The interactions and essential effects of intrinsic insulin-like growth factor-I on Leishmania (Leishmania) major growth within macrophages

Abstract

Previously, we showed in Leishmania infections that extrinsic insulin-like growth factor (IGF)-I favored Leishmania proliferation and leishmaniasis development. In this study, the interaction of intrinsically expressed IGF-I and Leishmania (Leishmania) major in macrophages was addressed, and a key finding was the observation, using confocal microscopy, of the co-localization of IGF-I and parasites within macrophages. Following stimulation with interferon-γ (IFN-γ), which is known to inhibit IGF-I production in macrophages, we observed a reduction in the expression of both IGF-I mRNA and protein. This reduced expression was accompanied by a reduction in the cellular parasite load that was completely recovered with the addition of extrinsic IGF-I, which suggests an essential role for IGF-I in Leishmania growth.

Keywords: Interferon-γ; Leishmania (Leishmania) major; confocal microscopy; insulin-like growth factor-I; macrophage.

Figure 1

Detection of IGF-I within RAW 264·7 macrophages following infection with Leishmania major promastigotes. Co-localization of IGF-I and Leishmania was measured using immunofluorescence. Anti-IGF-I antibody (recognized by the secondary antibody AlexaFluor-546, shown in red) and anti-Leishmania antibody (recognized by the antibody AlexaFluor-488, shown in green) were used to label cells (a, b). Free L. major promastigote culture immunofluorescence was measured using anti-IGF-I and the secondary antibody AlexaFluor-546 (c). No IGF-I staining was observed (red). 4′,6-diamidino-2-phenylindole (DAPI, shown in blue) was used to stain nuclei. Images were captured with a Leica LSM510 confocal microscope with a 63× objective and oil immersion.

Figure 2

Parasitism, nitric oxide (NO) production, IGF-I mRNA and IGF-I protein expression in RAW 264·7 cells that were infected with Leishmania major promastigotes. (a) Parasitism (shown as the number of parasites per 100 cells), (b) NO as nitrite levels evaluated in culture supernatants and (c) IGF-I mRNA expression (calculated as the ratio in relation to the control group without stimulation, ) with and without IFN-γ (200 U/mL) stimulus and with recombinant IGF-I (rIGF-I, 50 ng/mL) stimulus during a 48 h incubation. The data shown are representative of three independent assays. (d, e) Detection of IGF-I by confocal microscopy, using anti-IGF-I antibody (recognized by the secondary antibody AlexaFluor-546, shown in red) and anti-Leishmania antibody (recognized by the secondary antibody AlexaFluor-488, shown in green). 4′,6-diamidino-2-phenylindole (DAPI, shown in blue) was used to stain the nuclei. Images were captured using a confocal Leica LSM510 confocal microscope with a 63× objective and oil immersion. *P < 0·05 (anova and Tukey's test) in relation to the control group. **P < 0·05 (anova and Tukey's test) in relation to the IFN-γ group.