Recovery of the immune system in diabetic mice after transplantation of isolated islets of Langerhans

Diabetologia, Jul 1984

Summary Adult CBA/HZgb mice islets harvested by collagenase digestion were injected intraperitoneally in 52 singeneic diabetic recipients (CBA/HZgb→CBA/HZgb, 450–600 islets per mouse). Normal serum glucose levels, 24-h urine volume, insulin levels and body weight were completely restored to normal in all recipients during the next 2–5 months. Immunological function was assessed in control, diabetic and diabetic-transplanted mice by following their responses to sheep erythrocytes (expressed as the number plaque-forming cells in the spleen). In transplanted mice, the plaque-forming cell responses were as follows: 1 month after transplantation-43% of the plaque-forming cell counts in control (normal, non-diabetic) mice; 2 months after transplantation — 56% of the control value; and 94% of the control value after 5 months. Ten months after the transplantation, the plaque-forming cell counts were slightly above the control value (148%). It appears, therefore that transplantated islet tissue positively affects the immunological as well as the diabetic state of the recipients.

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Recovery of the immune system in diabetic mice after transplantation of isolated islets of Langerhans

Recovery of the immune system in diabetic mice after transplantation o f isolated islets of Langerhans M. Had~ija 0 M. Slijep~evir 0 V. Sverko 0 T. Marotti 0 a n d M. Poljak-Bla~i 0 0 Ruder Bogkovi6Institute , Zagreb , Yugoslavia Summary. Adult CBA/HZgb mice islets harvested by collagenase digestion were injected intraperitoneally in 52 singeneic diabetic recipients, (CBA/HZgb---~CBA/HZgb, 450-600 islets per mouse). Normal serum glucose levels, 24-h urine volume, insulin levels and body weight were completely restored to normal in all recipients during the next 2-5 months. Immunological function was assessed in control, diabetic and diabetic-transplanted mice by following their responses to sheep erythrocytes (expressed as the number plaque-forming cells in the spleen). In transplanted mice, the plaque-forming cell responses were as follows: 1 month after transplantation - 4 3 % of the plaque-forming cell counts in control (normal, non-diabetic) mice; 2 months after transplantation - 5 6 % of the control value; and 94% of the control value after 5 months. Ten months after the transplantation, the plaque-forming cell counts were slightly above the control value (148%). It appears, therefore that transplantated islet tissue positively affects the immunological as well as the diabetic state of the recipients. Islet transplantation; immune system; experimental diabetes; pancreatic islet isolation - 9 Springer-Verlag1984 Bacterial a n d m y c o t i c infections are c o m m o n in diabetes mellitus [ 1 ]. Such infections are difficult to eradicate, a n d complicate m a n a g e m e n t o f the diabetic state. Num e r o u s investigators have r e p o r t e d that the susceptibility o f diabetic patients to infections should be ascribed to defects o f p o l y m o r p h o n u c l e a r leucocytes [ 2, 3 ]. O t h e r investigators n o t e d decreased n u m b e r s o f T- a n d B-lymp h o c y t e s in peripheral b l o o d a n d a depression o f lymp h o c y t i c response to phytohaemagglutinin, suggesting i m p a i r e d cell-mediated i m m u n i t y in diabetic patients [ 4-6 ]. In a rat model, M a h m o u d et al. [7] also f o u n d suppression o f cell-mediated immunity. Paveli6 et al. [ 8 ] described a decreased n u m b e r o f nuclear cells in t h y m u s a n d spleen o f alloxan diabetic mice. B r o w n et al. [ 9 ] a n d o u r g r o u p [ 10 ] ,described decreased a n t i b o d y p r o d u c tion to sheep red b l o o d cells, a T-cell d e p e n d e n t antigen [ 9, 10 ]. Rejection o f skin allografts was delayed in animals with experimental diabetes [ 8, 11 ]. According to o u r d e t e r m i n a t i o n o f E, E37 or E A C rosettes in spleens o f diabetic mice [ 12 ], defect o f i