Pathophysiology of the lymphatic drainage of the central nervous system: Implications for pathogenesis and therapy of multiple sclerosis

In most organs of the body, immunological reactions involve the drainage of antigens and antigen presenting cells (APCs) along defined lymphatic channels to regional lymph nodes. The CNS is considered to be an immunologically privileged organ with no conventional lymphatics. However, immunological reactions do occur in the CNS in response to infections and in immune-mediated disorders such as multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). Here, we review evidence that cervical lymph nodes play a role in B and T cell mediated immune reactions in the CNS. Then we define the separate pathways by which interstitial fluid (ISF) and CSF drain to cervical lymph nodes. ISF and solutes drain from the brain along the 100–150 nm-wide basement membranes in the walls of capillaries and arteries. In humans, this perivascular pathway is outlined by the deposition of insoluble amyloid (Aβ) in capillary and artery walls in cerebral amyloid angiopathy in Alzheimer's disease. The failure of APCs to migrate to lymph nodes along perivascular lymphatic drainage pathways may be a major factor in immunological privilege of the brain. Lymphatic drainage of CSF is predominantly through the cribriform plate into nasal lymphatics. Lymphatic drainage of ISF and CSF and the specialised cervical lymph nodes to which they drain play significant roles in the induction of immunological tolerance and of adaptive immunological responses in the CNS. Understanding the afferent and efferent arms of the CNS lymphatic system will be valuable for the development of therapeutic strategies for diseases such as MS.     

Brain drains: new insights into brain clearance pathways from lymphatic biology

The lymphatic vasculature act as the drainage system for most of our tissues and organs, clearing interstitial fluid and waste and returning them to the blood circulation. This is not the case for the central nervous system (CNS), which is devoid of parenchymal lymphatic vessels. Nevertheless, the brain is responsible for 25% of the body’s metabolism and only compromises 2% of the body’s mass. This high metabolic load requires an efficient system to remove waste products and maintain homeostasis. Well-described mechanisms of waste clearance include phagocytic immune cell functions as well as perivascular fluid flow; however, the need for active drainage of waste from the brain is becoming increasingly appreciated. Recent developments in lymphatic vascular biology challenge the proposition that the brain lacks lymphatic drainage or an equivalent. In this review, we describe the roles of the glymphatic system (a key drainage mechanism in the absence of lymphatics), the recently characterized meningeal lymphatic vessels, and explore an enigmatic cell population found in zebrafish called mural lymphatic endothelial cells. These systems may play important individual and collective roles in draining and clearing wastes from the brain.     

Our current understanding of the lymphatics of the brain and spinal cord

The lymphatic system, segregated from the blood vascular system, is an essential anatomical route along which interstitial fluid, solutes, lipids, immune cells, and cellular debris, are conveyed. However, the way these mechanisms operate within the cranial compartment is mostly unknown. Herein, we review current understanding of the meningeal lymphatics, described anatomically over a century ago yet still poorly understood from a functional standpoint. We will delineate the cellular mechanisms by which the meningeal lymphatics are formed and discuss their unique anatomy. Furthermore, this review will discuss the recently‐coined “glymphatic system” and the manner by which cerebrospinal fluid (CSF) and interstitial fluid (ISF) are exchanged and thus drained by the meningeal lymphatic vasculature as a key route for conveying cellular waste, solutes, and immune traffic to the deep cervical lymph nodes. The clinical relevance of the meningeal lymphatics will also be described, as they are relevant to various common defects of the lymphatic system. Clin. Anat. 32:117–121, 2019. © 2018 Wiley Periodicals, Inc.     

The anatomy and metabolome of the lymphatic system in the brain in health and disease

Recent studies have demonstrated that the brain is equipped with a lymphatic drainage system that is actively involved in parenchymal waste clearance, brain homeostasis and immune regulation. However, the exact anatomic drainage routes of brain lymph fluid (BLF) remain elusive, hampering the physiological study and clinical application of this system. In this study, we systematically dissected the anatomy of the BLF pathways in a rat model. Moreover, we developed a protocol to collect BLF from the afferent lymphatic vessels of deep cervical lymph nodes (dcLNs) and cerebrospinal fluid (CSF) from the fourth ventricle. Nuclear magnetic resonance spectroscopy showed that BLF contains more metabolites than CSF, suggesting that BLF might be a more sensitive indicator of brain dynamics under physiological and pathological conditions. Finally, we identified several metabolites as potential diagnostic biomarkers for glioma, Parkinson's disease and CNS infectious diseases. Together, these data may provide insight into the physiology of the lymphatic system in the brain and into the clinical diagnosis of CNS disorders.     

