PubMed Health⌕ Search

Biomedical subjects

S R Watson

Publications and source records attributed to S R Watson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Subverting lymph node trafficking by treatment with the Mel-14 monoclonal antibody to L-selectin does not prevent an effective host response to Sendai virus.

A single 250-micrograms dose of the Mel-14 mAb to L-selectin greatly diminished the extent of L-selectin expression on lymphocytes and decreased (60 to 90%) the massive cellular recruitment to the cervical and mediastinal lymph nodes that follows intranasal infection of naive C57BL/6 mice with Sendai virus. The numbers of CD8+ CTL precursors in the mediastinal lymph nodes were considerably reduced on day 7, when compared with virus-infected mice given a control rat IgG2a, but potent CTL effectors were present in the lungs of both groups by day 10 after infection, and the overall magnitude of CTL precursor generation was not obviously compromised. The early dominance of Sendai virus-specific IgM Ab-forming cells was prolonged in the Mel-14-treated mice, whereas plasma cells producing virus-specific IgA were abnormally prominent in the lymph nodes but not in the spleen. The kinetics of virus-specific Ab-forming cells generation and the serum Ab response for the various IgG isotypes were also delayed. Thus, though L-selectin is clearly important for the localization of naive lymphocytes to regional lymph nodes, the Mel-14-treated mouse can still deal effectively with a virus that causes productive infection only in the respiratory tract. The spleen, where L-selectin does not determine lymphocyte trafficking, is a major site for the compensatory T cell and B cell responses.

Animals↗
PubMed1995

Selective modulation of the expression of L-selectin ligands by an immune response.

BACKGROUND: The adhesion molecule L-selectin is expressed on the cell surface of lymphocytes and mediates their migration from the bloodstream into lymph nodes. L-selectin is able to recognize four glycoprotein ligands, three of which--Sgp50, Sgp90, and Sgp200--are sulphated, bind specifically to L-selectin and are synthesized by the high endothelial venules of the peripheral and mesenteric lymph nodes. One of these three sulphated L-selectin ligands, Sgp90, has been shown to be identical to the known surface marker CD34 and is expressed on the cell surface of endothelial cells. The cDNA encoding Sgp50 has been cloned, and its product, which has been designated GlyCAM-1, is secreted. The third ligand, Sgp200, is both secreted and cell-associated. We have investigated how the expression of these sulphated glycoproteins is regulated during an immune response. RESULTS: Here we demonstrated that, during a primary immune response, the expression and secretion of both GlyCAM-1 and Sgp200 are reduced, recovering to normal levels 7-10 days after antigen stimulation. In contrast, the expression of cell-associated CD34 and Sgp200 is relatively unaffected. These results may account for the modest decreases in the binding of an L-selectin-IgG fusion protein to high endothelial venules of inflamed peripheral lymph nodes that have been observed after antigen exposure. In vivo experiments show that, following the decrease in the levels of secreted GlyCAM-1 and Sgp200, migration of lymphocytes from the blood stream into lymph nodes remains L-selectin-dependent, but more lymphocytes home to antigen-primed than unprimed peripheral lymph nodes. CONCLUSIONS: We suggest that the secreted forms of the L-selectin ligands GlyCAM-1 and Sgp200 act as modulators of cell adhesion, and that cell-associated CD34 and Sgp200 are the ligands that mediate the initial loose binding of lymphocytes to high endothelial venules.

Animals↗
PubMed1995

Reduced recruitment of inflammatory cells in a contact hypersensitivity response in P-selectin-deficient mice.

The inflammatory response at sites of contact hypersensitivity induced by oxazolone was examined in the ears of P-selectin-deficient and wild-type mice. Accumulation of CD4+ T lymphocytes, monocytes, and neutrophils was reduced significantly in the mutant mice, as well as mast cell degranulation. In contrast, there was no significant difference in vascular permeability or edema between the two genotypes. The results demonstrate a role for P-selectin in recruitment of CD4+ T lymphocytes and show that P-selectin plays a role in long-term inflammation as well as in acute responses.

