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Peripheral blood CD8αα+CD11c+MHC-II+CD3- cells attenuate autoimmune glomerulonephritis in rats.

Wu J, Zhou C, Robertson J, Carlock C, Lou YH - Kidney Int. (2013)

Bottom Line: Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25).When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats.Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic and Biomedical Sciences, SD, University of Texas Health Science Center at Houston, Houston, Texas, USA.

ABSTRACT
In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.

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Spontaneous differentiation of PBMC CD8αα+CD3− cells into DC-like cells after a short-term in vitro culture(a) Phase-contrast micrographs show morphological changes in purified PBMC CD8αα+CD3− cells after culture as indicated. (b) Anti-CD8α antibody reveals dendrite-like cellular projections of PBMC CD8αα+CD3− cells after a 3-day culture. (c) Comparison of morphological changes between PBMC CD8αα+CD3− cells (red and green) and PBMC CD8αα−RT1B+ monocytes (M)(red); PBMC CD8αα+CD3− cells become flattened at 36hr, while a nearby monocyte remains spherical shaped. (d) Western blot shows expression of MHC II (RT1D) in PBMC CD8αα+CD3− cells in comparison to monocytes. (e) Intracellular RT1D (green) was demonstrated by confocal immunofluorescence after permeablization of the cells; the cells were co-stained for CD8α (red). A CD8+ T cell (asterisk) is shown as a negative control for RT1D staining. (f) Active synthesis of RT1D was detected by comparison between the cells before (0hr) and after Golgi blockage (6hr); an arrow shows an accumulation of RT1D in the cell. DIC, differential interference contrast. (g) Up-regulation of surface RT1D expression in PBMC CD8αα+CD3− cells after incubation with LPS as indicated. Bars = 10 µm.
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Figure 3: Spontaneous differentiation of PBMC CD8αα+CD3− cells into DC-like cells after a short-term in vitro culture(a) Phase-contrast micrographs show morphological changes in purified PBMC CD8αα+CD3− cells after culture as indicated. (b) Anti-CD8α antibody reveals dendrite-like cellular projections of PBMC CD8αα+CD3− cells after a 3-day culture. (c) Comparison of morphological changes between PBMC CD8αα+CD3− cells (red and green) and PBMC CD8αα−RT1B+ monocytes (M)(red); PBMC CD8αα+CD3− cells become flattened at 36hr, while a nearby monocyte remains spherical shaped. (d) Western blot shows expression of MHC II (RT1D) in PBMC CD8αα+CD3− cells in comparison to monocytes. (e) Intracellular RT1D (green) was demonstrated by confocal immunofluorescence after permeablization of the cells; the cells were co-stained for CD8α (red). A CD8+ T cell (asterisk) is shown as a negative control for RT1D staining. (f) Active synthesis of RT1D was detected by comparison between the cells before (0hr) and after Golgi blockage (6hr); an arrow shows an accumulation of RT1D in the cell. DIC, differential interference contrast. (g) Up-regulation of surface RT1D expression in PBMC CD8αα+CD3− cells after incubation with LPS as indicated. Bars = 10 µm.

Mentions: The PBMC CD8αα+CD3− cells’ morphology was examined. Immunofluorescence confirmed co-expression of cell surface RT1B and CD8α in those cells (Figure 2h). The PBMC CD8αα+CD3− cells had a polygonic nucleus, in contrast to a kidney-shaped nucleus in monocytes (Figure 2h). They could also be easily distinguished from CD8+ T cells or NK cells by either their size or shape (Figure 2h). Morphological changes in PBMC CD8αα+CD3− cells, along with both surface and intracellular MHC-II expression, were observed after in vitro culture in comparison to monocytes. Freshly isolated PBMC CD8αα+CD3− cells were spherical. Many cells flattened after 12–36 hrs’ culture, and became irregularly shaped with various cellular projections at 60 hrs (Figure 3a). Staining with CD8α antibody revealed fine cellular projections in majority of cells, which resembled those of DCs (Figure 3b), suggesting that PBMC CD8αα+ cells were a type of phagocyte. On the other hand, most monocytes remained spherically shaped at 36 hrs (Figure 3c).


Peripheral blood CD8αα+CD11c+MHC-II+CD3- cells attenuate autoimmune glomerulonephritis in rats.

Wu J, Zhou C, Robertson J, Carlock C, Lou YH - Kidney Int. (2013)

Spontaneous differentiation of PBMC CD8αα+CD3− cells into DC-like cells after a short-term in vitro culture(a) Phase-contrast micrographs show morphological changes in purified PBMC CD8αα+CD3− cells after culture as indicated. (b) Anti-CD8α antibody reveals dendrite-like cellular projections of PBMC CD8αα+CD3− cells after a 3-day culture. (c) Comparison of morphological changes between PBMC CD8αα+CD3− cells (red and green) and PBMC CD8αα−RT1B+ monocytes (M)(red); PBMC CD8αα+CD3− cells become flattened at 36hr, while a nearby monocyte remains spherical shaped. (d) Western blot shows expression of MHC II (RT1D) in PBMC CD8αα+CD3− cells in comparison to monocytes. (e) Intracellular RT1D (green) was demonstrated by confocal immunofluorescence after permeablization of the cells; the cells were co-stained for CD8α (red). A CD8+ T cell (asterisk) is shown as a negative control for RT1D staining. (f) Active synthesis of RT1D was detected by comparison between the cells before (0hr) and after Golgi blockage (6hr); an arrow shows an accumulation of RT1D in the cell. DIC, differential interference contrast. (g) Up-regulation of surface RT1D expression in PBMC CD8αα+CD3− cells after incubation with LPS as indicated. Bars = 10 µm.
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Related In: Results  -  Collection