How well does the CSF inform upon pathology in the brain in Creutzfeldt-Jakob and Alzheimer's diseases?

Analysis of lumbar cerebrospinal fluid (CSF) plays a major role in the investigation of central nervous system disease, but how well do the changes in the CSF reflect pathology within the brain and spinal cord parenchyma? Both Creutzfeldt-Jakob (CJD) and Alzheimer's disease (AD) are characterized by the deposition of insoluble beta-pleated sheet peptides [prion protein (PrP) and beta-amyloid (Abeta), respectively] in the extracellular spaces of grey matter in the brain, but there is discordance in both diseases between the peptide levels in the brain and in the CSF. Experimental studies using tracers have shown that interstitial fluid (ISF) drains through very narrow intercellular spaces within grey matter into bulk flow perivascular channels that surround penetrating arteries. ISF then flows to the surface of the brain and joins CSF to drain to cervical lymph nodes. Such drainage of ISF and CSF to regional lymph nodes in the rat plays a significant role in B-cell and T-cell immune reactions within the brain. In man, the pia mater separates the periarterial ISF drainage pathways from the CSF in the subarachnoid space. The almost complete lack of insoluble protease-resistant PrP entering the CSF from the brain in patients with CJD, reported by Wong et al. in this issue of the Journal of Pathology, illustrates the limitations of ISF drainage pathways for the elimination of insoluble peptides from brain tissue. Insoluble Abeta accumulates in the extracellular spaces as plaques in AD and in periarterial ISF drainage pathways as cerebral amyloid angiopathy. Soluble Abeta appears to become entrapped by the insoluble Abeta in the ISF drainage pathways; thus, as the level of soluble Abeta in the brain rises in AD, the level in the CSF falls. Thus, the changes in the CSF do not accurately reflect the accumulation of the abnormal peptides in the brain parenchyma in either CJD or AD. In both diseases, facilitation of ISF drainage and elimination of PrP and Abeta peptides from the extracellular spaces of the brain may lead to practical therapeutic strategies for these devastating disorders.     

Pathways of Fluid Drainage from the Brain - Morphological Aspects and Immunological Significance in Rat and Man

There is firm physiological evidence for the lymphatic drainage of interstitial fluid and cerebrospinal fluid from the brains of rats, rabbits and cats. The object of this review, is to describe firstly the morphological aspects of lymphatic drainage pathways from the rat brain and secondly, to explore through scanning and transmission electron microscope techniques, the possibility of similar lymphatic drainage pathways in man. Interstitial and oedema fluid spreads diffusely through the white matter in the rat and appears to drain into the ventricular cerebrospinal fluid. In grey matter, however, tracers pass along perivascular spaces to the surface of the brain and into the cerebrospinal fluid. Paravascular compartments in the subarachnoid space follow the course of major arterial branches to the circle of Willis and thence along the ethmoidal arteries to the cribriform plate of the ethmoid bone. Particulate tracers, such as Indian ink, enter channels in the arachnoid beneath the olfactory bulbs and connect directly with nasal lymphatics through channels which pass through holes in the cribriform plate. Proteins and other solutes may also drain along other cranial nerves. Thus, there is a bulk flow pathway for interstitial and cerebrospinal fluid from the rat brain into cervical lymphatics. In man, it is probable that diffuse interstitial drainage of fluid from the white matter occurs in a similar way to that in the rat. Furthermore, the anatomical pathways exist by which bulk drainage of fluid could occur along perivascular spaces from the grey matter into perivascular spaces of the leptomeningeal arteries and thence into the cerebrospinal fluid (CSF).(ABSTRACT TRUNCATED AT 250 WORDS)     

家兔睾丸的淋巴流向

本文用活体淋巴管注射法,观察了家兔的淋巴流向。用29只家兔,随机分为两组。第一组13只家兔,由每侧睾丸实质注入普鲁士蓝氯仿溶液。第二组16只家兔,先结扎睾丸头侧端的集合淋巴管,然后进行注射。第一组家兔睾丸的集合淋巴管主要是沿睾丸血管向头侧走行,经过腰淋巴结或是直接进入腰淋巴干;但在8侧睾丸(30.8%),存有走向盆腔的淋巴流路。第2组阻断头侧端的淋巴流路后,则于29例睾丸(90.6%),出现走向盆腔的淋巴流路。     

Lymphocyte Targeting of the Central Nervous System: A Review of Afferent and Efferent CNS-Immune Pathways

