Human Eosinophilic Leukemia Cell Research Articles

Acute and Subacute Oral Toxicity of Deoxynivalenol Exposure in a Dermatophagoides farinae-Induced Murine Asthma Model.

Previously, researchers have demonstrated that mycotoxin deoxynivalenol (DON) significantly enhances immunocyte activation. However, the interaction between DON exposure and immune disorders remains unclear. In this study, we aimed to investigate whether acute and subacute oral exposure to DON exacerbates the development of respiratory allergy using a mite allergen (Dermatophagoides farina, Derf)-induced mouse model of asthma. The direct relationship between DON exposure and asthma development was examined following acute oral DON administration (0, 0.1, or 0.3 mg/kg body weight), immediately before the final mite allergen challenge. Simultaneously, the influence of subacute oral exposure via low dose DON contaminated wheat (0.33 ppm) was evaluated using the same settings. To detect the proinflammatory effects of DON exposure, we examined the total and Derf-specific serum IgE levels, histology, number of immunocytes, and cytokine and chemokine secretion. Acute oral DON significantly enhanced the inflammatory responses, including cellular infiltration into bronchoalveolar lavage fluid, infiltration of immunocytes and cytokine production in local lymph nodes, and cytokine levels in lung tissues. Corresponding proinflammatory responses were observed in a mouse group exposed to subacute oral DON. In vivo results were validated by in vitro experiments using the human bronchial epithelial (BEAS-2B) and human eosinophilic leukemia (EOL-1) cell lines. Following exposure to DON, the secretion of interleukin (IL)-1β, IL-6, IL-8, and/or tumor necrosis factor-α in BEAS-2B cells, as well as EoL-1 cells, increased significantly. Our findings indicate that DON exposure is significantly involved in the proinflammatory response observed in respiratory allergy.

Activation of aryl hydrocarbon receptor by benzo[a]pyrene increases interleukin 33 expression and eosinophil infiltration in a mouse model of allergic airway inflammation.

We recently demonstrated that benzo[a]pyrene (BaP), the aryl hydrocarbon receptor (AhR) ligand, directly contributes to aggravation of cutaneous allergy in a mouse model of allergic dermatitis. The present study aimed to determine whether BaP-induced AhR activation results in development of airway inflammation. Initially, the potential for a direct relationship between BaP-induced AhR activation and airway inflammation was investigated in vivo, using a mouse model of type 2 helper T cell (Th2) hapten toluene-2,4-diisocyanate (TDI)-induced airway inflammation. Mice were orally administered BaP at 48, 24, and 4 h before the final allergen challenge. Oral administration of BaP showed a significant increase in lung inflammation and eosinophil infiltration. While expression of Th2 cytokines such as interleukin 4 (IL-4) and IL-13 was not affected by exposure to BaP, AhR activation significantly increased IL-33 expression. To confirm the in vivo results, in vitro experiments were performed using the human eosinophilic leukemia cell line (EOL-1), human bronchial epithelial cell line (BEAS-2B), and human lung adenocarcinoma epithelial cell line (A549). Results indicated that pre-treatment with BaP increased expression of IL-8 in house dust mite-activated EOL-1, BEAS-2B, and A549 cells. In addition, IL-33 levels in BEAS-2B cells were significantly increased after BaP exposure. Our findings indicated that BaP-induced AhR activation is involved in the pro-inflammatory response in respiratory allergy, and that this effect may be mediated by increased IL-33 expression and eosinophil infiltration.

Activity of 2,6,9-trisubstituted purines as potent PDGFRα kinase inhibitors with antileukaemic activity

Receptor tyrosine kinase PDGFRα is often constitutively activated in various tumours and is regarded as a drug target. Here, we present a collection of 2,6,9-trisubstituted purines with nanomolar potency against PDGFRα and strong and selective cytotoxicity in the human eosinophilic leukaemia cell line EOL-1 that expresses the FIP1L1-PDGFRA oncogene. In treated EOL-1 cells, the example compound 14q inhibited the autophosphorylation of PDGFRα and the phosphorylation of STAT3 and ERK1/2. Interestingly, we observed pronounced and even increased effects of 14q on PDGFRα and some of its downstream signalling pathways after drug washout. In accordance with suppressed PDGFRα signalling, treated cells were arrested in the G1 phase of the cell cycle and eventually underwent apoptosis. Our results show that substituted purines can be used as specific modulators of eosinophilic leukaemia.

