RESUMO
BACKGROUND: Application of haptens to the skin induces release of immune stimulatory ATP into the extracellular space. This "danger" signal can be converted to immunosuppressive adenosine (ADO) by the action of the ectonucleotidases CD39 and CD73, expressed by skin and immune cells. Thus, the expression and regulation of CD73 by skin derived cells may have crucial influence on the outcome of contact hypersensitivity (CHS) reactions. OBJECTIVE: To investigate the role of CD73 expression during 2,4,6-trinitrochlorobenzene (TNCB) induced CHS reactions. METHODS: Wild type (wt) and CD73 deficient mice were subjected to TNCB induced CHS. In the different mouse strains the resulting ear swelling reaction was recorded along with a detailed phenotypic analysis of the skin migrating subsets of dendritic cells (DC). RESULTS: In CD73 deficient animals the motility of DC was higher as compared to wt animals and in particular after sensitization we found increased migration of Langerin+ DC from skin to draining lymph nodes (LN). In the TNCB model this led to a stronger sensitization as indicated by increased frequency of interferon-γ producing T cells in the LN and an increased ear thickness after challenge. CONCLUSION: CD73 derived ADO production slows down migration of Langerin+ DC from skin to LN. This may be a crucial mechanism to avoid over boarding immune reactions against haptens.
Assuntos
5'-Nucleotidase/metabolismo , Movimento Celular/imunologia , Células Dendríticas/imunologia , Dermatite Alérgica de Contato/imunologia , Pele/citologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Adenosina/imunologia , Adenosina/metabolismo , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Antígenos de Superfície/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Haptenos/administração & dosagem , Haptenos/imunologia , Interferon gama/metabolismo , Lectinas Tipo C/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cloreto de Picrila/administração & dosagem , Cloreto de Picrila/imunologia , Pele/imunologia , Pele/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Inflammatory bowel disease (IBD) is characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the etiology is poorly understood, it is widely accepted that loss of tolerance is involved in the development of IBD. Therefore, re-establishing tolerance or gut homeostasis is one of the key features in the development of new therapeutic strategies. Here we show that antigen targeting to DEC-205 on dendritic cells leads to an interleukin (IL)-10-dependent downregulation of C-X-C chemokine receptor 3 (CXCR3) expression on differentiated antigen-specific T helper type 1 (Th1) cells in vivo. This downregulation interferes with the migration of Th1 cells into the gut and protects mice against severe acute and relapsing intestinal inflammation. Moreover, CD4(+)CXCR3(+) T cells are highly enriched in the inflamed mucosa of IBD patients. Interference with this pathway may therefore be a promising approach for the treatment of IBD. In conclusion, we propose a hitherto undescribed mechanism by which IL-10 can act on effector T cells and orchestrate intestinal immune responses.
Assuntos
Antígenos CD/imunologia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Interleucina-10/imunologia , Lectinas Tipo C/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores CXCR3/imunologia , Receptores de Superfície Celular/imunologia , Células Th1/imunologia , Animais , Antígenos CD/genética , Antígenos CD4/genética , Antígenos CD4/imunologia , Movimento Celular , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Regulação da Expressão Gênica , Humanos , Tolerância Imunológica , Interleucina-10/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Lectinas Tipo C/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/genética , Receptores CXCR3/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Células Th1/patologiaRESUMO
Among antigen-presenting cells (APCs), dendritic cells as well as monocytes acquire immunostimulatory capacity only after appropriate maturation. Therefore, blockade of the maturation/activation results in a steady state or alternatively activated phenotype, which induces tolerance rather than immunity. Functional analyses revealed recently that steady-state dendritic cells and alternatively activated macrophages, respectively, actively induce regulatory T cells (Tregs) in the periphery of the body. Thus, production of Tregs does not rely exclusively on thymic development. Vice versa, Tregs respond to APCs by several means. Recent lines of evidence indicate that Tregs prevent terminal differentiation of subpopulations of APCs or lead to upregulation of surface expression of immunosuppressive molecules. Thus, Tregs foster an environment that further promotes their development. In conclusion, the mutual interaction of Tregs and APCs enables Tregs to sustain their immunosuppressive function(s), which in healthy individuals may be crucial for the maintenance of peripheral tolerance. Since macrophages bridge the innate and the acquired immune system, Tregs are able to gain influence on the innate immune system by interacting with macrophages beyond the mere interaction with effector T cells.