The central nervous system (CNS) is considered to be an immunological privileged site. However, inflammatory reactions in response to virus infections, in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAEl suggest that there are definite connections between the CNS and the immune system. In this review, we examine evidence for afferent and efferent pathways of communication between the CNS and the immune system, the pivotal role of regional lymph nodes in T-cell mediated autoimmune disease of the CNS, and the factors involved in lymphocyte targeting of the CNS. Afferent pathways of lymphatic drainage of the brain are well established in a variety of species, especially rodents. Fluid and antigens appear to drain along perivascular spaces populated by immunocompetent perivascular cells. Drainage pathways connect directly via the cribriform plate to nasal lymphatics and cervical lymph nodes. Soluble antigens draining from the brain induce antibody production in the cervical lymph nodes. Using a model of cryolesion-enhanced EAE, we review the role of lymphatic drainage and cervical lymph nodes in the enhancement of cerebral EAE. If a brain wound in the form of a cryolesion is produced 8 days post inoculation (dpi) of antigen in the induction of acute EAE, there is a 6-fold increase in severity of cerebral EAE by 15 dpi. Removal of the cervical lymph nodes significantly reduces such enhancement of EAE. These findings suggest that drainage of antigens from the brain to the cervical lymph nodes, in the presence of activated lymphocytes in the meninges or CNS, resuIts in an enhanced second wave of lymphocytes targeting the brain. In examining the efferent immune pathway by which lymphocytes home to the CNS, several studies have characterized the phenotype of infiltrating T lymphocytes by the use of immunocy-tochemistry or FACS analysis. T-cells infiltrating the CNS are recently activated/memory lymphocytes typified by their high expression of CD44, LFA-1 and ICAM-1 and low expression of CD45RB in the mouse. Following the induction of EAE in susceptible mice, ICAM-1 and VCAM-1 are dramatically upregulated on CNS vessels; lymphocytes bind to such vessels via the interaction of their known ligands, LFA-1/Mac-1 and 1times4-integrins, at least in vitro. It appears that 1times4-integrin plays a key role in lymphocyte recruitment across the blood-brain barrier and may be a major factor in lymphocyte targeting of the CNS. Definition of factors involved in the afferent and efferent connections between the CNS and the immune system may clarify mechanisms involved in immune privilege of the CNS and may open significant therapeutic opportunities for multiple sclerosis.     

Morphometric analysis of intralobular, interlobular and pleural lymphatics in normal human lung

In spite of their presumed relevance in maintaining interalveolar septal fluid homeostasis, the knowledge of the anatomy of human lung lymphatics is still incomplete. The recent discovery of reliable markers specific for lymphatic endothelium has led to the observation that, contrary to previous assumptions, human lymphatic vessels extend deep inside the pulmonary lobule in association with bronchioles, intralobular arterioles or small pulmonary veins. The aim of this study was to provide a morphometric characterization of lymphatic vessels in the periphery of the human lung. Human lung sections were immunolabelled with the lymphatic marker D2-40, followed by blood vessel staining with von Willebrand Factor. Lymphatic vessels were classified into: intralobular (including those associated with bronchovascular bundles, perivascular, peribronchiolar and interalveolar), pleural (in the connective tissue of the visceral pleura), and interlobular (in interlobular septa). The percentage area occupied by the lymphatic lumen was much greater in the interlobular septa and in the subpleural space than in the lobule. Most of the intralobular lymphatic vessels were in close contact with a blood vessel, either alone or within a bronchovascular bundle, whereas 7% were associated with a bronchiole and < 1% were not connected to blood vessels or bronchioles (interalveolar). Intralobular lymphatic size progressively decreased from bronchovascular through to peribronchiolar, perivascular and interalveolar lymphatics. Lymphatics associated with bronchovascular bundles had similar morphometric characteristics to pleural and interlobular lymphatics. Shape factors were similar across lymphatic populations, except that peribronchiolar lymphatics had a marginally increased roundness and circularity, suggesting a more regular shape due to increased filling, and interlobular lymphatics had greater elongation, due to a greater proportion of conducting lymphatics cut longitudinally. Unsupervised cluster analysis confirmed a marked heterogeneity of lymphatic vessels both within and between groups, with a cluster of smaller vessels specifically represented in perivascular and interalveolar lymphatics within the alveolar interstitium. Our data indicate that intralobular lymphatics are a heterogeneous population, including vessels surrounding the bronchovascular bundle analogous to the conducting vessels present in the pleural and interlobular septa, many small perivascular lymphatics responsible for maintaining fluid balance in the alveolar interstitium, and a minority of intermediate lymphatics draining the peripheral airways. These lymphatic populations could be differentially involved in the pathogenesis of diseases preferentially involving distinct lung compartments.     

