TNF-induced IL-8 and MCP-1 production in the eosinophilic cell line, EOL-1

The role of eosinophils in inflammation and their mode of activation is not well understood. Eosinophil accumulation and subsequent expression of cytokines at the site of inflammation may play a role in exacerbation of inflammatory responses. In the present study, we have examined the role of TNF-α in eosinophil activation and chemokine production using a human leukaemic eosinophil cell line, EOL-1. Initial studies demonstrated that TNF-α induced the upregulation of IL-8 and MCP-1 mRNA and protein. Kinetic studies indicated production of chemokines, IL-8 and MCP-1, as early as 4 h post-activation, with peak levels of chemokine produced at 8 h, and decreasing by 24 h post-TNF-α activation. When IL-10, a suppressive cytokine, was incubated with TNF-α and EOL-1 cells, no effect was observed on IL-8 and MCP-1 production. However, dexamethasone, a glucocorticoid, demonstrated potent inhibitory effects on the EOL-1-derived chemokines. These studies indicate that eosinophils may be a significant source of chemokines capable of participating in, and maintaining, leukocyte recruitment during inflammatory responses, such as asthma.


Introduction
Eosinophils are granule-containing cells that differentiate from stem cell precursors in the bone marrow, circulate in the blood, and then enter into peripheral tissues. 1'2 Normally present at low concentrations, increased levels of circulating and/or tissue eosinophils have been associated with diseases such as asthma and parasitic infections. 2'3 The presence of eosinophils during chronic inflammatory diseases has been associated with tissue pathology. In addition, eosinophils represent a potential source of cytokines that may influence chronic inflammatory reactions and other biological responses. The establishment of a human eosinophilic leukaemia cell line, EOL-1, has proved to be a useful in vitro model for studying the properties of eosinophils and the pathways of their inflammatory responses and released products.
Cytokines regulate immunologic and physiologic events by transmitting information to target cells through receptor-ligand interactions. 4 For example, it has been shown previously that interleukin-1 (IL-1) and tumour necrosis factor-or (TNF-cz) act as early response cytokines and can stimulate a variety of both immune and nonimmune cells to produce additional peptide mediators, some of which have been shown to 218 Mediators of Inflammation Vol 5 1996 be important leukocyte chemotactic factors. [4][5][6] These chemotactic cytokines, or chemokines, which recruit additional cells to the inflammatory site, can be divided into two polypeptide groups based on the position of two internal disulfide bonds formed from four cysteine amino acid residues. In one supergene family of chemokines (C-C chemokine family), exemplified by the protein monocyte chemoattractant protein-1 (MCP-1), two of the four cysteine amino acid residues are found in juxtaposition to each other. The members of this family are predominantly chemotactic for mononuclear cells, monocytes, and lymphocytes. In the other division of this polypeptide family (C-X-C chemotactic cytokines) two of the four cysteines are separated by one 67 different amino acid. This group, typified by interleukin-8 (IL-8), is predominantly chemotactic for neutrophils. Both MCP-1 and IL-8 attract leukocytes to sims of inflammation and have been identified in various diseases such as rheumatoid arthritis, atherosclerosis, as well as in multiple pulmonary diseases. ' In addition, eosinophils have recently demonstrated the ability to produce chemokines. 9 '1 In the present study, we determined the activation pathway of chemokine production of an eosinophilic leukemia cell line, EOL-1. Utilizing this eosinophilic leukaemic cell line we demon-(C) 1996 Rapid Science Publishers IL-8 and MCP-1 from EOL-1 cells strated that tumour necrosis factor-cz (TNF-cz) thiocyanate, 0.5% Sarkosyl, and 0.1 M 2induces the production of chemokines inter-mercaptoethanol was overlaid on eosinophil leukin-8 and monocyte chemoattractant protein-monolayers. After homogenization, the above 1. Interestingly, the production of these chemosuspension was added to a solution containing kines was not regulated by a suppressive cyto-an equal volume of 100 nM Tris, pH 8.0, 10 mM kine, interleukin-10, but could be regulated EDTA, and 1.0% SDS. Then, the mixture was nonspecifically by dexamethasone. Overall, these extracted using chloroform-phenol (1.1. v/v) studies suggest that eosinophils may contribute and chloroform-isoamyl alcohol (24:1 v/v). The to chronic inflammatory responses via their total RNA was precipitated by using alcohol and ability to produce chemotactic cytokines, thus the pellet dissolved in diethyl pyrocarbonate amplifying the leukocyte recruitment.
