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

Figures

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  Fig. 1. Collagen-induced arthritis fibroblast-like synoviocytes (CIA-FLS) modulate T cell migration and serve as antigen-presenting cells in a KCa1.1-dependent manner. a Effector memory T (TEM) cell migration toward CIA-FLS that were cultured for 72 h in the presence or absence of interferon (IFN)-γ, paxilline, or both. Data are presented as mean ± SEM (n = 3). b Flow cytometric images of ovalbumin-loaded, IFN-γ-stimulated CIA-FLS (violet) and ovalbumin-specific TEM cell (green) cocultures that were stained for CD3 (red) after cells were allowed to interact for 30 min. c Flow cytometric plots of mixtures of ovalbumin-loaded, IFN-γ-stimulated CIA-FLS (CellTrace Violet) and ovalbumin-specific TEM cells (carboxyfluorescein succinimidyl ester [CFSE]) that were allowed to interact for 30 min in the presence or absence of paxilline (Pax) or ShK-186. Flow cytometric gating was completed to exclude single cells. d Quantifications of the proportion of CIA-FLS and TEM cells that form conjugates, as defined by the proportion of CellTrace Violet+CFSE+ multiplets or nonsingle cells detected by flow cytometry, from experiments in (c). Data are presented as mean ± SEM (n = 4 separate experiments). e…

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  Fig. 2. Collagen-induced arthritis fibroblast-like synoviocytes (CIA-FLS) express major histocompatibility complex (MHC) class II molecules that are regulated through KCa1.1, as well as antigen-presenting proteins. a CIA-FLS plasma membrane expression of MHC class II protein following stimulation for 72 h with IFN-γ (gray circles), paxilline (Pax; black squares), or interferon (IFN)-γ and paxilline (open squares). Data are presented as mean ± SEM (n = 3–8 CIA-FLS donors). b Flow cytometric histogram of CIA-FLS stained for the plasma membrane expression of MHC class II protein in cells treated with paxilline for 72 h (dashed line), stimulated for 72 h with IFN-γ (solid line), or stimulated for 72 h with IFN-γ and paxilline (dotted line). The shaded histogram represents background staining. c MHC class II molecule expression of CIA-FLS that were permeabilized with saponin prior to staining for MHC class II in cells treated for 72 h with IFN-γ (gray circles), paxilline (black squares), or IFN-γ and paxilline (open squares). Data are presented as mean ± SEM (n = 3–6 CIA-FLS donors). d Flow cytometric histogram of CIA-FLS stained for MHC class II molecules following permeabilization with…

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  Fig. 3. Effector memory T (TEM) cells and cytokines regulate the phenotype of fibroblast-like synoviocytes (FLS). KCa1.1α (a) and major histocompatibility complex (MHC) class II molecule (b) plasma membrane expression levels by collagen-induced arthritis (CIA)-FLS cultured for 72 h in the conditioned medium of TEM cells that were antigen-stimulated in the presence or absence of ShK-186. Data are presented as mean ± SEM (n = 6–9 CIA-FLS donors). c KCa1.1α protein expression by rheumatoid arthritis (RA)-FLS, as determined by flow cytometry, following 24 h of stimulation with recombinant cytokines. Data are presented as mean ± SEM (n = 7–9 RA-FLS donors). d CIA-FLS invasion through Matrigel-coated Transwells toward the conditioned medium of TEM cells that were antigen-stimulated in the presence or absence of ShK-186. Data are presented as mean ± SEM (n = 3 CIA-FLS donors). *p < 0.05, **p < 0.01

