RESUMO
We show that cell surface glycans, sialic acid and mannose-containing species, are involved beside glycosaminoglycans (GAGs), heparan sulfate and chondroitin sulfate in the binding of full length (1--68) RANTES not only to CCR5 positive human primary lymphocytes or macrophages but also to CCR5 negative monocytic U937 cells. Pretreating the cells with neuraminidase, heparitinase, chondroitinase or adding soluble glycans such as mannan or GAGs (heparin or chondroitin sulfate), significantly inhibited RANTES binding. Such effects were not observed with truncated (10--68) RANTES. Heat-denaturation of (1--68) RANTES strongly decreased its binding to the cells, demonstrating involvement of the three-dimensional structure. Accordingly, full length, but not truncated (10--68) RANTES, specifically bound to soluble mannan as well as to mannose-divinylsulfone-agarose affinity matrix and to soluble heparin or chondroitin sulfate as well as to heparin-agarose. Soluble heparin exerts, depending on its concentration, inhibitory or enhancing effects on RANTES binding to mannose-divinylsulfone-agarose, which indicates that RANTES interaction with glycans is modulated by GAGs. These data demonstrate that full length RANTES, but not its (10--68) truncated counterpart, interacts with glycans and GAGs, in soluble forms or presented either by affinity matrices or CCR5 positive as well as CCR5 negative cells.
Assuntos
Quimiocina CCL5/metabolismo , Polissacarídeos/metabolismo , Receptores CCR5/metabolismo , Linhagem Celular , Quimiocina CCL5/antagonistas & inibidores , Sulfatos de Condroitina/farmacologia , Condroitinases e Condroitina Liases , Glicosaminoglicanos/metabolismo , Heparina/farmacologia , Humanos , Linfócitos/metabolismo , Macrófagos , Mananas/farmacologia , Neuraminidase , Polissacarídeo-Liases , Sefarose/análogos & derivadosRESUMO
We have reported previously that human alpha(1)-acid glycoprotein (AGP) inhibits the infection of human monocyte-derived macrophages (MDM) by R5 HIV-1, and that a disulphide-bridged peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds specifically to CCR5 (the major co-receptor of R5 HIV strains) on these cells [Seddiki, Rabehi, Benjouad, Saffar, Ferriere, Gluckman and Gattegno (1997) Glycobiology 7, 1229-1236]. The present study demonstrates that AGP binds specifically to MDM at high- and low-affinity binding sites with K(d) values of 16 nM and 4.9 microM respectively. The fact that heat denaturation of AGP only partly inhibited this binding (43%) suggests that protein-protein interactions are involved, as well as AGP glycans which are resistant to heat denaturation. Mannan, but not dextran, is a significant inhibitor (52%) of this binding, and sequential exoglycosidase treatment of AGP, which exposes penultimate mannose residues, has a strong stimulatory effect ( approximately 2.8-fold). Therefore AGP glycans (probably mannose residues) are involved, at least partly, in the binding of AGP to MDM. In addition, AGP inhibits the binding of V3Cs and macrophage inflammatory protein-1beta (MIP-1beta) to MDM. The anti-CCR5 monoclonal antibody 2D7, specific for the second extracellular loop of CCR5, also inhibited AGP binding (67%), whereas anti-CCR5 antibodies specific for the C-terminus of CCR5 region had no effect. Native AGP, like V3Cs (but not heat-denatured AGP), binds to 46 and 33-36 kDa electroblotted AGP-bound MDM membrane ligands, characterized as CCR5 by their interactions with anti-CCR5 antibodies and with MIP-1beta. Therefore both AGP glycans and MDM CCR5 are involved in the binding of AGP to MDM. This suggests that the inhibitory effect of AGP on the infection of human primary macrophages by R5 HIV-1 may be related to specific binding of AGP to a macrophage membrane lectin or lectin-like component and to CCR5.
