RESUMEN
BACKGROUND: 1α,25-dihydroxyvitamin D(3) (calcitriol), the biologically active form of vitamin D, is an immunomodulatory hormone, e.g. it inhibits IgE synthesis in B cells. As its clinical application is limited by hypercalcemia, synthetic vitamin D receptor (VDR) agonists that mediate immunomodulatory activities without adverse hypercalcemic effects are of great interest. This study aimed to investigate the impact of a low-calcemic VDR agonist on the IgE immune response in vitro and in vivo. METHODS: Human peripheral B cells were cultured under IgE inducing conditions in the presence of VDR ligands. B cells were analyzed by quantitative RT-PCR, enzyme-linked immunosorbent assays, enzyme-linked immunospot technique, and flow cytometry. BALB/c mice were sensitized with ovalbumin (OVA)/alum followed by the therapeutic VDR agonist treatment and analyzed regarding the humoral immunoglobulin profile. RESULTS: The natural VDR ligand calcitriol, but also a low-calcemic VDR agonist, profoundly suppressed IgE production by human peripheral B cells by 63.9 ± 5.9%. The potential mechanisms involved are the reduction of the transcript for activation-induced deaminase (AID) and the reduction of IgE immunoglobulin-secreting cells by 68.1 ± 12.7%. By using an in vivo approach, we finally demonstrate that the humoral IgE response in a type I allergy mouse model was impaired by the VDR agonist. CONCLUSION: Our results show that targeting the VDR modulates the humoral immune response including IgE. Whether it might be useful for clinical applications has to be determined in appropriate clinical trials.
Asunto(s)
Linfocitos B/inmunología , Hipersensibilidad/inmunología , Receptores de Calcitriol/inmunología , Animales , Linfocitos B/efectos de los fármacos , Calcitriol/farmacología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina E/biosíntesis , Inmunoglobulina E/inmunología , Ratones , Ratones Endogámicos BALB C , Receptores de Calcitriol/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vitaminas/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Glucocorticoids are highly effective in the therapy of inflammatory diseases. Their value, however, is limited by side effects. The discovery of the molecular mechanisms of the glucocorticoid receptor and the recognition that activation and repression of gene expression could be addressed separately opened the possibility of achieving improved safety profiles by the identification of ligands that predominantly induce repression. Here we report on ZK 245186, a novel, non-steroidal, low-molecular-weight, glucocorticoid receptor-selective agonist for the topical treatment of inflammatory dermatoses. EXPERIMENTAL APPROACH: Pharmacological properties of ZK 245186 and reference compounds were studied in terms of their potential anti-inflammatory and side effects in functional bioassays in vitro and in rodent models in vivo. KEY RESULTS: Anti-inflammatory activity of ZK 245186 was demonstrated in in vitro assays for inhibition of cytokine secretion and T cell proliferation. In vivo, using irritant contact dermatitis and T cell-mediated contact allergy models in mice and rats, ZK 245186 showed anti-inflammatory efficacy after topical application similar to the classical glucocorticoids, mometasone furoate and methylprednisolone aceponate. ZK 245186, however, exhibits a better safety profile with regard to growth inhibition and induction of skin atrophy after long-term topical application, thymocyte apoptosis, hyperglycaemia and hepatic tyrosine aminotransferase activity. CONCLUSIONS AND IMPLICATIONS: ZK 245186 is a potent anti-inflammatory compound with a lower potential for side effects, compared with classical glucocorticoids. It represents a promising drug candidate and is currently in clinical trials.
