Histamine

Serum-starved OA chondrocytes at P2 were treated with IL-6 (R&D Systems, Minneapolis, MN) plus sIL-6R (R&D Systems), IL-8 (R&D Systems), TNF-α (R&D Systems), IL-1α (R&D Systems), VEGF (R&D Systems), bFGF (Sigma-Aldrich), PGE2 (Sigma-Aldrich), IGF-1 (Sigma-Aldrich), histamine (Wako, Osaka, Japan) and TGF-β1 (R&D Systems), or vehicle alone in DMEM/F-12 containing 1% FBS for 24 h. The expression levels of HYBID and TMEM2 were determined by normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the THUNDERBIRD SYBR qPCR Mix (Toyobo) according to the ΔΔCt method^{47 (link)}. The primers were as follows: for HYBID 5′-AGGGAAGCAGGTCAGAGTGA-3′ (forward) and 5′-TCTCGGCTACAGACCCAGAG-3′ (reverse); for TMEM2 5′-ACTTGGTGGCTGGCATGTTC-3′ (forward) and 5′-CATGAGCTGGGCCTGAGTTG-3′ (reverse); and for GAPDH 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse)^{12 (link)}. Similarly, HYBID expression was also examined in OA chondrocytes treated with TNF-α and IL-6 in the presence of sIL-6R. Protein expression of HYBID was analyzed in chondrocytes stimulated with the cytokines for 48 h by immunoblotting, as described below.

LPS, endothelial cell growth supplement (ECGS), heparin, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Anti-TF-FITC and Actichrome^® TF activity assay kit was purchased from SEKISUI DIAGNOSTICS (Lexington, MA, USA). HUVECs from ATCC (Manassas, VA, USA). Coagulation factor VIII deficient human plasma was purchased from COSMO BIO Co., Ltd. (Tokyo, Japan). DMEM/F12 was purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Histamine was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Recombinant human TNF-α and VEGF were purchased from R & D systems (Minneapolis, MN, USA). RepCell^® was purchased from CellSeed Inc. (Tokyo, Japan)

For the Ca²⁺ measurements we used a N-2-hydroxy-ethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer containing (in mM) 10 HEPES, 136.9 NaCl, 5.4 KCl, 1.0 MgCl₂, 1.5 CaCl₂, 0.001 EDTA, and 5.5 glucose (HEPES-buffered solution). Dulbecco's modified eagles medium (DMEM), Hanks' balanced salt solution (HBSS), fetal bovine serum (FBS), and trypsin-EDTA were purchased from GIBCO, Grand Island, NY. Bradykinin, thrombin, ionomycin, albumin, cytochalasin B and 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were purchased from Sigma Chemical Co., St. Louis, MO. Fura 2-acetoxymethyl ester (fura 2-AM) was obtained from Dojindo Laboratories, Kumamoto, Japan. Histamine was purchased from Wako, Tokyo, Japan. Other materials and chemicals were obtained from commercial sources.

histamine-induced conjunctivitis was induced as described previously, with some modifications (Mochizuki et al., 2022 (link)). Briefly, histamine (4 μg in 4 μL/eye, FUJIFILM Wako) was dropped onto the surface of the eye. Fifteen minutes after histamine application, tear volume was measured using the Schirmer tear test. To investigate vascular permeability, Evans blue (SIGMA, Tokyo, Japan, 10 mg/mL, 0.1 mL) was injected intravenously 5 min after histamine challenge. The eyeballs (with lids and conjunctival tissue) were harvested 15 min after the histamine challenge, dried at 55°C and weighed.
In some experiments, (±)5(6)-DiHETE (300 μg/kg) or ketotifen (SIGMA, 10 mg/kg) was intraperitoneally injected into mice 15- or 30-min prior to histamine (4 μg/eye) administration, respectively. In other experiments, (±)5(6)-DiHETE (1 μg in 2 μL/eye) or ketotifen (0.1 μg in 2 μL/eye) was dropped onto the eye 15- and 45-min prior to histamine administration (8 μg in 4 μL/eye).