RESUMEN
Type 1 diabetes (T1D) in non-obese diabetic (NOD) mice may be favored by immune dysregulation leading to the hyporesponsiveness of regulatory T cells and activation of effector T-helper type 1 (Th1) cells. The immunoregulatory activity of natural killer T (NKT) cells is well documented, and both interleukin (IL)-4 and IL-10 secreted by NKT cells have important roles in mediating this activity. NKT cells are less frequent and display deficient IL-4 responses in both NOD mice and individuals at risk for T1D (ref. 8), and this deficiency may lead to T1D (refs. 1,6-9). Thus, given that NKT cells respond to the alpha-galactosylceramide (alpha-GalCer) glycolipid in a CD1d-restricted manner by secretion of Th2 cytokines, we reasoned that activation of NKT cells by alpha-GalCer might prevent the onset and/or recurrence of T1D. Here we show that alpha-GalCer treatment, even when initiated after the onset of insulitis, protects female NOD mice from T1D and prolongs the survival of pancreatic islets transplanted into newly diabetic NOD mice. In addition, when administered after the onset of insulitis, alpha-GalCer and IL-7 displayed synergistic effects, possibly via the ability of IL-7 to render NKT cells fully responsive to alpha-GalCer. Protection from T1D by alpha-GalCer was associated with the suppression of both T- and B-cell autoimmunity to islet beta cells and with a polarized Th2-like response in spleen and pancreas of these mice. These findings raise the possibility that alpha-GalCer treatment might be used therapeutically to prevent the onset and recurrence of human T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Galactosilceramidas/farmacología , Células Asesinas Naturales/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD1/genética , Ciclofosfamida/toxicidad , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/inmunología , Interferón gamma/metabolismo , Interleucina-4/metabolismo , Interleucina-7/farmacología , Células Asesinas Naturales/efectos de los fármacos , Selectina L/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Mutantes , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/inmunología , Receptores de Interleucina-10 , Bazo/efectos de los fármacos , Bazo/metabolismoAsunto(s)
Diabetes Mellitus Tipo 1/inmunología , Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Quimiocinas/inmunología , Citocinas/inmunología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/prevención & control , Humanos , Islotes Pancreáticos/inmunología , Células Asesinas Naturales/inmunología , Linfocitos T Reguladores/inmunología , Células Th2/inmunologíaRESUMEN
We have previously shown that systemic injection of multiple low doses of recombinant murine interleukin-4 (mIL-4) can prevent type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by activating regulatory T helper (Th) 2 cells in vivo. Here, we have developed a gene transfer approach to the prevention of T1D by testing the therapeutic potential of an adenovirus gene transfer vector engineered to express mIL-4. We found that only two systemic injections of a recombinant adenovirus type 5 vector-expressing mIL-4 (Ad5mIL-4) reduces destructive insulitis and protects NOD mice from the onset of diabetes by eliciting intrapancreatic Th2 cell responses. Host immune responses against the adenovirus vector were detectable; however, the levels of antibody production were insufficient to preclude Ad5mIL-4 treatment as a possible therapeutic agent against T1D. Thus, adenovirus-mediated delivery of IL-4 provides protection of NOD mice from T1D and represents a clinically viable therapeutic approach.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Terapia Genética/métodos , Inmunoterapia Activa/métodos , Interleucina-4/genética , Interleucina-4/uso terapéutico , Transfección/métodos , Adenoviridae/genética , Animales , Diabetes Mellitus Tipo 1/inmunología , Femenino , Vectores Genéticos/administración & dosificación , Inmunoglobulina E/inmunología , Inyecciones , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Páncreas/inmunología , Células Th2/inmunologíaRESUMEN
We tested the efficacy of biolistic-mediated gene transfer as a noninvasive therapy for type 1 diabetes (T1D) in nonobese diabetic (NOD) mice by expression of murine interleukin 4 (mIL-4) cDNA. Epidermal delivery of 2 microg of DNA yielded transient detection of serum mIL-4, using a conventional cDNA expression vector. A vector stabilized by incorporation of the Epstein-Barr virus (EBV) EBNA1/oriP episomal maintenance replicon produced higher levels of serum mIL-4 that persisted for 12 days after inoculation. Although biolistic inoculation of either vector reduced insulitis and prevented diabetes, the protracted mIL-4 expression afforded by the EBV vector resulted in Th2-type responses in the periphery and pancreas and more significant protection from the onset of diabetes. Our studies demonstrate the efficacy of biolistic gene delivery of stabilized cytokine expression as a viable therapeutic approach to prevent the onset of T1D.
