Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Expression of Human Leukocyte Antigen Class I in Endocrine and Exocrine Pancreatic Tissue at Onset of Type 1 Diabetes

2014; Elsevier BV; Volume: 185; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2014.09.004

ISSN

1525-2191

Autores

Oskar Skog, Stella Korsgren, Anna Wiberg, Angelika Danielsson, Bjørn Edwin, Trond Buanes, Lars Krogvold, Olle Korsgren, Knut Dahl‐Jørgensen,

Tópico(s)

Immune Cell Function and Interaction

Resumo

The cause of type 1 diabetes remains unknown. To dissect the link between hyperexpression of human leukocyte antigen (HLA) class I on the islet cells, we examined its expression in subjects with recent-onset type 1 diabetes. IHC showed seemingly pronounced hyperexpression in subjects with recent-onset type 1 diabetes, as well as in some nondiabetic subjects. In all subjects, HLA class I expression on exocrine tissue was low. However, no difference in the level of HLA class I expression was found between islet and exocrine tissue using Western blot, flow cytometry, real-time quantitative PCR, or RNA sequencing analyses. Also, the level of HLA class I expression on the messenger level was not increased in islets from subjects with recent-onset type 1 diabetes compared with that in nondiabetic subjects. Consistently, the HLA class I specific enhanceosome (NLRC5) and related transcription factors, as well as interferons, were not enhanced in islets from recent-onset type 1 diabetic subjects. In conclusion, a discrepancy in HLA class I expression in islets assessed by IHC was observed compared with that using quantitative techniques showing similar expression of HLA class I in islets and exocrine tissue in subjects with recent-onset type 1 diabetes, nor could any differences be found between type 1 diabetic and nondiabetic subjects. Results presented provide important clues for a better understanding on how this complex disease develops. The cause of type 1 diabetes remains unknown. To dissect the link between hyperexpression of human leukocyte antigen (HLA) class I on the islet cells, we examined its expression in subjects with recent-onset type 1 diabetes. IHC showed seemingly pronounced hyperexpression in subjects with recent-onset type 1 diabetes, as well as in some nondiabetic subjects. In all subjects, HLA class I expression on exocrine tissue was low. However, no difference in the level of HLA class I expression was found between islet and exocrine tissue using Western blot, flow cytometry, real-time quantitative PCR, or RNA sequencing analyses. Also, the level of HLA class I expression on the messenger level was not increased in islets from subjects with recent-onset type 1 diabetes compared with that in nondiabetic subjects. Consistently, the HLA class I specific enhanceosome (NLRC5) and related transcription factors, as well as interferons, were not enhanced in islets from recent-onset type 1 diabetic subjects. In conclusion, a discrepancy in HLA class I expression in islets assessed by IHC was observed compared with that using quantitative techniques showing similar expression of HLA class I in islets and exocrine tissue in subjects with recent-onset type 1 diabetes, nor could any differences be found between type 1 diabetic and nondiabetic subjects. Results presented provide important clues for a better understanding on how this complex disease develops. The cause of type 1 diabetes remains unknown. The disease seems to be a result of a complex interplay between genetic predisposition, the immune system, and environmental factors.1Eisenbarth G.S. Update in type 1 diabetes.J Clin Endocrinol Metab. 