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INFLAMMATION AND IMMUNOPHARMACOLOGY

Induction of C-X-C Chemokines, Growth-Related Oncogene Expression, and Epithelial Cell-Derived Neutrophil-Activating Protein-78 by ML-1 (Interleukin-17F) Involves Activation of Raf1-Mitogen-Activated Protein Kinase Kinase-Extracellular Signal-Regulated Kinase 1/2 Pathway

Johns Hopkins University, Asthma and Allergy Center, Baltimore Maryland (M.K., S.-K.H.); and First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan (M.K., F.K., S.M., K.I., M.O., S.W., S.S., M.A.)

Received June 30, 2003; accepted September 5, 2003.

	Abstract

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Neutrophil recruitment into the airway typifies pulmonary inflammation and is regulated through chemokine network, in which two C-X-C chemokines play a critical role. Airway epithelial cells and vein endothelial cells are major cell sources of chemokines. ML-1 (interleukin-17F) is a recently discovered cytokine and its function still remains elusive. In this report, we investigated the functional effect of ML-1 in the expression of growth-related oncogene (GRO) and epithelial cell-derived neutrophil activating protein (ENA)-78. The results showed first that ML-1 induces, in time- and dose-dependent manners, the gene and protein expressions for both chemokines in normal human bronchial epithelial cells and human umbilical vein endothelial cells. Furthermore, selective mitogen-activated protein kinase kinase (MEK) inhibitors 2'-amino-3'-methoxyflavone (PD98059), 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene (U0126), and Raf1 kinase inhibitor I partially inhibited Ml-1-induced GRO and ENA-78 production. In contrast, the combination of PD98059 and Raf1 kinase inhibitor I completely abrogated the chemokine production, whereas a protein kinase C inhibitor, 2-(1-(3-aminopropyl) indol-3-yl)-3-(1-methylindol-3-yl) maleimide, acetate (Ro-31-7549), and a phosphatidylinositol 3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), did not affect their production. Together, these data indicates a role for Raf1-MEK-extracellular signal-regulated kinase 1/2 pathway in ML-1 induced C-X-C chemokine expression, suggesting potential pharmacological targets for modulation.

Recently, we and others have independently discovered a novel cytokine, ML-1 (Kawaguchi et al., 2001) or IL-17F (Hymowitz et al., 2001; Starnes et al., 2001), belonging to the IL-17 gene family (Kawaguchi et al., 2001), but its function and signaling pathways remain as yet to be defined. ML-1 is expressed in activated CD4⁺ T cells, basophils, and mast cells, three important cell types involved in airway inflammation (Kawaguchi et al., 2001). We have previously shown that ML-1 is able to induce the expression of IL-6, IL-8, and (intercellular adhesion molecule-1 in bronchial epithelial cells (Kawaguchi et al., 2001) and that this activation process is mediated, in part, through the phosphorylation of ERK1/2, but not p38 and Jun-N-terminal kinase (JNK) (Kawaguchi et al., 2002). The importance of MAPKs in controlling cellular response to the environment and in regulating gene expression, cell growth, and apoptosis has made them a priority for research related to many human diseases (English and Cobb, 2002). The ERK1/2, p38, and JNK pathways are all molecular target for drug development, and inhibitors of MAPKs will be one of the next group of drugs developed for the treatment of human diseases (Johnson and Lapadat, 2002).

To gain further understanding of the function and signaling pathways of ML-1, the role of ML-1 in the expression of two critical chemokines, GRO and ENA-78, was investigated. In this communication, we provide evidence that ML-1 is a potent inducer of GRO and ENA-78, involving the activation of the Raf1-MEK-ERK1/2 signaling pathway in NHBEs and HUVECs, and suggest that the Raf1-MEK-ERK1/2 pathway may be a potential target for pharmacotherapeutical intervention of ML-1-induced C-X-C chemokine expression in the airway inflammatory diseases.