Assuntos
Fármacos Anti-HIV/farmacologia , Membrana Celular/metabolismo , HIV-1/efeitos dos fármacos , Macrófagos/metabolismo , Orosomucoide/metabolismo , Receptores CCR5/metabolismo , Ligação Competitiva , Quimiocina CCL4 , Humanos , Proteínas Inflamatórias de Macrófagos/metabolismo , Polissacarídeos/farmacologia , Ligação ProteicaRESUMO
We demonstrate that human immunodeficiency virus HIV-1(LAI) envelope glycoprotein 120 (gp120(LAi)) specifically interacts with several membrane ligands on lymphoid CEM or monocytic U937 cells in addition to its previously identified receptor, CD4, and CXCR4, its coreceptor. In its native state, gp120(LAI) is able to elicit specific multimolecular complexes with these membrane ligands at the surface of the cells; most of the interactions are abolished by mannan or heparin but not by dextran. Similarly, stromal cell-derived factor (SDF)-1alpha interacts not only with CXCR4 expressed by CXCR4(+) CD4(+) U937, CEM, and HOS-CD4(+) CXCR4(+) cells but also with CD4 expressed by intact U937, CEM, and HOS-CD4(+) CXCR4(+/-) cells or electroblotted onto Immobilon. SDF-1alpha binding to CD4(+) CXCR4(+/-) cells, or soluble CD4 electroblotted onto Immobilon, is significantly inhibited by sCD4, whereas truncated sCD4 lacking D3 and D4 domains had no significant effect, which indicates that SDF-1 binds to CD4 but at regions different from the HIV-gp120-binding site. Heparin and mannan also inhibit SDF-1alpha binding to intact CD4(+) CXCR4(+/-) cells, and electroblotted soluble CD4. Heparitinase treatment of such cells reduced SDF-1alpha binding. These data demonstrate that glycans and glycosaminoglycans are directly or indirectly involved in the interactions of HIV-1 gp120(LAI) and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells and thus could play a role both in HIV-1 infection and in the physiology of SDF-1alpha.
Assuntos
Antígenos CD4/metabolismo , Quimiocinas CXC/metabolismo , Glicosaminoglicanos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Polissacarídeos/metabolismo , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Quimiocina CXCL12 , Humanos , Ligantes , Dados de Sequência Molecular , Proteoglicanas/metabolismo , Proteínas Recombinantes/metabolismo , Células U937RESUMO
Sodium phenylacetate (NaPa), a physiological product of phenylalanine metabolism, present in micromolar concentrations in human plasma, has been shown to induce in vivo and in vitro cytostatic antiproliferative effects at millimolar concentrations. Cadherin molecules are powerful invasion suppressor molecules and the reduction of E-cadherin expression plays an important role in the invasion and metastasis of human breast cancer. In this study, we demonstrated, on one hand, that NaPa stimulated aggregation by increasing the expression of E-cadherin at the surface of breast cancer MCF-7ras cells transformed by Ha-ras oncogene and inhibited its expression in MCF-7 cells. We demonstrated that NaPa increased the formation of MCF-7ras cell aggregates and did not alter the formation of MCF-7 cell aggregates. By Northern blot, we demonstrated that the E-cadherin expression was not regulated at the transcriptional level. On the other hand, we analyzed the cell cycle of these 2 cell lines after NaPa treatment and showed that NaPa induced arrest at the G1/S phase in both MCF-7 and MCF-7ras cells. bFGF increased the growth of MCF-7 cells, but inhibited MCF-7ras cell proliferation. NaPa treatment suppressed the stimulation of MCF-7 cell proliferation and increased MCF-7ras cell growth inhibition. We have demonstrated a new target of NaPa action in blocking the cell cycle of tumor cells in G0/G1. We suggest that the anti-proliferative effect of NaPa associated to the restoration of the cadherin function in human mammary carcinoma cells indicates that NaPa could be a novel therapeutic agent in breast cancer.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Caderinas/metabolismo , Fenilacetatos/farmacologia , Caderinas/genética , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes ras , Humanos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fase de Repouso do Ciclo Celular/efeitos dos fármacosRESUMO
The present study demonstrates at the molecular level that dextran derivatives carboxymethyl dextran benzylamine (CMDB) and carboxymethyl dextran benzylamine sulfonate (CMDBS), characterized by a statistical distribution of anionic carboxylic groups, hydrophobic benzylamide units, and/or sulfonate moieties, interact with HIV-1 LAI gp120 and V3 consensus clades B domain. Only limited interaction was observed with carboxy-methyl dextran (CMD) or dextran (D) under the same conditions. CMDBS and CMDB (1 microM) strongly inhibited HIV-1 infection of primary macrophages and primary CD4+ lymphocytes by macrophage-tropic and T lymphocyte-tropic strains, respectively, while D or CMD had more limited effects on M-tropic infection of primary macrophages and exert no inhibitory effect on M- or T-tropic infection of primary lymphocytes. CMDBS and CMDB (1 microM) had limited but significant effect on oligomerized soluble recombinant gp120 binding to primary macrophages while they clearly inhibit (> 50%) such binding to primary lymphocytes. In conclusion, the inhibitory effect of CMDB and the CMDBS, is observed for HIV M- and T-tropic strain infections of primary lymphocytes and macrophages which indicates that these compounds interfere with steps of HIV replicative cycle which neither depend on the virus nor on the cell.
