Expression of the HNK-1 carbohydrate epitope in human retina and retinoblastoma

Summary Fifty formalin-fixed and paraffin-embedded retinoblastoma specimens and five normal human eyes were studied with the monoclonal anti-Leu-7 antibody, directed against the HNK-1 carbohydrate epitope that is shared by human natural killer cells and many neuronal, glial and neuroectodermal cells. The laboratory method was a sensitive immunohistochemical staining procedure, and neuroectodermal tumours that usually express this epitope were used as positive controls. In the human retina, Müiller cell membranes were positively stained, but additional staining of neuronal cells was not excluded at the light microscopical level. A positive cytoplasmic reaction was also seen in ciliary and retinal pigment epithelial cells. All but one intraocular retinoblastomata studied contained cells staining positively for the HNK-1 epitope, but these cells were probably not neoplastic. Although positive reaction has previously been reported in three retinoblastomata, the present results suggest that positive cells are derived from entrapped and infiltrated retina. Staining of adjacent sections against leukocyte common antigen suggested that the positively staining cells were not natural killer cells.This study was supported by a grant from the Emil Aaltonen Foundation     

Roles of HNK-1 carbohydrate epitope and its synthetic glucuronyltransferase genes on migration of rat neural crest cells

HNK-1 carbohydrate epitope is localized on the surface of avian neural crest cells (NCCs), and is necessary for their migration. However, it is still disputed whether the epitope works in similar ways in mammalian embryos. In this study, we found that HNK-1 carbohydrate epitope was specifically detected in some of the cranial ganglia, migrating trunk NCCs and some non-NCC derivatives in the rat embryo. Two genes encoding glucuronyltransferases that synthesize the HNK-1 epitope in vitro (GlcAT-P and GlcAT-D) were recently identified in the rat. Interestingly, the NCCs in the cranial ganglia expressed the GlcAT-D gene, whereas the migrating trunk NCCs expressed the GlcAT-P gene. To investigate in vivo functions of the GlcATs in the NCC migration further, we overexpressed GlcAT genes by electroporation in the cranial NCCs in cultured rat embryos. Transfection of both GlcAT genes resulted in efficient synthesis of the HNK-1 epitope in the NCCs. GlcAT-P overexpression increased distance of cranial NCC migration, whereas GlcAT-D overexpression did not show this effect. Our data suggest that the HNK-1 epitope synthesized by different GlcATs is involved in migration in the sublineages of the NCCs in the rat embryo, and that GlcAT-P and GlcAT-D mediate different effects on the NCC migration.     

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum, hippocampus and spinal cord using monoclonal antibodies with different epitope specificities

The HNK-1 carbohydrate, an unusual 3-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated 2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.     

Abnormal HNK-1 expression in the cerebellum of an N-CAM null mouse

Human natural killer antigen-1 (HNK-1) is a carbohydrate epitope associated with sulfoglucuronylglycolipids and glycoproteins. Biochemical analyses have demonstrated associations between the HNK-1 epitope and isoforms of the neural cell adhesion molecule (N-CAM) family. In the cerebellum, HNK-1 is prominently expressed in Purkinje cell dendrites and Golgi cells. Purkinje cell expression of HNK-1 reveals an array of parasagittal stripes and transverse zones. Interestingly, the parasagittal expression pattern of HNK-1 is different from those reported with several other markers such as zebrin II/aldolase C and the small heat shock protein HSP25. N-CAM null knockout mice were used to explore the possible role of the HNK-1/N-CAM interaction during the topographical organization of the cerebellar cortex. N-CAM null mice have no N-CAM immunoreactivity but otherwise the cerebellum appears morphologically normal. Further, in the N-CAM null HNK-1 immunoreactivity is abolished from Purkinje cell dendrites but is retained on Golgi cells and neurons of the cerebellar nuclei. Despite the absence of N-CAM/HNK-1, parasagittal stripes and transverse zones in the cerebellum as revealed by using zebrin II immunocytochemistry appear normal.     