	Materials and Methods

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Cell Culture. NHBEs were purchased from Clonetics (San Diego, CA) and cultured in bronchial epithelial basal medium (Clonetics) containing 0.5 ng/ml human recombinant epidermal growth factor, 52 µg/ml bovine pituitary extract, 0.1 ng/ml retinoic acid, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, and 50 pg/ml amphotericin-B (Clonetics). HUVECs were obtained from Clonetics. The cells were cultured for no more than three passages before the analysis.

Generation of Human Recombinant ML-1. Human recombinant ML-1 was generated as described previously (Kawaguchi et al., 2001). The coding sequence of ML-1 was amplified by polymerase chain reaction (PCR) and subcloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA) to generate a C-terminal His fusion gene. The vector pcDNA 3.1 was transfected into COS-7 cells by an Effectene reagent (QIAGEN, Chatsworth, CA) according to the manufacturer's instructions. ML-1 was purified with affinity purification by Ni²⁺-nitrilotriacetic acid agarose beads (QIAGEN) for His-tagged proteins. Then the concentration of ML-1 protein was quantified by Bradford assay (Bio-Rad, Hercules, CA) and stored at -80°C until used. Endotoxin levels were tested using Kinetic-QCL chromogenic LAL (Cambrex Bio Science Walkersville, Inc., Walkersville, MD). Endotoxin levels were undetectable. The cells were treated with ML-1 (10 and 100 ng/ml) for various time periods.

Gene Expression of GRO and ENA-78. Total RNA was extracted using RNeasy (QIAGEN) from 1 x 10⁶ cells at 0.5, 2, 4, 12, and 24 h after stimulation with 10 and 100 ng/ml ML-1. cDNAs were synthesized from 500 ng of total RNA in the presence of Moloney murine leukemia virus reverse transcriptase (1 U/reaction; Sigma-Aldrich, St. Louis, MO), oligo(dT) primer, and reaction buffer at 42°C for 90 min, followed by PCR. The sequences of PCR primers for GRO were as follows: forward, 5'-CGCTCCTCTCACAGCCGCCA-3' and reverse, 5'-AGGAACAGCCACCAGTGAGC-3'; ENA-78, forward, 5'-TGTGTTGAGAGAGCTGCGTTGCGTT-3' and reverse, 5'-TCAGTTTTCCTTGTTTCCACC-3'; and G3PDH, forward, 5'-ACCACAGTCCATGCCATCAC-3' and reverse, 5'-TCCACCACCCTGTTGCTGTA-3'. The amplification reaction was performed for 26 cycles with denaturation at 94°C for 45 s, annealing at 53°C for 45 s, and extension at 72°C for 45 s. The expected size for GRO was 400 bp, for ENA-78 was 222 bp, and for G3PDH was 450 bp. PCR products were detected by ethidium bromide staining, and quantified by video densitometry using Image 1.61 software (NIH Public Software, National Institutes of Health, Bethesda, MD). The level of GRO and ENA-78 gene expression was quantified by calculating the ratio of densitometric readings of the band intensity for chemokines and G3PDH from the same cDNA sample. The values are expressed as mean ± S.D. (n = 3 experiments).

Protein Levels of GRO and ENA-78. GRO and ENA-78 protein levels in the supernatants and cell lysate of ML-1-stimulated cells were determined with a commercially available ELISA kit (BioSource International, Camarillo, CA) according to the manufacturer's instruction. Cell supernatant was harvested from unstimulated or stimulated cultures with 10 and 100 ng/ml ML-1 at 2, 6, 12, 24, or 48 h after stimulation. The amount of secreted GRO and ENA-78 was determined by the ELISA and expressed as the amount recovered per 10⁶ cells. Cells corresponding to the supernatant samples described above were lysed into 0.5 ml Nonidet P-40 lysis buffer (20 mM Tris pH 7.4, 4 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 100 mg/ml aprotinin, 200 mg/ml leupeptin, 50 mM NaF, 5 mM Na₄P₂O₇, and 1% Nonidet P-40; all purchased from Sigma-Aldrich). The chemokine concentration of cell lysate was reported as the amount recovered per 10⁶ cells. The values are expressed as mean ± S.D. (n = 6 experiments).

