ARTICLE |
Correspondence to: Klemens Rappersberger, Dept. of Dermatology, Div. of General Dermatology, Waehringer Guertel 18-20, A-1090 Vienna, Austria.
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Summary |
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We performed a comparative investigation of the immunomorphological characteristics of lymphatic and blood microvascular endothelial cells in normal human skin, cutaneous lymphangiomas, and hemangiomas, employing a pre-embedding immunogold electron microscopic technique. We stained for cell membrane proteins that are commonly used for light microscopic characterization of blood endothelial cells. With blood microvascular endothelial cells, we observed uniform labeling of the luminal cell membranes with monoclonal antibodies (MAbs) JC70 (CD31), EN-4 (CD31), BMA120, PAL-E, and QBEND-10 (CD34), and strong staining of the vascular basal lamina for Type IV collagen under normal and pathological conditions. In contrast, lymphatic microvascular endothelial cells in normal human skin and in lymphangiomas displayed, in addition to a luminal labeling, pronounced expression of CD31 and CD34 along the abluminal cell membranes. Moreover, CD31 was preferentially detected within intercellular junctions. The expression of CD34 was mostly confined to abluminal endothelial microprocesses and was upregulated in lymphangiomas and hemangiomas. Type IV collagen partially formed the luminal lining of initial lymphatics and occasionally formed bridges over interendothelial gaps. Our findings suggest a function of transmigration protein CD31 in recruitment of dendritic cells into the lymphatic vasculature. CD34 labeling may indicate early endothelial cell sprouting. The distribution of Type IV collagen also supports its role as a signal for migration and tube formation for lymphatic endothelial cells. (J Histochem Cytochem 46:165176, 1998)
Key Words: immunoelectron, microscopy, immunomorphology, human dermal microvascular, endothelial cells, lymphatic capillaries, lymphatic endothelial cells, CD31, CD34, Type IV collagen
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Introduction |
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The DEVELOPMENT of endothelial cell (EC)-reactive antibodies has facilitated the investigation of antigenic and functional properties of EC and has clearly demonstrated a striking heterogeneity of these cells in different organs (
In contrast to our continuously increasing knowledge about the diverse functions of HDBMECs, little is known about human dermal lymphatic microvascular endothelial cells (HDLMECs). Previous morphological studies have identified the architecture of the dermal lymphatic microvascular system and have clearly defined the subcellular characteristics of HDLMECs (
In this study we focused our interests on the immunophenotypical and immunomorphological characteristics of HDLMECs to obtain a better understanding of the biology of this particular cellular component of the human skin. We performed a pre-embedding immunogold electron microscopic study with a panel of well-characterized EC reactive/specific monoclonal antibodies (MAbs) that are directed against cell membrane-associated proteins and Type IV collagen. After determination of the immunomorphological phenotype of the HDLMECs, we compared these findings with those on blood ECs. To assess whether the immunomorphological features of resting HDMECs in normal human skin are maintained during benign proliferation, we then investigated the ultrastructural immunomorphology of capillary hemangiomas and lymphangiomas.
Here we present the ultrastructural characteristics of resting and proliferating human dermal microvascular endothelial cells (HDMECs) in situ, that clearly allow the differentiation of these two cell populations by ultrastructural immunomorphological criteria. Because the distribution of certain surface proteins with lymphatic ECs indicates biological relevance, these findings contribute to our understanding of the function of these cells and may be helpful in clarification of the ontogeny of certain vascular skin tumors.
