1004 Diabetes Volume 65, April 2016 Erik Korsgren and Olle Korsgren An Apparent Deficiency of Lymphatic Capillaries in the Islets of Langerhans in the Human Pancreas Diabetes 2016;65:1004–1008 | DOI: 10.2337/db15-1285 The lymphatic system is crucial for efficient immune diagnosis, is 73% (4). These observations have led to the surveillance and for the maintenance of a physiolog- conclusion that T1D is an immune-mediated disease. ical pressure in the interstitial space. Even so, almost no Immune cells are constantly circulating in the body information is available concerning the lymph drainage to detect and evoke an immune response against invad- of the islets of Langerhans in the human pancreas. ing organisms and cells recognized as nonself. Antigen- Immunohistochemical staining allowed us to distinguish presenting cells leave the extracellular space of the lymphatic capillaries from blood capillaries. Almost no affected organ via the lymphatic capillaries and accumu- lymphatic capillaries were found within the islets in late in regional lymph nodes, where the encountered pancreatic biopsy specimens from subjects without antigens are presented to stimulate clonal expansion of diabetes or from subjects with type 1 or type 2 diabetes. T cells with affinity for the foreign peptides presented on Lymphatic capillaries were, however, found at the islet- the individual’s own HLA. exocrine interface, frequently located along blood capil- The b-cell is one of the most metabolically active cells laries and other fibrotic structures within or close to the islet capsule. Lymphatic capillaries were regularly found in the body and is critically dependent on a high supply of ISLET STUDIES b in the exocrine pancreas, with small lymphatic vessels oxygen and nutrients from the blood. Almost every -cell located close to and around acini. Larger collecting is in direct contact with a capillary that has a fenestrated lymphatic vessels were located in fibrotic septa between endothelial cell lining to allow optimal transport through the exocrine lobules and adjacent to the ductal system of the capillary wall, and in rodents, islet blood perfusion is the pancreas. In summary, we report a pronounced de- ;10 times higher than in the exocrine pancreas (5). In all ficiency of lymphatic capillaries in human islets, a finding organs, there is a net surplus in fluid transport over the with implications for immune surveillance and the regula- wall of the blood capillary that is correlated with the level tion of interstitial fluid transport in the endocrine pancreas of blood perfusion and the permeability of the capillary. as well as for the pathophysiology of both type 1 and type 2 This extracellular interstitial fluid (EIF) is transported diabetes. from the interstitial space via the lymphatic capillaries, and disturbances in this system can lead to the formation of edema (6). Type 1 diabetes (T1D) is caused by a continuing de- A well-organized lymphatic system is crucial for efficient struction of the insulin-producing cells that occurs over a immune surveillance and induction of T-cell–mediated period of several years after diagnosis (1). Autoantibodies immuneresponsesaswellasforthemaintenanceof with affinity for b-cells and for exocrine antigens usually physiological pressure in the interstitial space (6). An appear several years before the diagnosis (2,3). The in- absence of lymphatic capillaries has previously been cidence of insulitis, defined as the presence of more reported within the islets in rodents (7,8) and in the than two or five T cells infiltrating at least three islets fetal human pancreas (9); however, to the best of our in children (#14 years of age) dying within 1 month after knowledge, no information is available concerning the Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, © 2016 by the American Diabetes Association. Readers may use this article as Sweden long as the work is properly cited, the use is educational and not for profit, and Corresponding author: Olle Korsgren, [email protected]. the work is not altered. Received 12 September 2015 and accepted 7 January 2016. diabetes.diabetesjournals.org Korsgren and Korsgren 1005 lymphatic drainage of the islets of Langerhans in the Immunohistochemical Staining human pancreas. Formalin-fixed and paraffin-embedded tissues were cut Lymph capillaries can be distinguished from blood into 5-mm sections. Consecutive sections were processed capillaries by the expression of specific antigens (e.g., and labeled using a standard immunoperoxidase tech- podoplanin and LYVE-1) on the endothelial cells (10,11). nique, as previously described in detail (12). With the The present morphological study was conducted on hu- exception of insulin, all other antigens were unmasked man pancreases from subjects without diabetes and from by heat-induced epitope retrieval. Antibodies