(1/1220) Interaction of inflammatory cells and oral microorganisms. III. Modulation of rabbit polymorphonuclear leukocyte hydrolase release response to Actinomyces viscosus and Streptococcus mutans by immunoglobulins and complement.
In the absence of antiserum, rabbit polymorphonuclear leukocytes (PMNs) released lysosomal enzymes in response to Actinomyces viscosus (19246) but not to Streptococcus mutans (6715). Antibodies had a marked modulating influence on these reactions. PMN hydrolase release was significantly enhanced to both organisms when specific rabbit antiserum and isolated immunoglobulin G (IgG) were included in the incubations. Immune complex F(ab')2 fragments of IgG directed against S. mutans agglutinated bacteria. Immune complexes consisting of S. mutans and F(ab')2 fragments of IgG directed against this organism were not effective as bacteria-IgG complexes in stimulating PMN release. The intensity of the release response to bacteria-IgG complexes was also diminished when PMNs were preincubated with isolated Fc fragments derived from IgG. Fresh serum as a source of complement components had no demonstrable effect on PMN release either alone or in conjuction with antiserum in these experiments. These data may be relevant to the mechanisms and consequences of the interaction of PMNs and plaque bacteria in the pathogenesis of periodontal disease. (+info)
(2/1220) 2-Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages II. Dissociation of the inhibitory effects of 2-deoxyglucose on phagocytosis and ATP generation.
Macrophages incubated in 2-deoxy-D-glucose (2-dG)-containing medium showed a marked decrease in cellular ATP content, and were unable to ingest IgG- and complement-coated erythrocytes via the corresponding membrane receptors for these ligands. However, the inhibitory effects of 2-dG on Fc- and C3 receptor-mediated phagocytosis were not a consequence of lowered macrophage ATP levels since addition of glucose or mannose to the culture medium restored the capacity of the macrophages to ingest IgG- and C3-coated particles without increasing ATP levels. These results indicate that Fc- and C3 receptor-mediated phagocytosis (opsonin dependent) differs qualitatively from the ingestion of latex and zymosan particles (opsonin independent); they suggest that the same regulatory molecules govern the responses of phagocytic cells to signals initiated by both the Fc and C3 receptors. The possibility that these molecules are regulated by glycosylation is discussed. (+info)
(3/1220) The CTLA-4 gene is expressed in placental fibroblasts.
In order to elucidate the mechanisms that ensure survival of the allogeneic fetus, we are investigating the expression pattern of genes that are involved in peripheral self-tolerance in tissues at the maternal-fetal interface. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a negative regulator of T cell activation and may modulate peripheral self-tolerance. Previously, we reported the preferential transmission of maternally-inherited shorter alleles at a 3'-UTR microsatellite locus to liveborn children, but random transmission of paternally-inherited alleles, suggesting that CTLA-4 may be involved in the maintenance of tolerance at the maternal-fetal interface. In this report, we demonstrate that CTLA-4 mRNA and protein are indeed expressed in fetal tissues at the maternal-fetal interface throughout gestation. (+info)
(4/1220) Analysis of the interaction of monoclonal antibodies with surface IgM on neoplastic B-cells.
In vitro studies identified three Burkitts lymphoma cell lines, Ramos, MUTU-I and Daudi, that were growth inhibited by anti-IgM antibody. However, only Ramos and MUTU-I were sensitive to monoclonal antibodies (mAb) recognizing the Fc region of surface IgM (anti-Fc mu). Experiments using anti-Fc mu mAb (single or non-crossblocking pairs), polyclonal anti-mu Ab, and hyper-crosslinking with a secondary layer of Ab, showed that growth inhibition of B-cell lines was highly dependent on the extent of IgM crosslinking. This was confirmed by using Fab', F(ab')2 and F(ab')3 derivatives from anti-Fc mu mAb, where increasing valency caused corresponding increases in growth arrest and apoptosis, presumably as a result of more efficient BCR-crosslinking on the cell surface. The ability of a single mAb to induce growth arrest was highly dependent on epitope specificity, with mAb specific for the Fc region (C mu2-C mu4 domains) being much more effective than those recognizing the Fab region (anti-L chain, anti-Id and anti-Fd mu, or C mu1). Only when hyper-crosslinked with polyclonal anti-mouse IgG did the latter result in appreciable growth inhibition. Binding studies showed that these differences in function were not related to differences in the affinity, but probably related to intrinsic crosslinking capacity of mAb. (+info)
(5/1220) Expression and characterization of a DNase I-Fc fusion enzyme.
Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits. (+info)
(6/1220) Polymerization of IgA and IgM: roles of Cys309/Cys414 and the secretory tailpiece.
We have investigated how the secretory tailpiece (tp), Cys414 and the amino acids flanking Cys414 or Cys309 are involved in regulating the different polymerization of IgM and IgA to pentamers and dimers/monomers, respectively. Whereas changing the tp of IgM to that of IgA has little effect on IgM polymerization, introducing the mu tp to IgA leads to the formation of larger than wild-type IgA polymers, including pentamers and hexamer. This shows that the secretory tp can differentially regulate polymerization depending on the heavy chain context. Cys414, which is engaged in intermonomeric disulfide bonds in IgM, is not crucial for the difference in IgM and IgA polymerization; IgM with a C414S mutation forms more large polymers than IgA. Also, IgA with IgM-like mutations in the five amino acids flanking Cys309, which is homologous to Cys414, oligomerize similarly as IgA wild type. Thus, IgA appears to have an inherent tendency to form monomers and dimers that is partially regulated by the tp, while the Cys309 region has only a minor effect. We also show that complement activation by IgM is sensitive to alterations in the polymeric structure, while IgA is inactive in classical complement activation even for polymers such as pentamers and hexamers. (+info)
(7/1220) Small-angle X-ray studies of the Fab and Fc fragments from the human immunoglobulin molecule Kol.
The conformation of the Fab and Fc fragments from the human immunoglobulin molecule Kol [IgI I, chi2gamma2, Gm(f)+] was studied by small-angle x-ray scattering in solution. The fragments were studied in 0.02 M Tris-HCl buffer. For the Fab fragment the radius of gyration was found to be 3.15 +/- 0.15 nm, the volume to be 75 +/- 8 nm3. For the Fc fragment the respective values were 3.15 +/- 0.15 nm for the radius of gyration and 91 +/- 8 nm3 for the volume. A large number of models were calculated for both fragments to find models which fit these data and have the same scattering curve. The models with the best agreement were compared with the models found for the crystalline state by crystal x-ray studies. (+info)
(8/1220) A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells.
Human natural killer (NK) cells express several killer cell immunoglobulin (Ig)-like receptors (KIRs) that inhibit their cytotoxicity upon recognition of human histocompatibility leukocyte antigen (HLA) class I molecules on target cells. Additional members of the KIR family, including some that deliver activation signals, have unknown ligand specificity and function. One such KIR, denoted KIR2DL4, is structurally divergent from other KIRs in the configuration of its two extracellular Ig domains and of its transmembrane and cytoplasmic domains. Here we show that recombinant soluble KIR2DL4 binds to cells expressing HLA-G but not to cells expressing other HLA class I molecules. Unlike other HLA class I-specific KIRs, which are clonally distributed on NK cells, KIR2DL4 is expressed at the surface of all NK cells. Furthermore, functional transfer of KIR2DL4 into the cell line NK-92 resulted in inhibition of lysis of target cells that express HLA-G, but not target cells that express other class I molecules including HLA-E. Therefore, given that HLA-G expression is restricted to fetal trophoblast cells, KIR2DL4 may provide important signals to maternal NK decidual cells that interact with trophoblast cells at the maternal-fetal interface during pregnancy. (+info)