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Biomedical subjects

F W Symington

Publications and source records attributed to F W Symington.

13 recordsLinked to original sources
PubMed1992

Activation of human phagocytes through carbohydrate antigens (CD15, sialyl-CD15, CDw17, and CDw65).

The leukocyte carbohydrate (CHO) Ag CD15, sialyl-CD15, and CDw65 have recently been found to function as ligands for CD62 and ELAM-1 cell adhesion molecules on platelets and endothelium, respectively. Cell adhesion ligands also may act as receptors capable of signal transduction. We therefore investigated the possibility that these CHO Ag and CDw17, a glycolipid Ag whose expression is regulated by leukocyte activation, may have receptor-like characteristics. The effects of antibody cross-linking of CHO Ag on phagocyte activation were measured by using flow cytometry and fluorescent indicators for cytoplasmic calcium ions, oxidative burst, and the granule-associated proteins CD11b and CD67. Cross-linking of CD15, sialyl-CD15, CDw65, or CDw17 induced a moderate release of calcium ions into the cytoplasm of granulocytes, a strong activation of oxidative burst, and a low up-regulation of CD11b and CD67 compared to the effects of treatment with 4 microM FMLP. The results suggest a role for CHO Ag in leukocyte signal transduction and support the view that these molecules are involved in phagocyte activation.

Antibodies, Monoclonal↗
PubMed1992

The relationship of serum IL-6 levels to acute graft-versus-host disease and hepatorenal disease after human bone marrow transplantation.

The potential involvement of cytokines in acute graft-versus-host disease led us to analyze interleukin-6 in serial serum sets from 22 allogeneic marrow recipients who developed either grade 3 or 4 GVHD (n = 10), grade 2 GVHD (n = 6), or grade 1 or no diagnosed GVHD (n = 6). A total of 279 serial serum samples taken three times weekly before day 35 were analyzed. Maximum IL-6 levels were greater than 40 U/ml (range, 40-1536 U/ml), 11-40 U/ml, and less than or equal to 10 U/ml for six, eleven, and five patients, respectively. Serum IL-6 peaks were temporally related to onset of GVHD, onset of a syndrome of hepatorenal dysfunction (HRD), or bilateral lung infiltration. Eight of ten patients who developed grade 3 or 4 GVHD overall had IL-6 maxima of greater than 10 U/ml an average of 1.5 +/- 1.8 days before the clinical onset. Fifteen of 17 patients with peak IL-6 levels greater than 10 U/ml developed symptoms of hepatic and renal dysfunction within three days of the peak, while none of five patients with less than or equal to 10 U/ml of Il-6 developed HRD. Regression analysis demonstrated a linkage between the log magnitudes of the serum IL-6 peaks and onset of either GVHD or HRD within three days (P = 0.001). Furthermore, IL-6 peaks tended to precede GVHD onset for the 10 patients whose GVHD onset and IL-6 peak were within three days of each other (P = 0.02). These results, confirmed by both specific bioassay and by IL-6 ELISA, support the idea that acute GVHD in humans involves a cytokine cascade that includes production of IL-6 in addition to the previously reported involvement of tumor necrosis factor alpha and interferon-gamma.

Acute Disease↗
PubMed1992

Human epidermal keratinocyte expression of sialyl-Lewis X.

Cell-surface oligosaccharides can function as ligands for intercellular adhesion receptors, matrix proteins, and growth factors. We report that human neonatal and adult epidermal keratinocytes (KC) express sialyl Lewis X [s-Le(x); SA alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3R], a ligand for endothelial and platelet selectins. Freshly isolated or cultured KC bind FH6 monoclonal antibody (MoAb), which is specific for s-Le(x)-containing oligosaccharides. The relevant epitope is bona fide s-Le(x), because sialidase treatment of KC suspensions abrogates FH6 binding while generating de novo KC reactivity with anti-Le(x). KC stained in ice-cold suspension display a knobby membrane distribution of s-Le(x) detectable by immunofluorescence microscopy. As others have reported, FH6 appeared not to bind KC in perpendicular skin sections. However, basal KC in intact epidermal sheets exhibited obvious "honeycomb" reactivity with FH6 when stained and viewed en face, suggesting that s-Le(x) in intact epidermis may occur in bands that parallel the major tissue axis. FH6 specifically immunoprecipitated proteins of Mr 34 kd, 44 kd, and 56 kd from [35S]-labeled KC, and anti-Le(x) precipitated similar proteins from sialidase-treated KC. The enzymatic basis for KC s-Le(x) expression was studied by analyzing acceptor specificities and other properties of KC fucosyltransferases. Results indicate that KC express both Lewis- and myeloid-type alpha 1-3fucosyltransferases. KC s-Le(x) could be an important element of the epithelial milieu, because both epithelial cells and immune cells that home to epithelia express s-Le(x) and related structures, and because KC s-Le(x) is well positioned for selectin-mediated platelet binding after trans-cutaneous wounding. The apparent distributions of s-Le(x) in epidermis and on isolated KC are compatible with a functional role for s-Le(x) in these intercellular interactions.

