Study on pancreatic lymphatics in nonobese diabetic mouse with prevention of insulitis and diabetes by adjuvant immunotherapy

The present study has investigated the relationship between pancreatic lymphatics, infiltrating cells, and insulitic development after a single injection of complete Freund's adjuvant (CFA) given at an early age in the nonobese diabetic (NOD) mice. No CFA-treated NOD mice developed hyperglycemia, whereas most CFA-untreated mice died of diabetes at the age of 20-30 weeks. In untreated NOD mice, the increased infiltration of dendritic cells (DCs) and T-lymphocytes into the pancreatic islets appeared to be consistent with the increased expression of the secondary lymphoid chemokine (CCL21) and CD(31) by the endothelial cell lining of inter- and intralobular lymphatics. As the infiltration became severe, the reaction products of CCL21 and CD(31) were distributed in the nucleus and cytoplasm of lymphatic endothelial cells (LECs), through which DCs and T-lymphocytes migrated frequently. Administration of CFA reduced the number of infiltrating DCs and T-lymphocytes, but did not affect macrophage infiltration. The peri-insulitis occurred in numerous islets of CFA-treated NOD mice without the appearance of the intraislet infiltration and islet-associated lymphoid-like tissues. Furthermore, significant suppression of CCL21 and CD(31) was demonstrated on the infiltrating cells to the islets and islet-associated lymphatics. The abluminal endothelial cell lining of lymphatic vessels exhibited weaker immunoreactivity of CCL21 and CD(31) in comparison with the luminal surfaces. The reaction product of 5'-nucleotidase (5'-Nase) was evenly deposited on LECs, which were the absence of open junctions, cytoplasmic protrusions, and vesicles. CFA treatment influenced the migratory processes of the infiltrating cell, which were closely related with structural changes of pancreatic lymphatics and inhibited insulitic development. These findings suggest that in CFA-treated NOD mice, the suppression of insulitis and prevention of diabetes are secondary to the functional modulation of pancreatic lymphatics and infiltrating cells.     

小鼠胰腺淋巴管的分布及其与胰岛的关系

目的:探讨小鼠胰腺淋巴管的形态分布及其结构特点。方法:对小鼠胰组织切片进行HE染色,5核苷酸酶(5-Nase)和碱性磷酸酶(ALP)双重染色,光镜、透射电镜、扫描电镜二次电子和背散射电子图像(SEI/BEI)观察。结果:小鼠胰腺的淋巴管结构较典型,在胰腺的小叶间结缔组织内,较大淋巴管与血管和导管相互伴行;毛细淋巴管起自胰腺腺泡周围,并且均匀地分布于整个小叶内;小叶内有单独走行的集合淋巴管,亦存在与血管并行情况;在胰岛内部未发现毛细淋巴管,但胰岛周围可见丰富的毛细淋巴管。结论:小鼠胰腺小叶间和小叶内结缔组织中,均有淋巴管分布;胰岛内部虽无淋巴管,但胰岛与周围毛细淋巴管的关系较密切。     

Demonstration of the intralobular lymphatics in the guinea pig pancreas by an enzyme-histochemical method

Intralobular lymphatics in the guinea pig pancreas were demonstrated enzyme-histochemically showing the extent, distribution and fine structure by combined light and transmission electron microscopy. 5'-nucleotidase(5'-Nase)-positive lymphatic vessels were present throughout the pancreas. Intralobular lymphatics among the acini were comparatively rare and generally independent of the blood capillaries, pancreatic ducts and acini. These lymphatics revealed the usual structural features, such as typical intercellular junctions and very tenuous vascular walls without continuous basal laminae. Fine precipitates of the cerium-based reaction product for 5'-Nase activity were found to be associated with cell membranes of the lymphatic endothelium and pinocytotic vesicles. Lymphatics were not closely related to the endocrine islets, although alkaline phosphatase(ALPase)-positive blood capillaries were well developed. Collecting lymphatic vessels with valves with weaker 5'-Nase activity were also detected in the interlobular connective tissue. ALPase activity, absent in the lymphatics, was positive in the blood capillaries, suggesting that it is also a useful way of demonstrating, histochemically, the blood capillaries in the guinea pig pancreas.     

