Leukocyte integrin Mac-1 (CD11b/CD18, αMβ2, CR3) acts as a functional receptor for platelet factor 4

Abstract

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from α-granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (α_Mβ₂, CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by ∼4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the α_MI-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a K_d of 1.3 ± 0.2 μm Using the PF4-derived peptide library, synthetic peptides duplicating the α_MI-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for α_MI-domain were identified in the PF4 segments Cys¹²-Ser²⁶ and Ala⁵⁷-Ser⁷⁰ These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1.

Keywords: Mac-1; PF4; alarmins; chemokine; integrin; macrophage; phagocytosis; platelet.

Figure 1.

Identification of the α_MI-domain recognition motifs in PF4. A, the amino acid sequence of human PF4. The underlined sequences Cys¹²–Ser²⁶ and Ala⁵⁷–Ser⁷⁰ denote the α_MI-domain-binding sites and are colored in green and orange, respectively. B, the peptide library derived from the PF4 sequence (left column). The peptide energies (right column) that serve as a measure of probability each peptide can interact with the α_MI-domain were calculated as described (12). C, autoradiography analysis of binding of ¹²⁵I-labeled α_MI-domain to the PF4-derived peptide library. The membrane was blocked with 1% BSA and then incubated with 10 μg/ml of ¹²⁵I-labeled α_MI-domain in Tris-buffered saline containing 1 mm MgCl₂. After washing, the membrane was dried and the α_MI-domain binding was visualized by autoradiography. The α_MI-domain binding observed as dark spots was analyzed by densitometry. The numbers shown in the middle column in B indicate the relative binding of the α_MI-domain expressed as a percentage of the intensity of spot 6. The ribbon model of the PF4 monomer (D) and space-filling models of PF4 tetramer (E) were based on PDB code 1F9Q (45) with putative α_MI-domain-binding sites identified by screening of the PF4-derived peptide library. The Cys¹²–Ser²⁶ and Ala⁵⁷–Ser⁷⁰ sequences are shown in green and orange, respectively.

Figure 2.

Biolayer interferometry analysis of the PF4–α_MI-domain interaction. A, representative sensograms of binding of active α_MI-domain (0; 0.2; 0.5; 1.0; 1.9; 3.8; 5.7 μm) to PF4 immobilized on the ForteBio sensor. The 2:1 heterogeneous ligand fitting model for 1.9 μm of the α_MI-domain is shown in gray. B, the binding isotherm of the α_MI-domain–PF4 interaction. Data shown are mean ± S.E. from 3 separate experiments. C, effect of anti-α_M mAb 44a on the PF4–α_MI domain interaction. Data shown are mean ± S.E. from 3 separate experiments. **, p ≤ 0.01. D, comparison of different I-domains for their ability to bind PF4. Representative sensograms were obtained with active α_MI-domain, inactive α_MI-domain, and active α_LI-domain tested at 3.8 μm.

Figure 3.

