RESUMEN
Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min(-1) kg(-1), respectively. In mass balance studies using [(14)C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose. In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9. In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9. In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.
Asunto(s)
Acrilamidas/metabolismo , Acrilamidas/farmacocinética , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/farmacocinética , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Profármacos/metabolismo , Profármacos/farmacocinética , beta-Alanina/análogos & derivados , Absorción/efectos de los fármacos , Acrilamidas/química , Acrilamidas/farmacología , Animales , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/farmacología , Perros , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Haplorrinos , Humanos , Inyecciones Intravenosas , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Profármacos/química , Profármacos/farmacología , Ratas , Distribución Tisular/efectos de los fármacos , beta-Alanina/química , beta-Alanina/metabolismo , beta-Alanina/farmacocinética , beta-Alanina/farmacologíaRESUMEN
The protein-protein interaction between leukocyte functional antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is critical to lymphocyte and immune system function. Here, we report on the transfer of the contiguous, nonlinear epitope of ICAM-1, responsible for its association with LFA-1, to a small-molecule framework. These LFA-1 antagonists bound LFA-1, blocked binding of ICAM-1, and inhibited a mixed lymphocyte reaction (MLR) with potency significantly greater than that of cyclosporine A. Furthermore, in comparison to an antibody to LFA-1, they exhibited significant anti-inflammatory effects in vivo. These results demonstrate the utility of small-molecule mimics of nonlinear protein epitopes and the protein epitopes themselves as leads in the identification of novel pharmaceutical agents.
Asunto(s)
Inmunosupresores/farmacología , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Tiofenos/síntesis química , Tiofenos/farmacología , beta-Alanina/síntesis química , beta-Alanina/farmacología , Secuencia de Aminoácidos , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Ciclosporina/farmacología , Dermatitis Irritante/tratamiento farmacológico , Dinitrofluorobenceno , Diseño de Fármacos , Ensayo de Inmunoadsorción Enzimática , Epítopos , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/farmacología , Inmunosupresores/síntesis química , Inmunosupresores/química , Inmunosupresores/metabolismo , Molécula 1 de Adhesión Intercelular/química , Prueba de Cultivo Mixto de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos BALB C , Imitación Molecular , Mutagénesis , Estructura Secundaria de Proteína , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/metabolismo , beta-Alanina/análogos & derivados , beta-Alanina/química , beta-Alanina/metabolismoRESUMEN
The present study assesses the applicability of human skin-SCID (severe combined immunodeficiency) mouse chimeras in testing antipsoriatic therapeutics. Three agents were examined: (1) a monoclonal antibody to the alpha subunit of leukocyte function associated antigen-1 integrin (CD11a); (2) Cyclosporin A; and (3) clobetasol propionate (Temovate), a potent topical corticosteroid used clinically in the treatment of psoriasis. Skin transplanted to SCID mice from normal human volunteers or from psoriatic lesional skin was allowed to heal for 3 to 5 weeks before application of test reagents. During this period, psoriatic skin, which was 3.8-fold thicker than the corresponding normal skin before transplantation, maintained its phenotype (ie, increased epidermal thickness, rete ridges with blunted ends, and intralesional presence of T lymphocytes). Transplanted normal human skin, however, underwent a hyperplastic response during this period, resulting in a 2.4-fold increase in epidermal thickness. After the healing period, animals transplanted with normal or psoriatic skin were treated for 14 days by daily intraperitoneal injection of either Cyclosporin A or a monoclonal antibody to human CD11a, or by topical application of clobetasol propionate. At the end of the treatment period, the mice were killed and the tissue evaluated morphometrically for changes in epidermal thickness and immunohistologically for the presence of T lymphocytes. Both Cyclosporin A and anti-CD11a reduced the epidermal thickness of transplanted psoriatic skin, whereas neither reagent significantly reduced the thickness of transplanted normal skin. T lymphocytes were detected in the skin from treated animals; there did not seem to be any reduction in the number of T lymphocytes. Clobetasol propionate reduced the epidermal thickness of both normal and psoriatic skin. These data indicate that, in this model, therapies directed against pathophysiologic mechanisms that contribute to psoriasis can be distinguished from treatments that block epidermal hyperplasia occurring as a consequence of xenografting. Our observations provide evidence for the activity of anti-CD11a in an animal model of human psoriasis.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígeno-1 Asociado a Función de Linfocito/inmunología , Psoriasis/tratamiento farmacológico , Trasplante de Piel , Trasplante Heterólogo , Administración Tópica , Animales , Antiinflamatorios/uso terapéutico , Clobetasol/análogos & derivados , Clobetasol/uso terapéutico , Ciclosporina/uso terapéutico , Fármacos Dermatológicos/uso terapéutico , Glucocorticoides , Humanos , Ratones , Ratones SCID , Psoriasis/patología , Valores de Referencia , Piel/efectos de los fármacos , Piel/patologíaRESUMEN
A series of novel, highly potent alpha(v)beta(3) antagonists based on a thiophene scaffold and containing an acylguanidine as an Arg-mimetic is described. A number of structural features, such as cyclic versus open guanidine and a variety of lipophilic side chains, carbamates, sulfonamides and beta-amino acids were explored with respect to inhibition of alpha(v)beta(3) mediated cell adhesion and selectivity versus alpha(IIb)beta(3) binding. In addition, compound 19 was found to be active in the TPTX model of osteoporosis.