Assuntos
Apresentação de Antígeno , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Tolerância Imunológica , Macrófagos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Interleucina-10/metabolismo , Linfotoxina-alfa/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Dendritic cells (DC) utilize at least two pathways to process viral antigens onto MHC class I molecules. The conventional endogenous route is used to acquire antigens from both infectious and non-replicating virions. Exogenous pathways are used by DC to acquire and "cross-present" antigens derived from virus-infected donor cells that by themselves lack the ability to activate T cells directly. We analyzed the role of this pathway for antigens derived from vaccinia, a virus which inhibits DC maturation and causes extensive apoptosis of infected cells, yet is highly immunogenic. Using recombinant vaccinia virus encoding the influenza matrix protein as model vector, DC were shown to cross-present vaccinia-derived antigens from both apoptotic and necrotic infected cells to antigen-specific CD8(+) T cells. Efficient cross presentation required uptake of dead cells by immature DC and exposure to maturation stimuli, especially CD40 ligand. The responding CD8(+) T cells secreted IL-2 and IFN-gamma, proliferated and developed into cytotoxic effectors. Quantification of the cross presentation of vaccinia-derived antigens showed this pathway to be highly efficient, corresponding to a peptide pulse of 10-100 nM. While monocytes also phagocytosed apoptotic and necrotic cells, they were far less efficient at cross-presenting vaccinia-derived antigens to CD8(+) T cells. The ability of DC to cross-present vaccinia-derived antigens from infected apoptotic cells or necrotic cell lysates, bypasses the deleterious effects of direct infection of DC and provides one explanation for this pathogen's immunogenicity.
Assuntos
Apresentação de Antígeno , Antígenos Virais/imunologia , Células Dendríticas/fisiologia , Vaccinia virus/imunologia , Apoptose , Ligante de CD40/fisiologia , Humanos , Ativação Linfocitária , Necrose , Linfócitos T/imunologiaRESUMO
Dendritic cells (DCs) have the capacity to initiate immune responses, but it has been postulated that they may also be involved in inducing peripheral tolerance. To examine the function of DCs in the steady state we devised an antigen delivery system targeting these specialized antigen presenting cells in vivo using a monoclonal antibody to a DC-restricted endocytic receptor, DEC-205. Our experiments show that this route of antigen delivery to DCs is several orders of magnitude more efficient than free peptide in complete Freund's adjuvant (CFA) in inducing T cell activation and cell division. However, T cells activated by antigen delivered to DCs are not polarized to produce T helper type 1 cytokine interferon gamma and the activation response is not sustained. Within 7 d the number of antigen-specific T cells is severely reduced, and the residual T cells become unresponsive to systemic challenge with antigen in CFA. Coinjection of the DC-targeted antigen and anti-CD40 agonistic antibody changes the outcome from tolerance to prolonged T cell activation and immunity. We conclude that in the absence of additional stimuli DCs induce transient antigen-specific T cell activation followed by T cell deletion and unresponsiveness.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/imunologia , Antígeno B7-2 , Antígenos CD40/imunologia , Feminino , Linfócitos/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Muramidase/imunologia , Receptores de Superfície Celular/imunologia , Baço/citologiaRESUMO
Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Proteínas da Matriz Viral/imunologia , Apoptose/imunologia , Linfócitos B , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Transformada , Reações Cruzadas , Humanos , Necrose , Fagocitose/imunologia , Latência ViralRESUMO