Raman imaging highlights biochemical heterogeneity of human eosinophils versus human eosinophilic leukaemia cell line.

Eosinophils are acidophilic granulocytes that develop in the bone marrow. Although their population contributes only to approximately 1-6% of all leucocytes present in the human blood, they possess a wide range of specific functions. They play a key role in inflammation-regulating processes, when their numbers can increased to above 5×109 /l of peripheral blood. Their characteristic feature is the presence of granules containing eosinophil peroxidase (EPO), the release of which can trigger a cascade of events promoting oxidative stress, apoptosis or necrosis, leading finally to cell death. Raman spectroscopy is a powerful technique to detect EPO, which comprises a chromophore protoporphyrin IX. Another cell structure associated with inflammation processes are lipid bodies (lipid-rich organelles), also well recognized and imaged using high resolution confocal Raman spectroscopy. In this work, eosinophils isolated from the blood of a human donor were analysed versus their model, EoL-1 human eosinophilic leukaemia cell line, by Raman spectroscopic imaging. We showed that EPO was present only in primary cells and not found in the cell line. Eosinophils were activated using phorbol 12-myristate 13-acetate, which resulted in lipid bodies formation. An effect of cells stimulation was studied and compared for eosinophils and EoL-1.

Clinical validation of long-range PCR assay for MLL partial tandem duplications for acute myeloid leukemia.

7033 Background: Partial tandem duplication of the mixed lineage leukemia gene ( MLL-PTD) occurs in about 3% to 5% of adult acute myeloid leukemia (AML), mostly in patients with normal karyotypes, and is associated with poor prognosis. Real-time PCR on cDNA or RT-PCR followed by gel electrophoresis are the most commonly used methods for the detection of the mutation. However, those methods require RNA extraction, which is not often performed for molecular cytogenetic testing or next generation sequencing (NGS) for AML. In this study, we developed an MLL-PTD detection assay that uses long-range PCR on extracted DNA, a commonly processed sample type for AML molecular testing, to reduce sample processing requirements. Methods: DNA was extracted from de-identified 72 whole blood and 30 bone marrow aspirate specimens. Two PCR reactions were performed for each DNA sample: one to detect MLL-PTD and another to detect CHEK2 to monitor DNA integrity. The 3 most frequent forms of MLL-PTD (exons 2~8, 2~9, and 2~10) were detected using a forward DNA primer on exon 8 and a reverse primer on exon 2. MLL-PTD positivity was determined by the intensity of amplified DNA fragment at or above the lower detection limit control of this assay. Intra-assay precision, inter-assay precision, and limit of detection (LOD) were performed according to standard laboratory protocols. Results of this method were compared to those of NGS. Results: Intra-assay precision studies on 5 specimens yielded 100% genotype concordance. Inter-assay precision studies on 20 specimens yielded 100% concordance between replicates. LOD studies using a human eosinophilic leukemia cell line (EoL-1) with 12.5-kb PTD in size showed assay sensitivity of 5%; for smaller MLL-PTD positives (PTD ~3 kb and ~5 kb), LOD was 1%. For method comparison studies, 71 specimens (52 whole blood and 19 bone marrow) were also analyzed by NGS; concordance between the methods was 97.2% (69/71). Conclusions: We developed and validated long-range MLL-PTD detection assay for AML. The developed method uses DNA, which is commonly used for molecular testing for AML, and thus may reduce sample processing time by consolidating sample type across tests.

Different Protein Expression between Human Eosinophilic Leukemia Cells, EoL-1 and Imatinib-resistant EoL-1 Cells, EoL-1-IR

Different Protein Expression between Human Eosinophilic Leukemia Cells, EoL-1 and Imatinib-resistant EoL-1 Cells, EoL-1-IR

Role of estrogen receptors α and β in the development of allergic airway inflammation in mice: A possible involvement of interleukin 33 and eosinophils

