RESUMEN
Behçet's disease (BD) is a vasculitis of unknown aetiology typified by chronic recurrent oral ulcers and systemic inflammatory manifestations. Neutrophils, and specifically their protease neutrophil elastase (NE), have been implicated in its pathology. Although NE is an effective anti-microbial, excessive NE can damage host tissue. Recurrent oral ulceration is a primary BD symptom, therefore we hypothesized that excessive neutrophil infiltration evidenced by increased NE and a reduction in specific endogenous inhibitors, secretory leucocyte protease inhibitor (SLPI) and alpha1-anti-trypsin (α1AT) contributes to BD mucosal instability. NE, SLPI and α1AT were quantified in saliva from BD patients with active oral ulcers (BDa) and quiet without ulcers (BDq), recurrent aphthous stomatitis (RASa; RASq) and healthy controls (HC). Although BDq saliva had marginally higher median NE levels (1112 ng/ml) compared to both RASq (1043 ng/ml) and HC (999 ng/ml), SLPI was significantly reduced in BDq (P < 0·01). Despite decreased SLPI protein, mRNA expression was significantly increased in BDq buccal epithelial swabs compared to RASq and HC (P < 0·05, P < 0·001). NE remained enzymatically active, although α1AT levels were at least eight times higher than SLPI in all groups, suggesting that α1AT does not have a primary role in counteracting NE in saliva. Furthermore, NE levels in BDa patients medicated with both azathioprine (AZA) and colchicine (COLC) were significantly lower than those on COLC (P = 0·0008) or neither (P = 0·02), indicating that combining AZA + COLC may help to regulate excessive NE during ulceration. This study showed that enzymatically active NE coupled with reduced SLPI in BD saliva may contribute to recurrent oral ulcerations.
Asunto(s)
Síndrome de Behçet/metabolismo , Elastasa de Leucocito/metabolismo , Saliva/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , alfa 1-Antitripsina/metabolismo , Adulto , Síndrome de Behçet/patología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Behçet's disease (BD) is an autoinflammatory, chronic relapsing/remitting disease of unknown aetiology with both innate and acquired immune cells implicated in disease pathogenesis. Peripheral blood natural killer (NK) cells and their CD56Dim /CD56Bright subsets were surface phenotyped using CD27 and CD16 surface markers in 60 BD patients compared to 60 healthy controls (HCs). Functional potential was assessed by production of interferon (IFN)-γ, granzyme B, perforin and the expression of degranulation marker CD107a. The effects of disease activity (BDActive versus BDQuiet ) and BD medication on NK cells were also investigated. Peripheral blood NK cells (P < 0·0001) and their constituent CD56Dim (P < 0·0001) and CD56Bright (P = 0·0015) subsets were depleted significantly in BD patients compared to HCs, and especially in those with active disease (BDActive ) (P < 0·0001). BD patients taking azathioprine also had significantly depleted NK cells compared to HCs (P < 0·0001). A stepwise multivariate linear regression model confirmed BD activity and azathioprine therapy as significant independent predictor variables of peripheral blood NK percentage (P < 0·001). In general, CD56Dim cells produced more perforin (P < 0·0001) and granzyme B (P < 0·01) expressed higher CD16 levels (P < 0·0001) compared to CD56Bright cells, confirming their increased cytotoxic potential with overall higher NK cell CD107a expression in BD compared to HCs (P < 0·01). Interestingly, IFN-γ production and CD27 expression were not significantly different between CD56Dim /CD56Bright subsets. In conclusion, both BD activity and azathioprine therapy have significant independent depletive effects on the peripheral blood NK cell compartment.