Bridging extended nerve defects with an artifcial nerve graft containing Schwann cells pre-seeded on polyglactin filaments

A 24 mm long bioartificial nerve graft (BNG) was created to bridge extended peripheral nerve defects of the rat sciatic nerve. In our previous studies, an identical graft had demonstrated good results over nerve gaps of up to 15 mm. The BNG device comprised a collagen-I tube filled with ten Schwann-cell-seeded polyglactin filaments and 10(6) isogenic Schwann cells suspended in Matrigel which were implanted in 27 rats (group I). Schwann-cell-free grafts (27 rats) and nerve autografts (18 rats) served as controls. Functional recovery was followed over a period of six months using walking track analysis. Terminal analyses of graft efficacy included neurophysiology, muscle weight, and histological assessment of the implants and the distal nerve stumps. In 17/27 cases, axonal regeneration into the distal nerve stump could be detected across the BNG, but all animals in group I and II failed to regain motor function of the hindlimb upon completion of the experiment. Axon diameter and axonal density in the graft and distal nerve stump were greater in group I than in group II. Although Schwann cells had a significant positive effect on axonal regeneration, either granuloma formation or the amount of the inserted foreign material may have impaired nerve regeneration by acting as a physical impediment to nerve regeneration or negatively effecting cell function.     

Distribution of HNK-1+ cells in malignant lymphomas

HNK-1, a murine monoclonal antibody, is known to react with most of the natural killer (NK) and killer (K) cells in peripheral blood. Cells reacting with this antibody (HNK-1+ cells) were studied on tissue sections of ninety two cases of malignant lymphomas (MLs) by using immunoperoxidase technique, in an attempt to elucidate the role of this type of cells in MLs. Follicular lymphomas were found to be highly infiltrated with HNK-1+ cells. The mode of infiltration in follicular lymphomas is just like in normal germinal centers. Many cases of diffuse lymphomas with cleaved nuclei, indicative of diffuse B-cell lymphomas of follicular center cell origin, as well as diffuse ML with heavy fibrosis (sclerosis) or histiocytic reaction, were also found to be infiltrated with abundant HNK-1+ cells. Meanwhile, other types of B-cell ML and all types of T-cell ML, as well as Hodgkin's disease, were shown to be very poor in HNK-1+ cell reaction. From a prognostic viewpoint, the low grade malignancy group in the NCI Working Formulation or Kiel Classification was found to be infiltrated with significantly much more HNK-1+ cells as compared to the high grade malignancy group. The significance of these findings are discussed, with the stress on the possible suppressive function of HNK-1+ cells on proliferation and differentiation of follicular center cell type B-cell MLs.     

Specific expression of the neurofibromatosis type 1 gene (NF1) in the hamster Schwann cell.

The gene responsible for neurofibromatosis type 1 (NF1) has sequence homology to the GTPase-activating protein (GAP) and demonstrates GAP activity against ras p21. To study tissue-specific and/or tumor-specific expression of the NF1 gene product, now called neurofibromin, immunostaining and immunoblotting were applied to the N-nitroso-N-ethylurea (ENU)-induced Syrian hamster neurofibromatosis model using polyclonal antibodies against the NF1 fusion protein and a synthetic peptide. Strong expression was observed specific to the Schwann cells of the normal peripheral nerves by immunostaining. Neoplastic Schwann cells showed specific binding of anti-NF1; however, the frequency of positive cells was diminished. Immunoblotting also revealed positive expression of the 250-kd NF1 gene product in the brain, the normal peripheral nerves, and 7 of 14 ENU-induced neurofibromas. Although ENU-induced melanoma and Wilms' tumor were negative for neurofibromin, foci of Schwannian differentiation in both primary and transplanted melanomas were positive. These results suggest that neurofibromin plays some role in differentiation and growth regulation of the Schwann cell.     

Differential expression of lactoseries carbohydrate epitopes HNK-1, CD15, and NALA by olfactory receptor neurons in the developing chick

 The formation of the nasal lining with its sensory and its nonsensitive respiratory epithelium requires a spatially ordered pattern of cellular differentiation. Aiming at identifying cell recognition molecules that may be involved in cellular differentiation steps, we applied a panel of antibodies to terminal carbohydrate sequences of the lactoseries on the developing chick olfactory epithelium. This approach is based on the idea that these terminal sugar residues may be involved in certain steps of maturation. Restricted expression of three epitopes NALA, HNK-1, and CD15 was observed in olfactory receptor neurons. The first immature olfactory receptor neurons were observed by day 3 of incubation, expressing the HNK-1 epitope, whereas a total epithelial staining was observed for NALA. By day 9 of incubation high numbers of HNK-1 positive immature olfactory receptor neurons were observed. At the same time mature olfactory receptor neurons showed immunoreactivity for CD15, whereas NALA was still expressed throughout the whole epithelial cell population. However, there was a pronounced staining in the population of mature olfactory receptor neurons. Around hatching only CD15 was detectable in (mature) olfactory receptor neurons, whereas HNK-1 and NALA immunoreactivity have switched to glandular and sustentacular cells respectively. The differentiation-dependent expression patterns of these three cell surface molecules suggest them as suitable markers to explore mechanisms that determine embryonic olfactory receptor neurogenesis. Accepted: 15 October 1997     