Animals↗
PubMed1995

A P-selectin-immunoglobulin G chimera is protective in a rabbit ear model of ischemia-reperfusion.

BACKGROUND: Neutrophils have been shown to play a role in ischemia-reperfusion injury, and the initial interaction of neutrophils with the endothelium is mediated through the selectin family of adhesion molecules. Thus the purpose of these studies was to determine whether a P-selectin-IgG chimera was protective in a model of ischemia-reperfusion injury. METHODS: The model used was a rabbit ear model of ischemia-reperfusion. Selectin-IgG chimeras were given at the time of reperfusion of the tissue, and their efficacy was compared with an anti-CD18 antibody (MHM23). RESULTS: The P-selectin-IgG was as protective in this model as an anti-CD18 antibody. The chimera did not mediate its effect by causing the animals to become neutropenic. CONCLUSIONS: P-selectin plays a role in ischemia-reperfusion injury. This is in agreement with data from other groups. The fact that the chimera was effective in this model suggests that carbohydrates or small molecule mimics of carbohydrates would be effective in this model. Such antiinflammatory agents may have fewer side effects in terms of increased risk of sepsis.

Animals↗
PubMed1994

Entry of naive CD4 T cells into peripheral lymph nodes requires L-selectin.

Binding of L-selectin expressed on lymphocytes to carbohydrate ligand(s) on lymph node high endothelial venules is thought to initiate lymphocyte extravasation from blood to lymph during recirculation and localization to sites of antigen (Ag) exposure. Previous studies have shown that treatment of lymphocytes with antibody to L-selectin (MEL-14) ablates trafficking to peripheral lymph nodes (PLN). In mice, naive but not memory CD4 cells express L-selectin. To examine the role of L-selectin in helper T cell migration, we studied the effects of in vivo administration of MEL-14 on CD4 cell responses. Systemic exposure of mice to MEL-14 depleted CD4 cells expressing a naive phenotype (CD45RBhi, CD44lo) from PLN but not from spleen. The majority of residual lymph node CD4 cells exhibited the reciprocal, memory phenotype (CD45RBlo, CD44hi). MEL-14 treatment prevented priming of naive CD4 cells for proliferation and cytokine production (IL-2 and IL-4) to keyhole limpet hemocyanin in PLN draining the site of Ag injection, but not in the spleen. The results suggest that naive cells were not depleted, but rather diverted to other sites where priming occurred. The data demonstrate that L-selectin mediates extravasation of naive CD4 cells into PLN and that its function cannot be replaced by other homing receptors.

Animals↗
PubMed1994

Cell surface P- and E-selectin support shear-dependent rolling of bovine gamma/delta T cells.

The vascular selectins P- and E-selectin are inducible adhesion proteins expressed by endothelial cells that have been shown to support shear-dependent rolling of myeloid cells. This interaction is thought to be a prerequisite event for subsequent steps, such as tight adhesion/aggregation and transendothelial cell migration, involved in the accumulation of leukocytes into tissues. Certain lymphocyte subsets have also been shown to bind the vascular selectins, but the importance of this interaction in mediating shear-dependent rolling, as described for myeloid cells, has not been demonstrated. We expand on our earlier observation that bovine gamma/delta T cells bind E-selectin by showing that this interaction leads to a reproducible rolling event in assays done under shear forces that approximate those that occur in vivo. E-selectin, expressed by L cell transfectants or cytokine-stimulated human and bovine endothelial cells, equally supports the shear-dependent rolling interaction. The lymphocyte adhesion proteins L-selectin, CD44, and CD2 do not contribute to this event. Neuraminidase treatment of the gamma/delta T cells or addition of EDTA to the assay completely blocks the rolling interaction. We further show for the first time that P-selectin expressed by thrombin-activated platelets or a soluble P-selectin/human Ig chimera specifically binds gamma/delta T cells. The P-selectin interaction is similar to the rolling event mediated by E-selectin--it requires divalent cations and sialic acid on the lymphocyte, it lacks involvement of L-selectin and CD44, and rolling occurs under physiologic shear conditions. These results provide the documentation that the vascular selectins can support shear-dependent rolling of a lymphocyte subset and that P-selectin mediates the adhesion of gamma/delta T cells.

