RESUMO
Graphene oxide (GO) has been proposed as an hyperthermia agent for anticancer therapies due to its near-infrared (NIR) optical absorption ability which, with its small two-dimensional size, could have a unique performance when compared to that of any other nanoparticle. Nevertheless, attention should be given to the hyperthermia route and the kind of GO-cell interactions induced in the process. The hyperthermia laser irradiation parameters, such as exposure time and laser power, were investigated to control the temperature rise and consequent damage in the GOs containing cell culture medium. The type of cell damage produced was evaluated as a function of these parameters. The results showed that cell culture temperature (after irradiating cells with internalized GO) increases preferentially with laser power rather than with exposure time. Moreover, when laser power is increased, necrosis is the preferential cell death leading to an increase of cytokine release to the medium.
Assuntos
Morte Celular/efeitos dos fármacos , Citocinas/metabolismo , Grafite/farmacologia , Hipertermia Induzida/métodos , Nanopartículas , Morte Celular/imunologia , Linhagem Celular Tumoral , Tomografia com Microscopia Eletrônica , Humanos , Lasers , Microscopia Confocal , Osteoblastos , ÓxidosRESUMO
Graphene and more specifically, nanographene oxide (GO) has been proposed as a highly efficient antitumoral therapy agent. Nevertheless, its cell uptake kinetics, its influence in different types of cells and the possibility of controlling cellular internalization timing, is still a field that remains unexplored. Herein, different cell types have been cultured in vitro for several incubation periods in the presence of 0.075 mg ml(-1) pegylated GO solutions. GO uptake kinetics revealed differences in the agent's uptake amount and speed as a function of the type of cell involved. Osteoblast-like cells GO uptake is higher and faster without resulting in greater cell membrane damage. Moreover, the dependence on the commonly used PEG nature (number of branches) also influences the viability and cell uptake speed. These facts play an important role in the future definition of timing parameters and selective cell uptake control in order to achieve an effective therapy.
Assuntos
Grafite/farmacocinética , Nanoestruturas/química , Polietilenoglicóis/farmacocinética , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Grafite/química , Grafite/farmacologia , Histocitoquímica , Humanos , Camundongos , Microscopia Confocal , Nanoestruturas/ultraestrutura , Óxidos/química , Óxidos/farmacocinética , Óxidos/farmacologia , Tamanho da Partícula , Polietilenoglicóis/química , Polietilenoglicóis/farmacologiaRESUMO
The osteogenic capability of mesoporous bioactive nanoparticles (MBNPs) in the SiO2CaO system has been assessed in vivo using an osteoporotic rabbit model. MBNPs have been prepared using a double template method, resulting in spherical nanoparticles with a porous core-shell structure that has a high surface area and the ability to incorporate the anti-osteoporotic drug ipriflavone. In vitro expression of the pro-inflammatory genes NF-κB1, IL-6, TNF-α, P38 and NOS2 in RAW-264.7 macrophages, indicates that these nanoparticles do not show adverse inflammatory effects. An injectable system has been prepared by suspending MBNPs in a hyaluronic acid-based hydrogel, which has been injected intraosseously into cavitary bone defects in osteoporotic rabbits. The histological analyses evidenced that MBNPs promote bone regeneration with a moderate inflammatory response. The incorporation of ipriflavone into these nanoparticles resulted in a higher presence of osteoblasts and enhanced angiogenesis at the defect site, but without showing significant differences in terms of new bone formation. STATEMENT OF SIGNIFICANCE: Mesoporous bioactive glass nanoparticles have emerged as one of the most interesting materials in the field of bone regeneration therapies. For the first time, injectable mesoporous bioactive nanoparticles have been tested in vivo using an osteoporotic animal model. Our findings evidence that MBG nanoparticles can be loaded with an antiosteoporotic drug, ipriflavone, and incorporated in hyaluronic acid to make up an injectable hydrogel. The incorporation of MBG nanoparticles promotes bone regeneration even under osteoporotic conditions, whereas the presence of IP enhances angiogenesis as well as the presence of osteoblast cells lining in the newly formed bone. The injectable device presented in this work opens new possibilities for the intraosseous treatment of osteoporotic bone using minimally invasive surgery.
