus nephritis or rheumatoid arthritis: an update meta-analysis.
Rheumatol. Int.
35
:
825
827
.
26
Rosetti
,
F.
,
N.
Tsuboi
,
K.
Chen
,
H.
Nishi
,
T.
Ernandez
,
S.
Sethi
,
K.
Croce
,
G.
Stavrakis
,
J.
Alcocer-Varela
,
D.
Gómez-Martin
, et al
.
2012
.
Human lupus serum induces neutrophil-mediated organ damage in mice that is enabled by Mac-1 deficiency.
J. Immunol.
189
:
3714
3723
.
27
Chaves
,
L. D.
,
L.
Bao
,
Y.
Wang
,
A.
Chang
,
M.
Haas
,
R. J.
Quigg
.
2014
.
Loss of CD11b exacerbates murine complement-mediated tubulointerstitial nephritis.
PLoS One
9
: e92051.
28
Maiguel
,
D.
,
M. H.
Faridi
,
C.
Wei
,
Y.
Kuwano
,
K. M.
Balla
,
D.
Hernandez
,
C. J.
Barth
,
G.
Lugo
,
M.
Donnelly
,
A.
Nayer
, et al
.
2011
.
Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease.
Sci. Signal.
4
:
ra57
.
29
Dickinson
,
C. M.
,
B. W.
LeBlanc
,
M. M.
Edhi
,
D. S.
Heffernan
,
M. H.
Faridi
,
V.
Gupta
,
W. G.
Cioffi
,
X.
O’Brien
,
J. S.
Reichner
.
2018
.
Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients.
J. Intensive Care
6
:
19
.
30
Faridi
,
M. H.
,
M. M.
Altintas
,
C.
Gomez
,
J. C.
Duque
,
R. I.
Vazquez-Padron
,
V.
Gupta
.
2013
.
Small molecule agonists of integrin CD11b/CD18 do not induce global conformational changes and are significantly better than activating antibodies in reducing vascular injury.
Biochim. Biophys. Acta
1830
:
3696
3710
.
31
Khan
,
S. Q.
,
L.
Guo
,
D. J.
Cimbaluk
,
H.
Elshabrawy
,
M. H.
Faridi
,
M.
Jolly
,
J. F.
George
,
A.
Agarwal
,
V.
Gupta
.
2014
.
A small molecule β2 integrin agonist improves chronic kidney allograft survival by reducing leukocyte recruitment and accompanying vasculopathy.
Front. Med. (Lausanne)
1
:
45
.
32
Joshi
,
N.
,
A. K.
Kopec
,
J. L.
Ray
,
H.
Cline-Fedewa
,
A.
Nawabi
,
T.
Schmitt
,
R.
Nault
,
T. R.
Zacharewski
,
C. E.
Rockwell
,
M. J.
Flick
,
J. P.
Luyendyk
.
2016
.
Fibrin deposition following bile duct injury limits fibrosis through an αMβ2-dependent mechanism.
Blood
127
:
2751
2762
.
33
Jagarapu
,
J.
,
J.
Kelchtermans
,
M.
Rong
,
S.
Chen
,
D.
Hehre
,
S.
Hummler
,
M. H.
Faridi
,
V.
Gupta
,
S.
Wu
.
2015
.
Efficacy of leukadherin-1 in the prevention of hyperoxia-induced lung injury in neonatal rats.
Am. J. Respir. Cell Mol. Biol.
53
:
793
801
.
34
Panni
,
R. Z.
,
J. M.
Herndon
,
C.
Zuo
,
S.
Hegde
,
G. D.
Hogg
,
B. L.
Knolhoff
,
M. A.
Breden
,
X.
Li
,
V. E.
Krisnawan
,
S. Q.
Khan
, et al
.
2019
.
Agonism of CD11b reprograms innate immunity to sensitize pancreatic cancer to immunotherapies.
Sci. Transl. Med.
11
: eaau9240.
35
Means
,
T. K.
,
A. D.
Luster
.
2010
.
Integrins limit the toll.
Nat. Immunol.
