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Downmodulation of the inflammatory response to bacterial infection by gammadelta T cells cytotoxic for activated macrophages.

Egan PJ, Carding SR - J. Exp. Med. (2000)

Bottom Line: Furthermore, primary splenic gammadelta T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the gammadelta TCR.These results demonstrate a novel interaction between gammadelta T cells and activated macrophages in which gammadelta T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death.This interaction suggests that gammadelta T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Although gammadelta T cells are involved in the regulation of inflammation after infection, their precise function is not known. Intraperitoneal infection of T cell receptor (TCR)-delta(-/-) mice with the intracellular bacterium Listeria monocytogenes resulted in the development of necrotic foci in the livers. In contrast, the peritoneal cavities of infected TCR-delta(-/-) mice contained an accumulation of low density activated macrophages and a reduced percentage of macrophages undergoing apoptosis. gammadelta T cell hybridomas derived from mice infected with Listeria were preferentially stimulated by low density macrophages from peritoneal exudates of infected mice. Furthermore, primary splenic gammadelta T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the gammadelta TCR. These results demonstrate a novel interaction between gammadelta T cells and activated macrophages in which gammadelta T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death. This interaction suggests that gammadelta T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response.

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Stimulation of the γδ T cell hybridoma BC10 by PEC fractions from day 6 Listeria-infected mice. 5 × 104 BC10 cells were labeled with CFSE, cultured with 105 cells from each of the PEC fractions, isolated from wild-type mice, for 48 h, incubated with monensin for 4 h, and then stained for intracellular IFN-γ before flow cytometry. (A) Flow cytometric analysis of BC10 cells after culture with PEC fractions. Light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The percentage values shown for the histogram plots represent the frequency of IFN-γ–producing cells determined by comparing the level of intracellular staining obtained with an isotype-matched control antibody (dark solid lines and open plots) and the anti–IFN-γ antibody (light solid lines and filled-in plots). Similar results were obtained using nine other hybridoma lines. (B) IFN-γ–producing hybridoma cells exhibit blast-like morphology. Electronically gated CFSE+ cells were analyzed for IFN-γ synthesis, and the light scatter properties of cells that do (R2) or do not (R1) produce IFN-γ were compared. Note the uniform increase in granularity (SSC) and size (FSC) of IFN-γ–producing cells (R2). The results shown are representative of those obtained from six independent experiments. (C) Flow cytometric analysis of BC10 hybridomas after culture with PECs from day 3 or 9 Listeria-infected wild-type mice. Hybridomas were labeled with CFSE before culture, and light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The proportion of hybridomas with increased FSC and SSC after culture with PECs is shown in the gated population. The results shown are typical of those obtained from >10 independent experiments.
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Figure 4: Stimulation of the γδ T cell hybridoma BC10 by PEC fractions from day 6 Listeria-infected mice. 5 × 104 BC10 cells were labeled with CFSE, cultured with 105 cells from each of the PEC fractions, isolated from wild-type mice, for 48 h, incubated with monensin for 4 h, and then stained for intracellular IFN-γ before flow cytometry. (A) Flow cytometric analysis of BC10 cells after culture with PEC fractions. Light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The percentage values shown for the histogram plots represent the frequency of IFN-γ–producing cells determined by comparing the level of intracellular staining obtained with an isotype-matched control antibody (dark solid lines and open plots) and the anti–IFN-γ antibody (light solid lines and filled-in plots). Similar results were obtained using nine other hybridoma lines. (B) IFN-γ–producing hybridoma cells exhibit blast-like morphology. Electronically gated CFSE+ cells were analyzed for IFN-γ synthesis, and the light scatter properties of cells that do (R2) or do not (R1) produce IFN-γ were compared. Note the uniform increase in granularity (SSC) and size (FSC) of IFN-γ–producing cells (R2). The results shown are representative of those obtained from six independent experiments. (C) Flow cytometric analysis of BC10 hybridomas after culture with PECs from day 3 or 9 Listeria-infected wild-type mice. Hybridomas were labeled with CFSE before culture, and light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The proportion of hybridomas with increased FSC and SSC after culture with PECs is shown in the gated population. The results shown are typical of those obtained from >10 independent experiments.

