Interleukin-18 primes the oxidative burst of neutrophils in response to formyl-peptides: role of cytochrome b558 translocation and N-formyl peptide receptor endocytosis

Abstract

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O2 degrees-) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-alpha), which is a cytokine known to strongly prime O2 degrees- production, IL-18 did not induce either p47phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O2 degrees- production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O2 degrees- production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.

FIG. 1.

Effect of IL-18 on the PMN oxidative burst in whole blood. After preincubation with hydroethidine (1,500 ng/ml) for 15 min at 37°C, whole blood was pretreated with PBS or various concentrations of IL-18 for 45 min, and then with PBS or fMLP (10⁻⁶ M) for 5 min. The mean fluorescence intensity of the ethidium (E⁺) content was recorded as described in Materials and Methods. Values are expressed as means ± SEMs (n = 10). The mean fluorescence intensity of the sample preincubated with TNF-α (100 U/ml) and then stimulated with fMLP was 108.7 ± 14.7. *, significantly different from the results for the sample pretreated with PBS and then stimulated with fMLP (P < 0.05).

FIG. 2.

Effects of IL-18, IL-1, and TNF-α on p47^phox phosphorylation. ³²P-labeled neutrophils were incubated with IL-18 (500 ng/ml), TNF-α (100 U/ml), or IL-1β (50 ng/ml) for 45 min. p47^phox was immunoprecipitated and analyzed by SDS-PAGE transfer and revealed by autoradiography ([³²P]p47^phox) with an anti-p47^phox antibody. Data are representative of those from three experiments.

FIG. 3.

Effects of IL-18 on p47^phox and p67^phox translocation to the PMN membrane. Isolated neutrophils were incubated with IL-18 (500 ng/ml) or buffer. Following stimulation with fMLP (10⁻⁷ M) for 1 min, PMN fractions were prepared as described in Materials and Methods. Acrylamide gels (10%) were loaded with 20 μg of protein, and then Western blotting (A) of membranes and the cytosol was performed with antibodies directed against p47^phox and p67^phox. Equal amounts of protein were loaded and checked by Coomassie blue staining. (B) Quantification of p47^phox translocation at the membrane, as determined by densitometry. The data are representative of those from three experiments.

FIG. 4.

Immunocytochemical staining of p22^phox in resting and IL-18-stimulated PMNs in whole-blood smears. (A) Negative control, which consisted of no staining with nonimmune rabbit serum followed by incubation with a biotinylated goat anti-rabbit and alkaline phosphatase-labeled streptavidin, as described in Materials and Methods; (B) intracellular fuchsia-colored staining was observed with the rabbit anti-human p22^phox polyclonal antibody (0.8 μg/ml), revealed as described for panel A; (C) fuchsia-colored staining at the PMN membrane was observed with the rabbit anti-human p22^phox polyclonal antibody (revealed as described for panel A) after IL-18 stimulation. Smears were examined by light microscopy. Magnifications, ×750.

FIG. 5.

Effects of IL-18 on p22^phox and CD11b expression at the PMN surface. After preincubation with PBS (IL-18 at 0 ng/ml) or IL-18 (0.1 to 500 ng/ml) for 45 min, whole blood was incubated with anti-p22^phox and anti-CD11b antibodies at 4°C for 30 min. The mean fluorescence intensity was recorded as described in Materials and Methods. Values obtained with nonimmune rabbit serum or with an irrelevant antibody of the same isotype were subtracted. Values are expressed as means ± SEMs (n = 4). The mean fluorescence intensity of the sample incubated with TNF-α (100 U/ml) was 1,729 ± 89. *, significantly different from the results for the sample incubated with PBS (P < 0.05).

FIG. 6.

Effects of colchicine on CD11b expression and the PMN oxidative burst after IL-18 priming. Samples were preincubated at 37°C with colchicine at various concentrations for 5 min before the addition of IL-18 (500 ng/ml) for 45 min. CD11b expression was measured with a mouse anti-human FITC-anti-CD11b antibody. The oxidative burst was measured by incubation of the samples with hydroethidine for 15 min before the addition of IL-18 and then stimulation with fMLP (10⁻⁶ M) for 5 min. The mean fluorescence intensity of the FITC-anti-CD11b antibody and the ethidium content were recorded as described in Materials and Methods. Values are expressed as means ± SEMs (n = 3). *, significantly different from the results for the samples without colchicine (P < 0.05).

FIG. 7.

Visualization of IL-18-induced fMLP-R endocytosis by confocal microscopy. Whole-blood samples were incubated at 4°C for 1 h with FITC-anti-human CD15 and PE-anti-human fMLP-R antibodies. After the samples were washed once in ice-cold PBS, they were incubated at 37°C with IL-18 (500 ng/ml) for 45 min. Images were obtained by confocal laser scanning microscopy. (A) Horizontal (x-y) section; (B) vertical (x-z) section. Thirty optical sections were used for three-dimensional reconstruction.

FIG. 8.

Effects of kinase inhibitors on fMLP binding and CD11b expression after IL-18 activation. Samples were preincubated at 37°C with PBS or with various kinase inhibitors for 15 min at 37°C: genistein (Gen.) at 100 μM, SB203580 (SB) at 25 μM, wortmannin (Wort.) at 250 nM, PD98059 (PD) at 50 μM, or GF109203X (GFX) at 5 μM. Studies of CD11b expression and fMLP binding are described in the legend to Fig. 5 and in footnote a of Table 1, respectively. Values are expressed as means ± SEMs (n = 3). *, significantly different from the results for the sample pretreated with PBS and then incubated with IL-18.

FIG. 9.

Effects of IL-18 on intracellular tyrosine, p125^FAK, and p38MAPK phosphorylation. After preincubation of whole blood with PBS, IL-18 (500 ng/ml), or TNF-α (100 U/ml) for 2 to 45 min, the phosphotyrosine, phospho-p125^FAK, and phospho-p38MAPK contents were measured by flow cytometry with methanol-permeabilized cells, as described in Materials and Methods. Values obtained with an irrelevant antibody of the same isotype were subtracted. Values are expressed as means ± SEMs (n = 3). *, significantly different from the results for the sample incubated with PBS (P < 0.05).