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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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Functional C3 on BM-derived cells, and not systemic C3, is required for development of MLDS-induced diabetes. A: C3−/− BM→WT (n = 8), WT BM→C3−/−(n = 6), and WT BM→WT (n = 6) chimeric mice were stained for CD45.2 and CD45.1 and analyzed by flow cytometry to verify lethal irradiation and bone marrow engraftment on week 8. B: Zymosan C3 binding assay confirming presence of systemic C3 in WT and absence in C3−/− mice. C: Cumulative diabetes incidence and average blood glucose levels after administration of MLDS in chimeric mice. Results represent two independent experiments.
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Figure 5: Functional C3 on BM-derived cells, and not systemic C3, is required for development of MLDS-induced diabetes. A: C3−/− BM→WT (n = 8), WT BM→C3−/−(n = 6), and WT BM→WT (n = 6) chimeric mice were stained for CD45.2 and CD45.1 and analyzed by flow cytometry to verify lethal irradiation and bone marrow engraftment on week 8. B: Zymosan C3 binding assay confirming presence of systemic C3 in WT and absence in C3−/− mice. C: Cumulative diabetes incidence and average blood glucose levels after administration of MLDS in chimeric mice. Results represent two independent experiments.

Mentions: Because immune cell–derived complement plays an integral role in adaptive T responses (13,37), we transplanted C3−/− (CD45.2) BM into lethally irradiated CD45.1 WT B6 mice (C3−/− BM→WT) to produce animals that contained serum C3, but possessed C3−/− BM cells. Conversely, we transplanted WT B6 CD45.1 BM into lethally irradiated CD45.2 C3−/− mice (WT BM→C3−/−) to produce animals deficient in serum C3, but with C3+ BM–derived cells. WT BM→WT chimeras on the B6 background were produced as controls. Staining peripheral blood for CD45.1/CD45.2 and analyzing the cells by flow cytometry (Fig. 5A) verified that the immune cells were >90% donor BM-derived. Zymosan C3 uptake assays (Fig. 5B) validated the presence or absence of C3 in the serum of each animal. We did not detect C3 in the sera of any WT BM→C3−/− chimeras (same as C3−/− controls), but we observed that all sera from C3−/− BM→WT and WT BM→WT chimeras was C3+ comparable to WT controls (Fig. 5B). When we administered MLDS to the chimeric animals, we found that only 2 of 8 C3−/− BM→WT chimeras developed diabetes, whereas all of the WT BM→C3−/− chimeras and all of the control WT BM→WT chimeras developed sustained hyperglycemia (Fig. 5C).


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)

Functional C3 on BM-derived cells, and not systemic C3, is required for development of MLDS-induced diabetes. A: C3−/− BM→WT (n = 8), WT BM→C3−/−(n = 6), and WT BM→WT (n = 6) chimeric mice were stained for CD45.2 and CD45.1 and analyzed by flow cytometry to verify lethal irradiation and bone marrow engraftment on week 8. B: Zymosan C3 binding assay confirming presence of systemic C3 in WT and absence in C3−/− mice. C: Cumulative diabetes incidence and average blood glucose levels after administration of MLDS in chimeric mice. Results represent two independent experiments.
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Figure 5: Functional C3 on BM-derived cells, and not systemic C3, is required for development of MLDS-induced diabetes. A: C3−/− BM→WT (n = 8), WT BM→C3−/−(n = 6), and WT BM→WT (n = 6) chimeric mice were stained for CD45.2 and CD45.1 and analyzed by flow cytometry to verify lethal irradiation and bone marrow engraftment on week 8. B: Zymosan C3 binding assay confirming presence of systemic C3 in WT and absence in C3−/− mice. C: Cumulative diabetes incidence and average blood glucose levels after administration of MLDS in chimeric mice. Results represent two independent experiments.
Mentions: Because immune cell–derived complement plays an integral role in adaptive T responses (13,37), we transplanted C3−/− (CD45.2) BM into lethally irradiated CD45.1 WT B6 mice (C3−/− BM→WT) to produce animals that contained serum C3, but possessed C3−/− BM cells. Conversely, we transplanted WT B6 CD45.1 BM into lethally irradiated CD45.2 C3−/− mice (WT BM→C3−/−) to produce animals deficient in serum C3, but with C3+ BM–derived cells. WT BM→WT chimeras on the B6 background were produced as controls. Staining peripheral blood for CD45.1/CD45.2 and analyzing the cells by flow cytometry (Fig. 5A) verified that the immune cells were >90% donor BM-derived. Zymosan C3 uptake assays (Fig. 5B) validated the presence or absence of C3 in the serum of each animal. We did not detect C3 in the sera of any WT BM→C3−/− chimeras (same as C3−/− controls), but we observed that all sera from C3−/− BM→WT and WT BM→WT chimeras was C3+ comparable to WT controls (Fig. 5B). When we administered MLDS to the chimeric animals, we found that only 2 of 8 C3−/− BM→WT chimeras developed diabetes, whereas all of the WT BM→C3−/− chimeras and all of the control WT BM→WT chimeras developed sustained hyperglycemia (Fig. 5C).

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