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M L Hibbs

Publications and source records attributed to M L Hibbs.

35 records · Page 2Linked to original sources
PubMed1991

Regulation of adhesion of ICAM-1 by the cytoplasmic domain of LFA-1 integrin beta subunit.

Interactions between cytotoxic lymphocytes and their targets require the T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). LFA-1 is not constitutively avid for its counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. Cross-linking of the TCR transiently converts LFA-1 to a high avidity state and thus provides a mechanism for regulating cellular adhesion and de-adhesion in an antigen-specific manner. Truncation of the cytoplasmic domain of the beta, but not the alpha, subunit of LFA-1 eliminated binding to ICAM-1 and sensitivity to phorbol esters. Thus, LFA-1 binding to ICAM-1 was found to be regulated by the cytoplasmic domain of the beta subunit of LFA-1.

Amino Acid Sequence↗
PubMed1990

On the species specificity of the interaction of LFA-1 with intercellular adhesion molecules.

Species restrictions in immune cell interactions have been demonstrated both in Ag-specific responses of T lymphocytes and the phenomenon of natural attachment. To determine the possible contribution of adhesion receptors to these restrictions, we have studied binding between the murine and human homologues of LFA-1 (CD11a/CD18) and ICAM employing purified human LFA-1 and ICAM-1 (CD54) bound to solid substrates. Murine cell lines bind to purified human LFA-1 through ICAM-1 and at least one other counter-receptor. This provides evidence for multiple counter-receptors for LFA-1 in the mouse as well as in the human. In contrast to binding of murine ICAM-1 to human LFA-1, murine LFA-1 does not bind to human ICAM-1. The species specificity maps to the LFA-1 alpha subunit, because mouse x human hybrid cells expressing the human alpha subunit associated with a mouse beta subunit bind to human ICAM-1, whereas those with a human beta subunit associated with a murine alpha subunit do not. Increased adhesiveness for ICAM-1 stimulated by phorbol esters could be demonstrated for hybrid LFA-1 molecules with human alpha and murine beta subunits.

Animals↗
PubMed1990

Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

Amino Acid Sequence↗
PubMed1990

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.

Antibodies, Monoclonal↗
PubMed1990

The leukocyte integrin LFA-1 reconstituted by cDNA transfection in a nonhematopoietic cell line is functionally active and not transiently regulated.

The functional activity of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes can be regulated by T-cell receptor (TCR) stimulation and pharmacologic agents. It was of interest to determine if functionally active LFA-1 could be reconstituted on a nonhematopoietic, LFA-1-negative cell line. We report the expression of LFA-1 and diethylaminoethyl (DEAE) Mac-1 alpha beta heterodimers on the cell surface of a fibroblastoid cell line, COS, by DEAE dextran cotransfection of the alpha and beta subunit cDNAs. Immunoprecipitation studies demonstrated that the alpha and beta subunit was expressed in heterodimers. The alpha or beta subunit was expressed at lower levels after transfection with the alpha or beta subunit cDNA alone. Cotransfection of the alpha and beta subunit cDNAs, but not transfection of alpha or beta alone, was sufficient to reconstitute intercellular adhesion molecule-1 (ICAM-1) binding activity. Consistent with this observation, LFA-1 on the fibroblastoid cells possesses the activation epitope defined by the L16 monoclonal antibody (mAb). This epitope marks the conversion of LFA-1 from the low to high avidity state on peripheral blood T lymphocytes (PBLs) and is constitutively present on activated cell lines. In contrast to LFA-1 on leukocytes, the functional activity of LFA-1 on fibroblastoid cells was not influenced by phorbol ester treatment. Furthermore, the use of agents that interfere with intracellular signaling, a protein kinase C inhibitor, cAMP analogue, or the combination of a phosphodiesterase inhibitor and adenyl cyclase activator, did not affect the binding of COS cells expressing LFA-1 to purified ICAM-1.

Alkaloids↗
PubMed1990

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function.

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.

Animals↗
PubMed1989

Mechanisms for regulating expression of membrane isoforms of Fc gamma RIII (CD16).

Granulocyte and natural killer (NK) cell Fc receptors for immunoglobulin G (CD16) differ in only a few amino acids, yet have phosphatidylinositol glycan (PIG) or polypeptide membrane anchors, respectively. Mutagenesis shows that anchoring is regulated by a serine residue near the PIG anchor attachment site in the extracellular domain. The NK cell isoform was not expressed on the surface of COS cells unless cotransfected with a subunit that was expressed in NK cells and that was identical to the gamma subunit of the high affinity IgE Fc receptor (Fc epsilon RI). However, the CD16 sequence and not expression of the gamma subunit is dominant in regulating PIG reanchoring.

Animals↗
PubMed1989

Natural killer cell and granulocyte Fc gamma receptor III (CD16) differ in membrane anchor and signal transduction.