Assuntos
Fármacos Anti-HIV/farmacologia , Dextranos/farmacologia , HIV-1/efeitos dos fármacos , Linfócitos/virologia , Macrófagos/virologia , Sequência de Aminoácidos , Fármacos Anti-HIV/metabolismo , Células Cultivadas , Dextranos/metabolismo , Proteína do Núcleo p24 do HIV/biossíntese , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ligação ProteicaRESUMO
We previously demonstrated that gp120/160 (Env) of HIV-1 interact in a carbohydrate-specific manner with mannosyl/N-acetylglucosaminyl derivatives and that HIV-1LAI infection of monocytic U937 and lymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactive glycopeptides from U937 cells. We report here that the natural glycoproteins bovine fetuin and asialofetuin, human orosomucoid and alpha-fetoprotein, and mannan, which all specifically interact with Env, inhibited infection of primary macrophages by macrophage-tropic HIV-1 strains, whereas dextran had no such effect. This activity was conserved if fetuin, asialofetuin, or orosomucoid were heat-treated, which rules out the role of their three-dimensional structure. Orosomucoid and mannan partially inhibited Env binding to macrophages but not to U937 or CEM cells. This indicates that Env does not bind in the same manner to primary macrophages and to immortalized CD4+ cells, and that orosomucoid and mannan act at CD4-independent stages of virus binding to macrophages. Mannan also inhibited Env binding to surface glycopeptides obtained after trypsin treatment of macrophages. Furthermore, orosomucoid and fetuin interacted with, and they inhibited the binding of a V3 loop B clade consensus peptide to several macrophage membrane proteins, including two 36 and 42 kDa proteins. These data indicate that these glycoproteins interfere with post-binding events during HIV-1 infection of primary macrophages. In contrast, the compounds did not affect infection of U937 or CEM cells by T-cell tropic HIV-1LAI nor infection of peripheral blood lymphocytes by HIV-1LAI or HIV-1(Ba-L). Thus, carbohydrate-specific inhibition of HIV infection depends on the cell type more than on the viral strain, and differences in the glycan structure of cell-type-specific cofactors may be important for HIV entry into cells.
Assuntos
Glicoproteínas/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Linfócitos/virologia , Macrófagos/virologia , Manose , Animais , Assialoglicoproteínas/farmacologia , Ligação Competitiva , Bovinos , Células Cultivadas , Fetuínas , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Temperatura Alta , Humanos , Mananas/farmacologia , Glicoproteínas de Membrana/metabolismo , Orosomucoide/farmacologia , Fragmentos de Peptídeos/metabolismo , Tripsina/farmacologia , alfa-Fetoproteínas/farmacologiaRESUMO
The expression of the carbohydrate-binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte-derived macrophages (MDM) using an anti-CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N-acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60-derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti-CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 and p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin-3.
