Synergism of gamma interferon and interleukin-5 in the control of murine filariasis

Abstract

There has been a prevailing perception that Th1 and Th2 immune responses induce antagonistic immune effector mechanisms during an infection. We investigated the role of the Th1 cytokine gamma interferon (IFN-gamma) and the Th2 cytokine interleukin-5 (IL-5) in murine filariasis infections with the rodent filarial nematode Litomosoides sigmodontis with regard to immune responses to the parasite. Earlier data showed an important role for IL-5 and IFN-gamma in effective immune responses to filarial infection. Therefore, in this study it was asked whether IL-5 and IFN-gamma act synergistically or antagonistically. Indeed, IL-5 as well as IFN-gamma knockout (KO) mice show a higher worm load than the wild-type controls. IFN-gamma/IL-5 double-KO mice had a significantly higher worm load than any of the single-KO mice, suggesting a synergism between IFN-gamma and IL-5 in controlling worm infection. Neutrophils are known to play an important role for the containment and encapsulation process of the worms. In infected IFN-gamma KO, IL-5 KO, and IFN-gamma/IL-5 double-KO mice, neutrophils were significantly reduced in chemotactic activity levels compared to controls. In addition, the level of phagocytosis activity of neutrophils from IFN-gamma/IL-5 double-KO mice was further decreased in comparison to that of the single-KO mice. Levels of tumor necrosis factor alpha, which is an important factor for neutrophil activation, were found to be reduced in macrophages from KO mice. In conclusion, these results argue for immune effector mechanisms in murine filarial infection that are dependent on both IFN-gamma and IL-5. Synergistic effects of the two cytokines may be mediated, at least in part, by neutrophils for the control of adult worms.

FIG. 1.

Numbers of microfilariae in infected 5-KO and γ/5-KO mice were significantly increased in comparison to those seen with the γ-KO and BALB/c control mice at day 70 p.i. No significant differences were detectable between 5-KO and γ/5-KO mice. The graph shows the medians, and the numbers represent the range of microfilaria counts in each group. The data are representative of one of two experiments (*, P < 0.05 [Mann-Whitney U test with Bonferroni correction]), with five animals per group.

FIG. 2.

Numbers of accumulated cells in the thoracic cavity at day 80 p.i. All KO mice showed a significant reduction in levels of neutrophils and CD4⁺ T cells. Only the 5-KO and γ/5-KO mice showed a significant reduction in the numbers of macrophages, eosinophils, and NK cells. Each group contained five animals. The data are representative of one of two experiments (*, P < 0.05 [Mann-Whitney U test]). ND, not detectable. Bars show means ± standard deviations. B cells, macrophages, and CD4⁺ and CD8⁺ T cells were enumerated by FACS. Eosinophils and neutrophils were enumerated by a cytospin technique.

FIG. 3.

TNF-α production by thoracic cavity macrophages after stimulation with medium (grey bars), L. sigmodontis antigen (black bars), or LPS (hatched bars). Thoracic cavity macrophages were taken after 80 days p.i. The data show that the addition of S. sigmodontis antigen or LPS to macrophage cultures from single-KO and γ/5-KO mice does not restore the defective production of TNF-α. Furthermore, γ/5-KO mice had the lowest level of production of TNF-α in the macrophage culture of the medium control cell sample. Each group contained five animals. The data are representative of one of two experiments (*, P < 0.05 [Mann-Whitney U test with Bonferroni correction]). Bars show means ± standard deviations. *, significant difference in comparison to the results for the BALB/c group.

FIG. 4.

In vivo reconstitution of phagocytosis activity by injection of 0.25 μg of IL-5 and/or 0.25 μg of IFN-γ in the peritoneal cavity of the KO mice. Zymosan was used as a marker for the phagocytosis activity of the neutrophils. A reconstitution of the 5-KO mice with 0.25 μg of IL-5 and a reconstitution of the γ-KO mice with 0.25 μg of IFN-γ led to a significant increase in the level of phagocytosis activity of the neutrophils. Moreover, a reconstitution of the γ/5-KO mice with 0.25 μg of IL-5 or 0.25 μg of IFN-γ led to a partial increase in the level of phagocytosis activity of the neutrophils. Only the injection of 0.25 μg of IL-5 and 0.25 μg of IFN-γ restored the complete phagocytosis activity of the neutrophils from γ/5-KO mice. The results from one of two consistent experiments is shown. Five mice were used for each group (*, P < 0.05; **, P < 0.03 [Mann-Whitney U test]). n.s., not significant.