Lymphatic vascular endothelial hyaluronan receptor (lyve)-1- and ccl21-positive lymphatic compartments in the diabetic thymus

To explore the biological significance of the lymphatics in the autoimmune process, the thymus from non-obese diabetic (NOD) mice was evaluated by histochemistry and western blot analysis. Thymic lymphatic endothelial cells showed suggestive expression patterns of the functional molecules lymphatic vascular endothelial hyaluronan receptor (LYVE)-1, CCL21, CD31 and podoplanin. With increasing age, the expression of CCL21 was reduced in the medullary epithelial cells and lymphatics. Of note, LYVE-1-expressing lymphatics, filled with a cluster of thymocytes, increased in number and size and extended from the corticomedullary boundary into the medulla as the insulitis progressed. The development of lymphatic compartments was occasionally accompanied by a regional disappearance between the cortex and medulla. The CD4- and CD8-positive T cells frequently penetrated through the slender lymphatic walls. The epithelial reticular cell layer lining the perivascular spaces was extensively stained with cytokeratin, but the expression of cytokeratin showed an age-dependent decrease. These findings indicate that the occurrence of LYVE-1-expressing lymphatic compartments and the alteration of CCL21 expression in the lymphatics may be involved in defective thymocyte differentiation and migration, and play a significant role in insulitic and diabetic processes.     

Structural organization and fine distribution of lymphatic vessels in periodontal tissues of the rat and monkey: a histochemical study

The structural organization and fine distribution of lymphatic vessels in the periodontal tissues (gingiva, periodontium and alveolar process) were examined by light and electron microscopy using an enzyme-histochemical method. Whole mount preparations of periodontal membranes peeled from the teeth and cryostat sections of normal or decalcified tissues treated with EDTA were double-stained using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) and examined by light microscopy. This staining procedure allowed the lymphatic vessels in the periodontal tissue to be differentiated from blood vessels. Well-developed 5'-Nase-positive lymphatics were observed in the gingiva and periodontium. The histochemical aspects of 5'-Nase activity in lymphatic vessels are discussed in detail, with special reference to the supply of Mg++ ions. A network of 5'-Nase-positive lymphatics was observed in whole mount preparations of the periodontal membrane for the first time. This network was also observed in the tissue sections. More 5'-Nase-positive lymphatics were seen in the root area of the periodontium than in the cervical area. 5'-Nase-positive lymphatics in residual tissue blocks remaining after cryostat sectioning and in whole mount preparations were highlighted with good contrast and resolution on backscattered electron images produced by scanning electron microscopy. Dense granular precipitations resulting from the 5'-Nase reaction were observed on the luminal surface of the lymphatic endothelial cells as well as on the basal side but were absent in the blood vessels.     

Leukocyte attraction through the CCR5 receptor controls progress from insulitis to diabetes in non-obese diabetic mice

Lymphocyte infiltration to pancreatic islets is associated to chemoattraction, as are other inflammatory autoimmune processes. We examined whether development of insulitis and diabetes depends on chemoattraction of lymphocytes via the CCR5 chemokine receptor. In non-obese diabetic (NOD) mice, a substantial fraction of peripheral T cells and virtually all B cells expressed high CCR5 levels. CCR5 expression characterized the effector T cell phenotype, suggesting their potential involvement in disease development. In view of these findings and the CCL5 (RANTES, the CCR5 ligand) expression by pancreatic islets, we treated NOD mice with a neutralizing anti-CCR5 antibody. This did not influence peri-insulitis advancement, but inhibited beta-cell destruction and diabetes. These data demonstrate a role of CCR5-dependent chemoattraction in insulitis progression to islet destruction, suggesting the potential value of therapeutic intervention by CCR5 targeting.     

