The complement binding-like domains of the murine homing receptor facilitate lectin activity

The leukocyte homing receptor (HR), the endothelial leukocyte adhesion molecule, and gmp140/platelet activation-dependent granule membrane protein are members of a family of adhesion molecules, termed the lectin cell adhesion molecules (LEC-CAMS) which are unified by a multi-domain structure containing a lectin motif, an epidermal growth factor-like (egf) motif, and variable numbers of a complement binding-like (CB) motif. Previous data have indicated a predominant role for the lectin motif in cell adhesion directed by the LEC-CAMS, although the egf-like domain of the HR may also play a potential role in cell binding. While the role(s) of the CB domains in the LEC-CAMS is currently not understood, they have been hypothesized to act as rigid spacers or stalks for lectin and perhaps, egf domain presentation. In this paper, we analyze the functional characteristics of murine HR-IgG chimeras containing the lectin, lectin plus egf, and lectin plus egf plus CB domains. The Mel 14 mAb, an adhesion blocking antibody which recognizes a conformational determinant in the N-terminus of the HR lectin domain, shows a significantly decreased affinity for a HR construct which lacks the CB motifs, consistent with the possibility that the CB domains are involved with lectin domain structure. In agreement with this conjecture, HR mutants lacking the CB domains show a profound decrease in lectin-specific interaction with the carbohydrate polyphosphomannan ester, suggesting that the changes in Mel 14 affinity for the lectin domain are reflected in lectin functionality. Various assays investigating the interactions between the HR deletion mutants and the peripheral lymph node high endothelium, including cell blocking, immunohistochemical staining, and radioactively labeled ligand binding, all showed that removal of the CB domains results in a lack of HR adhesive function. These results imply that the CB domains of the HR, and, by analogy, the other members of the LEC-CAM family, may play important structural roles involving induction of lectin domain conformation and resultant functionality.     

A homing receptor-IgG chimera as a probe for adhesive ligands of lymph node high endothelial venules

The binding of lymphocytes to high endothelial venules (HEV) within peripheral lymph nodes (pln) is thought to be mediated by a lectinlike adhesion molecule termed the pln homing receptor (pln HR). The cloning and sequencing of cDNAs encoding both murine and human pln HR revealed that these adhesion molecules contain protein motifs that are homologous to C-type or calcium dependent lectin domains as well as to epidermal growth factor (egf) and complement-regulatory protein domains. We have produced a novel, antibody-like form of the murine HR by joining the extracellular region of the receptor to a human IgG heavy chain. This antibody-like molecule is capable of recognizing carbohydrates, blocking the binding of lymphocytes to pln HEV, and serving as a histochemical reagent for the staining of pln HEV. This murine HR-IgG chimera should prove useful in analyzing the distribution of the HR ligand(s) in normal as well as in inflammatory states.     

Lectin cell adhesion molecules (LEC-CAMs): a new family of cell adhesion proteins involved with inflammation.

The means by which leukocytes, including lymphocytes, monocytes, and neutrophils, migrate from the circulation to sites of acute and chronic inflammation is an area of intense research interest. Although a number of soluble mediators of these important cellular interactions have been identified, a major site of great importance to the inflammatory response is the physical interface between the white cell and the endothelium. This critical association is mediated by an array of cell surface adhesion molecules. Previous data have demonstrated that the integrin subfamily of heterotypic adhesion molecules was a major component of these adhesive interactions, although it was clear that other, non-integrin-like molecules of unknown identity also seemed to be involved during the inflammatory process. A number of these other cell-surface glycoproteins which may be involved with inflammation have recently been characterized by molecular cloning. These glycoproteins, including the peripheral lymph node homing receptor (pln HR), the endothelial cell adhesion molecule (ELAM), and PADGEM/gmp140, are all members of a family of proteins which are unified by the inclusion of three characteristic protein motifs: a lectin or carbohydrate recognition domain, an epidermal growth factor (egf) domain, and a variable number of short consensus repeats (scr) which are also found in members of the complement regulatory proteins. The appearance of lectin domains in all of these adhesion molecules is consistent with the possibility that these glycoproteins function by binding to carbohydrates which are expressed in a cell and/or region specific manner, and the members of this adhesion family have been given the generic name LEC-CAM (lectin cell adhesion molecules).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of a lymphocyte homing receptor reveals a lectin domain

Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs.     