2007; 92: 2403-2407Crossref PubMed Scopus (104) Google Scholar Pancreas is a difficult organ to study, and the methodological options in studying living islets are extremely limited. Most published studies describe pancreatic materials collected post-mortem. An updated review summarizes the present knowledge of the morphological characteristics and insulitis in type 1 diabetes.2In't Veld P. Insulitis in human type 1 diabetes: the quest for an elusive lesion.Islets. 2011; 3: 131-138Crossref PubMed Scopus (152) Google Scholar Although these studies have provided new insight in the inflammatory process in humans with type 1 diabetes, these materials have several disadvantages because of post-mortem autolysis, patient heterogeneity, and lack of clinical information. In The Diabetes Virus Detection (DiViD) Study, fresh pancreatic tissue was collected from living newly diagnosed type 1 diabetic patients shortly after diagnosis, and used in the present study.3Krogvold L. Edwin B. Buanes T. Ludvigsson J. Korsgren O. Hyoty H. Frisk G. Hanssen K.F. Dahl-Jorgensen K. Pancreatic biopsy by minimal tail resection in live adult patients at the onset of type 1 diabetes: experiences from the DiViD study.Diabetologia. 2014; 57: 841-843Crossref PubMed Scopus (116) Google Scholar The focus of type 1 diabetes research has mainly been concentrated on the endocrine pancreas. However, recently, the exocrine pancreas has attained more interest.4Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus.Diabetes. 1965; 14: 619-633Crossref PubMed Scopus (982) Google Scholar, 5Sarkar S.A. Lee C.E. Victorino F. Nguyen T.T. Walters J.A. Burrack A. Eberlein J. Hildemann S.K. Homann D. Expression and regulation of chemokines in murine and human type 1 diabetes.Diabetes. 2012; 61: 436-446Crossref PubMed Scopus (102) Google Scholar, 6Coppieters K.T. Dotta F. Amirian N. Campbell P.D. Kay T.W. Atkinson M.A. Roep B.O. von Herrath M.G. Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients.J Exp Med. 2012; 209: 51-60Crossref PubMed Scopus (463) Google Scholar A reduction in total pancreatic volume seems present already at the time of diagnosis when compared with nondiabetic healthy volunteers,7Silva M.E. Vezozzo D.P. Ursich M.J. Rocha D.M. Cerri G.G. Wajchenberg B.L. Ultrasonographic abnormalities of the pancreas in IDDM and NIDDM patients.Diabetes Care. 1993; 16: 1296-1297Crossref PubMed Scopus (34) Google Scholar, 8Gaglia J.L. Guimaraes A.R. Harisinghani M. Turvey S.E. Jackson R. Benoist C. Mathis D. Weissleder R. Noninvasive imaging of pancreatic islet inflammation in type 1A diabetes patients.J Clin Invest. 2011; 121: 442-445Crossref PubMed Scopus (158) Google Scholar, 9Williams A.J. Thrower S.L. Sequeiros I.M. Ward A. Bickerton A.S. Triay J.M. Callaway M.P. Dayan C.M. Pancreatic volume is reduced in adult patients with recently diagnosed type 1 diabetes.J Clin Endocrinol Metab. 2012; 97: E2109-E2113Crossref PubMed Scopus (97) Google Scholar and a subclinical exocrine deficiency has also been reported.10Frier B.M. Saunders J.H. Wormsley K.G. Bouchier I.A. Exocrine pancreatic function in juvenile-onset diabetes mellitus.Gut. 1976; 17: 685-691Crossref PubMed Scopus (143) Google Scholar, 11Frier B.M. Faber O.K. Binder C. Elliot H.L. The effect of residual insulin secretion on exocrine pancreatic function in juvenile-onset diabetes mellitus.Diabetologia. 1978; 14: 301-304Crossref PubMed Scopus (68) Google Scholar, 12Landin-Olsson M. Borgstrom A. Blom L. Sundkvist G. Lernmark A. Swedish Childhood Diabetes GroupImmunoreactive trypsin(ogen) in the sera of children with recent-onset insulin-dependent diabetes and matched controls.Pancreas. 1990; 5: 241-247Crossref PubMed Scopus (14) Google Scholar, 13Groger G. Layer P. Exocrine pancreatic function in diabetes mellitus.Eur J Gastroenterol Hepatol. 