Effects of TNF-alpha on leukocyte adhesion molecule expressions in cultured human lymphatic endothelium.

TNF-alpha alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-alpha stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-alpha treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-alpha leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-alpha treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-alpha concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-alpha-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-alpha-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.     

Sulphated endothelial ligands for L-selectin in lymphocyte homing and inflammation

Lymphocytes from the blood home to secondary lymphoid tissues through a process of tethering, rolling, firm adhesion and transmigration. Tethering and rolling of lymphocytes is mediated by the interaction of L-selectin on lymphocytes with sulphated ligands expressed by the specialized endothelial cells of high endothelial venules (HEVs). The sulphate-dependent monoclonal antibody MECA79 stains HEVs in peripheral lymph nodes and recognizes the complex of HEV ligands for L-selectin termed peripheral node addressin. High endothelial cell GlcNAc-6-sulphotransferase/L-selectin ligand sulphotransferase is a HEV-expressed sulphotransferase that contributes to the formation of the MECA79 epitope and L-selectin ligands on lymph node HEVs. MECA79-reactive vessels are also common at sites of chronic inflammation, suggesting mechanistic parallels between lymphocyte homing and inflammatory trafficking.     

Human and Murine High Endothelial Venule Cells Phagocytose Apoptotic Leukocytes

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0–6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosisin vivo.Mice were treated with a natural corticosterone (4-pregnene-11β,21-diol-3,20-dione) at levels detected during stress or malnutrition (93–180 μg serum cortisol/dl). At 4–12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.     

Gene expression profiling of mucosal addressin cell adhesion molecule-1+ high endothelial venule cells (HEV) and identification of a leucine-rich HEV glycoprotein as a HEV marker.

High endothelial venule (HEV) cells support lymphocyte migration from the peripheral blood into secondary lymphoid tissues. Using gene expression profiling of mucosal addressin cell adhesion molecule-1(+) mesenteric lymph node HEV cells by quantitative 3'-cDNA collection, we have identified a leucine-rich protein, named leucine-rich HEV glycoprotein (LRHG) that is selectively expressed in these cells. Northern blot analysis revealed that LRHG mRNA is approximately 1.3 kb and is expressed in lymph nodes, liver, and heart. In situ hybridization analysis demonstrated that the mRNA expression in lymph nodes is strictly restricted to the HEV cells, and immunofluorescence analysis with polyclonal Abs against LRHG indicated that the LRHG protein is localized mainly to HEV cells and possibly to some lymphoid cells surrounding the HEVs. LRHG cDNA encodes a 342-aa protein containing 8 tandem leucine-rich repeats of 24 aa each and has high homology to human leucine-rich alpha(2)-glycoprotein. Similar to some other leucine-rich repeat protein family members, LRHG can bind extracellular matrix proteins that are expressed on the basal lamina of HEVs, such as fibronectin, collagen IV, and laminin. In addition, LRHG binds TGF-beta. These results suggest that LRHG is likely to be multifunctional in that it may capture TGF-beta and/or other related humoral factors to modulate cell adhesion locally and may also be involved in the adhesion of HEV cells to the surrounding basal lamina.     

Dissection of murine lymphocyte-endothelial cell interaction mechanisms by SV-40-transformed mouse endothelial cell lines: novel mechanisms mediating basal binding, and alpha 4-integrin-dependent cytokine-induced adhesion.

Lymph node-derived endothelial cells were immortalized by infection with SV40 virus and subclones expressing the marker MECA 325 specific for high-endothelial venules (HEV) were selected. These transformed mouse endothelial (TME-) cell lines grow permanently without requirement for special growth factors. Staining of the selected clones with endothelium-specific antibodies and with anti-von Willebrand factor antiserum and uptake of acetylated low-density lipoprotein provide evidence for their endothelial origin. The vascular addressins identified by mAbs MECA 79 and MECA 367 on HEV are not detectable, indicating that the phenotype of the cells differs from that of HEV-type endothelium. The TME cells display a constitutive capacity to bind lymphocytes. An additional binding component is induced by treatment of the TME cells with TNF alpha. Antibodies against the homing receptor LECAM-1 (lectin-related leucocyte-endothelial cell adhesion molecule 1), alpha 4-integrins, vascular addressins, LFA-1, or ICAM-1 known to block lymphocyte interaction with particular types of HEV were unable to inhibit the basal adhesion to TME cells, indicating that a further binding mechanism in mice is displayed by this cell type. The adhesion component induced by TNF alpha is mediated by alpha 4-integrins since enhanced binding could be blocked by an antibody against mouse alpha 4 (lymphocyte-Peyer's patch adhesion molecule 1/2). TME cell lines therefore seem to be a useful model for the dissection and analysis of hitherto poorly characterized murine lymphocyte/endothelial cell interaction mechanisms.     

Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site.

Nasal-associated lymphoid tissue (NALT), a mucosal inductive site for the upper respiratory tract, is important for the development of mucosal immunity locally and distally to intranasally introduced Ag. To more fully understand the induction of nasal mucosal immunity, we investigated the addressins that allow for lymphocyte trafficking to this tissue. To investigate the addressins responsible for naive lymphocyte binding, immunofluorescent and immunoperoxidase staining of frozen NALT sections were performed using anti-mucosal addressin cell adhesion molecule-1 (MAdCAM-1), anti-peripheral node addressin (PNAd), and anti-VCAM-1 mAbs. All NALT high endothelial venules (HEV) expressed PNAd, either associated with MAdCAM-1 or alone, whereas NALT follicular dendritic cells expressed both MAdCAM-1 and VCAM-1. These expression profiles were distinct from those of the gut mucosal inductive site, Peyer's patches (PP). The functionality of NALT HEV was determined using a Stamper-Woodruff ex vivo assay. The anti-L-selectin MEL-14 mAb blocked >90% of naive lymphocyte binding to NALT HEV, whereas the anti-MAdCAM-1 mAb, which blocks almost all naive lymphocyte binding to PP, minimally blocked binding to NALT HEV. NALT lymphocytes exhibited a unique L-selectin expression profile, differing from both PP and peripheral lymph nodes. Finally, NALT HEV were found in increased amounts in the B cell zones, unlike PP HEV. These results suggest that NALT is distinct from the intestinal PP, that initial naive lymphocyte binding to NALT HEV involves predominantly L-selectin and PNAd rather than alpha4beta7-MAdCAM-1 interactions, and that MAdCAM-1 and VCAM-1 expressed by NALT follicular dendritic cells may play an important role in lymphocyte recruitment and retention.     

A SV-40 immortalized murine endothelial cell line from peripheral lymph node high endothelium expresses a new α-L-fucose binding protein

Summry— Endothelial cells from mouse peripheral lymph nodes were immortalized by cationic liposome-mediate transfection using a plasmid construct containing both the gene coding for the large T antigen of simian virus 40 and a geneticin resistance gene suitable for selection. A cell line (HECal10) was isolated on the basis of its capacity to specifically bind fucoside carrying glycoconjugates; these cells present the main characteristics of endothelial cells: production of angiotensin converting enzyme and of factor VII-related antigen. Upon stimulation, they express E-selectin which binds oligosaccharides containing the Lewis^x determinant (Fucα3[Galβ4 GlcNacβ3Galβ) and the MECA 79 addressin which is characteristics for the peripheral lymph node high endothelium and is a L-selectin. HECa10 cells, as well as peripheral lymph node high endothelial cells in primary culture, express a second fucoside binding protein which differs from E-selectin. Indeed, this new fucoside-binding protein is constitutively expressed on unstimulated cells while E-selectin is not. Furthermore, HECa10 cells mediate selective lymphoid cells adhesion in a selectin/addressin-dependent mechanism, mainly inhibited by MECA 79 antibody and, in a fucose-binding lectin-dependent manner, mainly inhibited by the specifc neoglycoprotein.     

Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO^•). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO^• to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration.Effects of NO^•, either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO^• down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO^• was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression.In conclusion, during angiogenesis and leukocyte recruitment, NO^• regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO^•, reflecting the functions of the tissue they originate from.     

Endothelial Cell E- and P-Selectin and Vascular Cell Adhesion Molecule-1 Function as Signaling Receptors

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca²⁺]_i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T.M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371–1380). To determine whether stimulation of [Ca²⁺]_i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca²⁺]_i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca²⁺]_i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti–P- and anti–E-selectin mAb, as well as anti–VCAM-1 mAb, induced transient increases in EC [Ca²⁺]_i that were comparable to those induced by 200 μM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca²⁺]_i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLe^x), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca²⁺]_i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLe^x and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca²⁺]_i changes in the 5 h–treated EC, whereas the anti–VLA-4 mAb alone was sufficient to inhibit [Ca²⁺]_i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti–PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca²⁺]_i, i.e., mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.     

The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells.