Asunto(s)
Síndrome de Behçet/inmunología , Circulación Sanguínea/inmunología , Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/inmunología , Adolescente , Adulto , Anciano , Azatioprina/efectos adversos , Azatioprina/uso terapéutico , Síndrome de Behçet/tratamiento farmacológico , Síndrome de Behçet/fisiopatología , Antígeno CD56/genética , Femenino , Proteínas Ligadas a GPI/genética , Granzimas/biosíntesis , Humanos , Interferón gamma/biosíntesis , Células Asesinas Naturales/química , Células Asesinas Naturales/clasificación , Proteína 1 de la Membrana Asociada a los Lisosomas/genética , Masculino , Persona de Mediana Edad , Perforina/biosíntesis , Receptores de IgG/genética , Adulto JovenRESUMEN
Behçet's disease (BD) is a chronic, multisystemic, recurrent vasculitis disease of unknown aetiology. Proinflammatory cytokines are a key feature of the disease, but the triggers for their induction are not well understood and/or controversial. Suppressor of cytokine signalling (SOCS) proteins which negatively regulate the JAK-STAT signalling pathway of cytokine induction may be dysregulated in BD. The expression of SOCS1 and 3 mRNA and protein was studied in peripheral blood mononuclear cells (PBMCs) and neutrophils of patients with BD and compared with healthy controls (HCs) and patients with recurrent aphthous stomatitis (RAS) using RT-PCR, Western blot and immunohistochemistry. SOCS1 and 3 mRNA was also measured in buccal mucosal cells (BMC) of patients with BD and HCs. SOCS1 and 3 mRNA was significantly upregulated in PBMCs of patients with BD compared with HCs (P = 0.0149; P = 0.0007). In addition, there were subtle differences between expression in active and symptom-free BD (quiescent BD). SOCS1 and SOCS 3 were also significantly upregulated in BMC from oral ulcers of BD compared with HCs (both at P = 0.0001). A differential expression of both SOCS1 and 3 was observed between PBMCs and neutrophils in patients with BD. Immunohistochemical analysis revealed differential expression of SOCS proteins in the buccal mucosa with an increased expression at the ulcer surface of ulcers than in the non-ulcerated tissue. These observations suggest a dysregulation of the expression of these important regulators not only between patients with BD and healthy controls but also between mucosal and systemic tissues, which may reflect the nature of the aetiopathology of the disease.
Asunto(s)
Síndrome de Behçet/genética , Estomatitis Aftosa/genética , Proteínas Supresoras de la Señalización de Citocinas/genética , Adulto , Síndrome de Behçet/inmunología , Citocinas/biosíntesis , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Mucosa Bucal/citología , Neutrófilos/metabolismo , Úlceras Bucales/metabolismo , ARN Mensajero/biosíntesis , Estomatitis Aftosa/inmunología , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/biosíntesis , Adulto JovenRESUMEN
The suppressor of cytokine signalling 3 (SOCS3) negatively regulates the Janus kinase (JAK)/signal transducer and activator of transcription-3 (STAT-3)/interleukin (IL)-17 pathway. The proinflammatory cytokine IL-17 is over-expressed in Sjögren's syndrome (SS) and is a key factor in its pathogenesis. We hypothesized that IL-17 over-expression in SS results from ineffective regulation by SOCS3. The expression of SOCS3 was analysed in peripheral blood mononuclear cells (PBMC) from SS cases, sicca controls (SC) and healthy controls (HC) and tissue samples from SS, SC and healthy salivary glands (HSG). PBMC and salivary gland tissue from SS and controls were dual-immunostained for SOCS3 and IL-17. IL-6-stimulated PBMC from SS and controls were evaluated for time-dependent STAT-3 activation and SOCS3 induction, and for IL-17 expression. Immunoblotting revealed greater levels of SOCS3 in PBMC from SS than SC (P = 0·017) or HC (P < 0·001). Similarly, the proportion of salivary-gland tissue cells staining for SOCS3 was significantly higher in SS than SC (P = 0·029) or HSG (P = 0·021). The cells in PBMC/salivary gland samples from controls predominantly expressed either SOCS3 or IL-17. However, there was a high frequency of SOCS3/IL-17 co-expression within cells of SS samples. IL-6-stimulation of PBMC from SS cases revealed prolonged activation of STAT-3 with reduced negative regulation by SOCS3, and enhanced expression of IL-17. This study showed that SOCS3 expression is up-regulated in SS. However, the absence in SS of the normal inverse relationship between SOCS3 and pSTAT-3/IL-17 indicates a functional disturbance in this signalling cascade. Consequently, a reduction in function, rather than a reduction in expression of SOCS3 accounts for the unregulated expression of IL-17 in SS, and may play a crucial role in aetiopathogenesis.