Mentions: Representative data, obtained with the BALB/c-derived hybridoma BC10, is shown in Fig. 4, although similar results were obtained with nine other hybridomas tested. Hybridomas cultured in the absence of peritoneal cells (Fig. 4 A) or with any of the fractions isolated from naive, noninfected mouse cells did not produce IFN-γ. In the presence of nonseparated PECs from day 6 infected wild-type mice, however, 11% of the hybridoma cells stained positive for IFN-γ. The percentage of IFN-γ–producing hybridomas was further increased to 22% in the presence of the 43% Percoll fraction of PECs, whereas the percentages of IFN-γ–positive hybridomas declined to 8 and 4% in the presence of 48 and 58% Percoll fractions, respectively. By contrast, higher density Percoll fractions were not stimulatory for any of the hybridomas tested. IFN-γ production was dependent upon direct cell–cell contact, as it was not possible to detect any hybridomas stained positive for IFN-γ after transwell culture in which hybridoma cells and PECs were cultured in individual chambers separated by a porous membrane (data not shown). Although the frequency of IFN-γ–secreting γδ T cells appears to be low, when the proportion of hybridoma cells that have spontaneously lost surface expression of the CD3–TCR complex (20–30%) is taken into account, the frequency of responding (TCR+) cells is higher (≥50%). Importantly, IFN-γ synthesis was restricted to TCRVδ6.3+ hybridoma cells, as it was not possible to detect any cytokine synthesis by TCR− variants isolated from the same cultures or by the parent line BW 5197 TCR-α−β− (data not shown).


Downmodulation of the inflammatory response to bacterial infection by gammadelta T cells cytotoxic for activated macrophages.

Egan PJ, Carding SR - J. Exp. Med. (2000)

Stimulation of the γδ T cell hybridoma BC10 by PEC fractions from day 6 Listeria-infected mice. 5 × 104 BC10 cells were labeled with CFSE, cultured with 105 cells from each of the PEC fractions, isolated from wild-type mice, for 48 h, incubated with monensin for 4 h, and then stained for intracellular IFN-γ before flow cytometry. (A) Flow cytometric analysis of BC10 cells after culture with PEC fractions. Light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The percentage values shown for the histogram plots represent the frequency of IFN-γ–producing cells determined by comparing the level of intracellular staining obtained with an isotype-matched control antibody (dark solid lines and open plots) and the anti–IFN-γ antibody (light solid lines and filled-in plots). Similar results were obtained using nine other hybridoma lines. (B) IFN-γ–producing hybridoma cells exhibit blast-like morphology. Electronically gated CFSE+ cells were analyzed for IFN-γ synthesis, and the light scatter properties of cells that do (R2) or do not (R1) produce IFN-γ were compared. Note the uniform increase in granularity (SSC) and size (FSC) of IFN-γ–producing cells (R2). The results shown are representative of those obtained from six independent experiments. (C) Flow cytometric analysis of BC10 hybridomas after culture with PECs from day 3 or 9 Listeria-infected wild-type mice. Hybridomas were labeled with CFSE before culture, and light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The proportion of hybridomas with increased FSC and SSC after culture with PECs is shown in the gated population. The results shown are typical of those obtained from >10 independent experiments.
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Related In: Results  -  Collection