Asunto(s)
Antiinflamatorios/farmacología , Benzofuranos/farmacología , Inflamación/tratamiento farmacológico , Pentanoles/farmacología , Quinolinas/farmacología , Receptores de Glucocorticoides/agonistas , Enfermedades de la Piel/tratamiento farmacológico , Piel/efectos de los fármacos , Administración Tópica , Animales , Antiinflamatorios/administración & dosificación , Benzofuranos/administración & dosificación , Evaluación Preclínica de Medicamentos , Ratones , Ratones Endogámicos , Pentanoles/administración & dosificación , Quinolinas/administración & dosificación , Ratas , Ratas Wistar , Sensibilidad y EspecificidadRESUMEN
AIM: To investigate the effects of ZK1916784, a low calcemic analog of calcitriol on intestinal inflammation. METHODS: Acute and chronic colitis was induced by dextran sodium sulfate (DSS) according to standard procedures. Mice were treated intraperitoneally with ZK1916784 or placebo and colonic inflammation was evaluated. Cytokine production by mesenterial lymph node (MLN) cells was measured by ELISA. Immunohistochemistry was performed to detect intestinal dendritic cells (DCs) within the colonic tissue, and the effect of the calcitriol analog on DCs was investigated. RESULTS: Treatment with ZK191784 resulted in significant amelioration of disease with a reduced histological score in acute and chronic intestinal inflammation. In animals with acute DSS colitis, down-regulation of colonic inflammation was associated with a dramatic reduction in the secretion of the proinflammatory cytokine interferon (IFN)-gamma and a significant increase in intereleukin (IL)-10 by MLN cells. Similarly, in chronic colitis, IL-10 expression in colonic tissue increased 1.4-fold when mice were treated with ZK191784, whereas expression of the Th1-specific transcription factor T-beta decreased by 81.6%. Lower numbers of infiltrating activated CD11c+ DCs were found in the colon in ZK191784-treated mice with acute DSS colitis, and secretion of proinflammatory cytokines by primary mucosal DCs was inhibited in the presence of the calcitriol analog. CONCLUSION: The calcitriol analog ZK191784 demonstrated significant anti-inflammatory properties in experimental colitis that were at least partially mediated by the immunosuppressive effects of the derivate on mucosal DCs.
Asunto(s)
Calcitriol/análogos & derivados , Colitis/tratamiento farmacológico , Células Dendríticas/patología , Intestinos/patología , Fenotipo , Enfermedad Aguda , Animales , Calcitriol/farmacología , Recuento de Células , Enfermedad Crónica , Colitis/inducido químicamente , Colitis/patología , Células Dendríticas/efectos de los fármacos , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Intestinos/efectos de los fármacos , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: The biologic role of 1,25-dihydroxyvitamin D(3), such as anti-inflammatory functions, reduction of cytokine production by T cells and immunoglobulin production by B cells, is well established. However, its clinical use as an immunosuppressive agent is limited because of the hypercalcemic toxicity occurring after systemic application. The purpose of this study was to investigate the immunmodulatory effects of 22-ene-25-oxa-vitamin D (ZK156979), a novel low calcemic vitamin D analogue. MATERIALS AND METHODS: Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using the Ficoll Hypaque technique, cultured for 24 h and treated with different concentrations of ZK156979 ranging from 10(-5) to 10(-10) mol L(-1) compared with 1,25-dihydroxyvitamin D(3)[10(-5)-10(-10) mol L(-1)] following phytohaemagglutinin (PHA) stimulation. Interferon gamma (IFNgamma), tumour necrosis factor alpha (TNFalpha), interleukin 1 beta (IL-1beta), interleukin 10 (IL-10) and interleukin 4 (IL-4) secretion in supernatants were measured by ELISA. RESULTS: ZK156979 inhibited the PHA-induced Th1-response (IFNgamma and TNFalpha levels) and the macrophage-product IL-1beta in a concentration-dependent manner (10(-10)-10(-5) mol L(-1)) with the efficiency on cytokine expression compared with 1,25-dihydroxyvitamin D(3) being slightly reduced. In contrast, ZK156979 and 1,25-dihydroxyvitamin D(3) both affected the Th2 response, leading to significantly increased IL-10- and IL-4 secretion. CONCLUSIONS: ZK156979 is a member of novel vitamin D analogues revealing prominent immunomodulatory and suppressive characteristics with distinctive inhibition of Th1-cytokines whereas the Th2 compartment is augmented, thus providing a considerable therapeutic potential in T-cell -mediated diseases.