1995; 7: 740-746PubMed Google Scholar, 14Hardt P.D. Ewald N. Exocrine pancreatic insufficiency in diabetes mellitus: a complication of diabetic neuropathy or a different type of diabetes?.Exp Diabetes Res. 2011; 2011: 761950Crossref PubMed Scopus (81) Google Scholar Morphological examinations of the pancreas from subjects with type 1 diabetes show considerable engagement of the exocrine pancreas.4Gepts W. Pathologic anatomy of the pancreas in juvenile diabetes mellitus.Diabetes. 1965; 14: 619-633Crossref PubMed Scopus (982) Google Scholar, 5Sarkar S.A. Lee C.E. Victorino F. Nguyen T.T. Walters J.A. Burrack A. Eberlein J. Hildemann S.K. Homann D. Expression and regulation of chemokines in murine and human type 1 diabetes.Diabetes. 2012; 61: 436-446Crossref PubMed Scopus (102) Google Scholar, 6Coppieters K.T. Dotta F. Amirian N. Campbell P.D. Kay T.W. Atkinson M.A. Roep B.O. von Herrath M.G. Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients.J Exp Med. 2012; 209: 51-60Crossref PubMed Scopus (463) Google Scholar Collectively, available information demonstrates that type 1 diabetes in humans is a pancreatic disease, with its main clinical manifestations emanating from the loss of the insulin-producing cells. Still, a consistently reported finding in subjects with type 1 diabetes is hyperexpression of human leukocyte antigen (HLA) class I in the islets of Langerhans, whereas this expression seems almost absent on the exocrine cells.6Coppieters K.T. Dotta F. Amirian N. Campbell P.D. Kay T.W. Atkinson M.A. Roep B.O. von Herrath M.G. Demonstration of islet-autoreactive CD8 T cells in insulitic lesions from recent onset and long-term type 1 diabetes patients.J Exp Med. 2012; 209: 51-60Crossref PubMed Scopus (463) Google Scholar, 15Bottazzo G.F. Dean B.M. McNally J.M. MacKay E.H. Swift P.G. Gamble D.R. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis.N Engl J Med. 1985; 313: 353-360Crossref PubMed Scopus (781) Google Scholar, 16Foulis A.K. Farquharson M.A. Hardman R. Aberrant expression of class II major histocompatibility complex molecules by B cells and hyperexpression of class I major histocompatibility complex molecules by insulin containing islets in type 1 (insulin-dependent) diabetes mellitus.Diabetologia. 1987; 30: 333-343Crossref PubMed Scopus (188) Google Scholar, 17Hanafusa T. Miyazaki A. Miyagawa J. Tamura S. Inada M. Yamada K. Shinji Y. Katsura H. Yamagata K. Itoh N. Asakawa H. Nakagawa C. Otsuka A. Kawata S. Kono N. Tarui S. Examination of islets in the pancreas biopsy specimens from newly diagnosed type 1 (insulin-dependent) diabetic patients.Diabetologia. 1990; 33: 105-111Crossref PubMed Scopus (102) Google Scholar, 18Hanninen A. Jalkanen S. Salmi M. Toikkanen S. Nikolakaros G. Simell O. Macrophages, T cell receptor usage, and endothelial cell activation in the pancreas at the onset of insulin-dependent diabetes mellitus.J Clin Invest. 1992; 90: 1901-1910Crossref PubMed Scopus (214) Google Scholar, 19Itoh N. Hanafusa T. Miyazaki A. Miyagawa J. Yamagata K. Yamamoto K. Waguri M. Imagawa A. Tamura S. Inada M. Mononuclear cell infiltration and its relation to the expression of major histocompatibility complex antigens and adhesion molecules in pancreas biopsy specimens from newly diagnosed insulin-dependent diabetes mellitus patients.J Clin Invest. 1993; 92: 2313-2322Crossref PubMed Scopus (290) Google Scholar, 20Somoza N. Vargas F. Roura-Mir C. Vives-Pi M. Fernandez-Figueras M.T. Ariza A. Gomis R. Bragado R. Marti M. Jaraquemada D. Pujol-Borrell R. Pancreas in recent onset insulin-dependent diabetes mellitus: changes in HLA, adhesion molecules and autoantigens, restricted T cell receptor V beta usage, and cytokine profile.J Immunol. 