Lymphangiogenesis,myeloid cells and inflammation

The lymphatic system is essential for the maintenance of tissue fluid balance, immune surveillance and the absorption of fatty acids in the gastrointestinal tract. The lymphatic circulation is also a key player in disease processes such as cancer metastasis, lymphedema and various inflammatory disorders. With the identification of specific growth factors for lymphatic endothelial cells and markers that distinguish blood and lymphatic vessels, as well as the development of in vivo imaging technologies that provide new tools to examine the lymphatic drainage function in real time, many advancements have been made in lymphatic vascular research during the past few years. Despite these significant achievements, our understanding of the role of lymphatics in disease processes other than cancer metastasis is still rather limited. The current review will focus on the recent progress made in studies of lymphatics in inflammatory disorders.     

Development of cerebrospinal fluid absorption sites in the pig and rat: connections between the subarachnoid space and lymphatic vessels in the olfactory turbinates

The textbook view that cerebrospinal fluid (CSF) absorption occurs mainly through the arachnoid granulations and villi is being challenged by quantitative and qualitative studies that support a major role for the lymphatic circulation in CSF transport. There are many potential sites at which lymphatics may gain access to CSF but the primary pathway involves the movement of CSF through the cribriform plate foramina in association with the olfactory nerves. Lymphatics encircle the nerve trunks on the extracranial surface of the cribriform plate and absorb CSF. However, the time during development in which the CSF compartment and extracranial lymphatic vessels connect anatomically is unclear. In this report, CSF–lymphatic connections were investigated using the silastic material Microfil and a soluble Evan’s blue-protein complex in two species; one in which significant CSF synthesis by the choroid plexus begins before birth (pigs) and one in which CSF secretion is markedly up regulated within the first weeks after birth (rats). We examined a total of 46 pig fetuses at embryonic (E) day E80–81, E92, E101, E110 (birth at 114 days). In rats, we investigated a total of 115 animals at E21 (birth at 21 days), postnatal (P) day P1–P9, P12, P13, P15, P22, and adults. In pigs, CSF–lymphatic connections were observed in the prenatal period as early as E92. Before this time (E80–81 fetuses) CSF–lymphatic connections did not appear to exist. In rats, these associations were not obvious until about a week after birth. These data suggest that the ability of extracranial lymphatic vessels to absorb CSF develops around the time that significant volumes of CSF are being produced by the choroid plexus and further support an important role for lymphatic vessels in CSF transport.     

CNS: Not an immunoprivilaged site anymore but a virtual secondary lymphoid organ

The cardinal dogma of central nervous system (CNS) immunology believed brain is an immune privileged site, but scientific evidences gathered so far have overturned this notion proving that CNS is no longer an immune privileged site, but rather an actively regulated site of immune surveillance. Landmark discovery of lymphatic system surrounding the duramater of the brain, made possible by high resolution live imaging technology has given new dimension to neuro-immunology. Here, we discuss the immune privilege status of CNS in light of the previous and current findings, taking into account the differences between a healthy state and changes that occur during an inflammatory response. Cerebrospinal fluid (CSF) along with interstitial fluid (ISF) drain activated T cells, natural killer cells, macrophages and dendritic cells from brain to regional lymph nodes present in the head and neck region. To keep an eye on inflammation, this system hosts an army of regulatory T cells (CD25+ FoxP3+) that regulate T cell hyper activation, proliferation and cytokine production. This review is an attempt to fill the gaps in our understanding of neuroimmune interactions, role of innate and adaptive immune system in maintaining homeostasis, interplay of different immune cells, immune tolerance, knowledge of communication pathways between the CNS and the peripheral immune system and lastly how interruption of immune surveillance leads to neurodegenerative diseases. We envisage that discoveries should be made not only to decipher underlying cellular and molecular mechanisms of immune trafficking, but should aid in identifying targeted cell populations for therapeutic intervention in neurodegenerative and autoimmune disorders.     