m m u n o l o g i c a l function appears to be due not only to metabolic disturbances, b u t possibly also to altered p r o p o r t i o n s o f the s u b p o p u lations o f T and B cells. Daily injections o f insulin into animals with experimental diabetes only partly restored the immunological function. This response was dep e n d e n t on duration o f the disease [ 13 ]. In this report we c o m p a r e reversal o f the diabetic state in mice after transplantation o f isolated adult islets f r o m syngeneic d o n o r s with recovery o f their i m m u n o l o g i c a l system. Materials and methods Animals and induction of experimental diabetes Female CBA/HZgb mice, aged 2 and 4 months, were used as donors or recipients of isolated pancreatic tissue. Recipients were made diabetic by intravenous injection of alloxan (50mg/kg body weight, Sigma, monohydrate, Lot. 50F-3823), prepared in phosphate-buffered saline (Hank's) solution. The drug was injected 14 days before planned transplantation. Mice received food and water ad libitum both before and after injection of the drug. Blood glucose was determined by the method of Hyvarinen and Nikkil/~, as modified by our laboratory [ 14 ]. Test-Tape strips (Gluketur-Test, Pliva, Zagreb) were used to check glycosuria. Only mice with glycosuria of> 28 mmol/1 were considered diabetic and were used in the experiment. Isolation of islets of Langerhans Islets of Langerhans were isolated from adult mice,aged 2 months, by the collagenase method of Lacy and Kostianovsky [ 16 ]. Following perfusion and distention of pancreatic tissue with 10ml of cold (4~ Hank's solution containing 0.68% neutral red, aprotinin (Trasylol, 500units/ml, Sigma), the pancreas was excised, chopped into fine pieces with scissors, and digested in Hank's solution containing col9 42o 30o- I | Time (month } lagenase (10 mg/ml, Sigma), bovine serum albumin (10 mg/ml, Sigma), and Hepes (20 mmol/1, Sigma). This was carried out within 12-17 min in a shaker bath at 36.5 ~ ~ operating at 95 cycles/min. Cold (4 ~ Hank's solution (50 ml) was then added. Fragments were further dispersed manually by repeated aspiration (40-55 times) with a siliconized Pasteur pipette, washed, and centrifuged at 200 g for 2 min. Sedimented tissue was dispersed with Hank's solution and free islets of Langerhans were collected by means of a Pasteur pipette under stereo microscope (Opton) at magnification x 20. Suspension of isolated islets was inoculated intraperitoneally, 450-600islets per mouse. Assay o fplaque-forming cells Washed sheep red blood cells (2 x 10 s in 0.5 ml) were injected intravenously. After 4 days plaque-forming cells were counted in the spleen, the haemolytic plaque test of Jerne et al. [15] as carried out in our laboratory [17]. Results Effect o f islet transplantation on recovery from the diabetic state In diabetic mice body weight decreased rapidly (Fig. 1), the volume o f urine increased to as high as 28.2+ 3.6 ml/day (compared with 0.33 ml/day in normal nondiabetic mice), and the concentration o f insulin in plasma (radioimmunoassay test) decreased to 8.0_+ 1.3mU/1 (compared with 12.9_+0.4mU/1 in normal mice). Transplantation o f islets resulted in rapid recovery o f body weight (Fig. 1), decrease o f urine volume to normal values within 2-5 months after transplantation, and restoration o f insulin secretion. Two months after islets transplantation, plasma insulin was higher than in control mice (15.1 + 0.22 mU/1; p < 0.025). In diabetic mice there was evident impairment o f immune response (Table 1). Transplantation o f the islets resulted in recovery o f immune competence: 2 months after transplantation, the mean plaque-forming cell 0 o 9 e., ~d ? t"q r +1 +1 +1 8 O'~ t"e9 'm +1 M. Had~ija et al.: Met transplantationand the immune system count was 56% of that in control non-diabetic mice; at 5 months, it approached the control value (96%), and at 10 months it was even increased in islet recipients (48% above control value). Discussion Alloxan exerts a highly specific cytotoxic effect on the fl cells of mouse pancreatic