CD16 is a low affinity Fc gamma R III expressed on granulocytes, macrophages and large granular lymphocytes, the mediators of antibody-dependent cellular cytotoxicity and NK. The occupancy of CD16 by aggregated IgG on large granular lymphocytes induces expression of activation markers, release of inflammatory mediators and triggering of effector functions such as antibody-dependent cellular cytotoxicity. Recently we and others described that CD16 is anchored to the membrane of granulocytes via a phosphatidylinositol glycan moiety. Here we show that the CD16 molecule expressed on NK cells, cultured monocytes, and lung macrophages is not phosphatidylinositol glycan moiety anchored. It is not released with phosphatidylinositol-specific phospholipase C, and after removal of N-linked carbohydrate is 5 to 7 kDa larger than the granulocyte CD16 molecule, strongly suggesting the presence of transmembrane and cytoplasmic protein domains. Redirected killing of hybridoma targets expressing anti-CD16 surface Ig shows that NK cell CD16 is unable to do so. These findings demonstrate that NK cell and granulocyte CD16 have different membrane anchors and indicate that the type of membrane anchor is an important biologic mechanism for regulating the functional capacity of surface receptors.

Antigens, Differentiation↗
PubMed1988

The structure of the murine Fc receptor for IgG. Assignment of intrachain disulfide bonds, identification of N-linked glycosylation sites, and evidence for a fourth form of Fc receptor.

The Fc receptor (Fc gamma R) of the murine macrophage cell line, J774, was purified by immunoaffinity chromatography then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequencing. FcR material judged to be pure by these criteria was digested with a number of enzymes to identify the cysteine residues engaged in disulfide bonds within the native structure. The results clearly establish that the mouse macrophage Fc gamma R contains two intrachain disulfide bonds, each of which connects adjacent cysteine residues within the two putative extracellular domains of the molecule. In addition, each disulfide-bonded domain was shown to contain two authentic sites of N-linked glycosylation. Extensive peptide sequencing resulted in the unexpected identification of peptide fragments from a fourth Fc gamma R whose sequences were highly homologous to sequences surrounding the two Cys residues in the amino-terminal domain of both alpha and beta 1 Fc gamma R. The fourth Fc gamma R contains a disulfide-bonded amino-terminal domain similar to beta 1 Fc gamma R.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of a human immunoglobulin G Fc receptor.

Human IgG Fc receptor (Fc gamma R) cDNA clones were isolated by cross-species hybridization by probing cDNA libraries with the low-affinity Fc gamma R beta 1 cDNA clone from mouse as well as a pool of oligonucleotides constructed from the nucleotide sequence of this Fc gamma R. Three cDNA clones were isolated and analysis of the predicted amino acid sequence indicated that the human Fc gamma R protein is synthesized with a 34-amino acid leader and the mature protein is composed of 281 amino acids. The extracellular region of this Fc gamma R was divided into two domains, which were very similar to each other and to the corresponding regions of both mouse alpha and beta Fc gamma Rs and showed a clear relationship to immunoglobulin variable regions. One possible N-linked glycosylation site was found in each of the extracellular domains. The human Fc gamma R leader sequence was shown to be similar to the mouse alpha Fc gamma R leader sequence, but the transmembrane region was most similar to the mouse beta 1 Fc gamma R. The intracellular domain of the human Fc gamma R was surprisingly different from both mouse Fc gamma Rs. RNA blot analysis of human cells demonstrated two transcripts (2.5 and 1.5 kilobases) that arise by use of different adenylylation signals. The cellular expression of these transcripts suggest that they encode the low-affinity p40 Fc gamma R protein.

Amino Acid Sequence↗
PubMed1987

The mouse Fc receptor for IgG (Ly-17): molecular cloning and specificity.

A cDNA clone encoding the mouse Ly-17+ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional Fc gamma R investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG gamma 1/2b and rabbit IgG complexes.

Amino Acid Sequence↗
PubMed1986

The murine Fc receptor for immunoglobulin: purification, partial amino acid sequence, and isolation of cDNA clones.

The murine Fc receptor for IgG (Fc gamma R) was purified to homogeneity by immunoaffinity chromatography from detergent lysates of the macrophage cell line J774. Microsequencing of intact protein yielded a single amino-terminal sequence, which was confirmed and extended to 20 residues by the isolation of an overlapping peptide. The isolation of additional proteolytic fragments obtained by using Staphylococcus aureus V8 protease, cyanogen bromide, and lysine C proteinase, facilitated sequence analysis of a total of 119 amino acid residues. Codon usage charts were used to construct oligonucleotide probes based on the amino acid sequences of three nonoverlapping peptides. These probes were used to screen a cDNA library derived from the WEHI-3B myelomonocytic cell line, and a single cDNA clone (pFc24) to which all three probes hybridized was isolated. This clone, containing a 1.02-kilobase cDNA insert, has been characterized by restriction mapping and partial DNA sequencing, and it has been shown to encode the Fc gamma R. The sequence at the 5' end of the clone contained the coding information for the amino-terminal sequence of the Fc gamma R as well as a putative 13-amino acid signal sequence. The 3' end of the clone encoded a peptide identified in purified receptor preparations. Thus, the presence of coding information at the 5' and 3' ends of this clone suggests that full-length Fc receptor cDNA spans greater than 1 kilobase.