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Materials and Methods |
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Tissue Specimens
Biopsies/excisions were taken under local anesthesia with 2% mepivacaine of normal human skin from 10 healthy volunteers, four patients with hemangiomas, and five with lymphangiomas. All tissue specimens underwent an identical technical procedure. Immediately after biopsy the tissue was rinsed in a precooled solution of physiological NaCl and was divided with a razor blade on a wax plate into pieces of approximately 1 mm3. From each biopsy specimen, one portion was fixed in 7.5% formaldehyde and further processed for routine histopathology. For light microscopic immunohistological studies, another part of the biopsy specimen was snap-frozen in Tissue-Tek OCT compound (Miles Scientific; Naperville, IL) using isopentane precooled in liquid nitrogen, and then stored at -70C until further use. Another part of the biopsy specimen was processed for immunoelectron microscopy. After fixation in a freshly prepared and slightly modified periodatelysineparaformaldehyde fixative for 4 hr at room temperature (RT) and a thorough rinse in PBS, pH 7.4, three times for 15 min at 4C, the specimens were infiltrated with 10% dimethylsulfoxide dissolved in PBS for 1 hr at 4C, snap-frozen, and stored in liquid nitrogen (
Antibodies
We selected a set of six MAbs (Table 1) that were all known to react with the cell membrane of EC or with the basal lamina: MAbs JC70 and, reportedly, EN-4 are directed against CD31, a glycoprotein of 130 kD that is found on various hematopoietic cells such as bone marrow stem cells, CD8+ cells, monocytes, neutrophils, platelets, and EC (
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For light microscopic immunomorphological studies, 4-µm-thick serial cryosections were mounted on gelatin-coated slides, air-dried, and fixed in acetone for 10 min at -20C. For blocking of Fc Ig receptors, sections were preincubated in PBS, pH 7.4, supplemented with 1% bovine serum albumin (BSA) for 2030 min at RT. For morphological orientation, the first section of each tissue block was stained with hematoxylin and eosin. For immunohistological staining the three-step avidinbiotinimmunperoxidase technique was employed using a Supersensitive Staining Kit (Bio Genex Laboratories; San Ramon, CA). Sections were incubated with the first-step antibodies (first-step antibodies used are listed in Table 1), appropriately diluted in PBS/1% BSA for 1 hr at 4C. Thereafter, the slides were thoroughly washed in PBS/0.1% BSA and consecutively reacted with an appropriately diluted biotinylated anti-mouse IgG. After three washes in PBS/0.1% BSA, the slides were incubated with peroxidase-conjugated streptavidin. Bound immunoreactants were visualized with AEC as chromogen.
Immunoelectron microscopic experiments were performed with 1520-µm-thick cryostat sections that were prepared with a Jung CM3000 cryomicrotome (Leica; Vienna, Austria) and were immediately rinsed in PBS at 4C for 20 min. To reduce nonspecific antibody binding via Fc Ig receptors, the specimens were preincubated in PBS/1% bovine serum albumin (BSA) for 30 min at RT in 5-ml glass tubes. Afterwards, the specimens were incubated with the first-step reagents appropriately diluted in PBS/0.1% BSA for 8 hr at 4C (Table 1). After thorough rinsing in PBS/0.1% BSA, three times for 60 min at 4C, the sections were incubated with goat anti-mouse IgG (Fc) conjugated to 15-nm colloidal gold particles (Amersham International; Poole, UK), diluted in PBS/1% BSA for another 8 hr at 4C. To remove unbound immunoreagents, the tissue was washed in PBS/0.1% BSA two times for 60 min at 4C and once in 0.1% cacodylate buffer. The specimens were consecutively fixed in 2% glutaraldehyde for 1 hr at RT, rinsed in 0.1% cacodylate buffer for 1 hr at RT, postfixed in Palade's osmium, and contrasted with uranyl acetate. After dehydration of the specimens in a graded series of ethanols and infiltration with propylenoxide and Epon 812, the resin was polymerized at 60C. Thin sections were cut with a Reichert Ultracut 2000, contrasted on grids with lead citrate/uranyl acetate, and examined with a JEOL 1200 EX electron microscope.
Controls
Negative controls included omission of either the first- or the second-step reagent and substitution of the first-step reagent with an irrelevant isotype-matched antibody.
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Results |
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Light microscopic Immunophenotype of the Dermal Microvasculature
In the papillary dermis of normal human skin, ECs of blood vessels such as arterioles, capillaries, and postcapillary venules were regularly stained with all antibodies employed (Figure 1). The most intense labeling was seen with MAbs EN-4 and JC70/CD31 and, to a lesser degree, with MAbs PAL-E, QBend10/CD34, and BMA120. Staining of Type IV collagen revealed a well-developed basement membrane around these vascular structures.
