The junctional adhesion molecule 3 (JAM-3) on human platelets is a counterreceptor for the leukocyte integrin Mac-1

Abstract

The recently described junctional adhesion molecules (JAMs) in man and mice are involved in homotypic and heterotypic intercellular interactions. Here, a third member of this family, human JAM-3, was identified and described as a novel counterreceptor on platelets for the leukocyte beta2-integrin Mac-1 (alphaMbeta2, CD11b/CD18). With the help of two monoclonal antibodies, Gi11 and Gi13, against a 43-kD surface glycoprotein on human platelets, a full-length cDNA encoding JAM-3 was identified. JAM-3 is a type I transmembrane glycoprotein containing two Ig-like domains. Although JAM-3 did not undergo homophilic interactions, myelo-monocytic cells adhered to immobilized JAM-3 or to JAM-3-transfected cells. This heterophilic interaction was specifically attributed to a direct interaction of JAM-3 with the beta2-integrin Mac-1 and to a lower extent with p150.95 (alphaXbeta2, CD11c/CD18) but not with LFA-1 (alphaLbeta2, CD11a/CD18) or with beta1-integrins. These results were corroborated by analysis of K562 erythroleukemic cells transfected with different heterodimeric beta2-integrins and by using purified proteins. Moreover, purified JAM-3 or antibodies against JAM-3 blocked the platelet-neutrophil interaction, indicating that platelet JAM-3 serves as a counterreceptor for Mac-1 mediating leukocyte-platelet interactions. JAM-3 thereby provides a novel molecular target for antagonizing interactions between vascular cells that promote inflammatory vascular pathologies such as in atherothrombosis.

Figure 2.

JAM-3 cDNA sequence and deduced amino acid sequence. (A) The NH₂-terminal amino acid sequence and internal sequences derived from amino acid sequence analysis are underlined. The transmembrane region is indicated by a double line. Putative N- (Asn₁₀₄, Asn₁₉₂) and O-glycosylation sites (Thr₆₀) are shown in bold. The protein kinase C phosphorylation site at Ser₂₈₁ is circled and PDZ binding motif is boxed. The GenBank/EMBL/DDBJ accession no. for the nucleotide sequence of the human platelet JAM-3 is AF448478. (B) Phylogenetic tree of homologous human (h) JAM-3 and murine (m) JAM-2. Phylogenetic distances obtained with the Clustal W program are indicated as values above the branches. (C) Alignment of hJAM-3 and mJAM-2 was done using the SMART program. Immunoglobulin C-2 domains and the transmembrane domain are indicated by single and double line, respectively. Cysteine residues are shown in bold and nonidentical amino acids are shaded in dark. The mJAM-2 sequence is accessible from the database (AJ300304).

Figure 2.

JAM-3 cDNA sequence and deduced amino acid sequence. (A) The NH₂-terminal amino acid sequence and internal sequences derived from amino acid sequence analysis are underlined. The transmembrane region is indicated by a double line. Putative N- (Asn₁₀₄, Asn₁₉₂) and O-glycosylation sites (Thr₆₀) are shown in bold. The protein kinase C phosphorylation site at Ser₂₈₁ is circled and PDZ binding motif is boxed. The GenBank/EMBL/DDBJ accession no. for the nucleotide sequence of the human platelet JAM-3 is AF448478. (B) Phylogenetic tree of homologous human (h) JAM-3 and murine (m) JAM-2. Phylogenetic distances obtained with the Clustal W program are indicated as values above the branches. (C) Alignment of hJAM-3 and mJAM-2 was done using the SMART program. Immunoglobulin C-2 domains and the transmembrane domain are indicated by single and double line, respectively. Cysteine residues are shown in bold and nonidentical amino acids are shaded in dark. The mJAM-2 sequence is accessible from the database (AJ300304).

Figure 1.

