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In type 1 diabetes a subset of anti-coxsackievirus B4 antibodies recognize autoantigens and induce apoptosis of pancreatic beta cells.

Bason C, Lorini R, Lunardi C, Dolcino M, Giannattasio A, d'Annunzio G, Rigo A, Pedemonte N, Corrocher R, Puccetti A - PLoS ONE (2013)

Bottom Line: Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet.We identified an immunodominant peptide recognized by the majority of individual patients'sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis.Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Internal Medicine, University of Verona, Verona, Italy.

ABSTRACT
Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients'sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients' sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.

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Related in: MedlinePlus

Antibodies against T1DM, COXSA, CA peptides induce apoptosis in beta-cell line through disruption of mitochondrial membrane potential and increase of intracellular calcium.A, NIT cells were incubated for 24 hours with antibodies against: T1DM peptide (bar 3,4,5), COXSA peptide (bar 6,7,8), CA peptide (bar 9,10,11), irrelevant peptide (bar 12,13,14), at three different concentrations (40, 20, 10 µg/ml) or incubated with cycloheximide (bar 1)(10 µg/ml) or TNF-α (bar 2)(50 ng/ml). The apoptotic index, reported in the vertical axis as mean ± s.d. of triplicates, shows the enrichment of nucleosomes released in the cytoplasm (value in control untreated cells = 1). An increase in the enrichment factor of 1.0 corresponds approximately to 10–12% of apoptotis. B, Apoptosis in NIT cells induced by affinity purified antibodies against T1DM peptide 10 µg/ml (bar 1) is inhibited by preincubation of these antibodies with T1DM peptide (bar 3,4), COXSA peptide (bar 5,6) or CA peptide (bar 7,8) at two different concentrations (100, 200 µg/ml) but not by the preincubation with the irrelevant control peptide (bar 2) at the maximum concentration (200 µg/ml). C, Induction of apoptosis of NIT cells by serum of five type I diabetes patients (bar 4,6,8,10,12) or by five controls sera (bar 3,5,7,9,11) at two different dilutions (1∶100, 1∶200) or by cycloheximide (bar 1)(10 µg/ml) or TNFα (bar 2) (50 ng/ml). Sera were heat-inactivated (56°C for 30 min). Data represent the mean ± s.d. of triplicate samples of three independent experiments. D, E, Anti-T1DM antibody alter the mitochondrial membrane potential (DΨm) of beta cells: D, Microscopy (magnification x40): Untreated beta cells (negative control) with well-polarized mitochondria are characterized by punctated red fluorescence (left slide); cells treated with anti-T1DM antibody show a population with intermediate mitochondrial transmembrane potential with a partial or complete loss of red fluorescence that is replaced by diffuse green fluorescence (center slide). Cells treated with CCCP (positive control), that causes fast mitochondrial membrane depolarization, show a diffuse green fluorescence (right slide). E, Flow cytometry. Untreated cells with well-polarized mithocondria are localized in the upper region of the plot (H = high DΨm, FL2 bright). Cells exposed to anti-T1DM antibody for 6 hours shift downward (I = intermediate DΨm and L = low DΨm, FL2 dim). This shift represents the progressive loss of mitochondrial JC-1 aggregates and an increase in the formation of green fluorescent cytoplasmic monomers, which indicates the disruption of the mitochondrial DΨm. Cells treated with CCCP completely shift downward (L = low DΨm, FL2 dim). F, G, Anti-T1DM antibody induce intracellular calcium mobilization: F, representative fluorescent traces showing the response to K+ addition in beta cells. Prior to assay, beta cells were stimulated with PBS alone (control; black), TNFα (50 ng/ml; gray), or the anti-T1DM antibody (20 µg/ml; red). The curves show the response of cells acutely treated with KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel). G, Bar graphs summarizing the results of experiments shown in panel F. The intracellular calcium increase was determined as the fluorescence mean triggered by KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel) in beta cells treated with PBS alone (control; black), TNFα (gray), or the anti-T1DM antibody (red); (mean ± S.E.M., n = 6). **, P<0.01 versus control.
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pone-0057729-g005: Antibodies against T1DM, COXSA, CA peptides induce apoptosis in beta-cell line through disruption of mitochondrial membrane potential and increase of intracellular calcium.A, NIT cells were incubated for 24 hours with antibodies against: T1DM peptide (bar 3,4,5), COXSA peptide (bar 6,7,8), CA peptide (bar 9,10,11), irrelevant peptide (bar 12,13,14), at three different concentrations (40, 20, 10 µg/ml) or incubated with cycloheximide (bar 1)(10 µg/ml) or TNF-α (bar 2)(50 ng/ml). The apoptotic index, reported in the vertical axis as mean ± s.d. of triplicates, shows the enrichment of nucleosomes released in the cytoplasm (value in control untreated cells = 1). An increase in the enrichment factor of 1.0 corresponds approximately to 10–12% of apoptotis. B, Apoptosis in NIT cells induced by affinity purified antibodies against T1DM peptide 10 µg/ml (bar 1) is inhibited by preincubation of these antibodies with T1DM peptide (bar 3,4), COXSA peptide (bar 5,6) or CA peptide (bar 7,8) at two different concentrations (100, 200 µg/ml) but not by the preincubation with the irrelevant control peptide (bar 2) at the maximum concentration (200 µg/ml). C, Induction of apoptosis of NIT cells by serum of five type I diabetes patients (bar 4,6,8,10,12) or by five controls sera (bar 3,5,7,9,11) at two different dilutions (1∶100, 1∶200) or by cycloheximide (bar 1)(10 µg/ml) or TNFα (bar 2) (50 ng/ml). Sera were heat-inactivated (56°C for 30 min). Data represent the mean ± s.d. of triplicate samples of three independent experiments. D, E, Anti-T1DM antibody alter the mitochondrial membrane potential (DΨm) of beta cells: D, Microscopy (magnification x40): Untreated beta cells (negative control) with well-polarized mitochondria are characterized by punctated red fluorescence (left slide); cells treated with anti-T1DM antibody show a population with intermediate mitochondrial transmembrane potential with a partial or complete loss of red fluorescence that is replaced by diffuse green fluorescence (center slide). Cells treated with CCCP (positive control), that causes fast mitochondrial membrane depolarization, show a diffuse green fluorescence (right slide). E, Flow cytometry. Untreated cells with well-polarized mithocondria are localized in the upper region of the plot (H = high DΨm, FL2 bright). Cells exposed to anti-T1DM antibody for 6 hours shift downward (I = intermediate DΨm and L = low DΨm, FL2 dim). This shift represents the progressive loss of mitochondrial JC-1 aggregates and an increase in the formation of green fluorescent cytoplasmic monomers, which indicates the disruption of the mitochondrial DΨm. Cells treated with CCCP completely shift downward (L = low DΨm, FL2 dim). F, G, Anti-T1DM antibody induce intracellular calcium mobilization: F, representative fluorescent traces showing the response to K+ addition in beta cells. Prior to assay, beta cells were stimulated with PBS alone (control; black), TNFα (50 ng/ml; gray), or the anti-T1DM antibody (20 µg/ml; red). The curves show the response of cells acutely treated with KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel). G, Bar graphs summarizing the results of experiments shown in panel F. The intracellular calcium increase was determined as the fluorescence mean triggered by KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel) in beta cells treated with PBS alone (control; black), TNFα (gray), or the anti-T1DM antibody (red); (mean ± S.E.M., n = 6). **, P<0.01 versus control.