Animals↗
PubMed1994

Eosinophil adhesion to nasal polyp endothelium is P-selectin-dependent.

Tissue eosinophilia is a characteristic feature of a number of inflammatory diseases including asthma and nasal polyposis. Eosinophil migration into tissues is controlled in part by interactions between eosinophil adhesion receptors and counter-structures on the vascular endothelium. To determine the receptors used by eosinophils to adhere to vascular endothelium in allergic inflammation we have adapted the Stamper-Woodruff frozen section assay (FSA) to study eosinophil adhesion to nasal polyp endothelium. Immunohistology indicated that intercellular adhesion molecule 1 (ICAM-1), E-selectin and P-selectin were well expressed by nasal polyp endothelium, whereas expression of vascular cell adhesion molecule 1 (VCAM-1) was weak or absent. Unstimulated human peripheral blood eosinophils adhered specifically to nasal polyp endothelium. Adherence was temperature and divalent cation-dependent and saturable at cell densities > 5 x 10(6) cells/ml. Eosinophil adhesion was almost completely inhibited by a monoclonal antibody (mAb) against P-selectin and by a chimeric molecule consisting of the Fc portion of human IgG and the lectin binding domain of P-selectin, which binds to the P-selectin ligand on leucocytes. Anti-Mac-1 mAb partially inhibited eosinophil adhesion whereas mAb against E-selectin, L-selectin, ICAM-1, VCAM-1, very late activation antigen 4, and lymphocyte function-associated antigen 1 had no effect. P-selectin is stored in intracellular granules within the endothelial cell and in vitro is only transiently expressed. To determine if P-selectin was expressed on the membrane of the nasal polyp endothelium we compared P-selectin expression in normal skin and nasal polyps after acetone fixation, which permeabilizes cells, and paraformaldehyde, which only allows staining of membrane expressed receptors. In the skin, good expression was seen with acetone fixation but no expression was seen after paraformaldehyde treatment, whereas in nasal polyps, similar expression was observed with both fixatives. In addition immunofluorescence with confocal microscopy demonstrated lumenal staining of nasal polyp endothelium indicating that P-selectin was located on the surface of endothelial cells while in skin only an intracellular granular distribution was apparent. Lastly, whereas eosinophils bound consistently to nasal polyp endothelium, no binding was observed to blood vessels in normal skin further supporting the idea that eosinophils were binding to membrane expressed and not intracellular P-selectin. The importance of P-selectin in eosinophil adhesion to nasal polyp endothelium suggests that P-selectin antagonists may be effective at inhibiting eosinophil accumulation at sites of allergic inflammation.

Adult↗
PubMed1994

Expression of fucosylated antigens and alpha 1,3 fucosyltransferases in human leukaemia cell lines.