Asunto(s)
Citocinas/inmunología , Inmunosupresores/farmacología , Linfocitos T Colaboradores-Inductores/inmunología , Vitamina D/análogos & derivados , Calcitriol/inmunología , Calcitriol/farmacología , Células Cultivadas , Citocinas/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Humanos , Interferón gamma/análisis , Interleucina-1/inmunología , Interleucina-10/análisis , Interleucina-4/análisis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/análisis , Vitamina D/farmacologíaRESUMEN
This work demonstrates that gp96 preparations isolated from cells infected with intracellular bacteria induce cytotoxic T-lymphocyte responses and confer protection. Our findings extend previous reports on the immunogenicity of gp96-associated peptides to antigens derived from intracellular bacteria. Immunization with gp96 may therefore represent a promising vaccination strategy against bacterial pathogens.
Asunto(s)
Proteínas de Choque Térmico/inmunología , Listeriosis/prevención & control , Péptidos/inmunología , Tuberculosis/prevención & control , Vacunación , Animales , Linfocitos T CD8-positivos/inmunología , Proteínas de Choque Térmico/metabolismo , Listeria monocytogenes/inmunología , Listeria monocytogenes/aislamiento & purificación , Listeria monocytogenes/metabolismo , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/inmunología , Péptidos/metabolismo , Bazo/microbiologíaRESUMEN
1Alpha,25-dihydroxyvitamin D3 diastereomer, differing from the parent compound in configuration at four asymmetric carbon atoms in the rings C/D and side chain (C13, C14, C17 and C20), was synthesized and shown to have a significant affinity for the vitamin D receptor.
Asunto(s)
Calcitriol/síntesis química , Calcitriol/metabolismo , Receptores de Calcitriol/metabolismo , Calcitriol/química , Conformación Molecular , Estructura Molecular , Unión Proteica , EstereoisomerismoRESUMEN
Besides the classical major histocompatibility complex (MHC) class I and MHC class II molecules, human CD1 molecules have been shown to present mycobacterial antigens in vitro. In this study, in vivo treatment of mice with anti-CD1 monoclonal antibodies resulted in exacerbated tuberculosis at very early time points. In CD1-modulated mice, Mycobacterium tuberculosis-specific production of the type 1 cytokines, IL-12, TNF, and IFN-gamma as well as of TGF beta was reduced. These findings suggest an antigen-presenting role of CD1 molecules in tuberculosis.
Asunto(s)
Antígenos CD1/fisiología , Citocinas/metabolismo , Tuberculosis Pulmonar/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD1/inmunología , Recuento de Colonia Microbiana , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II/genética , Interferón gamma/metabolismo , Interleucina-12/metabolismo , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Tuberculosis Pulmonar/microbiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD8-positivos/inmunología , Chaperonina 60/inmunología , Intestino Delgado/patología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos T CD8-positivos/metabolismo , Reacciones Cruzadas , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/metabolismo , Intestino Delgado/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Heat shock proteins (hsp) are conserved molecules that play an important role in protein folding and assembly and in translocation of proteins between different compartments. Under stress, hsp synthesis is drastically increased, representing a mechanism essential for cell survival. During infection or inflammation, numerous hsp are overexpressed. Not surprisingly, hsp represent dominant antigens in many infectious and autoimmune diseases that induce strong humoral and cellular immune responses. There is substantial evidence that hsp are dominant immune targets in a number of diseases, to the benefit or detriment of man. Nevertheless, findings also exist which argue against a universal role for hsp as target antigens in disease situations. It is suggested that hsp mainly serve as 'early' targets in the immune response, thus providing support for anti-infectious or autoaggressive immune responses directed against unique pathogen- or disease-associated antigens, respectively.
Asunto(s)
Enfermedades Transmisibles/inmunología , Proteínas de Choque Térmico/inmunología , Animales , Humanos , Inmunidad Innata/inmunología , VacunaciónRESUMEN
Increased synthesis of heat shock proteins (hsp) occurs in prokaryotic and eukaryotic cells when they are exposed to stress. By increasing their hsp content, cells protect themselves from lethal assaults, primarily because hsp interfere with the uncontrolled protein unfolding that occurs under stress. However, hsp are not produced only by stressed cells; some hsp are synthesized constitutively and perform important housekeeping functions. Accordingly, hsp are involved in the assembly of molecules which play important roles in the immune system. It is not surprising that due to their wide distribution and their homology among different species, hsp represent target antigens of the immune response. Frequent confrontation of the immune system with conserved regions of hsp which are shared by various microbial pathogens can potentiate antimicrobial immunity. However, long-term confrontation of the immune system with hsp antigens which are similar in the host and invaders may convert the immune response against these host antigens and promote autoimmune disease. This review provides an overview of the role of hsp in immunity with a focus on infectious and autoimmune diseases.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Infecciones/etiología , Infecciones/inmunología , Animales , Proteínas de Choque Térmico/inmunología , HumanosRESUMEN
Treating mice with an immunodominant T cell epitope from moth cytochrome c (MCC(88-103)) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC(88-103) in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC(88-103)-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC(88-103), were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.