Mentions: Based on these observations we decided to verify whether affinity-purified antibodies against the peptides had any functional effect on beta cells. To this aim we incubated beta cells for 24 hours with antibodies against the T1DM, COXSA, CA peptides and evaluated the ability of these antibodies to induce apoptosis. Indeed antibodies directed against the three peptides were able to induce internucleosomal DNA fragmentation in beta cells in a dose-dependent fashion reaching the maximum at an antibody concentration of 40 µg/ml (Fig. 5A and Fig. S1). Apoptosis was inhibited by a 1 hour preincubation of peptide-specific antibodies with each of the three peptides (Fig. 5B), further confirming the specificity of antibodies-beta-cell interaction. Moreover, patients’sera, incubated with beta cells for 24 hours, were able to induce beta-cell apoptosis (Fig. 5C).


In type 1 diabetes a subset of anti-coxsackievirus B4 antibodies recognize autoantigens and induce apoptosis of pancreatic beta cells.

Bason C, Lorini R, Lunardi C, Dolcino M, Giannattasio A, d'Annunzio G, Rigo A, Pedemonte N, Corrocher R, Puccetti A - PLoS ONE (2013)

Antibodies against T1DM, COXSA, CA peptides induce apoptosis in beta-cell line through disruption of mitochondrial membrane potential and increase of intracellular calcium.A, NIT cells were incubated for 24 hours with antibodies against: T1DM peptide (bar 3,4,5), COXSA peptide (bar 6,7,8), CA peptide (bar 9,10,11), irrelevant peptide (bar 12,13,14), at three different concentrations (40, 20, 10 µg/ml) or incubated with cycloheximide (bar 1)(10 µg/ml) or TNF-α (bar 2)(50 ng/ml). The apoptotic index, reported in the vertical axis as mean ± s.d. of triplicates, shows the enrichment of nucleosomes released in the cytoplasm (value in control untreated cells = 1). An increase in the enrichment factor of 1.0 corresponds approximately to 10–12% of apoptotis. B, Apoptosis in NIT cells induced by affinity purified antibodies against T1DM peptide 10 µg/ml (bar 1) is inhibited by preincubation of these antibodies with T1DM peptide (bar 3,4), COXSA peptide (bar 5,6) or CA peptide (bar 7,8) at two different concentrations (100, 200 µg/ml) but not by the preincubation with the irrelevant control peptide (bar 2) at the maximum concentration (200 µg/ml). C, Induction of apoptosis of NIT cells by serum of five type I diabetes patients (bar 4,6,8,10,12) or by five controls sera (bar 3,5,7,9,11) at two different dilutions (1∶100, 1∶200) or by cycloheximide (bar 1)(10 µg/ml) or TNFα (bar 2) (50 ng/ml). Sera were heat-inactivated (56°C for 30 min). Data represent the mean ± s.d. of triplicate samples of three independent experiments. D, E, Anti-T1DM antibody alter the mitochondrial membrane potential (DΨm) of beta cells: D, Microscopy (magnification x40): Untreated beta cells (negative control) with well-polarized mitochondria are characterized by punctated red fluorescence (left slide); cells treated with anti-T1DM antibody show a population with intermediate mitochondrial transmembrane potential with a partial or complete loss of red fluorescence that is replaced by diffuse green fluorescence (center slide). Cells treated with CCCP (positive control), that causes fast mitochondrial membrane depolarization, show a diffuse green fluorescence (right slide). E, Flow cytometry. Untreated cells with well-polarized mithocondria are localized in the upper region of the plot (H = high DΨm, FL2 bright). Cells exposed to anti-T1DM antibody for 6 hours shift downward (I = intermediate DΨm and L = low DΨm, FL2 dim). This shift represents the progressive loss of mitochondrial JC-1 aggregates and an increase in the formation of green fluorescent cytoplasmic monomers, which indicates the disruption of the mitochondrial DΨm. Cells treated with CCCP completely shift downward (L = low DΨm, FL2 dim). F, G, Anti-T1DM antibody induce intracellular calcium mobilization: F, representative fluorescent traces showing the response to K+ addition in beta cells. Prior to assay, beta cells were stimulated with PBS alone (control; black), TNFα (50 ng/ml; gray), or the anti-T1DM antibody (20 µg/ml; red). The curves show the response of cells acutely treated with KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel). G, Bar graphs summarizing the results of experiments shown in panel F. The intracellular calcium increase was determined as the fluorescence mean triggered by KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel) in beta cells treated with PBS alone (control; black), TNFα (gray), or the anti-T1DM antibody (red); (mean ± S.E.M., n = 6). **, P<0.01 versus control.