CD44 is a polymorphic integral membrane protein which recognizes hyaluronate and whose proposed roles encompass lymphocyte activation, matrix adhesion and the attachment of lymphocytes to lymph node high endothelial venules (HEVs). Immunochemical and RNA blot data have supported the existence of two forms of CD44: a hematopoietic form expressed by cells of mesodermal origin (and by some carcinoma cell lines) and an epithelial form weakly expressed by normal epithelium but highly expressed by carcinomas. This report describes the isolation of a cDNA encoding a distinct CD44 polypeptide expressed by epithelial cells. Re-expression of each form of CD44 in a B cell line allowed cells transfected with the hematopoietic but not the epithelial form to bind to viable rat lymph node HEV cells in primary culture.     

Endothelial-like cells derived from human CD14 positive monocytes

In the present study, we show that endothelial-like cells (ELCs) can develop from human CD14-positive mononuclear cells (CD14 cells) in the presence of angiogenic growth factors. The CD14 cells became loosely adherent within 24 h of culture and subsequently underwent a distinct process of morphological transformation to caudated or oval cells with eccentric nuclei. After 1 week in culture the cells showed a clear expression of endothelial cell markers, including von Willebrand factor (vWF), CD144 (VE-cadherin), CD105 (endoglin), acetylated low-density lipoprotein (AC-LDL)-receptor, CD36 (thrombospondin receptor), FLT-1, which is vascular endothelial cell growth factor (VEGF) receptor-1, and, to a weaker extent, KDR (VEGF receptor-2). Furthermore, in these cells structures resembling Weibel-Palade bodies at different storage stages were identified by electron microscopy, and upon culturing on three-dimensional fibrin gels the cells build network-like structures. In addition, cell proliferation and vWF expression was stimulated by VEGF, and the endothelial cell adhesion molecules CD54 (ICAM-1), and CD106 (VCAM-1) became transiently inducible by tumor necrosis factor-alpha (TNF-alpha). In contrast, the dendritic markers CD1a, and CD83 were not expressed to any significant extent. The expression of CD68, CD80 (B7-1), CD86 (B7-2), HLA-DR and CD36 may also suggest that ELCs might be related to macrophages, sinus lining or microvascular endothelial cells. Taken together, our observations indicate that ELCs can differentiate from cells of the monocytic lineage, suggesting a closer relationship between the monocyte/macrophage- and the endothelial cell systems than previously supposed.     

Trophoblast interactions with endothelial cells are increased by interleukin-1beta and tumour necrosis factor alpha and involve vascular cell adhesion molecule-1 and alpha4beta1

Interactions between fetal extravillous trophoblast cells and maternal uterine cells are of critical importance in successful placentation. In the first trimester, trophoblasts invade the uterine environment and reach the spiral arteries where they interact with vascular cells; however, little is known of the nature of these interactions. We have developed a fluorescent binding assay to investigate the contact between trophoblasts and endothelial cells and to determine its regulation by cytokines and adhesion molecules. Stimulation of an endothelial cell line (SGHEC-7) with interleukin-1beta or tumour necrosis factor-alpha significantly increased adhesion of the first-trimester extravillous trophoblast-derived cell line, SGHPL-4. Using blocking antibodies, vascular cell adhesion molecule-1 (VCAM-1) and integrin alpha4beta1 (VLA-4), but not intercellular adhesion molecule-1 (ICAM-1), were shown to be important in trophoblast binding to activated endothelial cells. SGHPL-4 cells were shown to express HLA-G, alpha4beta1 and ICAM-1 at high levels and LFA-1 and VCAM-1 at lower levels. ICAM-1 and VCAM-1 are expressed on SGHEC-7 cells and their expression was confirmed on primary decidual endothelial cells. In conclusion, we have demonstrated the importance of VCAM-1 and alpha4beta1 in trophoblasts-endothelial interactions. Improved knowledge of the nature of these fetal-maternal interactions will have implications for understanding situations when placentation is compromised.     

Induction by IL 1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1)

