Biomedical subjects

J Wilting

Publications and source records attributed to J Wilting.

At least 55 records · Page 3Linked to original sources

PubMed1997

Expression of the avian VEGF receptor homologues Quek1 and Quek2 in blood-vascular and lymphatic endothelial and non-endothelial cells during quail embryonic development.

We have studied the expression of Quek1 and Quek2 (VEGFR-2 and VEGFR-3, respectively) in quail embryos from day 2 to day 16 by in situ hybridization with digoxigenin-labelled riboprobes on whole-mounts and paraffin sections. Parallel sections were also stained with the QH1 antibody to detect all endothelial cells and with an antibody against alpha-smooth-muscle-actin to reveal the media of blood vessels. Quek1/VEGFR-2 is a marker of blood-vascular and lymphatic endothelial cells throughout development. In 2-day-old embryos, it is expressed in the intra-embryonic vascular plexus, in cells (most probably angioblasts) located in the paraxial head mesoderm and in the somites, and caudo-laterally from Hensen's node. Thereafter, until about day 9, Quek1 is expressed in all endothelial cells. Cells positive and negative for Quek1 can later be found within the same vessel. Quek1 is additionally expressed in lymphatic endothelial cells. Occasionally, some non-endothelial cell types express Quek1. Quek2/VEGFR-3 is also a marker of endothelial cells; however, its expression pattern differs from that of Quek1. In 2-day-old embryos, Quek2 is expressed in the notochord and the intra-embryonic vascular plexus. Whereas all endothelial cells are Quek2-positive in 3-day-old embryos, expression is subsequently reduced to a subset of endothelial cells: arteries become Quek2-negative and then expression of Quek2 is limited to a few vessels that appear to be lymphatic. Endothelial cells of lymph nodes and the periaortal lymphatic vessels are Quek2-positive in later stages. A few non-endothelial cells express Quek2.

Animals↗

PubMed1997

VEGF induces cardiovascular malformation and embryonic lethality.

The essential function of vascular endothelial growth factor (VEGF) in embryonic angiogenesis has clearly been documented in murine embryos with targeted deletions of either VEGF or its receptors. The effects of VEGF in the organogenetic phase of development have not been studied to date. Therefore, we applied 0.7 to 0.9 microgram of VEGF via methylcellulose carriers into the midbrain or onto the right forelimb of 4.5-day-old quail embryos. Another group of embryos was treated with 1 microgram of platelet-derived growth factor and controls were carried out using carriers without any growth factor. VEGF-induced cardiovascular malformations resulted in embryonic lethality. The venous area of the vasculature was dilated in almost all organs. The heart was most seriously affected and showed typical characteristics of insufficiency. VEGF strongly increased endocardial cell proliferation and obviously induced impairment of the growth rates of myocardium and endocardium. The myocardium of the ventricles was extremely thin, and septation defects were observed. As a result of the disturbed outflow, the atria were extremely dilated and thin-walled. The morphology of the hearts was reminiscent of that observed in congenital malformations such as Uhl's and Osler's syndromes. Our results show that expression of VEGF has to be tightly controlled during development.

Animals↗

PubMed1996

Scatter factor/hepatocyte growth factor (SF/HGF) induces emigration of myogenic cells at interlimb level in vivo.

The initiating event in the migration of myogenic cells to the limb buds is an epitheliomesenchymal transformation of cells located at the lateral edge of the dermomyotome. Recently, a targeted mutation of c-met in mice demonstrated an essential role of this tyrosine kinase receptor and its ligand, scatter factor/hepatocyte growth factor (SF/HGF), in the migration of myogenic cells to the limb buds. Here, we show that ectopic application of exogenous SF/HGF induces emigration of Pax-3-positive myogenic cells into the lateral plate mesoderm. During this process, the lateral portions of the dermomyotomes deepithelialize and the basement membrane disintegrates. Detaching myogenic cells do not lose N-cadherin from their surfaces. We conclude that an HGF/SF- and c-met-mediated signal detaches myogenic precursor cells from the somites and thus plays a necessary role in the initiation of myoblast migration.