Surfactant proteins A and D bind CD14 by different mechanisms

Surfactant proteins A (SP-A) and D (SP-D) are lung collectins that are constituents of the innate immune system of the lung. Recent evidence (Sano, H., Sohma, H., Muta, T., Nomura, S., Voelker, D. R., and Kuroki, Y. (1999) J. Immunol. 163, 387-395) demonstrates that SP-A modulates lipopolysaccharide (LPS)-induced cellular responses by direct interaction with CD14. In this report we examined the structural elements of the lung collectins involved in CD14 recognition and the consequences for CD14/LPS interaction. Rat SP-A and SP-D bound CD14 in a concentration-dependent manner. Mannose and EDTA inhibited SP-D binding to CD14 but did not decrease SP-A binding. The SP-A binding to CD14 was completely blocked by a monoclonal antibody that binds to the SP-A neck domain but only partially blocked by an antibody that binds to the SP-A lectin domain. SP-A but not SP-D bound to deglycosylated CD14. SP-D decreased CD14 binding to both smooth and rough LPS, whereas SP-A enhanced CD14 binding to rough LPS and inhibited binding to smooth LPS. SP-A also altered the migration profile of LPS on a sucrose density gradient in the presence of CD14. From these results, we conclude that 1) lung collectins bind CD14, 2) the SP-A neck domain and SP-D lectin domain participate in CD14 binding, 3) SP-A recognizes a peptide component and SP-D recognizes a carbohydrate moiety of CD14, and 4) lung collectins alter LPS/CD14 interactions.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.     

Production and characterization of a monoclonal antibody against the seed lectin of the Dolichos biflorus plant

Spleen cells from mice immunized with the Dolichos biflorus seed lectin were fused with cells from the mouse myeloma Sp2/O-Ag14 cell line to form hybridomas. Those hybridomas producing antibodies against the seed lectin were cloned at least four times and the monoclonal antibodies from clone C11/64-56.28 were characterized and found to be specific for Subunit I of the lectin; they do not react with the structurally similar Subunit II. In previous studies, we have shown that although these two subunits appear to differ only at their COOH-terminal ends, only Subunit I has carbohydrate binding activity. Using a solid phase enzyme immunoassay, the antigenic determinant fr the monoclonal antibody was found to be located on the COOH-terminal cyanogen bromide fragment of this subunit. The monoclonal antibody inhibits the ability of the lectin to agglutinate erythrocytes and N-acetyl-D-galactosamine, the specific hapten for the lectin, inhibits the ability of the antibody to combine with the lectin. These results suggest that the monoclonal antibody recognizes a determinant that is located either at or near the active site of the lectin or that is conformationally interdependent with the active site.     

Leukocyte-endothelial cell recognition: evidence of a common molecular mechanism shared by neutrophils, lymphocytes, and other leukocytes

The interaction of leukocytes with endothelial cells is intrinsic to the process of leukocyte extravasation, whether during the entry of blood polymorphonuclear leukocytes and monocytes into sites of acute and chronic inflammation, or during the homing of lymphocytes to lymphoid organs. A lymphocyte surface glycoprotein, defined by monoclonal antibody MEL-14, has been described that appears to mediate lymphocyte recognition of postcapillary venules in peripheral lymph nodes, and to control the migration of lymphocytes from the blood into these lymphoid organs. We now report that the antigenic determinant recognized by MEL-14 is present at high levels on other leukocytes as well, including neutrophils, monocytes, and eosinophils; and we demonstrate involvement of the MEL-14 antigen in neutrophil-endothelial cell interactions. MEL-14 immunoprecipitates a neutrophil surface protein of Mr approximately 100,000, similar in m.w. to the 80,000 to 90,000 dalton lymphocyte surface MEL-14 antigen, and it blocks the interaction of neutrophils with endothelial cells in an in vitro model of adhesion to postcapillary venules in lymph node frozen sections. Neutrophil binding to lymph node venules is also inhibited by PPME, a mannose-6-phosphate-rich yeast polysaccharide that is thought to mimic the endothelial cell ligand for the MEL-14-defined lymphocyte receptor. Interestingly, neither MEL-14 nor PPME exhibit a major effect on neutrophil binding to postcapillary venules in Peyer's patches, suggesting that as for lymphocytes, the neutrophil MEL-14 antigen is involved in recognition of tissue-specific endothelial determinants. Finally, we show that MEL-14 inhibits the capacity of neutrophils to migrate from the blood into sites of acute inflammation in the skin. These observations lead us to propose that receptors for tissue-specific endothelial determinants are utilized by neutrophils and lymphocytes and probably other leukocytes during the physiologic process of leukocyte extravasation in vivo.     

