Nitric oxide participation in the fungicidal mechanism of gamma interferon-activated murine macrophages against Paracoccidioides brasiliensis conidia

Abstract

Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungus Paracoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form. The macrophage (Mphi) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-gamma), murine Mphis kill the fungus by an oxygen-independent mechanism. Our goal was to determine the role of nitric oxide in the fungicidal effect of Mphis on P. brasiliensis conidia. The results revealed that IFN-gamma-activated murine Mphis inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mphis activated with 50 U/ml and incubated for 96 h at 37 degrees C. When Mphis were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect. The inhibitory effect was reversed by the addition of anti-IFN-gamma monoclonal antibodies. Activation by IFN-gamma also enhanced Mphi nitric oxide production, as revealed by increasing NO(2) values (8 +/- 3 microM in nonactivated Mphis versus 43 +/- 13 microM in activated Mphis). The neutralization of IFN-gamma also reversed nitric oxide production at basal levels (8 +/- 5 microM). Additionally, we found that there was a significant inverse correlation (r = -0.8975) between NO(2)(-) concentration and transformation of P. brasiliensis conidia. Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase, N(G)-monomethyl-L-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production. These results show that IFN-gamma-activated murine Mphis kill P. brasiliensis conidia through the L-arginine-nitric oxide pathway.

FIG. 1

Morphology of intracellular P. brasiliensis. (A) Nontransformed conidium; (B) transformed multiple budding yeast cell. Magnification, ×1,000.

FIG. 2

Intracellular transformation from conidium to yeast. Mean transformation values for intracellular P. brasiliensis conidia after 24, 48, 72, and 96 h (n = 6 experiments) are shown. Range bars represent standard deviation. ∗, significant (P < 0.05) increase after 48 to 96 h of coculture.

FIG. 3

Effect of rIFN-γ activation of Mφ on intracellular conidium-to-yeast transformation (A) and NO production by rIFN-γ-activated Mφs (B). (A) Mean transformation values of intracellular P. brasiliensis conidia after 96 h of coculture with murine Mφs activated with different concentrations of rIFN-γ (n = 6 experiments/concentration). Range bars represent standard deviation. ∗, significant decrease (P < 0.0001) compared to nonactivated Mφs (0 rIFN-γ). (B) Levels of NO₂⁻ production by murine Mφs activated with different concentrations of rIFN-γ and cocultured with P. brasiliensis conidia for 96 h (mean for six experiments). Range bars represent standard deviation. ∗, significant increase (P < 0.0001) in comparison to nonactivated-Mφs (0 rIFN-γ).

FIG. 4

Fungicidal activity of rIFN-γ-activated Mφs against P. brasiliensis conidia after 8 days of coculture (n = 4 experiments). Range bars represent standard deviation. ∗, significant increase (P < 0.001) versus nonactivated Mφs (0 rIFN-γ).

FIG. 5

Correlation between NO₂⁻ levels produced by rIFN-γ-activated Mφs and degree of conidium-to-yeast transformation. Data are from cocultures incubated for 96 h with or without IFN-γ (n = 55); r = −0.8975).

FIG. 6

Effect of addition of NO inhibitors to rIFN-γ-activated Mφs on intracellular conidium-to-yeast transformation (A) and NO production by rIFN-γ-activated Mφ (B). (A) Mean transformation values of intracellular P. brasiliensis conidia after 96 h of coculture with Mφs activated with 50 U of rIFN-γ per ml in the presence of different concentrations of ARG, AG, or LNMMA (n = 6 experiments/concentration). Range bars represent standard deviation. ∗, significant increase (P < 0.0005) compared to rIFN-γ-activated untreated Mφs (0 inhibitor). First columns (RPMI) represent the maximum transformation value observed inside nonactivated Mφs. (B) Levels of NO₂⁻ production by Mφs activated with different concentrations of rIFN-γ and cocultured with P. brasiliensis conidia for 96 h (mean for six experiments). Range bars represent standard deviation. ∗, significant increase (P < 0.0001) in comparison to nonactivated-Mφs (0 rIFN-γ).