Assuntos
Lectinas/metabolismo , Macrófagos/química , Diferenciação Celular , Núcleo Celular/química , Sobrevivência Celular , Citoplasma/química , Citometria de Fluxo , Células HL-60 , Humanos , Macrófagos/citologia , Microscopia de Fluorescência , Peso Molecular , Células Tumorais CultivadasRESUMO
The membrane glycolipids galactosylceramide (GalCer) and sulfatide (SGalCer) have been reported to act as receptors of human immunodeficiency virus (HIV) on CD4- cell lines. We show here that these glycolipids are present on CD4+ cells purified from human blood and on in vitro-differentiated monocyte-derived macrophages (MDMs). We investigated the role they could play in HIV infection. Glycolipids of MDMs were characterized at the molecular level by immunolabeling and thin-layer chromatography immune overlay, using a panel of human-, rabbit-, or murine-specific antibodies. GalCer and SGalCer were expressed at the surface of MDMs as assessed by indirect immunofluorescence and flow cytometry analysis, and they could be characterized with specific antibodies in the cellular glycolipid extracts in addition to GM1, GM3, and GD1b gangliosides. Recombinant 125-I-labeled gp160 specifically bound to GalCer, SGalCer, GM1, and GM3 as well as to phospholipids (phosphatidylethanolamine and phosphatidylserine) from MDM extracts. Anti-SGalCer monoclonal antibodies (MAbs), but not anti-GalCer antibodies, entailed limited (30-40%) but significant inhibition of gp160 binding to MDMs. However, the four human anti-SGalCer MAbs and the three murine or rabbit ant-GalCer antibodies tested did not inhibit HIV infection of MDMs, in contrast to CD4 antibody anti-Leu3a tested in parallel. These findings suggests that although HIV envelope glycoprotein can bind to SGalCer and GalCer from CD4+ MDM extracts, these glycolipids do not apparently act as HIV coreceptors nor are they involved in HIV infection of these cells.
Assuntos
Glicolipídeos/metabolismo , HIV-1/metabolismo , Macrófagos/virologia , Lipídeos de Membrana/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M3)/metabolismo , Galactosilceramidas/metabolismo , Produtos do Gene env/metabolismo , Proteína do Núcleo p24 do HIV/metabolismo , Proteína gp160 do Envelope de HIV , Humanos , Macrófagos/metabolismo , Camundongos , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Precursores de Proteínas/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Sulfoglicoesfingolipídeos/metabolismoRESUMO
We have previously demonstrated that human immunodeficiency virus (HIV) envelope glycoproteins have specific carbohydrate-binding properties for mannosyl/N-acetylglucosaminyl residues presented at high density on a carrier in vitro. Here, we investigated whether HIV envelope glycoprotein gp120 was able to interact with surface membrane carbohydrates of CD4+ cells by means of such lectin-carbohydrate interactions. CD4-free tryptic glycopeptides, prepared from the membrane of CD4+ monocytic U937 cells and partially purified by ConA-agarose affinity chromatography, could be eluted by mannan but not by methyl-alpha-mannose or methyl-alpha-glucose, which strongly suggests that they displayed oligomannosidic structures. These glycopeptides bound in a mannosyl-specific manner to radiolabeled recombinant gp120. Deglycosylation with N-glycanase which, as expected, strongly diminished binding of the glycopeptides to concanavalin A also abolished their interaction with gp120. In addition, the glycopeptides inhibited HIV infection of both U937 and CD4+ lymphoid CEM cells when preincubated with the virus. These findings indicate that, independently of the binding to CD4, mannosyl structures on CD4+ cells may play a role through lectin-carbohydrate interactions in envelope glycoprotein binding to a putative coreceptor(s) of HIV.
Assuntos
Antivirais/farmacologia , Linfócitos T CD4-Positivos/virologia , Glicopeptídeos/farmacologia , HIV-1/efeitos dos fármacos , Antivirais/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Glicopeptídeos/biossíntese , Glicopeptídeos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , HIV-1/crescimento & desenvolvimento , Humanos , Mananas/farmacologia , Monócitos/citologia , Proteínas Recombinantes/metabolismoRESUMO
Colony Stimulating Factor (CSF-1) and the CSF-1 receptor (the c-fms product) are expressed during the proliferation of L6 alpha 1 rat myogenic cell line and both are down regulated during the formation of myotubes. In this study, we demonstrated that the expression of c-fms antisense RNA in stably transfected myoblasts repressed the CSF-1 receptor (c-fms protein) and induced a G1-growth arrest. Expression of the cyclin genes, that control passage through the G1 phase and in particular the cyclins identified as genes induced late in G1 by CSF-1 in mouse macrophages was studied in comparative Northern blot analyses of RNAs of subpopulations prepared by centrifugal elutriation of L6 alpha 1 myoblasts and induced Antifms D5 cells expressing c-fms antisense RNA. Repression of the CSF-1 receptor (c-fms product) did not affect cyclins A, B and G expression during the cell cycle. However, D-type cyclins and, at a lesser extend, cyclin E expression were dramatically altered specifically during the late G1 and early S phases, in Antifms D5 cells. These results suggest a role for the CSF-1/c-fms autocrine loop in the control of the proliferation of L6 alpha 1 rat myogenic cell line at the G1/S boundary via the D-type and E cyclins expression.
