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Der F 2 Activates Phospholipase D in Human T Lymphocytes from Dermatophagoides Farinae Specific Allergic Individuals: Involvement of Protein Kinase C-Α

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Der F 2 Activates Phospholipase D in Human T Lymphocytes from Dermatophagoides Farinae Specific Allergic Individuals: Involvement of Protein Kinase C-Α EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 36, No. 5, 486-492, October 2004 Der f 2 activates phospholipase D in human T lymphocytes from Dermatophagoides farinae specific allergic individuals: Involvement of protein kinase C-α Jae-Won Oh1,*, Eun-Young Kim2,*, PLD activity through the PKC-α activation in T Bon-Suk Koo1, Ha Baik Lee1, Ki Sung Lee3, cells from Dermatophagoides farinae allergic Yong-Seok Kim2 and Joong-Soo Han2,3,4 individuals 1 Keywords: PKC-α, allergy; Der f2; phospholipase D; Department of Pediatrics T lymphocytes 2Institute of Biomedical Science Department of Biochemistry College of Medicine, Hanyang University Seoul 133-791, Korea Introduction 3The Research Center for Bio-Medicinal Resources The study of signal transduction is currently so broad PaiChai University that the only meaningful approach to describe its Daejon 302-735, Korea details is to focus on a specific example and where 4Corresponding author: Tel, 82-2-2290-0623; possible, note how the specifics can be generalized. Fax, 82-2-2294-6270; E-mail, [email protected] The hallmark of an allergic reaction also is the *The first two authors contributed equally to this work. secretion of mediators into the tissue or blood, where they act on a variety of organs to produce the Accepted 20 September 2004 hypersensitivity response. In the context of allergic diseases, T lymphocytes play a role in allergic inflam- Abbreviations: HDM, house-dust-mite; IP3, inositol 1,4,5-triphos- matory reaction. Signal transduction through T lym- phate; PA, phosphatidic acid; PBt, phosphatidy butanol; PC, phos- phocyte antigen receptor is believed to result in the phatidylcholine; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC activation of key signal transducing systems such as α, protein kinase C-α; PLC, phospholipase C; PLD, phospholipase the phosphatidylinositol 4,5-bisphosphate (PIP2)-speci- D; PMA, phorbol myristate acetate fic phospholipase C-γ (PLC-γ). Phospholipase C (PLC) generates the second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DG) by metabolizing inositol-containing phospholipids such as Abstract phophatidylinositol 4,5-biphosphate. IP3 releases cal- The major house-dust mite allergen, Der f 2, cium from intracellular storage vesicles, thereby in- stimulates the phospholipase D (PLD) in T lym- creasing cytosolic-free calcium. DG activates PKC, phocytes from Dermatophagoides farinae speci- which in turn induces the activation of various fic allergic individuals. PLD activity increased transcription factors. Calcium mobilization may also more than two-fold in T cells from allergic pa- take place through G protein-dependent activation of tients compared with those cells from normal PLC. However, T lymphocyte activation via the T cell controls with maximal responses within 30 min receptor (TCR) has been shown to occur in the after exposure of Der f 2. A well-known PLD absence of activation of PLC (O'Rourke and Mescher, activator PKC-α was found to be translocated to 1988) and it is demonstrated that PLC-mediated membrane from cytosol in Der f 2-treated T cells hydrolysis of PIP2 is not sufficient to stimulate T cell proliferation (Exton, 1994). Therefore, it remains un- from Dermatophagoides farinae specific allergic clear that the mechanism of signal transduction individuals. Down-regulation of PKC-α with phor- between the ligation of TCR with allergen and se- bol myristate acetate pretreatment for 24 h cretion of arachidonic acid in allergic inflammatory abolished Der f 2-induced PLD activation. Ro reaction. 320432, PKC inhibitor also reduced the effects House dust mites (HDMs) are the most important of Der f 2-induced PLD activation suggesting source of indoor aeroallergens (Plattis-Mills TAE and α that PKC- acts as upstream activator of PLD de Weck AL, 1989). Antigens from two common in Der f 2-treated T cells. Taken together, the house dust mite species are Dermatophagoides fa- present data suggest that Der f 2 can stimulate rinae (Der f) and Dermatophagoides pteronyssinus Der f 2 activates phospholipase D in human T lymphocytes 487 (Der p). These antigens are group 2 allergens mean of Der f specific IgE level was 89.7 IU/ml. Der produced by D. farinae and D. pteronyssinus, respec- f specific IgE was measured by using UniCap assay tively, having the amino acid sequence similar to each (Upjohn-Pharmacia, Sweden) as shown Table 1. other (Chua et al., 1990; Yuuki et al., 1991). Der f This study was permitted by Ethical Committee of and Der p are believed to be the major cause for Hanyang University, Guri Hospital. The mean age of allergic disease, because group 2 allergen-specific the patients was 20.3 years old. Among the healthy IgE antibodies are found in sera of allergic patients people who do not