Asunto(s)
Biolística , Diabetes Mellitus Tipo 1/prevención & control , Interleucina-4/genética , Animales , Citocinas/análisis , Citocinas/metabolismo , ADN Complementario/genética , Femenino , Citometría de Flujo , Vectores Genéticos , Herpesvirus Humano 4/genética , Inmunoglobulina E/sangre , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos NOD , Páncreas/metabolismo , Páncreas/patología , Linfocitos T/metabolismoRESUMEN
We investigated the biological role of CC chemokines in the Th1-mediated pathogenesis of spontaneous type I diabetes in nonobese diabetic (NOD) mice. Whereas an elevated ratio of macrophage inflammatory protein-1alpha (MIP-1alpha):MIP-1beta in the pancreas correlated with destructive insulitis and progression to diabetes in NOD mice, a decreased intrapancreatic MIP-1alpha:MIP-1beta ratio was observed in nonobese diabetes-resistant (NOR) mice. IL-4 treatment, which prevents diabetes in NOD mice by polarizing intraislet Th2 responses, decreased CCR5 expression in islets and potentiated a high ratio of MIP-1beta and monocyte chemotactic protein-1 (MCP-1): MIP-1alpha in the pancreas. Furthermore, NOD.MIP-1alpha-/- mice exhibited reduced destructive insulitis and were protected from diabetes. Neutralization of MIP-1alpha with specific Abs following transfer of diabetogenic T cells delayed the onset of diabetes in NOD.Scid recipients. These studies illustrate that the temporal expression of certain CC chemokines, particularly MIP-1alpha, and the CCR5 chemokine receptor in the pancreas is associated with the development of insulitis and spontaneous type I diabetes.
Asunto(s)
Quimiocinas CC/biosíntesis , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/inmunología , Páncreas/inmunología , Páncreas/metabolismo , Receptores CCR5/biosíntesis , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antagonistas de los Receptores CCR5 , Movimiento Celular/inmunología , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocinas CC/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/terapia , Progresión de la Enfermedad , Femenino , Interleucina-4/uso terapéutico , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Proteínas Inflamatorias de Macrófagos/deficiencia , Proteínas Inflamatorias de Macrófagos/genética , Proteínas Inflamatorias de Macrófagos/metabolismo , Proteínas Inflamatorias de Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Páncreas/patología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Receptores CCR5/genética , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
Fatty acid amide hydrolase is an integral membrane protein that hydrolyzes a novel and growing class of neuromodulatory fatty acid molecules, including anandamide, 2-arachidonyl glycerol, and oleamide. This activity is inhibited by serine and cysteine reactive agents, suggesting that the active site contains a serine or cysteine residue. Therefore serine and cysteine residues were mutated to alanine and the effects on activity were determined. Mutants were prepared using site-directed mutagenesis methods and expressed in COS-7 cells. Serine mutations S217A and S241A completely abolished enzymatic activity. Mutants S152A and C249A had no effect on activity, while S218A showed a slight decrease in activity. To confirm these results biochemically, the mutant enzymes were reacted with the irreversible inhibitor [(14)C]-diisopropyl fluorophosphate. All of the mutants except S217A and S241A were labeled. We therefore confirm that fatty acid amide hydrolase is a serine hydrolase and propose that both Ser-217 and Ser-241 are essential for enzyme activity.