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In addition, we observed flattened, thin cords of cells without any discernible lumen formation. In consecutive serial sections these structures displayed reactivity with MAbs EN-4, JC-70, and BMA-120, and also labeling with MAb QBend10, but the cords failed to react with MAb PAL-E. The additional observation of a Type IV collagen-positive basal lamina around these structures most likely indicated that they represent collapsed lymphatic capillaries.
Investigating the immunophenotype of proliferating vessels of cutaneous capillary hemangiomas and lymphangiomas, we found identical immunophenotypic features as for blood and lymphatic capillaries, respectively, in normal human skin (data not shown).
Ultrastructural Morphology of Blood and Lymphatic Capillaries in Normal Human Skin
ECs of blood and lymphatic capillaries were morphologically identified in accordance with previous investigations (
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Lymphatic capillaries originate as tiny, thin open tubes that are formed by extremely attenuated, almost spindle-shaped ECs and, occasionally, are completely devoid of an EC lining, but are enveloped by lamina densa-like structures that form the vessel wall at the most distal ending of the lymphatic channels. Along such laminar structures, lymphatic EC processes align and form the capillary tube (Figure 3). The cytoplasm of lymphatic ECs lacks cell-specific organelles such as WeibelPalade bodies. However, lipid droplets and dense bodies are frequently seen. The continuity of the endothelial lining is provided by cytoplasmic processes of lymphatic ECs that form interdigitating, overlapping, and end-to-end-type junctions (Figure 4). In addition, along these junctions the cell membranes may form characteristic intercellular adherens junctions that appear as desmosome-like structures and tight junctions. Nevertheless, large gaps are often observed between two neighboring ECs (Figure 3). Lymphatic capillaries are lined with a thin and discontinous but well-preserved basement membrane that may bridge over interendothelial gaps, thus forming the boundary between the lymphatic lumen and the interstitial connective tissue (Figure 3 and Figure 4). Another specific morphological feature of lymphatic capillaries is the intimate association with elastic and collagen fibers and fibrils that insert either directly or via microfilaments within the abluminal cell membrane. However, lymphatic capillaries lack the presence of accompanying pericytes (Figure 3 and Figure 4).
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Immunophenotype of Blood and Lymphatic Capillaries
Morphologically characterized blood and lymphatic microvascular ECs, but no other cellular component of the dermal microvasculature of normal human skin, exhibited labeling with MAbs JC70, EN-4, QBend10, and BMA120 (Figure 1 Figure 2 Figure 3 Figure 4).
The number of colloidal gold particles indicating the binding of MAbs and thus the expression of antigens is shown in detail in Table 2. The overall labeling intensity was higher with blood capillaries than with lymphatics. Quantitative analysis of bound immunogold particles per 100 µm abluminal and luminal EC cytoplasmic membrane displayed certain characteristic immunophenotypic differences between blood and lymphatic capillaries. The most intense labeling of blood capillaries was seen with MAbs JC70 and EN 4 and was somewhat weaker with QBend10, whereas the labeling with MAbs BMA-120 and PAL-E was much weaker. Immunolabeling with PAL-E was restricted to blood microvascular ECs. PAL-E labeling often revealed a preferential binding to coated pits and vesicles of the luminal cell membrane, as was reported previously for cultured blood ECs (
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As expected, staining for Type IV collagen demonstrated well-developed, occasionally reduplicated laminae densae around ECs and pericytes of blood capillaries. Moreover, it also confirmed the presence of a Type IV collagen-positive fragmented lamina densa with lymphatic capillaries. In addition, the laminar structures that were bridging over interendothelial gaps of lymphatic capillaries and forming the vessel walls of initial lymphatics, as shown in Figure 2, displayed a striking reactivity with anti-type IV collagen MAbs (Figure 5d).
