KCa1.1 and Kv1.3 channels regulate the interactions between fibroblast-like synoviocytes and T lymphocytes during rheumatoid arthritis.

BACKGROUND: Fibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA. METHODS: We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies. RESULTS: We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA. CONCLUSIONS: Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog.

Effector memory T lymphocytes (TEM cells) that lack expression of CCR7 are major drivers of inflammation in a number of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. The Kv1.3 potassium channel is a key regulator of CCR7- TEM cell activation. Blocking Kv1.3 inhibits TEM cell activation and attenuates inflammation in autoimmunity, and as such, Kv1.3 has emerged as a promising target for the treatment of TEM cell-mediated autoimmune diseases. The scorpion venom-derived peptide HsTX1 and its analog HsTX1[R14A] are potent Kv1.3 blockers and HsTX1[R14A] is selective for Kv1.3 over closely-related Kv1 channels. PEGylation of HsTX1[R14A] to create a Kv1.3 blocker with a long circulating half-life reduced its affinity but not its selectivity for Kv1.3, dramatically reduced its adsorption to inert surfaces, and enhanced its circulating half-life in rats. PEG-HsTX1[R14A] is equipotent to HsTX1[R14A] in preferential inhibition of human and rat CCR7- TEM cell proliferation, leaving CCR7+ naïve and central memory T cells able to proliferate. It reduced inflammation in an active delayed-type hypersensitivity model and in the pristane-induced arthritis (PIA) model of rheumatoid arthritis (RA). Importantly, a single subcutaneous dose of PEG-HsTX1[R14A] reduced inflammation in PIA for a longer period of time than the non-PEGylated HsTX1[R14A]. Together, these data indicate that HsTX1[R14A] and PEG-HsTX1[R14A] are effective in a model of RA and are therefore potential therapeutics for TEM cell-mediated autoimmune diseases. PEG-HsTX1[R14A] has the additional advantages of reduced non-specific adsorption to inert surfaces and enhanced circulating half-life.

KCa1.1 inhibition attenuates fibroblast-like synoviocyte invasiveness and ameliorates disease in rat models of rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) participate in joint inflammation and damage in rheumatoid arthritis (RA) and its animal models. The purpose of this study was to define the importance of KCa1.1 (BK, Maxi-K, Slo1, KCNMA1) channel expression and function in FLS and to establish these channels as potential new targets for RA therapy. METHODS: We compared KCa1.1 expression levels in FLS from rats with pristane-induced arthritis (PIA) and in FLS from healthy rats. We then used ex vivo functional assays combined with small interfering RNA-induced knockdown, overexpression, and functional modulation of KCa1.1 in PIA FLS. Finally, we determined the effectiveness of modulating KCa1.1 in 2 rat models of RA, moderate PIA and severe collagen-induced arthritis (CIA). RESULTS: We found that PIA FLS expressed the KCa1.1 channel as their major potassium channel, as has been found in FLS from patients with RA. In contrast, FLS from healthy rats expressed fewer of these channels. Inhibiting the function or expression of KCa1.1 ex vivo reduced proliferation and invasive properties of, as well as protease production by, PIA FLS, whereas opening native KCa1.1 or overexpressing the channel enhanced the invasiveness of both FLS from rats with PIA and FLS from healthy rats. Treatment with a KCa1.1 channel blocker at the onset of clinical signs stopped disease progression in the PIA and CIA models, reduced joint and bone damage, and inhibited FLS invasiveness and proliferation. CONCLUSION: Our results demonstrate a critical role of KCa1.1 channels in the regulation of FLS invasiveness and suggest that KCa1.1 channels represent potential therapeutic targets in RA.