11
:
691
693
.
36
Xiong
,
J. P.
,
R.
Li
,
M.
Essafi
,
T.
Stehle
,
M. A.
Arnaout
.
2000
.
An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain.
J. Biol. Chem.
275
:
38762
38767
.
37
Huth
,
J. R.
,
E. T.
Olejniczak
,
R.
Mendoza
,
H.
Liang
,
E. A.
Harris
,
M. L.
Lupher
Jr.
,
A. E.
Wilson
,
S. W.
Fesik
,
D. E.
Staunton
.
2000
.
NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding.
Proc. Natl. Acad. Sci. USA
97
:
5231
5236
.
38
Farley
,
F. W.
,
P.
Soriano
,
L. S.
Steffen
,
S. M.
Dymecki
.
2000
.
Widespread recombinase expression using FLPeR (flipper) mice.
Genesis
28
:
106
110
.
39
Bjørklund
,
M. M.
,
A. K.
Hollensen
,
M. K.
Hagensen
,
F.
Dagnaes-Hansen
,
C.
Christoffersen
,
J. G.
Mikkelsen
,
J. F.
Bentzon
.
2014
.
Induction of atherosclerosis in mice and hamsters without germline genetic engineering.
Circ. Res.
114
:
1684
1689
.
40
Celik
,
E.
,
M. H.
Faridi
,
V.
Kumar
,
S.
Deep
,
V. T.
Moy
,
V.
Gupta
.
2013
.
Agonist leukadherin-1 increases CD11b/CD18-dependent adhesion via membrane tethers.
Biophys. J.
105
:
2517
2527
.
41
Park
,
J. Y.
,
M. A.
Arnaout
,
V.
Gupta
.
2007
.
A simple, no-wash cell adhesion-based high-throughput assay for the discovery of small-molecule regulators of the integrin CD11b/CD18.
J. Biomol. Screen.
12
:
406
417
.
42
Zigmond
,
S. H.
1988
.
Orientation chamber in chemotaxis.
Methods Enzymol.
162
:
65
72
.
43
Altieri
,
D. C.
1991
.
Occupancy of CD11b/CD18 (Mac-1) divalent ion binding site(s) induces leukocyte adhesion.
J. Immunol.
147
:
1891
1898
.
44
Phillipson
,
M.
,
B.
Heit
,
P.
Colarusso
,
L.
Liu
,
C. M.
Ballantyne
,
P.
Kubes
.
2006
.
Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.
J. Exp. Med.
203
:
2569
2575
.
45
Sumagin
,
R.
,
H.
Prizant
,
E.
Lomakina
,
R. E.
Waugh
,
I. H.
Sarelius
.
2010
.
LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ.
J. Immunol.
185
:
7057
7066
.
46
Yao
,
X.
,
G.
Dong
,
Y.
Zhu
,
F.
Yan
,
H.
Zhang
,
Q.
Ma
,
X.
Fu
,
X.
Li
,
Q.
Zhang
,
J.
Zhang
, et al
.
2019
.
Leukadherin-1-mediated activation of CD11b inhibits LPS-induced pro-inflammatory response in macrophages and protects mice against endotoxic shock by blocking LPS-TLR4 interaction.
Front. Immunol.
10
:
215
.
47
Yang
,
M.
,
W.
Xu
,
Y.
Wang
,
X.
Jiang
,
Y.
Li
,
Y.
Yang
,
H.
Yuan
.
2018
.
CD11b-activated Src signal attenuates neuroinflammatory pain by orchestrating inflammatory and anti-inflammatory cytokines in microglia.
Mol. Pain
14
: 1744806918808150.
48
Sotiriou
,
S. N.
,
V. V.
Orlova
,
N.
Al-Fakhri
,
E.
Ihanus
,
M.
Economopoulou
,
B.
Isermann
,
K.
Bdeir
,
P. P.
Nawroth
,
K. T.
Preissner
,
C. G.
Gahmberg
, et al
.
2006
.
Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac-1 integrin.
FASEB J.
20
:
559
561
.