Recent studies have shown that the estrogen receptor α (ERα), but not ERβ, is involved in the proinflammatory and propruritic responses in cutaneous allergy. In addition, results from our recent study showed that while oral administration of the rather ERβ-selective agonist bisphenol A exacerbated the respiratory allergic inflammation, the potential inflammatory reaction in the skin was decreased after administration of bisphenol A. This study aimed to elucidate whether ERα and ERβ are involved in the progression of an allergic airway inflammation. We performed an in vivo experiment using an animal model of allergic airway inflammation using male BALB/c mice to confirm an increase in the proinflammatory response induced by propylpyrazoletriol (PPT), an ERα agonist, and diarylpropionitrile (DPN), an ERβ agonist. Oral administration of PPT or DPN showed a significant increase in the inflammation of the lung and infiltration of eosinophils. While the expression of Th2 cytokines such as interleukin 4 (IL-4) and IL-13 was not affected by exposure to PPT or DPN, administration of these agonists significantly increased the expression of IL-33. The mechanism underlying the development of such allergic inflammatory responses was determined by an in vitro study using the human bronchial epithelial cell line (BEAS-2B) and the human eosinophilic leukemia cell line (EoL-1). Activated cells were exposed to PPT or DPN for 24 h, and the cytokine levels were measured. The IL-33 levels in BEAS-2B cells increased significantly after exposure to PPT or DPN. In addition, pretreatment with PPT or DPN increased the expression of IL-8 in activated EoL-1 cells. Our findings indicate that ERα and ERβ are involved in the proinflammatory response in respiratory allergy, and their effects may be mediated by an increase in the expression of IL-33 and infiltration of eosinophils.

Chemotactic effect of S100A8 and S100A9 on human eosinophilic leukemia cells, EoL-1 through TLR4

S100A8 and S100A9 function as key factors in inflammatory responses including cell survival, differentiation and chemotactic activity. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy with eosinophilia. In this study, we investigated the contribution of S100A8 and S100A9 to chemotaxis of the human eosinophilic leukemia cell line, EoL-1. A chemotaxis assay, western blot analysis and a NF-κB transcription factor assay were conducted to investigate the chemotactic mechanism. S100A8 and S100A9 induced the migration of EoL-1 cells via the phosphorylation of PKCδ, AKT, and MAPKs and the translocation of NF-κB; however, they had no effect on eosinophils from normal peripheral blood. PKCδ, AKT, and MAPKs such as ERK, p38 MAPK, and JNK are upstream regulator molecules of NF-κB activation. Iκ-Bα degradation is needed for NF-κB activation. These findings suggest that S100A8 and S100A9 induce the cell movement and contribute to an understanding of S100A8 and S100A9 in eosinophil biology and the pathogenic mechanism of hematological malignancy.

Trisubstituted purine inhibitors of PDGFRα and their antileukemic activity in the human eosinophilic cell line EOL-1

Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1‐PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia.

(S)-(+)-Decursin and its Analogues as Inhibitors of Cytokines Secretion Induced by DpE in THP-1/EoL-1 Cell Line

(S)-(+)-Decursin and its analogues were synthesized and evaluated for their inhibitory effects on monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), and IL-6 secretion induced by Dermatophagoides pteronyssinus extract (DpE) in human acute monocytic leukemia (THP-1) cells and eosinophilic leukemia (EoL-1) cell lines. (S)-(+)- Decursin analogues with alkyl acryloyl groups, apart from (S)-(+)-decursin (1) and (S)-(+)-decursinol angelate (4a), did not influence the regulation of cytokine secretion. (S)-(+)-Decursin analogues (8d, 8f, 8g) with cinnamoyl groups carrying hydroxyl- and acetoxy-substitutes potently inhibited the secretion of MCP-1 and IL-6. The structure-activity relationship study revealed that increasing π-rich environment at the extensions from the eastern alcohol of (S)-(+)-decursin is a key element in building the pharmacophore for achieving the desired biological activity.

The Role of S100A8 and S100A9 in Differentiation of Human Eosinophilic Leukemia Cells, EoL-1

S100A8 and S100A9 are associated with myeloid cell differentiation, chemotactic activities, adhesion of neutrophils, and apoptosis. In this study, we investigated the contribution of S100A8 and S100A9 to differentiation of the human eosinophilic leukemia cell line, EoL-1. S100A8 and S100A9 increased the number of vacuole per one cell and the protein expression of EPO and MBP. Rottlerin, an inhibitor of protein kinase C delta (PKCδ), inhibited the EoL-1 cell differentiation induced by S100A8 and S100A9. These results suggest that S100A8 and S100A9 may regulate the differentiation of eosinophilic progenitors. Moreover, these findings may shed light on elucidation of eosinophil differentiation due to S100 proteins.