Adult↗
PubMed1991

Lysis of human keratinocytes by allogeneic HLA class I-specific cytotoxic T cells. Keratinocyte ICAM-1 (CD54) and T cell LFA-1 (CD11a/CD18) mediate enhanced lysis of IFN-gamma-treated keratinocytes.

Exposure of human KC to IFN-gamma increases their susceptibility to lysis by CTL. The mechanism of this enhanced lysis was investigated by analyzing interactions of IFN-gamma-treated and nontreated cultured KC with allogeneic class I-specific CTL clones. rIFN-gamma treatment augmented KC lysis in a time- and dose-dependent manner. Increased lysis of IFN-KC was detected after only 2 h of IFN-gamma treatment and was maximal by 12 h. Enhanced lysis of IFN-KC was Ag-specific, inasmuch as nonantigenic IFN-KC were not lysed either directly or as bystanders during the lysis of antigenic KC. Parallel immunofluorescence and cytotoxicity assays of KC treated with IFN-gamma for various intervals revealed a direct correlation between the degree of increased KC lysis and levels of cell surface ICAM-1 (CD54), but not of specific alloantigen or beta 2-microglobulin. Lysis of nontreated KC was blocked by mAb against class I or CD3, but not by mAb against ICAM-1 or LFA-1. In contrast, lysis of IFN-KC was partially inhibited by anti-ICAM-1 or anti-LFA-1 mAb, but resisted inhibition by anti-class I mAb except in the presence of anti-ICAM-1. These results indicate that both ICAM-1/LFA-1 and Ag/CD3-TcR interactions are important for Ag-specific lysis of IFN-KC, whereas lysis of nontreated KC depends on Ag/CD3-TcR but not ICAM-1/LFA-1 interactions. Equivalent inhibition of IFN-KC lysis by mAb against ICAM-1 or LFA-1 suggests that ICAM-1 is the only LFA-1 ligand involved in enhanced IFN-KC lysis. Furthermore, enhanced CTL lysis of KC after short-term IFN-gamma treatment can be explained solely on the basis of ICAM-1 induction, because all of the increase in specific lysis associated with IFN-gamma treatment could be blocked by mAb that block ICAM-1/LFA-1 interactions.

Cell Adhesion Molecules↗
PubMed1991

Lysis of keratinocytes by IL-2-activated peripheral blood lymphocytes.

Interleukin 2 (IL-2)-activated peripheral blood mononuclear cells (PBMC) have been reported to lyse tumor cells while essentially sparing normal cells in vitro. This report concerns IL-2-induced anti-keratinocyte (anti-KC) cytotoxic effectors that lyse normal human keratinocytes (KC) in vitro. Effectors were generated by culturing PBMC for 1-8 d in various concentrations of recombinant IL-2 and then assayed against 51Cr-labeled targets. Effectors stimulated with 10(3) U/ml of IL-2 for 8 d readily lysed adherent or trypsinized autologous or allogeneic KC cultured in serum-free medium. Induction of anti-KC effectors was IL-2 dose-dependent, with as little as 12-25 U/ml of IL-2 inducing increased anti-KC activity after 24 h of treatment. Although anti-KC activity was increased after overnight culture in IL-2, maximal effector potency in terms of lytic units (LU) per 10(6) effector cells required 4 d of IL-2 treatment. Maximal effector yield in terms of LU per input PBMC occurred after 8 d of IL-2 treatment. Antibody plus complement depletion studies showed that the anti-KC effectors predominantly have a CD16 --/CD3 --/CD2+ phenotype. A natural killer (NK)-like specificity of the effectors was suggested by two findings: unlabeled K562 cells totally inhibited lysis of 51Cr-KC in cold target competition assays, and interferon gamma (IFN-g) treatment (2.5 U/ml-500 U/ml of recombinant IFN-g for 48-72 h) down-regulated KC susceptibility to lysis by these effectors. Thus, IL-2 treatment of PBMC induces non-T cell, natural killer-like effectors that can lyse both autologous and allogeneic KC. Furthermore, KC resemble other cell types that become resistant to non-MHC-restricted lysis after treatment with IFN-g. Finally, the contrasting effects of IFN-g treatment on KC lysis by these effectors, as opposed to lysis by specific T cells, suggests that IFN-g could promote a shift from non-MHC-restricted to MHC-restricted KC lysis during epidermal immune responses in vivo.