ICAM-1 is a cell surface glycoprotein originally defined by a monoclonal antibody (MAb) that inhibits phorbol ester-stimulated leukocyte aggregation. Staining of frozen sections and immunofluorescence flow cytometry showed intercellular adhesion molecule-1 (ICAM-1) is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells, and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T lymphocyte blasts, and germinal center dendritic cells in tonsils, lymph nodes, and Peyer's patches. ICAM-1 staining on vascular endothelial cells is most intense in T cell areas in lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in low amounts on peripheral blood leukocytes. Phorbol ester-stimulated differentiation of myelomonocytic cell lines greatly increases ICAM-1 expression. ICAM-1 expression on dermal fibroblasts is increased threefold to fivefold by either interleukin 1 (IL 1) or interferon-gamma at 10 U/ml over a period of 4 or 10 hr, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ICAM-1 displays Mr heterogeneity in different cell types with a Mr of 97,000 on fibroblasts, 114,000 on the myelomonocytic cell line U937, and 90,000 on the B lymphoblastoid cell JY. ICAM-1 biosynthesis involves a Mr approximately 73,000 intracellular precursor. The non-N-glycosylated form resulting from tunicamycin treatment has a Mr of 55,000. ICAM-1 isolated from phorbol myristic acetate (PMA) stimulated U937 and from fibroblasts yields an identical major product of Mr = 60,000 after chemical deglycosylation. ICAM-1 MAb interferes with the adhesion of phytohemagglutinin blasts, and the adhesion of the cell line SKW3 to human dermal fibroblast cell layers. Pretreatment of fibroblasts but not lymphocytes with ICAM-1 MAb, and of lymphocytes but not fibroblasts with lymphocyte function-associated antigen 1 MAb inhibits adhesion. Intercellular adhesion is increased by prior exposure of fibroblasts to IL 1, and correlates with induction of ICAM-1.     

Expression of low levels of peripheral lymph node-associated vascular addressin in mucosal lymphoid tissues: possible relevance to the dissemination of passaged AKR lymphomas

Lymphoid tumors display a wide variety of growth patterns in vivo, from that of a solitary extralymphoid tumor, to a general involvement of all lymphoid organs. Normal lymphocytes are uniquely mobile cells continuously recirculating between blood and lymph throughout much of their life cycle. Therefore, it is reasonable to propose that disseminating malignant lymphocytes may express recirculation characteristics or homing properties consistent with that of their normal lymphoid counterparts. Trafficking of lymphocytes involves the expression and recognition of both lymphocyte homing receptors and their opposing receptors on endothelium, the vascular addressins. These cell surface elements direct the tissue-selective localization of lymphocyte subsets in vivo into organized lymphoid organs and sites of chronic inflammation where specific binding events occur between lymphocytes and the endothelium of specialized high endothelial venules (HEV). In a recent murine study of 13 lymphoma lines, we found that lymphomas that bind well to high endothelial venules, in the Stamper-Woodruff in vitro assay (an assay of lymphocyte binding to venules in frozen sections of peripheral lymph nodes or Peyer's patches), spread hematogenously to all high endothelial venule bearing lymphoid organs, whereas non-binding lymphomas did not. In some cases lymphomas that bound with a high degree of selectivity to peripheral lymph node (PLN) high endothelial venules exhibited only limited organ preference of metastasis, involving the mucosal lymphoid organs Peyer's patches (PP) in addition to the peripheral lymph nodes of adoptive recipients. Here we demonstrate that Peyer's patch high endothelial venules express a low but functional level of peripheral lymph node addressin (MECA-79) that can be recognized by lymphomas expressing the peripheral lymph node homing receptor (MEL-14 antigen).(ABSTRACT TRUNCATED AT 250 WORDS)     

PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells

Interactions between leukocytes and vascular endothelial cells are mediated by a complex set of membrane adhesion molecules which transduce bi-directional signals in both cell types. Endothelium of the cerebral blood vessels, which constitute the blood-brain barrier, strictly controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells, we previously documented the role of ICAM-1 in activation of the tyrosine phosphorylation of several actin-binding proteins and subsequent rearrangements of the actin cytoskeleton. In the present study, we show that, whereas PECAM-1 is known to control positively the trans-endothelial migration of leukocytes via homophilic interactions between leukocytes and endothelial cells, PECAM-1 engagement on brain endothelial surface unexpectedly counteracts the ICAM-1-induced tyrosine phosphorylation of cortactin and rearrangements of the actin cytoskeleton. We present evidence that the PECAM-1-associated tyrosine phosphatase SHP-2 is required for ICAM-1 signaling, suggesting that its activity might crucially contribute to the regulation of ICAM-1 signaling by PECAM-1. Our findings reveal a novel activity for PECAM-1 which, by counteracting ICAM-1-induced activation, could directly contribute to limit activation and maintain integrity of brain vascular endothelium.     