Assuntos
Nanopartículas , Osteoporose , Animais , Regeneração Óssea , Osso e Ossos , Vidro/química , Ácido Hialurônico/farmacologia , Hidrogéis/farmacologia , Interleucina-6 , Nanopartículas/química , Nanopartículas/uso terapêutico , Osteogênese , Osteoporose/tratamento farmacológico , Porosidade , Coelhos , Alicerces Teciduais/química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The use of nanoparticles for intracellular drug delivery could reduce the toxicity and side effects of the drug but, the uptake of these nanocarriers could induce adverse effects on cells and tissues after their incorporation. Macrophages play a central role in host defense and are responsible for in vivo nanoparticle trafficking. Assessment of their defense capacity against pathogenic micro-organisms after nanoparticle uptake, is necessary to prevent infections associated with nanoparticle therapies. In this study, the effects of hollow mesoporous SiO2-CaO nanospheres labeled with fluorescein isothiocyanate (FITC-NanoMBGs) on the function of peritoneal macrophages was assessed by measuring their ability to phagocytize Candidaalbicans expressing a red fluorescent protein. Two macrophage/fungus ratios (MOI1 and MOI5) were used and two experimental strategies were carried out: a) pretreatment of macrophages with FITC-NanoMBGs and subsequent fungal infection; b) competition assays after simultaneous addition of fungus and nanospheres. Macrophage pro-inflammatory phenotype markers (CD80 expression and interleukin 6 secretion) were also evaluated. Significant decreases of CD80+ macrophage percentage and interleukin 6 secretion were observed after 30 min, indicating that the simultaneous incorporation of NanoMBG and fungus favors the macrophage non-inflammatory phenotype. The present study evidences that the uptake of these nanospheres in all the studied conditions does not alter the macrophage function. Moreover, intracellular FITC-NanoMBGs induce a transitory increase of the fungal phagocytosis by macrophages at MOI 1 and after a short time of interaction. In the competition assays, as the intracellular fungus quantity increased, the intracellular FITC-NanoMBG content decreased in a MOI- and time-dependent manner. These results have confirmed that macrophages clearly distinguish between inert material and the live yeast in a dynamic intracellular incorporation. Furthermore, macrophage phagocytosis is a critical determinant to know their functional state and a valuable parameter to study the nanomaterial / macrophages / Candida albicans interface.
Assuntos
Compostos de Cálcio/administração & dosagem , Candida albicans , Macrófagos Peritoneais/efeitos dos fármacos , Nanosferas/administração & dosagem , Óxidos/administração & dosagem , Dióxido de Silício/administração & dosagem , Animais , Células Cultivadas , Macrófagos Peritoneais/fisiologia , Camundongos Endogâmicos C57BL , PorosidadeRESUMO
Mesoporous bioactive glasses (MBGs) are bioceramics designed to induce bone tissue regeneration and very useful materials with the ability to act as drug delivery systems. MBGs can be implanted in contact with bone tissue in different ways, as particulate material, in 3D scaffolds or as nanospheres. In this work, we assessed the effects of particles of mesoporous bioactive glass MBG-75S and mesoporous nanospheres NanoMBG-75S on RAW 264.7 and J774A.1 macrophages, which present different sensitivity and are considered as ideal models for the study of innate immune response. After evaluating several cellular parameters (morphology, size, complexity, proliferation, cell cycle and intracellular content of reactive oxygen species), the action of MBG-75S particles and NanoMBG-75S on the polarization of these macrophages towards the pro-inflammatory (M1) or reparative (M2) phenotype was determined by the expression of specific M1 (CD80) and M2 (CD206, CD163) markers. We previously measured the adsorption of albumin and fibrinogen on MBG-75S particles and the production of pro-inflammatory cytokines as TNF-α and IL-6 by macrophages in response to these particles. This comparative study demonstrates that particles of mesoporous bioactive glass MBG-75S and mesoporous nanospheres NanoMBG-75S allow the appropriated development and function of RAW 264.7 and J774A.1 macrophages and do not induce polarization towards the M1 pro-inflammatory phenotype. Therefore, considering that these mesoporous biomaterials offer the possibility of loading drugs into their pores, the results obtained indicate their high potential for use as drug-delivery systems in bone repair and osteoporosis treatments without triggering an adverse inflammatory response.