睾丸的淋巴流向观察

在32例足月胎儿尸体上,用淋巴管注射法观察了睾丸的淋巴流向。实验材料分为两组。第1组16例,由睾丸实质注入普鲁士蓝氯仿溶液,见到集合淋巴管沿睾丸血管上行,注入腰淋巴结,但一部分在途中直入髂总淋巴结和髂外淋巴结。第2组16例,先在第3腰椎高处结扎沿睾丸血管上行的集合淋巴管,然后再进行注射,结果见到上行的淋巴管在结扎处终止,有少数管也直入髂总淋巴结和髂外淋巴结;但在21侧睾丸,1、2条集合淋巴管沿输精管向下入盆腔,至膀胱底处消失或注入该处的髂内淋巴结。     

Surgical excision of CNS‐draining lymph nodes reduces relapse severity in chronic‐relapsing experimental autoimmune encephalomyelitis

Despite lack of classical lymphatic vessels in the central nervous system (CNS), cells and antigens do reach the CNS‐draining lymph nodes. These lymph nodes are specialized to mediate mucosal immune tolerance, but can also generate T‐ and B‐cell immunity. Their role in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) therefore remains elusive. We hypothesized that drainage of CNS antigens to the CNS‐draining lymph nodes is vital for the recurrent episodes of CNS inflammation. To test this, we surgically removed the superficial cervical lymph nodes, deep cervical lymph nodes, and the lumbar lymph nodes prior to disease induction in three mouse EAE models, representing acute, chronic, and chronic‐relapsing EAE. Excision of the CNS‐draining lymph nodes in chronic‐relapsing EAE reduced and delayed the relapse burden and EAE pathology within the spinal cord, which suggests initiation of CNS antigen‐specific immune responses within the CNS‐draining lymph nodes. Indeed, superficial cervical lymph nodes from EAE‐affected mice demonstrated proliferation against the immunizing peptide, and the deep cervical lymph nodes, lumbar lymph nodes, and spleen demonstrated additional proliferation against other myelin antigen epitopes. This indicates that intermolecular epitope spreading occurs and that CNS antigen‐specific immune responses are differentially generated within the different CNS‐draining lymphoid organs. Proliferation of splenocytes from lymphadenectomized and sham‐operated mice against the immunizing peptide was similar. These data suggest a role for CNS‐draining lymph nodes in the induction of detrimental immune responses in EAE relapses, and conclusively demonstrate that the tolerance‐inducing capability of cervical lymph nodes is not involved in EAE. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

输卵管的淋巴流向

用多色淋巴管间接注射方法，对３２例６４侧女性新生儿及婴儿输卵管的淋巴向及输卵管和卵巢淋巴管间的联系进行了观察。     

The cardiac lymphatic system

The lymphatic system, a network of vessels carrying clear interstitial fluid called lymph, is found throughout the human body. The system maintains homeostasis, receiving proteins and excess fluid from the interstitial tissues, and returning them to the venous system. Understanding of lymphatic drainage remains important in the diagnosis, prognosis, and treatment of diseases, including the metastasis of malignant diseases. Information specific to the cardiac lymphatics is scarce. Indeed, quite often the topic is not even mentioned in many medical textbooks. The goal of our review is to compile and analyze the information currently available concerning the cardiac lymphatics, hoping further to demonstrate the clinical importance of this neglected system.     

Functional anatomy of the lymphatics draining the skin: a detailed statistical analysis

Relatively little is known about the functional anatomy of the lymphatic vessels draining the skin. To address this issue, we previously created a three‐dimensional computer model of skin lymphatic drainage, using melanoma lymphoscintigraphy (LS) data from 5232 patients. In this study we sought to extend our model by performing a detailed statistical analysis of the mapped LS data to characterize the functional anatomy of the superficial lymphatics without any a‐priori spatial bias. We investigated the commonly held assumption that lymphatic drainage is symmetric between the two sides of the body. Results indicated that, with the exception of the lower anterior torso, posterior leg and a small section of the posterior torso, most skin regions with sufficient data showed symmetric drainage. LS data from each symmetric skin region were then reflected to the opposite side of the body to provide an increased LS dataset for subsequent analysis. Cluster analysis was then applied to this reflected LS dataset to group regions of skin that drained in a similar manner. Results defined nine large clusters of skin, largely draining to the dominant axillary, groin, cervical level II and preauricular node fields. Each of the four axillary and groin node fields defined large clusters of skin on the torso, dividing it into regions similar to the historical ‘Sappey’s lines’, although a fifth region of highly ambiguous drainage was also shown in the anterior and posterior center of the torso. Collectively, these results provide important new insights into skin lymphatic drainage, both improving and quantifying our understanding of functional lymphatic anatomy.     