1994; 153: 1360-1377PubMed Google Scholar, 21Imagawa A. Hanafusa T. Itoh N. Waguri M. Yamamoto K. Miyagawa J. Moriwaki M. Yamagata K. Iwahashi H. Sada M. Tsuji T. Tamura S. Kawata S. Kuwajima M. Nakajima H. Namba M. Matsuzawa Y. Immunological abnormalities in islets at diagnosis paralleled further deterioration of glycaemic control in patients with recent-onset type I (insulin-dependent) diabetes mellitus.Diabetologia. 1999; 42: 574-578Crossref PubMed Scopus (45) Google Scholar, 22Coppieters K.T. von Herrath M.G. Histopathology of type 1 diabetes: old paradigms and new insights: the review of diabetic studies.Rev Diabet Stud. 2009; 6: 85-96Crossref PubMed Scopus (42) Google Scholar, 23Tanaka S. Nishida Y. Aida K. Maruyama T. Shimada A. Suzuki M. Shimura H. Takizawa S. Takahashi M. Akiyama D. Arai-Yamashita S. Furuya F. Kawaguchi A. Kaneshige M. Katoh R. Endo T. Kobayashi T. Enterovirus infection, CXC chemokine ligand 10 (CXCL10), and CXCR3 circuit: a mechanism of accelerated beta-cell failure in fulminant type 1 diabetes.Diabetes. 2009; 58: 2285-2291Crossref PubMed Scopus (139) Google Scholar, 24Richardson S.J. Willcox A. Bone A.J. Morgan N.G. Foulis A.K. Immunopathology of the human pancreas in type-I diabetes.Semin Immunopathol. 2011; 33: 9-21Crossref PubMed Scopus (68) Google Scholar Also, in the area of solid organ transplantation, similar observations have been reported.25Daar A.S. Fuggle S.V. Fabre J.W. Ting A. Morris P.J. The detailed distribution of HLA-A, B, C antigens in normal human organs.Transplantation. 1984; 38: 287-292Crossref PubMed Scopus (352) Google Scholar HLA class I molecules are ubiquitously expressed on the surface of almost all nucleated cells, and their surface density determines, to a large extent, the function and strength of the CD8+ T-cell–dependent immune surveillance of all cells within our bodies. Expression of HLA class I is regulated by cell type–specific factors,26Meissner T.B. Li A. Biswas A. Lee K.H. Liu Y.J. Bayir E. Iliopoulos D. van den Elsen P.J. Kobayashi K.S. NLR family member NLRC5 is a transcriptional regulator of MHC class I genes.Proc Natl Acad Sci U S A. 2010; 107: 13794-13799Crossref PubMed Scopus (276) Google Scholar, 27Kobayashi K.S. van den Elsen P.J. NLRC5: a key regulator of MHC class I-dependent immune responses.Nat Rev Immunol. 2012; 12: 813-820Crossref PubMed Scopus (203) Google Scholar, 28Meissner T.B. Li A. Kobayashi K.S. NLRC5: a newly discovered MHC class I transactivator (CITA).Microbes Infect. 2012; 14: 477-484Crossref PubMed Scopus (51) Google Scholar, 29Meissner T.B. Liu Y.J. Lee K.H. Li A. Biswas A. van Eggermond M.C. van den Elsen P.J. Kobayashi K.S. NLRC5 cooperates with the RFX transcription factor complex to induce MHC class I gene expression.J Immunol. 2012; 188: 4951-4958Crossref PubMed Scopus (87) Google Scholar but can also be up-regulated by various cytokines [interferon (IFN) αβγ and tumor necrosis factor (TNF) α].30Campbell I.L. Bizilj K. Colman P.G. Tuch B.E. Harrison L.C. Interferon-gamma induces the expression of HLA-A, B,C but not HLA-DR on human pancreatic beta-cells.J Clin Endocrinol Metab. 1986; 62: 1101-1109Crossref PubMed Scopus (44) Google Scholar, 31Campbell I.L. Oxbrow L. West J. Harrison L.C. Regulation of MHC protein expression in pancreatic beta-cells by interferon-gamma and tumor necrosis factor-alpha.Mol Endocrinol. 1988; 2: 101-107Crossref PubMed Scopus (53) Google Scholar, 32Pujol-Borrell R. Todd I. Doshi M. Gray D. Feldmann M. Bottazzo G.F. Differential expression and regulation of MHC products in the endocrine and exocrine cells of the human pancreas.Clin Exp Immunol. 