Asunto(s)
Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Adulto , Anciano , Femenino , Humanos , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Glándulas Salivales/metabolismo , Síndrome de Sjögren/diagnóstico , Proteína 3 Supresora de la Señalización de CitocinasRESUMEN
Preventive immunization against HIV-1 infection requires a rapid immune response that does not rely exclusively on B or T cell memory. Innate immunity may fulfill this function as it may be activated directly at the time of HIV-1 transmission, inhibit early HIV-1 replication, stimulate adaptive immunity and enable specific antibodies followed by CD8(+) T cells to deal with the virus effectively. The three components of innate immunity - cellular, extracellular and intracellular - are presented, with an example given for each of these components; gammadelta T cells, CC chemokines and APOBEC3G. This brief account is presented to highlight the immuno-virological concept of coordinating activated innate immunity with adaptive antibody and T cell responses in preventive vaccination against HIV-1 infection.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunidad Innata , Desaminasa APOBEC-3G , Quimiocinas CC/inmunología , Citidina Desaminasa/inmunología , Infecciones por VIH/prevención & control , Humanos , Interferón Tipo I/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/inmunología , VacunaciónAsunto(s)
Bienestar del Animal/normas , Animales de Laboratorio , Publicaciones Periódicas como Asunto/normas , Edición/normas , Investigadores/normas , Investigación/normas , Bienestar del Animal/ética , Bienestar del Animal/legislación & jurisprudencia , Animales , Humanos , Publicaciones Periódicas como Asunto/ética , Edición/ética , Investigadores/ética , Investigadores/legislación & jurisprudenciaRESUMEN
The appalling toll on the populations of developing countries as a result of the HIV epidemic shows no signs of abatement. While costly drug therapies are effective in developed nations, the sheer scale of the epidemic elsewhere makes the need for a vaccine an ever more urgent goal. The prevalent DNA prime-viral boost strategy aims to elicit cytotoxic lymphocytes (CTL) against HIV, but this approach is undermined by the rapid mutation of HIV, which thereby escapes CTL control. Alloimmunity has been found to be protective in vertical transmission from infected mothers to their babies, in alloimmunization of women with their partners' mononuclear cells, and in monkeys immunized with SIV grown in human T-cells. Vaginal mucosal immunization, as a result of unprotected sex with a regular partner, induced in vitro protection against HIV infection, and this was confirmed in macaques. The second type of natural protection is found in persons with the homozygous 32 CCR5 mutation, a 32-base-pair deletion of the CCR5 gene, which results in a lack of cell-surface expression of CCR5, which is associated with an increase in CC chemokines and the development of CCR5 antibodies. These two 'experiments of nature' have been used to develop vaccine strategies--first, in vaginal immunization of macaques with CCR5 peptides, in addition to HIV envelope (env) and SIV core (gag) antigens, all of which were linked to the 70-kD heat-shock protein (HSP70); and second, in mucosal allo-immunization of macaques, which also gave rise to in vitro protection from infection. Immunization with this vaccine elicited serum and vaginal IgG and IgA antibodies, IFNgamma- and IL-12-producing cells, and increased concentrations of CCL-3 and CCL-4. Vaginal challenge with a simian immunodeficiency virus engineered to carry a human envelope protein (SHIV 89.6) showed significant clearance of SHIV in the immunized macaques. This platform strategy will now be developed to activate the co-stimulatory pathways with the aim of enhancing the primary allogeneic and CCR5-directed responses which are involved in natural protection against HIV infection.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH/inmunología , Infecciones por VIH/transmisión , Inmunidad Mucosa/fisiología , Vacunas contra el SIDAS/inmunología , Adyuvantes Inmunológicos , Animales , Quimiocinas/fisiología , Regulación hacia Abajo , Femenino , Antígenos VIH/inmunología , Infecciones por VIH/prevención & control , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Inmunidad Innata/fisiología , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Macaca , Masculino , Receptores del VIH/fisiología , Factores Supresores Inmunológicos/inmunología , Linfocitos T Citotóxicos/fisiología , Vagina/inmunologíaRESUMEN
