Role of protein tyrosine kinase p53/56lyn in diminished lipopolysaccharide priming of formylmethionylleucyl- phenylalanine-induced superoxide production in human newborn neutrophils

Abstract

Human newborns are more susceptible than adults to bacterial infection. With gram-negative bacteria, this may be due to a diminished response of newborn leukocytes to lipopolysaccharide (LPS). Since protein tyrosine kinase inhibition abolishes LPS priming in adult cells, we hypothesized that protein tyrosine kinases may have a critical role in LPS priming of polymorphonuclear neutrophils (PMNs) and that newborn PMNs may have altered protein tyrosine kinase activities. In the present study, we investigated the role of src family protein tyrosine kinases in the LPS response of newborn PMNs compared to adult cells. In a respiratory assay, the LPS-primed increase in formylmethionylleucylphenylalanine (fMLP)-triggered O2- release by adult PMNs was greatly decreased by PP1 [4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a src kinase inhibitor, to the level of untreated newborn PMNs, in which LPS failed to prime. LPS activated the src-like kinases p59hck (HCK) and p58fgr (FGR) in both adult and newborn PMNs but increased the activation of p53/56lyn (LYN) only in adult cells. In newborn PMNs, LYN was highly phosphorylated independent of LPS. We evaluated subcellular fractions of PMNs and found that the phosphorylated form of LYN was mainly in the Triton-extractable, cytosolic fraction in adult PMNs, while in newborn cells it was located mainly in Triton-insoluble, granule- and membrane-associated fractions. In contrast, the phosphorylated mitogen-activated protein kinases ERK1/2 and p38 were mainly detected in the cytosol in both adult and newborn PMNs. These data indicate a role for LYN in the regulation of LPS priming. The trapping of phosphorylated LYN in the membrane-granule fraction in newborn PMNs may contribute to the deficiency of newborn cells in responding to LPS stimulation.

FIG. 1.

LPS priming of fMLP-induced O₂⁻ release in adult PMNs is inhibited by PP1. PMNs from adults and newborns were resuspended at a density of 10⁶ cells/ml in HBSS^+/+ supplemented with 1% autologous plasma and 80 μM cytochrome c and treated with 1 μM PP1 or solvent (dimethyl sulfoxide [DMSO], 0.1%) for 10 min at room temperature. The cells were then incubated or not with 5 ng of LPS/ml for 20 min at 37°C before treatment or not with 1 μM fMLP for 15 min. The O₂⁻ released was calculated from measurement of the superoxide dismutase-inhibitable changes in absorbance at 550 nm as described in the text and expressed as mean ± standard deviation (n = 4). *, P < 0.05; **, P < 0.01 control versus PP1. NT, not treated.

FIG. 2.

Activation of HCK and FGR in adult and newborn PMNs. Adult and newborn PMNs were resuspended in HBSS^+/+ supplemented with 1% autologous plasma and incubated or not with LPS (5 ng/ml) for 20 min. HCK or FGR was immunoprecipitated from radioimmunoprecipitation assay lysates and assayed for kinase activity (autophosphorylation) with [γ-³²P]ATP, which was visualized by autoradiography following SDS-PAGE (upper two panels). Forty microliters (= 10 μg protein) of the supernatant of lysates after precipitation was analyzed by Western blotting for β-actin (lower panel). The results shown represent three independent experiments.

FIG. 3.

Activation of LYN in adult and newborn PMNs. Adult and newborn PMNs were resuspended in HBSS^+/+ supplemented with 1% autologous plasma and incubated with or without LPS (5 ng/ml) for 20 min. A. Total cellular protein was prepared and analyzed by Western blotting for phosphorylated LYN with a phosphorylation-specific antibody (upper panel). The membrane was then stripped and reblotted for LYN protein (middle panel). B. LYN was immunoprecipitated from radioimmunoprecipitation assay lysates and assayed for kinase activity (KA) (autophosphorylation) with [γ-³²P]ATP, which was visualized by autoradiography following SDS-PAGE (upper panel). The radioimmunoprecipitation assay lysates from the same donors were also analyzed by Western blotting (WB) for LYN protein (middle panel). The bottom panels of A and B graph the relative densities of phospho-LYN (dividing the density of the phospho-LYN band by that of the corresponding LYN protein band) pooled from four independent experiments. **, P < 0.01.

FIG. 4.

Triton solubility of LYN in adult and newborn PMNs. Adult and newborn PMNs were resuspended in HBSS^+/+ supplemented with 1% autologous plasma and incubated with or without LPS (5 ng/ml) for 20 min. PMNs were fractionated into Triton-soluble and Triton-insoluble fractions with a Triton-containing buffer, CSK, as described in Materials and Methods. Both fractions (25 μg/sample) were analyzed by Western blotting for phosphorylated LYN (upper panel) and for total LYN protein (middle panel). The bottom panel graphs the relative densities of phospho-LYN (dividing the density of the phospho-LYN band by that of the corresponding LYN protein band) pooled from three independent experiments. **, P < 0.01.

FIG. 5.

Distribution of protein markers among different fractions prepared from adult PMNs. Adult PMNs (50 × 10⁶ cells) were disrupted by sonication and then fractionated by centrifugation over discontinuous Percoll gradients as described in the text. Equal amounts of proteins (10 μg) from each fraction (Gα, α granules; Gβ, β granules; M, membrane; C, cytosol) were analyzed by Western blotting for elastase, lactoferrin, CD66b, CD14, and p47^PHOX. A representative result of three independent experiments is shown.

FIG. 6.

Subcellular distribution of LYN in adult and newborn PMNs. Adult and newborn PMNs (50 × 10⁶ cells for each) were resuspended in HBSS^+/+ supplemented with 1% autologous plasma and incubated with or without LPS (5 ng/ml) for 20 min. PMNs were disrupted by sonication and then fractionated by centrifugation over discontinuous Percoll gradients as described in the text. Upper panels: equal amounts of proteins (10 μg) from each fraction (Gα, α granules; Gβ, β granules; M, membrane; C, cytosol) were analyzed by Western blotting for LYN protein (A) and phosphorylated LYN (B) as described in the legend to Fig. 2. Lower panels: the corrected amounts of LYN protein and phosphorylated LYN in the cytosolic (C) and combined membrane and granule (M+G) fractions were calculated by multiplying the band densities (above) by the proportion of total protein in the corresponding fractions (see text). Results are expressed in arbitrary units (mean ± standard deviation for four independent experiments). **, P < 0.01.

FIG. 7.

Subcellular distribution of mitogen-activated protein kinases in adult and newborn PMNs. Subcellular fractions were prepared from LPS-stimulated PMNs of adults and newborns as described in the legend to Fig. 5. Proteins from each fraction (10 μg/sample) were analyzed by Western blotting for phosphorylated ERK1/2 or p38 and, as described in the legend to Fig. 2, the membranes were stripped and reblotted for ERK2 or p38 proteins. A representative result of three independent experiments is shown.