Assuntos
Vidro , Nanosferas , Proliferação de Células , Macrófagos , Porosidade , Alicerces TeciduaisRESUMO
Silicon-substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to optimize their bone regeneration properties, we have manufactured these scaffolds presenting different microstructures: nanocrystalline and crystalline. Moreover, their surfaces have been decorated with vascular endothelial growth factor (VEGF) to evaluate the potential coupling between vascularization and bone regeneration. In vitro cell culture tests evidence that nanocrystalline SiHA hinders pre-osteblast proliferation, whereas the presence of VEGF enhances the biological functions of both endothelial cells and pre-osteoblasts. The bone regeneration capability has been evaluated using an osteoporotic sheep model. In vivo observations strongly correlate with in vitro cell culture tests. Those scaffolds made of nanocrystalline SiHA were colonized by fibrous tissue, promoted inflammatory response and fostered osteoclast recruitment. These observations discard nanocystalline SiHA as a suitable material for bone regeneration purposes. On the contrary, those scaffolds made of crystalline SiHA and decorated with VEGF exhibited bone regeneration properties, with high ossification degree, thicker trabeculae and higher presence of osteoblasts and blood vessels. Considering these results, macroporous scaffolds made of SiHA and decorated with VEGF are suitable bone grafts for regeneration purposes, even in adverse pathological scenarios such as osteoporosis. STATEMENT OF SIGNIFICANCE: For the first time, the in vivo behavior of scaffolds made of silicon substituted hydroxyapatites (SiHA) has been evaluated under osteoporosis conditions. In order to optimize the bone regeneration properties of these bioceramics, 3D macroporous scaffolds have been manufactured by robocasting and implanted in osteoporotic sheep. Our experimental design shed light on the important issue of the biological response of nano-sized bioceramics vs highly crystalline bioceramics, as well as on the importance of coupling vascularization and bone growth processes by decorating SiHA scaffolds with vascular endothelial growth factor.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Durapatita/farmacologia , Osteoporose/patologia , Silício/farmacologia , Alicerces Teciduais/química , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adsorção , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Feminino , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoporose/diagnóstico por imagem , Porosidade , Ovinos , Suínos , Tomografia Computadorizada por Raios XRESUMO
The osteogenic and angiogenic responses to metal macroporous scaffolds coated with silicon substituted hydroxyapatite (SiHA) and decorated with vascular endothelial growth factor (VEGF) have been evaluated in vitro and in vivo. Ti6Al4V-ELI scaffolds were prepared by electron beam melting and subsequently coated with Ca10(PO4)5.6(SiO4)0.4(OH)1.6 following a dip coating method. In vitro studies demonstrated that SiHA stimulates the proliferation of MC3T3-E1 pre-osteoblastic cells, whereas the adsorption of VEGF stimulates the proliferation of EC2 mature endothelial cells. In vivo studies were carried out in an osteoporotic sheep model, evidencing that only the simultaneous presence of both components led to a significant increase of new tissue formation in osteoporotic bone. STATEMENT OF SIGNIFICANCE: Reconstruction of bones after severe trauma or tumors extirpation is one of the most challenging tasks in the field of orthopedic surgery. This scenario is even more complicated in the case of osteoporotic patients, since their bone regeneration capability is decreased. In this work we present a porous implant that promotes bone regeneration even in osteoporotic bone. By coating the implant with osteogenic bioceramics such as silicon substituted hydroxyapatite and subsequent adsorption of vascular endothelial growth factor, these implants stimulate the bone ingrowth when they are implanted in osteoporotic sheep.
