Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection

Abstract

The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.

FIGURE 1

Pyk2 expression is lost in PMNs and macrophages from pyk2^−/− mice. WT or pyk2^−/−PMNs and macrophages were lysed and probed with antibodies to Pyk2. 293T cells transfected with Pyk2 (indicated as 293T/P) served as a positive control. Data shown are representative of three independent experiments.

FIGURE 2

Analysis of integrin-mediated adhesion in pyk2^−/− PMNs. A, PMNs from WT or pyk2^−/− mice were allowed to adhere to pRGD-coated coverslips and fixed at the indicated time points. The fixed cells were stained for actin (red). Images shown are representative of three independent experiments. B, Fluorescently-labelled bone marrow PMNs from WT or pyk2^−/−mice were plated on pRGD coated wells and then exposed to a series of washes in a static adhesion assay. The decrease in fluorescence corresponding to the decrease in cell number was measured by fold-change from the baseline value was plotted over the series of washes. C, Following the assay design in B, PMNs from WT or pyk2^−/− mice were allowed to adhere to fibrinogen coated wells for 15 minutes. Error bars represent ± SEM of triplicate samples. Data shown are pooled from 3 independent experiments.

FIGURE 3

In vitro migration in pyk2^−/− PMNs. A, WT or pyk2^−/− PMNs were plated upon transwells and allowed to migrate toward 0.1 μM fMLF or 10 ng/mL MIP2, with RPMI used as a control. After 30 minutes, the cells that had migrated through the pores were lysed and stained for phosphatase. The amount of phosphatase remaining on the wells was quantitated through absorbance and plotted as a percentage of the total number of cells seeded originally on the transwell. B, Following the methodology described in A, PMNs from WT or pyk2^−/− mice were plated onto transwells that had been previously coated with fibrinogen. * = p< 0.05. Error bars represent ± SEM of duplicate samples from data averaged between three independent experiments.

FIGURE 4

Superoxide production in pyk2^−/− PMNs. A, PMNs from WT or pyk2^−/− mice were plated on pRGD coated surfaces for 2 hours in the presence of 100 ng/mL of TNF-α. The amount of superoxide released by the cells was measured via luminol reduction. B, PMNs from the indicated mice were plated on fibrinogen-coated surfaces for 2 hours in the presence of 100 ng/mL of TNF. C, PMNs from WT or pyk2^−/− mice were plated on fibronectin-coated surfaces for 2 hours in the presence of 100 ng/mL of TNF-α. D, PMNs from the indicated mice were exposed to complement-coated beads for 2 hours in the presence of 50 ng/mL of TNF-α. Data are the mean of at least 3 independent experiments each with n = 3. Error bars represent ± SEM.

FIGURE 5

Degranulation response in pyk2^−/− PMNs. To measure integrin-mediated degranulation responses, PMNs from WT or pyk2^−/− mice were isolated and plated onto pRGD-coated surfaces in the presence or absence of 100 ng/mL TNF-α for 1 hour. Supernatants from the stimulated PMNS were isolated for analysis. A, The ability of the PMNs to release MMP-9 upon integrin ligation was measured on a zymogram gel. B, Levels of MMP-9 were normalized to the level released by PMNs plated on milk-blocked wells. C, The amount of lactoferrin released into the supernatant by PMNs from WT or pyk2^−/− mice was measured via Ab plate-bound ELISA (n=18). D, Control immunoblot probing for levels of lactoferrin in WT or pyk2^−/− PMNs. Data are the mean of at least 3 independent experiments. Error bars represent ± SEM. * = p<0.05.

FIGURE 6

Antibacterial responses in pyk2^−/− PMNs. A, B, Rates of complement-mediated phagocytosis were compared in WT and pyk2^−/− PMNs using FITC-labelled complement-opsonized polystyrene beads, A, or complement-opsonized S. aureus, B. The rate of phagocytosis in PMNs was measured via FACs analysis using median FITC fluorescence as the degree of uptake over time. Data are representative of 3 independent experiments. C, The in vitro bactericidal activity of WT and pyk2^−/− PMNs was measured over time. Complement-opsonized S. aureus was fed to PMNs and then the amount of live bacteria over time was measured as fold-change from CFUs at timepoint zero. Data averaged from 3 independent experiments. Error bars represent ± SEM.

FIGURE 7

Signal activation following integrin ligation in pyk2^−/− PMNs. A, WT or pyk2^−/−bone marrow-derived PMNs were plated on pRGD in the presence or absence of 100 ng/mL TNF for 15 minutes. PMNs in suspension were used as control. Lysates were immunoblotted for phosphotyrosine (4G10 antibody) with Erk1 used as an equal loading control. B–D, WT or pyk2^−/− PMNs were activated on pRGD for 0, 5, 10 or 15 minutes (left panels) or by exposure to complement-opsonized S. aureus for 0, 10 or 30 minutes (right panels) and then lysed. Lysates were immunoblotted with Abs specific for phospho-Pyk2, phospho-paxillin or phospho-Vav. Arrows indicate the appropriate bands in each gel. Lower panels show the same lysates probed for total Pyk2, paxillin, and Vav as loading controls. Arrows indicate specific bands. Data are representative of 2–6 different experiments for each panel. E, Lysates from WT, pyk2^−/− and syk^−/− PMNs activated on pRGD for 15 minutes were probed for phospho-paxillin and phospho-Syk as indicated with Erk1 serving as an equal loading control.

FIGURE 8

In vivo host defense response to bacterial infection. WT or pyk2^−/− mice with dorsally-located air pouches were infected with S. aureus. Following 6 or 20 hours of infection, the air pouches were lavaged and the number of bacteria or infiltrated PMNs was determined. A, The number of live bacteria (CFU)/mL in the air pouch lavage shown as the fraction of the original amount of CFU loaded into the air pouch. B, The number of PMNs infiltrating the airpouch was quantitated. C, The levels of CDllb upon the surface of PMNs in the lavage (measured on Ly6G+ gated cells) was quantitated by median fluorescence intensity (data representative of at least 3 independent experiments). D, The amount of MMP-9 released into the air pouch following 6 hours of infection was quantitated via zymography (n=8). E, The levels of MPO released into the air pouch following 6 hours of infection was measured via absorbance assay. Data represent average of at least 3 independent experiments with n = 4 or n = 5. Error bars represent ± SEM. *=p<0.05.

FIGURE 9