(DEPC)-treated H20. Total RNA was separated by electrophoresis using denated formaldehyde, 1% Materials and Methods agarose gels, followed by transblotting to nitrocellulose. Blots were baked, prehybridized, and Cell culture: The eosinophilic cell line EOL-1 was hybridized with a P-5' end-labelled oligonucleomaintained in RPMI 1640 medium supplemented tide probe specific for IL-8 or MCP-1. The 30with 2 mM >glutamine, 24 mM Hepes, 100 I.tg/ml mer oligonucleotide probes for IL-8 and MCP penicillin, 100 I.tg/ml streptocycin (Hazelton were complementary to the nucleotide sequences Research Products) and 10% heat-inactivated 5'-GTT-GGC-GCA-GTG-TGG-TCC-ACT-CTC-AATfoetal calf serum (FCS) (Gibco Laboratories). CAC-3' and 5'-TTG-GGT-TTG-CTT-GTC-CAG-The isolation and characterization of EOL-1 have GTG-GTC-CAT-GGA-3', respectively. Blots were been previously described. stringently washed after hybridization and exposed to X-ray film. Specific chemokine mRNA Assessment of cytokine levels by specific ELISAs: was quantified using imaging analysis video den-Specific eosinophil-derived cytokines were quansitometry with a frame grabber (Image Capture tiffed using a modification of a double ligand 1000; Scion Corp., Walkersville, MD) and Image ELISA method as previously described. 2 Briefly, 1.49 software (National Institutes of Health flat-bottomed 96-well microtitre plates were Public Software, Bethesda, MD). coated with 50 I.d/well of rabbit antichemokine (IL-8 and MCP-1) antibodies (1 t.tg/ml in 0.6 Statistical analysis.. Data were analysed by Macinmol/1 NaCl, 0.26 ml/1 H3BO4, and 0.08 N NaOH, tosh II computer using a statistical software pH 9.6, for 16 h at 4C and then washed with package (Statview II; Abacus Concept, Inc., Ber-BPS (pH 7.5) 0.05% Tween-20 (wash buffer), keley, CA)and expressed as mean _+ S.E.M. Data Nonspecific binding sites on microtitre plates that appeared statistically significant were comwere blocked with 2% BSA in PBS and incubated pared by Student's t-test for comparing the for 90 min at 37C. Plates were rinsed three means of multiple groups and considered sigtimes with wash buffer and diluted eosinophilnificant if p values were <0.05. derived conditioned media (50 l.tl) was added, followed by incubation for i h at 37C. Plates Results were washed three times with wash buffer, 50 l.tl/well of biotinylated rabbit anticytokine (IL-8 Cytokine activation of EOL-I cells.. Previously, it and MCP-1) antibodies were added for 45 min has been shown that eosinophils produce cytoat 37C. After washing, 100 l.tl peroxidase con-kines, such as TNF-cz, which can act on a variety jugated Avidin (Dako Corp, 1:5 000 dilution)was of cell t.es to upregulate further cytokine proadded and incubated 30 min at 37C. Plates were Auction. In the present study, we assessed the again washed three times and then chromogen production of chemokines, IL-8 and MCP-1, from substrate OPD was added. The plates were incu-the eosinophilic cell line, EOL-1. Various conbated at room temperature to the desired extinccentrations, 0.1, 1.0, and 10 ng/ml, of IL-1, IFN-,, tion, the reaction terminated with 50 l.d/well of IL-4, IL-10, and TNF-z were incubated with the 3 M H3SO4 solution and samples were read at EOL-1 cells (1 x 10/ml). After 24h, super-490 nm in an ELISA reader. The sensitivity of natants were collected and analysed for IL-8 and this ELISA method is consistently greater than MCP-1 by specific ELISAs. Only TNF-cz-activated 50 pg/ml. EOL-1 cells showed significant cytokine production. None of the other cytokines examined Northern blot analysis of chemokine mRNA. Total induced chemokine production from the EOL-1 RNA from the EOL-1 cell line was isolated, as cells. Figures 1 and 2 show a dose-dependent previously described. 2' Briefly, a solution conincrease in IL-8 and MCP-1 production occurring taining 25 mM Tris, pH 8.0, 4.2 M guanidine iso-when EOL-1 cells were activated with TNF-t. By 24 h post-TNF<z activation cytokine levels had decreased (IL-8) or were maintained (MCP-1). Suppression of EOL-I-derived chemokines: To determine the regulation of IL-8 and MCP-1, EOL-1 cells were cultured with either IL-10 or dexamethasone. IL-10 is known to inhibit cytokine synthesis in other leukocyte populations, [5][6][7][8][9] whereas dexamethasone, a glucocorticoid, appears to nonspecifically shutdown cellular activation events. TNF-a (10 ng/ml)-stimulated EOL-1 cells were incubated with or without IL-10 (10 ng/ml). Supernatants were collected after 8 h and samples were assessed for cytokine production by specific ELISAs. TNF-a-stimulated EOL-1 cells incubated with IL-10 did not show significant differences in IL-8 or MCP-1 (Fig. 6) production. In fact, IL-10 appeared to augment the TNF driven It-8 production (p < 0.05). Northern blot analysis confirmed these data, demonstrating no decrease in either IL-8 or MCP-1 mRNA expression when IL-10 was added to the cultures (data not shown). In contrast, when EOL-1 cells were challenged with TNF-a in the presence of various doses of dexamethasone (10 -v to 10 -5 M), a significan decrease in both IL-8 and MCP-1 production was observed (Fig. 7). Altogether, these data indicate that although EOL-l-derived chemokine production can be inhibited by dexamethasone, it appears that the regulation of these chemocystic fibrosis, IPF, emphysema, ARDS, bronchiekines by IL-10 is different compared with other stasis, and chronic bronchitis. 