RESUMO
Dysregulation of mast cell function contributes to allergic and autoimmune disease that affects more than 70 million persons in the United States alone. Identifying novel mast cell targets that mediate disease or disease progression is required for the development of innovative therapeutics for the treatment of allergy/asthma and autoimmune disease. RNA interference technologies offer hope both as basic research tools for target identification and as potential, novel, specific therapeutics. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of evolutionarily conserved proteins that have been postulated to mediate the transport and fusion of inflammatory mediator-laden vesicles to the membrane in mast cells leading to their subsequent exocytosis. The functional role(s) of specific SNARE family member complexes in mast cell degranulation has not been fully elucidated. Here, we characterize the functional importance of SNARE complexes in FcεRI receptor-mediated degranulation of RBL-2H3 cells utilizing RNA interference. We demonstrate that ternary SNARE complexes of synaptosomal-associated protein-23, Syntaxin 4 and vesicle-associated membrane protein-7 (VAMP-7) or VAMP-8 are directly involved in mast cell degranulation. Additionally, we evaluate the siRNAs directed against these molecules as potential therapeutic agents for disease intervention. These studies have identified specific SNARE proteins and complexes that serve as novel targets for the development of siRNA therapies to treat allergic and autoimmune disease.
Assuntos
Degranulação Celular/imunologia , Regulação para Baixo/imunologia , Mastócitos/imunologia , RNA Interferente Pequeno/administração & dosagem , Proteínas SNARE/imunologia , Linhagem Celular , Humanos , Immunoblotting , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/imunologia , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/imunologia , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/imunologia , Proteínas R-SNARE/genética , Proteínas R-SNARE/imunologia , RNA/química , RNA/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/genéticaRESUMO
Lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 interactions mediate several important steps in the evolution of an immune response. LFA-1 is normally expressed in a quiescent state on the surface of leukocytes and interacts weakly with its ligands ICAM-1, -2, and -3. LFA-1 activity may be regulated by receptor clustering and by increasing the affinity of LFA-1 for its ligands. Affinity modulation of LFA-1 has been shown to occur via a conformational change in the LFA-1 heterodimer that can be detected by using monoclonal antibody 24 (mAb24). We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that demonstrates selective in vitro and in vivo inhibition of LFA-1/ICAM-1-mediated binding events. We now demonstrate that BIRT 377 blocks the induction of the mAb24 reporter epitope on LFA-1 on the surface of SKW-3 cells treated with various agonists known to induce high-affinity LFA-1. These data imply that BIRT 377 exerts its inhibitory effects by preventing up-regulation of LFA-1 to its high-affinity conformation.
Assuntos
Imidazóis/farmacologia , Imidazolidinas , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Regulação Alostérica , Adesão Celular , Relação Dose-Resposta a Droga , Epitopos/efeitos dos fármacos , Humanos , Imunossupressores/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/química , Antígeno-1 Associado à Função Linfocitária/imunologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/imunologia , Conformação Proteica/efeitos dos fármacos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacosRESUMO
LFA-1 (CD18,CD11a) is a cell-adhesion molecule that mediates critical immunological processes. In this paper we report the discovery and characterization of (R)-5-(4-bromobenzyl)-3-(3, 5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione (BIRT 377), an orally bioavailable small molecule that interacts specifically with LFA-1 via noncovalent binding to the CD11a chain and prevents LFA-1 from binding to its ligand, ICAM-1. BIRT 377 inhibits lymphocyte activity both in vitro and in vivo, in functional assays that require LFA-1-mediated cell adhesion. These results demonstrate that LFA-1-mediated leukocyte adhesion can be antagonized with noncharged, low m.w. molecules and suggest that the potential therapeutic value of adhesion inhibitors can be attained with a small, orally bioavailable compound.