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pone-0057729-g005: Antibodies against T1DM, COXSA, CA peptides induce apoptosis in beta-cell line through disruption of mitochondrial membrane potential and increase of intracellular calcium.A, NIT cells were incubated for 24 hours with antibodies against: T1DM peptide (bar 3,4,5), COXSA peptide (bar 6,7,8), CA peptide (bar 9,10,11), irrelevant peptide (bar 12,13,14), at three different concentrations (40, 20, 10 µg/ml) or incubated with cycloheximide (bar 1)(10 µg/ml) or TNF-α (bar 2)(50 ng/ml). The apoptotic index, reported in the vertical axis as mean ± s.d. of triplicates, shows the enrichment of nucleosomes released in the cytoplasm (value in control untreated cells = 1). An increase in the enrichment factor of 1.0 corresponds approximately to 10–12% of apoptotis. B, Apoptosis in NIT cells induced by affinity purified antibodies against T1DM peptide 10 µg/ml (bar 1) is inhibited by preincubation of these antibodies with T1DM peptide (bar 3,4), COXSA peptide (bar 5,6) or CA peptide (bar 7,8) at two different concentrations (100, 200 µg/ml) but not by the preincubation with the irrelevant control peptide (bar 2) at the maximum concentration (200 µg/ml). C, Induction of apoptosis of NIT cells by serum of five type I diabetes patients (bar 4,6,8,10,12) or by five controls sera (bar 3,5,7,9,11) at two different dilutions (1∶100, 1∶200) or by cycloheximide (bar 1)(10 µg/ml) or TNFα (bar 2) (50 ng/ml). Sera were heat-inactivated (56°C for 30 min). Data represent the mean ± s.d. of triplicate samples of three independent experiments. D, E, Anti-T1DM antibody alter the mitochondrial membrane potential (DΨm) of beta cells: D, Microscopy (magnification x40): Untreated beta cells (negative control) with well-polarized mitochondria are characterized by punctated red fluorescence (left slide); cells treated with anti-T1DM antibody show a population with intermediate mitochondrial transmembrane potential with a partial or complete loss of red fluorescence that is replaced by diffuse green fluorescence (center slide). Cells treated with CCCP (positive control), that causes fast mitochondrial membrane depolarization, show a diffuse green fluorescence (right slide). E, Flow cytometry. Untreated cells with well-polarized mithocondria are localized in the upper region of the plot (H = high DΨm, FL2 bright). Cells exposed to anti-T1DM antibody for 6 hours shift downward (I = intermediate DΨm and L = low DΨm, FL2 dim). This shift represents the progressive loss of mitochondrial JC-1 aggregates and an increase in the formation of green fluorescent cytoplasmic monomers, which indicates the disruption of the mitochondrial DΨm. Cells treated with CCCP completely shift downward (L = low DΨm, FL2 dim). F, G, Anti-T1DM antibody induce intracellular calcium mobilization: F, representative fluorescent traces showing the response to K+ addition in beta cells. Prior to assay, beta cells were stimulated with PBS alone (control; black), TNFα (50 ng/ml; gray), or the anti-T1DM antibody (20 µg/ml; red). The curves show the response of cells acutely treated with KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel). G, Bar graphs summarizing the results of experiments shown in panel F. The intracellular calcium increase was determined as the fluorescence mean triggered by KCl alone (top panel) or in combination with ionomycin (1 µM; bottom panel) in beta cells treated with PBS alone (control; black), TNFα (gray), or the anti-T1DM antibody (red); (mean ± S.E.M., n = 6). **, P<0.01 versus control.
Mentions: Based on these observations we decided to verify whether affinity-purified antibodies against the peptides had any functional effect on beta cells. To this aim we incubated beta cells for 24 hours with antibodies against the T1DM, COXSA, CA peptides and evaluated the ability of these antibodies to induce apoptosis. Indeed antibodies directed against the three peptides were able to induce internucleosomal DNA fragmentation in beta cells in a dose-dependent fashion reaching the maximum at an antibody concentration of 40 µg/ml (Fig. 5A and Fig. S1). Apoptosis was inhibited by a 1 hour preincubation of peptide-specific antibodies with each of the three peptides (Fig. 5B), further confirming the specificity of antibodies-beta-cell interaction. Moreover, patients’sera, incubated with beta cells for 24 hours, were able to induce beta-cell apoptosis (Fig. 5C).