Immunophenotype of Hemangiomas and Lymphangiomas
To prove our findings in normal human skin, we also investigated the immunomorphological characteristics of benign microvascular tumors with blood and lymphatic differentiation, i.e., hemangiomas and lymphangiomas. As with lymphatic capillaries in normal human skin, the most striking observation was the expression of CD31 and CD34 on the abluminal cell membranes of lymphatic EC in lymphangiomas (Figure 5c and Figure 6). Surprisingly, the most pronounced staining was seen with MAb QBend10, which indicated an increase in CD34 expression on both luminal and abluminal portions of lymphatic capillaries by a factor of 2. In addition, with hemangiomas there was a slight increase in CD34 expression (Table 2; Figure 5a and Figure 6). In situ labeling with antibodies QBend10, JC70, EN-4, and BMA120 did not disclose a localization to any defined subcellular structure but was evenly distributed along the cell membrane. PAL-E labeling was confined to blood vessels in lymphangiomas. The lamina densa of the basement membranes of both blood and lymphatic ECs, as well as pericytes, always displayed strong expression of Type IV collagen (Figure 5b and Figure 5d). It is of note that proliferating lymphatic capillaries, like normal lymphatic vessels, showed large interendothelial gaps. Frequently these gaps were bridged by a well-developed Type IV collagen-positive lamina densa that formed the boundary to the surrounding connective tissue (Figure 5d). As in normal skin, proliferating lymphatic ECs were intimately connected with the surrounding connective tissue by elastic and collagen fibers as well as by microfibrils that inserted into the abluminal cell membrane (Figure 5c).
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Discussion |
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Light microscopic immunohistochemistry is an adequate tool to grossly characterize immunophenotypic characteristics of cellular components in vitro and in vivo and therefore plays a major role in diagnostic pathology. However, because the expression of cell surface proteins is mostly associated with functional properties, identification of the exact immunolocalization of such molecules may be helpful in the understanding of biological functions. Such investigations are limited by the resolution of light microscopic techniques and therefore require immunoelectron microscopic techniques to define the ultrastructural localization of antigens.
Immunoelectron microscopic studies of human tissue taken ex vivo are largely hampered by technical problems, because the preservation of the ultrastructural morphology by fixation with denaturing and crosslinking aldehydes strikingly reduces the antigenicity of the structures studied. In this study we employed a technique that provides excellent preservation of both subcellular morphology and antigenicity of the tissue, allowing an unequivocal assignment of the immunolabel to ECs defined as blood or lymphatic microvascular ECs by morphological criteria. Using a panel of different MAbs directed against EC and Type IV collagen on normal human skin, hemangiomas, and lymphangiomas, we found a clear heterogeneity of the immunophenotype of blood and lymphatic HDMECs, in situ, at the ultrastructural level. The major findings of this study relate to the differential expression of CD31 and CD34 as well as Type IV collagen with lymphatic and blood microvascular ECs.
All dermal microvascular structures displayed a clear-cut labeling of the luminal cell membranes of ECs with anti-CD31 and anti-CD34 reagents as well as with MAb BMA-120, whereas PAL-E reactivity was restricted to blood microvascular ECs. The most striking observation of this study was the finding of concomitant abluminal and luminal expression of CD31 and CD34, respectively, by lymphatic microvascular endothelial cells. Abluminal expression of CD31 was never observed in blood capillaries, despite the fact that other investigators have detected CD31 on the abluminal cytoplasmic membranes of endothelial cells of cutaneous venous vessels (
Recent investigations have clearly confined the expression of the human progenitor cell marker CD34 to blood endothelial cells (
Most studies demonstrated CD34 along the luminal cell membrane. Occasionally however, abluminal expression of CD34 was found in vivo and in vitro (
In addition, angiogenesis and angioproliferation in vitro depend on the presence of the basement membrane proteins laminin and Type IV collagen, which promote the rapid alignment and differentiation of HDMECs into capillary-like tubes in vitro (
The labeling characteristics of ECs observed in normal human skin were identical to those in hemangiomas and lymphangiomas and therefore indicate a preservation of antigenic and functional properties of HDMECs in cases of benign proliferation.
Taken together, we present immunomorphological data that indicate a pronounced antigenic heterogeneity of HDMECs of blood and lymphatic origin in situ at the ultrastructural level. The characteristic immunolocalization of CD31 and CD34 by lymphatic ECs and the distribution of Type IV collagen with lymphatic microvessels allows a clear-cut immunophenotypic differentiation between these two distinct cell populations at the subcellular level and also indicates biological relevance.
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Acknowledgments |
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We are indebted to Frank J. Rietveld (Department of Pathology, University Hospital, Nijmegen, The Netherlands), who helped us with the staining procedure for monoclonal antibody PAL-E.