Asunto(s)
Amidohidrolasas/genética , Serina/química , Alanina/química , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Catálisis , Cisteína/química , Isoflurofato , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfolipasas A/metabolismo , Inhibidores de Proteasas , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
The mechanism of protection from type 1 diabetes conferred by regulatory T-cells induced by oral insulin treatment of NOD mice is not well understood. We demonstrate that oral insulin feeding of NOD mice induces the function of insulin B-chain reactive CD4+ regulatory T-cells, which compete with diabetogenic effector T-cells for the recognition of insulin in NOD.Scid recipient mice. These effector T-cells become deprived of interleukin (IL)-2 and interferon (IFN)-gamma and are unable to expand and migrate to the pancreas. Type 1 diabetes-protective splenic regulatory T-cells secrete relatively little transforming growth factor (TGF)-beta1, suggesting that TGF-beta may not contribute to the inactivation of effector T-cells in NOD.Scid recipients. The observed preferential infiltration of insulin-reactive regulatory T-cells rather than effector T-cells in the pancreas results in a nondestructive insulitis that correlates with an increased intrapancreatic expression of macrophage inflammatory protein-1beta. Thus, oral insulin therapy overcomes a deficiency in regulatory T-cells and protects against type 1 diabetes by inducing insulin B-chain reactive regulatory T-cells to block cytokine secretion and migration of diabetogenic effector T-cells to the pancreas. Our data emphasize that continuous oral insulin feeding over a prolonged period is required to prevent type 1 diabetes.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/prevención & control , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Administración Oral , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Hipoglucemiantes/química , Hipoglucemiantes/inmunología , Insulina/química , Insulina/inmunología , Ratones , Ratones Endogámicos NOD , Páncreas/inmunología , Fragmentos de Péptidos/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Fatty acid amide hydrolase contains a proline-rich sequence matching a consensus sequence for SH3-binding domains as well as a transmembrane domain. In this study, deletion mutants lacking the proline-rich region and the transmembrane domain were generated. Transfection experiments demonstrated that the proline-rich deleted amidase was enzymatically inactive. While immunostaining of the wild-type was always punctate with strong perinuclear staining characteristic for endoplasmic reticulum, the staining of the mutant was diffuse and distributed throughout the cytoplasm and perinuclear region. These observations along with the loss of activity suggest that the proline-rich region may play a role in the subcellular localization and enzymatic function. The transmembrane domain-deleted mutant was indistinguishable from the wild-type enzyme.
Asunto(s)
Amidohidrolasas/química , Prolina/fisiología , Animales , Células COS , Inmunohistoquímica , Mutagénesis , Dominios Homologos srcRESUMEN
Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.
Asunto(s)
Antígenos CD28/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Interleucina-4/fisiología , Linfocitos T/inmunología , Animales , Animales Recién Nacidos , Supervivencia Celular , Anergia Clonal , Femenino , Glutamato Descarboxilasa/inmunología , Inmunización Pasiva , Interleucina-2/biosíntesis , Islotes Pancreáticos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Transducción de Señal , Células Th2/inmunologíaRESUMEN