Liposomes have been proposed as a vehicle to deliver proteins to antigen-presenting cells (APC), such as dendritic cells (DC), to stimulate strong T cell-mediated immune responses. Unfortunately, because of their instability in vivo and their rapid uptake by cells of the mononuclear phagocyte system on intravenous administration, most types of conventional liposomes lack clinical applicability. In contrast, sterically stabilized liposomes (SL) have increased in vivo stability. It is shown that both immature and mature DC take up SL into neutral or mildly acidic compartments distinct from endocytic vacuoles. These DC presented SL-encapsulated protein to both CD4(+) and CD8(+) T cells in vitro. Although CD4(+) T-cell responses were comparable to those induced by soluble protein, CD8(+) T-cell proliferation was up to 300-fold stronger when DC had been pulsed with SL-encapsulated ovalbumin. DC processed SL-encapsulated antigen through a TAP-dependent mechanism. Immunization of mice with SL-encapsulated ovalbumin led to antigen presentation by DC in vivo and stimulated greater CD8(+) T-cell responses than immunization with soluble protein or with conventional or positively charged liposomes carrying ovalbumin. Therefore, the application of SL-encapsulated antigens offers a novel effective, safe vaccine approach if a combination of CD8(+) and CD4(+) T-cell responses is desired (ie, in anti-viral or anti-tumor immunity).
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Lipossomos/farmacocinética , Proteínas/imunologia , Transferência Adotiva/métodos , Transferência Adotiva/normas , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Composição de Medicamentos/métodos , Composição de Medicamentos/normas , Sistemas de Liberação de Medicamentos/normas , Estabilidade de Medicamentos , Corantes Fluorescentes , Humanos , Lipossomos/administração & dosagem , Linfonodos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/farmacologia , Proteínas/administração & dosagem , Proteínas/farmacologia , Toxina Tetânica/administração & dosagem , Toxina Tetânica/imunologia , Toxina Tetânica/farmacologiaRESUMO
Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-alpha) secretion and was significantly blocked in the presence of anti-TNF-alpha antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-alpha and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-alpha secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.
Assuntos
Apoptose , Avipoxvirus/fisiologia , Células Dendríticas/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Células Dendríticas/virologia , Humanos , Macaca mulatta , Fagocitose , Fator de Necrose Tumoral alfa/metabolismoRESUMO
DEC-205 is a multilectin receptor for adsorptive endocytosis, expressed in mouse dendritic cells (DC) and some epithelia. DEC-205 is homologous to the macrophage mannose receptor (MMR). A cDNA for murine DEC-205 was used to identify 3 overlapping human DEC-205 clones from a lymphocyte library. The human homologue is a transmembrane protein of 1722 aminoacids with 10 externally disposed C-type lectin domains having 77% identity to the mouse counterpart. The NH(2) terminal cysteine-rich and fibronectin type II domains were expressed and used to immunize mice. A hybridoma, MG38, which specifically recognized the immunogen was obtained from a DEC-205 knockout mouse. The antibody precipitated a 205 kD protein from metabolically labeled, monocyte-derived DCs. MG38 labeled mature monocyte-derived DCs but showed weak or no labeling of other peripheral blood mononuclear cells. In tissue sections, MG38 identified DEC-205 on thymic cortical epithelium and DCs in the thymic medulla and tonsillar T cell areas. In contrast, an anti-MMR antibody stained DEC-205 negative, macrophages in the thymus cortex, the trabeculae of the thymus and tonsil, as well as efferent lymphatics in the tonsil. Therefore, the MG38 anti-DEC-205 antibody is useful for identifying DCs and reveals clear differences in sites where MMR and DEC-205 are expressed in lymphoid tissues.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD , Células Dendríticas/imunologia , Endocitose/imunologia , Lectinas Tipo C , Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Animais , Linhagem Celular Transformada , Células Dendríticas/citologia , Citometria de Fluxo , Expressão Gênica , Humanos , Lipopolissacarídeos/imunologia , Tecido Linfoide , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Monócitos/citologia , Monócitos/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificaçãoRESUMO
Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.