Animals↗

PubMed1996

Expression of avian Pax1 and Pax9 is intrinsically regulated in the pharyngeal endoderm, but depends on environmental influences in the paraxial mesoderm.

Pax1 and Pax9 represent a subfamily of paired-box-containing genes. In vertebrates, Pax1 and Pax9 transcripts have been found specifically in mesodermal tissues and the pharyngeal endoderm. Pax1 expression in the sclerotomes has been shown to be indispensable for proper formation of the axial skeleton, but expression of Pax1 in the endoderm has not been studied in detail. We have cloned the chick homologue of the murine Pax9 gene. Our results show that transcripts of Pax1 and Pax9 are first detectable in the prospective foregut endoderm of headfold-stage avian embryos. Endodermal expression correlates with the highly proliferative zones of the folding foregut and evaginating pharyngeal pouches. In later stages, Pax1 and Pax9 are expressed in overlapping but distinct patterns within the developing sclerotomes and limb buds. From grafting experiments we conclude that activation of pharyngeal Pax1 and Pax9 expression is an intrinsic property of the endoderm, not requiring midline structures or head mesoderm. In contrast, notochord is required to induce Pax1 in competent sclerotomes. Here we show that in vitro there is a cranio-caudal gradient of inductive capacity in the notochord. This coincides with the graded expression of Pax1 and Pax9 along the cranio-caudal axis in 2- to 3-day-old embryos. Furthermore, paraxial head mesoderm shows no competence to express Pax1. Finally, in vitro we find counteracting influences on notochord signaling by lateral tissues (lateral plate, intermediate mesoderm), leading to an inhibition of Sonic hedgehog (Shh) expression in notochord and floor plate, as well as Pax1 and Pax9 expression in sclerotomes. Taken together, our results demonstrate that different mechanisms regulate expression of Pax1 and Pax9 in foregut and sclerotome, but suggest a common function for both genes in the two tissues that is promoting proliferation and preventing fusion of neighboring blastemas.

Amino Acid Sequence↗

PubMed1996

N-cadherin is involved in myoblast migration and muscle differentiation in the avian limb bud.

Limb muscle formation involves invasion of the limb bud mesoderm by myogenic precursor cells from the dermomyotomes at limb bud level. Directed cell migration, homing, and differentiation of myogenic cells are controlled by the stationary cells of the limb bud mesoderm. At the level of the extracellular matrix, the molecular basis of migration control has been suggested to be exerted by the distribution of hyaluronan. Here, we demonstrate that N-cadherin-mediated interactions play a role at cell-membrane level in myoblast distribution and differentiation. N-cadherin is strongly expressed by myogenic cells in the chick limb bud and more moderately expressed by stationary mesodermal cells in the myogenic zones and progress zone. After in vivo injection of antibodies and Fab-fragments against the homophilic binding site of N-cadherin into the wing bud mesoderm, aggregates of myoblasts are found predominantly in the dorsal myogenic zone 36 hr after injection apparently due to immobilization. In the same position, areas of myf-5-positive cells are also observed. In injected limb buds, Pax-3-positive cells are less evenly distributed than in uninjected limbs. They are found to spread up to the epidermis and also form loosely arranged aggregates. After prolonged reincubation periods, injected limbs show ectopic myoblasts that are rich in desmin and areas of strongly desmin-expressing myoblasts within muscle blastemas. These effects were not observed after application of antibodies against other parts of the N-cadherin molecule. We conclude that N-cadherin is involved in myoblast migration in the limb buds via homophilic interactions and that it plays a role in signal transduction during myogenesis.

Animals↗

PubMed1996

Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor.

Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PIGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PIGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions in vivo.

Alternative Splicing↗

PubMed1996

VEGF121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane.

We have studied the effect of VEGF(121) homodimer and VEGF(121/165) heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2-4 micrograms and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU-anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension, D, of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF(121) is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF(121) application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF(121)) and 1.41 (VEGF(121/165)). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF(165) and VEGF(121/165), VEGF(121) diffuses over a slightly greater distance. Using in situ hybridization, VEGF receptor-2 (flk-1/Quek1) and the homologous flt-4 (Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF(121) application on the CAM of 11-day-old quail embryos. During normal development, flk-1 expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF(121) application induces expression of flk-1 in capillaries that normally do not express the receptor. In the normal development of the CAM, flt-4 becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF(121) does not alter flt-4 expression.

