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Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo.

Richter-Dahlfors A, Buchan AM, Finlay BB - J. Exp. Med. (1997)

Bottom Line: Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively.These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease.This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Laboratory, University of British Columbia, Vancouver, B.C., Canada V6T 1Z3.

ABSTRACT
Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-micron-thick sections was used to detect bacteria after injection of approximately 100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

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Neutrophils are present in the foci at an early stage of infection, while macrophages dominate the tissue at later stages. Mouse liver  sections labeled with antineutrophil-FITC (purple), CD18-Cy5 (red), and  anti-Salmonella–TxR (green/light blue/yellow) in (a) uninfected liver, (b)  liver infected with 65 CFU SL1344 at day 3 after infection, and (c) the  same dose at day 5 after infection showing that neutrophils extravasate  into the liver as an early response, but disappear at a later stage of infection  when the foci consists of macrophages. The bacteria colocalize to the  phagocytes, not to the hepatocytes. Each image is a projection of an  8-μm z-stack collected through the ×20 objective on a Bio-Rad MRC-600 confocal microscope. Bar, 50 μm.
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Figure 4: Neutrophils are present in the foci at an early stage of infection, while macrophages dominate the tissue at later stages. Mouse liver sections labeled with antineutrophil-FITC (purple), CD18-Cy5 (red), and anti-Salmonella–TxR (green/light blue/yellow) in (a) uninfected liver, (b) liver infected with 65 CFU SL1344 at day 3 after infection, and (c) the same dose at day 5 after infection showing that neutrophils extravasate into the liver as an early response, but disappear at a later stage of infection when the foci consists of macrophages. The bacteria colocalize to the phagocytes, not to the hepatocytes. Each image is a projection of an 8-μm z-stack collected through the ×20 objective on a Bio-Rad MRC-600 confocal microscope. Bar, 50 μm.

Mentions: When an inflammatory response is triggered, neutrophils rapidly extravasate from the vasculature into the tissue and accumulate at the infectious foci (23, 24). It is not until the infection has progressed to later stages that the mononuclear cells begin to appear while the neutrophils disintegrate at the center of the lesions. The antibody RB6-8C5 recognizes a differentiation antigen specific for neutrophils (25) and has been used extensively in vivo to deplete mice of this cell type (24, 26–29). We used the FITC-tagged antibody RB6-8C5 (blue) in a triple labeling experiment together with CD18-Cy5 (red) and anti-Salmonella–TxR (green) to differentiate the neutrophils from the total leukocytes (CD18-positive) present at different times after infection. Fig. 4 a shows that there is an absence of neutrophils in the uninfected liver which contains resident macrophages (Kupffer cells) (red). Similar results were obtained at day 1 after infection. A few neutrophils were occasionally found in veins at day 2, but the liver parenchyma otherwise looked normal. At day 3 a marked infiltration of neutrophils into the tissue occurred and most of the CD18-positive foci consisted of centrally located neutrophils (Fig. 4 b). However, in some foci the neutrophils had already disintegrated and were replaced by mononuclear cells. By day 5, infiltration of mononuclear phagocytes into the lesions and disappearance of the neutrophils was complete, as all necrotic foci had transformed into granulomas (Fig. 4 c).


Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo.

Richter-Dahlfors A, Buchan AM, Finlay BB - J. Exp. Med. (1997)

Neutrophils are present in the foci at an early stage of infection, while macrophages dominate the tissue at later stages. Mouse liver  sections labeled with antineutrophil-FITC (purple), CD18-Cy5 (red), and  anti-Salmonella–TxR (green/light blue/yellow) in (a) uninfected liver, (b)  liver infected with 65 CFU SL1344 at day 3 after infection, and (c) the  same dose at day 5 after infection showing that neutrophils extravasate  into the liver as an early response, but disappear at a later stage of infection  when the foci consists of macrophages. The bacteria colocalize to the  phagocytes, not to the hepatocytes. Each image is a projection of an  8-μm z-stack collected through the ×20 objective on a Bio-Rad MRC-600 confocal microscope. Bar, 50 μm.
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Related In: Results  -  Collection

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Figure 4: Neutrophils are present in the foci at an early stage of infection, while macrophages dominate the tissue at later stages. Mouse liver sections labeled with antineutrophil-FITC (purple), CD18-Cy5 (red), and anti-Salmonella–TxR (green/light blue/yellow) in (a) uninfected liver, (b) liver infected with 65 CFU SL1344 at day 3 after infection, and (c) the same dose at day 5 after infection showing that neutrophils extravasate into the liver as an early response, but disappear at a later stage of infection when the foci consists of macrophages. The bacteria colocalize to the phagocytes, not to the hepatocytes. Each image is a projection of an 8-μm z-stack collected through the ×20 objective on a Bio-Rad MRC-600 confocal microscope. Bar, 50 μm.
Mentions: When an inflammatory response is triggered, neutrophils rapidly extravasate from the vasculature into the tissue and accumulate at the infectious foci (23, 24). It is not until the infection has progressed to later stages that the mononuclear cells begin to appear while the neutrophils disintegrate at the center of the lesions. The antibody RB6-8C5 recognizes a differentiation antigen specific for neutrophils (25) and has been used extensively in vivo to deplete mice of this cell type (24, 26–29). We used the FITC-tagged antibody RB6-8C5 (blue) in a triple labeling experiment together with CD18-Cy5 (red) and anti-Salmonella–TxR (green) to differentiate the neutrophils from the total leukocytes (CD18-positive) present at different times after infection. Fig. 4 a shows that there is an absence of neutrophils in the uninfected liver which contains resident macrophages (Kupffer cells) (red). Similar results were obtained at day 1 after infection. A few neutrophils were occasionally found in veins at day 2, but the liver parenchyma otherwise looked normal. At day 3 a marked infiltration of neutrophils into the tissue occurred and most of the CD18-positive foci consisted of centrally located neutrophils (Fig. 4 b). However, in some foci the neutrophils had already disintegrated and were replaced by mononuclear cells. By day 5, infiltration of mononuclear phagocytes into the lesions and disappearance of the neutrophils was complete, as all necrotic foci had transformed into granulomas (Fig. 4 c).

Bottom Line: Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively.These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease.This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Laboratory, University of British Columbia, Vancouver, B.C., Canada V6T 1Z3.

ABSTRACT
Salmonella typhimurium is considered a facultative intracellular pathogen, but its intracellular location in vivo has not been demonstrated conclusively. Here we describe the development of a new method to study the course of the histopathological processes associated with murine salmonellosis using confocal laser scanning microscopy of immunostained sections of mouse liver. Confocal microscopy of 30-micron-thick sections was used to detect bacteria after injection of approximately 100 CFU of S. typhimurium SL1344 intravenously into BALB/c mice, allowing salmonellosis to be studied in the murine model using more realistic small infectious doses. The appearance of bacteria in the mouse liver coincided in time and location with the infiltration of neutrophils in inflammatory foci. At later stages of disease the bacteria colocalized with macrophages and resided intracellularly inside these macrophages. Bacteria were cytotoxic for phagocytic cells, and apoptotic nuclei were detected immunofluorescently, whether phagocytes harbored intracellular bacteria or not. These data argue that Salmonella resides intracellularly inside macrophages in the liver and triggers cell death of phagocytes, processes which are involved in disease. This method is also applicable to other virulence models to examine infections at a cellular and subcellular level in vivo.

Show MeSH
Related in: MedlinePlus