Fetal Topographical Anatomy of the Upper Abdominal Lymphatics: Its Specific Features in Comparison With Other Abdominopelvic Regions

Using semiserial sections from 19 human fetuses of 8–30 weeks gestation, we examined the topohistology of the upper abdominal lymphatics and compared it with that of the lower abdominal and pelvic lymphatics. The upper abdominal lymphatics were characterized by an intimate relationship with the peritoneal lining, a common mesentery for the celiac trunk and superior mesenteric artery (SMA). Lymphatic connections from the upper abdominal viscera to the paraaortic and paracaval areas followed two routes: (1) from the intestinal mesentery, along the peritoneum on the left aspect of the proximal SMA, via the chain of lymph follicles (LFs) lying along the retropancreatic fusion fascia, to drain into the LFs around the left renal vein; (2) from sites along the peritoneum on the posterior wall of the omental bursa, via the root of the hepatoduodenal ligament, to drain into LFs around the vena cava. The development of these two posterior drainage routes seemed to be promoted by the peritoneum or a peritoneal remnant (i.e., fusion fascia) attaching to the great vessels, and inhibited or impeded by the developing nerves and diaphragm. No paraaortic, paracaval, or pelvic LFs lay along the peritoneum. The pelvic LFs were usually located along the bundle of lymphatic vessels originating from the femoral canal. Anat Rec, 2012. © 2011 Wiley Periodicals, Inc.     

Perivascular drainage of amyloid-beta peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer's disease.

Alzheimer's disease is the commonest dementia. One major characteristic of its pathology is accumulation of amyloid-beta (Abeta) as insoluble deposits in brain parenchyma and in blood vessel walls [cerebral amyloid angiopathy (CAA)]. The distribution of Abeta deposits in the basement membranes of cerebral capillaries and arteries corresponds to the perivascular drainage pathways by which interstitial fluid (ISF) and solutes are eliminated from the brain--effectively the lymphatic drainage of the brain. Theoretical models suggest that vessel pulsations supply the motive force for perivascular drainage of ISF and solutes. As arteries stiffen with age, the amplitude of pulsations is reduced and insoluble Abeta is deposited in ISF drainage pathways as CAA, thus, further impeding the drainage of soluble Abeta. Failure of perivascular drainage of Abeta and deposition of Abeta in the walls of arteries has two major consequences: (i) intracerebral hemorrhage associated with rupture of Abeta-laden arteries in CAA; and (ii) Alzheimer's disease in which failure of elimination of ISF, Abeta and other soluble metabolites from the brain alters homeostasis and the neuronal environment resulting in cognitive decline and dementia. Therapeutic strategies that improve elimination of Abeta and other soluble metabolites from the brain may prevent cognitive decline in Alzheimer's disease.     

The beta-chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors

The lymphatic vessels (lymphatics) play an important role in channeling fluid and leukocytes from the tissues to the secondary lymphoid organs. In addition to driving leukocyte egress from blood, chemokines have been suggested to contribute to leukocyte recirculation via the lymphatics. Previously, we have demonstrated that binding sites for several pro-inflammatory beta-chemokines are found on the endothelial cells (ECs) of lymphatics in human dermis. Here, using the MIP-1alpha isoform MIP-1alphaP, we have extended these studies to further support the contention that the in situ chemokine binding to afferent lymphatics exhibits specificity akin to that observed in vitro with the promiscuous beta-chemokine receptor D6. We have generated monoclonal antibodies to human D6 and showed D6 immunoreactivity on the ECs lining afferent lymphatics, confirmed as such by staining serial skin sections with antibodies against podoplanin, a known lymphatic EC marker. In parallel, in situ hybridization on skin with antisense D6 probes demonstrated the expression of D6 mRNA by lymphatic ECs. D6-immunoreactive lymphatics were also abundant in mucosa and submucosa of small and large intestine and appendix, but not observed in several other organs tested. In lymph nodes, D6 immunoreactivity was present on the afferent lymphatics and also in subcapsular and medullary sinuses. Tonsilar lymphatic sinuses were also D6-positive. Peripheral blood cells and the ECs of blood vessels and high endothelial venules were consistently nonreactive with anti-D6 antibodies. Additionally, we have demonstrated that D6 immunoreactivity is detectable in some malignant vascular tumors suggesting they may be derived from, or phenotypically similar to, lymphatic ECs. This is the first demonstration of chemokine receptor expression by lymphatic ECs, and suggests that D6 may influence the chemokine-driven recirculation of leukocytes through the lymphatics and modify the putative chemokine effects on the development and growth of vascular tumors.