Inhibiting HLA-B27 homodimer-driven immune cell inflammation in spondylarthritis.

OBJECTIVE: Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with ß(2) -microglobulin (ß(2) m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form ß(2) m-free heavy chain homodimers (HLA-B27(2) ), which, unlike classic HLA-B27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLA-B27(2) to KIR-3DL2-positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS: We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B27(2) -expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2-B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS: Monoclonal antibody HD6 specifically recognized recombinant HLA-B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA-B27(2) to KIR-3DL2 and the survival and proliferation of KIR-3DL2-positive NK cells. Finally, HD6 inhibited production of the proinflammatory disease-associated cytokine interleukin-17 by PBMCs from patients with AS. CONCLUSION: These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.

Endothelial nitric oxide synthase gene transfer inhibits human smooth muscle cell migration via inhibition of Rho A.

Smooth muscle cell (SMC) migration contributes to vascular remodeling. Nitric oxide (NO) produced via endothelial NO synthase (eNOS) inhibits SMC migration. This study analyzes signal transduction mechanisms of SMC migration targeted by NO. SMCs were cultured from human saphenous veins, and cell migration was studied using Boyden chambers. PDGF-BB (0.1 to 10 ng/ml) stimulated SMC migration in a concentration-dependent manner, which was inhibited by adenoviral-mediated overexpression of eNOS and by the NO donor diethylentriamine NONOate (DETANO, 10 to 10 mol/L). NO release was enhanced in eNOS-transduced SMCs, and L-NAME blunted the effect of eNOS overexpression on migration. PDGF-BB (10 ng/ml) activated Rho A, which was inhibited by the overexpression of eNOS by DETANO and by 8 bromo-cGMP. The inhibitory effect of DETANO on Rho A activity was prevented by the cGMP-dependant kinase inhibitor. Furthermore, inhibition of Rho A by C3 exoenzyme and inhibition of ROCK by Y-27632 diminished cell migration stimulated by PDGF-BB. Finally, in the cells overexpressing constitutively active ROCK mutant (CAT), DETANO failed to prevent PDGF-BB-induced SMC migration. In conclusion, NO inhibits human SMC migration via blockade of the Rho A pathway.

Toll-Like Receptor 9 Activation Rescues Impaired Antibody Response in Needle-free Intradermal DNA Vaccination.

The delivery of plasmid DNA to the skin can target distinct subsets of dermal dendritic cells to confer a superior immune response. The needle-free immunization technology offers a reliable, safe and efficient means to administer intradermal (ID) injections. We report here that the ID injection of DNA vectors using an NF device (NF-ID) elicits a superior cell-mediated immune response, at much lesser DNA dosage, comparable in magnitude to the traditional intramuscular immunization. However, the humoral response is significantly impaired, possibly at the stage of B cell isotype switching. We found that the NF-ID administration deposits the DNA primarily on the epidermis resulting in a rapid loss of the DNA as well as the synthesized antigen due to the faster regeneration rate of the skin layers. Therefore, despite the immune-rich nature of the skin, the NF-ID immunization of DNA vectors may be limited by the impaired humoral response. Additional booster injections are required to augment the antibody response. As an alternative and a viable solution, we rescued the IgG response by coadministration of a Toll-like receptor 9 agonist, among other adjuvants examined. Our work has important implication for the optimization of the emerging needle-free technology for ID immunization.

Cyclophilin A differentially activates monocytes and endothelial cells: role of purity, activity, and endotoxin contamination in commercial preparations.

BACKGROUND: Cyclophilin A (CyPA) is a cytoplasmic protein secreted under inflammatory conditions. Extracellular CyPA is detected in atherosclerotic plaques and has been observed to activate endothelial cells as well as monocytes. METHODS AND RESULTS: Commercially available recombinant CyPA-induced expression of tissue factor (TF) and vascular cell adhesion molecule-1 (VCAM-1) in human aortic endothelial cells (HAEC). However, CyPA from commercial sources contained lipopolysaccharide at concentrations up to 18.9 ng/ml; moreover, it exhibited low purity as determined by protein spectrum analysis and low activity as assessed by peptidyl prolyl cis-trans isomerase (PPIase) assay. An in-house preparation of pure, active, and uncontaminated CyPA failed to induce endothelial TF or VCAM-1 expression; moreover, it was not chemotactic for HAEC. In contrast, such CyPA exhibited potent chemotactic activity on monocytic THP-1 cells, with a maximal effect on migration occurring at a concentration of 5.5 x 10(-9)mol/l. Pretreatment of CyPA with cyclosporine A prevented its effect on THP-1 cell migration; similarly, PPIase-deficient mutant CyPA protein did not induce migration of these cells. In-house prepared CyPA induced the release of Il-6, but not TNF-alpha, from THP-1 cells. CONCLUSIONS: Commercially available CyPA exhibits low purity and activity and may be contaminated by endotoxin. Pure, active, and uncontaminated CyPA does not induce endothelial TF or VCAM-1 expression; instead, it acts as a potent monocyte chemoattractant and induces monocyte Il-6 release, implying a role for extracellular CyPA in the pathogenesis of atherosclerosis via activation of monocytes rather than endothelial cells.