Received for publication February 3, 1997; accepted August 5, 1997.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alles JU, Bosslet K (1986) Immunohistochemical and immunochemical characterization of a new endothelial cell specific antigen. J Histochem Cytochem 34:209-214[Abstract]
Barsky SH, Baker A, Siegal GP, Togo S, Liotto LA (1983) Use of anti basement membrane antibodies to distinguish blood vessel capillaries from lymphatic capillaries. Am J Surg Pathol 7:667-677[Medline]
Blank C, Fuchs H, Rappersberger K, Röllinghoff M, Moll H (1993) Parasitism of epidermal Langerhans cells in experimental cutaneous leishmaniasis. J Infect Dis 167:418-425[Medline]
Braverman IM (1989) Ultrastructure and organization of the cutaneous microvasculature in normal and pathological states. J Invest Dermatol 93:2S-9S[Abstract]
Burgio VL, Zupo S, Roncella S, Zocchi M, Ruco LP, Baroni CD (1994) Characterization of EN4 monoclonal antibody: a reagent with CD31 specificity. Clin Exp Immunol 96:170-176[Medline]
Daroczy J (1988) The Dermal Lymphatic Capillaries. Berlin, Heidelberg, Springer-Verlag
De Lisser HM, Newman PJ, Albelda SM (1994) Molecular and functional aspects of PECAM-1/CD31. Immunol Today 15:490-495[Medline]
Erhard H, Rietveld FJ, Bröcker EB, de Waal RM, Ruiter DJ (1996) Phenotype of normal cutaneous microvasculature. Immunoelectronmicroscopic observations with emphasis on the differences between blood vessels and lymphatics. J Invest Dermatol 106:135-140[Abstract]
Fina L, Moolgard HV, Robertson D, Bradley NJ, Monaghan P, Delia D, Sutherland RD, Baker MA, Greaves MV (1990) Expression of CD34 gene in vascular endothelial cells. Blood 75:2417-2426[Abstract]
Folkman J (1990) What is the evidence that tumors are angiogenesis dependent. J Natl Cancer Inst 82:4-6[Medline]
Form DM, Pratt BM, Madri JA (1986) Endothelial cell proliferation during angiogenesis: in vitro modulation by basement membrane components. Lab Invest 55:521-530[Medline]
Goerdt S, Walsh LJ, Murphy GF, Pober JS (1991) Identification of a novel high molecular weight protein preferentially expressed by sinusoidal endothelial cells in normal human tissue. J Cell Biol 113:1425-1437[Abstract]
Gröger M, Sarmay G, Fiebiger E, Wolff K, Petzelbauer P (1996) Dermal microvascular endothelial cells express CD32 receptors in vivo and in vitro. J Immunol 156:1549-1556[Abstract]
Hirschberg H, Gergh OJ, Thorby E (1980) Antigen presenting properties of human vascular endothelial cells. J Exp Med 152:249S-255S[Medline]
Kaiserling E, Kröber S (1994) Lymphatic amyloidosis, a previously unrecognized form of amyloid deposition in generalized amyloidosis. Histopathology 24:215-221[Medline]
Kubota Y, Kleidman HK, Martin GR, Lawley TJ (1988) Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J Cell Biol 197:1598-1598
Kuzu I, Bicknell R, Harris AL, Jones M, Gatter KC, Mason DY (1992) Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumors. J Clin Pathol 45:143-148[Abstract]
Larsen CP, Steinman RM, WitmerPack M, Hankins DDF, Morris PJ, Austyn JM (1990) Migration and maturation of Langerhans cells in skin transplants and explants. J Exp Med 172:1483-1493[Abstract]
Leak LV (1976) The structure of lymphatic capillaries in early lymph formation. Fed Proc 35:129-149
Liao F, Huynh HK, Eiroa A, Green T, Polizzi E, Muller WA (1995) Migration of monocytes across endothelium and passage through extracellular matrix involve separate molecular domains of PECAM-1. J Exp Med 182:1337-1343[Abstract]
Lukas M, Stössel H, Hefel L, Imamura S, Fritsch P, Schuler G, Romani N (1996) Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture. J Invest Dermatol 106:1287-1292[Abstract]
