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Immune cell-derived c3 is required for autoimmune diabetes induced by multiple low doses of streptozotocin.

Lin M, Yin N, Murphy B, Medof ME, Segerer S, Heeger PS, Schröppel B - Diabetes (2010)

Bottom Line: Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model.Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell-derived C3, and not serum C3, is involved in the induction of diabetes in this model.The data reveal a key role for immune cell-derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Mount Sinai School of Medicine, New York, New York, USA.

ABSTRACT

Objective: The complement system contributes to autoimmune injury, but its involvement in promoting the development of autoimmune diabetes is unknown. In this study, our goal was to ascertain the role of complement C3 in autoimmune diabetes.

Research design and methods: Susceptibility to diabetes development after multiple low-dose streptozotocin treatment in wild-type (WT) and C3-deficient mice was analyzed. Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model.

Results: Coincident with the induced elevations in blood glucose levels, we documented alternative pathway complement component gene expression within the islets of the diabetic WT mice. When we repeated the experiments with C3-deficient mice, we observed complete resistance to disease, as assessed by the absence of histologic insulitis and the absence of T-cell reactivity to islet antigens. Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell-derived C3, and not serum C3, is involved in the induction of diabetes in this model.

Conclusions: The data reveal a key role for immune cell-derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

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C3 deficiency protects from MLDS-induced diabetes. A: Cumulative diabetes incidence and average blood glucose levels of WT or C3−/− mice on both the B6 and Balb/c background after administration of MLDS (n = 7–11 per group) or Hi-STZ (n = 3–4); **P < 0.01 (comparing blood glucose level of WT versus C3−/− on day 17). B: Histopathology of pancreata on experimental day 19 of MLDS showing mononuclear cell infiltration in WT, but not in C3−/− mice. Arrowheads indicate areas of infiltration; original magnification ×200. Mean insulitis scores (% ± SEM) of mice on experimental day 19 were obtained from 3 mice for each group. P < 0.01 (C) qRT-PCR of CCL2, CCL3, IFN-γ, IL-6, and macrophages (CD68) from total pancreata (normalized to cyclophilin) on experimental days 0, 5, and 10 after initiation of MLDS (n = 6 per group and time point) with C3−/− mice with lower expression compared with WT mice. Box and whisker blots show the medians and percentile values (10, 25, 75, and 90) for normalized mRNA.
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Figure 3: C3 deficiency protects from MLDS-induced diabetes. A: Cumulative diabetes incidence and average blood glucose levels of WT or C3−/− mice on both the B6 and Balb/c background after administration of MLDS (n = 7–11 per group) or Hi-STZ (n = 3–4); **P < 0.01 (comparing blood glucose level of WT versus C3−/− on day 17). B: Histopathology of pancreata on experimental day 19 of MLDS showing mononuclear cell infiltration in WT, but not in C3−/− mice. Arrowheads indicate areas of infiltration; original magnification ×200. Mean insulitis scores (% ± SEM) of mice on experimental day 19 were obtained from 3 mice for each group. P < 0.01 (C) qRT-PCR of CCL2, CCL3, IFN-γ, IL-6, and macrophages (CD68) from total pancreata (normalized to cyclophilin) on experimental days 0, 5, and 10 after initiation of MLDS (n = 6 per group and time point) with C3−/− mice with lower expression compared with WT mice. Box and whisker blots show the medians and percentile values (10, 25, 75, and 90) for normalized mRNA.

Mentions: To test whether C3 is required to induce diabetes after MLDS, we administered MLDS to C3−/− mice on both C57BL/6 (n = 12) and BALB/c (n = 7) background. In contrast to the WTs (C57BL/6 n = 11, BALB/c n = 11) in which blood sugars exceeded 200 mg/dl at experimental day 11 in all mice, none of the C3−/− mice from either background developed hyperglycemia (Fig. 3A). Although we found histologic evidence of insulitis on experimental day 19 after MLDS in WT mice, we did not observe mononuclear infiltration within the islets of C3−/− animals (Fig. 3B). The mean insulitis score was 50.4 ± 6.5% in WT versus 5.3 ± 2.7% in C3−/− mice (P < 0.01) (Fig. 3B). In control experiments to test whether islets lacking C3 limits susceptibility to the effects of STZ, we cocultured WT and C3−/− islets overnight with 0.5 mg/ml STZ. When we then tested the cultured islets for apoptosis, we found similar levels of Annexin V staining in WT islets and C3−/− islets, 50.1% vs. 56.3%, respectively (Fig. S1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0044/DC1). In confirmation of equivalent susceptibility to STZ, all Hi-STZ treated C3−/− mice (n = 4) and WT mice (n = 7) developed diabetes with identical kinetics to the WT controls (Fig. 3A).


