NO contributes to proliferative suppression in a murine model of filariasis

Abstract

Infection of BALB/c mice with microfilariae (mf) of Brugia pahangi leads to the suppression of antigen (Ag)-specific proliferative responses in the spleen. The proliferative defect is dependent on inducible nitric oxide synthase (iNOS) activity, since inhibition of iNOS with either L-N-monomethyl arginine (L-NMMA) or aminoguanidine reversed defective proliferation. Splenocytes from mf-infected animals produce high levels of gamma interferon (IFN-gamma) upon in vitro restimulation with Ag, and experiments in IFN-gamma receptor-deficient (IFN-gamma R(-/-)) mice demonstrated that signaling via the IFN-gamma R is essential in the induction of NO production and subsequent proliferative suppression. Restimulation of splenocytes from mf-infected animals with an extract of Acanthocheilonema viteae, a related filarial worm which lacks endosymbiotic bacteria, also resulted in NO production and proliferative suppression, demonstrating that lipopolysaccharide of bacterial origin is not essential to the induction of iNOS activity. These results extend previous observations that infection with different life cycle stages of Brugia leads to the development of differentially polarized immune responses and demonstrate one method by which these differences may exert their effects on the proliferative potential of cells from infected animals.

FIG. 1

Infection of BALB/c mice with mf results in proliferative suppression at 96 h. (A and B) Ag-stimulated proliferative responses of splenocytes from BALB/c mice infected i.v. with 10⁵ mf, 50 L3 B. pahangi, or HBSS after 48 h (A) and 96 h (B) of in vitro restimulation with Ag (10 μg/ml) or medium only. Results are expressed as mean cpm incorporated in triplicate wells. (C) Ag-stimulated IFN-γ production after 48 h of culture. All values represent the mean and standard deviation for five animals per group.

FIG. 2

Proliferative suppression correlates with NO production by splenocytes from mf-infected animals. Nitrite production and proliferative responses of splenocytes from mf-infected BALB/c mice over a time course of in vitro restimulation with Ag (10 μg/ml) are shown. Data show the mean and standard deviation for five mice per group. Nitrite levels (right-hand axis) in culture display a strong negative correlation with proliferative responses (left-hand side) (r = −0.866, P = <0.001).

FIG. 3

Proliferative suppression can be reversed by inhibition of iNOS activity. (A and B) Proliferative responses of splenocytes from BALB/c mice given 10⁵ B. pahangi mf or HBSS after 96 h of Ag-stimulated culture in the presence or absence of 500 μM AMG (A) or 250 μg of l-NMMA per ml (B). (C) IFN-γ production by splenocytes from mf-infected mice cultured in the presence or absence of AMG at 96 h of culture. All data represent the mean and standard deviation for five mice per group. ∗, significantly different from unsupplemented cultures (P < 0.05).

FIG. 4

NO production and proliferative suppression are dependent on IFN-γ. Proliferative responses (A) and nitrite production (B) of splenocytes from wild-type (129/Sv) and IFN-γR^−/− mice given 10⁵ mf or 50 L3 B. pahangi or HBSS i.v. after 96 h in vitro restimulation with Ag are shown. All data show the mean and standard deviation for five mice per group. ∗, significantly different from wild-type counterparts (P < 0.05).

FIG. 5

Restimulation with A. viteae Ag induces NO production and proliferative suppression in splenocytes from mf-infected animals. (A) Nitrite production by splenocytes from B. pahangi mf-infected mice restimulated with 10 μg of A. viteae or B. pahangi Ag per ml, as shown. Cells from uninfected animals produced undetectable levels of nitrite in this experiment. (B) Proliferative responses of splenocytes from BALB/c mice given 10⁵ mf, 50 L3 B. pahangi, or HBSS i.v. after 96 h of in vitro restimulation with A. viteae Ag (10 μg/ml) or medium only. All data show the mean and standard deviation for five mice per group. ∗, significantly different from wild-type counterparts (P < 0.05).