Distribution of 5'-nucleotidase activity in greater omentum of rats

The aim of the present study is to demonstrate the cellular basis of 5'-nucleotidase (5'-Nase) activity in the greater omentum of rats. Enzyme histochemistry for 5'-Nase showed that lymphatic vessels in the omentum as well as lymphocytes in the milky spots were positively stained. Electron microscopic observation revealed-5'-Nase activity at the luminal surface of the lymphatic endothelial cells, pinocytotic vesicles in the endothelial cells and the surface of fibroblasts located at the intercellular space of adipose cells. Fibroblasts extended long cytoplasmic processes toward adipose cells and inflammatory cells. These findings suggest that lymphatic endothelial cells as well as fibroblasts in the omentum may play an important role in regulation of metabolism and immune mechanisms in the greater omentum by supplying adenosin.     

CCR7 directs the recruitment of T cells into inflamed pancreatic islets of nonobese diabetic (NOD) mice

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreatic islets. The migration of T cells from blood vessels into pancreas is critical for the development of islet inflammation and β cell destruction in T1D. To define the roles of C–C chemokine receptor type 7 (CCR7) in recruitment of T cells into islets, we used laser capture microdissection to isolate tissue from inflamed islets of nonobese diabetic (NOD) mice and uninflamed islets of BALB/c and young NOD mice. RT–PCR analyses detected mRNAs for CCR7 and its chemokine ligands CCL19 (ELC; MIP-3β) and CCL21 (SLC) in captures from inflamed, but not from uninflamed, islets. Immunohistology studies revealed that high endothelial venules in inflamed islets co-express CCL21 protein and MAdCAM-1 (an adhesion molecule that recruits lymphocytes into islets). Desensitization of lymphocyte CCR7 blocked about 75 % of T cell migration from the bloodstream into inflamed islets, but had no effect on B cell migration into islets. These results indicate that CCR7 and its ligands are important in the recruitment of T cells into inflamed islets and thus in the pathogenesis of T1D.     

Evidence for an enhanced adhesion of DC to fibronectin and a role of CCL19 and CCL21 in the accumulation of DC around the pre-diabetic islets in NOD mice

The non-obese diabetic (NOD) mouse is a widely used animal model for the study of human diabetes. The lymphocytic (peri-)insulitis is preceded by an early accumulation of dendritic cells (DC) around the islets of Langerhans. This DC accumulation is thought to derive from an influx of monocytes attracted by pro-inflammatory chemokines. Besides chemokines, extracellular matrix (ECM) proteins play an important role in the accumulation of leukocytes in tissues. We studied the expression of the chemokines CCL2, CCL5, CXCL10, CCL19 and CCL21 over time in pancreases of NOD and control mice by ELISA on pancreas lysates as well as by immunohistochemistry. In addition, we studied the adhesive capacity of bone marrow-derived DC (BMDC) to ECM components. DC in the NOD pancreas accumulated at sites with an intense expression of fibronectin. In vitro, NOD BMDC showed increased fibronectin adhesion and increased VLA-5 expression. At the time of early DC accumulation (<10 wk), the lymphoid tissue-related chemokines CCL19 and CCL21 were increased. Our findings support the view that the early accumulation of DC around the NOD islets is not the consequence of an enhanced attraction of precursors and immature DC by pro-inflammatory chemokines. It rather might be the consequence of an aberrantly enhanced adhesion and retention of NOD DC.     

Enzyme-histochemical study on the fine distribution of the intramural lymphatics at the ileocecal junction of the monkey intestine.