Beginning at the time of insulitis, nonobese diabetic (NOD) mice demonstrate a thymocyte and peripheral T cell proliferative hyporesponsiveness induced by TCR cross-linking, which is associated with reduced IL-2 and IL-4 secretion. We previously reported that NOD CD4+ T cell hyporesponsiveness is reversed completely in vitro by exogenous IL-4, and that administration of IL-4 to NOD mice prevents the onset of insulin-dependent diabetes mellitus (IDDM). This result suggested that T cell-mediated destruction of pancreatic islet beta cells may result from a hyporesponsiveness in regulatory Th2 cells favoring a Th1 cell-mediated environment in the pancreas. In the present study, we tested this possibility by analysis of the mechanisms of protection from IDDM afforded by IL-4 treatment in NOD mice. We show that IL-4 protects NOD mice from insulitis and IDDM when administered i.p. three times a week for 10 wk beginning at 2 wk of age. This occurs by the modulation of the homing of autoreactive cells to inflammatory sites and the stabilization of a protective Th2-mediated environment in the thymus, spleen, and pancreatic islets. Thus, IL-4 treatment favors the expansion of regulatory CD4+ Th2 cells in vivo and prevents the onset of insulitis and IDDM mediated by autoreactive Th1 cells.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/prevención & control , Interleucina-4/uso terapéutico , Islotes Pancreáticos/patología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/efectos de los fármacos , Inmunoglobulina E/sangre , Incidencia , Inyecciones Intraperitoneales , Interleucina-4/administración & dosificación , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/inmunología , Estudios Longitudinales , Ratones , Ratones Endogámicos NOD , Ratones SCID , Páncreas/efectos de los fármacos , Páncreas/inmunología , Páncreas/metabolismo , Sialadenitis/prevención & control , Bazo/citología , Bazo/inmunología , Timo/citología , Timo/inmunología , Tiroiditis/prevención & control , Factores de TiempoRESUMEN
Anandamide amidase is the hydrolytic enzyme responsible for the breakdown of anandamide, an endogenous cannabimimetic, to arachidonate and ethanolamine. Another enzymatic activity called anandamide synthase catalyzes the reverse reaction, that is the condensation of arachidonate and ethanolamine. Using a recently cloned rat fatty acid amidohydrolase (FAAH), we tested the hypothesis that the synthase and the amidase activities are catalyzed by the same enzyme. Untransfected and vector transfected (pcDNA3) COS-7 cells did not express detectable levels of either the amidase or synthase. However, when COS-7 cells were transiently transfected with a rat FAAH pcDNA3 construct, both amidase and synthase were concomitantly expressed. These results indicate that the enzymatic formation of anandamide from arachidonic acid and ethanolamine can be mediated by anandamide amidase acting in the reverse direction. The FAAH transfected cells expressed higher levels of enzyme than either rat brain homogenates or neuroblastoma cells in culture. Furthermore, the reaction rate for the amidase in FAAH transfected COS-7 cells, neuroblastoma cells and brain homogenate was always greater than the synthase reaction. These studies raise the question if this synthase reaction serves any physiological role, especially in view of the evidence that anandamide can be formed by a different pathway.
Asunto(s)
Amidohidrolasas/metabolismo , Ácido Araquidónico/metabolismo , Etanolamina/metabolismo , Animales , Encéfalo/metabolismo , Células COS , Catálisis , Clonación Molecular , Hidrólisis , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Bloqueadores de los Canales de Calcio/metabolismo , Riñón/irrigación sanguínea , Circulación Renal/efectos de los fármacos , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/farmacología , Southern Blotting , Bloqueadores de los Canales de Calcio/farmacología , Cannabinoides/antagonistas & inhibidores , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endocannabinoides , Etanolaminas/análisis , Indometacina/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Norepinefrina/metabolismo , Fosfatidiletanolaminas/análisis , Piperidinas/farmacología , Alcamidas Poliinsaturadas , Pirazoles/farmacología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Rimonabant , Sistema Nervioso Simpático/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