Asunto(s)
Osteoporosis/prevención & control , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Vitronectina/antagonistas & inhibidores , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Carbamatos/química , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Guanidina/química , Paratiroidectomía , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Ratas , Receptores de Vitronectina/metabolismo , Sensibilidad y Especificidad , Sulfonamidas/química , Tiofenos/síntesis química , Tiofenos/farmacología , TiroidectomíaRESUMEN
The synthesis of a series of RGD mimetic alpha(v)beta3 antagonists containing a hydantoin scaffold is shown. The results demonstrate some of the structural requirements for the design of selective alpha(v)beta3 antagonists (vs alpha(IIb)beta3) in terms of the Arg-mimetic, the distance between N- and C-terminus and the lipophilic side chain.
Asunto(s)
Hidantoínas/síntesis química , Oligopéptidos/síntesis química , Fibrinógeno/metabolismo , Humanos , Imidazoles/síntesis química , Estructura Molecular , Péptidos/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica/efectos de los fármacos , Receptores de Vitronectina/antagonistas & inhibidoresRESUMEN
Because of the lack of function-blocking anti-integrin antibodies that react with nonprimate species, the study of the role of integrins in in vivo animal models of atherosclerosis has been limited. In contrast, peptides or small molecules have shown less species specificity and thus may be better tools to use. In an attempt to identify integrin antagonists of potential use against smooth muscle response to injury, we investigated the role of human smooth muscle cell interactions with fibrin by using a panel of integrin antagonists consisting of the snake venom disintegrin, Kistrin, as well as cyclic peptides with well-defined integrin antagonists activities. We demonstrate that Kistrin, a disintegrin that inhibits beta1, beta2, beta3, and beta5 integrin interactions, had the most potent inhibitory effect. Based on our results, Kistrin or peptides with similar pan-integrin selectivity patterns are prime candidates for use as anti-integrin antagonists in further studies of atherosclerosis and restenosis.
Asunto(s)
Fibrina/antagonistas & inhibidores , Fibrina/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , Péptidos/farmacología , Aorta/citología , Coagulación Sanguínea/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Desintegrinas/farmacología , Fibrina/fisiología , Fibrinolíticos/farmacología , Humanos , Recién Nacido , Integrinas/antagonistas & inhibidores , Músculo Liso/fisiología , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Sulfóxidos/farmacologíaRESUMEN
The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins alphaIIbbeta3, alphavbeta3 and alpha5beta1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for beta3-chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to beta3-chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses alphavbeta3, but not to the parental cell line, which expresses no beta3-chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to beta3-chain integrins and the fact that p66 and B. burgdorferi bind to beta3-chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins alphaIIbbeta3 and alphavbeta3.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antígenos CD/metabolismo , Proteínas Bacterianas , Grupo Borrelia Burgdorferi/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Porinas/genética , Porinas/metabolismo , Antígenos CD/genética , Adhesión Bacteriana , Grupo Borrelia Burgdorferi/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Integrina beta3 , Ligandos , Proteínas de Unión a Maltosa , Biblioteca de Péptidos , Glicoproteínas de Membrana Plaquetaria/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The human pathogenic bacterium group A Streptococcus produces an extracellular cysteine protease [streptococcal pyrogenic exotoxin B (SpeB)] that is a critical virulence factor for invasive disease episodes. Sequence analysis of the speB gene from 200 group A Streptococcus isolates collected worldwide identified three main mature SpeB (mSpeB) variants. One of these variants (mSpeB2) contains an Arg-Gly-Asp (RGD) sequence, a tripeptide motif that is commonly recognized by integrin receptors. mSpeB2 is made by all isolates of the unusually virulent serotype M1 and several other geographically widespread clones that frequently cause invasive infections. Only the mSpeB2 variant bound to transfected cells expressing integrin alphavbeta3 (also known as the vitronectin receptor) or alphaIIbbeta3 (platelet glycoprotein IIb-IIIa), and binding was blocked by a mAb that recognizes the streptococcal protease RGD motif region. In addition, mSpeB2 bound purified platelet integrin alphaIIbbeta3. Defined beta3 mutants that are altered for fibrinogen binding were defective for SpeB binding. Synthetic peptides with the mSpeB2 RGD motif, but not the RSD sequence present in other mSpeB variants, blocked binding of mSpeB2 to transfected cells expressing alphavbeta3 and caused detachment of cultured human umbilical vein endothelial cells. The results (i) identify a Gram-positive virulence factor that directly binds integrins, (ii) identify naturally occurring variants of a documented Gram-positive virulence factor with biomedically relevant differences in their interactions with host cells, and (iii) add to the theme that subtle natural variation in microbial virulence factor structure alters the character of host-pathogen interactions.
