- Email: [email protected]
Early assessment of apoptosis in isolated islets of Langerhans
Early Assessment of Apoptosis in Isolated Islets of Langerhans P. Cattan, T. Berney, S. Schena, R.D. Molano, A. Pileggi, C. Vizzardelli, C. Ricordi, and L. Inverardi
T
HERE is substantial evidence to link early graft loss following islet transplantation to isolation-induced islet cell apoptosis. It has been established in vitro that apoptosis participates in the death of freshly isolated islets cultured under standard conditions.1 Isolation-induced cell death might be related in part to anoikis and lack of growth factors.2– 4 Additionally, chemicals and contaminants present during isolation and purification might contribute to the observed apoptosis.5,6 It would be of great value to define reliable tests that allow the prediction of islet cell viability prior to transplantation. The analysis of apoptosis based on DNA fragmentation by in situ labeling (TUNEL assay) and agarose gel electrophoresis (showing a characteristic ladderlike pattern of DNA fragments), although accepted as the gold standard, explores only late events of programmed cell death. Furthermore, these techniques are time-consuming and allow only retrospective analysis of the preparations after they have been transplanted. Therefore, newly available assays for early apoptosis detection (caspase 3 and Annexin V assays) were analyzed in this study and compared to assays based on the detection of DNA fragmentation. The identification of such early markers appears to be a valuable means to increase our analytical power to gain information on islet preparation quality. MATERIALS AND METHODS Islets were isolated from Lewis rats by intraductal collagenase digestion as previously described,7 purified over a discontinuous Euroficoll gradient, and cultured overnight before in vitro assays. Islet purity was ⬎95% in all isolations. Recombinant rat IL-1 (50 U/mL), IFN-␥ (103 U/mL), and TNF-␣ (103 U/mL) were utilized in combination to induce apoptosis. Untreated islets were used as controls. The fluorometric assay FluorAce Apopain Assay Kit (BIO-RAD, Hercules, Calif) was utilized to measure caspase 3 activity on cellular extracts. The fluorogenic peptide substrate Ac-DEVD-AFC was utilized at a concentration of 55 mol/L. The caspase 3 inhibitor Z-DEVD-FMK was utilized at a concentration of 15 mol/L to distinguish caspase 3 activity from nonspecific protease activity. Readings were made on a VersaFluor fluorometer (BIO-RAD) with excitation and emission wavelengths of 390 nm and 520 nm, respectively. An Annexin V assay (Annexin-VFLUOS Staining Kit, Roche Boehringer Mannheim Corp.) was performed after cell dissociation in trypsin-EDTA (0.05% trypsin, 0.53 mmol/L EDTA) by gentle pipetting for 4 minutes. Propidium iodide (PI) staining was performed simultaneously with the Annexin V staining to differentiate apoptotic cells (single Annexin V-positive) from necrotic cells (double Annexin V-PI-positive). 0041-1345/01/$–see front matter PII S0041-1345(00)02006-6
Readings were made on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif) using 488-nm excitation and a 530-nm bandpass filter for fluorescein detection and a 650-nm longpass filter for PI detection. Detection of DNA fragmentation in situ was performed on 5-m-thick paraffin sections of whole islets using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (In Situ Cell Death Detection, POD, Roche Boehringer Mannheim Corp). DNA fragmentation analysis by gel electrophoresis was performed after overnight DNA extraction (0.1 mg/mL proteinase K) and RNase treatment. Electrophoresis was run at 100 V for 90 minutes on a 1.2% agarose gel. The statistical analysis used a one-tailed Student’s t-test for the comparison of paired continuous variables. Values of P ⬍ .05 were considered significant.