Assuntos
Ciclo Celular , Ciclinas/metabolismo , Fator Estimulador de Colônias de Macrófagos/fisiologia , Músculos/citologia , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Animais , Linhagem Celular , Separação Celular , Células Cultivadas , Ciclinas/genética , Citometria de Fluxo , Expressão Gênica , Técnicas In Vitro , RNA Antissenso , RNA Mensageiro/genética , Ratos , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , TransfecçãoRESUMO
In the present study, we demonstrate a specific low-affinity interaction between recombinant precursor gp160 (rgp160) or surface unit gp120 (rgp120) of human immunodeficiency virus type 1 (HIV-1) and alpha 1-acid glycoprotein (AGP), a human glycoprotein displaying complex type N-glycans. Binding of rgp160/rgp120 to agarose-coupled AGP was dose-dependent, saturable, calcium-, pH- and temperature-dependent. Binding was inhibited by soluble AGP, asialo-AGP, fetuin, beta-D-GlcNAc47-BSA, alpha-D-Man20-BSA, mannan, complex-type asialo-agalacto-tetraanternary precursor oligosaccharide from human AGP and oligomannose 9 from porcine thyroglobulin; fully deglycosylated AGP was not inhibitory. The three AGP glycoforms separated on immobilized ConA bound rgp160 to the same extent as did unfractionated AGP. These findings extend our previous results on the carbohydrate-binding properties of HIV-1 envelope (Env) glycoprotein in that they demonstrate the involvement of AGP glycan moieties in the binding to rgp160/rgp120. Preincubation of rgp160 with AGP or mannan significantly reduced its binding to monocyte-derived macrophages (MDM), suggesting that AGP may play a role in preventing binding of soluble or virus-bound Env glycoprotein to CD4+ monocytic cells.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/química , Orosomucoide/metabolismo , Ligação Competitiva , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Metabolismo dos Carboidratos , Sequência de Carboidratos , Carboidratos/química , Proteína gp120 do Envelope de HIV/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/genética , Humanos , Dados de Sequência Molecular , Orosomucoide/química , Orosomucoide/farmacologia , Proteínas Recombinantes/metabolismo , Sefarose/química , Especificidade por SubstratoRESUMO
We show here that human immunodeficiency virus (HIV) envelope glycoproteins (gp160/gp120) bind to sulfatide and galactosyl ceramide. By immunofluorescence labeling with monoclonal antibody (mAb) A2B5, specific for ganglioside/sulfatide, we detect negatively charged glycolipids on CD4+ cells of the macrophage lineage and lymphocytes. Labeling of monocyte-derived macrophages (MDM) with mAb A2B5 was reproducibly found in 29 healthy donors, independently of the culture method and duration up to 11 days. The binding of the mAb to neuraminidase-treated MDM was unchanged relative to control cells, but mAb binding decreased after arylsulfatase treatment, which indicates that MDM membrane sulfatide is its major ligand. Preincubating MDM with the mAb partially (40-60%) but significantly inhibited the binding of HIV-1LAI radiolabeled recombinant gp160 to the cells. Similarly, the mAb entailed limited (32%) but significant inhibition of gp160 binding to cells of the monocytic U937 line but not to lymphoid CEM cells. However, mAb A2B5 did not inhibit the infection of CEM nor of U937 cells by HIV-1LAI strain, nor of MDM by monocytotropic HIV-1BaL. Thus, although sulfatide may be involved in the binding of HIV env glycoprotein to MDM or monocytic U937 cells, this does not play a significant role in HIV infection of these CD4+ cells.