Assuntos
Adjuvantes Imunológicos/genética , Apresentação de Antígeno/genética , Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Transfecção/imunologia , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/uso terapêutico , Células da Medula Óssea/metabolismo , Diferenciação Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Células Dendríticas/metabolismo , Feminino , Técnicas de Transferência de Genes , Haptenos/imunologia , Haptenos/metabolismo , Hemocianinas/imunologia , Hemocianinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Moluscos , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/terapia , Transfecção/métodos , Células Tumorais CultivadasRESUMO
The neuropeptide, alpha-melanocyte-stimulating hormone (alpha-MSH) is well known for its immunomodulating capabilities. alpha-MSH antagonizes the activity of numerous proinflammatory mediators; for example, Interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha (TNF alpha), and bacterial endotoxin. In vivo alpha-MSH has been shown to suppress a contact hypersensitivity reaction in mice, and to induce hapten-specific tolerance. Since antigen presenting cells (APC) represent key elements for tolerance induction, the effect of alpha-MSH, and the expression of its receptor-melanocortin receptor-1 (MC-1R), on human peripheral blood-derived monocytes and dendritic cells (DC), was investigated. Semiquantitative RT-PCR demonstrated that monocytes and DC express MC-1R, but none of the other members of the MC-receptor family. Moreover, the extent of MC-1R expression correlated with the state of activation of these cells. Since the major ligand of MC-1R is alpha-MSH the question of whether alpha-MSH affects the function of monocyte derived DC was further investigated. We found that the expression of the costimulatory molecules CD 86 and CD 40 was downregulated on DC in the presence of alpha-MSH. Thus, alpha-MSH may exert its immunosuppressive effects by altering the function of APC.
Assuntos
Células Dendríticas/fisiologia , Receptores da Corticotropina/genética , Receptores da Corticotropina/metabolismo , alfa-MSH/farmacologia , Animais , Antígenos CD/genética , Antígeno B7-2 , Antígenos CD40/genética , Membrana Celular/metabolismo , Células Cultivadas , Células Dendríticas/citologia , Regulação da Expressão Gênica/imunologia , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Monócitos/citologia , Monócitos/fisiologia , Receptores de Melanocortina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-MSH/fisiologiaRESUMO
Cells from the bone marrow can present peptides that are derived from tumors, transplants, and self-tissues. Here we describe how dendritic cells (DCs) process phagocytosed cell fragments onto major histocompatibility complex (MHC) class II products with unusual efficacy. This was monitored with the Y-Ae monoclonal antibody that is specific for complexes of I-Ab MHC class II presenting a peptide derived from I-Ealpha. When immature DCs from I-Ab mice were cultured for 5-20 h with activated I-E+ B blasts, either necrotic or apoptotic, the DCs produced the epitope recognized by the Y-Ae monoclonal antibody and stimulated T cells reactive with the same MHC-peptide complex. Antigen transfer was also observed with human cells, where human histocompatibility leukocyte antigen (HLA)-DRalpha includes the same peptide sequence as mouse I-Ealpha. Antigen transfer was preceded by uptake of B cell fragments into MHC class II-rich compartments. Quantitation of the amount of I-E protein in the B cell fragments revealed that phagocytosed I-E was 1-10 thousand times more efficient in generating MHC-peptide complexes than preprocessed I-E peptide. When we injected different I-E- bearing cells into C57BL/6 mice to look for a similar phenomenon in vivo, we found that short-lived migrating DCs could be processed by most of the recipient DCs in the lymph node. The consequence of antigen transfer from migratory DCs to lymph node DCs is not yet known, but we suggest that in the steady state, i.e., in the absence of stimuli for DC maturation, this transfer leads to peripheral tolerance of the T cell repertoire to self.
Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Fagocitose/imunologia , Animais , Células Dendríticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Recent studies showed that injection of interleukin (IL)-12 prevents ultraviolet (UV) light mediated suppression of contact hypersensitivity and breaks UV-induced hapten specific tolerance. UV-mediated suppression can be adoptively transferred by injecting splenocytes from UV-irradiated mice; however, suppression is not transferable when donor mice are treated with IL-12 after UV-irradiation. This study was performed to elucidate the mechanisms by which IL-12 counteracts this immunosuppression. To characterize the cells transferring suppression, depletion studies were performed revealing that UV-induced suppression is transferred via CD8+ T cells. To investigate whether IL-12 counteracts UV-induced suppression by either inhibiting the development of CD8+ suppressor T cells or inducing CD4+ effector T cells, splenocytes from mice, which were IL-12 treated and sensitized through UV-exposed skin, were depleted from CD4+ T cells and transferred into naive mice that were subsequently sensitized. Whereas transfer of splenocytes from UV-irradiated mice inhibited sensitization of recipients, no inhibition was observed after transfer of splenocytes from UV-exposed and IL-12 treated mice. Recipients that received CD4 depleted spleen cells from UV-exposed and IL-12 treated donors, were still fully sensitizable. IL-12 also blocked transfer of UV-induced suppression when it was injected into UV-exposed donor animals at a time point when suppressor cells had already developed. CD4 depletion of such splenocytes did not result in a loss of the reconstitutive effect of IL-12. This suggests that IL-12 may break UV-induced tolerance not by inducing CD4+ effector T cells, but rather by inhibiting or inactivating suppressor T cells belonging to the CD8 subtype.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos da radiação , Terapia de Imunossupressão/métodos , Interleucina-12/farmacologia , Raios Ultravioleta , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos da radiação , Linfócitos T CD8-Positivos/transplante , Dermatite de Contato/prevenção & controle , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/efeitos da radiação , Camundongos , Camundongos Endogâmicos C3HRESUMO
Irradiation with ultraviolet (UV) B light results in the formation of apoptotic keratinocytes called sunburn cells (SC). Although generation of SC appears to be one of the most characteristic features of UV-induced skin damage and has been a well-known phenomenon for a long time, the mechanisms involved are not quite clear. Recently, it was demonstrated that tumor necrosis factor alpha (TNF alpha) appears to be involved in the formation of SC since neutralization of TNF alpha both in vitro and in vivo reduced UVB-induced apoptosis of keratinocytes. Pentoxifylline is a methylxanthine derivative suppressing the release of TNF alpha. Therefore, we studied whether PTX is able to prevent the formation of SC. Addition of PTX to UVB-exposed HaCaT cells reduced DNA-fragmentation as examined by nick translation evaluated by flowcytometry. To prove whether PTX also reduces UVB-induced apoptosis in vivo, BALB/c mice were exposed to UVB on their abdomens, skin biopsies performed 24 h later and SC counted. A single dose of 2000 J/m2 caused a significant induction of SC which were remarkably reduced when PTX was injected intraperitoneally 3 h before and 12 h after UVB exposure. In summary, the data demonstrate that PTX can reduce the formation of SC both in vitro and in vivo and thus further support that TNF alpha is involved in UVB-induced apoptosis of keratinocytes.
Assuntos
Queratinócitos/efeitos da radiação , Pentoxifilina/uso terapêutico , Pele/patologia , Queimadura Solar/patologia , Vasodilatadores/uso terapêutico , Animais , Apoptose/efeitos da radiação , Linhagem Celular Transformada , Células Epidérmicas , Epiderme/efeitos dos fármacos , Feminino , Humanos , Injeções Intraperitoneais , Queratinócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Pentoxifilina/administração & dosagem , Queimadura Solar/prevenção & controle , Raios Ultravioleta/efeitos adversos , Vasodilatadores/administração & dosagemRESUMO
In vivo, dendritic cells (DC) reside in direct proximity to extracellular matrix (ECM) proteins. Because ECM proteins affect morphology and function of a number of cell types, this study investigated potential effects of ECM proteins on functional properties of DC. DC were generated from murine bone marrow cultures, supplemented with granulocyte-macrophage colony-stimulating factor, and subsequently cultured on tissue culture plates coated with various ECM proteins. Among the ECM proteins tested, collagen (COL) up-regulated the T cell stimulatory capacity of DC. This effect was accompanied by sustained surface expression of the co-stimulatory molecule heat stable antigen on DC and by enhanced release of interleukin-1 and interleukin-6, respectively. Because fibronectin or solubilized COL were unable to cause similar changes in DC phenotype or function, we conclude that adherence to COL interferes specifically with DC function. These data suggest that ECM proteins may be involved in regulation of DC phenotype as well as in their functional activation.