islets, and the resulting clinical condition closely resembles human insulin-dependent diabetes mellitus. In this model, using syngeneic mice and transplanting isolated islets, we demonstrated recovery of body weight, normalisation of glucose level in blood, disappearance of glycosuria, and rise of insulin concentration in serum. The immune response to bacterial antigens is reduced in diabetic patients [ 2, 3, 5 ]. In addition to the correction of metabolic derangement, islet transplantation also resulted in recovery of immune competence. Treatment with insulin corrects metabolic status but the immune competence is only partly restored [ 14 ]. This may indicate that the immune system requires a full complement of islet hormones for its proper function, and that impairment reflects not only metabolic, but also a hormonal defect. 1. Younger D , Hadley BW ( 1971 ) Infection in diabetes . In: Morbile A , White P , Bradley RF , Krall LP (eds) Joslins diabetes mellitus , Lea & Febiger , Philadelphia, pp 621 - 636 2. Nolan CM , Beaty HN , Bagdade JD ( 1978 ) Further characterization of the impaired bacterial function of granulocytes in patients with poorly controlled diabetes . Diabetes 27 : 889 - 894 3. Cech P , Stalder H , Widman JJ , Rohner A , Miescher PA ( 1979 ) Leukocyte myeloperoxidase deficiency and diabetes mellitus associated with candida albicans liver abscess . Am J Med 66 : 149 - 153 4. Cattaneo R , Saibene V , Pozza G ( 1976 ) Peripheral T-lymphocytes in juvenile-onsetdiabetes OOD) and in maturnity-onsetdiabetics (MOD) . Diabetes 25 : 223 - 226 5. Kadrnka-Lovren~ i6 M, Rabati6 S , Ma~uran R , Oberiter V , Dekaris D ( 1979 ) Cell mediated immunityin children with juvenile onset diabetes mellitus . Period Biol 81 : 255 - 256 6. Selam JL , Glot J , Mirouze J ( 1979 ) Circulating lymphocyte subpopulations in juvenile insulin-dependentdiabetes. Correction of abnormalities by adequate blood glucose control . Diabetologia 16 : 35 - 40 7. Mahmoud AF , Rodman HM , Mandel MA , Warren KS ( 1976 ) Induced and spontaneous diabetes mellitus and suppression of cellmediated immunologicresponses: granuloma formation, delayed dermal reactivity,and allograft rejection . J Clin Invest 57 : 362 - 367 8. Paveli6 K , Slijep~evi6 M, Paveli6 J ( 1978 ) Recovery of immune system in diabetic mice after treatment with insulin . Horm Metab Res 10 : 381 - 386 9. Brown DM , Yunis EJ , Fernandes G ( 1977 ) T-cell function in diabetic mice . Fed Porc 36 : 1258 (Abstract) 10. Maduna S , Slijeprevi6 M , Had~ija M, Stankovi6 V ( 1980 ) Immunological reactivity in diabetes mellitus-analysis of differences among various forms immunologicresponse . Diab Croat 9 : 61 - 74 11. Friedman EA , Beyer MM ( 1977 ) Immune competence of the streptozotocin-induced diabetic rats. I. Absent second-set skin allograft response . Transplantation24 : 367 - 371 12. Had2ija M , Benkovi6 B , Slijeprevi6 M ( 1979 ) Effect of insulinand of the oral antidiabeticdrugs Euglucon and Glurenorm on the immunological functions of diabetic mice . Period Biol 81 : 185 - 186 13. Slijeprevi6 M , Had~ija M, Stankovi6 V (1979) Long lasting experimental diabetes and immunologicalresponse in mice . Period Biol 81 : 183 - 184 14. Slijeprevi6 M , Rada~i6 M ( 1976 ) The growth of a transplantable leukemia in normal and alloxan treated mice . Period Biol 78 : 130 - 132 15. Jerne NK , Nordin RA , Henry O ( 1963 ) The agar plaque technique for recognizing antibody forming cells . In: Amos B , Koprowski H (eds) Cell bound antibodies . Wister Institute Press, Philadelphia, pp109- 122 16. Lacy PE , Kostianovsky M ( 1967 ) Method for the isolation of intact islets of Langerhans from the rat pancreas . Diabetes 16 : 35 - 39 17. Hrgak I , Marotti T ( 1973 ) Immunosuppressionmediated by Erlich ascites fluid . Eur J Cancer 9 : 717 - 724


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M. Hadžija, M. Slijepčević, V. Šverko, T. Marotti, M. Poljak-Blaži. Recovery of the immune system in diabetic mice after transplantation of isolated islets of Langerhans, Diabetologia, 1984, 93-95, DOI: 10.1007/BF00275657