Effect of Inhibitors on the Expression of GRO and ENA-78. For analysis of activation of the Raf1-MEK-ERK1/2 pathway, the cells were treated in the presence or absence of the following kinase inhibitors at varying doses: MEK1/2 inhibitors 2'-amino-3'-methoxyflavone (PD98059) (Calbiochem, La Jolla, CA) and 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene (U0126) (New England Bio Labs, Beverly, MA); p38 inhibitor 4-(4-fluoro-phenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl) 1H-imidazole (SB202190) (Calbiochem); a Raf1 kinase inhibitor I (5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone (Calbiochem); a PKC inhibitor, 2-(1-(3-aminopropyl) indol-3-yl)-3-(1-methylindol-3-yl) maleimide, acetate (Ro-31-7549) (Calbiochem); a PI3K inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) (Calbiochem); and a vehicle control, DMSO (Me₂SO) for 1 h before treatment with ML-1 (100 ng/ml) for 24 h. The final concentration of DMSO did not exceed 0.1% (v/v). GRO and ENA-78 protein levels in the supernatants were determined as described above. The values are expressed as mean ± S.D. (n = 4 experiments).

Data Analysis. The statistical significance of differences was determined by analysis of variance. The values are expressed as mean + S.D. from independent experiments. Any difference with p values less than 0.05 was considered. When analysis of variance indicated a significant difference, the Scheffe's F test was used to determine the difference between groups.

	Results

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To determine the functional role of ML-1 in the regulation of C-X-C chemokine expression, the mRNA expression of GRO and ENA-78 was examined. Although detectable gene expression for both GRO and ENA-78 was found in control cells at 2-h time point (Fig. 1A), the induction of chemokine gene expression was evident in both NHBEs and HUVECs (Fig. 1, A and B), and ML-1-induced gene expression peaked at 2-h time point and returned to baseline at 24-h time point in ML-1 (100 ng/ml)-treated NHBEs (Fig. 1, C and D). No increase of chemokine gene expression was seen in cells treated with a His-tag control protein (Positope, 10 or 100 ng/ml; Invitrogen; data not shown). A similar time course of gene expression in HUVECs was also found (data not shown).

View larger version (41K): [in this window] [in a new window]   Fig. 1. GRO and ENA-78 gene expression by different doses of ML-1. A, NHBEs and HUVECs were treated with 10 and 100 ng/ml ML-1 for 2 h. Reverse transcription-PCR is performed as described under Materials and Methods. B, GRO/G3PDH and ENA-78/G3PDH ratios were determined by densitometric analysis from the same cDNAs. *, p < 0.05 was considered significant versus control. Time-course study of GRO and ENA-78 gene expression. C, NHBEs were treated with 100 ng/ml ML-1 for 0.5 to 24 h. Reverse transcription-PCR is performed as described under Materials and Methods. D, GRO/G3PDH and ENA-78/G3PDH ratios were determined by densitometric analysis from the same cDNAs. The values are expressed as mean + S.D. (n = 3 experiments). *, p < 0.05 was considered significant versus the intensity of 0.5-h time point.

To investigate the protein expression for both chemokines, NHBEs and HUVECs were cultured in the absence or presence of varying doses of ML-1 at five different time points. GRO and ENA-78 proteins were detected in the absence of ML-1 in NHBEs. Cell lysate GRO protein level were significantly increased 6 h after stimulation with 10 and 100 ng/ml ML-1. Cell lysate ENA-78 protein level were significantly increased 12 h after stimulation with 10 and 100 ng/ml ML-1. They were significantly elevated at 24- and 48-h time points and attained their highest level 24 h after stimulation (Fig. 2A). Cell supernatant GRO and ENA-78 levels were significantly elevated 24 h after stimulation and were increased further at 48 h (Fig. 2B). A similar pattern was observed in HUVECs, except for significant induction of GRO secretion was seen at 12-h time point when HUVECs were stimulated with 100 ng/ml ML-1 (Fig. 3, A and B), suggesting that ML-1 is more potent in the induction of GRO expression compared with the ENA-78 expression in both cell types.