Adult↗
PubMed1990

Evidence for phosphatidylinositol-linked forms of human and avian fibronectin receptors.

Phosphatidylinositol (PI)-linked forms of surface molecules have been hypothesized to mediate the initial stages of cell adhesion or signal transduction. We report evidence for the occurrence of a functional PI-linked subset of cell surface fibronectin receptors (FNR). Treatment of human MG63 osteosarcoma cells or primary chicken embryo fibroblasts (CEF) with PI-specific phospholipase C (PI-PLC) reduced cell surface FNR expression by 30% as detected by immunofluorescence. PI-PLC treatment of cell membranes purified from [35S]methionine-labeled CEF or MG63 cells led to a similar loss of membrane-associated immunoprecipitable FNR from the pelleted membranes, while such treatment led to the appearance of FNR in the supernatant of treated MG63 membranes. Biosynthetic labeling of CEF FNR with [3H]palmitate and [3H]ethanolamine demonstrated the acylation and putative PI linkage of avian FNR subunits. PI-PLC treatment of CEF and MG63 cells also reduced fibronectin-specific adhesion in a short-term in vitro assay, suggesting that the avian and human FNR occur in PI-linked isoforms which appear to contribute to cell adhesion to fibronectin.

Animals↗
PubMed1990

Recognition of keratinocytes by cytotoxic T cells specific for conventional HLA class-I alloantigen.

This study analyzed whether human keratinocytes (KC) express conventional HLA class-I molecules as detected by class-I-specific cytotoxic T lymphocytes (CTL), and whether exposure of KC to interferon-gamma (IFN-g) is required for CTL recognition. Basal KC grown in serum-free medium and exposed to recombinant IFN-g for 24-96 h were used as targets in 51Cr-release assays. Target-cell susceptibilities to lysis were compared by analyzing the lytic unit (LU) activity of a given CTL population against IFN-g-treated and untreated KC. CTL effectors were cloned from alloantigen-primed cultures by limiting dilution in the presence of antigenic B lymphoblastoid cells (BCLL) and IL-2. These T-cell clones lysed appropriate BCLL and PHA blasts but not third-party BCLL or K562. Lysis of antigenic BCLL was specifically blocked by antibodies against CD3 or class-I antigens. Specificity of the clones for conventional class-I antigen was demonstrated by cytotoxicity tests employing a panel of HLA-typed BCLL. The clones specifically lysed KC syngeneic with the original effector immunogen, and lysis was also blocked by anti-class-I antibodies. The effect of IFN-g treatment was to increase KC susceptibility to lysis by these clones. From 3-25 times more LU were measured against IFN-g-treated KC than against nontreated KC, and the degree of enhancement was similar for KC treated with concentrations of IFN-g ranging from 2.5-200 U/ml. This effect of IFN-g treatment on KC lysis by CTL, which was detected after only 24 h at all doses tested, emphasizes the potential role of IFN-g in enhancing CTL-mediated antiviral epidermal immunity and in exacerbating epidermal disease mediated by specific lytic T cells. In addition, the finding that normal human KC can be recognized by MHC class-I-specific CTL demonstrates that KC do express conventional class-I-antigens and that KC lysis by CTL can occur independently of exogenous cytokines.

Adult↗
PubMed1989

Phorbol ester induces increased expression, altered glycosylation, and reduced adhesion of K562 erythroleukemia cell fibronectin receptors.

The human multipotential hematopoietic cell line K562 expresses fibronectin receptor (FNR) subunits of 160 kDa (alpha chain) and 120 kDa (beta chain). Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) led to reduced binding of K562 to immobilized fibronectin (FN), although treated cells expressed 10-fold more cell surface FNR than untreated cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis confirmed this and showed altered electrophoretic mobilities of FNR subunits from TPA-treated cells. TPA treatment affected N-linked glycosylation, as tunicamycin treatment of K562 cells abolished differences in FNR mobility. Sialidase treatment of FNR immunoprecipitates minimized and sialidase treatment of intact cells eliminated these mobility differences between subunits from control and TPA-treated cells. Reduced sialylation of FNR from TPA-treated cells was further demonstrated by chromatography with bead-coupled lectins and by the greater negative charge of untreated K562 FNR subunits in two-dimensional isoelectric focusing-polyacrylamide gel electrophoresis. A relationship between reduced FNR sialylation and reduced FN binding was suggested by adhesion assays of sialidase-treated K562 which showed that desialylation of cell surface FNR was associated with decreased cell adhesion. Thus, TPA treatment reduces the function, increases the expression, and alters the structure of K562 FNR, and these changes appear to involve FNR sialylation.

