Role of eosinophils and neutrophils in innate and adaptive protective immunity to larval strongyloides stercoralis in mice

Abstract

The goal of this study was to determine the roles of eosinophils and neutrophils in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice. The experimental approach used was to treat mice with an anti-CCR3 monoclonal antibody to eliminate eosinophils or to use CXCR2-/- mice, which have a severe neutrophil recruitment defect, and then determine the effect of the reduction or elimination of the particular cell type on larval killing. It was determined that eosinophils killed the S. stercoralis larvae in naïve mice, whereas these cells were not required for the accelerated killing of larvae in immunized mice. Experiments using CXCR2-/- mice demonstrated that the reduction in recruitment of neutrophils resulted in significantly reduced innate and adaptive protective immunity. Protective antibody developed in the immunized CXCR2-/- mice, thereby demonstrating that neutrophils were not required for the induction of the adaptive protective immune response. Moreover, transfer of neutrophil-enriched cell populations recovered from either wild-type or CXCR2-/- mice into diffusion chambers containing larvae demonstrated that larval killing occurred with both cell populations when the diffusion chambers were implanted in immunized wild-type mice. Thus, the defect in the CXCR2-/- mice was a defect in the recruitment of the neutrophils and not a defect in the ability of these cells to kill larvae. This study therefore demonstrated that both eosinophils and neutrophils are required in the protective innate immune response, whereas only neutrophils are necessary for the protective adaptive immune response to larval S. stercoralis in mice.

FIG. 1.

Effect of eosinophils and neutrophils on the survival of L3. (A) Eosinophils recovered from naïve IL-5 TG mice were transferred into diffusion chambers covered with 0.1-μm-pore-size membranes along with S. stercoralis L3, and the diffusion chambers were implanted in naïve C57BL/6J mice for 72 h. One asterisk indicates that there was a statistically significant difference between larval recovery from diffusion chambers in which there were no cells and larval recovery from diffusion chambers in which there were 5 × 10⁴ or 5 × 10⁵ eosinophils. Two asterisks indicate that there was a statistically significant difference between larval recovery from diffusion chambers in which there were 5 × 10⁴ eosinophils and larval recovery from diffusion chambers in which there were 5 × 10⁵ eosinophils. (B) Neutrophils recovered from the peritoneal cavity of C57BL/6J mice after injection of thioglycolate medium were transferred into diffusion chambers covered with 0.1-μm-pore-size membranes along with S. stercoralis L3, and the diffusion chambers were implanted in naïve C57BL/6J mice for 5 days. The asterisk indicates that there was a statistically significant difference between larval recovery from diffusion chambers in which there were no cells and larval recovery from diffusion chambers in which there were 5 × 10⁶ neutrophils. (C) Eosinophils and neutrophils, prepared as described above, were transferred into diffusion chambers covered with 0.1-μm-pore-size membranes along with S. stercoralis L3, and the diffusion chambers were implanted in naïve C57BL/6J mice for 5 days. One asterisk indicates that there was a statistically significant difference between larval recovery from diffusion chambers in which there were no cells and larval recovery from diffusion chambers in which there were eosinophils and/or neutrophils. Two asterisks indicate that the data were statistically significantly different from the data for all other groups. The data are means and standard deviations. PMN, neutrophils; Eos, eosinophils.

FIG. 2.

Role of eosinophils in adaptive immunity: cell recruitment (A) and larval recovery (B) in diffusion chambers implanted for 24 h in immunized C57BL/6J mice treated with anti-CCR3 MAb to eliminate eosinophils. The asterisk indicates that there was a statistically significant difference between larval recovery in naïve mice and larval recovery in immune mice. The data are means and standard deviations. PMN, neutrophils; Mf, macrophages; Eos, eosinophils; αCCR3, anti-CCR3 MAb.

FIG. 3.

Role of neutrophils in adaptive immunity: cell recruitment (A) and larval recovery (B) in diffusion chambers implanted in immunized BALB/cJ and CXCR2^−/− mice for 24 h. One asterisk indicates that there was a statistically significant difference between larval recovery from naïve mice and larval recovery from immune mice. Two asterisks indicate that there was a statistically significant difference between larval recovery from immunized BALB/cJ mice and larval recovery from immunized CXCR2^−/− mice. The data are means and standard deviations. PMN, neutrophils; Mf, macrophages; Eos, eosinophils.

FIG. 4.

Role of neutrophils in the development of protective IgM: parasite-specific IgM levels measured in diffusion chamber fluids recovered from immunized BALB/cJ and CXCR2^−/− mice. An asterisk indicates that there was a statistically significant difference between IgM levels in naïve mice and IgM levels in immune BALB/cJ or CXCR2^−/− mice. The data are means and standard deviations. O.D. (410 nm), optical density at 410 nm.

FIG. 5.

Passive transfer of immunity from immunized CXCR2^−/− mice to naïve BALB/cJ mice: larval recovery (A) and cell recruitment (B) in diffusion chambers implanted in mice for 24 h after transfer of serum from donor naïve and immunized BALB/cJ and CXCR2^−/− mice into naïve recipient BALB/cJ mice. An asterisk indicates that there was a statistically significant difference between larval recovery from naïve mice and larval recovery from immune mice. The data are means and standard deviations. PMN, neutrophils; Mf, macrophages; Eos, eosinophils.

FIG. 6.

Neutrophil-mediated killing in adaptive immunity: live larval recovery after neutrophil-enriched cell populations from BALB/cJ and CXCR2^−/− mice were transferred (4 × 10⁶ cells) into diffusion chambers (containing larvae) covered with 0.1-μm-pore-size membranes and implanted in naïve or immunized wild-type mice. An asterisk indicates that there was a statistically significant difference between larval survival levels in diffusion chambers containing cells in naïve recipients and larval survival levels in diffusion chambers containing cells in immune recipients. The data are means and standard deviations.