Purification and identification of Gi11 antigen from platelets. Purified Gi11 antigen was subjected to 10% SDS-PAGE under reducing conditions and was visualized by Coomasie blue staining (lane 2) in comparison to indicated molecular weight standards (lane 1). Gi11-antigen was then transferred onto nitrocellulose membrane, stained with mAb Gi11 or Gi13 (lanes 3 and 4) and visualized using streptavidin–chemiluminescence system.

Figure 3.

Flow cytometry analysis of JAM-3 expression on blood cells, U937 and K562 cell lines with mAb Gi11. The closed curves represent the reaction with mAb Gi11, the thin lines with normal mouse IgG (negative control) and the bold lines with different mAb specific for the indicated cell types: Gi9 (α2β1 integrin; platelets), 3G8 (CD16; granulocytes), MY4 (CD14; monocytes), w6/32 (HLA class I; lymphocytes), and Gi21 (CD55; erythrocytes, K562 cell line) and K20 (β1 integrin; U937 cell line).

Figure 4.

Characterization of CHO cells expressing stable JAM-3. (A) Flow cytometry analysis of JAM-3 transfectants using mAb Gi11 (anti-JAM3) and mAb Gi5 (anti-GPIIb/IIIa) is shown. The closed curves represent the reaction of antibodies with untransfected CHO cells. (B) Immunoblotting analysis using mAb Gi11 of lysates from untransfected cells (lane 1) or JAM-3 transfectants (lane 2) is presented.

Figure 5.

Leukocyte adhesion to JAM-3 is Mac-1 dependent. (A) The adhesion of PMA-stimulated (50 ng/ml) U937 cells to immobilized JAM-3 is shown in the absence (−) or presence of blocking mAb against the integrin β1-chain, β2-chain, LFA-1, Mac-1, p150.95, JAM-3 (each 10 μg/ml), or soluble fibrinogen (FBG, 20 μg/ml). (B) The adhesion of PMA-stimulated (50 ng/ml) U937 cells to immobilized fibrinogen is shown in the absence (−) or presence of blocking mAbs against integrins (each 10 μg/ml) or purified JAM-3 (20 μg/ml). Cell adhesion is expressed as absorbance at 590 nm. All data are mean ± SD (n = 3) of a typical experiment; similar results were obtained in at least three separate experiments. (C) U937 cell adhesion to nontransfected CHO cells (white bars) or JAM-3–transfected CHO cells (black bars) was studied without or with PMA (50 ng/ml) pretreatment as indicated, and in the absence (−) or presence of blocking mAb against integrins or against JAM-3 (each 10 μg/ml). The number of adherent cells is expressed as percentage of total added cells. All data are mean ± SD (n = 3) of a typical experiment; similar results were obtained in at least three separate experiments; *: P < 0.01 compared with respective control; ns, not significant.

Figure 6.

Characterization of the adhesion of K562 cells to JAM-3. (A) Adhesion of untransfected K562 cells (nt), or K562 cells transfected with LFA-1-, Mac-1-, or p150.95 to immobilized JAM-3 was studied in the absence (white bars) or presence of PMA (50 ng/ml; gray bars) or of the β2-integrin stimulating mAb Kim127 (10 μg/ml; black bars). (B) Adhesion of Mac-1- or p150.95-transfectant to immobilized JAM-3 after stimulation with mAb Kim127 in the absence (white bars) or presence of mAb against Mac-1 (dark gray bar), mAb against p150.95 (light gray bar), or mAb against JAM-3 (black bars; each 10 μg/ml). (C) Adhesion of K562 cells (nt), of LFA-1-, Mac-1-, or p150.95-transfectant to CHO cells or JAM-3–transfected CHO cells was studied without stimulation (open bars) or after stimulation with PMA (gray bars) or with mAb Kim127 (black bars). (D) Adhesion of Mac-1- or p150.95-transfectant to JAM-3–transfected CHO cells was studied in the absence (−) or presence of PMA or mAb Kim127 without (white bars) or with mAb against Mac-1 (dark gray bars), mAb against p150.95 (light gray bars) or mAb against JAM-3 (black bars; each 10 μg/ml). The number of adherent cells is expressed as percent of total added cells. All data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments.

