T cells, adhesion molecules and modulation of apoptosis in visceral leishmaniasis glomerulonephritis

Abstract

Background: Immune complex deposition is the accepted mechanism of pathogenesis of VL glomerulopathy however other immune elements may participate. Further in the present study, no difference was seen between immunoglobulin and C3b deposit intensity in glomeruli between infected and non-infected dogs thus T cells, adhesion molecules and parameters of proliferation and apoptosis were analysed in dogs with naturally acquired VL from an endemic area. The dog is the most important domestic reservoir of the protozoa Leishmania (L.) chagasi that causes visceral leishmaniasis (VL). The similarity of VL manifestation in humans and dogs renders the study of canine VL nephropathy of interest with regard to human pathology.

Methods: From 55 dogs with VL and 8 control non-infected dogs from an endemic area, kidney samples were analyzed by immunohistochemistry for immunoglobulin and C3b deposits, staining for CD4+ and CD8+ T cells, ICAM-1, P-selectin and quantified using morphometry. Besides proliferation marker Ki-67, apoptosis markers M30 and TUNEL staining, and related cytokines TNF-alpha, IL-1alpha were searched and quantified.

Results: We observed similar IgG, IgM and IgA and C3b deposit intensity in dogs with VL and non-infected control dogs. However we detected the Leishmania antigen in cells in glomeruli in 54, CD4+ T cells in the glomeruli of 44, and CD8+ T cells in 17 of a total of 55 dogs with VL. Leishmania antigen was absent and T cells were absent/scarse in eight non-infected control dogs. CD 4+ T cells predominate in proliferative patterns of glomerulonephritis, however the presence of CD4+ and CD8+ T cells were not different in intensity in different patterns of glomerulonephritis. The expression of ICAM-1 and P-selectin was significantly greater in the glomeruli of infected dogs than in control dogs. In all patterns of glomerulonephritis the expression of ICAM-1 ranged from minimum to moderately severe and P-selectin from absent to severe. In the control animals the expression of these molecules ranged from absent to medium intensity. It was not observed any correlation between severity of the disease and these markers. There was a correlation between the number of Leishmania antigen positive cells and CD4+ T cells, and between the number of CD4+ T cells and CD8+ T cells. In dogs presenting different histopathological patterns of glomerulonephritis, parameters of proliferation and apoptosis were studied. Ki-67, a proliferative marker, was not detected locally, but fewer apoptotic cells and lower TNF-alpha expression were seen in infected animals than in non-infected controls.

Conclusion: Immunopathogenic mechanisms of VL glomerulonephritis are complex and data in the present study suggest no clear participation of immunoglobulin and C3b deposits in these dogs but the possible migration of CD4+ T cells into the glomeruli, participation of adhesion molecules, and diminished apoptosis of cells contributing to determine the proliferative pattern of glomerulonephritis in VL.

Figure 1

Immunoglobulin and C3b deposits in the glomeruli of dogs with visceral leishmaniasis. Intensity of IgG (A), IgM (B), IgA (C) and C3b (D) deposits in 21 dogs with visceral leishmaniasis and in five non-infected control dogs.

Figure 2

Leishmania antigen, CD4⁺ T cells and CD8⁺ T cells in the glomeruli in dogs with and without visceral leishmaniasis. Detection of Leishmania antigen (A), CD4⁺ T cells (B) and CD8⁺ T cells (C) in glomeruli in dogs with visceral leishmaniasis. Bar = 16 μm). Absence of staining of Leishmania antigen (D), CD4⁺ T cells (E) and CD8⁺ T cells (F) in glomeruli in non-infected control dogs. bar = 25 μm. Immunohistochemistry. Different molecules when present appear stained in brown.

Figure 3

Quantitative analysis of T cells and Leishmania antigen⁺ cells and their correlation in glomeruli in dogs with visceral leishmaniasis. (A) Number of CD4⁺ T cells in glomeruli in VL dogs with different patterns of GN, and non-infected control animals. (B) Number of CD8⁺ T cells in glomeruli in VL dogs with different patterns of GN, and non-infected control animals. (C) Number of Leishmania antigen⁺ cells in VL infected and non-infected control dogs by glomerulonephritis pattern. (D) Correlation between the number of CD4⁺ T cells and Leishmania antigen+ cells. (E) Correlation between the number of CD4⁺ T and CD8⁺ T cells.

Figure 4

Expression of adhesion molecules in the glomeruli in dogs with or without visceral leishmaniasis. Expression of ICAM-1 (A) and P-selectin (B) in glomeruli in canine visceral leishmaniasis, and of ICAM-1 (C) and P-selectin (D) in glomeruli in non-infected animals. Figures A and B. Bar = 16 μm. Figures C and D. Bar = 25 μm. Immunohistochemistry. Different molecules when present appear stained in brown.

Figure 5

Detection of apoptotic cells in the glomeruli in dogs with or without visceral leishmaniasis. Detection of cytodeath marker M30 in glomerular cells in non-infected animals (A), and in dogs with visceral leishmaniasis (B). TUNEL staining in glomerular cells in non-infected animals (C), and in dogs with visceral leishmaniasis (D) Figure A. Bar = 25 μm. Figures B, C and D. Bar = 16 μm. Different molecules when present appear stained in dark brown.

Figure 6

Quantitative analysis of expression of cytodeath marker M30 and its correlation with cell number and Leishmania antigen⁺ cells in the glomeruli in dogs with or without visceral leishmaniasis. (A) Number of M30⁺ cells in glomeruli in VL and non-infected dogs by glomerulonephritis pattern. (B) Correlation between the M30⁺ cells and Total number of cells per glomerulus. (C) Correlation between the M30⁺ cells and Leishmania antigen⁺ cells.

Figure 7

Expression of TNF-α and IL-1 in the glomeruli in dogs with or without visceral leishmaniasis. Expression of TNF-α in glomerular cells in non-infected dogs (A), and dogs with visceral leishmaniasis (B). Expression of IL-1α in glomerular cells in non-infected dogs (C), and in dogs with visceral leishmaniasis (D). Figures A and B. Bar = 25 μm. Figures C and D. Bar = 16 μm. Immunohistochemistry. Different molecules when present appear stained in brown.

Figure 8

Quantitative analysis of cells expressing TNF-α and its correlation with M30⁺ cells. (A) Number of cells expressing TNF-α in glomeruli in infected and in non-infected dogs. (B) Correlation between the number of cells expressing TNF-α and M30⁺cells.