Allantois↗

PubMed1996

Fibroblast growth factor receptor 1 in skeletal and heart muscle cells: expression during early avian development and regulation after notochord transplantation.

Basic fibroblast growth factor (bFGF, FGF-2) mediates several biological functions during embryonic development. With regard to skeletal muscle formation, it has been suggested that FGF-2 is involved in the growth and differentiation of myogenic precursor cells. To identify the FGF-responsive cells we studied the expression of FGF receptor type I (FGFR-1) during early embryonic development of the chick. FGFR-1 immunoreactivity is present at all stages examined (embryonic day [E] 2-E5). Expression of FGFR-1 is found in the somite myotome, limb bud muscle cells, eye and tongue muscle cells, and myocardium. Transplantation of an additional notochord into the paraxial mesoderm, which prevents the formation of a myotome, reveals the absence of FGFR-1 immunoreactivity on the operated side. The distinct expression pattern of FGFR-1 in migrating and differentiating muscle cells indicates that in addition to the stimulation of proliferation of myoblasts, FGF-2 exerts other (nonmitogenic) effects on postmitotic myocytes.

Animals↗

PubMed1996

Embryonic angiogenesis: a review.

Supply with nutrients is essential from early embryonic stages onwards. Therefore, circulatory organs form the first functioning organ system. With the exception of the heart, this system is at first formed by only one cell type, the endothelial cell. Emergence, behavior, and differentiation of endothelial cells are discussed in this review. At first, endothelial cells develop from angioblasts (primary angiogenesis/angioblastic development), later they develop from preexisting endothelial cells (secondary angiogenesis/angiotrophic growth). The composition of the extracellular matrix may promote or inhibit angiogenesis. Various growth factors which can be bound to the extracellular matrix may have been found, but only two of them (VEGF, P1GF) seem to influence endothelial cell behavior directly. Heterogeneity and organ-typical differentiation of endothelial cells seem to be dependent on cell-cell signaling within each organ.

Angiogenesis Inducing Agents↗

PubMed1996

Function of somite and somitocoele cells in the formation of the vertebral motion segment in avian embryos.

We have studied the distribution of thoracic somite and somitocoele-derived cells using homotopical grafting between quail and chicken embryos and reincubation periods of 2-6 days. Serial sections were evaluated with antibodies against quail cells, quail hemangiopoietic cells and desmin. With the exception of neural crest cells in the cranial sclerotome half, all cells of the operated segment are quail cells derived from a single somite. These cells differentiate into sclerotome, myotome and the anlage of the dermis of the back. After longer reincubation periods, the somite-derived quail cells form the neighboring halves of 2 adjacent vertebral bodies and the intervening (disc-homologous) tissue. Resegmentation is furthermore visible in the lamina and the spinous process. Somite cells also form the articular and transverse processes, and the intertransverse muscle including its insertion to the next cranial transverse process. One thoracic somite forms the proximal part of 1 rib. In more distal parts, 1 somite forms the cranial half of 1 rib and the caudal half of the next cranial rib, and the intercostal muscle and part of the connective tissue. Somite-derived quail cells are found in muscle that bridges over 2 segments cranial and caudal from the operated segment. The craniocaudal distribution of endothelial cells is approximately the same. Somitocoele cells that are located centrally in the epithelial somite express the sclerotome-markers Pax-1 and Pax-9. After 2-3 days of reincubation, grafted thoracic somitocoele cells are found mainly in the cranial part of the caudal sclerotome half. They form an area representing the anlagen of the intervertebral disc and the rib. After longer reincubation periods, the grafted quail somitocoele cells form the intervertebral disc-homologous tissue and the proximal part of the rib. In more distal parts of the rib they are located in the cranial half of 1 rib and the caudal half of the next cranial rib. The somitocoele cells also form the surface of the intervertebral joint, and give rise to a small number of endothelial cells that are found up to 1 segment cranial and caudal to the operation site. Our studies show that resegmentation is found in most parts of the vertebra and in the distal ribs. One somite forms the origin and insertion of the segmental muscle. Therefore, the somite can be regarded as the ancestor of the vertebral motion segment. Somitocoele cells are located centrally both in the epithelial somite and in the vertebral motion segment.