Inhibitory effect of Panax ginseng and Pleurotus osteratus complex on expression of cytokine genes induced by extract of Dermatophagoides pteronissinus in human monocytic THP-1 and EoL-1 cells

A recent study reported that Pleurotus ostreatus has the potential to be used as a β-glucan- based cream for supportive complementary therapy of atopic dermatitis. KH054 is a new herbal prescription consisting of P. ostreatus and Panax ginseng. The effects of atopic dermatitis-induced materials on the expression of cytokine genes in human monocytes (THP-1, EoL- 1) have been examined. Some reports demonstrated that P. ginseng augments the activity of natural killer cells, which plays an important role in innate immunity against infection and tumor development. Monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-6, and IL-8 have important roles in mediating the infiltration of various cells into the skin of atopic dermatitis and psoriasis. The present study investigated whether KH054 on induced IL-6, IL-8, and MCP-1 secretion by house dust mite (Dermatophagoides pteronissinus) in THP-1 (human acute monocytic leukemia) and EoL-1 (Human eosinophilic leukemia) cell. D. pteronissinus functions in the pathogenesis of allergic diseases, including atopic dermatitis and asthma. The inhibitory effect of KH054 on the induction of IL-6, IL-8, and MCP-1 secretion by D. pteronissinus extract in THP-1 and EoL-1 cells was examined. KH054 potently suppressed the elevated production of IL-6 and IL-8 induced by D. pteronissinus treatment in THP-1 and EoL-1 cells. Based on the present results, KH054 may be useful for developing functional foods to treat atopic dermatitis.

Mechanisms of human eosinophil migration induced by the combination of IL-5 and the endocannabinoid 2-arachidonoyl-glycerol

Mechanisms of human eosinophil migration induced by the combination of IL-5 and the endocannabinoid 2-arachidonoyl-glycerol

SL-401, a Targeted Therapy Directed To The Interleukin-3 Receptor (IL-3R), Possesses Preclinical Cytotoxic Activity Against Chronic Eosinophilic Leukemia

BackgroundHypereosinophilic syndrome (HES), a rare myeloproliferative disorder characterized by a persistently elevated eosinophil count, results in damage to the heart, lungs, peripheral nervous system, and other organs. An acquired (non-familial) form of HES is particularly aggressive and debilitating. Acquired forms of HES are subclassified as secondary (reactive), idiopathic, and clonal HES, the latter often transitioning into chronic eosinophilic leukemia (CEL), or hypereosinophilic leukemia, which can result in myocardial fibrosis and congestive heart failure. Patients may respond to corticosteroids, the monoclonal antibodies mepolizumab and alemtuzumab, and the tyrosine kinase inhibitor imatinib, which is registered for HES patients who express the FIP1L1-PDGFRA fusion protein. However, several of the aforementioned agents also induce severe toxicity. Since eosinophils ubiquitously express the IL-3R, SL-401, a novel IL-3R-targeted therapeutic, which is comprised of IL-3 conjugated to a truncated diphtheria toxin, represents a rational and selective agent for development in hypereosinophilic disorders. MethodsExpression of the IL-3R (CD123) on a human eosinophilic leukemia cell line (EOL-1) was determined by flow cytometry. Cells were washed with phosphate-buffered saline (PBS) and stained with anti-CD123 PE (BD Biosciences) and control IgG for 20 minutes at 4oC and then washed with PBS again. Stained cells were acquired using the LSR II (BD) cytometer and data were analyzed using Flow Jo (Tree Star). To analyze patient samples, PBMCs were prepared and stained using a similar method and then analyzed using Cytopaint software. The sensitivity of the cells to SL-401 was assessed using a CellTiter Glo in vitro cytotoxicity assay. A CD123 positive leukemia cell line (TF-1), which is known to be sensitive to SL-401, was used as a positive control. The cells were cultured in the presence of absence of SL-401 for 48 hours and assessed for viability at concentrations ranging from 5x10-6 to 21 µg/ml (8.7x10-5 to 368 nM). ResultsPrimary eosinophils, elevated in number and CD16 negative, were harvested from a patient with HES and found to be positive for IL-3R. EOL-1 cells, similarly, were found to express high levels of IL-3R, with greater than 98% of cells expressing the receptor compared to isotype control. Based on the IL-3R expression pattern, the sensitivity of EOL-1 cells to SL-401 was then tested. Cells were shown to be highly sensitive to SL-401 after 48-hour incubation in a concentration-dependent manner, with IC50 values of 1, 0.6, and 0.47 pM from triplicate experiments (mean: 0.69 pM). Cell viability was also reduced by 96% at 48 hours post-treatment with a picomolar concentration of SL-401 (350 pM). ConclusionThese results indicate SL-401 possesses potent in vitro anti-cancer activity against CEL, which expresses high levels of the IL-3R. Importantly, SL-401 exhibited potent activity against these cells at concentrations that were significantly lower (>10-fold) than peak plasma concentrations achieved in clinical studies. These findings, together with the need for novel clinical strategies to treat CEL and HES, warrant further exploration of SL-401 for the treatment of patients with hypereosinophilic diseases. Disclosures:Brooks: Stemline Therapeutics: Employment, Equity Ownership. Macri:Stemline Therapeutics, Inc., New York, NY USA : Employment. Bergstein:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. Rowinsky:Stemline Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Inhibitory effects of casticin on migration of eosinophil and expression of chemokines and adhesion molecules in A549 lung epithelial cells via NF-κB inactivation