Nonobese diabetic (NOD) mouse thymocytes are hyporesponsive to T cell antigen receptor (TCR)-mediated stimulation of proliferation, and this T cell hyporesponsiveness may be causal to the onset of autoimmune diabetes in NOD mice. We previously showed that TCR-induced NOD T cell hyporesponsiveness is associated with a block in Ras activation and defective signaling along the PKC/Ras/MAPK pathway. Here, we report that several sequential changes in TCR-proximal signaling events may mediate this block in Ras activation. We demonstrate that NOD T cell hyporesponsiveness is associated with the (a) enhanced TCR-beta-associated Fyn kinase activity and the differential activation of the Fyn-TCR-zeta-Cbl pathway, which may account for the impaired recruitment of ZAP70 to membrane-bound TCR-zeta; (b) relative inability of the murine son of sevenless (mSOS) Ras GDP releasing factor activity to translocate from the cytoplasm to the plasma membrane; and (c) exclusion of mSOS and PLC-gamma1 from the TCR-zeta-associated Grb2/pp36-38/ZAP70 signaling complex. Our data suggest that altered tyrosine phosphorylation and targeting of the Grb2/pp36-38/ZAP70 complex to the plasma membrane and cytoskeleton and the deficient association of mSOS with this Grb2-containing complex may block the downstream activation of Ras and Ras-mediated amplification of TCR/CD3-mediated signals in hyporesponsive NOD T cells. These findings implicate mSOS as an important mediator of downregulation of Ras signaling in hyporesponsive NOD T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Enfermedades Autoinmunes/inmunología , Diabetes Mellitus Tipo 1/inmunología , Linfocitos T/inmunología , Ubiquitina-Proteína Ligasas , Animales , Enfermedades Autoinmunes/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Proteína Adaptadora GRB2 , Factores de Intercambio de Guanina Nucleótido , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Fosforilación , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-cbl , Proteínas Proto-Oncogénicas c-fyn , Receptores de Antígenos de Linfocitos T/metabolismo , Autotolerancia , Transducción de Señal , Tirosina/metabolismo , Factores de Intercambio de Guanina Nucleótido ras , Proteínas ras/metabolismoRESUMEN
Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonoyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF3), have been described (Koutek et al., J Biol Chem 269: 22937-22940, 1994). In the current study, methyl arachidonyl fluorophosphonate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approximately 1000-fold more potent than Arach-CF3. MAFP demonstrated selectivity towards anandamide amidase for which it was approximately 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, respectively. MAFP displaced [3H]CP-55940 binding to the CB1 cannabinoid receptor with an IC50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addition, can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonoyl ethanolamide amidase.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Ácidos Araquidónicos/farmacología , Inhibidores Enzimáticos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Sitios de Unión , Ciclohexanoles/metabolismo , Inhibidores Enzimáticos/metabolismo , Organofosfonatos , Ratas , Receptores de Cannabinoides , Receptores de Droga/metabolismoRESUMEN
Arachidonoyl ethanolamide (anandamide) is an endogenous ligand for cannabinoid receptors (CB1, CB2) and a putative neurotransmitter. Phenylmethylsulfonyl fluoride (PMSF) is an inhibitor of the enzyme (an amidase) which hydrolyzes anandamide to arachidonic acid and ethanolamine. We report here that fatty acid sulfonyl fluorides are potent inhibitors of anandamide metabolism. In order to investigate the SAR of these anandamide amidase inhibitors we tested a series of fatty acid (C12 to C20) sulfonyl fluorides both as inhibitors of anandamide degradation and as ligands for the central cannabinoid receptor (CB1). AM374 (palmitylsulfonyl fluoride, C16) was approximately 20 times more potent than PMSF and 50 times more potent than arachidonyltrifluoromethyl ketone in preventing the hydrolysis of anandamide in brain homogenates. AM374 was over a thousand-fold more effective than PMSF in inhibiting the amidase in cultured cells. The C12 to C18 sulfonyl fluoride analogs were equipotent as inhibitors of the amidase and the reverse reaction (the synthase) with nanomolar IC50 values. These compounds generally showed decreasing affinity for the CB1 receptor as the chain length increased; thus, C12 sulfonylfluoride had an IC50 of 18 nM and C20 sulfonylfluoride had an IC50 of 78 microM. The C14, C16, and C18 sulfonyl fluorides showed high selectivity for the amidase over the CB1 receptor and thus are potentially useful selective anandamide amidase inhibitors.