Assuntos
Regeneração Óssea/efeitos dos fármacos , Durapatita , Osteoporose , Silício , Titânio , Fator A de Crescimento do Endotélio Vascular , Ligas , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Durapatita/química , Durapatita/farmacologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Camundongos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/patologia , Ovinos , Silício/química , Silício/farmacologia , Suínos , Titânio/química , Titânio/farmacologia , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Graphene-based materials are revealing a great promise for biomedical applications and demonstrating attractiveness for neural repair. In the context of neural tissue damage, the dialogue between neural and immune cells appears critical for driving regeneration, thus making the understanding of their relations pivotal. Herein, the acute response of RAW-264.7 macrophages on nanostructured reduced graphene oxide (rGO) microfibers has been evaluated through the analysis of cell parameters including proliferation, viability, intracellular content of reactive oxygen species, cell cycle, apoptosis, and cell size and complexity. The influence of the direct contact of rGO microfibers on their polarization towards M1 and M2 phenotypes has been studied by analyses of both M1 (CD80) and M2 (CD163) markers and the secretion of the inflammatory cytokines TNF-α and IL-6. Finally, the capability of these rGO microfibers to regulate neural stem cell differentiation has been also evaluated. Findings reveal that rGO microfibers inhibit the proliferation of RAW-264.7 macrophages without affecting their viability and cell cycle profiles. The presence of M1 and M2 macrophages on these microfibers was confirmed after 24 and 48 h, respectively, accompanied by a decrease in TNF-α and an increase in IL-6 cytokine secretion. These rGO microfibers were also able to support the formation of a highly interconnected neural culture composed of both neurons (map2+ cells) and glial cells (vimentin+ cells). These findings encourage further investigation of these microfibers as attractive biomaterials to interact with immune and neural cells, attempting to support wound healing and tissue repair after implantation.
Assuntos
Grafite/química , Grafite/farmacologia , Macrófagos/efeitos dos fármacos , Nanofibras/química , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Óxidos/química , Animais , Apoptose/efeitos dos fármacos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Interleucina-6/metabolismo , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Fenótipo , Células RAW 264.7 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Macrophages, as effector cells involved in the innate and adaptive immunity, play a key role in the response to nanomaterials as graphene oxide (GO) and in their cellular uptake. The interactions at the interface of GO nanosheets, macrophages and microbial pathogens need to be assessed to determine the possible impairment of the immune system induced by biomedical treatments with this nanomaterial. Here, we have evaluated by flow cytometry and confocal microscopy the ability of murine peritoneal macrophages to phagocytose the fungal pathogen Candida albicans, alive or heat-killed, after treatment with poly(ethylene glycol-amine)-derivatized GO nanosheets (PEG-GO). After GO treatment, differences in fungal phagocytosis were observed between macrophages that had taken up GO nanosheets (GO+ population) and those that had not (GO- population). GO treatment increased the ingested alive yeasts in GO- macrophages, whereas phagocytosis diminished in the GO+ population. Ingestion of heat-killed yeasts was slightly higher in both GO- and GO+ populations when comparing with control macrophages. For the first time, we show that GO uptake by macrophages modulates its phagocytic capability, affecting differentially the subsequent ingestion of either alive or heat-killed yeasts. Enhanced ingestion of heat-killed yeast by GO-treated macrophages suggests a beneficial role of this nanomaterial for the clearance of dead microorganisms during infection.
Assuntos
Candida albicans/imunologia , Grafite/química , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Nanoestruturas/administração & dosagem , Fagocitose/imunologia , Animais , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Células Cultivadas , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Nanoestruturas/química , Fagocitose/efeitos dos fármacosRESUMO
A mesoporous bioactive glass (MBG) of molar composition 75SiO2-20CaO-5P2O5 (MBG-75S) has been synthetized as a potential bioceramic for bone regeneration purposes. X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), nitrogen adsorption studies and transmission electron microscopy (TEM) demonstrated that MBG-75S possess a highly ordered mesoporous structure with high surface area and porosity, which would explain the high ionic exchange rate (mainly calcium and silicon soluble species) with the surrounded media. MBG-75S showed high biocompatibility in contact with Saos-2 osteoblast-like cells. Concentrations up to 1â¯mg/ml did not lead to significant alterations on either morphology or cell cycle. Regarding the effects on osteoclasts, MBG-75S allowed the differentiation of RAW-264.7 macrophages into osteoclast-like cells but exhibiting a decreased resorptive activity. These results point out that MBG-75S does not inhibit osteoclastogenesis but reduces the osteoclast bone-resorbing capability. Finally, in vitro studies focused on the innate immune response, evidenced that MBG-75S allows the proliferation of macrophages without inducing their polarization towards the M1 pro-inflammatory phenotype. This in vitro behavior is indicative that MBG-75S would just induce the required innate immune response without further inflammatory complications under in vivo conditions. The overall behavior respect to osteoblasts, osteoclasts and macrophages, makes this MBG a very interesting candidate for bone grafting applications in osteoporotic patients.