1986; 65: 128-139PubMed Google Scholar In contrast, HLA class I down-regulation occurs via an array of mechanisms by viruses (adenovirus, cytomegalovirus, human papilloma virus, HIV, and hepatitis C virus) and constitutes a viable strategy to promote immune escape.33Seliger B. Ritz U. Ferrone S. Molecular mechanisms of HLA class I antigen abnormalities following viral infection and transformation.Int J Cancer. 2006; 118: 129-138Crossref PubMed Scopus (110) Google Scholar, 34Ploegh H.L. Viral strategies of immune evasion.Science. 1998; 280: 248-253Crossref PubMed Scopus (686) Google Scholar However, this down-regulation is a double-edged sword because it may also trigger natural killer cell–mediated lysis of the infected cells.35Karre K. Welsh R.M. Viral decoy vetoes killer cell.Nature. 1997; 386: 446-447Crossref PubMed Scopus (24) Google Scholar Hyperexpression of HLA class I on the islet cells has so far only been reported using immunohistochemical (IHC) techniques. IHC is not a quantitative technique, and results obtained depend on several factors other than the actual level of expression of the targeted antigen.36Education Guide: Immunohistochemical (IHC) Staining Methods.in: Kumar G. Rudbeck L. Dako North America, Carpinteria, CA2009Google Scholar Therefore, the aim of the present study was to examine the expression of HLA class I on the insulin-producing cells using quantitative techniques on both protein and messenger levels in eight subjects examined 1 to 60 days after diagnosis of type 1 diabetes and to compare obtained findings with those in the exocrine cells and with those in nondiabetic control islets. All work involving human tissue was conducted according to the principles expressed in the Declaration of Helsinki and in the European Council's Convention on Human Rights and Biomedicine. Written informed consent was given by the patients with type 1 diabetes (T1D), and the DiViD Study (http://www.clinicaltrials.gov, study NCT01129232, last accessed August 28, 2014) was approved by the Governmental Regional Ethics Committee (Oslo, Norway). Consent for organ donation (for clinical transplantation and for use in research) was obtained verbally from the deceased's next of kin by the attending physician and documented in the medical records of the deceased in accordance with Swedish law and as approved by the Regional Ethics Committee. The study was approved by the Regional Ethics Committee (Uppsala, Sweden), according to the Act Concerning the Ethical Review of Research Involving Humans. Laparoscopic pancreatic tail resections were performed on six live patients with recent-onset T1D in the DiViD Study (donors T1-3 to T1-8) (Table 1). Biopsy specimens from the pancreatic tail resections and from the head region of the organ donor pancreases were immediately fixed in 4% paraformaldehyde or immersed in liquid nitrogen and subsequently stored at −80°C. The clinical characteristics of these patients have been described previously.3Krogvold L. Edwin B. Buanes T. Ludvigsson J. Korsgren O. Hyoty H. Frisk G. Hanssen K.F. Dahl-Jorgensen K. Pancreatic biopsy by minimal tail resection in live adult patients at the onset of type 1 diabetes: experiences from the DiViD study.Diabetologia. 2014; 57: 841-843Crossref PubMed Scopus (116) Google Scholar Pancreatic specimens from multiorgan donors that died at onset of T1D (n = 2),37Korsgren S. Molin Y. Salmela K. Lundgren T. Melhus A. Korsgren O. On the etiology of type 1 diabetes: a new animal model signifying a decisive role for bacteria eliciting an adverse innate immunity response.Am J Pathol. 