雪旺细胞促进周围神经再生机制的研究进展

雪旺细胞(SCs)具有强大的可塑性。周围神经损伤后,雪旺细胞去分化为修复表型,主导了修复过程。c-Jun氨基末端激酶、有丝分裂原激活的蛋白激酶(MAPK)等多种信号通路和转录调节因子参与调控雪旺细胞介导的修复程序。本综述讨论了雪旺细胞促进周围神经再生的主要信号通路与转录调节因子。     

Immunocytochemical localization of the L1 and N-CAM cell adhesion molecules and their shared carbohydrate epitope L2/HNK-1 in the developing and differentiated gustatory papillae of the mouse tongue

Summary The localization of the cell adhesion molecules L1 and N-CAM, and their shared carbohydrate epitope L2/HNK-1, was investigated at the light and electron microscopic levels in developing and adult fungiform and circumvallate gustatory papillae of the mouse tongue.At embryonic day 13, the earliest stage investigated, the tongue epithelium was still undifferentiated and was not yet innervated by sensory fibres. At this stage none of the three molecules was detectable within the tongue epithelium. At embryonic day 15 the primordia of the gustatory papilla became unequivocally discernible when the papillary epithelium was already innervated by few sensory axons. At this stage N-CAM was the first molecule expressed on epithelial cells and was confined to those parts of the papillary epithelium destined to become the chemosensory cells of the taste buds. The sensory axons were N-CAM-, L1- and L2/HNK-1-positive when fasciculating or contacting their accompanying Schwann cells or the cells of the papillary epithelium. Contacts between Schwann cells were also prominently labelled by antibodies to the three antigens. The mesenchymal tissue underlying the prospective sensory epithelium expressed N-CAM at all embryonic stages, but ceased to be N-CAM positive within the first six postnatal days. From embryonic day 16 onward a weak L1 immunoreactivity was detectable within the basal and intermediate layers of the lingual epithelium and remained present in adulthood.Cytodifferentiation of epithelial cells into spindle-shaped sensory cells and organization into taste buds began at postnatal day two. Simultaneously, L1 and L2/HNK-1 immunoreactivity increased on taste bud cells and N-CAM disappeared from the non-sensory extragemmal parts of the papillary epithelium. At approximately postnatal day six, taste bud formation was complete and the pattern of cell adhesion molecule expression was comparable to that found in the adult in that L1 was strongly expressed on the apposing surfaces of all cells, whereas N-CAM was confined to cell contacts between a subpopulation of intragemmal cells. The L2/HNK-1 epitope was visible on the surfaces of taste bud cells, on intragemmal axons, and in a small portion of extracellular matrix directly underlying the taste buds, but was no longer expressed on those parts of the sensory fibres embedded in the subepithelial mesenchyme. The L2/HNK-1 epitope may thus be regarded as a cell surface marker for the cellular elements of mature taste buds. The highly sialylated form of N-CAM was not detectable at any stage investigated.The observations suggest that the expression of the three molecules within the papillary epithelium follows rather than precedes the innervation by sensory axons and does not, therefore, reflect the gustatory epithelium's susceptibility to innervation as found for N-CAM in the neuromuscular system. The spatio-temporal expression of N-CAM, however, is suggestive of its influence on the differentiation of taste bud cells. Apart from axon-axon and axon-Schwann cell interactions L1 might be involved in interactions between gustatory cells and sensory nerve terminals and, surprisingly, also between non-sensory epithelial cells, whereas the L2/HNK-1 epitope may be implicated in the maintenance of the characteristic cytoarchitecture of the differentiated taste buds.     

