RESUMO
The leukocyte beta2 integrin CR3 (CD11/CD18), is a surface heterodimeric glycoprotein that functions as a divalent cation-dependent adhesive complex. It mediates several important cell-substrate and cell-cell adhesive interactions among which the interaction with vascular endothelial cells that lead to leukocyte transmigration. We have isolated cDNA clones-coding for the rat complement receptor type 3 (CR3) alphaM subunit (CD11b) from a cDNA library. The cDNA sequence showed respectively 89.4% and 74.6% homology with its mouse and human counterpart. We have expressed the sequence coding for the VA module or Von Willebrand type domain (A-domain) and produced it in E. coli as a soluble recombinant fusion protein with GST. Simultaneously, we have cloned DNA fragments specific to the rat ICAM-1 domain 1 and domain 3 and expressed each clone in E. coli as recombinant soluble (rs) fusion proteins with GST. Recombinant CD11b A-domain was released from the fusion protein by thrombin cut. Purified ICAM-1 fusion peptides and CD11b A-domain were used to develop a direct binding assay that showed a specific binding between the rat ICAM-1 Ig like domain 3 and CD11b A-domain. These data demonstrate that the IgSF modules can be produced as a soluble recombinant fusion protein and used to study direct binding to the VA module displayed by members of the integrin superfamily.
Assuntos
Antígeno CD11b/genética , Antígenos CD18/genética , Clonagem Molecular/métodos , Expressão Gênica/genética , Molécula 1 de Adesão Intercelular/genética , Fator de von Willebrand/genética , Sequência de Aminoácidos , Animais , Adesão Celular/genética , Quimiotaxia de Leucócito/genética , DNA Complementar/genética , Escherichia coli/genética , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de SequênciaRESUMO
The beta2 integrin CD11b/CD18 (CR3) is a major adhesion receptor of neutrophils, normally utilized to fend off infections. This receptor contributes, however, to multiple forms of non-infectious inflammatory injury when dysregulated as shown in gene knock-outs and through the use of blocking monoclonal antibodies. The major ligand recognition site of CR3 has been mapped to the A-domain in the CD11b subunit (CD11bA). The recombinant form of this domain exhibits a ligand binding profile similar to that of the holoreceptor. To assess the potential anti-inflammatory activity of CD11bA as a competitive antagonist of CR3 in vivo, we assessed its effects on a developed animal model of traumatic skeletal muscle injury in the rat. Recombinant soluble rat CD11bA-domain fused to glutathione-S-transferase (GST) was administered intravenously in a single dose at 1 mg/kg to nine groups of Wistar rats, five in each group, 30 min before inducing traumatic skeletal muscle injury. Control animals received either a function-blocking anti-CD11b/CD18 monoclonal antibody (1 mg/kg), non-functional mutant forms of the CD11bA (D140GS/AGA, T209/A, D242/A), recombinant GST or buffer alone. In control animals, the wounded muscle showed oedema, erythrocyte extravasation and myonecrosis both within and outside the immediate wounded area (5-10 mm zone) and influx of neutrophils was detected 30 min post-wound, followed by a second wave 3 hr later. Wild-type CD11bA- or anti-CD11b monoclonal antibody (mAb)-treated rats showed a comparable and significant decrease in the number of infiltrating PMN (78 + 4%, n = 70 and 86 +/- 2%, n = 50, respectively) and preservation of the muscular fibres outside the immediate zone of necrosis (75 + 4%, n = 70, 84 +/- 1%, n = 50, respectively), compared to controls. These data demonstrate that CD11bA can be an effective tissue-preserving agent in acute inflammatory muscular injury.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Antígeno CD11b/uso terapêutico , Músculo Esquelético/imunologia , Miosite/prevenção & controle , Infiltração de Neutrófilos/imunologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios não Esteroides/imunologia , Anticorpos Monoclonais/imunologia , Antígeno CD11b/imunologia , Modelos Animais de Doenças , Feminino , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/imunologia , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Miosite/imunologia , Ratos , Ratos Wistar , Proteínas Recombinantes/uso terapêutico , Alinhamento de SequênciaRESUMO
An experimental model to study the effects of viral inactivators on the biological properties of DNA was developed. Beta-propiolactone (betaPL) was used in this model and its effects on ligation, transfer and gene expression of naked DNA were assessed. Evidence that betaPL impairs these two major DNA functions are presented. The amounts of betaPL that alter or abolish gene expression and prevent DNA cohesive ends ligation were determined. Based on these observations, it was concluded that this experimental approach could be used to study the effects on the biological properties of DNA of other inactivators used in vaccine preparations.