Asunto(s)
Grupo Citocromo c/inmunología , Epítopos de Linfocito T/inmunología , Tolerancia Inmunológica/inmunología , Fragmentos de Péptidos/inmunología , Animales , Columbidae , Reacciones Cruzadas , Grupo Citocromo c/administración & dosificación , Epítopos de Linfocito T/administración & dosificación , Epítopos Inmunodominantes/administración & dosificación , Epítopos Inmunodominantes/inmunología , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos C57BL , Imitación Molecular/inmunología , Mariposas Nocturnas , Fragmentos de Péptidos/administración & dosificaciónRESUMEN
Heat shock protein 60 (hsp60)-specific CD8 T cells lysed Mycobacterium bovis BCG-infected macrophages in vitro and adoptively transferred protection against mycobacterial infection. Moreover, CD8 T cells with this hsp60 specificity were activated in vivo by BCG vaccination. Our studies suggest there is participation of hsp60-specific CD8 T cells in BCG-induced immunity.
Asunto(s)
Vacuna BCG/inmunología , Linfocitos T CD8-positivos/inmunología , Chaperonina 60/inmunología , Activación de Linfocitos , Mycobacterium bovis/inmunología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Citotoxicidad Inmunológica , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Bazo/inmunologíaRESUMEN
Potential anti-leukemia effects mediated by T cells or by natural killer (NK) cells were investigated in chronic myelogenous leukemia (CML) patients treated with interferon-alpha. Therapy-associated modulation of T cell and NK reactivity was monitored for one year from initiation in autologous mixed lymphocyte-tumor cell reactions and cytotoxicity directed against autologous CML cells, respectively. During the course of IFN-therapy, NK activity against autologous CML cells increased steadily, whereas T cell reactivity fluctuated randomly. Despite the high level of T cell reactivity to autologous tumor cells in short-term (6 days) culture, 1) they failed to respond to synthetic peptides corresponding to the bcr/abl fusion sequence of the patient, and 2) only one proliferative T cell clone (TCC) was isolated which specifically recognized HLA-DR-matched CML cells. This TCC appeared not to recognize synthetic peptides corresponding to the bcr/abl fusion sequence of the patient; the antigen to which it responds remains unknown. To assess potential immunogenicity of bcr/abl peptides, it was attempted to sensitize T cells from normal donors in vitro. Of 109 cell lines obtained from seven different donors, eleven showed peptide-dependent proliferation. Therefore, although these results show that it is possible to isolate apparently CML-specific T cells from patients, as well as to prime T cells against tumor-specific peptide in vitro, the frequency of such T cell-mediated reactivity appears low and its relevance to anti-leukemic effects questionable. On the other hand, the strong time-dependent enhancement of natural killing of autologous CML blasts during IFN-alpha treatment, a phenomenon not observed for T cell reactivity, suggests that natural immunity may be more important in controlling disease.
Asunto(s)
Interferón-alfa/administración & dosificación , Interleucina-2/administración & dosificación , Células Asesinas Naturales/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Linfocitos T/inmunología , Secuencia de Aminoácidos , Proteínas de Fusión bcr-abl/inmunología , Humanos , Células Asesinas Activadas por Linfocinas/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Activación de Linfocitos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunologíaRESUMEN
Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H-2Db)-restricted CD8 T cell receptor (TcR) alpha/beta cells. A CD8 TcR alpha/beta clone with specificity for the mycobacterial hsp60 peptide499-508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon-gamma (IFN-gamma) stimulation. The stress-induced, self peptide could be extracted from IFN-gamma-stressed macrophages by acid elution, suggesting that the IFN-gamma-induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60-specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9-mer to 11-mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H-2Db binding were tested in CTL assays. The overlapping murine hsp60 peptides162-170/171 were stimulatory at a concentration as low as 10-100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10-100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN-gamma-stressed macrophages to the cross-reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.
