ABSTRACT
Background: Pneumococci are the major cause of bacterial meningitis globally. To cause meningitis pneumococci interact with the 2 endothelial receptors, polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1), to penetrate the blood-brain barrier (BBB) and invade the brain. Methods: C57BL/6 mice were infected intravenously with bioluminescent pneumococci, and treated with ceftriaxone (1 hour postinfection) and anti-pIgR and PECAM-1 antibodies (1 or 5 hours postinfection), then monitored for 5 and 10 days. Bacterial brain invasion was analyzed using IVIS imaging and bacterial counts. Results: Ceftriaxone, given early after pneumococcal challenge, cleared pneumococci from the blood but not from the brain. After combining ceftriaxone with receptor blockade, using anti-pIgR and PECAM-1 antibodies, we found 100% survival after 5 and 10 days of infection, in contrast to 60% for ceftriaxone alone. Combined antibiotic and antibody treatment resulted in no or few viable bacteria in the brain and no microglia activation. Antibodies remained bound to the receptors during the study period. Receptor blockade did not interfere with antibiotic permeability through the BBB. Conclusions: We suggest that adjunct treatment with pIgR and PECAM-1 antibodies to antibiotics may prevent pneumococcal meningitis development and associated brain damages. However, further evaluations are required.
Subject(s)
Meningitis, Pneumococcal/drug therapy , Platelet Endothelial Cell Adhesion Molecule-1/antagonists & inhibitors , Receptors, Polymeric Immunoglobulin/antagonists & inhibitors , Animals , Anti-Bacterial Agents/administration & dosage , Antibodies/administration & dosage , Bacterial Load , Ceftriaxone/administration & dosage , Disease Models, Animal , Drug Therapy, Combination , Intravital Microscopy , Mice, Inbred C57BL , Survival Analysis , Treatment OutcomeABSTRACT
Human polymeric immunoglobulin receptor (pIgR) protein was expressed in the adeno-carcinoma cell line HT-29 using a recombinant vaccinia virus transfection method. The pIgR protein was detected as 110- and 120-kDa bands by immunoprecipitation after metabolic labeling. PIgR was released as a free secretory component into the culture supernatant and was detected as a 110-kDa band. PIgR cleavage was investigated by adding the proteinase inhibitor leupeptin or protein kinase C activator PMA. Consistent with previous observations in the Madin Darby canine kidney cell system, cleavage of pIgR was inhibited by leupeptin and enhanced by PMA stimulation, thus indicating that it is regulated by common mechanisms. This experimental system should be very useful for pIgR investigation.
Subject(s)
Receptors, Polymeric Immunoglobulin/biosynthesis , Culture Media , Cysteine Proteinase Inhibitors/metabolism , Enzyme-Linked Immunosorbent Assay , HT29 Cells/metabolism , Humans , Immunoprecipitation , Leupeptins/metabolism , Protein Kinase C/metabolism , Protein Transport , Receptors, Polymeric Immunoglobulin/antagonists & inhibitors , Secretory Component/metabolism , Tetradecanoylphorbol Acetate/metabolism , Transfection/methods , Vaccinia virusABSTRACT
The polymeric IgR (pIgR) mediates transport of dimeric IgA and pentameric IgM across mucosal epithelia, thereby generating secretory Abs. Its expression is up-regulated at the transcriptional level by IL-4 in HT-29 cells. In this study, we demonstrate that IL-4 mediates up-regulation of human pIgR through a 554-bp IL-4-responsive enhancer in intron 1. Mutation of a binding site for STAT-6 within this region abolished IL-4-induced enhancement, while an adjacent putative C/EBP site was dispensable. IL-4 treatment induced binding of STAT6 to the intronic STAT6 site, but cooperation with nearby upstream and downstream DNA elements was required for IL-4 responsiveness. Furthermore, IL-4-mediated increased transcription of the pIgR-derived enhancer, like the endogenous pIgR gene, required de novo protein synthesis. Interestingly, a conditionally active form of STAT6 sufficed to activate a pIgR-derived enhancer in HT-29 cells, but not in Cos-1 cells, suggesting a requirement for cell type-specific factors. Thus, STAT6 activation mediates a delayed transcriptional enhancement of pIgR by induction of a de novo synthesized protein that cooperates with STAT6 itself bound to its cognate DNA element in intron 1. This mechanism may represent a general strategy for how pleiotropic cytokines elicit cell type-specific transcriptional responses.
Subject(s)
Interleukin-4/physiology , Receptors, Polymeric Immunoglobulin/biosynthesis , Response Elements/immunology , Trans-Activators/physiology , Transcription, Genetic/immunology , Up-Regulation/immunology , Animals , Binding Sites/genetics , Binding Sites/immunology , COS Cells , Chlorocebus aethiops , Cycloheximide/pharmacology , Enhancer Elements, Genetic/immunology , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Genes, Reporter/immunology , Genetic Vectors/chemical synthesis , Genetic Vectors/immunology , HT29 Cells , Humans , Interleukin-4/antagonists & inhibitors , Introns/immunology , Kinetics , Luciferases/antagonists & inhibitors , Luciferases/genetics , Molecular Sequence Data , RNA, Messenger/antagonists & inhibitors , RNA, Messenger/biosynthesis , Receptors, Polymeric Immunoglobulin/antagonists & inhibitors , Receptors, Polymeric Immunoglobulin/genetics , Regulatory Sequences, Nucleic Acid/immunology , STAT6 Transcription Factor , Time Factors , Trans-Activators/metabolism , Up-Regulation/drug effectsABSTRACT
Phosphatidyl inositol 3-kinase (PI3K) plays an essential role in numerous signaling events, and increasingly has been implicated in regulation of certain membrane traffic events. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) across epithelial cells and into external secretions, where the dIgA forms the first specific immunological defense against infection. We show here that wortmannin, a highly specific inhibitor of PI3K, inhibits transcytosis of dIgA by the pIgR. Instead, the dIgA is recycled back to the basolateral surface of the epithelial cell. PI3K therefore plays an essential role in regulating the transcytosis of dIgA, a key step in the mucosal immune response.