Development of mammalian nerve-muscle synapses in culture: lack of interference by antibodies to the neural cell adhesion molecule N-CAM and its L2/HNK-1 carbohydrate epitope

Rat myotubes were cultured with embryonic mouse spinal cord explants. After 4 days of co-culture, (miniature) endplate potentials, (M)EPPs, could be detected in 20-40% of the nerve-contacted myotubes. Polyclonal antibodies against the neural cell adhesion molecule N-CAM and monoclonal antibodies to its L2/HNK-1 carbohydrate epitope were tested for possible effects on synapse formation in this system. Co-cultures that were started and maintained in the presence of Fab fragments of these antibodies developed functional synapses to the same extent as did control cultures. Staining with an anti-IgG antibody demonstrated that added Fab fragments had bound to neurites and that an excess was present in the medium. These findings suggest that even if N-CAM and the L2/HNK-1 epitope were involved in early stages of muscle-nerve contact, they are not essential for the formation of electrophysiologically active synapses.     

Myelination of regenerated axons in goldfish optic nerve by Schwann cells

Summary This study uses immunohistochemistry and EM to examine the site of injury in goldfish optic nerve during axonal regeneration. Within seven days of nerve crush axons begin to regrow and a network of GFAP⁺ reactive astrocytes appears in the nerve on either side of the injury. However, the damaged area remains GFAP^–. By 42 days after nerve crush, the sheaths of new axons acquire myelin marker 6D2, and the crush area becomes populated by a mass of longitudinally-orientated S-100⁺ cells. Ultrastructurally, the predominant cells in the crush area bear a strong resemblance to peripheral nerve Schwann cells; they display a one-to-one association with myelinated axons, have a basal lamina and are surrounded by collagen fibres. It is proposed that these cells are Schwann cells which enter the optic nerve as a result of crush, where they become confined to the astrocyte-free crush area.     

In vivo and electron microscopic observations on Schwann cells in developing tadpole nerve fibers

The migration of Schwann cells and their early association with axons were studied in transparent tadpole tail fins. Nomarksi optics revealed that in vivo these cells are pleomorphic, changing their shape by extending and withdrawing long, blunt pseudopods. Daily observations of the same fiber fascicles for a month or more combined with intensive short-term studies of other tadpoles showed that migrating Schwann cells move sporadically at rates of up to 114 μm/day. They are usually, although not always, in contact with one or more axons. In the electron microscope, these migrating cells are similar in cytoplasmic structure to others that have settled down and begun to spread along axons; however, they possess no basal lamina. Later, Schwann cells become more spindle-shaped and acquire a basal lamina. Schwann cell surface characteristics and the changes imposed by the presence of the basal lamina may be important in the establishment of permanent axon-Schwann cell relationships. In our living material we were unable to visualize the intricate, rapidly changing associations between Schwann cells and small axonal fascicles that precede myelination. However, they are probably more complex than Speidel's studies would indicate.     

周围神经损伤与再生中施万细胞可塑性的研究进展

周围神经损伤后具有适宜轴突再生的微环境,这与周围神经损伤后施万细胞的可塑性密不可分,现阐述施 万细胞可塑性的研究新进展,为促进周围神经良好的再生修复提供基础知识。     

Polysialylated NCAM expression on endocardial cells of the chick primary atrial septum

Septation of the tubular heart to form the multi-chambered heart involves endocardial cell mesenchymal transformation at discrete sites. These sites include the crests of endocardial cushions at the atrioventricular junction, crests of the spiral ridges within the outflow tract, and the leading edge of the atrial septum. The factors involved in this multi-step inductive process appear to include the neural cell adhesion molecule (NCAM). The down-regulation of NCAM coincident with mesenchymal transformation has been documented at the atrioventricular cushion tissue. In view of the function-regulating properties of polysialylated NCAM (PSA-NCAM), we hypothesized that this form of NCAM would be playing a role during the dramatic changes in cell-cell interactions occurring in the endocardium at the leading edge of the primary atrial septum. Chicken hearts at stages during primary atrial septum development were fixed with paraformaldehyde and either immunofluorescently stained for the light microscope analysis or immunoperoxidase stained for ultrastructural analysis. A monoclonal antibody to an NCAM polypeptide epitope (5E) was used to detect all forms of NCAM, while a monoclonal to the polysialic acid (5A5) was used to detect that subset of NCAM which is highly polysialylated (PSA-NCAM). By light microscope level analysis, an increase in immunostaining for NCAM and the appearance of PSA-NCAM was detected on embryonic chicken endocardial cells at the leading edge of the growing atrial septum. The ultrastructural analysis revealed that there is also a change in the pattern of NCAM and PSA-NCAM from a polarized localization to a more ubiquitous distribution over the endocardial cell surface as these cells send out processes, form multiple layers, and sink or move into the underlying extracellular matrix. PSA-NCAM was also detected along cell appositions of cells within the matrix. Both NCAM and PSA-NCAM levels were reduced on cells deep within the matrix. These findings indicate that during primary atrial septation, PSA-NCAM may be deployed on endocardial epithelial cells in order to down-regulate cell-cell interactions and allow the detachment and migration of some of these cells into the underlying matrix. Anat. Rec. 247:71–84 © 1997 Wiley-Liss, Inc.     