The fine distribution of the intramural lymphatics at the ileocecal junction of the monkey intestine, especially in the lamina propria of the ileocecal valve, was examined by light and electron microscopy using enzyme-histochemical staining. The distinction between the lymphatics and the blood vessels was made by light microscopy on cold glycol methacrylate resin (JB-4) sections using 5'-nucleotidase (5'-Nase)-alkaline phosphatase (ALPase) double staining. The lymphatics were found to show strong 5'-Nase activity and to comprise irregularly shaped vessels or spaces. The central lymphatic vessels (central lacteals) in low villi were seen to lie deep within the ALPase-positive subepithelial capillary network. In the ileum side of the ileocecal junction, the 5'-Nase-positive lymphatics were seen both in the superficial layer and the deep layer of the lamina propria. On the contrary, in the cecum side the mucosal lymphatics were less numerous in the superficial layer and were distributed mainly in the deep layer near the lamina muscularis mucosae. These lymphatics ran through the lamina muscularis and merged into the lymphatic network in the submucosa. The submucosal lymphatics communicated with each other at the ileocecal junction and formed a well-developed network. Collecting lymphatics with valves were also seen near the tunica muscularis (sphincter muscle) in the deep submucosa. These lymphatics traversed the muscle layer and drained into the subserosal lymphatics.     

Predominance of T lymphocytes in pancreatic islets and spleen of pre-diabetic non-obese diabetic (NOD) mice: a longitudinal study.

We examined sequential changes in the subsets of mononuclear cells infiltrating the pancreatic islets and splenic lymphocytes in pre-diabetic non-obese diabetic (NOD) mice, an animal model for type I diabetes, using immunofluorescent techniques. In the pancreas, a predominant infiltration by activated T lymphocytes, including helper inducer and cytotoxic suppressor T cells, was observed in the early stage of insulitis. Natural killer cells were also detected in the lesions. Immunoglobulin bearing cells tended to increase in number with the progression of insulitis. T lymphocytes were localized close to islet cells, while immunoglobulin bearing cells appeared adjacent to blood vessels and around T cell clusters. Immunoglobulin deposition or Ia expression on islet cells was not observed. The percentage of splenic T lymphocytes was markedly increased in the initial stage of insulitis as compared with control ICR mice and this elevated proportion of T cells continued throughout the observation period. As for splenic T cell subsets, cytotoxic suppressor T cells were increased in NOD mice. These results suggest that T lymphocytes play an important role in the initiation of insulitis long before the onset of overt diabetes. Moreover, NOD mice seem to have characteristic immunological features different from the BB rat or a reported case with human type I diabetes.     

Differential expression of CCL19 by DC-Lamp+ mature dendritic cells in human lymph node versus chronically inflamed skin

De novo formation of lymphoid tissue is one of the characteristic features of chronic inflammation. The formation of T cell–mature dendritic cell (DC) clusters has been previously demonstrated in chronically inflamed skin infected with Candida albicans. A functional similarity was also found between chronic inflammation and the T‐cell zone of lymph nodes (LNs), since a substantial fraction of phenotypically mature DCs in both tissues expressed CCL22 (macrophage‐derived chemokine; MDC) and were closely surrounded by memory‐type T cells expressing its receptor, CCR4. To analyse the nature of T cell–mature DC interactions further in chronically inflamed skin and LNs, the present study focuses on another chemokine system, namely CCL19 (EBI1 ligand chemokine; ELC), CCL21 (secondary lymphoid tissue chemokine; SLC) and their shared receptor, CCR7. RT‐PCR analysis revealed expression of CCL19, CCL21, and CCR7 at high levels in LNs and at low levels in inflamed skin. Using immunohistochemistry, the majority of DC‐Lamp⁺ mature DCs in the T‐cell area of LNs expressed CCL19 and were surrounded by CCR7⁺ naïve‐type lymphocytes, while CCL21 was expressed in reticular stromal cells and vascular endothelial cells. Very few mature DCs in LNs were found to express CCR7. In contrast, the majority of DC‐Lamp⁺ mature DCs in inflamed skin were totally negative for CCL19 and were surrounded by CCR7^? memory‐type T cells. Furthermore, CCL21 expression in the inflamed skin was detected in dermal lymphatic endothelial cells and rare CCR7⁺ mature DCs were mostly seen within the lymphatic vessels. In normal skin, on the other hand, no cells immunoreactive for CCL19, CCL21, or CCR7 were found. The present study thus reveals a striking difference in the function of mature DCs between LNs and chronically inflamed skin. Copyright © 2002 John Wiley & Sons, Ltd.     