Assuntos
Imidazóis/química , Imidazóis/farmacologia , Imidazolidinas , Imunossupressores/química , Imunossupressores/farmacologia , Antígeno-1 Associado à Função Linfocitária/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/imunologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Agregação Celular/efeitos dos fármacos , Agregação Celular/imunologia , Feminino , Humanos , Imidazóis/isolamento & purificação , Imidazóis/metabolismo , Imunossupressores/isolamento & purificação , Imunossupressores/metabolismo , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Teste de Inibição de Aderência Leucocítica , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/imunologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.
Assuntos
N-Formilmetionina Leucil-Fenilalanina/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Peptídeos/metabolismo , Antígenos/biossíntese , Sítios de Ligação , Feminino , Humanos , Imuno-Histoquímica , Masculino , Neuroblastoma/química , Neuroblastoma/metabolismo , Especificidade de Órgãos , Ligação Proteica , Receptores de Formil Peptídeo , Receptores Imunológicos/biossíntese , Receptores Imunológicos/imunologia , Receptores de Peptídeos/biossíntese , Receptores de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
The interactions of intercellular adhesion molecules-1 and -3 (ICAM-1 and ICAM-3) with lymphocyte function-associated antigen-1 (LFA-1) have been characterized and compared on the molecular and cellular level. Enzyme-linked immunosorbent-based molecular assays have been utilized to calculate the binding affinities of soluble ICAM-1 (sICAM-1) and soluble ICAM-3 (sICAM-3) for LFA-1. Consistent with previously published data, we found that sICAM-1 binds to LFA-1 with an affinity of approximately 60 nM. In contrast, sICAM-3 binds to LFA-1 with an affinity approximately 9 times weaker ( approximately 550 nM). Both sICAM-1 and sICAM-3 require divalent cations for binding. Specifically, both Mg2+ and Mn2+ support high affinity adhesion, although interestingly, high concentrations of Ca2+ decrease the affinity of each molecule for LFA-1 substantially. Furthermore, a panel of anti-LFA-1 monoclonal antibodies were characterized for their ability to block sICAM-1 and sICAM-3/LFA-1 interactions in molecular and cellular assays to help distinguish binding sites on LFA-1 for both molecules. Finally, molecular and cellular competition experiments demonstrate that sICAM-1 and sICAM-3 compete with each other for binding to LFA-1. The above data demonstrate that sICAM-1 and sICAM-3 share a common binding site or an overlapping binding site on LFA-1 and that the apparent differences in binding sites can be attributed to different affinities of sICAM-1 and sICAM-3 for LFA-1.
Assuntos
Antígenos CD , Antígenos de Diferenciação , Moléculas de Adesão Celular/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Ligação Competitiva , Moléculas de Adesão Celular/genética , Ensaio de Imunoadsorção Enzimática , Molécula 1 de Adesão Intercelular/genética , Magnésio/metabolismo , Manganês/metabolismo , Mutagênese , Células Tumorais CultivadasRESUMO
CD26 and ecto-adenosine deaminase (ADA) are found associated on the plasma membrane of T lymphocytes and each possess distinct catalytic activities. CD26 has a proteolytic activity identical to dipeptidylpeptidase IV (DPPIV; E.C. 3.4.14.5), and ecto-ADA (E.C. 3.5.4.4) degrades extracellular adenosine. The cell surface expression of CD26 and ecto-adenosine deaminase (ecto-ADA) is regulated on stimulated T lymphocytes, and ADA binding to CD26 produces a synergistic costimulatory response with T cell receptor activation. This study addresses the potential regulation by allosteric interactions of the catalytic activities of CD26 associated with ecto-ADA, which could define the mechanism of the synergism observed in T cell signaling. Cell lines genetically deficient in ADA, ligands for ADA such as adenosine, and a specific inhibitor of ADA, deoxycoformycin, were used to define the effect of ADA activity on CD26 DPPIV activity and affinity for dipeptide substrate. Conversely, a recombinant Chinese hamster ovary cell line expressing human CD26 with or without a mutation in the DPPIV catalytic domain, and the boronic acid inhibitor Val-boroPro, were used to determine the effect of DPPIV activity on ecto-ADA activity and association with CD26. These studies found no significant allosteric interaction between the catalytic activities of CD26 and ecto-ADA when associated. Therefore, signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules.