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Ácidos Araquidónicos/metabolismo , Cannabinoides/metabolismo , Ácidos Grasos/farmacología , Palmitatos/farmacología , Receptor Cannabinoide CB2 , Receptores de Droga/metabolismo , Sulfonas/farmacología , Animales , Encéfalo/metabolismo , Endocannabinoides , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/química , Neuronas/metabolismo , Palmitatos/química , Fluoruro de Fenilmetilsulfonilo/análogos & derivados , Fluoruro de Fenilmetilsulfonilo/farmacología , Alcamidas Poliinsaturadas , Ratas , Ratas Sprague-Dawley , Receptores de Cannabinoides , Relación Estructura-Actividad , Sulfonas/química , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To compare human thyroid xenografts from patients with Graves' disease in severe combined immunodeficient (SCID) mice and triple immunodeficient NIH-beige-nude-xid (NIH-3) mice to obtain an improved animal model for studying these xenografts. DESIGN: Animal study. PARTICIPANTS AND ANIMALS: Patients with Graves' disease; SCID and NIH-3 mice. INTERVENTIONS: Thyroid tissue from six patients with Graves' disease was xenografted to SCID and NIH-3 mice; in addition, peripheral blood mononuclear cells (PBMC) from 12 patients with Graves' disease were grafted intraperitoneally to separate SCID and NIH-3 mice. OUTCOME MEASURES: Levels of human immunoglobulin (IgG), thyroperoxidase antibodies (TPO-Ab), thyroglobulin (Tg-Ab), and expression of thyrocyte intercellular adhesion molecule-1 (ICAM-1) and histocompatibility leukocyte antigen (HLA-DR) in mice after xenografting. RESULTS: IgG was detected in all mice grafted with Graves' thyroid tissue and some mice grafted with PBMC; levels of human IgG peaked 6 to 10 weeks after xenografting. Human IgG levels reached a mean of 500 mg/L (standard error of the mean [SEM] 150 mg/L) in the NIH-3 mice with thyroid xenografts. This was similar to results in SCID mice with thyroid xenografts, which had a mean level of human IgG of 640 mg/L (SEM 230 mg/L). PBMC xenografting resulted in a mean IgG level of 1200 mg/L (SEM 250 mg/L) in NIH-3 mice, which was similar to the mean level of 1000 mg/L (SEM 280 mg/L) in SCID mice. The rate of rise in human IgG in the sera of the NIH-3 mice with thyroid xenografts was similar to that in the SCID mice. TPO-Ab were also detected in some mice with Graves' thyroid grafts and in a few mice injected with PBMC, with levels peaking 4 to 6 weeks after xenografting. TPO-Ab levels reached a mean 109.3 U/mL (SEM 57.2 U/mL) in the NIH mice with thyroid xenografts, which were similar to the mean level of 91.7 U/mL (SEM 34.2 U/mL) in the SCID mice. There were no significant differences in the Tg-Ab levels in each type of mice (13.9 [SEM 12.1] U/mL v. 17.9 [SEM 7.9] U/mL). Eight weeks after xenografting into mice, the expression of xenograft thyrocyte ICAM-1 decreased significantly in both the SCID and NIH-3 mice (from 43.4%, SEM 4.9%, to 35.9%, SEM 4.6%, in the NIH-3 mice, p < 0.05, and from 43.4%. SEM 4.9%, to 32.5%, SEM 5.2%, in the SCID mice, p < 0.05). However, the expression of thyrocyte HLA-DR did not change significantly in the NIH-3 mice (from 11.5%, SEM 3.3%, to 10.8%, SEM 3.3%), whereas it decreased significantly in the SCID mice (from 11.5%, SEM 3.3%, to 4.2%, SEM 2.0%, p < 0.02). CONCLUSIONS: Not only SCID mice but also NIH-3 mice may be useful as animal models for xenografted thyroid tissue, which will help us elucidate the pathogenesis of autoimmune thyroid disease. NIH-3 mice are superior to SCID mice in maintaining the expression of thyrocyte HLA-DR in Graves' thyroid xenografts at levels as high as those before xenografting; this maintenance of expression may be due to the lack of natural killer cells in NIH-3 mice.
