Specificity and mechanism of immunoglobulin M (IgM)- and IgG-dependent protective immunity to larval Strongyloides stercoralis in mice

Abstract

Protective immunity in mice to the infective third-stage larvae (L3) of Strongyloides stercoralis was shown to be dependent on immunoglobulin M (IgM), complement activation, and granulocytes. The objectives of the present study were to determine whether IgG was also a protective antibody isotype and to define the specificity and the mechanism by which IgG functions. Purified IgG recovered from mice 3 weeks after a booster immunization with live L3 was shown to transfer high levels of protective immunity to naïve mice. IgG transferred into mice treated to block complement activation or to eliminate granulocytes failed to kill the challenge larvae. Transfer of immune IgG into IL-5 knockout (KO) mice, which are deficient in eosinophils, resulted in larval attrition, while transfer into FcRgamma KO mice did not result in larval killing. These findings suggest that IgG from mice immunized with live L3 requires complement activation and neutrophils for killing of L3 through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. This is in contrast to the results of investigations using IgM from mice immunized with live L3 and IgG from mice immunized with larval antigens soluble in deoxycholate in which protective immunity was shown to be ADCC independent. Western blot analyses with immune IgM and IgG identified few antigens recognized by all protective antibody isotypes. Results from immunoelectron microscopy demonstrated that the protective antibodies bound to different regions in the L3. It was therefore concluded that while IgM and IgG antibodies are both protective against larval S. stercoralis, they recognize different antigens and utilize different killing mechanisms.

FIG. 1.

Mean percentage of live larval recovery from mice that received the transfer of whole immune sera, purified IgM, and purified IgG recovered 3 weeks (A) and 5 weeks (B) after booster immunization with live L3. The data represent the means and standard deviations of the results for 10 animals per group. The asterisks indicate statistical differences (P ≤ 0.05) from control values.

FIG. 2.

Effect of CVF treatment of mice that have received transfer of naïve and immune IgG on the survival of challenge larvae. The data represent the means and standard deviations of the results for 10 animals per group. The asterisks indicate statistical differences (P ≤ 0.05) from control values.

FIG. 3.

Effect of granulocyte depletion with MAb RB6-8C5 on immunity transferred with purified immune IgG. The data represent the means and standard deviations of the results for 10 animals per group. The asterisks indicate statistical differences (P ≤ 0.05) from control values.

FIG. 4.

Transfer of naïve and immune IgG into wild-type C57BL/6J mice and IL-5 KO mice to determine whether eosinophils are required for protective immunity. The data represent the means and standard deviations of the results for 10 animals per group. The asterisks indicate statistical differences (P ≤ 0.05) from control values.

FIG. 5.

Effect of immunization of C57BL/6J and FcRγ KO mice on the survival of challenge larvae at 1 week (A) and 3 weeks (B) after booster immunization. The data represent the means and standard deviations of the results for 12 animals per group for the experiment at 1 week after booster immunization and 5 animals per group for the experiment at 3 weeks after booster immunization. The asterisks indicate statistical differences (P ≤ 0.05) from control values.

FIG. 6.

Effect on larval survival of transfer of immune IgG from mice immunized with live L3 (A) or with soluble DOC antigen (B) into C57BL/6J and FcRγ KO mice. The data represent the means and standard deviations of results for 10 animals per group. The asterisks indicate statistical differences (P ≤ 0.05) from control values.

FIG. 7.

Antigen recognition in Western blot analysis of protective IgM and IgG (recovered from mice 3 weeks after immunization with live L3) from solubilized S. stercoralis larval antigens. The bars represent bands recognized by both IgM and IgG.

FIG. 8.

Immunoelectron microscopy performed to determine where protective IgM recovered 1 week after booster immunization bound to the larvae of S. stercoralis. Cu, cuticle; g, glandular esophagus granules. The thin arrows indicate labeling in the basal cuticle-hypodermis area, the thick arrows indicate labeling on the surface of the cuticle, and the arrowheads indicate labeling in the coelomic cavity. Bar, 250 nm.

FIG. 9.

Immunoelectron microscopy performed to determine where protective IgG, recovered at 3 weeks after booster immunization, bound to the larvae of S. stercoralis. Cu, cuticle; g, glandular esophagus granules. The thin arrows indicate labeling in the basal cuticle-hypodermis area; the arrowheads indicate labeling in the coelomic cavity. Bar, 250 nm.