Chemokine production and leukocyte recruitment to the lungs of Paracoccidioides brasiliensis-infected mice is modulated by interferon-gamma

Abstract

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.

Figure 1.

Lack of IFN-γ alters the leukocyte subsets infiltrating the lungs of P. brasiliensis-infected mice. Lung leukocytes from uninfected (day 0) and infected WT and GKO mice on days 3, 7, and 14 were obtained by mechanical and enzymatic dispersion. The total cell number (A) was determined in hemacytometer chamber and the differential leukocyte counts (B, C, D) was done after Rosenfeld staining of samples cytospun onto slides. Results are means ± SD of triplicate counts from three mice per group, and are representative of five independent experiments. A: *P ≤ 0.05 compared with non-infected mice. B and D: *P ≤ 0.05 compared with infected GKO mice. C: *P ≤ 0.05 compared with infected WT mice.

Figure 2.

Absence of IFN-γ induces chronic polymorphonuclear cell infiltration in the lung of P. brasiliensis-infected mice. Lung leukocytes obtained from Pb18-infected WT and GKO mice at day 14 were immunostained with fluorescein isothiocyanate-conjugated antibody to mouse Mac-1 or phycoerythrin-conjugated antibody to mouse GR-1 and analyzed by flow cytometry. Results are the means ± SD of the number of each leukocyte subset from three mice per group and are representative of three independent experiments. *P ≤ 0.05 compared with infected WT animals and **P ≤ 0.05 compared with non-infected WT mice.

Figure 3.

Chemokines are differentially expressed in the lungs of P. brasiliensis-infected WT and GKO mice. The expression of RANTES, MIP-1α, MCP-1, and KC was evaluated by RT-PCR in the lungs of Pb18-infected WT and GKO mice at different times after the infection. The amplification products were separated by electrophoresis in an acrylamide gel and silver-stained. These data are representative of three independent experiments.

Figure 4.

IFN-γ regulates the chemokine production in the lungs of P. brasiliensis-infected mice. The levels of RANTES (A), MCP-1 (B), MIP-1α (C), and KC (D) were determined by ELISA in lung homogenates of WT and GKO mice on days 7 and 14 of infection and in non-infected mice (zero). Data represent the means ± SD of three animals for each group at each time point. *P ≤ 0.05 compared with infected GKO animals and **P ≤ 0.05 compared with infected WT mice.

Figure 5.

Paracoccidioides brasiliensis induces a Th1 chemokines and chemokine receptor patterns in the lungs of infected mice. Lungs of WT and GKO mice were harvested at different times after Pb18 infection and total RNA was extracted with Trizol reagent. The mRNA expression of chemokines and chemokine receptors was evaluated by RT-PCR, the amplification products were separated by electrophoresis in acrylamide gel and silver-stained. These data are representative of three independent experiments.

Figure 6.

Lungs of P. brasiliensis-infected mice preferentially produce chemokine chemoattractants of Th1 lymphocytes. Frozen sections of lungs harvested on day 14 of infection were fixed in acetone and immunostained with antibodies against Mig, IP-10, CCR3, and CCR5 (green fluorescence) and the T cell marker CD90.2 (Thy1.2, red fluorescence). The double-stained is yellow (magnification, ×400).

Figure 7.

Migratory response of leukocytes from P. brasiliensis-infected mice was regulated by IFN-γ. Lung cells of WT and GKO on day 14 of Pb18 infection were added to the upper chamber of transmigration (trans-well) system in contact with serum-free medium, IP-10 (100 ng/ml), eotaxin (100 ng/ml), or fMLP (10⁻⁸M) in the lower compartment. The plate was incubated for 3 hours in a 5% CO₂ incubator at 37°C and the number of transmigrating cells was determined on hemacytometer chamber. These data are representative of three independent experiments. *P ≤ 0.05 compared with cells from infected GKO or non-infected WT and GKO mice.