The fruits of Vitex rotundifolia L. have long been used for the treatment of inflammation of the respiratory tract in East Asia. To determine if casticin, one of the constituents of Vitex rotundifolia L., has anti-allergic and anti-inflammatory effects in asthma. The in vitro anti-inflammatory activity of casticin was studied in A549 human type II-like epithelial lung cells using an eotaxin inhibition assay. Additionally, its effects on eotaxin, regulated on activation normal T cell expressed and secreted (RANTES), vascular cell adhesion molecule (VCAM)-1, and inter-cellular adhesion molecule (ICAM)-1 expression were investigated by real time-polymerase chain reaction (real time-PCR). The inhibition of nuclear factor κB (NF-κB) activity in the presence of casticin was determined by analyzing confocal microscopy images of fluorescence immunocytochemical analysis while the suppression of inhibitory κB (IκB)-α phosphorylation was studied using Western blot analysis. Finally, the inhibitory effect of casticin on eosinophil migration toward prestimulated A549 cell media was measured using the human eosinophilic leukemia cell line. Casticin significantly suppressed eotaxin production in cytokine activated A549 lung epithelial cells. Casticin also suppressed the mRNA expression levels of eotaxin, RANTES, VCAM-1, and ICAM-1, which subsequently contributed to the inhibition of eosinophil migration. Furthermore, casticin inhibited IκB-α phosphorylation and nuclear translocation of p65 in A549 cells. Casiticin inhibited the eosinophil migration and activity of chemokines and adhesion molecules involved in the inflammatory process of asthma by suppressing the NF-κB pathway. These results suggest that casticin has the potential for use in the treatment of allergic asthma.

Leukotactin-1/CCL15 induces cell migration and differentiation of human eosinophilic leukemia EoL-1 cells through PKCδ activation

Leukotactin-1 (Lkn-1)/CCL15 is a CC chemokine that binds to the CCR1 and CCR3. Lkn-1 functions as an essential factor in the migration of monocytes, lymphocytes, and neutrophils. Although eosinophils express both receptors, the role of Lkn-1 in immature eosinophils remains to be elucidated. In this present study, we investigated the contribution of the CCR1-binding chemokines to chemotactic activity and in the differentiation in the human eosinophilic leukemia cell line EoL-1. Lkn-1 induced the stronger migration of EoL-1 cells than other CCR1-binding chemokines such as RANTES/CCL5, MIP-1alpha/CCL3 and HCC-4/CCL16. Lkn-1-induced chemotaxis was inhibited by pertussis toxin, an inhibitor of G(i)/G(o) protein; U73122, an inhibitor of phospholipase C and rottlerin, an inhibitor of protein kinase C delta (PKCdelta). Lkn-1 increased PKCdelta activity, which was partially blocked by the pertussis toxin and U73122. Lkn-1 enhanced the butyric acid-induced differentiation via PKCdelta after binding to the increased CCR1 because Lkn-1 caused EoL-1 cells to change morphologically into mature eosinophil-like cells. Likewise, Lkn-1 increased the expression of both eosinophil peroxidase (EPO) and the major basic protein (MBP). PKCdelta activation due to Lkn-1 is involved in migration, as well as the butyric acid-induced differentiation. This finding contributes to an understanding of CC chemokines in eosinophil biology and to the development of novel therapies for the treatment of eosinophilic disorders. This study suggests the pivotal roles of Lkn-1 in the regulation of the movement and development of eosinophils.