Immune cell-derived c3 is required for autoimmune diabetes induced by multiple low doses of streptozotocin.

Lin M, Yin N, Murphy B, Medof ME, Segerer S, Heeger PS, Schröppel B - Diabetes (2010)

C3 deficiency protects from MLDS-induced diabetes. A: Cumulative diabetes incidence and average blood glucose levels of WT or C3−/− mice on both the B6 and Balb/c background after administration of MLDS (n = 7–11 per group) or Hi-STZ (n = 3–4); **P < 0.01 (comparing blood glucose level of WT versus C3−/− on day 17). B: Histopathology of pancreata on experimental day 19 of MLDS showing mononuclear cell infiltration in WT, but not in C3−/− mice. Arrowheads indicate areas of infiltration; original magnification ×200. Mean insulitis scores (% ± SEM) of mice on experimental day 19 were obtained from 3 mice for each group. P < 0.01 (C) qRT-PCR of CCL2, CCL3, IFN-γ, IL-6, and macrophages (CD68) from total pancreata (normalized to cyclophilin) on experimental days 0, 5, and 10 after initiation of MLDS (n = 6 per group and time point) with C3−/− mice with lower expression compared with WT mice. Box and whisker blots show the medians and percentile values (10, 25, 75, and 90) for normalized mRNA.
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Figure 3: C3 deficiency protects from MLDS-induced diabetes. A: Cumulative diabetes incidence and average blood glucose levels of WT or C3−/− mice on both the B6 and Balb/c background after administration of MLDS (n = 7–11 per group) or Hi-STZ (n = 3–4); **P < 0.01 (comparing blood glucose level of WT versus C3−/− on day 17). B: Histopathology of pancreata on experimental day 19 of MLDS showing mononuclear cell infiltration in WT, but not in C3−/− mice. Arrowheads indicate areas of infiltration; original magnification ×200. Mean insulitis scores (% ± SEM) of mice on experimental day 19 were obtained from 3 mice for each group. P < 0.01 (C) qRT-PCR of CCL2, CCL3, IFN-γ, IL-6, and macrophages (CD68) from total pancreata (normalized to cyclophilin) on experimental days 0, 5, and 10 after initiation of MLDS (n = 6 per group and time point) with C3−/− mice with lower expression compared with WT mice. Box and whisker blots show the medians and percentile values (10, 25, 75, and 90) for normalized mRNA.
Mentions: To test whether C3 is required to induce diabetes after MLDS, we administered MLDS to C3−/− mice on both C57BL/6 (n = 12) and BALB/c (n = 7) background. In contrast to the WTs (C57BL/6 n = 11, BALB/c n = 11) in which blood sugars exceeded 200 mg/dl at experimental day 11 in all mice, none of the C3−/− mice from either background developed hyperglycemia (Fig. 3A). Although we found histologic evidence of insulitis on experimental day 19 after MLDS in WT mice, we did not observe mononuclear infiltration within the islets of C3−/− animals (Fig. 3B). The mean insulitis score was 50.4 ± 6.5% in WT versus 5.3 ± 2.7% in C3−/− mice (P < 0.01) (Fig. 3B). In control experiments to test whether islets lacking C3 limits susceptibility to the effects of STZ, we cocultured WT and C3−/− islets overnight with 0.5 mg/ml STZ. When we then tested the cultured islets for apoptosis, we found similar levels of Annexin V staining in WT islets and C3−/− islets, 50.1% vs. 56.3%, respectively (Fig. S1, available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db10-0044/DC1). In confirmation of equivalent susceptibility to STZ, all Hi-STZ treated C3−/− mice (n = 4) and WT mice (n = 7) developed diabetes with identical kinetics to the WT controls (Fig. 3A).

Bottom Line: Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model.Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell-derived C3, and not serum C3, is involved in the induction of diabetes in this model.The data reveal a key role for immune cell-derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Mount Sinai School of Medicine, New York, New York, USA.

ABSTRACT

Objective: The complement system contributes to autoimmune injury, but its involvement in promoting the development of autoimmune diabetes is unknown. In this study, our goal was to ascertain the role of complement C3 in autoimmune diabetes.

Research design and methods: Susceptibility to diabetes development after multiple low-dose streptozotocin treatment in wild-type (WT) and C3-deficient mice was analyzed. Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model.

Results: Coincident with the induced elevations in blood glucose levels, we documented alternative pathway complement component gene expression within the islets of the diabetic WT mice. When we repeated the experiments with C3-deficient mice, we observed complete resistance to disease, as assessed by the absence of histologic insulitis and the absence of T-cell reactivity to islet antigens. Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell-derived C3, and not serum C3, is involved in the induction of diabetes in this model.

Conclusions: The data reveal a key role for immune cell-derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

Show MeSH
Related in: MedlinePlus