ABSTRACT
Fas ligand (L)/CD95L, a proapoptotic member of the TNF family, is a potential target for clinical intervention in various diseases. In the present study, we generated a humanized anti-human FasL mAb and characterized the epitopes of neutralizing mAbs by extensive alanine-scanning mutagenesis of human FasL. The predicted molecular model of FasL trimer revealed that the mAbs recognize largely overlapped conformational epitopes that are composed of two clusters, one around the outer tip-forming D-E loop and another near the top of FasL. Both of these sites on FasL are critically involved in the direct interaction with the corresponding receptor, Fas. These results suggest that the mAbs efficiently neutralize FasL cytotoxicity by masking both of these FasL/Fas contact sites.
Subject(s)
Antibodies, Monoclonal/immunology , Epitope Mapping , Membrane Glycoproteins/immunology , Amino Acid Sequence , Amino Acid Substitution , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibody Specificity , Apoptosis , Binding Sites, Antibody , CHO Cells , COS Cells , Chlorocebus aethiops , Computer Simulation , Cricetinae , Cricetulus , Cytotoxicity, Immunologic , Epitopes/chemistry , Epitopes/immunology , Fas Ligand Protein , Gene Rearrangement, B-Lymphocyte , Genes, Immunoglobulin , Humans , Macromolecular Substances , Membrane Glycoproteins/chemistry , Mice , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Neutralization Tests , Protein Conformation , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Structure-Activity Relationship , fas Receptor/immunologyABSTRACT
Activin A, a member of the transforming growth factor-beta (TGF-beta) family, is produced by a variety of cells and implicated in the regulation of the reproductive endocrine system, mesoderm induction, and erythropoiesis. In the present study, we showed that activin A inhibited the production of interleukin-1beta (IL-1beta), a potent proinflammatory cytokine, and enhanced the production of IL-1 receptor antagonist (IL-1ra), in activated THP-1 and U-937 human monocytic cells, resulting in the reduction of IL-1 biologic activity. Northern blot analysis revealed that activin A had no effect on mRNA accumulation of IL-1beta and IL-1ra, indicating that activin A regulates IL-1beta and IL-1ra production at a posttranscriptional level. As it is well known that an inactive precursor form of IL-1beta (pro-IL-1beta) is converted to an active mature form (mature IL-1beta), we examined the expression levels of pro-IL-1beta and mature IL-1beta by immunoblot analysis. Although activin A inhibited the production of mature IL-1beta in activated U-937 cells, the relative protein expression of pro-IL-1beta was unaltered by activin A, suggesting that activin A inhibits IL-1beta production by blocking proteolytic conversion of pro-IL-1beta into mature IL-1beta. Taken together, these findings suggest that activin A may function as an anti-inflammatory cytokine by modulating mature IL-1beta and IL-1ra production in inflammatory sites.