have any clinical and family history (Holck et al., 1986; Yasueda et al., 1986, 1989; van of allergy, three healthy volunteers with serum IgE der Zee et al., 1988; Heymann et al., 1989; Baldo levels (mean value: 32.8 IU/ml) were included. and Donovan, 1990; Smith et al., 2001; Jeong et al., 2002). Skin prick tests and serum IgE levels Phospholipase D (PLD) is a widely distributed enzyme that hydrolyzes phosphatidylcholine, a major Skin prick tests were performed with allergen extracts phospholipids in the cell membrane, to form phos- of house dust mite (ALK, Copenhagen, Denmark; phatidic acid (PA) which acts by itself as a cellular Greer Lab). Histamine was used as a positive control messenger (Exton, 1990, 1999) or can be trans- (10 mg/ml, ALK, Copenhagen, Denmark), and a di- formed by PA phosphohydrolase into diacyglycerol luent of an unbuffered saline containing 0.03% human (DAG), which is essential for the activation of protein serum albumin (ALK, Copenhagen, Denmark), was kinase C (PKC) (Nishizuka, 1995). Recently, PLD has used as a negative control. Reactions to each skin been shown to be coupled to TCR in the leukemic test solution were measured 15 min after the pricks. T cell line, Jurkat, resulting in the induction of pro- The contours of each wheal reaction were outlined liferation of T cells (Reid et al., 1997) and activated with a fine filter tip pen. The contours were then by Der p 1 in peripheral blood mononuclear cells from transferred to the record sheet by means of trans- allergic patients (Oh et al., 2000). lucent tapes. The size of each wheal was docu- In this study, we evaluated whether Der f 2 induces mented as the mean of the longest diameter (a) and PLD activation in T cells isolated from allergic indi- the diameter perpendicular to it at its mid-point (b): viduals. In addition, we have explored an insight of (a + b)/2. Wheals with more than half of the histamine the mechanism of PLD activation by confirming the reaction after substracting the response to the diluent involvement of PLD activity in the allergic reaction control were considered as expressions of clear-cut induced by Der f 2 in allergic T-cells. positively. Total serum IgE was measured by the paper radioimmunosorbent test (Kabi-Pharmacia, Up- psala, Sweden). The lower detection limit of IgE was Materials and Methods ＜1.5 IU/ml. Subjects CD4＋ T lymphocytes purification and culture Six patients who referred with symptoms of allergic Fifty ml of whole blood was collected from each diseases had positive skin prick tests to house dust subject at each setting and total 10 times. Fifty µl mite (Dermatophagoides farinae) extracts and mean of Rossette Sep antibody cocktail per ml of blood was of total serum IgE level was 765.3 IU/ml and the mixed well with blood and then incubated for 20 min Table 1. The clinical features of allergic individuals. ꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚꠚ Serum IgE Der f specific IgE Der f specific Patient Age (yr) Sex Diagnosis (IU/mL) (IU/mL) Skin prick test ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ A 21 F AD, AR, AC 566 85 +++ B 18 F AS, AR 1,446 ＞100 +++ C 19 M AS 238 95 +++ D 21 F AS, AD 328 72 +++ E 23 M AS, AR, AC 284 86 ++ F 20 M AS, AR 1,730 ＞100 ++++ ꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏꠏ AD, atopic dermatitis; AR, allergic rhinitis; AC, allergic conjunctivitis; AS, asthma; Dp, Dermatophagoides pternyssinus; Df, Dermatophagoides farinae; M, male; F, female; Der f 2 specific IgE was measured by using UniCap assay (Upjohn-Pharmacia, Sweden) 488 Exp. Mol. Med. Vol. 36(5), 486-492, 2004 at room temperature. After incubation, sample was cultured on 6-well plate were metabolically labeled diluted with an equal volume of PBS containing 2% with 2 µCi/ml of [3H]palmitate in serum free medium FBS and mixed thoroughly. This diluted sample was for 18 h. The cells were then pretreated with 0.3 % carefully layered on the top of 15 ml of Hypaque 1-butanol for 15 min before the treatment with Der Ficoll density medium to isolate autologous CD4+ T f2. In some experiments the cells were treated with lymphocytes. The sample was centrifuged for 20 min phorbol myristate acetate (PMA) (200 nM) for 24 h at 1,200 g at room temperature, with the brake off. to down-regulate PKC before labeling with [3H]pal- The enriched cells were removed from the interface mitate. Also, the cells were pre-incubated with Ro between the density medium and plasma and washed 320432 for 30 min, after being labeled with [3H]pal- with PBS containing 2% FBS. The isolated cells were mitate and serum-starved for 18 h. After treatment of washed again with PBS containing 2% FBS. One× Der f 2 for 30 min, the cells were quickly washed with 106 cells of CD4＋ T lymphocytes were loaded on ice-cold PBS and suspended in ice-cold methanol. each well of 24 well culture plate with 5 mg of Der Lipids were extracted according to the method of f2 recombinant protein and 2×106 macrophages as Bligh and Dyer (Bligh and Dyer, 1959), and PBt was antigen presenting cell (APC) and then incubate for separated by a thin layer chromatography using a 7 days.
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