Molecular mapping of functional domains of the leukocyte receptor for endothelium, LAM-1

The human lymphocyte homing receptor LAM-1, like its murine counterpart MEL-14, functions as a mammalian lectin, and mediates the binding of leukocytes to specialized high endothelial cells in lymphoid organs (HEV). LAM-1 is a member of a new family of cell adhesion molecules, termed selectins or LEC-CAMs, which also includes ELAM-1 and PAD-GEM (GMP-140/CD62). To localize the regions of LAM-1 that are involved in cell adhesion, we developed chimeric selectins, in which various domains of PAD-GEM were substituted into LAM-1, and used these chimeric proteins to define the domain requirements for carbohydrate binding, and to localize the regions recognized by several mAb which inhibit the adhesion of lymphocytes to lymph node HEV. The binding of PPME or fucoidin, soluble complex carbohydrates that specifically define the lectin activity of LAM-1 and MEL-14, required only the lectin domain of LAM-1. The LAM1-1, LAM1-3, and LAM1-6 mAb each strongly inhibit the binding of lymphocytes to HEV in the in vitro frozen section assay, and defined three independent epitopes on LAM-1. Blocking of PPME or fucoidin binding by LAM1-3 indicated that this site is identical, or in close proximity, to the carbohydrate binding site, and analysis of the binding of LAM1-3 to chimeric selectins showed that the epitope detected by LAM1-3 is located within the lectin domain. Although the LAM1-6 epitope is also located in the lectin domain, LAM1-6 did not affect the binding of PPME or fucoidin. The LAM1-1 epitope was located in, or required, the EGF domain, and, importantly, binding of LAM1-1 significantly enhanced the binding of both PPME and fucoidin. These results suggest that adhesion mediated by LAM-1 may involve cooperativity between functionally and spatially distinct sites, and support previous data suggesting a role for the EGF domain of LAM-1 in lymphocyte adhesion to HEV.     

Distribution of saccharides in pig lymph-node high-endothelial venules and associated lymphocytes visualized using fluorescent lectins and confocal microscopy

Summary The distribution of saccharides in pig lymph nodes, particularly on high-endothelial venule (HEV) endothelium and on lymphocytes in these vessels, was studied by examining the binding of fluorescent conjugates of 18 different lectins. Eight of the lectins, particularly with glycan specificity restricted to mannose and polyacetyllactosamine determinants, were found to bind with a high affinity to these structures. Competitive inhibition experiments revealed that polylactosamine-containing glycans were present on endothelia and lymphocytes using lectins from Lycopersicon esculentum and Solanum tuberosum, the latter lectin reacting with lymphocytes only when apparently adherent to the luminal endothelium. The absence on pig endothelium of the Ulex europaeus binding, shown by human endothelia due to the presence of certain fucose epitopes, was confirmed. Pig lymph-node endothelium, however, bound the fucose-specific lectin of Tetragonolobus purpureas, indicating the presence of fucose on pig endothelia in a different conformation to that seen on human endothelia. The results suggested that pig lymph-node HEV endothelium expressed a core fucosylated tri- or tetra-antennary complex glycan with polylactosamine extensions and expressing an Le^y determinant.Abbreviations used BS-I Bandeiraea simplicifolia BS-I - BS-I-B B. simplicifolia isolectin B₄ - BS-II B. simplicifolia, lectin II - FACS fluorescence-activated cell sorter - FITC fluorescein isothiocyanate - HEV high-endothelial venule - LN lymph node - MLR mixed lymphocyte reaction - PBS phosphate-buffered saline - PPME phosphomannan - UEA-I Ulex europeaus lectin I     

Further characterization of the interaction between L-selectin and its endothelial ligands.

