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D L Simmons

Publications and source records attributed to D L Simmons.

At least 73 records · Page 4Linked to original sources
PubMed1995

The role of ICAM expression in immunity and disease.

The ICAM family consists of three members. All are ligands for LFA1, and ICAM1 can also bind Mac1. However, the complete repertoire of other ligands has not yet been defined. The ICAMs are not functionally redundant, since they have quite distinct expression profiles and can bind LFA1 differentially. ICAM3 is constitutively and abundantly expressed by all leucocytes, whereas ICAM1 and ICAM2 are expressed at very low levels. ICAM1 is rapidly upregulated by inflammatory cytokines. A current working model for the interplay of the ICAMs is that ICAM3 is the major ligand for LFA1 in the initiation of immune responses and that ICAM1 takes over after these early stages. ICAM3 not only functions as an adhesion molecule, but also is clearly emerging as a potent signalling molecule.

Animals↗
PubMed1994

Normal human tissues, in addition to some tumors, express multiple different CD44 isoforms.

At least 20 different isoforms of the human CD44 lymphocyte-homing receptor/hyaluronan receptor have been described to date that arise from the differential splicing of up to 10 alternative exons (termed v1-v10) encoding the membrane-proximal extracellular domain. Although numerous analyses at the mRNA level have indicated tissue-specific expression of CD44 variants, few analyses have been performed at the protein level because of limited availability of suitable monoclonal antibodies. Recently, however, exon-specific monoclonal antibodies have been generated using bacterial fusion proteins, and these have been reported to detect high levels of vCD44 containing the v6 exon on human tumors. Together with earlier evidence linking this particular exon with tumor metastasis in the rat, these latter experiments have led to the interpretation that v6 splice variants play a causative role in tumor dissemination. In this paper we describe the use of a new and comprehensive panel of CD44 exon-specific monoclonal antibodies generated against a recombinant CD44(v3-10)-immunoglobulin chimera to study vCD44 expression in a large number of normal and neoplastic tissues. We show that the expression of vCD44 varies greatly among different human tumors and that some express either very low levels of vCD44 or no CD44 at all. Furthermore, we demonstrate that expression is not limited to isoforms containing the v6 exon but includes variants carrying v3, v4/5, and v8/9. Additionally, normal epithelial tissues are shown to express considerable levels of these same vCD44 isoforms. Such results argue against a ubiquitous role for vCD44 isoforms in promoting tumor growth and metastasis.

Antibodies, Monoclonal↗
PubMed1994

The N-domain of the biliary glycoprotein (BGP) adhesion molecule mediates homotypic binding: domain interactions and epitope analysis of BGPc.

The biliary glycoproteins (BGPs) represent a group of at least eight differentially spliced molecules belonging to the carcinoembryonic antigen (CEA) subgroup of the CEA family. These molecules are recognized by the CD66 monoclonal antibodies (MoAbs) and function as homotypic and heterotypic adhesion molecules. The extracellular region of the BGPc splice variant comprises an N-terminal IgV-like domain and three IgC2-set domains (A1, B1, and A2). Using soluble recombinant BGP domain variants, we demonstrate in this report that the N-terminal domain mediates homotypic adhesion. Furthermore, this adhesion is both temperature- and cation-dependent. The soluble domain variants of BGP are ideal molecules for epitope mapping. Using these constructs, we have mapped 11 MoAbs that react with the CEA family to different domains of BGPc and have shown that the CD66 MoAbs, YTH71.3.2 and CLBgran 10 (M38), recognize epitopes in the N-terminal domain.

Animals↗
PubMed1994

CD66 identifies the biliary glycoprotein (BGP) adhesion molecule: cloning, expression, and adhesion functions of the BGPc splice variant.