Animals↗

PubMed1996

Pax1 is expressed during development of the thymus epithelium and is required for normal T-cell maturation.

Pax1 is a transcriptional regulatory protein expressed during mouse embryogenesis and has been shown to have an important function in vertebral column development. Expression of Pax1 mRNA in the embryonic thymus has been reported previously. Here we show that Pax1 protein expression in thymic epithelial cells can be detected throughout thymic development and in the adult. Expression starts in the early endodermal epithelium lining the foregut region and includes the epithelium of the third pharyngeal pouch, a structure giving rise to part of the thymus epithelium. In early stages of thymus development a large proportion of thymus cells expresses Pax1. With increasing age, the proportion of Pax1-expressing cells is reduced and in the adult mouse only a small fraction of cortical thymic stromal cells retains strong Pax1 expression. Expression of Pax1 in thymus epithelium is necessary for establishing the thymus microenvironment required for normal T cell maturation. Mutations in the Pax-1 gene in undulated mice affect not only the total size of the thymus but also the maturation of thymocytes. The number of thymocytes is reduced about 2- to 5-fold, affecting mainly the CD4+8+ immature and CD4+ mature thymocyte subsets. The expression levels of major thymocyte surface markers remains unchanged with the exception of Thy-1 which was found to be expressed at 3- to 4-fold higher levels.

Animals↗

PubMed1996

The formation of somite compartments in the avian embryo.

The somites develop from the unsegmented paraxial mesoderm that flanks the neural tube. They form in an intrinsic process which lays down the primary segmental pattern of the vertebrate body. We review the processes of somitogenesis and somite differentiation as well as the mechanisms involved in these developmental events. Long before overt differentiation occurs, different compartments of the still epithelial somites give rise to special cell lines and to particular derivatives. By means of isotypic grafting between quail and chick embryos, it is possible to follow the fate of groups of somitic cells. In this way, the development of the myotome and the back dermis from the dorsomedial quadrant and of the hypaxial body wall and limb musculature from the dorsolateral quadrant was established. The two ventral quadrants and the somitocoele give rise to the chondrogenic/fibroblastic lineage of the sclerotome and form the vertebral column. Somite compartments can first be visualized by the expression pattern of Pax genes. Pax-3 is expressed in the dorsal part of the epithelial somite, while the ventral two thirds express Pax-1, a marker of sclerotome development. Pax-3 expression is retained also in the premitotic myogenic cells that migrate into the limb buds. In differentiating myoblasts, Pax-3 expression is turned down and taken over by the activation of MDF's. This initial event in myogenesis occurs in the absence of local signals, whereas the expression of Pax-1 in the sclerotome can be shown to be induced by signals from the notochord and floor-plate of the neural tube. Epaxial myotome differentiation is supported by the neural tube, after the neural tube has received patterning signals from the notochord. The hypaxial musculature and limb musculature differentiate independently of the axial structures. The myogenic cells migrating within the limb buds respond to signals of the lateral plate mesoderm which guide their distalward migration and pattern the muscle.

Animals↗

PubMed1995

Angiogenic potential of the avian somite.

We have studied the angiogenic potential of the unsegmented paraxial mesoderm and epithelial somites of the trunk with homotopical grafts between quail and chick embryos. Quail endothelial cells of the grafts were stained with the QH-1 antibody after 1-6 days of reincubation. The unsegmented paraxial mesoderm and all parts of the epithelial somite were found to contain angioblasts which develop into QH-1 positive endothelial cells. These cells are incorporated into the lining of the host's blood vessels such as the perineural vascular plexus and the dorsal branches of the aorta. There is a certain preference as concerns the location of endothelial cells derived from different parts of the somites. Angioblasts from ventral somite halves are mainly found in ventrolateral blood vessels. Those from dorsomedial quadrants form vessels in the dermis of the back, and those from dorsolateral quadrants can be found in the ventrolateral body wall and the wing. With the exception of the dorsal perineural vascular plexus, angioblasts do not cross the median plane of the body. This shows that, although angioblasts migrate extensively, there is bilaterality of the vascular system in the trunk. It remains to be studied whether the notochord plays a role in the establishment of this bilaterality.