25 IL-8 levels have leukocyte populations, also been detected during late-phase reactions of asthma. 26 In addition, IL-8 is known to be a Diuion chemoattractant for neutrophils, 27'28 eosinophils, 29 and has been implicated as a lymphocyte Eosinophilic infiltrations are found in chronic chemotactic protein. 1 These findings suggest inflammatory responses, such as those observed that eosinophil-derived IL-8 may recruit additional during parasitic and allergic inflammation 2'3 and leukocytes into tissue during an inflammatory/ may play a role in exacerbation of inflammatory/ allergic reaction, thus exacerbating the overall allergic reactions by perpetuation of leukocyte response. recruitment. A better understanding of the eosi-MCP-1 is a potent monocyte chemoattractant 3 nophil activation pathway and the subsequent and recently has been found to act as a T-lymcytokine production cascade would provide phocyte chemoattractant. 1 Activated T-lymphouseful information for application in immunocytes, in addition to eosinophils, have been therapy during chronic inflammation. Our studies documented to accumulate in tissue during have confirmed the importance of TNF-cz as a chronic inflammation and more specifically key activator of eosinophil-like EOL-1 cell inducduring late-phase asthmatic reactions. 31 MCP-1 tion of IL-8 and MCP-1 mRNA expression and made by eosinophils may augment T-lymphocyte protein production. Dose response studies mediated responses via its ability to recruit demonstrated that TNF-cz was capable of sigvarious leukocyte populations to the site of nificant activation of the IL-8 and MCP-1 from inflammation. Furthermore, eosinophils found at EOL-1 cells. Time course studies showed that sites of active fibrosis have been shown to cytokine production occurred within 4 h of incu-express MCP-1 mRNA 1 suggesting that they may bation with TNF and that culture of eosinophils play an important role in chronic fibrosing diswith TNF-cz for 8 h induced peak levels of IL-8 eases. and MCP-1. Interestingly, incubation of IL-10 with Finally, we determined IL-10 had a minimal TNF-cz and EOL-1 cells did not affect IL-8 and role in regulating EOL-l-derived IL-8 and MCP-1.
MCP-1 production, while EOL-1 cells could be IL-10 is a cytokine produced by CD4 + cells, B regulated by dexamethasone. These results cells, certain populations of CD8 + cells, suggest that EOL-1 cells are regulated by IL-10 Epstein-Barr Virus (EBF)-transformed lymphodifferently than other leukocyte populations, blastoid cell lines, monocytes and macro-Overall, these studies suggest that eosinophils phages. 25'32-34 In addition, IL-10 demonstrates may significantly contribute to the exacerbation varied immunosuppressive bioactivity on cytokine of chronic inflammation and upregulate the leuproduction and, along with IL-4 and IL-5, is pro- 35 kocyte extravasation through the production of duced during allergic responses. IL-10 downchemokines, regulates IFN-7 production associated with Thl Activation of eosinophils and the subsequent responses as well as monocyte and polymorphorelease of IL-8 and MCP-1 may be an important nuclear (PMN)-derived cytokine production, contributing factor during a chronic inflammatory including IL-8. 6 In this study we have demonresponse. In particular, the infiltration of eosino-strated that IL-10 does not inhibit cytokine prophils into the lung tissue is known to occur duction from TNF-activated EOL-1 cells. Since during the late phase of asthma.  It has been eosinophil accumulation appears to be enhanced previously shown that BAL cells from asthmatics, during Th2 responses and IL-10 does not regas compared to normal controls, express ulate this response, eosinophils may play a sig- 24 increased levels of TNF-specific mRNA. This nificant role in maintaining chronic allergic and increased production of TNF, which occurs in parasitic responses. However, since IL-10 strongly conjunction with the presence of eosinophils, regulates the production of TNF from leukocyte may be a contributing factor for activating the populations IL-10 may indirectly affect the cytoeosinophils leading to upregulated chemokine ldne production in vivo from eosinophilic cells. production. Interestingly, recent studies have A final possibility may be that the EOL-1 cells do identified eosinophils as a source of TNF sugnot have IL-10 receptors and therefore cannot gesting that they can also contribute to the respond to the cytokine. Unlike IL-10, dexaoverall activation of inflammatory responses, methasone did inhibit chemokine production, The chemokines, IL-8 and MCP-1, which were indicating that the regulation within these cells is found to be produced by the EOL-1 cells, have similar to other leukocyte populations. The dexabeen associated with a number of inflammatory methasone induced inhibition of the chemokines diseases. IL-8 production has been observed in correlates well with the successful use of steroids IL-8 and MCP-I from EOL-I cells in treatment of chronic eosinophilic disease states, such as asthma.
Our findings suggest that eosinophils may participate in inflammatory/allergic reactions through the generation of chemokines, IL-8 and MCP-1, further augmenting leukocyte recruitment. In addition, IL-10, an inhibitor of monocyte and PMN-derived cytokines, did not regulate EOL-1derived cytokine production, suggesting altered regulation of this leukocyte subset.