The expression of alpha 1,3 fucosylated type 2 antigens is generally thought to be restricted to myeloid cells among normal human haemopoietic tissue. The distribution of three fucosylated antigens [Lewis X (Le(x)), sialyl Lewis X (sLex) and VIM2] was investigated among nine human leukaemia cell lines by fluorescence activated cell sorting (FACS) analysis. As expected, all myeloid cell lines were positively stained by antibodies against these three fucosylated antigens. Unexpectedly, two T-lymphocytic cell lines (CCRF-CEM and MOLT4) were found to express Le(x) and VIM2, and the plasma, B-cell line, RPMI 8226, expressed all three fucosylated antigens. Enzymatic and RNA analyses [Northern blot and reverse transcription polymerase chain reaction (RT-PCR)] were used to evaluate possible control points in the biosynthetic pathway for Le(x) and sLex. beta 1,4 Galactosyltransferase (beta 1,4GalT, an enzyme involved in the synthesis of the core oligosaccharide of the three fucosylated antigens) activity and the corresponding mRNA were found in all of the leukaemia cell lines, regardless of whether or not they expressed the fucosylated antigens. In contrast, alpha 1,3 fucosyltransferase (GDP-fucose:beta-D-N-acetylglucosaminide 3-alpha-L-fucosyltransferase; alpha 1,3FT) activity and the corresponding mRNA were found only in those cell lines expressing fucosylated antigens. Based on RNA analysis, acceptor specificity and N-ethylmaleimide inhibition studies, it was concluded that all of the cell lines expressing fucosylated antigens contained alpha 1,3FTIV (myeloid alpha 1,3FT). This appeared to be the major alpha 1,3FT in the myeloid and T-lymphocytic cell lines. Interestingly, even though both types of cell lines expressed the same alpha 1,3FT, only the myeloid cell lines expressed sLex, whereas all of the myeloid and T-lymphocytic cell lines expressed a structural analogue of sLex (i.e. VIM2). In contrast to the myeloid and T-cell lines, RPMI 8226 cells contained more than one fucosyltransferase activity. Acceptor specificity analysis demonstrated that this cell line contains alpha 1,3 and alpha 1,4FTs. Among the fucosyltransferases expressed by RPMI 8226, alpha 1,3FTIV accounted for only a small amount of the total activity. The results of this study demonstrate that fucosylated antigens, which are generally considered to be myeloid specific antigens, are also expressed by lymphocytic leukaemia cell lines, and that the types of fucosylated antigens and fucosyltransferases expressed in these cell lines vary.

Antigens, Neoplasm↗
PubMed1993

Expression of GlyCAM-1, an endothelial ligand for L-selectin, is affected by afferent lymphatic flow.

The interaction of naive, L-selectin-bearing lymphocytes with counterreceptors on the surface of high endothelial venules (HEV) is the initial step in the extravasation of these cells from the bloodstream into the peripheral lymph node. Recently, two sulfated glycoprotein ligands, 50 and 90 kDa, respectively, have been identified as ligands for L-selectin using an L-selectin-IgG chimera. cDNA cloning of one of these molecules, the 50-kDa sulfated glycoprotein (glycosylation-dependent cell adhesion molecule 1 [GlyCAM-1]), has shown it to be a mucinlike scaffold that presents a carbohydrate ligand(s) to the lectin domain of L-selectin. Herein, we analyze the factors that might regulate the expression of these ligands. Ligation of afferent lymphatics results in a complete loss of the mRNA for GlyCAM-1. In addition, L-selectin-mediated adhesion, as inferred by binding of an L-selectin-IgG chimera, is also lost on interruption of afferent flow. It thus appears that a soluble and/or cellular component(s) of afferent lymph regulates the expression of GlyCAM-1 mRNA and the resultant HEV adhesiveness for lymphocytes.

Animals↗
PubMed1993

Protective effects of selectin chimeras in neutrophil-mediated lung injury.

Recombinant selectin chimeric molecules featuring the joining of the extracellular domains of L-, P-, and E-selectin to the CH2 and CH3 domains of human IgG1 have been evaluated for their ability to protect against neutrophil-dependent lung injury in rats after systemic activation of C caused by vascular infusion of cobra venom factor (CVF) or lung injury that follows intrapulmonary deposition of IgG immune complexes. Previous studies using anti-selectin antibodies have suggested that the former model is P-selectin dependent, whereas the latter is E-selectin dependent. Requirements for L-selectin have not been identified because of lack of reagents. For the current studies employing the CVF model of lung injury, infusion of P-selectin-Ig chimera reduced injury (as assessed by changes in permeability and hemorrhage) in a dose-dependent manner, with parallel reductions in lung myeloperoxidase (MPO) content. Similar results were obtained with the L-selectin-Ig chimera, whereas the E-selectin-Ig chimera was not protective and failed to alter MPO content. In contrast, in the IgG immune complex model of lung injury, the L- and E-selectin-Ig chimeras both showed dose-related protective effects and reductions in MPO content, whereas the P-selectin-Ig chimera failed to protect against injury and did not alter MPO content in this model of lung injury. In all cases of blocking of injury, this was incomplete, suggesting multi-selectin engagement or inadequate amounts of selectin-Ig chimeras employed. These data indicate that neutrophil recruitment and attendant lung injury in the CVF model are L- and P-selectin dependent and E-selectin-independent, whereas in the IgG immune complex model, neutrophil recruitment and lung injury are L- and E-selectin-dependent but independent of P-selectin. Thus, differing selectin requirements for acute inflammatory lung injury have been identified.