Animals↗

PubMed1995

Blood vessel formation in the avian limb bud involves angioblastic and angiotrophic growth.

The vasculature of the avian limb bud takes its origin from the intersomitic vessels as can be shown by ink perfusion of the embryo. While the primitive vessels form a central network in the early limb bud, an area of about 100 microns in width from the ectoderm inward remains free from lumenized vessels. However, this subectodermal avascular zone contains isolated angioblasts, which can be demonstrated by confocal laser scanning microscopy in connection with QH-1-staining. QH-1-positive cells from the avascular zone are capable of giving rise to endothelial cells when grafted ectopically into a "permissive" environment such as the dorso-lateral paraxial mesoderm. Several grafting sites are compared regarding their permissiveness for capillary formation. In order to investigate the origin of the QH-1-positive angioblasts we carried out injections of DiI-Ac-LDL, which is specifically taken up by endothelial cells and macrophages, and found the lumenized vessels and a few isolated cells in the peripheral limb mesoderm stained. In double-labelling studies combining DiI-Ac-LDL and QH-1, it can be shown that there exists a pool of isolated angioblasts that are only QH-1-positive, but have not incorporated DiI-Ac-LDL. In contrast to the lumenized vessels in the core of the limb bud, we found that angioblasts in the avascular zone do not proliferate, as shown by proliferation studies applying the BrdU-method to semithin sections in connection with QH-1-labelled parallel sections. We conclude that the vascularization of the avian limb bud is achieved by a combination of angiotrophic growth (sprouting of vessels) and angioblastic growth (recruitment of angioblasts from the limb mesoderm.

Animals↗

PubMed1995

Proliferation pattern of capillary endothelial cells in chorioallantoic membrane development indicates local growth control, which is counteracted by vascular endothelial growth factor application.

The density and distribution of whole mount BrdU-anti-BrdU labeled endothelial cells (days 6-15) in the chick chorioallantoic membrane (CAM) was analyzed with computer-assisted microscopy. A significant loss of proliferative activity was noted after day 10: the density of labeled nuclei (in 10(-2) mm-2) decreased from a median 7.78 (days 6, 8, 10) to 2.42 (days 12, 14, 15). CAMs initially showed random patterns of labeled endothelial cells, but changed to clearly focal patterns after day 12. A regular arrangement of labeled nuclei was never seen. After application of vascular endothelial growth factor (VEGF) to the day 13 CAM, a significant increase in proliferative activity (11.50) and a random distribution of labeled endothelial cells was observed on day 15. Development of CAM precapillary vessels was assessed in terms of length density (in mm-1, mean +/- standard deviation), which was augmented three-fold from day 6 (1.22 +/- 0.05) to day 14 (3.54 +/- 0.23) and then remained nearly constant. VEGF application from day 13 to 15 raised arterial length per unit area to 4.53 +/- 0.77. It is concluded that normally a local regulation of endothelial proliferation and differentiation develops in the CAM, which doubles capillary endothelial cell density but simultaneously adapts to the decreasing need for endothelial cells, and thus maintains the quasi two-dimensional vessel pattern. However, proliferative foci persist in the capillary layer after day 10, and precapillary vessel density continues to increase until day 14. VEGF enhances DNA synthesis in all capillary endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Allantoin↗

PubMed1995

Pax-1 in the development of the cervico-occipital transitional zone.