The roles of MCP-1 and protein kinase Cδ activation in human eosinophilic leukemia EoL-1 cells

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-α (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKCδ in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G i/G o protein, phospholipase C (PLC), PKCδ, p38 MAPK and NF-κB. MCP-1 activates p38 MAPK via G i/G o protein, PLC and PKCδ cascade. MCP-1 also induces NF-κB translocation and the activation is inhibited by PKCδ activation. The increase in the basal expression and activity of PKCδ in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKCδ is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKCδ functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils.

2-arachidonoylglycerol (2-AG) is an endogenous cannabinoid receptor ligand. To date, two types of cannabinoid receptors have been identified: the CB1 receptor, abundantly expressed in the brain, and the CB2 receptor, expressed in various lymphoid tissues such as the spleen. The CB1 receptor has been assumed to play an important role in the regulation of synaptic transmission, whereas the physiological roles of the CB2 receptor remain obscure. In this study, we examined whether the CB2 receptor is present in human eosinophils and found that the CB2 receptor is expressed in human peripheral blood eosinophils. In contrast, human neutrophils do not contain a significant amount of the CB2 receptor. We then examined the effect of 2-AG on the motility of eosinophils. We found that 2-AG induces the migration of human eosinophilic leukemia EoL-1 cells. The migration evoked by 2-AG was abolished in the presence of SR144528, a CB2 receptor antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB2 receptor and Gi/o are involved in the 2-AG-induced migration. The migration of EoL-1 cells induced by 2-AG was suggested to be a result of chemotaxis. In contrast to 2-AG, neither anandamide nor free arachidonic acid elicited the migration. Finally, we examined the effect of 2-AG on human peripheral blood eosinophils and neutrophils and found that 2-AG induces migration of eosinophils but not neutrophils. These results suggest that the CB2 receptor and its endogenous ligand 2-AG may be closely involved in allergic inflammation accompanied by the infiltration of eosinophils.

Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL-1.

The human eosinophilic leukemia cell line, EoL-1, differentiated with butyrate as an eosinophilic cellular model was evaluated for peroxidase-dependent tyrosine nitration. Butyrate suppressed cell growth and induced eosinophilic granules in EoL-1 cells after 9 days of culture. Peroxidase activity was detected biochemically and histochemically from 3-day cultures and it increased in a time dependent manner. This peroxidase activity was inhibited by cyanide. Nitrotyrosine formation catalysed by peroxidase using hydrogen peroxide and nitrite was detected at a high level similar to that of mature eosinophils. However, no expression of eosinophil peroxidase (EPO) was detected by RT-PCR or immunocytochemistry. In contrast, the induction of myeloperoxidase (MPO) by butyrate was clearly detected by RT-PCR, Northern blot, and immunocytochemical staining. These results suggest that butyrate induces MPO rather than EPO in EoL-1 cells and that the formation of nitrotyrosine in butyrate-induced cells is dependent on MPO.

Troglitazone Suppresses Cell Growth of Myeloid Leukemia Cell Lines by Induction of p21WAF1/CIP1 Cyclin-Dependent Kinase Inhibitor

In a human eosinophilic leukemia cell line, EoL-1, cell proliferation was suppressed by 2-day treatment with troglitazone. EoL-1 cells treated with troglitazone were arrested and maintained in the G0/G1 phase in the cell cycle. This suppression correlated with the up-regulation of mRNA for p21WAF1/CIP1 cyclin-dependent kinase (Cdk) inhibitor. The inhibitory effects of troglitazone on cell proliferation and expression of p21 mRNA were observed in a human myelomonocytic cell line, U937, and a human myelomonoblastic cell line, KPB-M15. In addition, in EoL-1 cells, p21 protein was induced by troglitazone treatment and the induction was inhibited by protein synthesis inhibitor, cycloheximide. These data suggest that troglitazone inhibits cell proliferation in myeloid leukemia cell lines at least in part by induction of p21 Cdk inhibitor.