PF4 supports adhesion of the α_Mβ₂-expressing cells. A, aliquots (100 μl; 5 × 10⁵/ml) of Mac-1-expressing HEK293 (Mac-1–HEK293), WT HEK293 (HEK293), and HEK293 cells expressing the I-less Mac-1 labeled with calcein were added to microtiter wells coated with different concentrations of PF4 and post-coated with 1% PVP. After 30 min at 37 °C, nonadherent cells were removed by washing and fluorescence of adherent cells was measured in a fluorescence plate reader. The number of adherent cells was determined by using the fluorescence of 100-μl aliquots with a known number of labeled cells. Data are expressed as a percentage of added cells and are mean ± S.E. from 3 separate experiments with triplicate measurements. **, p ≤ 0.01. B, adhesion of human neutrophils, monocytes, and U937 monocytic cells to microtiter wells coated with different concentrations of PF4. Data are expressed as a percentage of added cells and are mean ± S.E. from 3 separate experiments with triplicate measurements. C, Mac-1–HEK293 cells were preincubated with anti-α_M mAb 44a (10 μg/ml), heparin (10 μg/ml; 2 units/ml), or their mixture (5 μg/ml of mAb44a + 5 μg/ml of heparin (1 units/ml)) and added to wells coated with 5 μg/ml of PF4. Adhesion in the absence of Mac-1 inhibitors and heparin was assigned a value of 100%. Data shown are mean ± S.E. from 3 separate experiments with triplicate measurements. **, p ≤ 0.01; ***, p ≤ 0.001 compared with control adhesion in the absence of inhibitors. D, calcein-labeled neutrophils were preincubated with 10 μg/ml of each anti-α_M mAb 44a, heparin, CsA, or CsB for 15 min. Cells were also preincubated with the mixtures of mAb 44a with each glycosaminoglycan or the mixture of all three reagents. In addition, neutrophils were preincubated with 1 μg/ml of NIF. Adhesion in the absence of Mac-1 inhibitors and glycosaminoglycans was assigned a value of 100%. Data shown are mean ± S.E. from 3 separate experiments with duplicate measurements. **, p ≤ 0.01; ***, p ≤ 0.001 compared with control adhesion in the absence of inhibitors. E, binding of PF4 to human neutrophils assessed by flow cytometry. Human neutrophils were incubated with PF4 (20 μg/ml) in the presence or absence of NIF (1 μg/ml). PF4 binding was detected using polyclonal anti-PF4 antibody and Alexa 488-conjugated goat anti-rabbit antibody.

Figure 4.

PF4-derived peptides Cys¹²–Ser²⁶ and Ala⁵⁷–Ser⁷⁰ support adhesion of the Mac-1-expressing cells. A, aliquots (100 μl; 5 × 10⁵/ml) of Mac-1–HEK293 cells were labeled with calcein and added to microtiter wells coated with different concentrations of the Cys¹²–Ser²⁶ and Ala⁵⁷–Ser⁷⁰ peptides (0–6.5 μm). After 30 min at 37 °C, nonadherent cells were removed and adhesion was measured. The data shown are mean ± S.E. from four experiments with triplicate determinations at each point. B, calcein-labeled Mac-1–HEK293 cells were incubated with soluble Cys¹²–Ser²⁶ and Ala⁵⁷–Ser⁷⁰ (30 μm) or their mixture for 15 min at 22 °C and added to wells coated with 5 μg/ml of PF4 and post-coated with 1% PVP. Adhesion in the absence of peptides was assigned a value of 100%. The data shown are the mean ± S.E. from three experiments each with triplicate determinations. **, p ≤ 0.01; ***, p ≤ 0.001. C, adhesion of Mac-1–HEK293 cells to WT and mutant PF4 immobilized at 10 μm. Cell adhesion to WT PF4 was assigned a value of 100%. The data shown are the mean ± S.E. from three experiments each with triplicate determinations. **, p ≤ 0.01; ***, p ≤ 0.001.

Figure 5.

Migration of Mac-1-expressing cells to PF4 in a Transwell system. A, Transwell inserts were coated with different concentrations of PF4 (1–5 μg/ml) for 60 min at 37 °C. Mac-1-expressing or WT HEK293 cells (100 μl at 3 × 10⁶/ml) were added to the upper wells of the Transwell chamber, and their ability to migrate was analyzed. After 16 h at 37 °C, the cells were labeled with calcein for 30 min at 37 °C. The cells from the upper chamber of the Transwells were removed by wiping with a cotton-tipped applicator, and images of the cells on the underside of the Transwell filter were taken. The figure is representative of 5 experiments. The scale bar is 100 μm. B, images from A were analyzed, and cells that migrated were counted. Data are presented as numbers of migrated cells per field ± S.E. for five random fields per well from five individual experiments. ***, p ≤ 0.001. C, calcein-labeled human neutrophils were placed in the upper chamber and allowed to migrate to PF4 (5 μg/ml) for 90 min. Data are expressed as the fluorescence of c-labeled cells migrated to the lower chamber. Results are the mean ± S.E. from three independent experiments with triplicate samples. ***, p ≤ 0.001. D, migration of macrophages isolated from WT and Mac-1-deficient mice. Macrophages (3 × 10⁵) were placed in the upper chamber and allowed to migrate to PF4 (5 μg/ml) for 90 min. The number of migrated cells was determined as in A. Data are presented as number of migrated cells per field ± S.E. for five random fields per well from 3 experiments. ***, p ≤ 0.001.