2012; 181: 1735-1748Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar with type 2 diabetes (T2D) (n = 9), with autoantibodies against IA2 (insulinoma associated protein 2) and/or glutamic acid decarboxylase 65 (n = 6), and without pancreatic disease or diabetes-related autoantibodies (n = 14), were obtained through the Nordic Network for Clinical Islet Transplantation (Table 1). These cases were used for analysis of HLA class I protein expression by IHC. All cases with T1D and three nondiabetic controls were used for the analysis of HLA class I RNA expression in handpicked islets. Two T1D donors (cases T1 and T2) and four nondiabetic controls were used for analysis of HLA class I RNA expression in laser-captured islets. Five nondiabetic controls were used for analysis of HLA class I expression by flow cytometry.Table 1Donor CharacteristicsDonor no.T1D (weeks from diagnosis)T2DAutoantibodiesAge, yearsBMI, kg/m2HbA1c, % (mmol/mol)HLA exo∗Intensity of staining was evaluated visually and graded from 0 (negative) to 4 (intense).HLA endo∗Intensity of staining was evaluated visually and graded from 0 (negative) to 4 (intense).T1-1Y (0)NNeg4027.2ND01–2T1-2Y (0)NNeg2924.210.412–4T1-3Y (4)NGAD/INS/ZnT8/IA225216.7 (50)04T1-4Y (3)NGAD/ZnT8/IA22420.910.3 (89)02–4T1-5Y (9)NGAD/ZnT8/IA23423.77.1 (54)24T1-6Y (5)NGAD/INS/IA23125.67.4 (57)02–4T1-7Y (5)NGAD/INS/IA22428.67.4 (57)01–4T1-8Y (5)NGAD3526.77.1 (54)24T2-1NYND5435.1ND03–4T2-2NYNeg7434.6ND02T2-3NYND7029.4ND02T2-4NYNeg1629.2ND01–3T2-5NYNeg7423.96.3 (45)0–12–4T2-6NYNeg6433.37.3 (56)12–3T2-7NYNeg4523.47.3 (56)42T2-8NYNeg6928.46.4 (46)1–22T2-9NYNeg6722.96.3 (45)1–22–4Ab-1NNGAD/IA23819.45.3 (34)13–4Ab-2NNGAD/IA27022.55.2 (33)0–13–4Ab-3NNGAD5232.15.8 (40)0–12–4Ab-4NNGAD3124.85.7 (39)0–12–4Ab-5NNGAD7427.25.8 (40)1–23–4Ab-6NNGAD5228.45.5 (37)NDNDC-1NNNeg4722.26.1 (43)0–13–4C-2NNNeg7225.24.2 (22)0–13–4C-3NNNeg6522.95.6 (38)1–23C-4NNND6642.2ND02–3C-5NNND5441.8ND02C-6NNND5640.7ND0–13–4C-7NNNeg1819.6ND01–2C-8NNND2024.1ND03C-9NNND1923.1ND02–4C-10NNND2224.4ND02C-11NNNeg820.45.2 (33)12C-12NNNeg2228.95.5 (37)23C-13NNNeg2522.95.9 (41)0–13C-14NNNeg2027.85.8 (40)02–3BMI, body mass index; endo, endocrine; exo, exocrine; GAD, glutamic acid decarboxylase; HbA1c, glycated hemoglobin; IA2, insulinoma associated protein 2; N, no; ND, not determined; Neg, negative; Y, yes.∗ Intensity of staining was evaluated visually and graded from 0 (negative) to 4 (intense). Open table in a new tab BMI, body mass index; endo, endocrine; exo, exocrine; GAD, glutamic acid decarboxylase; HbA1c, glycated hemoglobin; IA2, insulinoma associated protein 2; N, no; ND, not determined; Neg, negative; Y, yes. The most distal part (0.5 to 1 cm) of laparoscopic pancreatic tail resections performed at Oslo University Hospital (Oslo, Norway) was shipped in cold organ preservation solution (Viaspan, UW solution; Bristol-Myers Squibb AB, Solna, Sweden) to Uppsala University for islet isolation. The islets were isolated by a method based on the procedure used for clinical islet isolation that has been described previously.38Goto M. Eich T.M. Felldin M. Foss A. Kallen R. Salmela K. Tibell A. Tufveson G. Fujimori K. Engkvist M. Korsgren O. Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.Transplantation. 2004; 78: 1367-1375Crossref PubMed Scopus (180) Google Scholar Basically, the pancreatic duct was located under a surgical microscope and cannulated with a fine catheter, and collagenase (Liberase; Roche, Indianapolis, IN) was injected and digested at 37°C for 30 minutes. Islets (300 to 700) from each patient were handpicked under a microscope. Islets from the two brain-dead organ donors with acute-onset T1D and donors without pancreatic disease were isolated and cultured as described previously.37Korsgren S. Molin Y. Salmela K. Lundgren T. Melhus A. Korsgren O. On the etiology of type 1 diabetes: a new animal model signifying a decisive role for bacteria eliciting an adverse innate immunity response.Am J Pathol. 