Bottom Line: Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet.We identified an immunodominant peptide recognized by the majority of individual patients'sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis.Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Internal Medicine, University of Verona, Verona, Italy.

ABSTRACT
Type 1 diabetes is characterized by autoimmune destruction of pancreatic beta cells. The role played by autoantibodies directed against beta cells antigens in the pathogenesis of the disease is still unclear. Coxsackievirus B infection has been linked to the onset of type 1 diabetes; however its precise role has not been elucidated yet. To clarify these issues, we screened a random peptide library with sera obtained from 58 patients with recent onset type 1 diabetes, before insulin therapy. We identified an immunodominant peptide recognized by the majority of individual patients'sera, that shares homology with Coxsackievirus B4 VP1 protein and with beta-cell specific autoantigens such as phogrin, phosphofructokinase and voltage-gated L-type calcium channels known to regulate beta cell apoptosis. Antibodies against the peptide affinity-purified from patients' sera, recognized the viral protein and autoantigens; moreover, such antibodies induced apoptosis of the beta cells upon binding the L-type calcium channels expressed on the beta cell surface, suggesting a calcium dependent mechanism. Our results provide evidence that in autoimmune diabetes a subset of anti-Coxsackievirus antibodies are able to induce apoptosis of pancreatic beta cells which is considered the most critical and final step in the development of autoimmune diabetes without which clinical manifestations do not occur.

Show MeSH
Related in: MedlinePlus