Animals↗
PubMed1993

L- and E-selectin can recognize the same naturally occurring ligands on high endothelial venules.

The selectin family of cell adhesion molecules consists of three members, E-selectin (ELAM-1), L-selectin (LECAM-1), and P-selectin (CD62, GMP140, or PADGEM), which are all involved in binding of leukocytes to endothelial cells. All members have structural similarities and they can all bind to a common carbohydrate epitope, sialyl Lewis X in in vitro assays. To study cross-reactivity of the selectins in more detail we used Ig chimeras of murine and human L- and human E-selectin. All three chimeras bound to the natural ligands of L-selectin on specialized high endothelial venules in both human and murine lymphoid organs as determined by immunohistochemistry and were able to precipitate 50- and 90-kDa sulfated ligands from organ cultures of murine peripheral and mesenteric lymph nodes. This recognition was calcium dependent and inhibitable by mAb specific for the lectin domain of the respective selectin. L- and E-selectin binding to high endothelial venules and to the sulfated ligands was inhibitable by the carbohydrates, fucoidan and sialyl Lewis X, respectively. Although immunoprecipitations showed that both murine and human L-selectin could recognize the sulfated ligands from high endothelial venules more efficiently than human E-selectin at the concentrations used, both L- and E-selectin chimeras could inhibit in vivo lymphocyte trafficking into peripheral lymph nodes equally well.

Animals↗
PubMed1993

Cloning of a rat homologue of mouse GlyCAM 1 reveals conservation of structural domains.

Recently we described the isolation of a mouse cDNA clone encoding a mucin-like endothelial glycoprotein that appears to function as an adhesive ligand for L selectin. This ligand has been named GlyCAM 1 (Gly-cosylation-dependent Cell Adhesion Molecule 1) because its adhesive interactions with the L selectin lectin domain require that the GlyCAM 1 polypeptide chain be appropriately modified with carbohydrates. These carbohydrate modifications include the addition of sialic acid as well as sulfate residues to O-linked carbohydrate side chains that are clustered in two serine/threonine-rich domains of the mucin. An additional interesting structure that may have relevance to the association of GlyCAM 1 with the lumenal surface of the endothelium was a potential amphipathic helix at the C terminus of the glycoprotein. In order to examine the importance of the postulated O-linked domains as well as the potential amphipathic helix, we have cloned the rat homologue of GlyCAM 1. The sequence of this clone reveals a serine/threonine-rich protein that is highly homologous with the mouse GlyCAM 1. As was found for the mouse GlyCAM 1, the rat homologue shows a clustering of these potential O-linked carbohydrate acceptors in two domains of the protein. Interestingly, many of the serines and threonines are found to be spaced identically in the two homologues, consistent with the possibility that both density and position of the O-linked side chains may be important for appropriate L selectin-mediated adhesion. In support of its postulated functional importance, the C-terminal potential amphipathic helix is conserved in the rat homologue. Finally, immunoprecipitation analysis of [35S]sulfate-labeled rat lymph nodes with either a mouse L selectin IgG chimera or a peptide antiserum directed against a relatively conserved portion of mouse GlyCAM 1 demonstrates a approximately 45-kDa sulfated ligand in rat lymph nodes that is analogous to that previously described for mouse lymph nodes.

Amino Acid Sequence↗
PubMed1993

P- and E-selectin use common sites for carbohydrate ligand recognition and cell adhesion.

