Tyrosine kinase Btk regulates E-selectin-mediated integrin activation and neutrophil recruitment by controlling phospholipase C (PLC) gamma2 and PI3Kgamma pathways

Abstract

Selectins mediate leukocyte rolling, trigger beta(2)-integrin activation, and promote leukocyte recruitment into inflamed tissue. E-selectin binding to P-selectin glycoprotein ligand 1 (PSGL-1) leads to activation of an immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway, which in turn activates the spleen tyrosine kinase (Syk). However, the signaling pathway linking Syk to integrin activation after E-selectin engagement is unknown. To identify the pathway, we used different gene-deficient mice in autoperfused flow chamber, intravital microscopy, peritonitis, and biochemical studies. We report here that the signaling pathway downstream of Syk divides into a phospholipase C (PLC) gamma2- and phosphoinositide 3-kinase (PI3K) gamma-dependent pathway. The Tec family kinase Bruton tyrosine kinase (Btk) is required for activating both pathways, generating inositol-3,4,5-trisphosphate (IP(3)), and inducing E-selectin-mediated slow rolling. Inhibition of this signal-transduction pathway diminished Galpha(i)-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster. Galpha(i)-independent neutrophil recruitment into the inflamed peritoneal cavity was reduced in Btk(-/-) and Plcg2(-/-) mice. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by E-selectin engagement.

Figure 1

The Tec family kinase Btk is required for E-selectin–mediated slow rolling and Gα_i-independent adhesion, but not for chemokine-induced arrest in vivo. (A) The carotid artery of chimeric mice reconstituted with bone marrow from WT mice (n = 3) or Btk^−/− mice (n = 3) was cannulated with a catheter, which was connected to autoperfused flow chambers. Average rolling velocity of neutrophils on E-selectin (left) and E-selectin and ICAM-1 (right) is presented as means ± SEM. The wall shear stress in all flow chamber experiments was 5 to 6 dyn/cm². (B) Isolated bone marrow neutrophils were resuspended in plasma, and the rolling velocity on either E-selectin alone or E-selectin plus ICAM-1 was measured. In these experiments, a shear stress of 3 dyn/cm² was used (n = 3). (C) Mixed chimeric mice were generated by injecting bone marrow cells from LysM-GFP⁺ WT mice and Btk^−/− mice into lethally irradiated WT mice. Cumulative histogram of rolling velocities of 100 GFP⁺ (WT; ●) and 100 GFP⁻ (Btk^−/−; ○) leukocytes in inflamed cremaster muscle venules of mixed chimeric mice (n = 4) treated with PTx and a monoclonal blocking P-selectin antibody (RB40.34). Inset data are means ± SEM. (D) Numbers of adherent cells per square millimeter in murine cremaster muscle venules. The cremaster muscle was exteriorized 2 hours after intrascrotal injection of 500 ng TNF-α in chimeric mice reconstituted with bone marrow from WT mice or Btk^−/− mice. The dotted line indicates the number of adherent cells in WT mice treated with PTx and monoclonal blocking E-selectin antibody (9A9). #P < .05; *P < .05 vs other groups.

Figure 2

Elimination of PLCγ2 partially abrogates E-selectin–mediated slow rolling and consequently reduces leukocyte adhesion in vivo. (A) Carotid cannulas were placed in chimeric mice reconstituted with bone marrow from Plcg2^−/− mice (n = 3) or WT mice (n = 3) and connected to autoperfused flow chambers. Average rolling velocity of neutrophils on E-selectin (left) and E-selectin and ICAM-1 (right) is presented as means ± SEM. The wall shear stress in all flow chamber experiments was 5 to 6 dyn/cm². (B) Mixed chimeric mice were generated by injecting bone marrow cells from LysM-GFP⁺ WT mice and Plcg2^−/− mice or Syk^−/− mice into lethally irradiated WT mice. Cumulative histogram of rolling velocities of 150 WT leukocytes (●), 150 Plcg2^−/− leukocytes (▾), and 150 Syk^−/− leukocytes (○) in inflamed cremaster muscle venules of mixed chimeric mice (n = 4) treated with PTx and a monoclonal blocking P-selectin antibody (RB40.34). Inset data are means ± SEM. (C) Numbers of adherent cells per square millimeter in murine cremaster muscle venules. Cremaster muscle exteriorized 2 hours after intrascrotal injection of 500 ng TNF-α in chimeric mice reconstituted with bone marrow from WT mice or Btk^−/− mice. Dotted line indicates the number of adherent cells in WT mice treated with PTx and monoclonal blocking E-selectin antibody (9A9). #P < .05.