Assuntos
Anticorpos Monoclonais/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Macrófagos/química , Sulfoglicoesfingolipídeos/análise , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Produtos do Gene env/metabolismo , Proteína gp160 do Envelope de HIV , HIV-1/metabolismo , Humanos , Radioisótopos do Iodo , Macrófagos/imunologia , Macrófagos/microbiologia , Ligação Proteica/efeitos dos fármacos , Precursores de Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Sulfoglicoesfingolipídeos/imunologia , Sulfoglicoesfingolipídeos/metabolismoRESUMO
The aim of this study was to determine whether mannosyl-specific lectins, especially Concanavalin A (ConA), may bridge HIV-1 env glycoproteins to cell membranes to increase virus binding to its targets, and to what extent this lectin-carbohydrate interaction can modify HIV-1 infectivity for monocytic compared with lymphoid cells. Monocytic U937 and lymphoid CEM cells, which both express surface mannose, were utilized. Whether first incubated with env glycoprotein or with the cells, lectins bound both to the cells and to radiolabeled recombinant gp160 (rgp160). Thus, they enhanced rgp160 adsorption to the cells in a methyl-alpha-mannose inhibitable manner. ConA did not appear to bind to the V1 domain of CD4 at the U937 cell surface since Leu3a binding was not blocked in the presence of ConA, nor was recombinant CD4 retained on a ConA-agarose affinity matrix. Moreover, enhanced rgp160 binding to the cells was CD4 independent, since it was not modified by preincubating the cells with Leu3a. Finally, ConA did not inhibit the binding of CD4-IgG3 chimeric molecules to virions immobilized on nitrocellulose membrane, which argues against the possibility that it interferes with the interaction of gp120 and CD4. However, both when incubated with the virus or with the cells and despite mediating enhanced binding of env glycoprotein, ConA neutralized HIV-1 infectivity for monocytic U937 as well as for lymphoid CEM cells. In this respect, ConA behaves like neutralizing antibodies which do not interfere with CD4 binding of gp120 but rather with some later event that leads to virus entry. These findings obtained with plant lectins may be of relevance in vivo, inasmuch as endogenous mannosyl-binding proteins, which are known to function as opsonins, have been reported to inhibit in vitro infection by HIV-1.
Assuntos
Síndrome da Imunodeficiência Adquirida/microbiologia , HIV-1/metabolismo , Lectinas/metabolismo , Síndrome da Imunodeficiência Adquirida/metabolismo , Linhagem Celular , Produtos do Gene env/metabolismo , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV , Humanos , Lectinas/fisiologia , Linfócitos , Monócitos , Testes de Neutralização , Precursores de Proteínas/metabolismo , Receptores Mitogênicos/efeitos dos fármacos , Receptores Virais/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
The effect of carbohydrate structures on the adsorption of HIV-1 or of recombinant envelope glycoprotein gp 160 (rgp 160) to cells of the CEM line was investigated with an indirect immunofluorescence assay using gp 120-specific mouse monoclonal antibodies (mAbs) directed to envelope gp 120. The beta-D-galactosyl, alpha-D-mannosyl, beta-D-glucosyl, N-acetyl-beta-D-glucosaminyl, sialosyl, and L-fucosyl derivatives tested had no effect on this binding. However, preincubation of HIV-1 (or rgp 160) with the neoglycoprotein, beta-D-GlcNAc47-BSA, specifically inhibited the labeling, by some of the mAb used, of HIV-1 (or rgp 160) bound at the cell membrane. This inhibition occurred only with mAbs that were specific for the immunodominant "neutralizing" third variable region (V3) of gp 120. Competition for the binding to rgp 160 between beta-D-GlcNAc47-BSA and mAb was further demonstrated by use of affinity matrices substituted with one of the relevant mAb (110-4), or with beta-D-GlcNAc47-BSA. Besides beta-D-GlcNAc47-BSA-Sepharose, rgp 160 also bound with low affinity, but high specificity, to two other N-acetyl-beta-D-glucosaminyl affinity matrices, beta-D-GlcNAc-divinylsulfone-agarose and asialoagalactothyroglobulin-agarose. Conversely, beta-D-[125I]GlcNAc47-BSA bound specifically to gp 160-Sepharose. These results indicated that rgp 160 behaves as a N-acetyl-beta-D-glucosaminyl-binding protein for GlcNAc residues presented at high density on a carrier, the carbohydrate-binding site of which is close to, or located on the V3 region of gp 120.