2012; 181: 1735-1748Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Goto M. Eich T.M. Felldin M. Foss A. Kallen R. Salmela K. Tibell A. Tufveson G. Fujimori K. Engkvist M. Korsgren O. Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture.Transplantation. 2004; 78: 1367-1375Crossref PubMed Scopus (180) Google Scholar RNA was extracted with the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Stockholm, Sweden) from 50 to 100 islets per subject, immediately after handpicking from the digested pancreatic tail resections, or after storage of isolated islets from the multiorgan donors on day 1 after isolation at −80°C in RNAlater (Qiagen, Stockholm, Sweden). The extracted RNA was of good quality (RNA Integrity Number values between 7.1 and 9.5) and enough quantity (>1 μg) for performing whole transcriptome sequencing. Fragment library construction, sequencing on an AB SOLiD 5500xl-W system, and mapping of reads were performed by the Uppsala Genome Center (Rudbeck Laboratory, Uppsala, Sweden).39Ameur A. Zaghlool A. Halvardson J. Wetterbom A. Gyllensten U. Cavelier L. Feuk L. Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain.Nat Struct Mol Biol. 2011; 18: 1435-1440Crossref PubMed Scopus (213) Google Scholar Data for HLA-A, HLA-B, HLA-C, and selected other genes are presented herein as reads per kilobase per million mapped reads. The entire data set will be published in a manuscript currently in preparation and made publicly available. Expression levels for HLA-A, HLA-B, HLA-C, and selected other genes were extracted from the transcriptome data, acquired at the Human Protein Atlas (Uppsala, Sweden) by Illumina (San Diego, CA),40Fagerberg L. Hallstrom B.M. Oksvold P. Kampf C. Djureinovic D. Odeberg J. Habuka M. Tahmasebpoor S. Danielsson A. Edlund K. Asplund A. Sjostedt E. Lundberg E. Szigyarto C.A. Skogs M. Takanen J.O. Berling H. Tegel H. Mulder J. Nilsson P. Schwenk J.M. Lindskog C. Danielsson F. Mardinoglu A. Sivertsson A. von Feilitzen K. Forsberg M. Zwahlen M. Olsson I. Navani S. Huss M. Nielsen J. Ponten F. Uhlen M. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.Mol Cell Proteomics. 2014; 13: 397-406Crossref PubMed Scopus (1964) Google Scholar from exocrine and endocrine pancreatic tissue isolated from four subjects without pancreatic disease. The data are presented as fragments per kilobase per million mapped reads. Sections (6 μm thick) from formalin-fixed, paraffin-embedded pancreatic biopsy specimens were processed and stained using a standard immunoperoxidase technique. After heat-induced epitope retrieval, the sections were stained with an anti-HLA class I ABC antibody (clone EMR8-5; dilution 1:300; Abcam, Cambridge, UK) visualized with Dako EnVision (Dako, Glostrup, Denmark). The dilution was optimized to minimize unspecific staining, and no staining was observed after removal of the primary antibody. The sections were counterstained with hematoxylin and analyzed by light microscopy. Intensity of staining was evaluated visually and graded from 0 (negative) to 4 (intense) by a blinded investigator (O.S.). Consecutive sections (8 μm thick) from frozen pancreatic biopsy specimens were treated and stained in the same way (but without heat-induced epitope retrieval) or mounted on PEN-membrane glass slides (Arcturus; Life Technologies, Paisley, UK) and used for laser-capture microdissection (LCM). LCM was performed using a protocol modified from Marselli et al.41Marselli L. Sgroi D.C. Bonner-Weir S. Weir G.C. Laser capture microdissection of human pancreatic beta-cells and RNA preparation for gene expression profiling.Methods Mol Biol. 2009; 560: 87-98Crossref PubMed Scopus (19) Google Scholar Briefly, frozen pancreatic sections were fixed in 70% ethanol