L-Selectin is a lectin-like receptor on lymphocytes which mediates their attachment to high endothelial venules (HEV) within lymph nodes. Previous work has identified HEV-associated endothelial ligands for L-selectin as sialylated, fucosylated and sulphated glycoproteins of approximately 50 kDa and approximately 90 kDa (Sgp50 and Sgp90). The interaction of L-selectin with these ligands is carbohydrate directed, reflecting the involvement of its amino-terminal, calcium-type lectin domain. It has been reported, and we have confirmed, that anti-Ly22 blocks the adhesive function of L-selectin without reducing its binding to a carbohydrate- based ligand PPME (phosphomannan monoester core from Hansenula hostii). The epitope for this monoclonal antibody depends on the epidermal growth factor (EGF) domain of L-selectin. We demonstrate that anti-Ly22 inhibits the interaction of L-selectin with both of the Sgps, thus establishing that the interaction of L-selectin with HEV can be accounted for by the Sgps. Furthermore, the interaction of trypsin fragments of Sgp50 with L-selectin is inhibitable both by an antibody that maps to the lectin domain and by anti-Ly22. These findings raise the possibility that anti-Ly22 is affecting the function of the lectin domain of L-selectin rather than directly antagonizing the EGF domain. Toward a further characterization of L-selectin's carbohydrate specificity, we show that Sgp50 is partially inactivated by the linkage-specific Newcastle Disease virus sialidase (alpha 2,3 linkage). We additionally demonstrate that a sialyl Lewis x-related tetrasaccharide can interact with L-selectin, as has also been demonstrated for E-selectin and P-selectin.     

Oligomerisation and carbohydrate binding in an Atlantic salmon serum C-type lectin consistent with non-self recognition

A C-type lectin was previously isolated from the blood of healthy Atlantic salmon (Salmo salar) and this salmon serum lectin (SSL) was found to opsonise bacteria. Selective binding to bacteria in vivo requires that the lectin be able to recognise a carbohydrate pattern on the bacterial surface distinguishable from that of the host. In order to investigate this selectivity in the lectin, a phage-display antibody was prepared and then used for detection of lectin by Western blotting. A carbohydrate binding-inhibition assay with Western blot detection of the lectin showed mannose to be the primary ligand and related sugars including glucose, N-acetylglucosamine and methyl alpha-D-mannopyranoside to be additional ligands of this lectin. The SSL in serum detected by Western blotting was shown to form a complex oligomer. These results show that the salmon serum lectin is oligomeric in blood and that it recognizes a broad spectrum of carbohydrates with optimal binding to mannose. The lectin might therefore be an ideal opsonin for multiple salmon pathogens with carbohydrate arrays on their surfaces. No similar lectins were identified in the sera of other fish by Western blotting using the phage-display antibody. Molecular analysis will be required in order to determine whether homologous lectins are expressed in related fish species. It is anticipated that similar lectins might have related pathogen recognition roles in divergent fish species.     

Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.     

Species-Specific and Conserved Epitopes on Mouse and Human E-Selectin Important for Leukocyte Adhesion

Selectins are C-type, cell surface lectins that are key players in leukocyte adhesion to the blood vessel wall endothelium. We describe here epitopes for a series of novel monoclonal antibodies (moAbs), UZ4-UZ7, directed against mouse E-selectin. All four antibodies specifically bind to mouse E-selectin, but not to P- or L-selectin, and all inhibit the adhesion of granulocytes, peripheral blood lymphocytes, and promyelocytic HL-60 cells to cytokine-activated mouse endothelium. Three moAbs, UZ5, UZ7, and UZ6, specifically inhibit mouse E-selectin-mediated adhesion by binding to epitopes in domains CR1 or CR2. moAb UZ4 inhibits leukocyte adhesion to both human and murine endothelium activated with IL-1 or other proinflammatory stimuli. UZ4 is the first described moAb that detects an epitope in the lectin domain which is conserved in both murine and human E-selectin (CXKKKL), but is not present in the other members of the selectin family, P- and L-selectin. Interestingly, UZ5, UZ6, and UZ7 more efficiently interfere with lymphocyte than with granulocyte adhesion to cytokine-activated endothelium, while UZ4 completely blocks adhesion of PMN, lymphocytes, and HL-60 and U937 cell lines. The data suggest that E-selectin–ligand engagement differs between lymphocytes and PMN, and that these differences may be accentuated by the CR1 and CR2 domains in the E-selectin cell adhesion molecule.     