Assuntos
Materiais Biocompatíveis/metabolismo , Cerâmica/metabolismo , Macrófagos/citologia , Osteoblastos/citologia , Osteoclastos/citologia , Animais , Apoptose , Ciclo Celular , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Tamanho Celular , Humanos , Macrófagos/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Porosidade , Células RAW 264.7RESUMO
Silicon substituted and nanocrystalline hydroxyapatites have attracted the attention of many researchers due to their up-regulation in osteoblast cell metabolism and enhanced bioreactivity, respectively. On the other hand, the biomaterial success or failure depends ultimately on the immune response triggered after its implantation. Macrophages are the main components of the innate immune system with an important role in healing and tissue remodelling due to their remarkable functional plasticity, existing in a whole spectrum of functional populations with varying phenotypic features. The effects of nanocrystalline hydroxyapatite (nano-HA) and nanocrystalline silicon substituted hydroxyapatite (nano-SiHA) on the macrophage populations defined as pro-inflammatory (M1) and reparative (M2) phenotypes have been evaluated in the present study using RAW 264.7 cells and mouse peritoneal macrophages as in vitro models. M1 and M2 macrophage phenotypes were characterized by flow cytometry and confocal microscopy by the expression of CD80 and CD163, known as M1 and M2 markers, respectively. The polarization of primary macrophages towards the M1 or M2 phenotype was induced with the pro-inflammatory stimulus LPS or the anti-inflammatory stimulus IL-10, respectively, evaluating the biomaterial effects under these conditions. Our results show that both nano-HA and nano-SiHA favour the macrophage polarization towards an M2 reparative phenotype, decreasing M1 population and ensuring an appropriate response in the implantation site of these biomaterials designed for bone repair and bone tissue engineering.
RESUMO
CD4 cross-linking by HIV gp120 triggers CD4+ T cell death. Several authors have suggested that this effect is mediated by CD95, but this possibility is debated by other authors. In a previous work, we found by co-capping that gp120(451) and gp120MN, but not gp120(IIIB), induce lateral association of CD4 with CD95 on the T cell surface. In this work, we used fluorescence resonance energy transfer to confirm that CD4/CD95 lateral association is induced by gp120(451), but not gp120(IIIB). Moreover, we found that gp120 ability to induce the CD4/CD95 association correlates with ability to induce cell death, since gp120(451) and gp120MN induced higher levels of cell death than did gp120(IIIB) in PHA-derived CD4+ T cell lines. CD95 involvement in gp120-induced cell death was confirmed by showing that gp120(451) and gp120MN did not induce death in CD4+ T cells derived from patients with autoimmune/lymphoproliferative disease (ALD) and decreased CD95 function. Cell death induced by gp120MN was inhibited by a recombinant CD95/IgG.Fc molecule blocking the CD95/CD95L interaction. However, inhibition was late and only partial. These data suggest that the gp120-induced CD4/CD95 association exerts a dual effect: an early effect that is independent of CD95L and may be due to direct triggering of CD95 by gp120, and a late effect that may be due to sensitization of CD95 to triggering by CD95L. In line with the former effect, cell treatment with gp120MN activated caspase 3 in the presence of Fas/IgG.Fc, which shows that cell death induced by gp120MN independently of CD95L uses the same pathway as CD95.