Summaryand interleukin (IL)-12 by dendritic cells (DC) from patients with Crohn's disease. TNF-alpha concentration was increased significantly when DC from Crohn's disease were stimulated with HSP70 or CD40L and this was associated with signalling by the extracellular signal regulated kinase (ERK) 1/2 and p38 mitogen activated protein (MAP) kinase pathway. IL-12 production was also increased when DC were stimulated with HSP70. Cells eluted from inflamed intestinal mucosa from Crohn's disease, stimulated with HSP70, CD40L or lipopolysaccharide produced significantly greater TNF-alpha and IL-12 concentrations than cells from uninflamed mucosa. Significant inhibition of TNF-alpha production was demonstrated when DC from peripheral blood mononuclear cells or cells eluted from intestinal mucosa of Crohn's disease were treated with either the HSP70 inhibitory peptide (aa 457-496) or peptides derived from CD40 and CD40L. These inhibitory peptides target the CD40-CD40L and the emerging CD40-HSP70 co-stimulatory pathway. Our findings offer a novel strategy to prevent excessive production of TNF-alpha in Crohn's disease.
Asunto(s)
Enfermedad de Crohn/inmunología , Células Dendríticas/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Mucosa Intestinal/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Colitis Ulcerosa/inmunología , Humanos , Inmunidad Mucosa , Interleucina-12/biosíntesis , Lipopolisacáridos/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Monocitos/inmunología , Fragmentos de Péptidos/inmunologíaRESUMEN
Cell-surface CCR5 is a major coreceptor with CD4 glycoprotein, mediating cellular entry of CCR5 strains of HIV-1 or SIV. We targeted the SIV CCR5 coreceptor in a combined CCR5-SIV antigen immunization strategy. Rhesus macaques were immunized i.m. with the 70 kDa heat shock protein (HSP70) covalently linked to the CCR5 peptides, SIV gpl20 and p27. Intravenous challenge with SIV mac 8980 prevented SIV infection or decreased the viral load with the CCR5-SIV combined vaccine. CC chemokines and antibodies which block and downmodulateCCR5 were induced, as well as immune responses to the subunit SIV antigens. This novel vaccination strategy complements cognate immunity to SIV with innate immunity to the CCR5 coreceptor of SIV.
Asunto(s)
Receptores CCR5/inmunología , Vacunas contra el SIDAS/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/biosíntesis , Antígenos de Superficie/inmunología , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Productos del Gen rex/genética , Productos del Gen rex/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Esquemas de Inmunización , Inmunoglobulina A/análisis , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/análisis , Inmunoglobulina G/biosíntesis , Cinética , Macaca mulatta , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Behcet's disease (BD) specific peptide (p336-351) was identified within the human 60 kD heat shock protein (HSP60). Oral p336-351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB). This strategy was adopted in a phase I/II clinical trial by oral administration of p336-351-CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD. The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations. Oral administration of p336-351-CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen. After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10-18 months after cessation of all treatment. Control of uveitis and extra-ocular manifestations of BD was associated with a lack of peptide-specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN-gamma and TNF-alpha production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed. The efficacy of oral peptide-CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.