for 30 seconds, rinsed by five dips in RNase-free water, stained with HistoGene Staining Solution (Life Technologies) containing 500 U/mL SuperAse (Life Technologies) for 1 minute, rinsed again in RNase-free water as above, dehydrated twice in 100% ethanol, and once in xylene for 5 minutes. After letting the slides air dry for 5 minutes, an ArcturusxT microdissection instrument was used to microdissect islets and exocrine tissue. Microdissected tissue was extracted from the LCM cap by incubation in buffer RLT plus (Qiagen) with β-mercaptoethanol at 42°C for 30 minutes, followed by RNA extraction using an RNeasy Plus Microkit (Qiagen). The RNA was primed with random primers (Invitrogen, Carlsbad, CA) and transcribed to cDNA by SuperScript II RT (Invitrogen), according to the manufacturer's instructions. Locus-specific quantitative real-time PCR (qPCR) for HLAs A, B, and C was performed, as described by Garcia-Ruano et al,42Garcia-Ruano A.B. Mendez R. Romero J.M. Cabrera T. Ruiz-Cabello F. Garrido F. Analysis of HLA-ABC locus-specific transcription in normal tissues.Immunogenetics. 2010; 62: 711-719Crossref PubMed Scopus (18) Google Scholar with Power SYBR Green master mix (Life Technologies) on a Step One Plus Real-Time PCR system (Life Technologies). Predesigned gene-specific primer sets (QuantiTect Primer Assays; Qiagen) were used for detection of INS, RRN18S, and glyceraldehyde-3-phosphate dehydrogenase cDNA. PCR specificity was verified by melt curve analysis of all PCR products. Islet preparations containing 35% to 85% endocrine cells (estimated by dithizone staining and computer-assisted digital image analysis43Friberg A.S. Brandhorst H. Buchwald P. Goto M. Ricordi C. Brandhorst D. Korsgren O. Quantification of the islet product: presentation of a standardized current good manufacturing practices compliant system with minimal variability.Transplantation. 2011; 91: 677-683Crossref PubMed Scopus (30) Google Scholar), and exocrine preparations containing <5% endocrine cells, from four organ donors without pancreatic disease were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO) supplemented with protease inhibitors (Sigma-Aldrich) for 20 minutes at 4°C while rocking. Protein concentration was measured by Qubit (Invitrogen), and 30 μg of each protein lysate was loaded onto a 10% to 20% Criterion Precast SDS-PAGE gel (BioRad, Hercules, CA). Proteins were then transferred to a polyvinylidene fluoride membrane (BioRad). The membrane was first incubated with a mouse anti-HLA class I ABC antibody (clone EMR8-5, 1:500; Abcam) and after stripping (0.2 mol/L NaOH for 5 minutes) with a rabbit anti-insulin/proinsulin antibody (HPA4932, dilution 1:500; Atlas Antibodies, Stockholm, Sweden). The primary antibodies were incubated at room temperature for 1 hour. After washing, horseradish peroxidase–conjugated secondary antibodies (anti-rabbit, dilution 1:3000, and anti-mouse, dilution 1:7000; Dako) were added for 1 hour. Immobilon Western chemiluminescent horseradish peroxidase substrate from Millipore (Billerica, MA) was used. Chemiluminescence was detected by a charge-coupled device camera (BioRad). The membrane was also stained for total protein using amido black, confirming that similar amounts of protein were loaded from the endocrine and exocrine preparations from each donor and allowing a comparison of HLA class I expression between the two types of tissue. The intensities of the bands were quantified by densitometric scanning using Kodak Digital Science 1D software version 3.0 (Eastman Kodak, New Haven, CT). To test the effect of substances that could potentially be responsible for the up-regulation of HLA class I in T1D islets, isolated pancreatic tis

Referência(s)