The selectins are a family of three calcium-dependent lectins that mediate adhesive interactions between leukocytes and the endothelium during normal and abnormal inflammatory episodes. Previous work has implicated the carbohydrate sialyl Lewis(x) (sLe(x); sialic acid alpha 2-3 galactose beta 1-4 [Fucose alpha 1-3] N-acetyl glucosamine) as a component of the ligand recognized by E- and P-selectin. In the case of P-selectin, other components of the cell surface, including 2'6-linked sialic acid and sulfatide (galactose-4-sulfate ceramide), have also been proposed for adhesion mediated by this selectin. We have recently defined a region of the E-selectin lectin domain that appears to be directly involved with carbohydrate recognition and cell adhesion (Erbe, D. V., B. A. Wolitzky, L. G. Presta, C. R. Norton, R. J. Ramos, D. K. Burns, R. M. Rumberger, B. N. N. Rao, C. Foxall, B. K. Brandley, and L. A. Lasky. 1992. J. Cell Biol. 119:215-227). Here we describe a similar analysis of the P-selectin lectin domain which demonstrates that a homologous region of this glycoprotein's lectin motif is involved with carbohydrate recognition and cell binding. In addition, we present evidence that is inconsistent with a biological role for either 2'6-linked sialic acid or sulfatide in P-selectin-mediated adhesion. These results suggest that a common region of the E- and P-selectin lectin domains appears to mediate carbohydrate recognition and cell adhesion.

Amino Acid Sequence↗
PubMed1993

L-selectin-IgG chimera--in vitro and in vivo.

In order to perform their role as mediators of inflammation neutrophils must be able to extravasate from the blood into the surrounding tissue. The initial step in this process is the rolling of neutrophils along the endothelium and is mediated by adhesion molecules known as selectins. This review will discuss the in vitro and in vivo characterization of an L-selectin-IgG chimera. The aim of the studies being to understand the role that the selectins might play in inflammatory responses and hopefully control those that are chronic or lifethreatening.

Animals↗
PubMed1992

Identification of a soluble form of a ligand for the lymphocyte homing receptor.

Lymphocytes are engaged in constant trafficking from the blood into secondary lymphoid tissues, such as peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), and Peyer's patches (PP). The initial step in this process is the binding of lymphocytes to high endothelial venules (HEV), and in the case of trafficking of cells to the PLN, it is required that they bear the L-selectin surface receptor. Using a chimeric protein, combining the extracellular domains of L-selectin with a human immunoglobulin (Ig) G1 Fc region (L-selectin-IgG), we have probed the expression of ligands for this receptor on HEV and in cell lysates. Two sulfated glycoproteins of 50 and 90 kD have been identified in lysates from PLN and MLN, but not PP. Here we show that the 50-kD molecule is secreted in organ cultures in vitro and is present in the blood of normal animals. Indeed, normal serum inhibits lymphocyte binding to HEV by approximately 50% in an in vitro assay. This inhibitory activity can be removed by passage of the serum over an L-selectin-IgG column and has a molecular mass of approximately 50 kD. We speculate on the possible reasons for secretion of a homing receptor ligand.

Amino Acid Sequence↗
PubMed1992

An endothelial ligand for L-selectin is a novel mucin-like molecule.

The adhesive interaction between circulating lymphocytes and the high endothelial venules (HEV) of lymph nodes (LN) is mediated by lymphocyte L-selectin, a member of the selectin family of cell adhesion proteins. Previous work has identified a sulfated 50 kd glycoprotein (Sgp50) as an HEV ligand for L-selectin. We now report the purification of this glycoprotein and the utilization of the derived N-terminal amino acid sequence to clone a cDNA. The predicted sequence reveals a novel, mucin-like molecule containing two serine/threonine-rich domains. The mRNA encoding this glycoprotein is preferentially expressed in LN. Antibodies against predicted peptides immunoprecipitate Sgp50 and stain the apical surface of LN HEV. These results thus define a tissue-specific mucin-like endothelial glycoprotein that appears to function as a scaffold that presents carbohydrates to the L-selectin lectin domain.

Amino Acid Sequence↗
PubMed1992

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide.

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.

Animals↗