Expression of the intercellular adhesion molecule-1 on high endothelial venules and on non-lymphoid antigen handling cells: interdigitating cells,antigen transporting cells and follicular dendritic cells

Intercellular adhesion molecule-1 (ICAM-1)¹ has been implicated in the development of germinal center reactions in vitro, and the present study was undertaken to determine the distribution of ICAM-1 in active germinal centers in vivo and in murine secondary lymphoid tissues in general. Anti-ICAM-1-specific monoclonal antibodies were used in conjunction with immunohistochemistry at both the light and ultrastructural levels of resolution. Examination of cryostat sections of lymph nodes, spleens, and Peyer's patches revealed that anti-ICAM-1 distinctly labeled cells in the light zones of germinal centers, a few cells in the T cell zones (e.g. paracortex of lymph nodes), cells in the sinus floor of the subcapsular sinuses of lymph nodes, and high endothelial venules (HEV). Ultrastructural studies revealed that the cells labeling with anti-ICAM-1 in germinal centers were follicular dendritic cells (FDC) which appeared to have more ICAM-1 than any other cell type. The surfaces of well-developed, intricate, convoluted FDC processes were intensely labeled even under conditions where B cells appeared negative. Interdigitating cells (IDC) were also labeled as were certain endothelial cells in the HEV. The cells in the subcapsular sinus floor labeling with anti-ICAM-1 were the antigen transporting cells (ATC) that carry antigen-antibody complexes into lymph node follicles. We suspect ATC are FDC precursors which mature into FDC in the follicles. Interestingly, FDC, IDC, and ATC are 3 important accessory cells known to handle antigens in specific compartments of lymphoid tissues. The marked localization of this adhesion molecule on these critical antigen handling cells supports the concept that ICAM-1 is important in providing the intercellular adhesion necessary for optimal initiation of immune responses in vivo.Abbreviations ICAM-1 Intercellular adhesion molecule-1 - LFA-1 leukocyte functional antigen-1 - IDC interdigitating cells - ATC antigen transporting cells - FDC follicular dendritic cells - HEV high endothelial venules - DC dendritic cells - PBS phosphate-buffered saline - PLP periodate-lysine-4% paraformaldehyde - GPLP periodate-lysine-0.1% glutaraldehyde-2% paraformaldehyde - EM electron microscopy - HRP horseradish peroxidase - DAB diaminobenzidine tetrahydrochloride - HSA human serum albumin     

Distinct roles for PECAM-1, ICAM-1, and VCAM-1 in recruitment of neutrophils and eosinophils to the cornea in ocular onchocerciasis (river blindness)

Infiltration of granulocytes into the transparent mammalian cornea can result in loss of corneal clarity and severe visual impairment. Since the cornea is an avascular tissue, recruitment of granulocytes such as neutrophils and eosinophils into the corneal stroma is initiated from peripheral (limbal) vessels. To determine the role of vascular adhesion molecules in this process, expression of platelet endothelial cell adhesion molecule 1 (PECAM-1), ICAM-1, and VCAM-1 on limbal vessels was determined in a murine model of ocular onchocerciasis in which Ags from the parasitic worm Onchocerca volvulus are injected into the corneal stroma. Expression of each of these molecules was elevated after injection of parasite Ags; however, PECAM-1 and ICAM-1 expression remained elevated from 12 h after injection until 7 days, whereas VCAM-1 expression was more transient, with peak expression at 72 h. Subconjunctival injection of Ab to PECAM-1 significantly inhibited neutrophil recruitment to the cornea compared with eyes injected with control Ab (p = 0.012). Consistent with this finding, corneal opacification was significantly diminished (p < 0.0001). There was no significant reduction in eosinophils. Conversely, subconjunctival injection of Ab to ICAM-1 did not impair neutrophil recruitment, but significantly inhibited eosinophil recruitment (p = 0.0032). Injection of Ab to VCAM-1 did not significantly inhibit infiltration of either cell type to the cornea. Taken together, these results demonstrate important regulatory roles for PECAM-1 and ICAM-1 in recruitment of neutrophils and eosinophils, respectively, to the cornea, and may indicate a selective approach to immune intervention.     

Lymphatic endothelium expresses PECAM-1

The expression of adhesion molecules on the lymphatic endothelium of human small intestine and submandibular lymph node was studied immunohistochemically with the antibodies for selectin family and Ig superfamily members. In both small intestine and submandibular lymph node, lymphatic endothelium did not express intercellular adhesion molecule-1 and endothelial cell-selectin but expressed platelet-endothelial cell adhesion molecule-1 (PECAM-1). Though lymphatic vessels may not have a positive function in leukocyte rolling and adhesion, lymphatic endothelium may interact with leukocytes, with PECAM-1 playing a role.     

The Expression and Release of Adhesion Molecules by Human Endothelial Cell Lines and Their Consequent Binding of Lymphocytes

We report the characterization of a novel series of human endothelial cell lines (designated SGHEC) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1; CD54) which increased after stimulation with tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), or interferon-γ (IFN-γ). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNFα or IL-1β but not with IFN-γ. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNFα-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.