View larger version (27K): [in this window] [in a new window]   Fig. 2. GRO and ENA-78 protein levels in NHBEs. NHBEs were treated with 10 and 100 ng/ml ML-1 for 2 to 48 h. ELISA was performed as described under Materials and Methods. A, GRO and ENA-78 protein was measured in NHBE cell lysates harvested at the indicated time points after stimulation with ML-1. The cells were lysed as described under Materials and Methods. B, GRO and ENA-78 proteins were measured in the supernatants at the indicated time points stimulated with ML-1. The values are expressed as mean + S.D. (n = 6). *, p < 0.05 was considered significant versus control.

View larger version (27K): [in this window] [in a new window]   Fig. 3. GRO and ENA-78 protein levels in HUVECs. HUVECs were treated with 10 and 100 ng/ml ML-1 for 2 to 48 h. ELISA was performed as described under Materials and Methods. A, GRO and ENA-78 protein was measured in HUVEC cell lysates harvested at the indicated time points after stimulation with ML-1. The cells were lysed as described under Materials and Methods. B, GRO and ENA-78 proteins were measured at the indicated time points after stimulation with ML-1 in the supernatants. The values are expressed as mean + S.D. (n = 6).*, p < 0.05 was considered significant versus control.

We next investigated whether the activation of the Raf1-MEK-ERK1/2 pathway was necessary for the stimulation of chemokine production. As shown in Figs. 4 and 5, 1-h pretreatment of selective MEK inhibitors PD98059, U0126, and Raf1 kinase inhibitor I significantly attenuated, in a dosedependent manner, the production of GRO and ENA-78 in HHBEs and HUVECs, respectively, whereas 1-h pretreatment of the cells with vehicle alone (0.05% DMSO) did not affect the protein release. In addition, the protein levels of GRO and ENA-78 were unchanged in ML-1-treated cells in the presence of a p38 kinase inhibitor SB202190, even at a dose of 10 µM (Figs. 4 and 5). Although induction of GRO and ENA-78 is partially inhibited by PD98059, U0126, or Raf1 kinase inhibitor I even at relatively high dose (50, 10, and 10 nM, respectively), the combination with 10 µM PD98059 and 1 nM Raf1 kinase inhibitor I completely inhibited the production of these two chemokines in NHBEs and HUVECs.

View larger version (34K): [in this window] [in a new window]   Fig. 4. Effect of PD98059, U0126, and Raf1 kinase inhibitor I on GRO and ENA-78 expression in NHBEs. NHBEs were preincubated with 1 to 50 µM PD98059 (PD), 1 to 10 µM U0126, 0.01 to 10 nM of Raf1 kinase inhibitor I (Raf I), 1 to 10 µM SB202190, or a combination of 10 µM PD and 1 nM Raf I for 1 h, followed by stimulation with 100 ng/ml ML-1 for 24 h. The results are expressed as the mean ± S.D. (n = 4 experiments). *, p < 0.05 was considered significant versus ML-1-stimulated cells.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Effect of PD98059, U0126, and Raf1 kinase inhibitor I on GRO and ENA-78 expression in HUVECs. The cells were preincubated with various inhibitors same as described in Fig. 4 legend, followed by stimulation with 100 ng/ml ML-1 for 24 h. The results are expressed as the mean ± S.D. (n = 4 experiments). *, p < 0.05 was considered significant versus ML-1-stimulated cells.

We also investigated whether other signaling molecules, such as PKC and PI3K, are involved in upstream signaling pathway of the C-X-C chemokine expression. The results showed that no significant inhibitory effect on ML-1-induced chemokine expression in NHBEs and HUVECs was found when a selective PKC inhibitor, Ro-31-7549 (0.01–0.5 µM), or a PI3K inhibitor, LY294002 (0.1–20 µM), was used (Fig. 6, A and B, respectively).