Cell Adhesion↗
PubMed1989

CDw17: a neutrophil glycolipid antigen regulated by activation.

The plasma membrane and intracellular granules of human polymorphonuclear neutrophils (PMN) contain large amounts of the glycolipid, lactosylceramide (LacCer; Gal beta 1----4Glc beta 1----1Cer). Despite its abundance, novel subcellular distribution, and lineage-restricted expression, nothing of PMN LacCer function is known. We examined the relationship between LacCer and PMN activation by assessing binding of anti-LacCer mAb (T5A7; anti-CDw17) to PMN during and after cell stimulation. CDw17 expression markedly decreased after treatment with PMA, dioctanoylglycerol, calcium ionophore, FMLP (with or without cytochalasin B or added Ca2+), TNF-alpha, or lymphotoxin. Depending on the stimulus, CDw17 declined to levels ranging from 70% (TNF, lymphotoxin) to less than 5% (phorbol ester, dioctanoylglycerol) of levels detected on untreated PMN. Loss of CDw17 from PMA-treated PMN followed dose- and temperature-dependent kinetics, with loss being detected after PMA treatment for 1 min. Membrane internalization explained PMA-induced loss of CDw17, as cell-associated 125I-anti-CDw17 became inaccessible to fluorescent anti-Ig after PMA treatment. CDw17 on PMN cytoplasts or retinoic acid-induced HL-60 cells was only slightly affected by stimulation, suggesting that down-regulation of the epitope is associated with granule exocytosis rather than superoxide production. Results with PMN from a patient with chronic granulomatous disease confirmed that normal superoxide production is not required for CDw17 loss induced by PMA or FMLP treatment. The data collectively demonstrate that reduced levels of cell-surface CDw17 are associated with granule exocytosis after PMN activation.

Antigens, CD↗
PubMed1989

Lymphotoxin, tumor necrosis factor, and gamma interferon are cytostatic for normal human keratinocytes.

The effects of crude lymphokine-enriched supernatants, purified recombinant lymphotoxin (LT), tumor necrosis factor-alpha (TNF), and gamma interferon (gamma IF) on proliferating human keratinocytes were assessed using two in vitro culture systems. Activated splenocyte supernatants inhibited keratinocyte colony growth on fibroblast feeder layers and arrested basal keratinocyte DNA synthesis within 24 h. Purified recombinant LT, TNF, and gamma IF inhibited cell proliferation in serum-free medium without noticeably affecting viability. Cytostasis was dose-dependent (up to 90% with LT or TNF and 99% with gamma IF) and was maximal within 24-36 h. Specific antibodies neutralized TNF- and gamma IF-mediated cytostasis. Combined treatment with LT (or TNF) and gamma IF increased the degree of cytostasis, particularly at low lymphokine concentrations. Maximum inhibition of DNA synthesis and the duration of exposure required for this inhibition were comparable for LT and TNF and differed for gamma IF. Each of these lymphokines induced cell enlargement, flattening, and vesiculation, with gamma IF apparently more potent in this respect than LT or TNF. Fusiform keratinocytes with diffusely distributed cytokeratin were observed after prolonged treatment with gamma IF alone or gamma IF plus either LT or TNF. Flow cytometric studies of lymphokine-treated keratinocytes indicated that LT, TNF, and gamma IF could enhance beta-2 microglobulin expression 1.5-fold to threefold, whereas only gamma IF induced class II antigens. Staining for class II and beta-2 microglobulin was reduced on cells treated with high concentrations of gamma IF compared with either optimally treated or untreated cells. The potential relevance of these findings to cutaneous immune defense and disease is discussed.

Antineoplastic Agents↗
PubMed1979

Rapid magnetic purification of rosette-forming lymphocytes.

High gradient magnetic separation, which as previously been shown effective in extracting erythrocytes from a flowing cell suspension, has been used to separate rosetted and unrosetted human peripheral blood lymphocytes. The hemoglobin in the sheep red cells used to form rosettes was first oxidizied to the paramagnetic methemoglobin form. Samples of 50 x 10(6) lymphocytes could be processed in 10 min under sterile conditions with greater than 90% purity of the rosetted cell fraction and maintenance of T cell function in mixed lymphocyte cultures.

Animals↗