49
Yakubenko
,
V. P.
,
A.
Bhattacharjee
,
E.
Pluskota
,
M. K.
Cathcart
.
2011
.
αMβ2 integrin activation prevents alternative activation of human and murine macrophages and impedes foam cell formation.
Circ. Res.
108
:
544
554
.
50
Stöckl
,
J.
,
O.
Majdic
,
W. F.
Pickl
,
A.
Rosenkranz
,
E.
Prager
,
E.
Gschwantler
,
W.
Knapp
.
1995
.
Granulocyte activation via a binding site near the C-terminal region of complement receptor type 3 alpha-chain (CD11b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol-anchored Fc gamma RIIIB (CD16) molecules.
J. Immunol.
154
:
5452
5463
.
51
Lefort
,
C. T.
,
Y. M.
Hyun
,
J. B.
Schultz
,
F. Y.
Law
,
R. E.
Waugh
,
P. A.
Knauf
,
M.
Kim
.
2009
.
Outside-in signal transmission by conformational changes in integrin Mac-1.
J. Immunol.
183
:
6460
6468
.
52
Hind
,
L. E.
,
W. J.
Vincent
,
A.
Huttenlocher
.
2016
.
Leading from the back: the role of the uropod in neutrophil polarization and migration.
Dev. Cell
38
:
161
169
.
53
Smith
,
L. A.
,
H.
Aranda-Espinoza
,
J. B.
Haun
,
M.
Dembo
,
D. A.
Hammer
.
2007
.
Neutrophil traction stresses are concentrated in the uropod during migration.
Biophys. J.
92
:
L58
L60
.
54
Kumar
,
S.
,
J.
Xu
,
C.
Perkins
,
F.
Guo
,
S.
Snapper
,
F. D.
Finkelman
,
Y.
Zheng
,
M. D.
Filippi
.
2012
.
Cdc42 regulates neutrophil migration via crosstalk between WASp, CD11b, and microtubules.
Blood
120
:
3563
3574
.
55
Szczur
,
K.
,
Y.
Zheng
,
M. D.
Filippi
.
2009
.
The small Rho GTPase Cdc42 regulates neutrophil polarity via CD11b integrin signaling.
Blood
114
:
4527
4537
.
56
Hughes
,
B. J.
,
J. C.
Hollers
,
E.
Crockett-Torabi
,
C. W.
Smith
.
1992
.
Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion.
J. Clin. Invest.
90
:
1687
1696
.
57
Hyun
,
Y. M.
,
Y. H.
Choe
,
S. A.
Park
,
M.
Kim
.
2019
.
LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) distinctly regulate neutrophil extravasation through hotspots I and II.
Exp. Mol. Med.
51
:
1
13
.
58
Gray
,
J. L.
,
R.
Shankar
.
1995
.
Down regulation of CD11b and CD18 expression in atherosclerotic lesion-derived macrophages.
Am. Surg.
61
:
674
680
.
59
Yakubenko
,
V. P.
,
L. C.
Hsi
,
M. K.
Cathcart
,
A.
Bhattacharjee
.
2013
.
From macrophage interleukin-13 receptor to foam cell formation: mechanisms for αMβ2 integrin interference.
J. Biol. Chem.
288
:
2778
2788
.
60
Kubo
,
N.
,
W. A.
Boisvert
,
C. M.
Ballantyne
,
L. K.
Curtiss
.
2000
.
Leukocyte CD11b expression is not essential for the development of atherosclerosis in mice.
J. Lipid Res.
41
:
1060
1066
.
61
Szpak
,
D.
,
L.
Izem
,
D.
Verbovetskiy
,
D. A.
Soloviev
,
V. P.
Yakubenko
,
E.
Pluskota
.
2018
.
αMβ2 is antiatherogenic in female but not male mice.
J. Immunol.
200
:
2426
2438
.

V.G. is founder of Adhaere Pharmaceuticals (now part of Gossamer Bio), which is developing CD11b-targeted therapeutics, and has significant financial interest in it. The other authors have no financial conflicts of interest.