against subjects with T1D or type 2 diabetes (T2D) to describe chromogranin A (1:150, clone LK2H10; NeoMarkers, the extent of the intraislet lymph capillaries and their Thermo Fisher Scientific, Inc., Fremont, CA), insulin relation to the insulin-producing cells and intraislet (1:200, clone A564; Dako, Glostrup, Denmark), and syn- blood capillaries. aptophysin (1:50, clone DAK-SYNAP; Dako) were used to identify islets. A monoclonal antibody against CD34 RESEARCH DESIGN AND METHODS (1:50, clone EP373Y; Dako) was used to detect blood en- Ethics dothelial cells and antibodies against podoplanin (1:50, All work involving human tissue was conducted according clone D2-40; Dako) or LYVE-1 (1:200, clone ab 36993; to the principles expressed in the Declaration of Helsinki Abcam, Cambridge, U.K.) were used to detect lymphatic and in the Council of Europe’s Convention on Human endothelial cells. Bound antibodies were visualized using Rights and Biomedicine. Consent for organ donation EnVision or an EnVision DuoFLEX Doublestain System (for clinical transplantation and for use in research) was (both Dako) and diaminobenzidine-based substrate or obtained from the relatives of the deceased donors by the 3-amino-9-ethylcarbazole (Dako). Sections were counter- donor’s physicians and documented in the medical re- stained with hematoxylin, scanned, and analyzed by cords of the deceased patient. The Regional Ethics Com- Aperio ImageScope and by light microscopy by two inves- mittee in Uppsala, Sweden, approved the study according tigators who were blinded to their origin. to the Act Concerning the Ethical Review of Research In- volving Humans (2003:460; permit number Dnr 2009/043, RESULTS 2009/371). Immunohistochemical Staining Optimization of the staining for lymph and blood capil- Human Pancreatic Specimens laries was performed on sections of the intestinal wall and Biopsy specimens from 35 human pancreases were on human pancreases. Double staining of lymph and included in the study. Before islet isolation, a clamp blood capillaries revealed that the two types of capillaries was used to compress the main pancreatic duct at the were distinctly and specifically stained, without overlap or head of the pancreas, and the tissue proximal to the background. clampwastakenasaspecimenandstoredinformalin. Donors were chosen based on factors such as weight, Blood and Lymph Capillaries in the Pancreas age, and health (Table 1). Two pancreases were obtained Larger collecting lymphatic vessels (Fig. 1A, B, M, and O) at the onset of T1D, as previously described in detail were often located in fibrotic septa between the exocrine (12); seven were obtained from patients with longstand- lobules and adjacent to the ductal system of the pancreas. ing T1D; eight were obtained from patients with long- Lymphatic capillaries were frequently found in the exo- standing T2D; and the remaining pancreases were crine pancreas (Fig. 1E, F, K, and L) located close to and collected from multiorgan donors without any known around acini. pancreatic disease, divided according to age and BMI A total of 4,365 islets from 35 subjects were examined (Table 1). (Table 1). Lymphatic capillaries were found in only 24 Table 1—Characterization of the groups of subjects included and the number of lymphatic capillaries found Total number of islets Subjects n Age (years) BMI (kg/m2) Total number of islets examined with lymph capillaries Without diabetes BMI ,18.8 kg/m2 4 48.5 6 17.4 18.2 6 0.5 508 0 BMI .40.7 kg/m2 4 58.5 6 5.3 42.6 6 2.2 187 1 in 1 subjects Age ,24 years 5 20.6 6 2.4 22.7 6 1.9 452 0 Age .70 years 5 75.6 6 1.5 26.0 6 3.2 810 4 in 3 subjects With T2D 8 52.9 6 18.1 30.6 6 8.0 765 17 in 3 subjects With T1D Longstanding 7 38.4 6 20.7 23.9 6 3.7 1,121 1 in 1 subject Recent-onset 2 34.5 6 7.8 25.7 6 2.1 522 1 in 1 subject Values are presented as means 6 SD. 1006 Deficiency of Lymph Capillaries in Human Islets Diabetes Volume 65, April 2016 Figure 1—Blood and lymph capillaries in the human pancreas. A: Immunohistochemistry showing distinct staining of several large lymph vessels (LYVE-1, brown) close to a vein and an artery in a subject with longstanding T1D. B: Several large lymph vessels (D2-40, brown) close to a small duct in a subject with T2D. C: A network of lymph capillaries (D2-40, brown) in the islet-exocrine interface in a subject with T2D. The arrow indicates a tiny lymph capillary (D2-40, brown) in a fibrotic strand in the center of the islet (SYN, red). D: Lymph capillaries (D2-40, brown) surrounding a small duct, but not in the islet (SYN, red), in a subject with high age. E: Lymph capillaries (D2-40, brown) in the exocrine parenchyma, but not in the islets (SYN, red), in a subject with high age. F: Lymph capillaries (D2-40, brown) in the exocrine parenchyma, but not in the islet (SYN, red), in a subject with recent-onset T1D.