Asunto(s)
Síndrome de Behçet/inmunología , Toxina del Cólera/administración & dosificación , Uveítis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Administración Oral , Adulto , Antígenos CD/inmunología , Síndrome de Behçet/complicaciones , Linfocitos T CD4-Positivos , División Celular/inmunología , Humanos , Tolerancia Inmunológica , Interferón gamma/inmunología , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos , Fenotipo , Receptores de Quimiocina/inmunología , Linfocitos T/inmunología , Células TH1/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Uveítis/complicaciones , Uveítis/inmunologíaRESUMEN
The need for an effective vaccine against HIV has prompted a refocusing of attention on mucosal immunity. More than 75% of all infections are acquired across a mucosal surface. It is therefore a prerequisite for a vaccine to target directly the mucosal tissues or indirectly the regional lymph nodes in order to prevent or control viral replication. Although mucosal immunization has induced responses at the genital or rectal surfaces, immune mechanisms alone have not been shown to be sufficient to contain infections in macaques. A growing body of evidence suggests that a dual mechanism may be required for effective mucosal protection, mediated by specific CD4 and CD8 T cell and antibody responses to the immunizing agents, plus innate antiviral factors and beta chemokines that down-regulate CCR5 coreceptors. Targeted iliac lymph node immunization with SIV gp 120 and p27 in alum prevents SIV infection or significantly decreases the viral load when immunized macaques were challenged with SIV by the rectal route. Indeed, in addition to specific immunity, including significant SIgA antibody secreting cells in the iliac lymph nodes, CD8-suppressor factor and the 3beta chemokines (RANTES, MIP-1alpha and MIP-1beta) are significantly associated with protection against rectal mucosal SIV infection.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Animales , Anticuerpos Antivirales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/inmunología , Quimiocinas CC/inmunología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Productos del Gen gag/uso terapéutico , Inmunidad Mucosa , Inmunización , Inmunoglobulina A Secretora/inmunología , Ganglios Linfáticos/inmunología , Macaca , Proteínas Inflamatorias de Macrófagos/inmunología , Glicoproteínas de Membrana/uso terapéutico , Receptores CCR5/inmunología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Factores Supresores Inmunológicos/inmunología , Proteínas del Envoltorio Viral/uso terapéutico , Carga ViralRESUMEN
The C-C chemokine receptor CCR5 serves an important function in chemotaxis of lymphocytes, monocytes, and dendritic cells. CCR5 is also the major coreceptor in most macrophage-tropic HIV-1 infections. Immunization of rhesus macaques with a baculovirus-generated CCR5 construct or peptides derived from the sequences of the four extracellular domains of CCR5 elicited IgG and IgA Abs, inhibition of SIV replication, and CD4+ T cell proliferative responses to three of the extracellular domains of CCR5. The immune sera reacted with cell surface CCR5 expressed on HEK 293 cells. T and B cell epitope mapping revealed major and minor T and B cell epitopes in the N-terminal, first, and second loops of CCR5. The three C-C chemokines, RANTES, macrophage-inflammatory protein-1alpha, and macrophage-inflammatory protein-1beta, were up-regulated by immunization with the CCR5-derived peptides, and the cell surface expression of CCR5 was decreased. The CCR5 Abs were complementary to the C-C chemokines in inhibiting HIV replication in vitro. Immunization with the four extracellular domains of CCR5 suggests that three of them are immunogenic, with maximal T cell responses being elicited by the second loop peptide. However, maximal Abs to the cell surface CCR5 or viral inhibitory Abs in vitro were induced by the N-terminal peptide. Up-regulation of the three C-C chemokines and down-modulation of cell surface CCR5 were elicited by the second loop, N-terminal, and first loop peptides. The data suggest that a dual mechanism of C-C chemokines and specific Abs may engage and down-modulate the CCR5 coreceptors and prevent in vitro HIV or SIV replication.