Asunto(s)
Enfermedad de Graves/inmunología , Glándula Tiroides/trasplante , Animales , Anticuerpos/sangre , Antígenos HLA-DR/biosíntesis , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulinas Estimulantes de la Tiroides/metabolismo , Molécula 1 de Adhesión Intercelular/biosíntesis , Yoduro Peroxidasa/inmunología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/trasplante , Masculino , Ratones , Ratones SCID , Ratas , Tiroglobulina/sangre , Tiroglobulina/inmunología , Glándula Tiroides/citología , Glándula Tiroides/inmunología , Trasplante HeterólogoRESUMEN
T cells from NOD mice display an age-dependent, TCR-inducible proliferative hyporesponsiveness that may be causal to IDDM. Exogenous IL-4 completely restores this hyporesponsiveness in vitro and prevents IDDM in vivo when administered to NOD mice. We therefore tested the hypothesis that stimulation of a Th2 response by either IL-4 or CD28 costimulation may block progression to IDDM. Low-dose IL-4 treatment beginning at 2 weeks of age (pre-insulitis) protects NOD mice from insulitis, sialitis, and thyroiditis, indicating that IL-4 modulates T cell migration to these inflammatory sites. Cytokine secretion profiles of stimulated T cells and assays of intrapancreatic cytokine concentrations revealed that IL-4 treatment prevents IDDM by stabilizing a protective Th2-mediated environment in the thymus, spleen, and pancreatic islets. Whereas treatment of NOD mice with an anti-CD28 mAb between 2 to 4 weeks of age inhibits destructive insulitis and protects against IDDM by enhancing IL-4 production by T cells, anti-CD28 treatment between 5 to 7 weeks of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the protective effect conferred by anti-CD28 treatment. Our data demonstrate that stimulation of a Th2-cell-enriched environment in the pancreas during the inductive phase of disease development blocks progression to IDDM in NOD mice.
Asunto(s)
Antígenos CD28/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Interleucina-4/farmacología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD28/farmacología , División Celular , Quimiocinas CC/inmunología , Citocinas/inmunología , Citocinas/farmacología , Diabetes Mellitus Tipo 1/inmunología , Modelos Animales de Enfermedad , Humanos , Interleucina-4/inmunología , Ratones , Ratones Endogámicos NOD , Linfocitos T/citología , Células Th2/inmunologíaRESUMEN
To investigate the effect of adding a surfeit of CD8+ T cells as a potential immunoregulator in Graves' disease (GD), thyroid tissues from 4 patients with GD and 2 normal subjects (N) were initially xenografted into nude mice. Eight weeks after xenografting, the thyroid tissues, which were then devoid of lymphocytes and appeared normal, were retrieved from the nude mouse, and rexenografted (rexenografts) into severe combined immuno-deficient (SCID) mice; 20 x 10(6) of autologous peripheral blood mononuclear cells (PMBC) or 20 x 10(6) of CD8(+)-depleted PBMC ("non-CD8 cells," i.e., CD4-enriched PBMC) were simultaneously engrafted into SCID mice with thyroid rexenografts. In addition, 20 x 10(6) of CD8(+)-enriched PBMC ("CD8-doubled" cells, which were prepared to double the percentage of CD8+ T cells compared to that of PBMC) were engrafted into SCID mice with rexenografts from 2 GD and 2 N; finally, 20 x 10(6) of PBMC plus an extra 10 x 10(6) of CD8+ T cells ("extra-CD8 added" cells, total 30 x 10(6) of CD8-enriched cells) were engrafted into separate SCID mice with rexenografts from 2 GD. The reengraftment of GD rexenografts or N rexenografts alone did not result in the detection of thyroperoxidase (TPO)-antibodies (Abs), thyroglobulin (Tg)-Abs, thyroid-stimulating Ab (TSAb) production, human IgG, or lymphocytic infiltration in the xenografts. However, the engraftment of either autologous PBMC or non-CD8 cells from patients with GD and N into SCID mice with rexenografts caused human IgG to become detectable and then rise further in 10 of 17 SCID mice; when human IgG, TPO-Ab, Tg-Ab, and TSAb were quantitated, GD rexenografts plus non-CD8 cells engrafted into SCID mice showed a higher production of each antibody and human IgG than in GD rexenografts plus PBMC, or GD rexenografts plus CD8-doubled cells, or GD rexenografts plus extra "CD8-added" cells. Moreover, when CD8-doubled cells or extra CD8-added cells with rexenografts were engrafted to SCID mice with rexenografts, they showed generally lower production of human IgG and thyroid antibodies compared to SCID mice into which PBMC were engrafted with rexenografts, despite the fact that 50% more cells (30 x 10(6)) were engrafted in the preparations of extra CD8-added cells. In conclusion, CD8+ T cells from patients with GD appeared to suppress the induction of thyroid antibodies, TSAb, and human IgG. The CD8+ cells thus are acting as suppressor or regulatory T cells. Such cells might be important in the pathogenesis of autoimmune thyroid disease.