Within the hematopoietic lineage, the monoclonal antibody (MoAb) CD66 reacts with cells of the granulocyte lineage, but not with the majority of progenitor cells from human bone marrow. Our previous studies have shown that CD66 binds specifically to at least three carcinoembryonic antigen (CEA) superfamily members, ie, CEA itself, nonspecific cross-reacting antigen (NCA), and CGM1, but not to CGM6 (NCA-95). In this report, we show that CD66 will also identify the biliary glycoproteins (BGP). A full-length cDNA for the BGPc molecule (a cytoplasmic splice variant of BGPa) was isolated by expression cloning using the CD66 MoAbs. This protein has an identical extracellular and transmembrane sequence to BGPa with one N-terminal IgV like domain, three IgC-like extracellular domains (A1, B1, and A2), plus a transmembrane domain, but the cytoplasmic domain is spliced by 53 nucleotides. Reverse transcriptase-polymerase chain reaction experiments show that this splice variant can be detected in colonic carcinoma cell lines, in primary colonic adenocarcinomas, and in myeloid and B-cell lines to varying degrees. Quantitative analyses of BGPc RNA expression by RNase protection indicate that abundant levels occur only in the colonic, but not in the hematopoietic, cell lines tested. Studies presented here show that BGPc mediates homotypic adhesion and suggest that the cytoplasmic splicing does not alter the initial homotypic adhesion properties of BGPa.

Amino Acid Sequence↗
PubMed1994

Distinct binding of T lymphocytes to ICAM-1, -2 or -3 upon activation of LFA-1.

LFA-1 (CD11a/CD18) mediates leukocyte adhesion by binding to one of its ligands: ICAM-1, ICAM-2 or ICAM-3. Here, we investigated whether stimuli known to induce adhesion to ICAM-1 were also capable of inducing LFA-1-mediated adhesion of T lymphocytes to ICAM-2 and -3 transfectants. We observed that phorbol 12-myristate 13-acetate, Mn2+, cross-linking of CD3 or activating antibodies against LFA-1 enhanced LFA-1-mediated T cell adhesion to ICAM-2 and -3, although to a lesser extent than to ICAM-1. These results indicate that, similar to what has been reported for adhesion to ICAM-1, activation of LFA-1 is also required for adhesion to ICAM-2 and -3. Furthermore, the results suggest that ICAM-1 is the major ligand for LFA-1 on activated T lymphocytes. Interestingly, we observed that in contrast to activating antibodies against CD18, activating antibodies against CD11a were incapable of inducing adhesion of LFA-1 to all three ligands. The antibody MEM-83 stimulated binding to ICAM-1, while at the same time inhibiting the interaction of LFA-1 with ICA8M-2 and -3. The antibody NKI-L16 selectively induced adhesion to ICAM-1 and -2, but not to ICAM-3. Our results suggest that different conformations of LFA-1 are required to support adhesion to ICAM-1, -2 or -3, and that ligands may bind on different sites of the LFA-1 molecule.

Animals↗
PubMed1994

The human intercellular adhesion molecule 3 (ICAM3) gene is located in the 19p13.2-p13.3 region, close to the ICAM1 gene.

The chromosomal location of the intercellular adhesion molecule 3 (ICAM3) gene, coding for a lymphocyte function-associated antigen (LFA)-1 counterreceptor and selectively expressed by human leukocytes, was analyzed by in situ hybridization with the cDNA coding sequence as a probe. This sequence mapped to the p13.2-p13.3 region of chromosome 19, close to the ICAM1 gene chromosomal location.

Animals↗
PubMed1994

Involvement of the "I" domain of LFA-1 in selective binding to ligands ICAM-1 and ICAM-3.

