Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils

Abstract

The insect immune response has a number of structural and functional similarities to the innate immune response of mammals. The objective of the work presented here was to establish the mechanism by which insect hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils. Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were immunoprecipitated and analyzed by matrix-assisted laser desorption ionization--time of flight analysis. The results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.

FIG. 1.

Kinetics of phagocytosis by neutrophils and hemocytes: in vitro phagocytosis of serum- or hemolymph-opsonized C. albicans (2 × 10⁶ cells) by neutrophils (1 × 10⁷ cells) (A) and hemocytes (1 × 10⁷ cells) (B). The scale on the right y axis indicates the number of internalized yeast cells per 100 phagocytic cells. Over 60 min, the number of phagocytosing neutrophils (bars) or internalized Candida cells (○) was not significantly different from the number of hemocytes (bars) (P = 0.374 and P = 0.26). Similar results were obtained in two independent experiments. The data are means ± standard errors for three determinations.

FIG. 2.

Kinetics of bacterial and fungal killing by human neutrophils and insect hemocytes. Serum- or hemolymph-opsonized S. aureus cells (1 × 10⁷ cells) (A and B) or C. albicans cells (2 × 10⁶ cells) (C and D) were exposed to 1 × 10⁷ neutrophils (A and C) or hemocytes of G. mellonella (B and D). Killing was also measured in the presence (□ and ▪) and in the absence (▵ and ▴) of DPI (5 μM) added 3 min prior to initiation of phagocytosis. Microbial survival is expressed as a percentage of the control at time zero, and the data are means ± standard errors. The data are from a representative experiment performed in triplicate.

FIG. 3.

Inhibition of the hemocyte oxidative burst by DPI. The rate of oxygen consumption by neutrophils (1 × 10⁷ cells [•] or 1 × 10⁶ cells [▵]) was compared to the rate of oxygen consumption by insect hemocytes (1 × 10⁷ cells) (□) after stimulation with PMA (1 μg/ml). Oxygen consumption by neutrophils (1 × 10⁶ cells) (○) and hemocytes (1 × 10⁷ cells) (▪) was also measured in the presence of DPI (5 μM) added 3 min prior to elicitation. The data are means of three trials carried out on separate days.

FIG. 4.

Effect of an NADPH oxidase inhibitor on in vitro PMA-stimulated generation of O₂⁻ in hemocytes of G. mellonella. The production of O₂⁻ by unstimulated hemocytes (1 × 10⁶ cells/ml) (Control) and PMA (1 μg/ml)-activated cells was measured using the reduced cytochrome c assay. Production of O₂⁻ by hemocytes was inhibited in the presence of DPI (5 μM) and scavenged in the presence of SOD (50 μg/ml).

FIG. 5.

Immunoblotting of hemocytes of G. mellonella with antibodies to human neutrophil phox proteins. (A) Coomassie blue-stained SDS-PAGE gel (12.5% polyacrylamide) of neutrophil cytosol (lane 1) and membranes (lane 2). The protein profile of neutrophil PNS (lane 3) was compared to the protein profile of PNS of insect hemocytes (lane 4). The positions of molecular weight markers are indicated on the left. (B) Electrophoretically separated neutrophil membrane proteins and insect hemocyte PNS transferred to nitrocellulose and probed with rabbit antiserum to gp91^phox. The results show that there was an immunoreactive band at 90 kDa for neutrophil membranes and a band at 77 kDa for insect hemocyte PNS. (C to G) Neutrophil cytosol and insect hemocyte PNS probed with rabbit antisera to p67^phox (C), p47^phox (D), p40^phox (E), rac 2 (F), and PKC δ (G). The results revealed immunologically related proteins having the same molecular mass for all probed proteins except p40^phox, which was not detected in insect hemocytes.

FIG. 6.

Distribution of p47^phox and p67^phox homologues in unstimulated hemocytes: distribution of 47-kDa (A) and 67-kDa (B) insect proteins in hemocytes adhering to glass slides. The distribution of the proteins was predominantly perinuclear (indicated by an arrow) and throughout the cytosol. (magnification, ×400).

FIG. 7.

Immunoprecipitation of p67^phox and p47^phox from human neutrophils and hemocytes of G. mellonella. Postnuclear supernatants were prepared from 2 × 10⁸ neutrophils or hemocytes and solubilized in buffer as described in the text. (A) Immunoprecipitation was carried out using rabbit polyclonal sera against p67^phox and p47^phox, the preparation was resuspended in 60 μl of Laemmli sample buffer, and 25 μl was analyzed by SDS-PAGE and Coomassie blue staining. The positions of immunoprecipitated p67^phox and p47^phox from neutrophils (N) and hemocytes (H) are indicated by the arrows. (B) Immunoprecipitation preparations were Western blotted using goat polyclonal sera against p67^phox and p47^phox. In two other experiments similar immunoprecipitation results were obtained.

FIG. 8.

Matching peptides of hemocyte 67-kDa and 47-kDa proteins to human neutrophil phox proteins. (A) Matching peptides of hemocyte 67-kDa protein to human p67^phox that strongly reacted with both rabbit and goat anti-human p67^phox in immunoprecipitation. (B) Regions of p67^phox involved in protein-protein interactions with matching peptides of hemocyte 67-kDa proteins (indicated by arrows). (C) Matching peptides of hemocyte 47-kDa protein to human p47^phox that strongly reacted with both rabbit and goat anti-human p47^phox. (D) Regions of p47^phox involved in protein-protein interactions. Matching peptides of hemocyte 47-kDa protein to neutrophil p47^phox in the PX domain is indicated by an arrow.