The Pax-1 gene has been found to play an important role in the development of the vertebral column. The cervico-occipital transitional zone is a specialized region of the vertebral column, and malformations of this region have frequently been described in humans. The exact embryonic border between head and trunk is a matter of controversy. In order to determine a possible role of Pax-1 in the development of the cervico-occipital transitional zone we studied the expression of this gene in a series of quail embryos and murine fetuses with in situ hybridization and immunohistochemistry. Pax-1 is expressed in all somites of the embryo, including the first five occipital ones. During embryonic days 3-5 the gene is down-regulated in the caudal direction within the first five somites, whereas more caudally Pax-1 is strongly expressed in the cells of the perinotochordal tube. In 5-day-old quail embryos, the cartilaginous anlage of the basioccipital bone has developed and ther is no more expression of Pax-1 in this region. The fusion of the dens axis with the body of the axis also coincides with switching off of the Pax-1 gene. More caudally, the gene is continuously expressed in the intervertebral discs of murine embryos and therefore seems to be important for the process of resegmentation. Quail embryos do not possess permanent intervertebral discs. ¿Hyper-¿ or ¿hyposegmentation¿ defects may be explained by an over- or under-expression of Pax-1 during development. We also reinvestigated the border between the head and trunk in chick embryos by performing homotopical grafting experiments of the 5th somite between chick and quail embryos.

Animals↗

PubMed1995

Pax-1, a regulator of sclerotome development is induced by notochord and floor plate signals in avian embryos.

Pax-1 encodes for a DNA-binding transcriptional activator that was originally discovered in murine embryos using a probe from the Drosophila paired-box-containing gene, gooseberry-distal. We have cloned the avian Pax-1 gene as a basis for experimental studies of the induction of Pax-1 in the paraxial mesoderm. The amino acid sequence of the paired-domain is exactly the same in the quail and mouse, whereas outside the paired-domain there is 61% homology. Starting at about the eight-somite stage, quail Pax-1 is expressed in the paraxial mesoderm in a craniocaudal sequence. The unsegmented paraxial mesoderm and the two most recently formed somites do not express Pax-1. In the epithelial somite, the somitocoele cells and the cells of the ventral two-thirds of the epithelial wall are positive. As soon as the sclerotome is formed, only a subset of sclerotome cells expresses Pax-1. These are the cells that migrate towards the notochord to form the perinotochordal tube. Expression then becomes restricted to the intervertebral discs, the perichondrium of the vertebral bodies and the connective tissue surrounding the spinal ganglia. Additional expression domains are found in the scapula and the pelvic region, distinct areas of the head, and the epithelium of the second to the fourth visceral pouch. In later stages the thymus is positive. In vitro and in vivo experiments show that the notochord induces Pax-1 in the paraxial mesoderm, but limb bud mesoderm is not competent to respond to notochordal signals. Floor plate is also capable of inducing Pax-1 expression in sclerotome cells. Our studies show that in competent cells of the paraxial mesoderm, Pax-1 is a mediator of signals emanating from the notochord and the floor plate.

Amino Acid Sequence↗

PubMed1995

Rapid purification of human Langerhans cells using paramagnetic microbeads.

Detailed studies on the biology of Langerhans cells (LC), which account for only 1-3% of all epidermal cells, require isolation from their cutaneous symbionts. Several techniques of LC isolation have been reported, including positive enrichment with mAb coupled to immunomagnetic beads. The disadvantage of this technique is the size of the beads (approximately 2-5 microns), which can interfere with subsequent phenotypic and functional analyses. This limitation prompted us to test whether paramagnetic microbeads (15 nm) employed by the MACS system could be used to purify LC from human skin. To isolate fresh LC (fLC), epidermal cell suspensions (EC) were stained with anti-CD1a mAb and with appropriate secondary reagents conjugated to microbeads and to FITC. They were then passed over a separation column and exposed to a strong magnetic field. Thereafter both CD1a-depleted and CD1a-enriched cells were collected. Cultured LC (cLC) were isolated by staining 72-h cultured EC with anti-HLA-DR mAb followed by the same isolation procedure. Using this technique, we could routinely isolate viable EC that were 45-88% CD1a+ or HLA-DR+ as determined by FACS. Two-color FACS analysis demonstrated the majority of MACS-purified cells to be CD1a+/HLA-DR+, indicating that they were indeed LC. By transmission electron microscopy (TEM), the MACS-purified CD1a+/HLA-DR+ cells showed typical ultrastructural characteristics of LC. Furthermore, MACS-purified fLC or cLC were functionally intact, because they stimulated the proliferation of alloreactive T cells in a primary, one-way, mixed epidermal cell leukocyte reaction (MECLR). We conclude that MACS-separation is an efficient and rapid method to isolate human fLC and cLC of high purity and unimpaired function.

Bacterial Proteins↗