Asunto(s)
Proteínas Bacterianas/inmunología , Chaperonina 60/inmunología , Mycobacterium/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Chaperonina 60/química , Reacciones Cruzadas , Femenino , ISCOMs/inmunología , Interferón gamma/farmacología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Homología de Secuencia de AminoácidoRESUMEN
The peptides recognized by an H-2Db-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2Db binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2Db was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2Db with an efficacy similar to that of the nonapeptide corresponding to the H-2Db motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2Db cleft. Because the carboxy-terminal part is thus larger than predicted, this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.
Asunto(s)
Antígenos H-2/inmunología , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/química , Células Cultivadas , Chaperonina 60/inmunología , Citotoxicidad Inmunológica , Mapeo Epitopo , Inmunidad Celular , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Mycobacterium/inmunología , Péptidos/químicaRESUMEN
CD8+ T cells recognize antigenic peptides in the context of MHC class I molecules that encompass two distinct polypeptide chains, the MHC-encoded alpha-chain and the non-MHC-encoded beta 2-microglobulin (beta 2-m). The beta 2-m is considered essential for the stability and function of the MHC class I peptide complex and, hence, for peptide presentation to CD8+ T cells. In this study, we describe peptide presentation by macrophages from beta 2-m-deficient mice to a CD8+ CTL clone tht cross-recognizes an H-2Db-restricted peptide of the mycobacterial heat shock protein 60 (hsp60) and a self-peptide presented by IFN-gamma-stressed macrophages. Specific lysis of stressed or hsp60 peptide-pulsed beta 2-m-/- macrophages was inhibited by the nucleoprotein peptide with high affinity to H-2Db. Brefeldin A, a known inhibitor of MHC class I processing, interfered with lysis of IFN-gamma-stressed, but not of hsp60 peptide-pulsed, beta 2-m-/- macrophages. The hsp60 peptide failed to stimulate surface expression of H-2Db in beta 2-m-/- macrophages, and slightly increased MHC class I expression in the transporter mutant cell line RMA-S, as detected by cytofluorometry. We concLude that presentation of endogenously processed cytosolic epitopes and exogenously added foreign peptides by the MHC class I alpha-chain can occur independent from beta 2-m. Presumably, H-2Db peptides, but not H-2Kb peptides, have the capacity to induce and/or stabilize surface expression of a small number of MHC class I alpha-chains, and this low density is sufficient for recognition by CD8+ CTL, although it need not be detected by serologic means.
Asunto(s)
Presentación de Antígeno/fisiología , Antígenos H-2/química , Antígenos H-2/inmunología , Linfocitos T Citotóxicos/inmunología , Microglobulina beta-2/fisiología , Secuencia de Aminoácidos , Animales , Autoantígenos/inmunología , Células Cultivadas , Chaperonina 60/inmunología , Reacciones Cruzadas/inmunología , Pruebas Inmunológicas de Citotoxicidad , Epítopos/inmunología , Femenino , Antígenos H-2/biosíntesis , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Péptidos/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
T lymphocytes with specificity for the bacterial heat shock protein (hsp) 60 recognize stressed host cells, thus possibly promoting pathogenesis of certain infectious and autoimmune diseases. Here, we show that autoimmune destruction of stressed Schwann cells and macrophages by cytotoxic T lymphocytes raised against mycobacterial hsp60 can be inhibited by the use of hsp60-specific antisense oligodeoxynucleotides (A-ODNs). The inhibitory effect of hsp60 A-ODNs was specific because lysis of murine cytomegalovirus-infected host cells by virus-specific cytotoxic lymphocytes was not affected. Immunoblot analysis and immunoprecipitation studies suggest that different forms of stress increase hsp60 synthesis in Schwann cells and that this neosynthesis is reduced by hsp60 A-ODNs. These findings (i) provide evidence for participation of endogenous hsp60 in the recognition of stressed host cells by mycobacterial hsp60-crossreactive T cells and (ii) suggest the feasibility of inhibiting autoimmune reactions by target-cell treatment with specific A-ODNs.