Asunto(s)
Espacio Extracelular/inmunología , VIH-1/inmunología , Receptores CCR5/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacología , Antivirales/farmacología , Baculoviridae/genética , Baculoviridae/inmunología , Línea Celular , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Quimiocinas CC/biosíntesis , Quimiocinas CC/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/análisis , Epítopos de Linfocito T/análisis , Humanos , Sueros Inmunes/farmacología , Inmunoglobulina A/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/farmacología , Inyecciones Intramusculares , Activación de Linfocitos/inmunología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Macaca mulatta , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína/genética , Receptores CCR5/administración & dosificación , Receptores CCR5/biosíntesis , Receptores CCR5/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Spodoptera/genética , Spodoptera/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Transfección , Regulación hacia Arriba/inmunología , Replicación Viral/inmunologíaRESUMEN
It has been suggested that the presence of immunoglobulin and complement receptors on rectal epithelium may facilitate the entry of HIV complexed to nonneutralizing antibody. We tested this hypothesis using simian immunodeficiency virus (SIV) infection of rhesus macaques. First, in a pilot study, a nonneutralizing IgG fraction of macaque anti-SIV gp120 was shown to enhance the immunogenicity of SIV envelope following rectal immunization. The same antibody was then mixed with a subinfectious dose of SIV and the occurrence of rectal infection was compared with virus alone. Animals were not infected overtly and were rechallenged with a 10-fold higher dose of virus with and without addition of antibody. There was no evidence of antibody-mediated infection, since equal numbers of macaques became infected, regardless of the presence of antibody. In addition, the application of immune complexes did not alter significantly the subsequent virus load or the immune responses generated. In seronegative animals, in which virus and proviral DNA were undetectable in PBMC and tissues, SIV-specific T-cell responses and antibody-secreting cells were found in systemic and gut-associated sites. Our results show that nonneutralizing antibody neither facilitated nor enhanced rectal infection with SIV, in the small number of animals used, despite the consistent trend for this antibody to enhance antibody responses to gp120 following rectal immunization with immune-complexed antigen. However, mucosal exposure to subinfectious doses of virus primed both systemic and local immunity, regardless of addition of nonneutralizing antibody.
Asunto(s)
Recto/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios , Animales , Anticuerpos Antivirales/administración & dosificación , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Citotoxicidad Inmunológica , Proteína gp120 de Envoltorio del VIH/inmunología , Inmunidad Activa , Inmunidad Celular , Inmunoglobulina G/análisis , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Leucocitos Mononucleares/virología , Macaca mulatta , Recto/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Carga ViralRESUMEN
The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.
Asunto(s)
Antígenos CD40/fisiología , Quimiocina CCL5/biosíntesis , Células Dendríticas/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas Inflamatorias de Macrófagos/biosíntesis , Monocitos/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Proteínas Bacterianas , Antígenos CD40/química , Antígenos CD40/genética , Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Membrana Celular/metabolismo , Quelantes/farmacología , Quimiocina CCL4 , Quimiocina CCL5/genética , Células Dendríticas/metabolismo , Ácido Egtácico/farmacología , Proteínas de Escherichia coli/farmacología , Proteínas HSP70 de Choque Térmico/farmacología , Humanos , Riñón , Lipopolisacáridos/farmacología , Sustancias Macromoleculares , Proteínas Inflamatorias de Macrófagos/genética , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes de Fusión/fisiología , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
In view of the role of gammadelta(+) T cells in mucosal protection against infection, the proportion of gamma delta T cells was examined in cells eluted from lymphoid and mucosal tissues of macaques immunized with simian immunodeficiency virus (SIV) gp120 and p27 in alum and challenged with live SIV by the rectal mucosal route. This revealed a significant increase in gammadelta T cells eluted from the rectal mucosa (p < 0.01) and the related iliac lymph nodes (p < 0.0001) in protected as compared with infected macaques. Preferential homing of PKH-26-labeled gammadelta(+) T cells from the primed iliac lymph nodes to the rectal and cervico-vaginal mucosa was demonstrated after targeted iliac lymph node as compared with i. m. immunization. Investigations of the mechanism of protection revealed that gammadelta(+) T cells can generate antiviral factors, RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta which can prevent SIV infection by binding to the CCR5 coreceptors. Up-regulation of gammadelta(+) T cells was demonstrated by immunization of macaques with heat shock protein (HSP)70 linked to peptides and with granulocyte-macrophage colony-stimulating factor (GM-CSF). This was confirmed by in vitro studies showing that GM-CSF can up-regulate gammadelta(+) T cells from macaques immunized with HSP-linked peptides but not those from naive animals. We suggest that a novel strategy of immunization with HSP70 linked to antigen may generate both cognate immunity to the antigen and innate immunity by virtue of up-regulation of gammadelta(+) T cells. These cells generate antiviral factors and the three beta-chemokines that prevent binding and transmission of SIV or M-tropic HIV by the CCR5 coreceptor.
