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Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Beattie L, Peltan A, Maroof A, Kirby A, Brown N, Coles M, Smith DF, Kaye PM - PLoS Pathog. (2010)

Bottom Line: Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens.Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells.This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Centre for Immunology and Infection, Hull York Medical School, University of York, York, United Kingdom.

ABSTRACT
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

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Kupffer Cell redistribution as a result of L. donovani infection.A) Whole mount immuno-histochemistry showing the distribution of F4/80+ (green) KCs in naïve (1 unit  = 23.3 µm) and B) L. donovani infected liver (1 unit  = 19.5 µm). C) Image from A) demonstrating the method used to determine the volume of F4/80 positive cells with Volocity software. D) Comparison of cell volumes of single F4/80+ cells in naïve and non-granuloma associated KC in infected livers (mean ± SEM). E) Hepatic mononuclear cell preparations showing 4 populations of cells based on expression of F4/80 and CD11c from naïve and F) day 14 infected mice. G–J) expression of MHCII on the surface of R1 (G), R2 (H), R3 (I) and R4 (J) populations from naïve (black lines) and infected (red lines) mice. Data is representative of 21 replicates from 5 individual mice.
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ppat-1000805-g002: Kupffer Cell redistribution as a result of L. donovani infection.A) Whole mount immuno-histochemistry showing the distribution of F4/80+ (green) KCs in naïve (1 unit  = 23.3 µm) and B) L. donovani infected liver (1 unit  = 19.5 µm). C) Image from A) demonstrating the method used to determine the volume of F4/80 positive cells with Volocity software. D) Comparison of cell volumes of single F4/80+ cells in naïve and non-granuloma associated KC in infected livers (mean ± SEM). E) Hepatic mononuclear cell preparations showing 4 populations of cells based on expression of F4/80 and CD11c from naïve and F) day 14 infected mice. G–J) expression of MHCII on the surface of R1 (G), R2 (H), R3 (I) and R4 (J) populations from naïve (black lines) and infected (red lines) mice. Data is representative of 21 replicates from 5 individual mice.

Mentions: Although macrophages are acknowledged to be a central feature of granulomatous inflammation, the precise origin of these cells has not been directly determined. To address this issue, we first studied the distribution of liver resident and inflammatory phagocytes in naïve and L. donovani-infected mice. KCs in the liver of uninfected mice show a characteristically uniform distribution, lining the sinusoids and forming a reticular surveillance network [34]. To more fully determine the spatial context in which KCs line the sinusoids, we performed whole mount immuno-histochemistry, using F4/80 as a marker of mature KCs (Figure 2). In naïve mice, large KCs with extensive projections were readily apparent within sinusoidal spaces (Figure 2A, and Video S1) forming a regular uniformly distributed phagocytic network. In contrast, in mice infected for 14 days with L. donovani, many KCs were aggregated within granulomas, leaving large areas of the sinusoidal network devoid of detectable KCs (Figure 2B and Video S1). Strikingly, although not participating in the granulomatous inflammatory response, KCs that remained isolated within the sinusoidal network nevertheless displayed morphological changes, which could be quantified as a reduced total cell volume compared to KCs in uninfected mice (Figure 2C, D). Although losing the spatial information provided by whole mount immunohistochemistry, we isolated hepatic mononuclear cells and labeled with F4/80 and CD11c to identify four populations of cells in both naive (Figure 2E) and L. donovani infected (Figure 2F) livers. While all four populations were present in both naïve and infected mice, the proportions changed with infection. CD11c−F4/80− cells (Figure 2E and F, R1) accounted for 51.7+/− 5.13% of F4/80+ cells in naïve mice and 38.88 +/− 4.34% in infected mice. CD11chiF4/80int cells (Figure 2E and F, R2) accounted for 17.11 +/− 3.12% in naïve mice and 19.29 +/− 3.31% in infected mice. CD11chiF4/80hi cells (Figure 2E and F, R3) accounted for 13.39 +/− 2.51% in naïve mice and 13.5 +/− 2.96% in infected mice. Finally, CD11cintF4/80int cells (Figure 2E and F, R4) accounted for 7.83 +/− 0.87% in naïve mice and 14.67 +/− 4.82% in infected mice. MHCII expression, used as a surrogate marker for macrophage activation, was shown to be upregulated on all four populations upon infection (Figure 2G–J). These data suggest that most KCs in the infected liver, even if not recruited into granulomas, had responded to the developing inflammatory environment.


Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Beattie L, Peltan A, Maroof A, Kirby A, Brown N, Coles M, Smith DF, Kaye PM - PLoS Pathog. (2010)

Kupffer Cell redistribution as a result of L. donovani infection.A) Whole mount immuno-histochemistry showing the distribution of F4/80+ (green) KCs in naïve (1 unit  = 23.3 µm) and B) L. donovani infected liver (1 unit  = 19.5 µm). C) Image from A) demonstrating the method used to determine the volume of F4/80 positive cells with Volocity software. D) Comparison of cell volumes of single F4/80+ cells in naïve and non-granuloma associated KC in infected livers (mean ± SEM). E) Hepatic mononuclear cell preparations showing 4 populations of cells based on expression of F4/80 and CD11c from naïve and F) day 14 infected mice. G–J) expression of MHCII on the surface of R1 (G), R2 (H), R3 (I) and R4 (J) populations from naïve (black lines) and infected (red lines) mice. Data is representative of 21 replicates from 5 individual mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837408&req=5

ppat-1000805-g002: Kupffer Cell redistribution as a result of L. donovani infection.A) Whole mount immuno-histochemistry showing the distribution of F4/80+ (green) KCs in naïve (1 unit  = 23.3 µm) and B) L. donovani infected liver (1 unit  = 19.5 µm). C) Image from A) demonstrating the method used to determine the volume of F4/80 positive cells with Volocity software. D) Comparison of cell volumes of single F4/80+ cells in naïve and non-granuloma associated KC in infected livers (mean ± SEM). E) Hepatic mononuclear cell preparations showing 4 populations of cells based on expression of F4/80 and CD11c from naïve and F) day 14 infected mice. G–J) expression of MHCII on the surface of R1 (G), R2 (H), R3 (I) and R4 (J) populations from naïve (black lines) and infected (red lines) mice. Data is representative of 21 replicates from 5 individual mice.
Mentions: Although macrophages are acknowledged to be a central feature of granulomatous inflammation, the precise origin of these cells has not been directly determined. To address this issue, we first studied the distribution of liver resident and inflammatory phagocytes in naïve and L. donovani-infected mice. KCs in the liver of uninfected mice show a characteristically uniform distribution, lining the sinusoids and forming a reticular surveillance network [34]. To more fully determine the spatial context in which KCs line the sinusoids, we performed whole mount immuno-histochemistry, using F4/80 as a marker of mature KCs (Figure 2). In naïve mice, large KCs with extensive projections were readily apparent within sinusoidal spaces (Figure 2A, and Video S1) forming a regular uniformly distributed phagocytic network. In contrast, in mice infected for 14 days with L. donovani, many KCs were aggregated within granulomas, leaving large areas of the sinusoidal network devoid of detectable KCs (Figure 2B and Video S1). Strikingly, although not participating in the granulomatous inflammatory response, KCs that remained isolated within the sinusoidal network nevertheless displayed morphological changes, which could be quantified as a reduced total cell volume compared to KCs in uninfected mice (Figure 2C, D). Although losing the spatial information provided by whole mount immunohistochemistry, we isolated hepatic mononuclear cells and labeled with F4/80 and CD11c to identify four populations of cells in both naive (Figure 2E) and L. donovani infected (Figure 2F) livers. While all four populations were present in both naïve and infected mice, the proportions changed with infection. CD11c−F4/80− cells (Figure 2E and F, R1) accounted for 51.7+/− 5.13% of F4/80+ cells in naïve mice and 38.88 +/− 4.34% in infected mice. CD11chiF4/80int cells (Figure 2E and F, R2) accounted for 17.11 +/− 3.12% in naïve mice and 19.29 +/− 3.31% in infected mice. CD11chiF4/80hi cells (Figure 2E and F, R3) accounted for 13.39 +/− 2.51% in naïve mice and 13.5 +/− 2.96% in infected mice. Finally, CD11cintF4/80int cells (Figure 2E and F, R4) accounted for 7.83 +/− 0.87% in naïve mice and 14.67 +/− 4.82% in infected mice. MHCII expression, used as a surrogate marker for macrophage activation, was shown to be upregulated on all four populations upon infection (Figure 2G–J). These data suggest that most KCs in the infected liver, even if not recruited into granulomas, had responded to the developing inflammatory environment.

Bottom Line: Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens.Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells.This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Centre for Immunology and Infection, Hull York Medical School, University of York, York, United Kingdom.

ABSTRACT
Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Show MeSH
Related in: MedlinePlus