RESULTS AND DISCUSSION
Measurable caspase 3 activity and Annexin V positivity were detected after a 24-hour culture period under standard conditions, whereas no substantial DNA fragmentation was observed at this time with a TUNEL assay and DNA gel electrophoresis. As soon as 3 hours after cytokine stimulation, a statistically significant increase in caspase 3 activity was observed in the treated group when compared to the control group (1.4 ⫾ 0.45 U/100 IEQ vs 0.8 ⫾ 0.3 U/100 IEQ, P ⬍ .05). This difference remained significant at 6 hours (P ⬍ .05) and 12 hours (P ⬍ .01) after stimulation. Annexin V assay showed a significant increase in the percentage of apoptotic cells (Annexin V-positive cells) after 6 hours of stimulation (22.4 ⫾ 7.2% in the control group vs 27.5 ⫾ 8.1% in the treated group; P ⬍ .01). Cytokine treatment also resulted in an increased percentage of necrotic cells (Annexin V-PI-positive cells) as soon as 3 hours after cytokine incubation (6.2 ⫾ 1.7% in the control group vs 10.6 ⫾ 2.6% in the treated group; P ⬍ .05). Percentages of TUNEL-positive nuclei were 43 ⫾ 5% and 69 ⫾ 11% after 24 hours and 48 hours of cytokine stimuFrom the Diabetes Research Institute, University of Miami School of Medicine, Miami, Florida, USA. P.C. is supported by grants from the Glaxo Wellcome Laboratories and from Assistance Publique-Hopitaux de Paris. T.B. is supported by a grant from the Swiss Foundation for BiologicalMedical Grants. Address reprint requests to Luca Inverardi, MD, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136. E-mail: linverar@med. miami.edu. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
264
Transplantation Proceedings, 33, 264–265 (2001)
ASSESSMENT OF APOPTOSIS IN ISLET CELLS
lation and 17 ⫾ 5% and 19 ⫾ 5% in control islets at the same time points, respectively. Gel electrophoresis showed a distinct DNA laddering after 24 hours of cytokine stimulation, but not in the control islets. DNA fragmentation was no longer detectable after 48 hours in either preparation. The increase in TUNEL-positive nuclei and the appearance of DNA laddering after cytokine stimulation clearly validate the signal observed earlier with the caspase 3 and Annexin V assays. Whereas caspase 3 measurement has the advantage of being detectable as early as 3 hours after the delivery of an apoptotic signal, Annexin V-PI staining is more suitable for evaluating the viability of the preparations in a quantitative fashion. These two assays are easy to perform and have good reproducibility. If the analysis of apoptosis/necrosis is to be a powerful assay for the prospective analysis of islet function within time frames compatible with a transplant procedure, the early assays seem to
265
provide a concrete advantage, in terms of both baseline sensitivity and kinetics.
REFERENCES 1. Paraskevas S, Duguid WP, Maysinger D, et al: Transplant Proc 29:750, 1997 2. Petrik J, Arany E, McDonald TJ, et al: Endocrinology 139: 2994, 1998 3. Ilieva A, Yuan S, Wang RN, et al: J Endocrinol 161:357, 1999 4. Thomas FT, Contreras JL, Bilbao G, et al: Surgery 126:299, 1999 5. Vargas F, Vives-Pi M, Somoza N, et al: Transplantation 65:722, 1998 6. Berney T, Molano RD, Cattan P, et al: Transplantation (in press) 7. Rabinovitch A, Suarez-Pinzon WL, Shi Y, et al: Diabetologia 37:733, 1994
T
HERE is substantial evidence to link early graft loss following islet transplantation to isolation-induced islet cell apoptosis. It has been established in vitro that apoptosis participates in the death of freshly isolated islets cultured under standard conditions.1 Isolation-induced cell death might be related in part to anoikis and lack of growth factors.2– 4 Additionally, chemicals and contaminants present during isolation and purification might contribute to the observed apoptosis.5,6 It would be of great value to define reliable tests that allow the prediction of islet cell viability prior to transplantation. The analysis of apoptosis based on DNA fragmentation by in situ labeling (TUNEL assay) and agarose gel electrophoresis (showing a characteristic ladderlike pattern of DNA fragments), although accepted as the gold standard, explores only late events of programmed cell death. Furthermore, these techniques are time-consuming and allow only retrospective analysis of the preparations after they have been transplanted. Therefore, newly available assays for early apoptosis detection (caspase 3 and Annexin V assays) were analyzed in this study and compared to assays based on the detection of DNA fragmentation. The identification of such early markers appears to be a valuable means to increase our analytical power to gain information on islet preparation quality. MATERIALS AND METHODS Islets were isolated from Lewis rats by intraductal collagenase digestion as previously described,7 purified over a discontinuous Euroficoll gradient, and cultured overnight before in vitro assays. Islet purity was ⬎95% in all isolations. Recombinant rat IL-1 (50 U/mL), IFN-␥ (103 U/mL), and TNF-␣ (103 U/mL) were utilized in combination to induce apoptosis. Untreated islets were used as controls. The fluorometric assay FluorAce Apopain Assay Kit (BIO-RAD, Hercules, Calif) was utilized to measure caspase 3 activity on cellular extracts. The fluorogenic peptide substrate Ac-DEVD-AFC was utilized at a concentration of 55 mol/L. The caspase 3 inhibitor Z-DEVD-FMK was utilized at a concentration of 15 mol/L to distinguish caspase 3 activity from nonspecific protease activity. Readings were made on a VersaFluor fluorometer (BIO-RAD) with excitation and emission wavelengths of 390 nm and 520 nm, respectively. An Annexin V assay (Annexin-VFLUOS Staining Kit, Roche Boehringer Mannheim Corp.) was performed after cell dissociation in trypsin-EDTA (0.05% trypsin, 0.53 mmol/L EDTA) by gentle pipetting for 4 minutes. Propidium iodide (PI) staining was performed simultaneously with the Annexin V staining to differentiate apoptotic cells (single Annexin V-positive) from necrotic cells (double Annexin V-PI-positive). 0041-1345/01/$–see front matter PII S0041-1345(00)02006-6
Readings were made on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif) using 488-nm excitation and a 530-nm bandpass filter for fluorescein detection and a 650-nm longpass filter for PI detection. Detection of DNA fragmentation in situ was performed on 5-m-thick paraffin sections of whole islets using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay (In Situ Cell Death Detection, POD, Roche Boehringer Mannheim Corp). DNA fragmentation analysis by gel electrophoresis was performed after overnight DNA extraction (0.1 mg/mL proteinase K) and RNase treatment. Electrophoresis was run at 100 V for 90 minutes on a 1.2% agarose gel. The statistical analysis used a one-tailed Student’s t-test for the comparison of paired continuous variables. Values of P ⬍ .05 were considered significant.