View larger version (34K): [in this window] [in a new window]   Fig. 6. Effect of Ro-31-7549 and LY294002 on GRO and ENA-78 expression. NHBEs and HUVECs were preincubated with 0.01 to 0.5 µM Ro-31-7549 and 0.1 to 20 µM LY294002 for 1 h, followed by stimulation with 100 ng/ml ML-1 for 24 h. The results are expressed as the mean ± S.D. (n = 4). *, p < 0.05 was considered significant versus ML-1-stimulated cells.

	Discussion

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GRO has been shown to be a potent neutrophil chemoattractant and activator in vitro, and this chemotactic activity is equivalent to that of IL-8 (Balentien et al., 1990). Similarly, ENA-78 is as equally potent as IL-8 in inducing neutrophil chemotaxis; however, it is consistently less active in inducing the release of granules from neutrophils (Walz et al., 1991). Besides eosinophils, it is reported that neutrophils are also involved in the features of bronchial asthma, airway hyper-reactivity, airway hypersecretion, and airway wall remodeling (Molet et al., 2001). In addition, pulmonary neutrophilia has also been found in severe asthmatic airways, and at sites of allergen challenge in asthmatic subjects (Ordonez et al., 2000). Several inflammatory stimuli such as tumor necrosis factor , lipopolysaccharide, diesel exhaust exposure, and respiratory syncytial virus infection can induce GRO and/or ENA-78 (Lukacs et al., 1995; Matsukawa et al., 1999; Salvi et al., 2000; Nasu et al., 2001; Zhang et al., 2001). The expression of GRO and ENA-78 has been found in several inflammatory models and diseases (Luster, 1998), strongly implicating its role in the pathogenesis of inflammation.

ML-1 is derived from activated CD4⁺ T cells, basophils, and mast cells, which are important regulatory cells for the inflammation (Kawaguchi et al., 2001). It is thus a strong possibility that ML-1-induced GRO and ENA-78 are involved in neutrophilic inflammation. It is of interest to note that ML-1 induces C-X-C chemokines, but not C-C chemokines, such as eotaxin and regulated on activation normal T cell expressed and secreted, which are potent chemoattractants for eosinophil (data not shown), suggesting a selective role of ML-1 in neutrophil recruitment and activation. As a corollary, a recent study has suggested an in vivo role of human IL17F in recruiting neutrophils into the pulmonary mucosa in mice after adenoviral gene transfer (Hurst et al., 2002), further suggesting a potential role of ML-1 in the pathogenesis of neutrophilic inflammation.

Raf-1 is a MAP kinase kinase kinase (MAP3K), which functions downstream of the Ras family of membrane associated GTPases, and is able to activate the dual specificity protein kinases MEK1 and MEK2, which in turn activate the serine/threonine-specific protein kinases ERK1 and ERK2 (English and Cobb, 2002). Our previous and current data demonstrated that ML-1-induced IL-8, GRO, and ENA-78 production is dependent on the activation of ERK1/2, but not p38 and JNK in the MAPK signaling pathway (Kawaguchi et al., 2002). ERK1/2 is known to be involved in the regulation of cell proliferation and apoptosis. In this study, the inhibitors used did not affect both the cell number and viability (data not shown), suggesting an effect on gene and protein expression.

Finally, we used inhibitors for other signal molecules, such as PKC and PI3K, because several studies have demonstrated both PKC and PI3K are linked to MAP kinase pathway. PKC is a key activator of the Raf1/MAP kinase cascade at multiple steps. It is known that PKC can regulate Raf1 signaling through phosphorylation of Raf kinase inhibitory protein (Corbit et al., 2003) and also phosphorylates Raf1 at serine 499 (Kolch et al., 1993). On the other hand, Ras is likely to act through additional proteins besides Raf1. PI3K is a candidate Ras effector (Rodriguez-Viciana et al., 1997). Activation of PI3K by a variety of extracellular stimuli leads to the accumulation of the second messenger phosphatidylinositol 3,4,5-trisphosphate. Its final target is the serine/threonine kinase Akt/PKB. Activated Ras promotes cell survival in epithelial cell through activation of PI3K and Akt/PKB because at high dose, 20 µM, LY294002 induces apoptosis (Khwaja et al., 1997). In our study, however, LY294002 did not show any effect on the cell number and viability (data not shown). This is likely due to different stimuli and cell types used.

