The Kv1.3 voltage-gated potassium channel is selectively upregulated upon activation in effector memory T (TEM) cells in inflamed tissue, and plays an important role in maintenance of T-cell activation. Although Kv1.3 blockers have been shown to ameliorate allergic contact dermatitis (ACD) in a rat model, it remains unknown whether the effect of Kv1.3 blockers on ACD is mediated by suppressing TEM cell function and/or whether naive T-cells or central memory T (TCM) cells are influenced.
To analyse the detailed mechanism of Kv1.3 blockers in a rat model of ACD.
We examined the effects of a Kv1.3 blocker on inflammation and production of the effector cytokine interferon (IFN)-γ in inflamed tissue in rat ACD. Single-cell suspensions were isolated from inflamed rat ears (TEM cells), and regional lymph nodes (naive T/TCM cells), and the effect of Kv1.3 blockers on anti-CD3-stimulated IFN-γ production in vitro was measured.
The Kv1.3 blocker significantly suppressed ear inflammation and IFN-γ production at the protein level in vivo. It also suppressed in vitro IFN-γ production from TEM cells from inflamed tissues, but did not suppress the function of naive T/TCM cells from lymph nodes.
We found that the Kv1.3 blocker ameliorated ACD by inhibiting TEM cell functions only, thus Kv1.3 blockers could be a potentially selective therapeutic agent for TEM cell-mediated inflammatory skin diseases without producing harmful side-effects.
The voltage-gated potassium channel Kv1.3 and the Ca2+-dependent intermediate-conductance calcium-activated K+ channel (IKCa1) are expressed in T cells, and are important in maintaining T cell activation by allowing influx of extracellular calcium.[1-3] All quiescent human T cells express approximately 10-fold more Kv1.3 than IKCa1 channels, but activation induces distinctive channel phenotypes in naive T, central memory T (TCM) and effector memory T (TEM) cell subsets. Naive T/TCM cells express C-C chemokine receptor (CCR)7, which is required for lymph-node (LN) homing. Upon antigen challenge, naive T/TCM cells home to LNs, and then migrate to sites of inflammation. TEM cells lack CCR7, and rapidly enter inflamed tissues, secrete inflammatory cytokines and exhibit rapid effector functions.[5, 6] Activation of naive T/TCM cells transcriptionally augments IKCa1 expression without changing Kv1.3 expression. By contrast, TEM cells upregulate Kv1.3, but not IKCa1, during activation.
Recent studies have shown that disease-associated autoreactive T cells from patients with autoimmune disease were mainly CCR7-negative TEM cells with increased Kv1.3 levels, and that Kv1.3 blockers specifically suppressed the proliferation and cytokine production of autoantigen-specific TEM cells in vitro.[4, 7, 8] In addition, selective blockade of Kv1.3 inhibited inflammation in various animal models of autoimmune disease, allergic contact dermatitis (ACD) and psoriasis.[7-12] However, the direct effect of Kv1.3 blockers on primary TEM cells in the treatment of animal models of disease has not been reported.
In ACD, inflammation is mediated by the IFN-γ-producing CD8+ effector T cells.[13-15] Azam et al. showed by immunohistochemistry that ear-infiltrating cells from the oxazolone-induced ACD rat were Kv1.3-expressing CD8+ memory T cells, suggesting that they were of an effector memory phenotype. Furthermore, a small-molecule Kv1.3 blocker, PAP-1 [5-(4-phenoxybutoxy) psoralen] inhibited the elicitation phase of ACD. Thus, it has been suggested that ear inflammation in ACD is mediated by TEM cells. In addition, the mechanism of inhibition by PAP-1 in ACD was shown to be due to reduced numbers of ear-infiltrating CD8+ T cells and IFN-γ mRNA levels.
In this study, we established a rat model of oxazolone-induced ACD and investigated the effects of Kv1.3 blocker treatment in vivo and in vitro in more detail.
The study was approved by the local animal ethics committee and conducted according to the guidelines for animal experimentation at Shionogi and Co., Ltd.
Female Lewis rats (6–9-weeks old; Charles River Japan, Inc., Kanagawa Japan) were used for all experiments.
