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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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Related in: MedlinePlus

Increased GIL CD8αα+ cells and T cell apoptosis in the glomeruli of PBMC CD8αα+CD3− recipient WKY rats at d23(a) Immunofluorescence shows increased CD8α+ cells (green) in a glomerulus of a PBMC CD8αα+CD3+ cell-recipient WKY rat (WKY-LEW CD8) as compared to immunized WKY (WKY) at d23 (n=3). (b) Summary of CD8α+ cells and ED1+ macrophages in the glomeruli in experimental groups as indicated. (c) Genotyping of glumeruli-infiltrating cells using DNA microsatellite based PCR. Pooled GIL CD8αα+ cells (shown as CD8α+CD3−, left panel) and CD3+ pan T cells (second to the left) pooled from three PBMC CD8αα+CD3+ cell-recipient WKY rats. The right panel shows PCR products with primers for microsatellite (D7R24). Note that GIL CD8αα+ cells are predominantly LEW type while T cells are absolutely WKY type. (d) DNA fragmentation assay for the pan T cells (refer to c) isolated from the glomeruli of immunized WKY rats (W) or PBMC CD8αα+CD3+ cell-recipient WKY rats (WLCD8). The T cells were incubated for one day before the assay. (e) Histogram shows comparison of TUNEL+ population among T cells isolated from glomeruli of indicated rats. (f) Immunoperoxidase staining for caspase 3 in immunized WKY which received no cells (WKY) or PBMC CD8αα+CD3− cells (WKY+CD8α+). Clustered caspase 3+ cells in glomeruli are present in PBMC CD8αα+CD3− cell-recipient WKY rat. Bars = 50 µm.
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Figure 7: Increased GIL CD8αα+ cells and T cell apoptosis in the glomeruli of PBMC CD8αα+CD3− recipient WKY rats at d23(a) Immunofluorescence shows increased CD8α+ cells (green) in a glomerulus of a PBMC CD8αα+CD3+ cell-recipient WKY rat (WKY-LEW CD8) as compared to immunized WKY (WKY) at d23 (n=3). (b) Summary of CD8α+ cells and ED1+ macrophages in the glomeruli in experimental groups as indicated. (c) Genotyping of glumeruli-infiltrating cells using DNA microsatellite based PCR. Pooled GIL CD8αα+ cells (shown as CD8α+CD3−, left panel) and CD3+ pan T cells (second to the left) pooled from three PBMC CD8αα+CD3+ cell-recipient WKY rats. The right panel shows PCR products with primers for microsatellite (D7R24). Note that GIL CD8αα+ cells are predominantly LEW type while T cells are absolutely WKY type. (d) DNA fragmentation assay for the pan T cells (refer to c) isolated from the glomeruli of immunized WKY rats (W) or PBMC CD8αα+CD3+ cell-recipient WKY rats (WLCD8). The T cells were incubated for one day before the assay. (e) Histogram shows comparison of TUNEL+ population among T cells isolated from glomeruli of indicated rats. (f) Immunoperoxidase staining for caspase 3 in immunized WKY which received no cells (WKY) or PBMC CD8αα+CD3− cells (WKY+CD8α+). Clustered caspase 3+ cells in glomeruli are present in PBMC CD8αα+CD3− cell-recipient WKY rat. Bars = 50 µm.

Mentions: In two other experiments, three immunized WKY rats each were transferred with the PBMC CD8αα+CD3− cells of LEW rats at d17 and 20; three other immunized WKY rats without transfer served as a positive control. The cell recipients or controls were sacrificed at d23. In the first experiment, immunofluorescence showed a significant increase in GIL CD8αα+ cells in the cell recipients as compared to the controls (Figure 7a and b). The recipients also showed a slightly lower number (though not significantly different) of ED1+ macrophages in the glomeruli than those in the immunized controls (Figure 7b). In the second experiment, glomerular T cells and GIL CD8αα+ cells were isolated for genotyping (Figure 7c). Due to a low number, glomeruli-infiltrating pan-T cells and GIL CD8αα+ isolated from the three cell recipients were pooled. PCR genotyping using three polymorphic DNA microsatellites showed that the majority of the isolated GIL CD8αα+ cells were of LEW origin, while CD3+ T cells were of WKY origin (Figure 7c). This experiment was repeated once and a similar result was yielded. T cells from the controls were of WKY origin. We failed to yield sufficient GIL CD8αα+ cells for analysis due to their low number at d23.16 A portion of glomerular T cells were used to test if they were undergoing apoptosis (Figure 7c). After incubation for one day, apoptosis in CD3+ T cells was determined. First, DNA fragmentation assay showed apoptotic T cells in PBMC CD8αα+CD3− cell recipient WKY rats, but not in the immunized controls (Figure 7d). Flow cytometry after TUNEL staining confirmed that about 41% of CD3+ T cells were apoptotic in PBMC CD8αα+CD3− cell recipients, as compared to less than 5% in immunized WKY control (Figure 7e). Finally, renal sections from PBMC CD8αα+CD3− cell recipients or immunized controls were stained for caspase 3 using the immunoperoxidase method.16 PBMC CD8αα+CD3− cell recipients showed clustered caspase 3+ cells in their glomeruli (Figure 7f), which was similar to those observed in LEW rats during recovery at d23.16 As expected, no caspase 3+ cells were observed in the immunized WKY rats at d23 (Figure 7f).