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Figure 3: Spontaneous differentiation of PBMC CD8αα+CD3− cells into DC-like cells after a short-term in vitro culture(a) Phase-contrast micrographs show morphological changes in purified PBMC CD8αα+CD3− cells after culture as indicated. (b) Anti-CD8α antibody reveals dendrite-like cellular projections of PBMC CD8αα+CD3− cells after a 3-day culture. (c) Comparison of morphological changes between PBMC CD8αα+CD3− cells (red and green) and PBMC CD8αα−RT1B+ monocytes (M)(red); PBMC CD8αα+CD3− cells become flattened at 36hr, while a nearby monocyte remains spherical shaped. (d) Western blot shows expression of MHC II (RT1D) in PBMC CD8αα+CD3− cells in comparison to monocytes. (e) Intracellular RT1D (green) was demonstrated by confocal immunofluorescence after permeablization of the cells; the cells were co-stained for CD8α (red). A CD8+ T cell (asterisk) is shown as a negative control for RT1D staining. (f) Active synthesis of RT1D was detected by comparison between the cells before (0hr) and after Golgi blockage (6hr); an arrow shows an accumulation of RT1D in the cell. DIC, differential interference contrast. (g) Up-regulation of surface RT1D expression in PBMC CD8αα+CD3− cells after incubation with LPS as indicated. Bars = 10 µm.
Mentions: The PBMC CD8αα+CD3− cells’ morphology was examined. Immunofluorescence confirmed co-expression of cell surface RT1B and CD8α in those cells (Figure 2h). The PBMC CD8αα+CD3− cells had a polygonic nucleus, in contrast to a kidney-shaped nucleus in monocytes (Figure 2h). They could also be easily distinguished from CD8+ T cells or NK cells by either their size or shape (Figure 2h). Morphological changes in PBMC CD8αα+CD3− cells, along with both surface and intracellular MHC-II expression, were observed after in vitro culture in comparison to monocytes. Freshly isolated PBMC CD8αα+CD3− cells were spherical. Many cells flattened after 12–36 hrs’ culture, and became irregularly shaped with various cellular projections at 60 hrs (Figure 3a). Staining with CD8α antibody revealed fine cellular projections in majority of cells, which resembled those of DCs (Figure 3b), suggesting that PBMC CD8αα+ cells were a type of phagocyte. On the other hand, most monocytes remained spherically shaped at 36 hrs (Figure 3c).

Bottom Line: Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25).When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats.Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Diagnostic and Biomedical Sciences, SD, University of Texas Health Science Center at Houston, Houston, Texas, USA.

ABSTRACT
In an anti-glomerular basement membrane (GBM) glomerulonephritis (GN) model, GN-resistant Lewis rats naturally recover from early glomerular inflammation. Here we investigated recovery mechanisms for development of a potential immunotherapy for autoimmune GN. Our previous studies suggested that glomeruli-infiltrating leukocytes with a phenotype of CD8αα+CD11c+MHC-II+CD3- (GIL CD8αα+ cells) were responsible for recovery through induction of T-cell apoptosis. Now, we identified peripheral blood CD8αα+CD11c+MHC-II+CD3- cells (PBMC CD8αα+CD3- cells), which shared 9 markers with GIL CD8αα+ cells. Upon incubation, PBMC CD8αα+CD3- cells displayed a morphology resembling that of dendritic cells. Similar to GIL CD8αα+ cells, PBMC CD8αα+CD3- cells were capable of inducing T-cell apoptosis in vitro. Hence, PBMC CD8αα+CD3- cells were likely the precursor of GIL CD8αα+ cells. We next tested their potential in vivo function. PBMC CD8αα+CD3- cells were able to infiltrate inflamed but not normal glomeruli. Isolated PBMC CD8αα+CD3- cells of Lewis rats were transferred into GN-prone Wistar-Kyoto rats at early inflammatory stage (days 17-25). When examined at day 45, both histopathology and blood urea nitrogen/serum creatinine level showed significantly attenuated GN in 80% of cell recipient Wistar-Kyoto rats. Separate experiments verified infiltration of transferred Lewis PBMC CD8αα+CD3- into the glomeruli, accompanied with apoptotic CD4+ T cells in the glomeruli of the recipient Wistar-Kyoto rats. Thus, PBMC CD8αα+CD3- cells of Lewis rats were able to terminate ongoing autoimmune inflammation in the glomeruli.

Show MeSH
Related in: MedlinePlus