Induction of allergic contact dermatitis and administration of PAP-1
We established a rat model of oxazolone-induced ACD to elucidate IFN-γ production at the protein level. The abdomen of each rat was shaved and then sensitized with 300 μL of a freshly prepared 1% solution of oxazolone (Sigma Chemical Co., St. Louis, MO, USA) in a 4 : 1 ratio of acetone:olive oil (AOO). Seven days later, 60 μL of 0.3% oxazolone in AOO was applied to each side of both ears. Ear thickness was measured using a dial thickness gauge, before and 6 or 24 h after challenge, and ear swelling was calculated as the difference between the ear thickness before and that after challenge.
We evaluated the effect of PAP-1, which was synthesized as previously described, dissolved in peanut oil and administered by oral gavage at 100 mg/kg at 28, 20 and 4 h before the challenge. The control rats received the same volume of the vehicle (peanut oil).
Cytokine production in ear tissue
The rats were killed in a CO2 chamber, then ears were harvested 6 or 24 h after challenge and homogenized in PBS (Life Technologies, Grand Island, NY, USA) and separated by centrifugation. An ELISA (BD Biosciences, San Diego, CA, USA) was used to determine the concentration of IFN-γ in the ear extracts, in accordance with the manufacturer's protocol.
Detection of IFN-γ-producing cells in ear tissue
To detect IFN-γ-producing cells directly ex vivo, single-cell suspensions were prepared from inflamed ears 24 h after challenge, and analyzed by flow cytometry. The harvested rat ears were minced and digested with 2.7 mg/mL collagenase IV (Worthington Biochemical, Lakewood, NJ, USA), 1 mg/mL DNase I and 0.25 mg/mL hyaluronidase (both Sigma Chemical Co.) in RPMI medium containing 10 mmol/L HEPES (both Life Technologies) at 37 °C for 60 min. The remaining undigested tissue was carefully poured through a 100 μm cell strainer, and disintegrated using a syringe plunger. Cell suspensions were collected and washed, then aliquots of 1 × 106 cells/mL were incubated for 3–4 h at 37 °C with a commercial mix (Leucocyte Activation Cocktail; BD Biosciences) in culture medium: RPMI supplemented with 1% fetal bovine serum (Equitech-Bio Inc., Kerrville, TX, USA), with the addition of 100 U/mL penicillin and 100 μg/mL streptomycin (both Life Technologies).
Cells were harvested, washed, and labelled with allophycocyanin (APC) anti-CD3 antibody (1F4; BD Biosciences). Cells were subsequently fixed and permeabilized in buffer (Cytofix/Cytoperm; BD Biosciences), and intracellular IFN-γ was labelled with R-phycoerythrin (RPE) anti-IFN-γ antibody (DB-1; BD Biosciences). Cells were analysed using flow cytometry (FACSAria; BD Biosciences).
Cell culture and cytokine production
To assess whether the effect of the Kv1.3 inhibitor on ear inflammation was caused by suppression of TEM cell function, single-cell suspensions containing TEM cells were prepared from the inflamed ears, and were directly assayed in vitro.
Single-cell suspensions from ears were prepared as described by Plitz et al. Briefly, rat ears were harvested 24 h after challenge and pooled by treatment group (n = 5–10). After splitting the ears with forceps, the dermal sides of the ear halves were treated with 1% trypsin (Life Technologies) for 40 min at 37 °C. The reaction was stopped by transferring the ear halves to 80% fetal bovine serum in Hanks balanced salt solution (Life Technologies). The cells were dislodged from the tissue by gently meshing the ear halves, dermal side down, through a 100 μm cell strainer, and washed with cold culture medium.
For analysis of the T-cell population in prepared ear cells, isolated cells were incubated with APC anti-CD3 antibody, peridinin chlorophyll protein complex (PerCP) anti-CD8a antibody (OX-8) and RPE anti-CD45RC antibody (OX-22) (both BD Biosciences), then the cells were washed and analysed using flow cytometry (FACSAria; BD Bioscience).
