Recombinant IgG1 Fc hexamers block cytotoxicity and pathological changes in experimental in vitro and rat models of neuromyelitis optica

Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM μ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50 mg/kg taken at 8 h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50 mg/kg administered before and at 12 h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO

Aquaporin water channels in transepithelial fluid transport

Aquaporins (AQPs) are membrane water channels that are involved in a diverse set of functions in mammalian physiology including epithelial fluid transport, brain water balance, cell migration, cell proliferation, neuroexcitation, fat metabolism, epidermal hydration, and others. Phenotype analysis of knockout mice has demonstrated an important role for AQPs in transepithelial fluid transport in kidney tubules, salivary and airway submucosal glands, choroid plexus and ciliary epithelium. The physiological functions of these epithelia, such as absorption of glomerular filtrate by proximal tubule and secretion of saliva by salivary gland, involve rapid transcellular water transport across epithelial cell barriers. Studies in knockout mice have also provided evidence that AQPs are not physiologically important in some epithelia where they are expressed, including lacrimal gland, sweat gland, gallbladder, alveoli and airways. Rates of transepithelial fluid transport per unit membrane surface area in these epithelia are substantially lower than transepithelial fluid transport rates in proximal tubule and salivary gland. Pharmacological inhibition of AQP water permeability in epithelia, with consequent reduced fluid transport, offers potential therapy for human diseases involving water imbalance such as congestive heart failure, hypertension and glaucoma

Fractionation of a Herbal Antidiarrheal Medicine Reveals Eugenol as an Inhibitor of Ca2+-Activated Cl− Channel TMEM16A

The Ca2+-activated Cl− channel TMEM16A is involved in epithelial fluid secretion, smooth muscle contraction and neurosensory signaling. We identified a Thai herbal antidiarrheal formulation that inhibited TMEM16A Cl− conductance. C18-reversed-phase HPLC fractionation of the herbal formulation revealed >98% of TMEM16A inhibition activity in one out of approximately 20 distinct peaks. The purified, active compound was identified as eugenol (4-allyl-2-methoxyphenol), the major component of clove oil. Eugenol fully inhibited TMEM16A Cl− conductance with single-site IC50∼150 µM. Eugenol inhibition of TMEM16A in interstitial cells of Cajal produced strong inhibition of intestinal contraction in mouse ileal segments. TMEM16A Cl− channel inhibition adds to the list of eugenol molecular targets and may account for some of its biological activities

Therapeutic Cleavage of Anti–Aquaporin-4 Autoantibody in Neuromyelitis Optica by an IgG-Selective Proteinase

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of pathogenic IgG autoantibodies (NMO-IgG) to astrocyte water channel aquaporin-4 (AQP4). Astrocyte damage and downstream inflammation require NMO-IgG effector function to initiate complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we evaluated the potential therapeutic utility of the bacterial enzyme IdeS (IgG-degrading enzyme of Streptococcus pyogenes), which selectively cleaves IgG antibodies to yield Fc and F(ab')(2) fragments. In AQP4-expressing cell cultures, IdeS treatment of monoclonal NMO-IgGs and NMO patient sera abolished CDC and ADCC, even when IdeS was added after NMO-IgG was bound to AQP4. Binding of NMO-IgG to AQP4 was similar to that of the NMO-F(ab')(2) generated by IdeS cleavage. NMO-F(ab')(2) competitively displaced pathogenic NMO-IgG, preventing cytotoxicity, and the Fc fragments generated by IdeS cleavage reduced CDC and ADCC. IdeS efficiently cleaved NMO-IgG in mice in vivo, and greatly reduced NMO lesions in mice administered NMO-IgG and human complement. IgG-selective cleavage by IdeS thus neutralizes NMO-IgG pathogenicity, and yields therapeutic F(ab')(2) and Fc fragments. IdeS treatment, by therapeutic apheresis or direct administration, may be beneficial in NMO