Asunto(s)
Quimiocinas CC/fisiología , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T/fisiología , Animales , Proteínas HSP70 de Choque Térmico/farmacología , Inmunidad Mucosa , Inmunización , Interleucina-2/farmacología , Macaca mulatta , Receptores CCR5/fisiología , Vacunas Virales/inmunologíaRESUMEN
A non-cognate mechanism of protection against human immunodeficiency virus-1 (HIV-1) infection involves up-regulation of beta-chemokines, which bind and may down-modulate the CCR5 co-receptors, thereby preventing transmission of M-tropic HIV-1. The objective of this investigation was to evaluate this mechanism in vivo in non-human primates. Rhesus macaques were immunized by a modified targeted lymph nodes (TLN) route with recombinant simian immunodeficiency virus (SIV) glycoprotein 120 (gp120) and p27 in alum, and adsorbed recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) with either interleukin (IL)-2 or IL-4. Immunization induced significant increases in the concentrations of CD8 cell-derived suppressor factor (CD8-SF), regulated on activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and down-modulation of the proportion of cells expressing CCR5 (r = 0.737, P<0.05). The macaques were then challenged with SIVmac 220 by the rectal mucosal route. The plasma SIVmac RNA showed a significant inverse correlation with the CD8-SF or the concentration of the three beta-chemokines (r = 0.831 and 0.824, P<0.01), but a positive correlation between the proportion of CCR5+ cells and SIVmac RNA (r = 0.613, P = 0.05). These results demonstrate for the first time in vivo that immunization up-regulates beta-chemokines, which may down-modulate CCR5 co-receptors, and both functions are significantly correlated with the viral load. Hence, the non-cognate beta-chemokine-CCR5 mechanism should be considered as complementary to specific immunity in vaccination against HIV.
Asunto(s)
Quimiocinas CC/metabolismo , Glicoproteínas de Membrana , Receptores CCR5/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios , Proteínas del Envoltorio Viral , Animales , Antígenos CD8/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/metabolismo , Productos del Gen env/genética , Productos del Gen gag/administración & dosificación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/uso terapéutico , Ingle , Proteína gp120 de Envoltorio del VIH/administración & dosificación , Inyecciones Subcutáneas , Interleucina-2/uso terapéutico , Interleucina-4/uso terapéutico , Macaca mulatta , Proteínas Inflamatorias de Macrófagos/metabolismo , Membrana Mucosa/inmunología , ARN/análisis , Proteínas Recombinantes , Recto , Proteínas Oncogénicas de Retroviridae/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Proteínas Virales de Fusión/genética , Carga Viral , Vacunas Virales/administración & dosificaciónRESUMEN
Heat shock proteins (HSP) are widely distributed and highly immunogenic molecules. A novel property reported here is that stimulation with HSP70 of CD8-enriched T cells derived from naive non-human primates caused a dose-dependent increase in concentrations of the beta-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha or MIP-1beta. However, the concentrations of these beta-chemokines were greatly increased when the CD8 T cells derived from HSP70-immunized non-human primates were stimulated with HSP70. HSP linked to peptides or proteins combined generation of beta-chemokines with an adjuvant function by enhancing specific T cell proliferative responses and IgG and IgA antibodies. The beta-chemokine and adjuvant functions were also elicited by topical mucosal administration of HSP linked to an antigen. We postulate that microbial HSP can stimulate beta-chemokine production which may be responsible for innate adjuvanticity, as was found in cells eluted from normal rectal mucosal tissue, and constitutes a significant component of the mucosal-associated lymphoid system. Furthermore, stimulation of innate immunity may drive adaptive immunity and account for the protective effects of HSP against tumors and viruses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Quimiocinas CC/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Inmunidad , Adyuvantes Inmunológicos , Animales , Presentación de Antígeno , Antígenos Bacterianos/inmunología , Antígenos Virales/inmunología , Linfocitos T CD8-positivos/microbiología , Linfocitos T CD8-positivos/virología , Relación Dosis-Respuesta Inmunológica , PrimatesRESUMEN