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)

Increased GIL CD8αα+ cells and T cell apoptosis in the glomeruli of PBMC CD8αα+CD3− recipient WKY rats at d23(a) Immunofluorescence shows increased CD8α+ cells (green) in a glomerulus of a PBMC CD8αα+CD3+ cell-recipient WKY rat (WKY-LEW CD8) as compared to immunized WKY (WKY) at d23 (n=3). (b) Summary of CD8α+ cells and ED1+ macrophages in the glomeruli in experimental groups as indicated. (c) Genotyping of glumeruli-infiltrating cells using DNA microsatellite based PCR. Pooled GIL CD8αα+ cells (shown as CD8α+CD3−, left panel) and CD3+ pan T cells (second to the left) pooled from three PBMC CD8αα+CD3+ cell-recipient WKY rats. The right panel shows PCR products with primers for microsatellite (D7R24). Note that GIL CD8αα+ cells are predominantly LEW type while T cells are absolutely WKY type. (d) DNA fragmentation assay for the pan T cells (refer to c) isolated from the glomeruli of immunized WKY rats (W) or PBMC CD8αα+CD3+ cell-recipient WKY rats (WLCD8). The T cells were incubated for one day before the assay. (e) Histogram shows comparison of TUNEL+ population among T cells isolated from glomeruli of indicated rats. (f) Immunoperoxidase staining for caspase 3 in immunized WKY which received no cells (WKY) or PBMC CD8αα+CD3− cells (WKY+CD8α+). Clustered caspase 3+ cells in glomeruli are present in PBMC CD8αα+CD3− cell-recipient WKY rat. Bars = 50 µm.
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Figure 7: Increased GIL CD8αα+ cells and T cell apoptosis in the glomeruli of PBMC CD8αα+CD3− recipient WKY rats at d23(a) Immunofluorescence shows increased CD8α+ cells (green) in a glomerulus of a PBMC CD8αα+CD3+ cell-recipient WKY rat (WKY-LEW CD8) as compared to immunized WKY (WKY) at d23 (n=3). (b) Summary of CD8α+ cells and ED1+ macrophages in the glomeruli in experimental groups as indicated. (c) Genotyping of glumeruli-infiltrating cells using DNA microsatellite based PCR. Pooled GIL CD8αα+ cells (shown as CD8α+CD3−, left panel) and CD3+ pan T cells (second to the left) pooled from three PBMC CD8αα+CD3+ cell-recipient WKY rats. The right panel shows PCR products with primers for microsatellite (D7R24). Note that GIL CD8αα+ cells are predominantly LEW type while T cells are absolutely WKY type. (d) DNA fragmentation assay for the pan T cells (refer to c) isolated from the glomeruli of immunized WKY rats (W) or PBMC CD8αα+CD3+ cell-recipient WKY rats (WLCD8). The T cells were incubated for one day before the assay. (e) Histogram shows comparison of TUNEL+ population among T cells isolated from glomeruli of indicated rats. (f) Immunoperoxidase staining for caspase 3 in immunized WKY which received no cells (WKY) or PBMC CD8αα+CD3− cells (WKY+CD8α+). Clustered caspase 3+ cells in glomeruli are present in PBMC CD8αα+CD3− cell-recipient WKY rat. Bars = 50 µm.
Mentions: In two other experiments, three immunized WKY rats each were transferred with the PBMC CD8αα+CD3− cells of LEW rats at d17 and 20; three other immunized WKY rats without transfer served as a positive control. The cell recipients or controls were sacrificed at d23. In the first experiment, immunofluorescence showed a significant increase in GIL CD8αα+ cells in the cell recipients as compared to the controls (Figure 7a and b). The recipients also showed a slightly lower number (though not significantly different) of ED1+ macrophages in the glomeruli than those in the immunized controls (Figure 7b). In the second experiment, glomerular T cells and GIL CD8αα+ cells were isolated for genotyping (Figure 7c). Due to a low number, glomeruli-infiltrating pan-T cells and GIL CD8αα+ isolated from the three cell recipients were pooled. PCR genotyping using three polymorphic DNA microsatellites showed that the majority of the isolated GIL CD8αα+ cells were of LEW origin, while CD3+ T cells were of WKY origin (Figure 7c). This experiment was repeated once and a similar result was yielded. T cells from the controls were of WKY origin. We failed to yield sufficient GIL CD8αα+ cells for analysis due to their low number at d23.16 A portion of glomerular T cells were used to test if they were undergoing apoptosis (Figure 7c). After incubation for one day, apoptosis in CD3+ T cells was determined. First, DNA fragmentation assay showed apoptotic T cells in PBMC CD8αα+CD3− cell recipient WKY rats, but not in the immunized controls (Figure 7d). Flow cytometry after TUNEL staining confirmed that about 41% of CD3+ T cells were apoptotic in PBMC CD8αα+CD3− cell recipients, as compared to less than 5% in immunized WKY control (Figure 7e). Finally, renal sections from PBMC CD8αα+CD3− cell recipients or immunized controls were stained for caspase 3 using the immunoperoxidase method.16 PBMC CD8αα+CD3− cell recipients showed clustered caspase 3+ cells in their glomeruli (Figure 7f), which was similar to those observed in LEW rats during recovery at d23.16 As expected, no caspase 3+ cells were observed in the immunized WKY rats at d23 (Figure 7f).

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