Key role of proline-rich tyrosine kinase 2 in interleukin-8 (CXCL8/IL-8)-mediated human neutrophil chemotaxis

Abstract

The signalling pathways leading to CXCL8/IL-8-induced human neutrophil migration have not been fully characterized. The present study demonstrates that CXCL8 induces tyrosine phosphorylation as well as enzymatic activity of proline-rich tyrosine kinase 2 (Pyk2), a non-receptor protein tyrosine kinase (PTK), in human neutrophils. Induction of Pyk2 tyrosine phosphorylation by CXCL8 is regulated by Src PTK activation, whereas it is unaffected by phosphatidylinositol 3-kinase activation. Inhibition of Pyk2 activation by PP1, a Src PTK inhibitor, is paralleled by the inhibition of CXCL8-mediated neutrophil chemotaxis. Among CXCL8 receptors, Src protein tyrosine kinase activation selectively regulates CXCR1-mediated polymorphonuclear neutrophil (PMN) chemotaxis. Overexpression of PykM, the kinase-dead mutant of Pyk2, blocks CXCL8-induced chemotaxis of HL-60-derived PMN-like cells, thus pinpointing the key role of Pyk2 in CXCL8-induced chemotaxis.

Figure 1

Interleukin-8 (CXCL8) induces tyrosine phosphorylation of an intracellular protein that corresponds to proline-rich tyrosine kinase 2 (Pyk2) in human polymorphonuclear neutrophils (PMNs). (a) PMNs were stimulated with vehicle or increasing concentrations of CXCL8 for 15 seconds at 37°. (b) PMNs were stimulated for different periods of time with vehicle or CXCL8 (400 ng/ml). The lysates were analysed by immunoblotting with antibody to phosphotyrosine (anti-pTyr) (upper panels) immunoglobulin. The same filters were probed with anti-Pyk2 (N19) immunoglobulin (lower panels). Data shown in the figure represent one of five independent experiments, with similar results obtained in each. MW, molecular weight; WB, Western blot.

Figure 2

Interleukin-8 (CXCL8) induces tyrosine phosphorylation and tyrosine kinase activity of proline-rich tyrosine kinase 2 (Pyk2) in human polymorphonuclear neutrophils (PMNs). PMNs were stimulated with vehicle, CXCL8 (400 ng/ml; 15 seconds at 37°) or saturating concentrations of anti-β₂ (30 min at 4°). The lysate was immunoprecipitated using antibody to proline-rich tyrosine kinase 2 (anti-Pyk2) (600). (a) The immunoprecipitate was immunoblotted by antibody to phosphotyrosine (anti-pTyr) (upper panel). The same filter was probed with anti-Pyk2 (N19; lower panel). (b) Tyrosine kinase activity was evaluated in the immunoprecipitate, as described in the Materials and methods. Data shown in the figure represent one of three independent experiments, with similar results obtained in each. IP, immunoprecipitate; MW, molecular weight.

Figure 3

Src protein tyrosine kinases (PTKs), but not phosphatidylinositol 3-kinase (PI3K), is involved in interleukin-8 (CXCL8)-induced proline-rich tyrosine kinase 2 (Pyk2) phosphorylation. (a) Human polymorphonuclear neutrophils (PMNs) were pretreated with vehicle or PP1 (10 µm) for 15 min at 37°. PMNs were then stimulated with vehicle or CXCL8 (400 ng/ml) for 15 seconds at 37° in serum-free RPMI-1640. (b) PMNs were pretreated with vehicle, PP1 (10 µm) or LY294002 (25 µm or 50 µm) for 15 min at 37°. PMNs were then stimulated with vehicle, CXCL8 (400 ng/ml; 15 seconds at 37°) or endotoxin [lipopolysaccharide (LPS)] (100 ng/ml, 30 min at 37°) in RPMI supplemented with 1% fetal bovine serum (FBS). Cell lysates were immunoprecipitated using anti-Pyk2 (600) and analysed by Western blotting using anti-pTyr (upper panels). The same filter was probed with anti-Pyk2 (N19) (lower panels). Data shown in the figure represent one of three independent experiments, with similar results obtained in each. IP, immunoprecipitate; WB, Western blot.