For culture of ear cells, isolated cells (1 × 105 cells/well in 96-well plates) were stimulated with 3 μg/mL plate-bound anti-CD3 (G4.18; BD Biosciences) with or without drug treatment. PAP-1 was dissolved to 10 mmol/L in dimethyl sulfoxide. Psora-4 [5-(4-phenylbutoxy)psoralen], the IKCa1 blocker clotrimazole, and the immunosuppressant cyclosporin A (all from Sigma Chemical Co.) were also dissolved to 10 mmol/L in dimethyl sulfoxide. The specific peptidyl Kv1.3 blocker ShK (Stichodactyla helianthus K-channel toxin; Peptide Institute Inc., Osaka, Japan) was reconstituted to 10 μmol/L in distilled water. These drugs were diluted with culture medium and added to the cell culture at the indicated concentration. To avoid cell damage, clotrimazole was used at concentrations below 3 μmol/L. Supernatants were collected after 24 h, and assayed for IFN-γ by ELISA.
To compare the ability of K+ channel blockers to suppress the function of TEM cells vs. naive T/TCM cells, we isolated LN cells as naive T/TCM cells from the same ACD rats from which we isolated the ear cells. LN cells were added at 1 × 105 cells/well with an equal number of syngeneic, mitomycin-treated, T-cell-depleted splenocytes, and then cultured in the presence of anti-CD3-stimulating antibody. IFN-γ production was measured with and without drug treatment as described above for ear cells.
Data are presented as means ± SEM unless otherwise stated. Differences between groups were determined using the Welch t-test, and P < 0.05 was considered significant.
The Kv1.3 blocker suppresses allergic contact dermatitis by reducing interferon-γ production from effector memory T cells in vivo
Measurement of the ear swelling response and IFN-γ production at 0, 6 and 24 h after challenge showed a gradual increase in IFN-γ protein production, which correlated with the ear swelling (Fig. 1). PAP-1 treatment reduced ear swelling at 24 h after challenge (Fig. 2). Concurrently, the IFN-γ protein levels in ears and the number of infiltrating IFN-γ-producing T cells were also decreased (Fig. 2).
Kv1.3 blockers suppress interferon-γ production by effector memory T cells from allergic contact dermatitis rats in vitro
We found that 2.5% of isolated ear cells were T cells, all of which had a CD45RC-negative memory phenotype (Fig. 3a). When stimulated by anti-CD3, IFN-γ was rapidly produced in the cell-culture supernatant after overnight incubation (Fig. 3b). PAP-1, Psora-4 and ShK significantly suppressed this in vitro IFN-γ secretion from TEM cells in a dose-dependent manner. Clotrimazole had no significant inhibitory effect, but ciclosporin A exhibited a strong inhibitory effect.
Kv1.3 blockers do not suppress the function of naive T cells or central memory T cells
PAP-1 and Psora-4 preferentially inhibited IFN-γ production from TEM cells without affecting naive T/TCM cells (Fig. 4). By contrast, clotrimazole suppressed naive T/TCM cell functions more effectively than TEM cells. Ciclosporin A strongly suppressed the function of both naive T/TCM cells TEM cells.
In this study, we first investigated the contribution of IFN-γ production at the protein level in an oxazolone-induced ACD rat model. Treatment with Kv1.3 blockers reduced the ear swelling and the protein levels of the effector cytokine IFN-γ, and also reduced the number of infiltrating IFN-γ producing T cells. This suggests that IFN-γ produced by infiltrating T cells acts as an effector cytokine, and that PAP-1 treatment alleviated inflammation by suppressing IFN-γ production from the infiltrating T cells in the rat ACD model. Next, we evaluated the direct effect of Kv1.3 blocker on TEM cells in inflamed tissue and naive T/TCM cells in LNs by analysing IFN-γ production as an indicator of T cell function. In vitro assays using single-cell suspensions prepared from inflamed ears and LNs showed that the Kv1.3 blockers suppressed the function of infiltrating TEM cells without affecting naive T/TCM cell functions.