To analyze the binding requirements of LFA-1 for its two most homologous ligands, ICAM-1 and ICAM-3, we compared the effects of various LFA-1 activation regimes and a panel of anti-LFA-1 mAbs in T cell binding assays to ICAM-1 or ICAM-3 coated on plastic. These studies demonstrated that T cell binding to ICAM-3 was inducible both from the exterior of the cell by Mn2+ and from the interior by an agonist of the "inside-out" signaling pathway. T cells bound both ICAM ligands with comparable avidity. A screen of 29 anti-LFA-1 mAbs led to the identification of two mAbs specific for the alpha subunit of LFA-1 which selectively blocked adhesion of T cells to ICAM-3 but not ICAM-1. These two mAbs, YTH81.5 and 122.2A5, exhibited identical blocking properties in a more defined adhesion assay using LFA-1 transfected COS cells binding to immobilized ligand. Blocking was not due to a steric interference between anti-LFA-1 mAbs and N-linked carbohydrate residues present on ICAM-3 but not ICAM-1. The epitopes of mAbs YTH81.5 and 122.2A5 were shown to map to the I domain of the LFA-1 alpha subunit. A third I domain mAb, MEM-83, has been previously reported to uniquely activate LFA-1 to bind ICAM-1 (Landis, R. C., R. I. Bennett, and N. Hogg. 1993. J. Cell Biol. 120:1519-1527). We now show that mAb MEM-83 is not able to stimulate binding of T cells to ICAM-3 over a wide concentration range. Failure to induce ICAM-3 binding by mAb MEM-83 was not due to a blockade of the ICAM-3 binding site on LFA-1. This study has demonstrated that two sets of functionally distinct mAbs recognizing epitopes in the I domain of LFA-1 are able to exert differential effects on the binding of LFA-1 to its ligands ICAM-1, and ICAM-3. These results suggest for the first time that LFA-1 is capable of binding these two highly homologous ligands in a selective manner and that the I domain plays a role in this process.

Antibodies, Monoclonal↗
PubMed1994

Human leukemia inhibitory factor inhibits development of experimental atherosclerosis.

The effect of human leukemia inhibitory factor (hLIF) on the development of atherosclerosis was investigated in an experimental animal model. Two conditions were examined: one in which lesions could arise because of the influence of both "injury" (cuffed vessel) and diet and one in which only the effect of diet could be significant in other areas of the vasculature (aorta). At time zero, the right carotid artery of rabbits (n = 32) was ensheathed in a soft Silastic cuff, and an osmotic minipump (2-mL capacity; 2.5 microL/h; 28 days) containing either hLIF or saline was inserted into the peritoneal cavity. Rabbits were divided into four groups (n = 8): group 1 received normal diet/saline; group 2, normal diet/LIF (30 micrograms.kg-1.d-1); group 3, 1% cholesterol diet/saline; and group 4, 1% cholesterol diet/LIF (30 micrograms.kg-1.d-1). After 28 days, the cholesterol diet (group 3) resulted in a sixfold increase in plasma cholesterol level compared with group 1 rabbits on a normal diet (3.80 +/- 0.50 versus 0.55 +/- 0.01 mmol/L). This was significantly lower (P = .01) with hLIF treatment in group 4 rabbits (2.80 +/- 0.44 mmol/L). Group 2 rabbits had higher aortic tissue cholesterol levels (1.40 +/- 0.35 mg/g) compared with group 1 rabbits on a normal diet (0.10 +/- 0.06 mg/g) (P = .01), whereas hLIF treatment decreased tissue cholesterol levels by 60% in group 4 rabbits (0.60 +/- 0.05 mg/g) (P = .01). Group 3 rabbits developed lipid-filled lesions covering 63.25 +/- 17.66% of the thoracic aorta surface, whereas lesions were significantly reduced (9.88 +/- 8.79%) (P = .01) with LIF treatment (group 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes.

The platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), defined by the CD31 monoclonal antibody (MoAb), was initially described as a cell-cell adhesion molecule mediating both homotypic and heterotypic adhesion. In this report, we show that enriched CD34+ human hematopoietic progenitor cell populations, containing early myeloid, erythroid, and multipotential progenitor cells, are CD31+. Analyses of CD34+ cell lines representing early myeloid, multipotential, and pre-pre-B-lymphoid progenitors indicate that precursors of both myeloid and B-lymphoid cells express PECAM-1 at high levels. Three-color flow-cytometric analyses also show that normal human bone marrow CD31+ CD34+ subsets coexpress myeloid (CD33) or B-lymphoid (CD19, CD10) markers. Except for the monocytic cell line, U937, all CD34- cell lines tested, which represent more mature stages of the myeloid, erythroid, and lymphoid lineages, expressed substantially lower or negligible levels of PECAM-1. Western blotting studies indicated that the CD31 MoAb, JC/70A, detected molecules in the 120- to 140-kD molecular weight range on the monocytic CD34- CD33+ CD31+ cell line, U937; on the CD34+ CD31+ CD33+ CD19- multipotential/lymphomyeloid precursor cell lines, KG1 and KG1B; on the CD34+ CD31+ CD19+ CD10+ CD33- precursor pre-pre-B-cell line, MIK-ALL; and on a CD34(+)-enriched precursor cell population from normal human bone marrow. A single molecular weight species was generally observed with enriched membrane preparations, whereas two PECAM-1 molecules were present in whole-cell lysates of cell lines and the CD34+ bone marrow cell subset. Preliminary studies show that a proportion of the PECAM-1 molecules on the lymphomyeloid/multipotential progenitor cell line, KG1, and on the monocytic cell line, U937, binds to heparin-sepharose. A soluble form of PECAM-1 also binds heparin-sepharose. The high level of expression of PECAM-1 on CD34+ cells suggests that this glycoprotein may function as a heterotypic adhesion molecule, possibly mediating multipotential, myeloid, and early-B-lymphoid precursor cell interactions with stromal cells and extracellular matrix molecules via heparan sulfate proteoglycans. It may also act as a homotypic adhesion molecule by interacting with PECAM-1 on bone marrow stromal macrophage-like cells and endothelial cells or on endothelial cells during stem/progenitor cell migration. Thus, this molecule has the potential importance of directing both lineage commitment and trafficking of early hematopoietic progenitor cells.

Adult↗
PubMed1993

Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions.

Prostaglandin endoperoxide synthase isoform 2 (PGS-2) mRNA and protein are transiently induced by gonadotropins in granulosa cells of preovulatory follicles prior to ovulation. To better understand the hormonal regulation of the rat PGS-2 (rPGS-2) gene in these cells, genomic clones containing rPGS-2 as well as up to 6 kilobases of 5'-flanking DNA were isolated by screening a rat liver genomic library with a labeled 5'-fragment of the mouse PGS-2 cDNA. Primer extension analysis using ovarian follicular mRNA identified the presence of a single rPGS-2 transcription initiation site located 144 nucleotides upstream of the ATG translation initiation codon. To test for promoter activity within the 5'-flanking region of the rPGS-2 gene, a genomic fragment, -2698/32 (1 = cap site), as well as a series of 5'-deletion mutants, were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into primary cultures of granulosa cells. Forskolin (7.5 microM), follicle-stimulating hormone (500 ng/ml) and luteinizing hormone (500 ng/ml) induced CAT activity following transfection with the -2698/32PGS.CAT, whereas gonadotropin-releasing hormone (10(-6) M) and interleukin-1 beta (30 ng/ml) had no effect. Deletion mutants delineated the region spanning from -192 to -54 of the transcription start site to be essential for both basal and forskolin-regulated expression of the reporter gene. The same DNA fragment (-192/-54) exhibited specific binding to granulosa cell nuclear extract proteins as analyzed by electrophoretic mobility shift assays. Additional specific bands were observed in extracts prepared from granulosa cells exposed to an ovulatory dose of gonadotropin. Collectively, these results provide the first structural and functional evidence that the transcriptional regulation of the rat PGS-2 gene by gonadotropins and forskolin in granulosa cells involves 5'-flanking DNA sequences, specifically a region between -192 and -54 of the transcription initiation site.

Animals↗
PubMed1993

Macrosialin, a mouse macrophage-restricted glycoprotein, is a member of the lamp/lgp family.