Assuntos
Apoptose , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptor fas/metabolismo , Apoptose/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Proteína Ligante Fas , Humanos , Ligação ProteicaRESUMO
HYPOTHESIS: Graphene oxide (GO) has attracted the scientific community attention due to its novel properties and wide range of potential applications including hyperthermia cancer therapy. However, little is known about the GO effects on the immune function which involves both innate and adaptive defence mechanisms through the activation of different cell populations and secretion of several cytokines. The effect of different GO nanosheets designed for hyperthermia cancer therapy on macrophage and lymphocyte function should be determined before using GO for this application. EXPERIMENTS: The effects of GO nanosheets with 1 (1-GOs) and 6 arms (6-GOs) of polyethylene glycol on RAW-264.7 macrophages and primary splenocytes (as approximation to the in vivo situation) were evaluated through the proinflammatory cytokine secretion and the modulation of cell proliferation in the presence of specific stimuli for either T-lymphocytes (concanavalin A, anti-CD3 antibody) or B-lymphocytes/macrophages (lipopolysaccharide). FINDINGS: 6-GOs significantly increased the secretion of TNF-α by RAW-264.7 macrophages without alteration of IL-6 and IL-1ß levels. The treatment of primary splenocytes with 1-GOs and 6-GOs in the presence of concanavalin A, anti-CD3 antibody and lipopolysaccharide, produced significant dose-dependent decreases of cell proliferation and IL-6 levels, revealing weak inflammatory properties of GOs which are favourable for hyperthermia cancer therapy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Grafite/farmacologia , Macrófagos/imunologia , Nanopartículas/química , Linfócitos T/imunologia , Animais , Linhagem Celular , Citocinas/imunologia , Grafite/química , Macrófagos/citologia , Camundongos , Baço/citologia , Baço/imunologia , Linfócitos T/citologiaRESUMO
Tyrosine phosphorylation of different substrates is the earliest intracellular signal detected after T cell receptor (TcR) ligation. Several tyrosine kinases have been detected associated to the CD3-TcR complex in stimulated or unstimulated cells, including p56lck, p59fyn and ZAP-70. We have observed, in one mouse T helper CD4 T cell line, that most TcR- or CD3-associated tyrosine kinase activity comes from CD4:p56lck (Diez-Orejas, R., Ballester, S., Feito, M. J., Ronda, M., Ojeda, G., Criado, G., Portolés, P. and Rojo, J. M., EMBO J. 1994. 13: 90). To analyze whether this is a major way of tyrosine kinase association to the TcR in normal CD4+ T cells, we examined the nature and mode of association of tyrosine kinases to the TcR complex in normal spleen CD4+ T lymphocytes. Our results show that, in normal CD4+ T lymphocytes, as in CD4+ T cell lines, there is a stable and readily detectable association between CD4:p56lck and the TcR/CD3 complex, as determined by in vitro kinase activity in immunoprecipitates from cell lysates. However, TcR/CD3 complexes from nature CD4+ lymphocytes have detectable amounts of p56lck associated in a CD4-independent manner, as shown by immunodepletion of the lysates with anti-CD4 antibodies. In addition, TcR/CD3 also bind p59fyn regardless of the presence of CD4. Conversely, we have observed that CD4 co-precipitates small quantities of p56fyn in a TcR/CD3-independent manner. Overall, our data suggest the existence of different possible molecular complexes between TcR/CD3, CD4 and their attending kinases, as well as some quantitative and qualitative differences between CD4+ T cells and CD4+ T cell lines in kinase association to the TcR/CD3 complex.
Assuntos
Complexo CD3/química , Antígenos CD4/química , Linfócitos T CD4-Positivos/enzimologia , Proteínas Tirosina Quinases/química , Animais , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-fyn , Transdução de Sinais , Quinases da Família src/químicaRESUMO
This work extends our previous finding that lymphocyte treatment with gp120IIIB specifically induces CD4 association with several surface molecules to other molecules and to three other gp120s from different HIV-1 strains. The ability to induce this association was displayed by the four gp120s employed, i.e. gp120IIIB, gp120SF2, gp120MN and gp120(451), and the association patterns were different, as shown by both co-capping and immunoprecipitation. Co-capping showed that all four gp120s significantly potentiated CD4 association with CD3, CD45RA, CD45RB, CD38, CD26, CD59 and class I MHC molecules. By contrast, CD4 association with CD95 was induced only by gp120(451) and gp120MN; that with CD11a only by gp120SF2 and gp120MN; and that with CD27 and CD45RO only by gp120MN and gp120(451) respectively. All gp120s induced significant CD4 association with CD49d, but gp120SF2 displayed a significantly weaker effect than gp120IIIB. Induction of association was not mediated by inside-out signaling via the CD4-associated tyrosine kinase p58lck, since it was not inhibited by the tyrosine kinase inhibitors herbymicin and genistein, nor by CD45 bridging between CD4 and the associating molecule, since similar patterns of association were detected IN cells expressing different CD45 isoform patterns. Moreover, it was not mediated by chemokine receptors interacting with the gp120 V3 loop, since RANTES did not alter the gp120-induced CD4 association pattern. By contrast, the observation that gp120s from four HIV-1 strains induce different CD4 association patterns suggests that gp120 directly interacts with the associating molecules, possibly via their hypervariable regions.