Possible role for cell-surface carbohydrate-binding molecules in lymphocyte recirculation

We are investigating the hypothesis that carbohydrate-binding molecules on the cell surface are involved in the recirculation of lymphocytes from the bloodstream into lymphoid organs. This phenomenon requires the specific attachment of circulating lymphocytes to the endothelial cells of postcapillary venules. Using an in vitro assay to measure the adhesive interaction between lymphocytes and postcapillary venules, we have found that L-fucose, D mannose, and the L-fucose-rich, sulfated polysaccharide fucoidin specifically inhibit this binding interaction. L-fucose shows stereo-selective inhibitory activity at concentrations greater than 18 mM while fucoidin produces 50% inhibition at approximately 1-5 X 10(-8) M. Fucoidin appears to interact with the lymphocyte, and not the postcapillary venule, to inhibit binding. These data suggest that cell surface carbohydrates (fucoselike) and carbohydrate-binding molecules (cell surface lectins) may contribute to the specific attachment of lymphocytes to postcapillary venules.     

The human peripheral lymph node vascular addressin is a ligand for LECAM-1, the peripheral lymph node homing receptor

The trafficking of lymphocytes from the blood and into lymphoid organs is controlled by tissue-selective lymphocyte interactions with specialized endothelial cells lining post capillary venules, in particular the high endothelial venules (HEV) found in lymphoid tissues and sites of chronic inflammation. Lymphocyte interactions with HEV are mediated in part by lymphocyte homing receptors and tissue-specific HEV determinants, the vascular addressins. A peripheral lymph node addressin (PNAd) has been detected immunohistologically in mouse and man by monoclonal antibody MECA-79, which inhibits lymphocyte homing to lymph nodes and lymphocyte binding to lymph node and tonsillar HEV. The human MECA-79 antigen, PNAd, is molecularly distinct from the 65-kD mucosal vascular addressin. The most abundant iodinated species by SDS-PAGE is 105 kD. When affinity isolated and immobilized on glass slides, MECA-79 immunoisolated material binds human and mouse lymphocytes avidly in a calcium dependent manner. Binding is blocked by mAb MECA-79, by antibodies against mouse or human LECAM-1 (the peripheral lymph node homing receptor, the MEL-14 antigen, LAM-1), and by treatment of PNAd with neuraminidase. Expression of LECAM-1 cDNA confers PNAd binding ability on a transfected B cell line. We conclude that LECAM-1 mediates lymphocyte binding to PNAd, an interaction that involves the lectin activity of LECAM-1 and carbohydrate determinants on the addressin.     

Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion [published erratum appears in J Cell Biol 1993 Feb;120(4):1071]

E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.     

A family of cell-surface glycoproteins defined by a putative anti-endothelial cell receptor antibody in man

An efficient immune response depends directly on the ability of lymphocytes and other blood borne leukocytes to leave the blood and enter lymphoid organs and other sites of immune response or inflammation. A new murine mAb termed 515 recognizes a 85- to 90-kDa structure involved in lymphocyte binding to specialized endothelium in postcapillary venules of lymphoid organs (high endothelial venules). In crossed immunoprecipitations followed by SDS-PAGE analysis, 515 appears to be identical to the antilymphocyte homing receptor mAb, Hermes-1. We show here that 515 recognizes peripheral blood leukocytes of all classes examined, but, like Hermes-1, fails to recognize germinal center cells or nearly all cortical (but not medullary) thymocytes. In addition, we demonstrate that 515 recognizes both fibroblasts and epidermal keratinocytes, both in situ and in cultured cell lines. Immunoprecipitation and SDS-PAGE analysis reveals that 515-defined Ag from T lymphocytes, neutrophils, and the epidermal keratinocyte cell line HaCaT are clearly distinct, both before and after treatment with peptide N-glycosidase F, which removes all asparagine-linked oligosaccharides. In contrast, the Mr of 515-defined Ag from T cells and fibroblasts appear indistinguishable. Thus, the 515 mAb recognizes a broadly distributed family of glycoproteins which may be involved in a variety of adhesive functions.     

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)