Figure 4

Src protein tyrosine kinase (PTK) activation is involved in interleukin-8 (CXCL8)-mediated CXCL8 chemotaxis. (a) Human polymorphonuclear neutrophils (PMNs) were preincubated with vehicle or PP1 (3 µm, 10 µm or 30 µm) for 15 min at 37°. (b) PMNs were preincubated with neutralizing anti-CXCR2 immunoglobulin (3 µg/ml), in the presence or absence of PP1 (3 µm or 10 µm), for 15 min at 37°. (c) PMNs were preincubated with neutralizing anti-CXCR1 (3 µg/ml) immunoglobulin, in the presence or absence of PP1 (3 µm or 10 µm), for 15 min at 37°. Cells were then tested for their ability to migrate in response to CXCL8 (10 ng/ml). Data are expressed as mean values ± standard deviation (SD) of three independent experiments. Spontaneous PMN migration was 13 ± 4. *P < 0·05 versus CXCL8 alone (Mann–Whitney U-test); **P < 0·01 versus CXCL8 alone (Mann–Whitney U-test); ‡P < 0·05 versus anti-CXCR2 alone pretreated group (Mann–Whitney U-test).

Figure 5

Phosphatidylinositol 3-kinase (PI3K) activation is involved in interleukin-8 (CXCL8)-induced human polymorphonuclear neutrophil (PMN) chemotaxis. PMNs were preincubated with vehicle or LY294002 (25 µm or 50 µm) for 15 min at 37°. Cells were then tested for their ability to migrate in response to CXCL8 (10 ng/ml). Data are expressed as mean values ± standard deviation (SD) of three independent experiments. Spontaneous PMN migration: 23 ± 5 **P < 0·01 versus CXCL8 alone (Mann–Whitney U-test).

Figure 6

Overexpression of PykM (the kinase-dead mutant of proline-rich tyrosine kinase 2)-blocked interleukin-8 (CXCL8) chemotaxis in HL-60-derived human polymorphonuclear neutrophil (PMN)-like cells. (a) Membrane expression of CXCR1 and CXCR2 receptors in differentiated HL-60 cells, or differentiated HL-60 cells transiently transfected with pcDNA3-derived plasmid expressing PykM or empty pcDNA3, was determined by flow cytometry analysis. Cells were labelled with anti-CXCR1, anti-CXCR2 or isotype-matched control monoclonal antibodies (mAbs) followed by incubation with fluorescein isothiocyanate (FITC) goat anti-mouse Ab. Dotted, solid and heavy type lines represent isotype-control Ab, anti-CXCR1 Ab and anti-CXCR2 Ab, respectively. Mean channel fluorescence (MCF) is reported. (b) Differentiated HL-60 (5 × 10⁷/ml) were stimulated with CXCL8 (400 ng/ml) for 15 seconds at 37°. The lysate was analysed by immunoblotting with anti-active proline-rich tyrosine kinase 2 (Pyk2) (upper panel). The same filter was probed with anti-total-Pyk2 (N19; lower panel). (c) Differentiated HL-60 cells were transfected with pcDNA-derived plasmid expressing PykM or with the empty pcDNA3. Overexpression of PykM was demonstrated by Western blotting with anti-Pyk2 (N19), 40 hr after electroporation (low). Transfected cells were then tested for their ability to migrate in response to increasing concentrations of CXCL8. Data are expressed as mean values ± standard deviation (SD) of three independent experiments. **P < 0·01 versus empty pcDNA3-transfected cells [analysis of variance (anova)]. MW, molecular weight.

Figure 7

Src protein tyrosine kinase (PTK) activation is involved in interleukin-8 (CXCL8)-induced HL-60-derived PMN-like cell migration. HL-60-derived human polymorphonuclear neutrophil (PMN)-like cells were transiently transfected with pcDNA-derived plasmid expressing PykM, or with empty pcDNA3. Cells were preincubated with vehicle or PP1 (10 µm) for 15 min at 37° and then tested for their ability to migrate in response to CXCL8 (100 ng/ml). Data are expressed as mean values ± standard deviation (SD) of three independent experiments. **P < 0·01 versus the corresponding vehicle-treated group (Mann–Whitney U-test).