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  Fig. 4. Kv1.3 and KCa1.1 blockers synergize to reduce disease severity in collagen-induced arthritis (CIA). a Clinical scores of rats with CIA treated with vehicle (open squares), iberiotoxin (IbTX; black triangles), ShK-186 (gray squares), or both IbTX and ShK-186 (open triangles) every other day (EOD) starting at disease onset. Data are presented as mean ± SEM (n = 11 or 12 rats per group). b Left: X-rays of hind paws from a healthy rat and from rats with CIA treated with vehicle, IbTX, ShK-186, or both IbTX and ShK-186 EOD for 14 days after disease onset. Center and right: Safranin O/Fast Green staining (center) and H&E staining (right) of tissue sections of hind paw joints of a healthy rat and of rats in each treatment group. Arrows indicate areas of cartilage erosions (Safranin O/Fast Green) or hyperplasia (H&E). Scale bar = 100 μm. c Scoring of disease parameters from tissue sections of paws from rats of each treatment group (n = 3 rats per group). *p < 0.05, ****p < 0.0001

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  Fig. 5. Potassium channel blockers alter serum cytokines in collagen-induced arthritis (CIA). a–i Serum concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-12, IL-17A, monocyte chemoattractant protein (MCP)-1, IL-2, IL-1β, IL-6, and IL-10 from healthy rats or rats with CIA treated with vehicle, ShK-186, iberiotoxin (IbTX), or both ShK-186 and IbTX every other day for 14 days after disease onset. Data are presented as mean ± SEM (n = 5 or 6 rats per group). *p < 0.05

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  Fig. 6. Fibroblast-like synoviocytes (FLS) from collagen-induced arthritis (CIA) rats treated with potassium channel blockers have reduced pathogenic phenotypes ex vivo. a Invasion through Matrigel-coated Transwell inserts of FLS isolated from healthy rats or from rats with CIA treated with either vehicle, ShK-186, iberiotoxin (IbTX), or ShK-186 and IbTX every other day (EOD) for 14 days after disease onset. Data are presented as mean ± SEM (n = 3 FLS donors per group). b KCa1.1α expression by FLS isolated from healthy rats and from rats with CIA treated with vehicle, ShK-186, IbTX, or ShK-186 and IbTX EOD for 14 days after disease onset. Data are presented as mean ± SEM (n = 5 FLS donors per group). c Data from (b) plotted against the clinical scores on the 14th day after disease onset of the rats with CIA from which the FLS were isolated, along with a linear regression plot. *p < 0.05, **p < 0.01

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  Fig. 7. T cell populations are altered in rats with collagen-induced arthritis (CIA) treated with potassium channel blockers. a–o Expression of CD3, CD4, CD8, CD25, and CD45RC of inguinal lymph node cells of rats with CIA treated with vehicle, ShK-186, iberiotoxin (IbTX), or ShK-186 and IbTX every other day for 14 days after disease onset (n = 6 rats per group). *p < 0.05, **p < 0.01

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  Fig. 8. KCa1.1 and Kv1.3 blockers work in tandem to reduce disease severity in pristane-induced arthritis (PIA). a Clinical scores of paw inflammation in rats with PIA treated with vehicle (open squares), paxilline (Pax; black triangles), ShK-186 (gray squares), or both paxilline and ShK-186 (open triangles) every other day for 21 days after disease onset. Data are presented as mean ± SEM (n = 18–27 rats per group). b Ex vivo invasiveness of fibroblast-like synoviocytes (FLS) isolated from healthy rats and from rats with PIA treated with vehicle, paxilline, ShK-186, or both paxilline and ShK-186. Data are presented as mean ± SEM, N = 3 FLS donors per group. c Left, example X-rays of hind paws of a healthy rat and from rats with PIA treated with vehicle, paxilline, ShK-186, or paxilline and ShK-186 every other day for 21 days after disease onset. Center and right: Safranin O/Fast Green staining (center) and H&E staining (right) of tissue sections of hind paw joints from a healthy rat and of rats from each treatment group. Arrows indicate areas of cartilage erosions (Safranin O/Fast Green) or hyperplasia (H&E). Scale bar = 100 μm. *p < 0.05, ****p < 0.0001

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  Fig. 9. Schematic summarizing the model by which fibroblast-like synoviocytes (FLS) and T cells interact, within the context of previous studies showing the role of KCa1.1 in regulating integrin-mediated invasion in FLS and Kv1.3 in regulating calcium-mediated T cell activation [7, 16]