Assuntos
Adenosina Desaminase/metabolismo , Ácidos Borônicos/farmacologia , Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/farmacologia , Pentostatina/farmacologia , Inibidores de Adenosina Desaminase , Regulação Alostérica , Animais , Células CHO , Linhagem Celular , Cricetinae , Humanos , Linfócitos/enzimologiaRESUMO
The ability of soluble ICAM-1 (sICAM-1) to inhibit ICAM-1/LFA-1 adhesion events has been reported previously by numerous investigators. sICAM-1 has been demonstrated to inhibit various in vitro assays at concentrations ranging from 2 nM to greater than 40 microM. Given the hypothesis that circulating ICAM-1 modulates immune functions, the ability of sICAM-1 to inhibit cellular functions may have significant ramifications. Considering the potential clinical importance of the interaction between ICAM-1 and its receptor, LFA-1, it is necessary to understand this receptor-ligand interaction at a molecular level. In this study, direct binding experiments were utilized to determine the affinity between biotinylated monomeric sICAM-1 and immobilized LFA-1 (approximately 130 nM). Competitive binding experiments with unlabeled sICAM-1 and a truncated form of sICAM-1 (D1D2) yielded similar affinities. The specificity of this interaction was characterized using mAbs directed against sICAM-1 or LFA-1. This assay system was extended to include multimeric species using nonblocking mAbs directed against domains D4 and D5 of sICAM-1. Dimerizing sICAM-1 with a mAb alphaD4 or alphaD5 increased the affinity for immobilized LFA-1 by two orders of magnitude (approximately 4 nM), an effect presumably due to avidity. These results indicate that while the monomeric sICAM-1/LFA-1 interaction may involve only a moderate binding affinity, multimeric ICAM-1 present on a cell surface may bind cell surface-immobilized LFA-1 with very high avidity. These sICAM-1/LFA-1 molecular assays should be useful in defining the efficacy of potential antagonists.
Assuntos
Molécula 1 de Adesão Intercelular/química , Antígeno-1 Associado à Função Linfocitária/química , Ligação Competitiva , Biotina , Sistema Livre de Células , Molécula 1 de Adesão Intercelular/metabolismo , Cinética , Antígeno-1 Associado à Função Linfocitária/metabolismo , Ligação Proteica , Proteínas Recombinantes , Solubilidade , Relação Estrutura-AtividadeRESUMO
In solution, intercellular adhesion molecule-1 (ICAM-1) exhibits extremely low affinity for its receptor, LFA-1, as direct binding to LFA-1 has not been reported. Furthermore, there are conflicting reports on the ability of ICAM-1 in solution to inhibit cell adhesion events. These differences could be due to the valency or an oligomeric native biochemical form of membrane-bound and soluble ICAM-1, which may correlate with its ability to bind to integrins. To test this, stimulated adenocarcinoma (A549) cells or HUVEC were labeled with 35S-methionine/cysteine and treated with a chemical cross-linker. A high m.w. form (200 kDa) of ICAM-1 but not ICAM-2 was specifically immunoprecipitated from cross-linked cell lysates and supernatants. Affinity purification of crosslinked supernatants revealed that the majority of ICAM-1 was dimeric as opposed to recombinant soluble ICAM-1, which contains a minor fraction of dimer. Gel filtration chromatography was used to isolate monomeric and dimer-rich fractions of recombinant soluble ICAM-1, and tested for direct binding to affinity-purified LFA-1. Dimer-rich fractions demonstrated an enhanced ability and estimated affinity, compared with monomeric protein, to bind to purified LFA-1. These data suggest that ICAM-1 exists in its native membrane-bound and shed form as a non-covalent dimer, and that dimerization directly correlates with enhanced binding to LFA-1.