Asunto(s)
Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Integrinas/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Receptores de Vitronectina/metabolismo , Streptococcus pyogenes/patogenicidad , Alelos , Animales , Proteínas Bacterianas , Células CHO , Adhesión Celular/efectos de los fármacos , Cricetinae , Endotelio Vascular/citología , Variación Genética , Humanos , Integrinas/genética , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Unión Proteica , Receptores de Vitronectina/genética , Proteínas Recombinantes , Streptococcus pyogenes/enzimologíaRESUMEN
By extensive mutagenic analysis of the inserted domain (I-domain) of the alpha-chain (CD11a) of the leukocyte function-associated antigen-1 (LFA-1), we have defined a putative binding surface for intercellular adhesion molecules 1 and 2 (ICAM-1 and -2). This analysis showed that individually mutating Leu-205 or Glu-241 to alanine completely abolished LFA-1 binding to ICAM-1 or -2 without affecting I-domain structure, as assayed by antibody binding. Mutating Thr-243 to alanine also had a profound effect on LFA-1 binding to ICAM-1 and -2, as seen by complete loss of binding to ICAM-1 and a significant reduction (70% decrease) in binding to ICAM-2. Mutating Glu-146 to alanine reduced LFA-1 binding to ICAM-1 or -2 by 70%, and mutating His-264 or Glu-293 to alanine reduced binding to ICAM-1 or -2 by about 30-40%. Mutating Thr-175 to alanine reduced binding to ICAM-1 by about 30% and binding to ICAM-2 by about 70%. Interestingly, mutating Lys-263 to alanine preferentially abolished LFA-1 binding to ICAM-2. Using these data, we have generated a model of the interface between the LFA-1 I-domain and residues in the first domain of ICAM-1 that have been shown to be critical for this interaction. In addition, this model, together with the ICAM-2 crystal structure, has been used to map residues that are likely to mediate LFA-1 I-domain binding to ICAM-2.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Secuencia de Aminoácidos , Antígenos CD/química , Antígenos CD/genética , Sitios de Unión , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Epítopos/metabolismo , Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/genética , Antígeno-1 Asociado a Función de Linfocito/química , Antígeno-1 Asociado a Función de Linfocito/genética , Modelos Moleculares , Datos de Secuencia Molecular , MutagénesisRESUMEN
Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin alpha(IIb)beta3 was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins alpha(v)beta3 and alpha5beta1, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified alpha(v)beta3 and alpha5beta1. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified alpha(v)beta3 and alpha5beta1 was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to alpha(v)beta3 was also shown by using a transfected cell line that expresses this receptor but not alpha(IIb)beta3. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both alpha5beta1 and alpha(v)beta3. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.
Asunto(s)
Adhesión Bacteriana , Grupo Borrelia Burgdorferi/fisiología , Receptores de Fibronectina/fisiología , Receptores de Vitronectina/fisiología , Línea Celular , HumanosRESUMEN
Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.