McLean IW, Nakane PK (1974) Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectronmicroscopy. J Histochem Cytochem 22:1077-1082[Medline]
Muller WA (1995) The role of PECAM-1 (CD31) in leucocyte emigration: studies in vitro and in vivo. J Leukocyte Biol 57:523-528[Abstract]
Muller WA, Weigl SA, Deng X, Phillips DM (1993) PECAM-1 is required for transendothelial migration of leucocytes. J Exp Med 178:449-460[Abstract]
Newman PJ (1994) The role of PECAM-1 in vascular cell biology. Ann NY Acad Sci 714:165-174[Abstract]
Page C, Rose M, Yacoub M, Pigott R (1992) Antigenic heterogeneity of vascular endothelium. Am J Pathol 141:673-683[Abstract]
Parums DV, Cordell JL, Micklem K, Heryet AR, Gatter KC, Mason DY (1990) JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections. J Clin Pathol 43:752-757[Abstract]
Petzelbauer P, Bender JR, Wilson J, Pober JS (1993) Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture. J Immunol 151:5062-5072
Pober JS, Cotran RS (1990) The role of endothelial cells in inflammation. Transplantation 50:537-544[Medline]
Pober JS, Gimbrone MA (1982) Expression of Ia-like antigens by human vascular endothelial cells is inducible in vitro. Demonstration by monoclonal antibody binding and immunoprecipitation. Proc Natl Acad Sci USA 79:6641-6645[Abstract]
Ramani P, Bradley NJ, Fletcher CDM (1990) QBend10, a new monoclonal antibody to endothelium: assessment of its diagnostic utility in paraffin sections. Histopathology 17:237-242[Medline]
Ruiter D, Schlingemann RO, Rietveld FJ, de Waal RM (1989) Monoclonal antibody defined human endothelial antigens as vascular markers. J Invest Dermatol 93:25S-32S[Abstract]
Ruiter DJ, Schlingeman RO, Westphal JR, Denijn M, Rietveld FJ, de Waal RM (1993) Angiogenesis in wound healing and metastasis. Behring Inst Mitt 92:258-272[Medline]
Ryan TJ (1989) Structure and function of lymphatics. J Invest Dermatol 93:18S-24S[Abstract]
Ryan TJ, Mortimer PS, Jones RL (1986) Lymphatics of the skin: neglected but important. Int J Dermatol 7:411-419
Schlingemann RO, Dingijan GM, Emeis JJ, Blok J, Warnaar SO, Ruiter DJ (1985) Monoclonal antibody PAL-E specific for endothelium. Lab Invest 52:71-76[Medline]
Schlingemann RO, Rietveld FJ, de Waal RM, Bradley NJ, Skene AI, Davies AJ, Greaves MF, Denekamp J, Ruiter DJ (1990) Leucocyte antigen CD34 is expressed by a subset of cultured endothelial cells and on endothelial abluminal microprocesses in the tumor stroma. Lab Invest 62:690-696[Medline]
Schlingemann RO, Rietveld FJ, Kwaspen F, van de Kerkhof PC, de Waal RM, Ruiter DJ (1991) Differential expression of markers for endothelial cells, pericytes and basal lamina in the microvasculature of tumors and granulation tissue. Am J Pathol 138:1335-1347[Abstract]
Silberberg I, Baer RL, Rosenthal SA (1976) The role of Langerhans cells in allergic contact hypersensitivity. A review of findings in man and guinea pigs. J Invest Dermatol 66:210-226[Abstract]
Sterniczky B, Födinger D, Sauter B, Rappersberger K (1996) Anwendung der Elektronenmikroskopie in der Dermatologie. Hautarzt 47:148-165[Medline]
Thorgeierson G, Robertson AL (1978) The vascular endotheliumpathobiologic significance. Am J Pathol 93:803-846[Medline]
Vartanian RK, Weidner N (1994) Correlation of intratumoral endothelial cell proliferation with microvessel density (tumor angiogenesis) and tumor cell proliferation in breast carcinoma. Am J Pathol 144:1188-1194[Abstract]
Weiss JM, Sleeman J, Renkl AC, Dittmar H, Termeer CC, Taxis S, Howells N, Hofmann M, Köhler G, Schöpf E, Ponta H, Herrlich P, Simon JC (1997) An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function. J Cell Biol 137:1137-1147