家兔舌淋巴管分布的酶组织化学法观察

应用５＇－核苷酸酶－碱性磷酸酶（５＇－Ｎａｓｅ－ＡＫＰａｓｅ）双重染色法，观察了兔淋巴管的微细分布。光镜下可见舌的毛细淋巴管和淋巴管呈５＇－核苷酸酶染色强阳性反应，管壁显示明显的棕色或深棕色，而毛细血管或血管的碱性磷酸酶的反应则显示强阳性，管壁呈现明显的蓝色。研究证明，在舌粘膜固有层存有丰富的毛细淋巴管和淋巴管；于舌肌层肌纤维束间的结缔组织内也可见毛细淋巴管及淋巴管。     

Intra-islet proliferation of cytotoxic T lymphocytes contributes to insulitis progression

Infiltration of pancreatic islets by immune cells, termed insulitis, increases progressively once it begins and leads to clinical type 1 diabetes. But even after diagnosis some islets remain unaffected and infiltration is patchy rather than uniform. Traffic of autoreactive T cells into the pancreas is likely to contribute to insulitis progression but it could also depend on T-cell proliferation within islets. This study utilizes transgenic NOD mice to assess the relative contributions of these two mechanisms. Progression of insulitis in NOD8.3 TCR transgenic mice was mildly reduced by inhibition of T-cell migration with the drug FTY720. In FTY720-treated mice, reduced beta cell MHC class I expression prevented progression of insulitis both within affected islets and to previously unaffected islets. CTL proliferation was significantly reduced in islets with reduced or absent beta cell expression of MHC class I protein. This indicates that intra-islet proliferation, apparently dependent on beta cell antigen presentation, in addition to recruitment, is a significant factor in progression of insulitis.     

Lymphatic precollectors contain a novel, specialized subpopulation of podoplanin low, CCL27-expressing lymphatic endothelial cells

Expression of the lymphoendothelial marker membrane mucoprotein podoplanin (podo) distinguishes endothelial cells of both blood and lymphatic lineages. We have previously discovered two distinct subpopulations of lymphatic endothelial cells (LECs) in human skin that were defined by their cell surface densities of podoplanin and were designated LEC^podo-low and LEC^podo-high. LEC^podo-low is restricted to lymphatic precollector vessels that originate from initial LEC^podo-high-containing lymphatic capillaries and selectively express several pro-inflammatory factors. In addition to the chemokine receptor protein Duffy blood group antigen receptor for chemokines, these factors include the constitutively expressed chemokine CCL27, which is responsible for the accumulation of pathogenic CCR10⁺ T lymphocytes in human inflammatory skin diseases. In this study, we report that CCR10⁺ T cells accumulate preferentially both around and within CCL27⁺ LEC^podo-low precollector vessels in skin biopsies of human inflammatory disease. In transmigration assays, isolated CCR10⁺ T lymphocytes are chemotactically attracted by LEC^podo-low in a CCL27-dependent fashion, but not by LEC^podo-high. These observations indicate that LEC^podo-low-containing precollector vessels constitute a specialized segment of the initial lymphatic microvasculature, and we hypothesize that these LEC^podo-low-containing vessels are involved in the trafficking of CCR10⁺ T cells during skin inflammation.     