The function of Kv1.3 in TEM cells has been investigated previously in humans, using a chronically activated TEM cell line and primary disease-associated autoreactive TEM cells from patients with autoimmune disease. Kv1.3 levels expressed in TEM cells after activation were 2–3 times higher in peripheral blood T cells (mainly naive T/TCM cells).[4, 8, 18] In rats, a chronically activated TEM cell line (PAS T cell) exhibited 10-fold larger Kv1.3 currents than splenic or LN T cells (mainly naive T/TCM cells) when activated.[7, 18] Kv1.3 blockers were reported to suppress the TCR-stimulated proliferation of human or rat TEM cell lines,[4, 7, 9, 19] and cytokine production from human RA-SF T cells (prepared from the synovial fluid of a patient with rheumatoid arthritis). However, to our knowledge, there are no published studies examining Kv1.3 functions on rat primary TEM cells from inflamed tissues.
In our experiments using ear cells from ACD rats, the suppression of IFN-γ production from TEM cells by ShK was 200–300 times more effective than the suppression by PAP-1 and Psora-4, which is consistent with the effect on Kv1.3 currents as evaluated by patch-clamp assays. However, compared with the effect on the TEM cell line and human RA-SF T cells, Kv1.3 blockers were less effective in our study, where the maximum efficacy was weak (50–60%) and the effective concentration was high. This might be because Kv1.3 expression levels in TEM cells increase only slightly with a single antigen challenge. This might also explain why PAP-1 does not completely suppress ear swelling and IFN-γ production in vivo. A recent study using naive T/TCM cells from patients with multiple sclerosis showed that repeated stimulation caused a decrease in IKCa1 expression and a gradual increase in Kv1.3 levels. Furthermore, Kv1.3 blockers were reported to almost completely inhibit the function of human primary autoreactive TEM cells subjected to repeated antigen challenge. Therefore, when Kv1.3 blockers are given to patients with chronic inflammatory disease, who are probably undergoing repeated antigen challenge, inflammation is expected to be dramatically suppressed.
We also evaluated the effect of Kv1.3 blockers on naive T/TCM cells. FACS analysis confirmed that LN T cells and splenic T cells expressed CCR7, showing that they were naive T/TCM cells (data not shown). We found that the LN T cells were less sensitive to Kv1.3 blockers than the TEM cells from ears, similar to splenic T cells (data not shown), indicating that naive T/TCM cells might be less affected by treatment of Kv1.3 blockers in vivo.
We investigated the direct effect of Kv1.3 blockers on infiltrating TEM cells in inflamed tissues in a rat model of ACD, which is the first such study to our knowledge. We found that Kv1.3 blockers ameliorate disease by preferentially suppressing the function of TEM cells infiltrating ear tissues, whereas they do not have inhibitory effects on naive T/TCM cells. Thus, specific Kv1.3 blockers may be beneficial for treatment of inflammatory diseases in which TEM cells that are chronically activated by repeated antigen challenge play a role, without harmful side-effects.
What's already known about this topic?
Kv1.3 blockers can ameliorate inflammation in a rat model of ACD.
Kv1.3 blockers can suppress the TCR-stimulated proliferation of chronically activated rat TEM cell lines.
What does this study add?
This is the first study to investigate the direct effect of Kv1.3 blockers on infiltrating TEM cells in inflamed tissue in an animal model of inflammatory disease.
Kv1.3 blockers ameliorated disease by preferentially suppressing TEM cell function without affecting naive T/TCM cells in a rat ACD model.
We thank Dr K. Takaya for the synthesis of PAP-1 and critical comments.
To demonstrate understanding of the role of effector memory T cells and Kv1.3.
What is Kv1.3?
Calcium-release-activated calcium channel.
G protein-coupled receptor.
Voltage-gated potassium channel.
Calcium-activated potassium channel.
Sodium-potassium adenosine triphosphatase.
Which of the following characteristics apply to effector memory T (TEM) cells?
They exhibit rapid effector functions.
They express C-C chemokine receptor (CCR)7.
They home to lymph nodes upon antigen challenge.
Their function can be suppressed by blockers of intermediate-conductance calcium-activated K+ channel (IKCa)1.
They induce immune tolerance.
Where do effector memory T (TEM) cells mainly exist?
Which subtypes of T cells upregulate Kv1.3 expression during activation?
Effector memory T (TEM) cells.
Central memory T (TCM) cells.
Naive CD4 T cells.
Naive CD8 T cells.
Regulatory T cells.
In which of the following diseases is Kv1.3 not thought to be involved ?