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Figure 4: Stimulation of the γδ T cell hybridoma BC10 by PEC fractions from day 6 Listeria-infected mice. 5 × 104 BC10 cells were labeled with CFSE, cultured with 105 cells from each of the PEC fractions, isolated from wild-type mice, for 48 h, incubated with monensin for 4 h, and then stained for intracellular IFN-γ before flow cytometry. (A) Flow cytometric analysis of BC10 cells after culture with PEC fractions. Light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The percentage values shown for the histogram plots represent the frequency of IFN-γ–producing cells determined by comparing the level of intracellular staining obtained with an isotype-matched control antibody (dark solid lines and open plots) and the anti–IFN-γ antibody (light solid lines and filled-in plots). Similar results were obtained using nine other hybridoma lines. (B) IFN-γ–producing hybridoma cells exhibit blast-like morphology. Electronically gated CFSE+ cells were analyzed for IFN-γ synthesis, and the light scatter properties of cells that do (R2) or do not (R1) produce IFN-γ were compared. Note the uniform increase in granularity (SSC) and size (FSC) of IFN-γ–producing cells (R2). The results shown are representative of those obtained from six independent experiments. (C) Flow cytometric analysis of BC10 hybridomas after culture with PECs from day 3 or 9 Listeria-infected wild-type mice. Hybridomas were labeled with CFSE before culture, and light scatter properties and synthesis of IFN-γ by γδ T cell hybridomas was determined by electronically gating on CFSE+ cells. The proportion of hybridomas with increased FSC and SSC after culture with PECs is shown in the gated population. The results shown are typical of those obtained from >10 independent experiments.
Mentions: Representative data, obtained with the BALB/c-derived hybridoma BC10, is shown in Fig. 4, although similar results were obtained with nine other hybridomas tested. Hybridomas cultured in the absence of peritoneal cells (Fig. 4 A) or with any of the fractions isolated from naive, noninfected mouse cells did not produce IFN-γ. In the presence of nonseparated PECs from day 6 infected wild-type mice, however, 11% of the hybridoma cells stained positive for IFN-γ. The percentage of IFN-γ–producing hybridomas was further increased to 22% in the presence of the 43% Percoll fraction of PECs, whereas the percentages of IFN-γ–positive hybridomas declined to 8 and 4% in the presence of 48 and 58% Percoll fractions, respectively. By contrast, higher density Percoll fractions were not stimulatory for any of the hybridomas tested. IFN-γ production was dependent upon direct cell–cell contact, as it was not possible to detect any hybridomas stained positive for IFN-γ after transwell culture in which hybridoma cells and PECs were cultured in individual chambers separated by a porous membrane (data not shown). Although the frequency of IFN-γ–secreting γδ T cells appears to be low, when the proportion of hybridoma cells that have spontaneously lost surface expression of the CD3–TCR complex (20–30%) is taken into account, the frequency of responding (TCR+) cells is higher (≥50%). Importantly, IFN-γ synthesis was restricted to TCRVδ6.3+ hybridoma cells, as it was not possible to detect any cytokine synthesis by TCR− variants isolated from the same cultures or by the parent line BW 5197 TCR-α−β− (data not shown).

Bottom Line: Furthermore, primary splenic gammadelta T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the gammadelta TCR.These results demonstrate a novel interaction between gammadelta T cells and activated macrophages in which gammadelta T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death.This interaction suggests that gammadelta T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Although gammadelta T cells are involved in the regulation of inflammation after infection, their precise function is not known. Intraperitoneal infection of T cell receptor (TCR)-delta(-/-) mice with the intracellular bacterium Listeria monocytogenes resulted in the development of necrotic foci in the livers. In contrast, the peritoneal cavities of infected TCR-delta(-/-) mice contained an accumulation of low density activated macrophages and a reduced percentage of macrophages undergoing apoptosis. gammadelta T cell hybridomas derived from mice infected with Listeria were preferentially stimulated by low density macrophages from peritoneal exudates of infected mice. Furthermore, primary splenic gammadelta T cells isolated from Listeria-infected mice were cytotoxic for low density macrophages in vitro, and cytotoxicity was inhibited in the presence of antibodies to the gammadelta TCR. These results demonstrate a novel interaction between gammadelta T cells and activated macrophages in which gammadelta T cells are stimulated by terminally differentiated macrophages to acquire cytotoxic activity and which, in turn, induce macrophage cell death. This interaction suggests that gammadelta T cells regulate the inflammatory response to infection with intracellular pathogens by eliminating activated macrophages at the termination of the response.

Show MeSH
Related in: MedlinePlus