RESULTS AND DISCUSSION
Measurable caspase 3 activity and Annexin V positivity were detected after a 24-hour culture period under standard conditions, whereas no substantial DNA fragmentation was observed at this time with a TUNEL assay and DNA gel electrophoresis. As soon as 3 hours after cytokine stimulation, a statistically significant increase in caspase 3 activity was observed in the treated group when compared to the control group (1.4 ⫾ 0.45 U/100 IEQ vs 0.8 ⫾ 0.3 U/100 IEQ, P ⬍ .05). This difference remained significant at 6 hours (P ⬍ .05) and 12 hours (P ⬍ .01) after stimulation. Annexin V assay showed a significant increase in the percentage of apoptotic cells (Annexin V-positive cells) after 6 hours of stimulation (22.4 ⫾ 7.2% in the control group vs 27.5 ⫾ 8.1% in the treated group; P ⬍ .01). Cytokine treatment also resulted in an increased percentage of necrotic cells (Annexin V-PI-positive cells) as soon as 3 hours after cytokine incubation (6.2 ⫾ 1.7% in the control group vs 10.6 ⫾ 2.6% in the treated group; P ⬍ .05). Percentages of TUNEL-positive nuclei were 43 ⫾ 5% and 69 ⫾ 11% after 24 hours and 48 hours of cytokine stimuFrom the Diabetes Research Institute, University of Miami School of Medicine, Miami, Florida, USA. P.C. is supported by grants from the Glaxo Wellcome Laboratories and from Assistance Publique-Hopitaux de Paris. T.B. is supported by a grant from the Swiss Foundation for BiologicalMedical Grants. Address reprint requests to Luca Inverardi, MD, Diabetes Research Institute, University of Miami School of Medicine, 1450 NW 10th Avenue, Miami, FL 33136. E-mail: linverar@med. miami.edu. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
264
Transplantation Proceedings, 33, 264–265 (2001)
ASSESSMENT OF APOPTOSIS IN ISLET CELLS
lation and 17 ⫾ 5% and 19 ⫾ 5% in control islets at the same time points, respectively. Gel electrophoresis showed a distinct DNA laddering after 24 hours of cytokine stimulation, but not in the control islets. DNA fragmentation was no longer detectable after 48 hours in either preparation. The increase in TUNEL-positive nuclei and the appearance of DNA laddering after cytokine stimulation clearly validate the signal observed earlier with the caspase 3 and Annexin V assays. Whereas caspase 3 measurement has the advantage of being detectable as early as 3 hours after the delivery of an apoptotic signal, Annexin V-PI staining is more suitable for evaluating the viability of the preparations in a quantitative fashion. These two assays are easy to perform and have good reproducibility. If the analysis of apoptosis/necrosis is to be a powerful assay for the prospective analysis of islet function within time frames compatible with a transplant procedure, the early assays seem to
265
provide a concrete advantage, in terms of both baseline sensitivity and kinetics.
REFERENCES 1. Paraskevas S, Duguid WP, Maysinger D, et al: Transplant Proc 29:750, 1997 2. Petrik J, Arany E, McDonald TJ, et al: Endocrinology 139: 2994, 1998 3. Ilieva A, Yuan S, Wang RN, et al: J Endocrinol 161:357, 1999 4. Thomas FT, Contreras JL, Bilbao G, et al: Surgery 126:299, 1999 5. Vargas F, Vives-Pi M, Somoza N, et al: Transplantation 65:722, 1998 6. Berney T, Molano RD, Cattan P, et al: Transplantation (in press) 7. Rabinovitch A, Suarez-Pinzon WL, Shi Y, et al: Diabetologia 37:733, 1994