Amino Acid Sequence↗
PubMed1985

Gm-3.2, a new granulocyte/macrophage alloantigen.

A monoclonal antibody defining a unique mouse neutrophil cell-surface antigen, Gm-3.2, is described. Gm-3.2 is found on all neutrophils in peritoneal exudates and in bone marrow, and is also present on macrophages activated by thioglycolate but is absent from lymphoid, kidney, liver, heart, and red cells. Gm-3.2 is a differentiation antigen of myeloid cells, as granulocyte/macrophage colony-forming cells are Gm-3.2- while mature neutrophils are Gm-3.2+. Strain distribution pattern analysis shows linkage of the Gm-3 locus to the Ly-4, B2m, H-3 complex on chromosome 2.

Animals↗
PubMed1985

The mouse Ly-17 locus identifies a polymorphism of the Fc receptor.

The mouse Ly-17.2 alloantigen has recently been defined with both conventional and monoclonal antibodies; it identifies a locus, sited on chromosome 1, the products of which were considered to be specific for B cells. Using another Ly-17.2-specific monoclonal antibody (described herein), the tissue distribution of the Ly-17.2 antigen was shown to extend to a subpopulation of T lymphocytes and to neutrophils. This distribution is remarkably similar to that of the Fc receptor for immunoglobulin. Indeed, we now demonstrate that the Ly-17 locus codes for a polymorphism of the Fc receptor, a conclusion based upon (a) an identical tissue distribution of Ly-17.2 and FcR on both normal and tumor tissue; (b) specific inhibition of EA rosette formation by F(ab')2 fragments of anti-Ly-17.2; (c) inhibition of the binding of the 2.4G2 monoclonal rat antimouse Fc receptor antibody by Ly-17.2 antibody; (d) precipitation of an identical series of molecules by our Ly-17.2-specific antibody and by the recognized Fc receptor-specific antibody (2.4G2); and (e) the demonstration by coprecipitation that the Ly-17.2 specificity is present on Fc receptor molecules. The studies suggest that the xenogeneic monoclonal antibody (2.4G2) which recognizes an invariant site on the FcR molecule and the polymorphic site are closely associated. In addition, the studies firmly map a gene coding for or regulating the expression of the FcR to chromosome 1.

Alleles↗
PubMed1984

Monoclonal antibody to murine neutrophils: identification of the Gm-2.2 specificity.

A neutrophil-specific alloantigen (Gm-2.2) was defined by a monoclonal antibody, 5119-4/7. The Gm-2.2 antigen is found only on bone marrow neutrophils or calcium caseinate-induced neutrophils and is absent from all lymphoid cells examined as well as adherent thioglycollate-induced peritoneal exudate cells and the nonhemopoietic tissues, kidney, liver, heart, brain, and red blood cells. Furthermore, unlike mature neutrophils, granulocyte/macrophage progenitor cells are Gm-2.2-, suggesting that Gm-2.2 is a differentiation antigen for the neutrophil series. The Gm-2 locus is linked to, but distinct from, the Ly-6 locus.

Animals↗
PubMedDate not supplied

The cell surface phenotype of mouse neutrophils.

Using monoclonal antibodies, mouse peritoneal neutrophils were typed for the presence of 23 cell surface alloantigens, the expression of which was quantitated by flow cytofluorometry and compared with that of lymphocytes. The H-2K and H-2D alloantigens and beta 2-microglobulin were present on all neutrophils, but Ia and Qa antigens were not detected. It was found that Ly-5.1, Ly-15.2, Ly-21.2, Ly-24.2 (Pgp-1) and Ly-25.1 were present on greater than 90% of neutropils; Ly-6.2 and Ly-27.2 were absent, but Ly-28.2 (encoded by an Ly-6 linked gene), was present on greater than 90% of neutrophils. As expected, the lymphocyte-specific antigens Ly-1.1, Ly-2.2, Ly-3.1, Ly-7.2, Ly-12.1 and Thy-1.2 were absent from the neutrophils. When compared with lymphocytes, marked differences in alloantigen expression on neutrophils were seen for Ly-5.1, Ly-24.2 and Ly-28.2. These studies should be of value in the study of neutrophil structure and function.

Animals↗