Temporal and spatial patterns of expression of laminin, chondroitin sulphate proteoglycan and HNK-1 immunoreactivity during regeneration in the goldfish optic nerve

Summary Current views suggest that the extracellular environment is critically important for successful axonal regeneration in the CNS. The goldfish optic nerve readily regenerates, indicating the presence of an environment that supports regeneration. An analysis of changes that occur during regeneration, in this model may help identify those molecules that contribute to a favourable environment for axonal regrowth. We examined the distribution and expression of two extracellular matrix molecules, laminin and chondroitin sulphate proteoglycan, and a carbohydrate epitope shared by a family of adhesion molecules (HNK-1), using immunocytochemical detection in sections from the normal adult goldfish optic nerve and in nerves from one hour to five months following optic nerve crush. We also usedin vitro preparations to determine if neurites in retinal explants could express these same molecules.The linear distributions of laminin and chondroitin sulphate proteoglycan immunoreactivity in control optic nerves are co-extensive with the glia limitans, suggesting both are expressed by non-neuronal components surrounding the axon fascicles. Between one and three weeks postoperatively when axons elongate and reach their target, laminin and chondroitin sulphate proteoglycan immunoreactivity increases around the crush site and distally. At six weeks postoperatively the pattern of immunoreactivity has returned to normal. While the temporal pattern of changes in immunoreactivity is similar, the spatial pattern of these two extracellular proteins in the regenerating nerve differs. Chondroitin sulphate proteoglycan immunoreactivity is organized in discrete columns associated with regenerating axons while laminin immunoreactivity is more diffusely distributed. Examination of retinal explants reveals growing neurites express chondroitin sulphate proteoglycan but not laminin. Our results suggest that laminin is only associated with non-neuronal cells, while chondroitin sulphate proteoglycan is associated with axons as well as non-neuronal cells.HNK-1 immunoreactivity is co-extensive with both the glia limitans and axon fascicles and is more extensively distributed in the intact nerve than either laminin or chondroitin sulphate proteoglycan immunoreactivity. In contrast to laminin and chondroitin sulphate proteoglycan, HNK-1 immunoreactivity is substantially decreased at the crush site within one week following optic nerve crush. HNK-1 immunoreactivity reappears through the crush site during the next several weeks, although non-immunoreactive regions, co-extensive with areas predominantly containing non-neuronal cells, persist both proximal and distal to the crush, up to six weeks postoperatively. The pattern suggests that HNK-1 epitope expression by these non-neuronal cells is decreased during axonal regeneration.Our results show that each of these molecules is constitutively expressed with a unique distribution in the normal goldfish optic nerve and each exhibits different patterns of change during regeneration. It thus appears that each may contribute to modifications of the environment that supports axonal regeneration. Both neurons and non-neuronal cells contribute to these changes.     

Schwann细胞发生发育过程中和周围神经损伤后的表型表达及其影响因素

Schwann细胞（SCs）起源于胚胎的神经嵴细胞,是周围神经系统（PNS）的髓鞘形成细胞,包绕或包裹PNS的轴突形成有髓或无髓神经纤维。SCs不仅在整个发育过程中与PNS存在着密切联系,如对发育中周围神经元的存活、成熟有髓纤维的跳跃式传导等,而且在周围神经损伤后,还可逆分化为幼稚状态形成Btingner带,分泌神经营养因子,促进离断轴突的出芽再生,并重新包绕再生的轴突形成髓鞘,从而在中枢及周围神经的损伤再生及修复中发挥重要作用。因此,SCs具有广阔的研究及应用前景。本文就SCs的生长发育过程以及各阶段的表型表达和影响因素加以综述。