Assuntos
Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/farmacologia , Anticorpos Monoclonais , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1 , Antígenos HLA/metabolismo , Humanos , Subpopulações de LinfócitosRESUMO
The effect of CD4 expression on the activation threshold of mouse T lymphocytes has been analysed. To do this, the authors studied the response to antigen and other T cell receptor (TCR) ligands in a series of CD4- mutants obtained from the SR.D10 clone. This non-tumour clone spontaneously arose from the Th2 clone D10.G4.1, and characteristically shows a low threshold for antigen activation as well as reactivity to syngeneic antigen presenting cells (APC). Although SR.D10 CD4- mutant cells can be stimulated by antigen, they need higher antigen concentration or more APC than SR.D10 or CD4 transfectants to yield optimal antigen responses. Furthermore, CD4- clones are not activated by syngeneic APC or by clonotypic antibodies. These effects do not correlate with changes in the expression of cell surface molecules implicated in antigen recognition, like TCR/CD3, CD2, LFA-1, or CD45, or with lower p56lck or p59fyn activity in the mutant cells. Since inhibition experiments using anti-CD4 antibodies have previously shown that activation of the CD4+ T cell clone D10.G4.1 by antigen or alloantigens is largely dependent on CD4, our results indicate that activation by antigen-plus self MHC may become CD4-independent if the activation threshold is lowered enough, e.g. in cells like SR.D10. Expression of CD4 further lowers the activation threshold of the cells, allowing the detection of low-affinity TCR reactivities like those directed at self MHC. Moreover, by using anti-TCR/CD3 antibodies, the authors have confirmed the importance of CD4-associated tyrosine kinase activity in early TCR/CD3 signalling in this Th2 cell line, as (1) upon TCR/CD3 ligation, tyrosine phosphorylation is detected only in those CD3 chains co-precipitating with CD4; and (2) CD4 expression is needed for efficient early tyrosine phosphorylation and detectable p56lck-TCR co-precipitation.
Assuntos
Antígenos CD4/genética , Antígenos CD4/imunologia , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Linfócitos T/imunologia , Animais , Anticorpos/imunologia , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais , Apresentação de Antígeno , Northern Blotting , Antígenos CD2/imunologia , Complexo CD3/imunologia , Células Cultivadas , Células Clonais/imunologia , Clonagem Molecular , Citometria de Fluxo , Regulação da Expressão Gênica , Immunoblotting , Interleucina-1/genética , Antígenos Comuns de Leucócito/imunologia , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Plasmídeos , Testes de Precipitina , Proteínas Quinases/metabolismo , Proteínas Recombinantes/imunologia , Células Th2/imunologia , TransfecçãoRESUMO
Current models of the TCR / CD3 complex assume that, in mature peripheral T lymphocytes, variability is restricted to the alpha beta (or gamma delta) chains of the TCR heterodimer responsible for antigen recognition, whereas the CD3 polypeptides involved in signal transmission are invariant. Here we show that mouse CD4(+) T lymphocytes and T cell lines are bound with different avidity by anti-CD3 monoclonal antibodies. These findings cannot be accounted for by allelic differences between CD3 chains, by the nature of the TCR chains, or by the ratio of CD3epsilon delta to CD3epsilon gamma chain pairing. Rather, they are linked to heterogeneity of the N-terminal region of CD3epsilon chains, as detected by peptide-specific antibodies. In turn, these differences among CD3epsilon chains correlate with variations in the strength of TCR / CD3 interaction. N-terminal CD3epsilon heterogeneity is not due to alternative splicing mechanisms, but rather involves digestion by metalloproteases, as suggested by reverse transcription-PCR amplification and by the effect of protease inhibitors, respectively. Based on these data, we propose a model linking CD3epsilon N-terminal variability with altered CD3 recognition by monoclonal antibodies and TCR / CD3 interaction. This model suggests the possibility of distinct spatial arrangements of the TCR / CD3 complex.
