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. 2003 Jan 6;197(1):63-75.
doi: 10.1084/jem.20021638.

Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation

Hyunsil Han  1 , Michele Fuortes, Carl Nathan
Affiliations

Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation

Hyunsil Han et al. J Exp Med. .

Abstract

Transduction of Tat-tagged fusion proteins confirmed a hypothesis based on pharmacologic inhibitors (Fuortes, M., M. Melchior, H. Han, G.J. Lyon, and C. Nathan. 1999. J. Clin. Invest. 104:327-335) that proline-rich tyrosine kinase (Pyk2) plays a critical role in the activation of adherent human neutrophils, and allowed an analysis of individual Pyk2 domains not possible with chemical inhibitors. Acting as a dominant negative, the COOH terminus of Pyk2 fused to a Tat peptide (Tat-CT), but not other regions of Pyk2, specifically inhibited the respiratory burst of cells responding to tumor necrosis factor (TNF), Salmonella, or Listeria, while sparing responses induced by phorbol ester. Tat-CT suppressed TNF-triggered cell spreading and the phosphorylation of endogenous Pyk2 and the associated tyrosine kinase Syk without blocking the ability of neutrophils to degranulate and kill bacteria. Thus, separate signals control the respiratory burst and degranulation, and a normal rate of killing of some bacteria can be sustained by granule products in conjunction with a minimal residual respiratory burst. Inhibition of select inflammatory functions without impairment of antibacterial activity may commend the Pyk2 pathway as a potential target for antiinflammatory therapy.

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Figures

Figure 1.
Figure 1.
Schematic representation of the Pyk2 regions expressed as Tat fusion proteins. Pro denotes the proline-rich regions critical for some of the protein–protein interactions associated with the COOH terminus of Pyk2. The ball and stick represent the Tat peptide, hexahistidine, and hemagglutinin tag. Amino acid residue numbers are shown in parentheses.
Figure 2.
Figure 2.
Uptake of Tat fusion proteins by human PMNs, FACS® analysis. Flow cytometric analysis of the uptake of Tat fusion proteins by neutrophils. The cells were incubated with labeled Tat-AP (A and D) or Tat-CT (B, C, and E), either with 1 μM Tat construct for the indicated time at 37°C (A, B, and C), or for 60 min at 37°C with the indicated concentration of Tat construct (D and E). In C, cells were also preincubated with 100 μM chloroquine for 30 min at 37°C.
Figure 3.
Figure 3.
Distribution of Tat fusion proteins within human PMNs, confocal microscopy. Neutrophils were incubated with 1 μM fluorescent Tat-CT (rows 1 and 3), Tat-AP (row 2), or no protein (row 4), and stained for the lysosomal marker LAMP-1 (rows 2 and 3). Neutrophils were treated without (row 1, left) or with 100 μM chloroquine (row 1, right, and rows 2–4) along with the Tat fusion protein for 1 h at 37°C and then permeabilized and stained. Differential interference contrast microscopy (DIC) demonstrates the presence of cells. ×800 for row 1 and ×2,000 for rows 2–4.
Figure 4.
Figure 4.
Effect of Pyk2 domains on the respiratory burst of adherent neutrophils. (A) Concentration-dependent effect of Tat-CT. Neutrophils were preincubated with the indicated concentrations of Tat-CT at 37°C for 30 min before stimulation with 100 ng/ml TNF, 100 ng/ml PMA, or an equivalent volume of KRPG buffer as a control. H2O2 release was measured at 15-min intervals. Results are displayed for 75 min and expressed as means ± SEM for triplicates in one representative experiment. (B) Summary of experiments with Tat-CT and other Pyk2 constructs. Results are means ± SEM for results at 100–120 min from the number of experiments with different blood donors shown in parentheses, each in triplicate. Percent inhibition was calculated in comparison to results for TNF without Tat fusion proteins in the same experiments.
Figure 5.
Figure 5.
Inhibition of TNF-triggered neutrophil spreading by Tat-CT. Neutrophils were plated on FBS-coated glass coverslips and preincubated or not with 500 nM Tat-CT at 37°C for 30 min before stimulation with 100 ng/ml TNF, 100 ng/ml PMA, or an equal volume of KRPG (Cont.), and fixed and photographed with phase-contrast microscope. ×1,000.
Figure 6.
Figure 6.
Effect of Tat-CT on TNF-triggered neutrophil degranulation. Neutrophils were incubated as indicated (test proteins were added at 500 nM) and their supernate was assayed for the presence of lactoferrin (LF) as a measure of specific granule release and myeloperoxidase (MPO) for azurophil granule release. LDH was measured as an indicator of spontaneous cell lysis. Specific LF (A) and MPO (B) release (corrected for cell lysis) are shown normalized to that seen with TNF alone in the absence of ovalbumin (ova) or Tat-CT. TNF alone led to the specific release of an average of 67 and 27% of total detergent-releasable LF and MPO content of the cells, respectively, over and above the mean 5% release that could be attributed to cell death. Results are the mean ± SEM in three independent experiments for LF release and two for MPO release.
Figure 7.
Figure 7.
Inhibition of TNF-induced phosphorylation of endogenous Pyk2 by Tat-CT. Neutrophils were preincubated with Tat-CT or ovalbumin (ova; each 500 nM) at 37°C for 30 min and stimulated with TNF (T) or buffer alone as a control (C). Total cell lysates were separated by SDS-PAGE and western-blotted (WB) with (A) antiphosphotyrosine Ab or (B) Abs specific for individual phosphotyrosine (PY) residues of Pyk2. One membrane was stripped and reprobed with anti-Pyk2 Ab, demonstrating equal loading of the lanes.
Figure 8.
Figure 8.
TNF-induced association of Pyk2 and Syk, and the inhibition of tyrosine phosphorylation of Syk by wortmannin and Tat-CT. (A) Pyk2 was immunoprecipitated (IP) from neutrophils stimulated with TNF (T) or treated with buffer alone as a control (C) and Western blotted (WB) for Syk. (B) Syk was immunoprecipitated from neutrophils after preincubation with or without TNF (T) and/or 10 nM wortmannin and Western blotted for phosphotyrosine (PY; top) or Syk (bottom). (C) Syk was immunoprecipitated from neutrophils after preincubation with or without TNF (T) and/or with Tat-CT or ovalbumin (ova; each 500 nM) at 37°C for 30 min and probed with antiphosphotyrosine Ab (PY) or anti-Syk.
Figure 9.
Figure 9.
Effect of Tat-CT on interaction of neutrophils with bacteria. (A) Impact of Tat-CT or ovalbumin (ova; each 500 nM, 30 min preincubation) on the respiratory burst triggered by S. typhimurium or L. monocytogenes, measured at 150 min. Results are means ± SEM for triplicates in a representative experiment of two performed. (B) Survival of bacteria in culture only (open symbols) or in culture with neutrophils (closed symbols). Survival of bacteria in the presence of Ova (▪ and □) or Tat-CT (▴ and ▵) are shown in CFU/ml. Results are means ± SEM for triplicates in a representative experiment of three performed. Some of the error bars fall within the symbols.
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