Distinct Leishmania braziliensis isolates induce different paces of chemokine expression patterns

Abstract

Inflammatory events during Leishmania braziliensis infection in mice were investigated. Large lesions were directly correlated with the inflammatory reaction but not with parasite burden. Different L. braziliensis strains induce different paces of chemokine expression patterns, leading to diverse cell recruitment and differential inflammatory responses.

FIG. 1.

Course of infection with L. braziliensis in BALB/c mice. The inset shows kinetics of lesion development in BALB/c mice during the course of infection with six L. braziliensis isolates. The main figure illustrates the time course of infection with the two isolates used here, showing a polar pattern of infection. Mice were inoculated in the hind footpads with 10⁶ stationary-phase L. braziliensis promastigotes, and lesions were measured weekly for 30 days p.i. The footpads of three to five animals per group were measured. The data shown, reported as the mean and standard error of the mean, are from a single experiment representative of three separate experiments. The asterisk indicates a significant difference between values at the indicated time point, as determined by Student's t test (P < 0.05). Experiments with all L. braziliensis isolates were repeated three times, with similar results.

FIG. 2.

Kinetics of leukocyte recruitment (A), expressed as total numbers of neutrophils, macrophages, eosinophils, and lymphocytes, in pouch exudates in response to lipopolysaccharide (LPS) or L. braziliensis (BA788 and H3227) and comparison of results at 12 h after inoculation (B). Air pouches were prepared by injecting 3 ml of air into the dorsal surface of mice under light anesthesia. Stationary-phase L. braziliensis promastigotes (10⁷) were injected immediately following the air injection. Control mice were injected with endotoxin-free saline (negative control) and LPS (20 μg/ml; positive control). Mice were killed at the indicated time points, and the pouch contents were washed several time with saline. Exudate cells were centrifuged and stained; proportions of neutrophils, macrophages, eosinophils, and lymphocytes/200 cells were enumerated; and relative cell numbers were calculated from the total number of exudate leukocytes. Data represent the mean and standard error of the mean for three to five mice. The asterisk indicates a significant difference between values at the indicated time point, as determined by Student's t test or one-way analysis of variance (P < 0.05). Results are representative of two independent experiments.

FIG. 3.

Chemokine (A) and chemokine receptor (B) mRNA expression and protein production, determined by immunohistochemical analysis (C), in lesions of L. braziliensis-infected BALB/c mice. (A and B) Mice were infected with 10⁶ H3227 or BA788 promastigotes and killed at 6 h, 3 days, and 15 days p.i. The infected hind footpads were used in assays of mRNA expression by reverse transcription-PCR. Densitometric analysis was performed, and quantification was normalized to the levels of β-actin expression. Results are expressed as n-fold increases over results obtained with uninfected control animals (0 h). Upper and lower rows in the gels show the expression of chemokines and β-actin, respectively, at 0 h, 6 h, 3 days, and 15 days p.i. (lanes from left to right). The profiles are representative of at least three independent experiments. In each experiment, mRNA was prepared from pools of three or four mice per time point. Each point in the graphs represents the mean and standard error of the mean for a pool of three or four mice per time point in three experiments. CCL5/RANTES, CXCL9/MIG, and CCL22/MDC expression did not show significant modulation in this model of L. braziliensis infection (data not shown). (C) Frozen 5-μm sections of infected and uninfected foot tissues were used to perform immunohistochemical analysis for chemokines. Immunoperoxidase staining clearly showed at 3 days p.i. strong expression of CCL2/MCP-1 in H3227-infected sections (a) and weak expression in BA788-infected sections (b). Strong anti-CXCL10/IP-10 immunoreactivity was seen in sections of BA788-infected mice (d) but not in sections of H3227-infected mice (c). Magnification, ×34.

FIG. 3.

Chemokine (A) and chemokine receptor (B) mRNA expression and protein production, determined by immunohistochemical analysis (C), in lesions of L. braziliensis-infected BALB/c mice. (A and B) Mice were infected with 10⁶ H3227 or BA788 promastigotes and killed at 6 h, 3 days, and 15 days p.i. The infected hind footpads were used in assays of mRNA expression by reverse transcription-PCR. Densitometric analysis was performed, and quantification was normalized to the levels of β-actin expression. Results are expressed as n-fold increases over results obtained with uninfected control animals (0 h). Upper and lower rows in the gels show the expression of chemokines and β-actin, respectively, at 0 h, 6 h, 3 days, and 15 days p.i. (lanes from left to right). The profiles are representative of at least three independent experiments. In each experiment, mRNA was prepared from pools of three or four mice per time point. Each point in the graphs represents the mean and standard error of the mean for a pool of three or four mice per time point in three experiments. CCL5/RANTES, CXCL9/MIG, and CCL22/MDC expression did not show significant modulation in this model of L. braziliensis infection (data not shown). (C) Frozen 5-μm sections of infected and uninfected foot tissues were used to perform immunohistochemical analysis for chemokines. Immunoperoxidase staining clearly showed at 3 days p.i. strong expression of CCL2/MCP-1 in H3227-infected sections (a) and weak expression in BA788-infected sections (b). Strong anti-CXCL10/IP-10 immunoreactivity was seen in sections of BA788-infected mice (d) but not in sections of H3227-infected mice (c). Magnification, ×34.