Asunto(s)
Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Sitios de Unión de Anticuerpos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/aislamiento & purificación , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/aislamiento & purificación , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The extracellular matrix of bone is composed mainly of type I collagen. In this report we studied the role and collagen-binding properties of osteoclast integrins (alpha v, alpha 2, beta 1, and beta 3). Cell adhesion assays with rat osteoclasts and affinity chromatography/SDS-PAGE analysis with purified human osteoclast membranes demonstrated adhesion of osteoclasts to native type I collagen in a divalent cation and Arg-Gly-Asp (RGD)-dependent way via alpha 2 beta 1 integrin, whereas osteoclast adhesion to denatured collagen predominantly involved alpha v beta 3. In receptor-binding assays, the involvement of human recombinant alpha v beta 3 in adhesion to denatured collagen was confirmed. Additionally, osteoclasts adhered to type I collagen fibers and to monomeric types II-V collagen with characteristics similar to those on native monomeric type I collagen. Osteoclastic bone resorption in vitro was inhibited (> 40%) in the presence of alpha 2 and beta 1 antibodies. Using scanning laser confocal microscopy, alpha v beta 3, alpha 2, and beta 1 integrin were detected within podosomes in nonresorbing osteoclasts and in the ruffled border area and basolateral membrane in resorbing osteoclasts, but not in the sealing zone of resorbing osteoclasts. These results demonstrate that alpha 2 beta 1, in addition to alpha v beta 3, has an important role in osteoclast function and acts as a receptor for native, but not denatured, collagen.
Asunto(s)
Resorción Ósea/fisiopatología , Adhesión Celular/fisiología , Integrinas/metabolismo , Osteoclastos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/uso terapéutico , Unión Competitiva , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Cationes Bivalentes/metabolismo , Cromatografía de Afinidad , Colágeno/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunohistoquímica , Integrinas/genética , Integrinas/inmunología , Integrinas/fisiología , Proteínas de la Membrana/metabolismo , Oligopéptidos/metabolismo , Osteoclastos/citología , Fragmentos de Péptidos/metabolismo , Ratas , Receptores Inmunológicos/metabolismoRESUMEN
Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.
Asunto(s)
Adhesión Celular , Integrinas/fisiología , Receptores de Superficie Celular/metabolismo , Receptores de Citoadhesina/metabolismo , Secuencia de Aminoácidos , Antígenos CD18/metabolismo , Línea Celular , Fibronectinas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Integrina beta1/metabolismo , Integrinas/metabolismo , Ligandos , Datos de Secuencia Molecular , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Vitronectina/metabolismoRESUMEN
Several studies indicate that the I domain located in the alpha chain (CD11a) of leukocyte function-associated antigen-1 (LFA-1; CD11a/CD18) plays an essential role in ligand recognition. We recently identified three distinct epitopes (IdeA, IdeB, and IdeC) within the CD11a I domain, recognized by antibodies that block binding of LFA-1 to intercellular adhesion molecules (ICAM) 1, 2, and 3. In the present study, we used a series of human/murine CD11a I domain chimeras, to localize a fourth I domain epitope (IdeD), recognized by three independently derived anti-CD11a antibodies that selectively block the binding of LFA-1 to ICAM-3, but not to ICAM-1. The IdeD epitope depended on human CD11a residues Asp182 and Ser184 and was not present in CD11b or CD11c. Although mutation of Asp182 and Ser184 failed to abolish ICAM-3 adhesion of LFA-1 transfectants, alignment of these residues with the crystal structure of the CD11a I domain suggested that the IdeD epitope is located in close proximity to residues (Ile126 and Asn129) recently implicated in the ICAM-3 binding site. Interestingly, the IdeB and IdeC epitopes appeared to be in close proximity of a divalent cation binding pocket within the CD11a I domain that regulates both ICAM-1 and ICAM-3 adhesion. Taken together, these data indicate that distinct regions of the CD11a I domain contain epitopes for antibodies that either selectively inhibit binding of LFA-1 to ICAM-3, or interfere with both ICAM-1 and ICAM-3 binding of LFA-1.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación , Moléculas de Adhesión Celular/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Adhesión Celular , Activación Enzimática , Mapeo Epitopo , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
We have identified amino acid residues within the evolutionarily conserved I domain of the alpha-chain (CD11a) of the leukocyte integrin leukocyte function-associated antigen (LFA) 1 that are critical for intercellular adhesion molecule (ICAM) 3 (CD50) binding. ICAM-3, a ligand of LFA-1, is thought to mediate intercellular adhesion essential for the initiation of immune responses. Using a panel of human/murine I domain chimeras and point mutants, we observed that the Ile-Lys-Gly-Asn motif, located in the NH2-terminal part of the CD11a I domain, is required for ICAM-3 but not ICAM-1 binding. These findings demonstrate that the I domain of CD11a contains distinct functional subdomains for ligand specific binding. An aspartic acid located at position 137, which is essential to ICAM-1/LFA-1 interactions (Edwards, C.P., M. Champe, T. Gonzalez, M.E. Wessinger, S.A. Spencer, L.G. Presta, P.W. Berman, and S.C. Bodary. 1995. J. Biol. Chem. 270:12635-12640), was also critical for ICAM-3 binding, whereas Ser at position 139 did not effect ICAM-1 or ICAM-3 binding. A synthetic peptide containing the Ile-Lys-Gly-Asn motif inhibited ICAM-3-dependent adhesion and proliferation of T cells at micromolar concentrations, suggesting that this peptide interferes with immune recognition. These observations underscore the importance of ICAM-3 in leukocyte function, and may lead to development of a new category of immunosuppressive agents.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Antígeno-1 Asociado a Función de Linfocito/química , Antígeno-1 Asociado a Función de Linfocito/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Evolución Biológica , Adhesión Celular/efectos de los fármacos , Línea Celular , Secuencia Conservada , Expresión Génica , Humanos , Cinética , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacología , Péptidos/química , Péptidos/farmacología , Mutación Puntual , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhesion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM-1, -2, -3). Using human/murine chimeras of the I-domain of the LFA-1 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ in human and murine I-domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I-domain. Mutation of the aspartic acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.