Macrosialin is a heavily glycosylated transmembrane protein of 87-115 kDa, highly and specifically expressed by mouse tissue macrophages, and to a lesser extent by dendritic cells. We have isolated cDNA clones encoding macrosialin from a thioglycollate-elicited peritoneal macrophage cDNA library by transient expression in COS cells and panning with the anti-macrosialin monoclonal antibody FA/11. A single 1.3-kilobase macrosialin transcript was detected in both untreated and phorbol 12-myristate 13-acetate-stimulated RAW cells. The cDNA sequence predicts a type I integral membrane protein of 326 residues with a heavily glycosylated extracellular domain of 306 residues containing nine potential N-linked glycosylation sites and numerous potential O-linked glycosylation sites. The extracellular domain consists of two distinct regions, separated by an extended 12 residue proline-rich hinge; a membrane-distal mucin-like domain of 89 residues containing short peptide repeats and consisting of 44% serine and threonine residues; and a membrane proximal domain of 170 residues, which has significant sequence homology to a family of lysosomal associated glycoproteins known as the lamp-1 group. Macrosialin is the murine homologue of the human macrophage glycoprotein CD68 (72% identity, 80% similarity). Both proteins are preferentially expressed by macrophages and share the same bipartite structure having a mucin-like domain and a domain common to the lamp family. Macrosialin and CD68 are the first examples of a lamp family protein with a restricted cell-type-specific expression. They may have evolved from the lamps to carry out specialized functions in dedicated phagocytic cells.

Amino Acid Sequence↗
PubMed1993

Structure of the human 3-methyladenine DNA glycosylase gene and localization close to the 16p telomere.

We recently reported the presence of four genes lying between the human alpha-globin gene cluster and the telomere of the short arm of chromosome 16 (16p). We now report that one of these genes encodes 3-methyladenine DNA glycosylase, an enzyme important in the repair of DNA after damage by alkylating agents. The gene comprises five exons, representation of which differs in independently isolated cDNA clones. Although the gene is widely expressed, the abundance of its mRNA is considerably higher in a colon adenocarcinoma cell line (HT29) than in other cell lines that were tested. The major positive erythroid-specific regulatory element controlling alpha-globin gene expression lies equidistant between the promoters of the alpha-globin genes and the 3-methyladenine DNA glycosylase gene. Interestingly, in contrast to the alpha-globin genes, expression of the 3-methyladenine DNA glycosylase gene is not influenced by the regulatory element in the human erythroleukemia cell line K562.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins.

CD68 is a 110-Kd transmembrane glycoprotein of unknown function highly expressed by human monocytes and tissue macrophages. We have isolated cDNA clones encoding CD68 from a U937 cDNA library by transient expression in COS cells and panning with the anti-CD68 monoclonal antibodies (MoAbs) Y2/131, Y1/82A, EBM11, and Ki-M6. CD68 transcripts are constitutively present in the promonocyte cell line U937 and are upregulated by phorbol myristic acid (PMA). By contrast, CD68 transcripts are absent or present at very low levels in many hematopoietic lines including KG1, CEM, and K562, but can be induced by exposure to PMA. The cDNA sequence predicts a type I integral membrane protein of 354 residues with a heavily glycosylated extracellular domain of 298 residues containing nine potential N-linked glycosylation sites and numerous potential O-linked glycosylation sites. The extracellular domain consists of two distinct regions separated by an extended proline hinge: a membrane-distal mucin-like domain containing short peptide repeats and consisting of 54% serine and threonine residues; and a membrane proximal domain that has significant sequence homology to a family of lysosomal/plasma membrane shuttling proteins known as the lamp 1 group. CD68 is a member of a growing family of hematopoietic mucin-like molecules, including leukosialin/CD43, the stem cell antigen CD34, and the lymph node high endothelial ligand for L-selectin GlyCAM-1.

Amino Acid Sequence↗
PubMed1993

Structural determination and promoter analysis of the chicken mitogen-inducible prostaglandin G/H synthase gene and genetic mapping of the murine homolog.