Assuntos
Antígenos CD/metabolismo , Colágeno/fisiologia , Citocinas/metabolismo , Células Dendríticas/fisiologia , Matriz Extracelular/fisiologia , Ativação Linfocitária , Glicoproteínas de Membrana , Animais , Células da Medula Óssea , Antígeno CD24 , Adesão Celular , Células Cultivadas , Feminino , Imunidade Celular , Camundongos , Camundongos Endogâmicos BALB C , Regulação para CimaRESUMO
Irradiation with ultraviolet (UV) B radiation results in the formation of apoptotic keratinocytes called sunburn cells. Recently, it was demonstrated that keratinocytes can release tumor necrosis factor-alpha (TNF-alpha), which is known to cause apoptosis in particular cells. In addition, it has been shown that UVB light induces the release of TNF-alpha by keratinocytes and that keratinocytes express the 55-kD receptor for TNF-alpha. Therefore, we investigated whether TNF-alpha is involved in UV-induced apoptosis of keratinocytes. Normal human keratinocytes and HaCaT cells were exposed to UVB light, and apoptosis was examined by nick translation evaluated by fluorescence-activated cell sorter analysis. UVB induced apoptosis in a dose-dependent manner, which was confirmed by electron microscopy. Addition of a polyclonal antibody directed against human TNF-alpha immediately after UVB exposure was able to reduce DNA fragmentation. However, it was not possible to rescue all cells from apoptosis. To prove whether TNF-alpha is also involved in vivo in UVB-induced apoptosis of keratinocytes, Balb/c mice were exposed to UVB on their abdomens, skin biopsies were performed 24 h later, and sunburn cells were counted. A single dose of 2000 J/m2 caused a significant induction of sunburn cells. Subcutaneous injection of a polyclonal antibody directed against murine TNF-alpha immediately after UVB treatment resulted in a significant but incomplete reduction of sunburn cells, whereas injection of a rabbit IgG as a control had no effect. In both the in vitro and in vivo systems, application of recombinant TNF-alpha alone either to untreated keratinocytes or into normal murine skin did not induce sunburn cells. Thus, these data demonstrate that TNF-alpha is involved in UVB-induced apoptosis, but by itself is not able to induce sunburn cells. This further supports the notion that UVB-induced apoptosis of keratinocytes is a multifactorial event.
Assuntos
Apoptose/efeitos da radiação , Queratinócitos/citologia , Queimadura Solar/etiologia , Fator de Necrose Tumoral alfa/fisiologia , Raios Ultravioleta , Animais , Anticorpos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The 27E10 antigen is a heterodimer of MRP8 and MRP14, two Ca(2+)-binding proteins related to the S-100 protein family. Previous studies have shown that 27E10 epitope-bearing monocyte subsets are prevalent in early acute but absent in chronic inflammatory conditions. These observations further provide an impetus for identifying the cellular mechanisms responsible for the appearance of different monocyte subpopulations during inflammation. Therefore this in vitro study was carried out to investigate the influence of adhesion in inducing 27E10-positive subsets. In adhesion assays the role of 27E10 antigen in spontaneous adherence was obvious, as a monoclonal antibody directed against the 27E10 antigen significantly inhibited the adherence of monocytes to collagen and fibronectin. In contrast, these extracellular matrix (ECM) proteins induce the cell surface expression and association of 27E10 antigen with cytoskeleton (CSK), detected by flow cytometry and confocal laser scan microscopy, respectively. Similar results were obtained on cross-linking with specific antibodies, thus showing involvement of the integrin molecules VLA-2 and VLA-4. In addition, the association with CSK could be confirmed by differential detergent extraction. The observed redistribution of 27E10 antigen guided by collagen compared with fibronectin was also paralleled by an augmented release of inflammatory cytokines interleukin-6, tumor necrosis factor alpha, and superoxide anions. Thus, this study demonstrates that under inflammatory conditions the interactions of extravasating monocytes with the ECM may induce an activated phenotype of monocytes marked by 27E10.