The lack of success in the development of an effective conventional vaccine against HIV has focused attention on mucosal immunity. This is a rational move, since HIV is transmitted mostly by the mucosal route. The mucosal strategy is based on the concept that: a) HIV/SIV has to cross the mucosal-regional lymph node-blood barriers, each of which can prevent viral transmission or decrease the viral load. b) Immunization has to target directly the mucosal tissues or indirectly the regional lymph nodes, in order to prevent or control viral replication. This strategy is consistent with antigen localization and effective entry into the lymph nodes, driving the immune response. c) A dual immune mechanism may be necessary for effective mucosal protection, mediated by specific CD4 and CD8 T-cell and antibody responses to the immunizing antigens, and innate antiviral factors and beta-chemokines which down-modulate CCR5 co-receptors. Targeted iliac lymph node immunization with SIVgp120 and p27 in alum prevents SIV infection or significantly decreases the viral load when challenged by the rectal route. Indeed, in addition to specific immunity, including significant sIgA antibody-forming cells in the iliac lymph nodes, CD8-suppressor factor and the three beta-chemokines (RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta) are significantly associated with protection against rectal mucosal SIV infection.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Infecciones por VIH/terapia , Inmunidad Mucosa , Animales , Quimiocinas CC/inmunología , Femenino , Infecciones por VIH/inmunología , Humanos , Ganglios Linfáticos/inmunología , Masculino , Membrana Mucosa/inmunología , Recto/inmunología , Vacunas contra el SIDAS/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Vagina/inmunologíaRESUMEN
The seven-transmembrane G-protein-linked CCR5 molecule functions as a major coreceptor for HIV or simian immunodeficiency virus (SIV) infection. Antibodies to CCR5 were studied in rhesus macaques immunized with SIV grown in human CD4(+) T cells. These macaques were completely protected against i.v. challenge with live SIV. Sera from the protected macaques showed significantly greater inhibition of SIV replication (p < 0.001) and macrophage inflammatory protein-1beta-generated CCR5-dependent chemotaxis (p < 0.01) than sera from unprotected macaques, in the absence of significant neutralizing antibodies to SIV. These two functional assays demonstrate serum antibodies to the CCR5 receptors which were specifically inhibited by CCR5-transfected HEK-293 cells. We postulate that anti-CCR5 antibodies may be complementary to beta-chemokines in blocking CCR5 coreceptors to HIV or SIV binding and fusion of CD4(+) cells.
Asunto(s)
Receptores CCR5/inmunología , Receptores Virales/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Animales , Anticuerpos Monoclonales , Formación de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Quimiotaxis de Leucocito/inmunología , Humanos , Inmunización , Inmunoglobulina G/sangre , Técnicas In Vitro , Macaca mulatta , Receptores CCR5/genética , Receptores del VIH/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/fisiología , Transfección , Replicación ViralRESUMEN
In macaques, the route of immunization has a profound effect on the immune response. Augmenting rectal or vaginal immunization with oral or nasal immunization enhanced the secretory IgA, serum IgG, and T cell responses. However, targeted iliac lymph node (TILN) immunization with recombinant simian immunodeficiency virus (SIV) gp120 and p27 elicited the most consistent mucosal antibody responses in the rectum, vagina, urine, seminal fluid, and blood. Both mucosal and TILN immunization induced specific CD4+ T cell proliferative responses in the iliac lymph nodes, which drain these mucosal surfaces, and in the splenic and circulating T cells. Rectal mucosal challenge with cell-free SIV induced total protection in 4 of 7 macaques that were immunized by the TILN route, and, compared with unimmunized macaques or those immunized by the mucosal route (P<.001), it induced a >90% decrease in virus load in 3 of them. Protection from mucosal rectal infection with SIV was significantly associated with an increase in the CD8 suppressor factor (which was generated by the iliac lymph node), RANTES, and MIP-1beta (P<.01).