Assuntos
Acetilglucosamina/metabolismo , Produtos do Gene env/metabolismo , HIV-1/metabolismo , Precursores de Proteínas/metabolismo , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Ligação Competitiva , Linfócitos T CD4-Positivos/metabolismo , Metabolismo dos Carboidratos , Linhagem Celular , Membrana Celular/metabolismo , Anticorpos Anti-HIV/metabolismo , Proteína gp160 do Envelope de HIV , Humanos , Lectinas/metabolismo , Receptores de HIV/metabolismo , Soroalbumina Bovina/metabolismoAssuntos
Anticorpos Monoclonais/imunologia , Carboidratos/farmacologia , Adesão Celular/efeitos dos fármacos , Envelhecimento Eritrocítico , Membrana Eritrocítica/efeitos dos fármacos , Antígeno de Macrófago 1/imunologia , Macrófagos/fisiologia , Proteínas Opsonizantes/fisiologia , Fagocitose/efeitos dos fármacos , Receptores de Complemento/imunologia , Biomarcadores , Depressão Química , Membrana Eritrocítica/imunologia , HumanosRESUMO
Human mononuclear cells were isolated from peripheral blood by centrifugation over Ficoll Hypaque, followed by adherence to plastic dishes. Monocyte-derived macrophages were obtained after culture for 3 or 5 days of the adherent cells in RPMI medium containing 20% heat-inactivated foetal calf serum. The sialidase activities were assayed in the whole homogenate using sodium 4-methyl-umbelliferyl-alpha-D-neuraminate as substrate, at various pHs, ranging from 3.6 to 6. The in vitro differentiation of monocytes into macrophages from day 0 up to day 5 was accompanied by a significant (P less than or equal to 0.01) increase in the sialidase activity on both a per-cell (+360%) and a per-mg protein in the homogenate (+125%) basis.
Assuntos
Macrófagos/enzimologia , Monócitos/enzimologia , Neuraminidase/metabolismo , Diferenciação Celular/fisiologia , Humanos , Macrófagos/citologia , Monócitos/citologiaRESUMO
The effect of different murine monoclonal antibodies (Mab) specific for the glycoprotein complement receptor type 1 (CR1), type 2 (CR2), and type 3 (CR3) on the adhesion to and on the phagocytosis of human senescent red blood cells (S-RBC) by monocytes or by monocyte-derived macrophages (M phi) was investigated. Murine Mab anti-CR3 (anti-Leu 15 and OKM1) were found to inhibit, in the same order of magnitude, on one hand, the Fc receptors (FcR)-dependent rosetting and phagocytosis, and, on the other hand, the S-RBC rosetting and phagocytosis by adherent monocytes. Thus, the specific involvement of the CR3 epitopes recognized by Mab anti-Leu 15 or by OKM1 in the interactions between S-RBC and monocyte/macrophage could not be demonstrated. Murine Mab anti-CR1 was found to be a significant inhibitor of binding to and of phagocytosis of S-RBC (but not of young [Y] RBC) by monocytes or M phi, whereas Mab OKM5 carrying the same isotype as Mab anti-CR1, but a different specificity, was devoid of any significant inhibitory effect. Furthermore, Y-RBC or S-RBC opsonized with Mab anti-CR1 did not form FcR-dependent rosettes and were not internalized by monocytes; in addition, preincubation of phagocytes with Mab anti-CR1 did not inhibit FcR-dependent rosetting and phagocytosis. These results suggest that the effect of anti-CR1 is mediated through a specific binding to CR1 and not through an FcR blockade. As the role of specifically bound IgG on phagocytosis of human S-RBC by macrophages has previously been demonstrated by several authors, the present study suggests that monocyte-macrophage complement receptor type 1 may act in synergy with Fc receptors in the recognition of S-RBC by macrophages. It is shown in addition that the tripeptide Arg-Gly-Asp, identical to the region of iC3b recognized by CR3 and by several adhesion-promoting receptors that are structurally similar to CR3, such as fibronectin or vitronectin, is a significant inhibitor of the binding to and the phagocytosis of S-RBC by monocytic-macrophagic cells.
Assuntos
Anticorpos Monoclonais/farmacologia , Eritrócitos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Receptores de Complemento/imunologia , Adesão Celular , Separação Celular , Envelhecimento Eritrocítico , Humanos , Técnicas In Vitro , Oligopeptídeos/farmacologia , FagocitoseRESUMO
Two immunostimulating peptides, Val-Glu-Pro-Ile-Pro-Tyr and Gly-Leu-Phe, obtained from human caseins, were demonstrated to significantly stimulate binding of human senescent red blood cells to human monocytic-macrophagic cells and their phagocytosis by these cells.