Figure 3

Blocking PI3Kγ in Plcg2^−/− neutrophils completely abolishes E-selectin–mediated slow rolling. (A) Rolling velocity of WT neutrophils on E-selectin alone or E-selectin/ICAM-1 of WT mice pretreated with either a PI3Kδ-inhibitor (PI3Kδ-inh.) or DMSO. (B) Rolling velocity of WT and Plcg2^−/− neutrophils on E-selectin alone or E-selectin/ICAM-1 of either PI3Kγ-inhibitor (PI3Kγ inh.)– or DMSO-pretreated mice. (C) Rolling velocity of WT and Pik3cg^−/− neutrophils on E-selectin or E-selectin/ICAM-1 of either untreated or PLC-inhibitor–pretreated mice. Data are presented as means ± SEM from 3 mice. (D) Mixed chimeric mice were generated by injecting bone marrow cells from Pik3cg^−/− mice and LysM-GFP⁺ WT mice into lethally irradiated WT mice. Cumulative histogram of rolling velocities of 100 GFP⁺ (WT; ●) and 100 GFP⁻ (Pik3cg^−/−; ○) leukocytes in inflamed cremaster muscle venules of mixed chimeric mice (n = 3) treated with PTx and a monoclonal blocking P-selectin antibody (RB40.34). Inset data are means ± SEM. (E) Numbers of adherent cells per square millimeter in murine cremaster muscle venules. The cremaster muscle was exteriorized 2 hours after intrascrotal injection of 500 ng TNF-α in chimeric mice reconstituted with bone marrow from Pik3cg^−/− mice or WT mice. The dotted line indicates the number of adherent cells in WT mice treated with PTx and monoclonal blocking E-selectin antibody (9A9). #P < .05.

Figure 4

Gα_i-independent neutrophil recruitment is defective in Btk^−/−and Plcg2^−/− mice. (A) Number of extravasated leukocytes in cremasteric venules of TNF-α–treated chimeric mice reconstituted with bone marrow from WT mice (n = 4), Btk^−/− mice (n = 4), Pik3cg^−/− mice (n = 3), or Plcg2^−/− mice (n = 4) per 1.5 × 10⁴ μm² tissue area. The measurements were performed 2 hours after intrascrotal TNF-α injection. The same groups were also analyzed after pretreatment with 4 μg PTx intravenously (+PTx; WT mice + PTx [n = 4], Btk^−/− mice + PTx [n = 4], Pik3cg^−/− mice + PTx [n = 3], Plcg2^−/− mice + PTx [n = 4]). In addition to this, we analyzed WT mice after pretreatment with a blocking E-selectin antibody alone (+ anti–E-sel. ab [n = 3]) and in combination with PTx (WT mice + anti–E-sel. ab + PTx [n = 4]). (B) Neutrophil influx into the peritoneal cavity 8 hours after 1 mL injection of 3% thioglycollate into chimeric mice reconstituted with bone marrow from WT mice (n = 5), Btk^−/− mice (n = 5), or Plcg2^−/− mice (n = 5). The same groups were also analyzed after pretreatment with 4 μg PTx intravenously (+PTx; WT mice + PTx [n = 5], Btk^−/− mice + PTx [n = 4], and Plcg2^−/− mice + PTx [n = 5]). Total numbers of neutrophils in the peritoneal lavage fluid were determined by flow cytometry and hemocytometer count. #P < .05.

Figure 5

E-selectin engagement induces phosphorylation of Btk, PLCγ2, and PI3K. Bone marrow–derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 minutes, and then lysates were prepared. (A) Lysates were immunoprecipitated with anti-Btk, followed by immunoblotting (IB) with a general phosphotyrosine (PY; 4G10) antibody. (B) Lysates were immunoblotted with antibody to phosphorylated PLCγ2 (phospho-PLCγ2 [Tyr1217]), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated p38 MAPK (phospho-p38), or total p38 (n = 3).

Figure 6

Btk is required for the activation of the PLCγ2- and PI3Kγ-dependent pathways. Bone marrow–derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 minutes, and then lysates were prepared. (A) Lysates were immunoblotted with antibodies to phosphorylated PLCγ2 (phospho-PLCγ2 [Tyr1217]), total PLCγ2 (n = 3), phosphorylated Akt (n = 3), total Akt (n = 3), phosphorylated MAPK (phospho-p38), or total p38 (n = 3). (B) Lysates were immunoprecipitated with anti-Syk, followed by immunoblotting (IB) with a general phosphotyrosine (PY; 4G10) antibody. (C) Bone marrow–derived neutrophils were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 minutes, and then intracellular IP₃ levels were determined using a competitive binding assay. *P < .05 vs other groups.

Figure 7

PLCγ2, but not PI3Kγ, is required for p38 MAPK phosphorylation. Bone marrow–derived neutrophils from Plcg2^−/− mice or Pik3cg^−/− mice were plated on uncoated (unstimulated) or E-selectin–coated wells for 10 minutes, and then lysates were prepared (A-B). Lysates were immunoblotted with antibody to phosphorylated p38 MAPK (phospho-p38) or total p38 (n = 3).