Neutrophil lactoferrin release induced by IgA immune complexes differed from that induced by cross-linking of fcalpha receptors (FcalphaR) with a monoclonal antibody, MIP8a

Abstract

The human IgA Fc receptor (FcalphaR, CD89) plays an important role in host defence against invading pathogens. To study the properties of the receptor, 12 MoAbs, namely, MIP7c, MIP8a, MIP9a, MIP10c, MIP11c, MIP14b, MIP15b, MIP38c, MIP59c, MIP65c, MIP68b and MIP71a, were generated. The inhibitory effects of the antibodies on FcalphaR functions were tested. Three of the antibodies, MIP7c, MIP8a and MIP59c, were able to block up to 90% of soluble FcalphaR binding to IgA-coated beads and 70-80% of neutrophil phagocytosis of IgA immune complexes (IC). MIP8a could also inhibit IgA IC-induced neutrophil lactoferrin release, while cross-linking of FcalphaR with MIP8a and anti-mouse IgG could elicit neutrophil lactoferrin release. However, IgA IC-induced lactoferrin release required both extracellular calcium and magnesium, whereas MIP8a-induced release did not require extracellular magnesium and only partially required extracellular calcium. In addition, the time course of IgA IC-induced lactoferrin release was slow. Lactoferrin was not detectable if the incubation time was less than 0.5 h. In contrast, MIP8a-induced lactoferrin release was fast. Lactoferrin could be detected within 5 min of incubation. Therefore, neutrophil lactoferrin release induced by IgA IC differed from that induced by cross-linking of FcalphaR with MIP8a.

Fig. 1

Binding profiles of MoAbs to FcαR. (a) ELISA analysis of MoAb binding to soluble FcαR. Soluble FcαR (200 ng/well) was coated on 96-well plates. Tissue culture supernatant (25 μl) was tested. The negative control sample was 25 μl supernatant from cells which did not secrete antibodies. Peroxidase-conjugated anti-mouse IgG was used as the secondary antibody. Each data point was the mean of triplicate samples. (b) Flow cytometry analysis of MoAb binding to cell surface FcαR. Neutrophils (2 × 10⁵) were first incubated with 50 μl of tissue culture supernatant in 500 μl of PBS containing 2 mm sodium azide and 1% bovine serum albumin. The negative control sample was 50 μ l supernatant from cells which did not secrete antibodies. The cells were stained with FITC-conjugated F(ab′)₂ fragments of goat anti-mouse IgG. After fixing with 2% paraformaldehyde, 10 000 cells were analysed. (c) Western blotting of MoAbs. Soluble FcαR was run on non-reducing 7–17% SDS–PAGE and transferred on to nitrocellulose paper. The paper was then cut into strips and incubated separately with 1 ml of the antibody tissue culture supernatant. Anti-mouse IgG (Fc-specific) peroxidase conjugate was used as the secondary antibody. The bands were visualized using ECL system (Amersham Int. plc, Aylesbury, UK). The negative control sample was supernatant from cells that did not secrete antibodies.

Fig. 2

Blocking soluble FcαR binding to IgA beads by MoAbs. Tissue culture supernatant (1 ml) from cells secreting MoAb against FcαR was first incubated with 7 μl of ¹²⁵I-FcαR. Then 50 μl of IgA beads were added. After washing, the remaining radioactivity from the beads was counted (see Materials and Methods). The positive sample was 1 ml of culture medium instead of cell supernatant. The negative control sample was NIP beads instead of IgA beads. The results are representative of three separate experiments.

Fig. 3

Inhibition of neutrophil phagocytosis of IgA immune complexes (IC) by MoAbs. Cell supernatant (37·5 μl) was used to inhibit neutrophil (3·75 × 10⁵ cells) phagocytosis of 5 μg IgA IC (see Materials and Methods). The control sample was culture medium instead of cell supernatant. The results are representative of three separate experiments.

Fig. 4

Inhibition of neutrophil lactoferrin release by MIP8a and MIP10c. Neutrophils (100 μl) (4 × 10⁶ cells/ml) were incubated with MIP8a F(ab′)₂ fragments or MIP10c F(ab′)₂ fragments in 96-well plates at room temperature for 10 min. Then 50 μl of IgA/NIP-BSA IC (60 μg/ml) were added to each well. After incubation at 37°C for 2 h, lactoferrin released from neutrophils was measured. ▪, MIP10c; ▴, MIP8a. Results are representative of three separate experiments.

Fig. 5

Lactoferrin release induced by MIP8a and IgA immune complexes (IC). (a) Dose–response of MIP8a-induced lactoferrin release. Neutrophils (100 μl) (4 × 10⁶ cells/ml) were incubated at room temperature for 10 min with 25 μl MIP8a F(ab′)₂ fragments as indicated. Then 25 μl of F(ab′)₂ fragments of rabbit anti-mouse IgG (120 μg/ml) were added to each well. After incubation at 37°C for 2 h, lactoferrin released from neutrophils was measured. (b) Time course of MIP8a- and IgA IC-induced lactoferrin release. For MIP8a-induced lactoferrin release, 100 μl of neutrophils (4 × 10⁶ cells/ml) were incubated at room temperature for 10 min with 25 μl of MIP8a F(ab′)₂ fragments (40 μg/ml). Then 25 μl of F(ab′)₂ fragments of rabbit anti-mouse IgG (120 μg/ml) were added to each well. For IgA IC-induced lactoferrin release, 100 μ l of neutrophils (4 × 10⁶ cells/ml) were incubated with 50 μl of IgA/NIP-BSA IC (60 μg IgA/ml). After incubation at 37°C for the times indicated, lactoferrin released from neutrophils was measured. ▪, IgA IC; ▴, MIP8a. Results are representative of three separate experiments.

Fig. 6

Requirement of divalent cations for MIP8a- and IgA immune complex (IC)-induced neutrophil lactoferrin release. For MIP8a-induced lactoferrin release, 100 μl of neutrophils (4 × 10⁶ cells/ml) were incubated with 25 μl of MIP8a F(ab′)₂ fragments (40 μg/ml) at room temperature for 10 min. Then 25 μl of F(ab′)₂ fragments of rabbit anti-mouse IgG (120 μ g/ml) were added to each well. For IgA IC-induced lactoferrin release, 100 μl of neutrophils (4 × 10⁶ cells/ml) were incubated with 50 μl of IgA/NIP-BSA IC (60 μ g IgA/ml). After incubation at 37°C for 3 h, lactoferrin released from neutrophils was measured. Hanks' balanced salt solution (HBSS) (EDTA), medium containing 1 mm Ca²⁺, 1·5 mm Mg²⁺ and 6 mm EDTA; HBSS (EGTA), medium containing 1 mm Ca²⁺, 1·5 mm Mg²⁺ and 6 mm EGTA; HBSS (Ca, Mg), medium containing 1 mm Ca²⁺ and 1·5 mm Mg²⁺; HBSS (Ca), medium containing 1 mm Ca²⁺; HBSS (Mg), medium containing 1·5 mm Mg²⁺; HBSS, Ca²⁺-and Mg²⁺-free medium. Results are representative of three separate experiments.