Deep splenic lymphatic vessels in the mouse: A route of splenic exit for recirculating lymphocytes

A study of pathways of lymphocyte migration through mouse spleen revealed lymphatic channels closely following arteries in trabeculae and white pulp. Because there is no detailed record of the layout of deep splenic lymphatics in the mouse, or other species, we present our observations in this paper, relating our findings to normal migratory pathways of lymphocytes through the spleen. Lymphatics draining the spleen are so inconspicuous that they often are not mentioned in anatomical discussions. The data presented clearly demonstrate (1) the existence and layout of deep lymphatic vessels in the mouse spleen, and (2) that migrating lymphocytes exit white pulp via these lymphatic vessels. CD4⁺ and CD8⁺ T cell subsets migrated proximally along the central artery from distal (dPALS) to proximal periarterial lymphatic sheaths (pPALS) and exited via deep lymphatic vessels that originate there. B cells migrated from dPALS to enter lymphatic nodules (NOD), thus segregated from T cells. B cells then migrated toward and exited via deep lymphatics. The appearance of labelled lymphocytes in lymph coincided with their disappearance from white pulp compart-ments. Labelled T cells were observed in splenic lymphatics as early as 1 hr after intravenous infusion but took, on average, about 6 hr. B cells took somewhat longer. Thus T and B cells entered and left white pulp through shared pathways, but took divergent intermediate routes through dedicated zones, pPALS for T cells, NOD for B cells.     

Enzyme-histochemical visualization of lymphatic capillaries in the mouse tongue: light and electron microscopic study

The distinction between lymphatic capillaries and blood capillaries in the mouse tongue was studied enzyme-histochemically by light and electron microscopy. The lymphatic walls are characterized by a strong 5'-nucleotidase (5'-Nase) activity, whereas those of the blood capillaries reveal a significantly lower or no activity. The alkaline phosphatase (ALPase) activity, on the other hand, is markedly higher in the blood capillaries than in the lymphatic capillaries. The specific reaction of 5'-Nase activity in the lymphatic capillaries is obtained by simultaneous inhibition of ALPase on incubation in a medium (Wachstein and Meisel, 1957) with L-tetramisole for 5'-Nase histochemistry. The distribution and intensity of 5'-Nase activity in the lymphatic capillaries can be adequately visualized by comparison with serial cryostat sections for histochemical detection under light and backscattered imaging scanning electron microscopes. The reaction products of the 5'-Nase activity are localized on the outer surface of the cell membrane of the lymphatic endothelial cells, whereas those in the blood capillaries reveal a weak or no reaction. The present results demonstrates satisfactory isolated visualizations of 5'-Nase activity in the lymphatic capillaries and of ALPase activity in the blood capillaries.     

Role of L-selectin in the development of autoimmune diabetes in non-obese diabetic mice

Autoimmune diabetes is characterized by an early mononuclear infiltration of pancreatic islets and later selective autoimmune destruction of insulin-producing beta cells. Lymphocyte homing receptors have been considered candidate targets to prevent autoimmune diabetes. L-selectin (CD62L) is an adhesion molecule highly expressed in naive T and B cells. It has been reported that blocking L-selectin in vivo with a specific antibody (Mel-14) partially impairs insulitis and diabetes in autoimmune diabetes-prone non-obese diabetic (NOD) mice. In the present study we aimed to elucidate whether genetic blockade of leukocyte homing into peripheral lymph nodes would prevent the development of diabetes. We backcrossed L-selectin-deficient mice onto the NOD genetic background. Surprisingly NOD/L-selectin-deficient mice exhibited unaltered islet mononuclear infiltration, timing of diabetes onset and cumulative incidence of spontaneous diabetes when compared to L-selectin-sufficient animals. CD4, CD8 T cells and B cells were present in islet infiltrates from 9-week-old L-selectin-sufficient and -deficient littermates. Moreover, total splenocytes from wild-type, heterozygous or NOD/L-selectin-deficient donor mice showed similar capability to adoptively transfer diabetes into NOD/SCID recipients. On the other hand, homing of activated, cloned insulin-specific autoaggressive CD8 T cells (TGNFC8 clone) is not affected in NOD/L-selectin-deficient recipients. We conclude that L-selectin plays a small role in the homing of autoreactive lymphocytes to regional (pancreatic) lymph nodes in NOD mice.