To date little is known about the upstream signaling pathway of GRO and ENA-78 expression. However, the results of current study suggest that Raf is predominantly associated with the activation of MEK-ERK1/2 pathway. Therefore, we concluded that the Raf-MEK-ERK1/2 pathway is a central upstream pathway of ML-1 induced GRO and ENA-78 expression in NHBEs and HUVECs. In fact, MAP kinases are important molecules in the airway epithelial activation in response to various stimuli such as tumor necrosis factor-, IL-1, diesel exhaust particles, and influenza virus infection (Griego et al., 2000; Hashimoto et al., 2000a,b; Reibman et al., 2000). Also, MAP kinases, including ERK1/2, are involved in cytokine signaling in HUVECs (May et al., 1998; Goebeler et al., 1999; Surapisitchat et al., 2001). On the other hand, the downstream signaling pathway is currently unclear. IL-17 is known to activate transcription factor nuclear factor-B in chondrocytes and intestinal epithelial cells (Shalo-Barak et al., 1998; Awane et al., 1999). Because of high homology between IL-17 and ML-1, it is possible that ML-1 is able to activate nuclear factor-B in the downstream signaling pathway.

It is noted that a delay between the synthesis and release of both GRO and ENA-78 chemokines was observed. For example, the level of ML-1-induced GRO in cell lysate was noted at the 6-h time point, whereas significant increase of GRO in the supernatants was seen at 24 h after stimulation. The significance of this delay is at present unclear. It is noted, however, that a trend of increase for GRO secretion is seen at the 12-h time point, although it did not reach statistical significance. Additional time intervals between the 12- and 24-h time points will be needed to identify its release kinetics. Of interest, we have also found previously a similar "delay" phenomenon for ML-1-induced IL-6 secretion (Kawaguchi et al., 2002). The delay in protein expression may be as a result of the required time frame for protein modification and/or transport, or alternatively, but not mutually exclusively, a faster synthesis/secretion kinetic requires an additional factor induced by ML-1. It is also noted that chemokine gene expression is induced by ML-1 at the 2-h time point, suggesting a direct effect of ML-1 on de novo synthesis of transcripts. However, until a ML-1-inducible factor, if it exists, is found, a possible secondary (or perhaps additive) effect of ML-1 on the induction of chemokine gene and protein expression cannot be ruled out in the current study.

In conclusion, this study reports that ML-1 induces C-X-C chemokines GRO and ENA-78 via the activation of the Raf1-MEK-ERK1/2 pathway. These results suggest a potential role of ML-1 in the pathogenesis of the airway inflammatory diseases, such as chronic obstructive pulmonary disease, bronchial asthma, and bacterial pneumonia, and the Raf1-MEK-ERK1/2 pathway is a potential pharmacotherapeutical target for inhibition of ML-1-induced neutrophil recruitment and activation in the airway inflammatory diseases.

	Acknowledgements

 
We thank Hiroko Takeuchi, Tomoko Shinbara, and Makoto Murakami for excellent technical assistance.

	Footnotes

 
This work was supported, in part, by National Institutes of Health Grant AI-40274 and Astra Zeneca Asthma Research Award.

DOI: 10.1124/jpet.103.056341.

ABBREVIATIONS: ELR, Glu-Leu-Arg; IL, interleukin; GRO growth-related oncogene ; ENA-78, epithelial-cell derived neutrophil activating protein-78; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; MAPK, mitogen-activated protein kinase; NHBE, normal human bronchial epithelial; HUVEC, human umbilical vein endothelial cell; MEK, mitogen-activated protein kinase kinase; PCR, polymerase chain reaction; bp, base pair; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; PKC protein kinase C; PI3K phosphatidylinositol 3-kinase; DMSO, dimethyl sulfoxide.

Address correspondence to: Dr. Shau-Ku Huang, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. E-mail: skhuang{at}jhmi.edu

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