Assuntos
Proteínas de Ligação ao Cálcio/biossíntese , Proteínas da Matriz Extracelular/metabolismo , Interleucina-6/biossíntese , Monócitos/metabolismo , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Antígenos/análise , Antígenos de Diferenciação/análise , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/química , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/química , Calgranulina A , Calgranulina B , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Colágeno/farmacologia , Proteínas da Matriz Extracelular/fisiologia , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Citometria de Fluxo , Humanos , Microscopia Confocal , Monócitos/citologiaRESUMO
Helicobacter pylori colonization of the human gastric mucosa causes a long-term, not self-limiting inflammation, suggesting that the microbe has properties to protect itself against the host immune defence system. Recently we were able to demonstrate that H. pylori suppresses the in vitro proliferative response of human peripheral blood mononuclear cells to antigens as well as to mitogens without affecting cell viability. The purpose of this study was to clarify which cell subsets of mononuclear cells are influenced by H. pylori. The use of monocytes which had been pretreated with a soluble cytoplasmic fraction of H. pylori (30 micrograms ml-1) led to a suppressed proliferation of T cells after PHA-activation. Activation of isolated T cells with PHA and PMA revealed that the proliferative response of lymphocytes could also be inhibited independently of monocytes. The anti-proliferative effect was associated with a reduction of IL-2 receptor (CD25) expression as well as an inhibition of blastogenesis. Furthermore, the spontaneous proliferation of EBV-transformed B cell lines was suppressed in a dose-dependent manner. FACS-analysis of HLA-DR, ICAM-1 and CD14 expression on the surface of monocytes revealed an influence of H. pylori on CD14 expression at a concentration of 30 micrograms ml-1, while the expression of HLA-DR and ICAM-1 was not affected at this concentration.
Assuntos
Helicobacter pylori/imunologia , Monócitos/imunologia , Linfócitos T/imunologia , Antígenos CD/biossíntese , Antígenos de Superfície/biossíntese , Linfócitos B/imunologia , Moléculas de Adesão Celular/biossíntese , Transformação Celular Viral , Citoplasma/imunologia , Antígenos HLA-DR/biossíntese , Humanos , Tolerância Imunológica/imunologia , Técnicas In Vitro , Molécula 1 de Adesão Intercelular , Ativação Linfocitária/efeitos dos fármacos , Fito-Hemaglutininas/farmacologia , Frações Subcelulares/imunologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Monocytes/macrophages expressing an epitope recognized by a monoclonal antibody 27E10 are present in acute but are absent in chronic inflammatory disorders. This report shows that the 27E10 antigen is formed by noncovalent association of the two Ca(2+)-binding proteins MRP8 and MRP14 which belong to the S100 protein family. Identification has been confirmed immunochemically, by matrix-assisted UV-laser desorption/ionization spectrometry and by partial amino acid sequencing. Surface expression of the MRP8/MRP14 complex on a subset of monocytes is reported for the first time and shown to be up-regulated in a Ca(2+)-dependent manner. The 27E10 surface-positive monocytes isolated by cell separation techniques release high amounts of tumor necrosis factor-alpha and interleukin-1 beta in contrast to their 27E10 surface-negative counterparts thus emphasizing their role in inflammation.