Figure 6.

Effect of PF4 on the redistribution of Mac-1 on the cell surface. A–C, upper panels: adherent Mac-1–HEK293 cells (A), neutrophils (B), and monocytes (C) were treated with soluble PF4 (100 μg/ml) for 30 min, fixed, and incubated with anti-α_M mAb M1/70 followed by Alexa 488-conjugated goat anti-rat secondary antibody. Bottom panels in A, B, and C, control cells were incubated with medium alone. In addition, cells were stained with Alexa Fluor 546-conjugated phalloidin and 4′,6-diamidino-2-phenylindole. The scale bars are 15 μm. D, analysis of Mac-1 expression on the surface of neutrophils and monocytes. Data are mean ± S.E. from the measurement of fluorescence of 30 cells using ImageJ software. ***, p ≤ 0.001.

Figure 7.

Effect of PF4 on phagocytosis of bacteria and latex beads by various macrophages. A, fluorescently labeled E. coli were incubated with PF4 (40 μg/ml) for 30 min at 37 °C. Soluble peptide was removed by centrifugation and PF4-coated bacteria were subsequently incubated with adherent IC-21 mouse macrophages for 60 min at 37 °C. Part of IC-21 macrophages were preincubated with anti-α_M mAb M1/70 (20 μg/ml) for 15 min before adhesion. Phagocytosis was determined as described under “Experimental Procedures.” Data are expressed as mean ratios of bacteria per macrophage ± S.E. **, p ≤ 0.01. B, fluorescent latex beads (2.5 × 10⁷/ml) were preincubated with PF4 (40 μg/ml) for 30 min at 37 °C. Soluble PF4 was removed from beads by high-speed centrifugation. PF4-coated latex beads were incubated with adherent IC-21 mouse macrophages, mouse peritoneal macrophages, or differentiated THP-1 human macrophages for 60 min at 37 °C. C, adherent IC-21 macrophages (10⁶/ml) were preincubated with anti-α_M mAb M1/70 (20 μg/ml), heparin (20 μg/ml), or NIF (2 μg/ml) for 20 min at 22 °C. PF4-coated latex beads were incubated with cells for 60 min at 37 °C, and nonphagocytosed beads were removed and phagocytosis was measured. Phagocytosis of PF4-coated latex beads in the absence of Mac-1 inhibitors and heparin was assigned a value of 100%. Data shown are mean ± S.E. of five random fields determined for each condition and are representative of 3 separate experiments. **, p ≤ 0.01; ***, p ≤ 0.001. D, a representative experiment showing fluorescence of IC-21 macrophages exposed to PF4-coated latex beads. Shown are bright field (a and d), fluorescence (b and e), and merge (c and f) images of IC-21 macrophages incubated with PF4-coated (a–c) or uncoated (d–f) control beads. The scale bar is 15 μm. E, PF4-coated beads were incubated with adherent mouse peritoneal macrophages isolated from WT and Mac-1^−/− mice for 30 min at 37 °C. Nonphagocytosed beads were removed, and the ratio of beads per macrophage was quantified from three fields of fluorescent images. Data shown are mean ± S.E. of triplicate measurements from three experiments. **, p ≤ 0.01 compared with untreated control beads. F, representative confocal image showing phagocytosed beads inside a macrophage. Horizontal (b) and vertical (c) cross-sections of the macrophage were taken at the positions shown by white lines. The scale bar is 5 μm.

Figure 8.

Basic residues in PF4 important for the α_MI-domain and heparin binding. A and B, electrostatic potential surface representation (positive, blue; negative, red) of the PF4 monomer (A) and tetramer (B) based on PDB code 1F9Q (45). Basic residues Arg²⁰, Arg²² in the ¹²Cys–Ser²⁶ segment and Lys⁶¹, Lys⁶², Lys⁶⁵, and Lys⁶⁶ in the ⁵⁷Ala–Ser⁷⁰ segment in the PF4 monomer (A) and PF4 tetramer (B) that have been identified as critical for α_MI-domain binding are shown. The same residues have been demonstrated to be important for heparin binding (45).