Figure 6.

Characterization of the adhesion of K562 cells to JAM-3. (A) Adhesion of untransfected K562 cells (nt), or K562 cells transfected with LFA-1-, Mac-1-, or p150.95 to immobilized JAM-3 was studied in the absence (white bars) or presence of PMA (50 ng/ml; gray bars) or of the β2-integrin stimulating mAb Kim127 (10 μg/ml; black bars). (B) Adhesion of Mac-1- or p150.95-transfectant to immobilized JAM-3 after stimulation with mAb Kim127 in the absence (white bars) or presence of mAb against Mac-1 (dark gray bar), mAb against p150.95 (light gray bar), or mAb against JAM-3 (black bars; each 10 μg/ml). (C) Adhesion of K562 cells (nt), of LFA-1-, Mac-1-, or p150.95-transfectant to CHO cells or JAM-3–transfected CHO cells was studied without stimulation (open bars) or after stimulation with PMA (gray bars) or with mAb Kim127 (black bars). (D) Adhesion of Mac-1- or p150.95-transfectant to JAM-3–transfected CHO cells was studied in the absence (−) or presence of PMA or mAb Kim127 without (white bars) or with mAb against Mac-1 (dark gray bars), mAb against p150.95 (light gray bars) or mAb against JAM-3 (black bars; each 10 μg/ml). The number of adherent cells is expressed as percent of total added cells. All data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments.

Figure 7.

Interaction between purified JAM-3 and Mac-1 proteins. (A) The binding of ICAM-1, fibrinogen (FBG), or JAM-3 to immobilized Mac-1 (black bars) and the binding of JAM-3 or ICAM-1 to immobilized LFA-1 (white bars) was studied. (B) Dose-dependent specific binding of JAM-3 to immobilized Mac-1 is shown. (C) The binding of JAM-3 (white bars) to immobilized Mac-1 was analyzed in the absence (−) or presence of fibrinogen (FBG, 20 μg/ml), mAb against Mac-1 or mAb against JAM-3 (each 10 μg/ml). The binding of fibrinogen (black bars) to immobilized Mac-1 was studied in the absence (−) or presence of purified JAM-3 (20 μg/ml) or of mAb against Mac-1 (10 μg/ml). Specific binding is expressed as absorbance at 405 nm. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments.

Figure 8.

JAM-3 mediates platelet–leukocyte adhesive interactions. (A) PMA-stimulated adhesion of human neutrophils to surface-adherent platelets was studied in the absence (−) or presence of mAb against Mac-1, LFA-1, JAM-3, or GPIbα as well as a mixture of mAbs against JAM-3 and GPIbα (each 10 μg/ml) or in the presence of purified JAM-3 or soluble glycocalicin (GPIbα; each 10 μg/ml), respectively. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in at least three separate experiments. (B) Adhesion of human neutrophils to surface-adherent platelets from a patient with Bernard-Soulier-Syndrome was studied in the absence (−) or presence of mAb against Mac-1, GPIbα, or JAM-3 (10 μg/ml), or of purified JAM-3 (10 μg/ml). The number of adherent cells is expressed as percent of total added cells. Data are mean ± SD (n = 3) of a typical experiment; similar results were observed in 2 separate experiments; *: P < 0.01 compared with control; ns, not significant.

Figure 9.

Role of JAM-3 in platelet–neutrophil aggregate formation in whole blood. The platelet–neutrophil adhesion in whole blood stimulated with the stable thromboxane A₂ mimetic U46619 (5 μM) is shown in the absence or presence of purified JAM-3 (20 μg/ml), soluble glycocalicin (20 μg/ml), or Fab-fragments of blocking mAb GA6* against P-selectin (PS; 10 μg/ml). Adhesion experiments were performed in a micro-couette at low (20 s⁻¹) and high (2,000 s⁻¹) shear rates. The data represent mean ± SD of four independent experiments.