Assuntos
Complexo CD3/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Alelos , Animais , Complexo CD3/genética , Variação Genética , CamundongosRESUMO
Cancer patients are often treated with biological response modifiers to enhance immunological functions. However, little is known about the actual mechanism of action of many of these substances. Therefore, we were interested in the effect of i.p. treatment with porcine low-molecular-weight spleen peptides, which are used during supportive cancer therapy, on lymphoid cell populations and function in mice. After treatment with 0.5 microgram peptides/kg body weight for 14 consecutive days, lymphokine secretion and the generation of cytotoxic T-cells were significantly enhanced as compared with controls. However, there was no effect on the number of cells or the percentage of cells expressing functional surface markers in secondary lymphoid organs.
Assuntos
Glicopeptídeos/imunologia , Fatores Imunológicos/imunologia , Fenóis/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais , Células Cultivadas , Combinação de Medicamentos , Injeções Intraperitoneais , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Linfocinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologiaRESUMO
Recent observations suggest that the tyrosine kinase p56lck is involved in the transduction of transmembrane signals through the antigen specific T cell receptor (TCR) in CD4+ T cells. By means of in vitro kinase assays, we have found that p56lck coprecipitated with the TCR from lysates of a murine CD4+ T cell line in the absence of TCR-mediated stimuli. Analysis of CD4- mutants and CD4-transfected cells shows that p56lck-TCR association occurred only when CD4 was present. The functional importance of CD4:p56lck-TCR association was demonstrated by low activating potential of rare clonotypic antibodies which did not coprecipitate CD4:p56lck, as well as by total or partial loss of anti-TCR or antigen induced stimulation in CD4- cells, which could be recovered by CD4 transfection. Complementation assays using different anti-TCR antibodies suggest that cross linking of TCR-p56lck:CD4 plus structural changes in the complex are needed for efficient transduction of activating signals through the TCR in these cells.
Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais , Animais , Complexo CD3/metabolismo , Antígenos CD4/genética , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Citometria de Fluxo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Camundongos , Camundongos Endogâmicos C3H , Mutação , Testes de Precipitina , TransfecçãoRESUMO
We have previously shown that HIV-1 glycoprotein 120 (gp120) induces CD4 association with several molecules on the surface of CD4+ lymphocytes. Since one of these molecules was CD38, involved in lymphocyte/endothelium interaction, this article examines the possibility that gp120/CD4 binding alters CD4+ T cell interaction with vascular endothelium in vitro and in vivo. Cocapping experiments showed that gp120 induced CD4 association with CD38, CD29, CD49d, and CD11a in peripheral blood CD4+ T cells. Two in vitro binding assays were used to evaluate the effect of gp120. A static binding assay, performed at 37 degrees C, evaluated stable interactions mediated by integrins, and a dynamic binding assay, performed at 4 degrees C on a rocking shelf, evaluated weak interactions mediated by constitutively active molecules such as selectins and CD38. Gp120 increased dynamic binding and inhibited static binding to the endothelium of peripheral blood CD4+ T cells and SUPT-1 cells. Binding inhibition with mAbs suggested that the gp120 effect on dynamic binding involved CD38, CD31, and CD49d, whereas the effect on static binding involved CD11a and CD49d. In vivo experiments showed that treatment of 2D4 cells, a CD4- CD8- mouse T cell clone transfected with the human CD4, with gp120 increased their homing into the spleen, intestine, and mesenteric lymph nodes, whereas it decreased homing into peripheral lymph nodes. Alteration of lymphocyte homing may contribute to immune deficiency in HIV-1+ patients by decreasing the probability of an encounter between Ags and lymphocytes and inhibiting the spread of effector lymphocytes into tissues.