We have previously reported the isolation and characterization of a new form (PGHS-2) of prostaglandin G/H synthase (PGHS, cyclooxygenase) from chicken embryo fibroblasts. To further study the regulation and structure of the gene, we have cloned the entire chicken PGHS-2 (previously termed miPGHSch) gene with its 5'-flanking region from a chicken genomic library. A genomic Southern blot showed the existence of a single PGHS-2 gene. The size of the gene was estimated at 8.9 kb through DNA sequencing and polymerase chain reaction analysis. The PGHS-2 gene was found to contain 10 exons, giving it a structure similar to that of the human PGHS-1 and murine PGHS-2 genes. The transcription start site was determined by primer extension, and the nucleotide sequence of 1.6 kb of the 5'-flanking region immediately upstream of the transcription start site was determined. The promoter sequence contained a TATA box and a variety of enhancer elements, including a serum response element, an AP-1, an NF-kappa B, and several SP-1 and AP-2 sites. Chloramphenicol acetyltransferase (CAT) assays showed that the first 158 nucleotides of the promoter efficiently drove transcription of the CAT reporter gene in serum-stimulated cells. Dexamethasone, a potent inhibitor of prostaglandin synthesis, had no effect on CAT activity, although this drug is known to markedly decrease PGHS-2 mRNA in vivo. This suggests that dexamethasone may inhibit PGHS-2 mRNA expression at the post-transcriptional level. Analysis of hamster/mouse somatic cell hybrids with radiolabeled cDNA probes demonstrated that PGHS-1 mapped to chromosome 2 and PGHS-2 mapped to chromosome 1 of the mouse genome.

3T3 Cells↗
PubMed1993

Prostaglandin G/H synthase isoenzyme 2 expression in fibroblasts: regulation by dexamethasone, mitogens, and oncogenes.

Mitogens increase prostaglandin synthesis in fibroblasts. In the present studies, transcriptional activation of the prostaglandin G/H synthase isoenzyme 2 (PGHS-2) gene is shown to be responsible for this induction. Transcription of the PGHS-2 gene maximally increased within 15 min of stimulation, and elevated cellular PGHS-2 mRNA, protein, and prostaglandin synthase activity were detected shortly thereafter. Pulse-chase experiments showed induced isoenzyme 2 is short lived, with a half-life of 22 min in chicken embryo fibroblasts. Neoplastic transformation of fibroblasts by a variety of oncogenes induced either or both PGHS-1 and PGHS-2 mRNAs in immortalized murine NIH3T3 cells. Some preference of oncogenes such as v-src to induce PGHS-2 mRNA has been previously observed. However, in this study exceptions in which v-src did not induce PGHS-2 mRNA were noted. Analysis of independent cell lines transformed by v-fes showed this oncogene induced both PGHS-1 and PGHS-2. Dexamethasone, a synthetic glucocorticoid, selectively decreased both basal and induced levels of PGHS-2 mRNA. Simultaneous addition of the steroid with mitogen reduced mitogen-induced PGHS-2 mRNA levels by 80%. However, nuclear run-on experiments failed to detect any decrease in PGHS-2 mRNA transcription. This suggested dexamethasone rapidly caused PGHS-2 mRNA destabilization. Cycloheximide blocked PGHS-2 mRNA decrease. A fibroblast cell line, RS2, was identified that contained only isoenzyme 2 and possessed PGHS activity that was more highly mitogen-inducible than in any other cell line yet described. Mitogen-inducible activity in RS2 cells was inhibited by aspirin, indicating that PGHS-2, like PGHS-1, can be inactivated by this drug.

3T3 Cells↗
PubMed1993

Dissecting the modes of interactions amongst cell adhesion molecules.

The process of cell adhesion can be mediated by more than 50 molecules. Fortunately, most of these can be grouped into a small number of superfamilies. For example, more than half of all leukocyte adhesion molecules are members of the immunoglobulin superfamily. The principles of cell-cell adhesion are reviewed including: kinetics and equilibria; on/off rates; affinities/avidities; homotypic/heterotypic interactions; mapping and delineation of binding sites. These principles are illustrated with two CAMs: firstly the interaction of the homotypic epithelial/myeloid adhesins CD66, and the endothelial adhesin, CD31, and secondly the heterotypic adhesins ICAM-1, 2 and 3, which interact with the leukocyte integrin LFA-1.

Antigens, CD↗