Asunto(s)
Molécula 1 de Adhesión Intercelular/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Animales , Adhesión Celular/genética , Adhesión Celular/fisiología , Cloruros/farmacología , Análisis Mutacional de ADN , Epítopos , Humanos , Ligandos , Antígeno-1 Asociado a Función de Linfocito/genética , Antígeno-1 Asociado a Función de Linfocito/inmunología , Cloruro de Magnesio/farmacología , Compuestos de Manganeso/farmacología , Ratones , Mutación Puntual , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la Especie , Relación Estructura-ActividadRESUMEN
Laminins may be encountered by osteoclasts and their precursors in basement membranes when they migrate from periosteal vasculature during skeletal development and in pathological situations. We have examined the recognition by osteoclasts of intact laminins and their proteolytic derivatives, and analysed the mechanism of adhesion. Rat osteoclasts fail to bind intact mouse Engelbreth-Holm-Swarm (EHS) laminin (3% adhesion relative to adhesion to foetal calf serum proteins) and bind only weakly to native human placental laminin (13%) or human merosin (9%). Pepsin treatment of native mouse EHS and human laminins increased osteoclast adhesion. Rat osteoclasts adhered to mouse EHS laminin-derived P1 fragment (70%), but failed to bind the E8 fragment, which contains adhesion sites recognised by some integrins. Binding to human and mouse P1 laminins was abolished by treatment with RGD-containing peptides and required divalent cations, but not by YIGSR peptide. Combinations of monoclonal antibodies to rat beta 3 and alpha v integrins reduced binding to P1 fragment by 91% and to human laminin by 72%, demonstrating that the major integrin involved in rat osteoclast adhesion to proteolysed laminin is alpha v beta 3. Antiserum to beta 1 integrin inhibited adhesion to human laminin by 40%, but to P1 fragment by only 8%; this suggests that beta 1 integrins(s) contribute to osteoclast adhesion to human laminin but probably not to P1 fragment. The involvement of alpha v beta 3 integrin was confirmed using a recombinant human alpha v beta 3 solid phase binding assay, alpha v beta 3 bound to mouse P1 fragment and proteolytically digested human laminin, but not intact laminins.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Integrinas/metabolismo , Laminina/metabolismo , Osteoclastos/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Laminina/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Unión Competitiva , Cationes Bivalentes , Adhesión Celular/efectos de los fármacos , Humanos , Integrinas/aislamiento & purificación , Laminina/química , Ratones , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Ratas , Receptores de Citoadhesina/aislamiento & purificación , Receptores de Citoadhesina/metabolismo , Receptores de Vitronectina , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Venenos de Serpiente/metabolismoRESUMEN
In many cell systems, cell-cell and cell-matrix interactions are mediated by integrins, a family of cell surface heterodimeric glycoprotein receptors. Osteoclast integrins may play a role in the process of bone resorption. Osteoclasts express the alpha v and beta 3 subunits of the vitronectin receptor and adhere to a wide range of proteins in vitro, all which contain the amino acid sequence Arg-Gly-Asp (RGD), an adhesion site recognition sequence common to many protein ligands that bind to integrins. The effect of kistrin, an RGD-containing snake venom protein, on osteoclast-mediated bone resorption was investigated in vivo and in vitro. When kistrin was infused into normocalcemic and hypercalcemic mice, serum calcium was significantly lowered at 3 and 6 h after the start of infusion, indicating an inhibitory effect on osteoclast activity in vivo. In vitro, kistrin potently inhibited bone resorption by isolated rat osteoclasts cultured on slices of bovine bone, and kistrin also inhibited the attachment of 293 cells expressing recombinant human alpha v beta 3 to fibrinogen (IC50 = 1 nM). These results indicate the potential therapeutic use of RGD-containing molecules for hypercalcemia of malignancy or for other disorders associated with bone loss.
Asunto(s)
Resorción Ósea/fisiopatología , Calcio/sangre , Osteoclastos/fisiología , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Venenos de Crotálidos/farmacología , Venenos de Crotálidos/uso terapéutico , Relación Dosis-Respuesta a Droga , Fibrinógeno/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Osteoclastos/efectos de los fármacos , Péptidos/química , Péptidos/uso terapéutico , RatasRESUMEN
Integrins are a family of cell surface glycoproteins that promote cell adhesion. The integrin alpha V beta 3, vitronectin receptor, is a major integrin expressed by osteoclasts. To further investigate the role of alpha V beta 3 in cell adhesion, we generated and characterized monoclonal antibodies to alpha V beta 3 by immunizing BALB/c mice with purified alpha V beta 3 protein. Three monoclonal antibodies (mAbs), 9D4.9.1, 9G2.1.3, and 10C4.1.3, from a total of more than 1100 positive cultures which bound alpha V beta 3, were characterized extensively: mAbs 9G2.1.3 and 10C4.1.3 recognize the alpha V beta 3 complex whereas mAb 9D4.9.1 reacts with the beta 3-chain shared between the alpha V beta 3 complex and gpIIbIIIa. Further epitope mapping using flow microfluorometry analysis and histochemical staining of various tissues showed that 9D4.9.1 and 10C4.1.3 recognized distinct epitopes. Ligand-binding studies using cell-bound and purified alpha V beta 3 demonstrated that all three mAbs blocked fibrinogen binding. Vitronectin binding was blocked by mAb 9D4.9.1 and, less effectively, by mAb 10C4.1.3; mAb 9G2.1.3 was without effect. All three mAbs recognized osteoclasts from human tissues; mAb 9G2.1.3 also stained osteoclasts from a wide range of nonhuman species. Monoclonal antibodies 9D4.9.1 and 9G2.1.3 bound to a panel of cultured cell lines and various tissues. In contrast, mAb 10C4.1.3 bound only weakly or not at all to tissues expressing alpha V beta 3 with the exception of osteoclasts. Thus, mAb 10C4.1.3 showed a very narrow tissue specificity being restricted to high-level expression on human osteoclasts.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Integrinas/inmunología , Osteoclastos/inmunología , Receptores de Citoadhesina/inmunología , Secuencia de Aminoácidos , Animales , Adhesión Celular , Epítopos , Fibrinógeno/metabolismo , Citometría de Flujo , Glicoproteínas/metabolismo , Humanos , Integrinas/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Receptores de Citoadhesina/metabolismo , Receptores de Vitronectina , Proteínas Recombinantes/inmunología , Especificidad de la Especie , Transfección , Células Tumorales Cultivadas , VitronectinaRESUMEN
Cyclic RGD-containing peptides caused a dose-dependent inhibition of binding of human embryonic kidney cells transfected with recombinant GpIIb/IIIa (r293 clone B) to human fibrinogen coated on to non-tissue culture plates. The inhibitory activity, IC50, of a panel of seventeen RGD-containing peptides ranged from 0.12 to 89.2 microM. These IC50 values correlated with those determined by the inhibition of platelet aggregation (r = 0.99). Even though there was a correlation, there were differences between the platelet aggregation and the bioadhesion assay. The binding of r293 clone B to fibrinogen was not increased by ADP suggesting that GpIIb/IIIa expressed on the surface of r293 clone B cells may be in the 'activated' form. Moreover, preincubation of r293 clone B cells with a monoclonal antibody (mAb) specific for GpIIIa (4B12) resulted in a dose-dependent decrease of binding to fibrinogen while a mAb specific for GPIIb (2D2) had no effect. Neither of these mAbs inhibited platelet aggregation. The binding of r293 clone B cells to fibrinogen required Ca2+ or Mg2+. This cell-based bioadhesion method can provide a tool for screening potential GpIIb/IIIa antagonists and investigating the interaction of GpIIb/IIIa and fibrinogen not possible with platelet aggregation.