Mycobacterium bovis BCG induces CXC chemokine ligand 8 secretion via the MEK-dependent signal pathway in human epithelial cells

ellular Immunology Cellular Immunology 234 (2005) 9–15 www.elsevier.com/locate/ycimm

Mycobacterium bovis BCG induces CXC chemokine ligand 8 secretion via the MEK-dependent signal pathway in human epithelial cells Patricia Méndez-Samperio ¤, Artemisa Trejo, Elena Miranda Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, IPN, Carpio y Plan de Ayala, Mexico, D.F. 11340, Mexico Received 23 February 2005; accepted 15 April 2005 Available online 13 May 2005

Abstract Current knowledge of the cellular signaling by Mycobacterium bovis bacillus Calmette-Guerin (BCG) in epithelial cells is still limited. In this study, we provide evidence that the signaling events induced by M. bovis BCG in these cells included phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Our data also demonstrate that M. bovis BCG-induced CXC chemokine ligand (CXCL)8 release in epithelial cells was reduced by a mitogen-activated protein/ERK kinase (MEK) inhibitor (PD98059), but not by a p38 MAPK (SB203580) inhibitor. In addition, we found that a second and more potent MEK inhibitor (U0126) signiWcantly blocked CXCL8 release in epithelial cells by M. bovis BCG. Evaluation of CXCL8 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory eVect of PD98059 and U0126 was associated with a reduction in this parameter. Moreover, the induction of CXCL8 secretion in epithelial cells by M. bovis BCG occurs at the transcription level. Collectively, the Wndings reported in the present study suggest that MEK signaling is essential for the induction of CXCL8 in epithelial cells in response to M. bovis BCG.  2005 Elsevier Inc. All rights reserved. Keywords: CXCL8; Epithelial cells; Mycobacterium bovis; MEK; p38 MAP kinase

1. Introduction Worldwide, one of the major causes of death by infectious disease is Mycobacterium tuberculosis infection [1]. Bacillus Calmette-Guérin (BCG) is a live, attenuated strain of Mycobacterium bovis widely used as a vaccine against M. tuberculosis infection [2]. During infection, mycobacteria induces increased expression of chemokines, including the CC chemokine subfamily members and the CXC chemokine subfamily members, such as interleukin (IL)-8 (CXCL8) [3,4]. CXCL8 is an important chemokine which stimulates chemotactic action in T cells, neutrophils, and basophils in tuberculosis [5,6]. *

Corresponding author. Fax: +5 396 35 03. E-mail address: [email protected] (P. Méndez-Samperio). 0008-8749/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2005.04.002

At a cellular level, phagocytosis of M.tuberculosis by monocytic cells or epithelial cells is an important stimulus to CXCL8 production [7,8]. Recently, we reported that human monocytes infected with M. bovis induce the secretion of CXCL8 [9]. Since it has been reported that the respiratory epithelium is a primary target for microbial infection [10,11], and that epithelial cells are considered as an important cellular source of chemokines [8], we also have investigated the induction of CXCL8 by human epithelial cells infected with M. bovis [12]. However, the molecular mechanisms underlying the M. bovisinduced CXCL8 secretion in the human epithelial cells are not completely understood. Cellular signaling by mycobacteria is mediated through activation of mitogen-activated protein (MAP) kinases [13,14]. These kinases include three major members: the extracellular signal-regulated kinases (ERKs),

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the c-Jun N-terminal kinase (JNK)/stress-activated protein kinases, and the p38 kinase [15,16]. The role of MAPK pathways in generation of cytokines and chemokines during mycobacterial infection has been investigated in vitro. For example, infection of human monocytes with Mycobacterium avium induces phosphorylation of all three MAP kinases, and subsequent ERK1/2 activation is required for TNF-
secretion [17,18]. In addition, we have demonstrated that M. bovis BCG induction of TNF-
is dependent on the ERK pathway [19]. Recently, it has been demonstrated that mannosylated phosphatidylinositol, isolated from M. tuberculosis, stimulated ERK1/2 phosphorylation in human alveolar macrophages and activation of this MAP kinase was required for production of CC-chemokines [20]. Little is known, however, about the cellular signaling pathways by which M. bovis mediate CXCchemokine induction. In this study, we have investigated the involvement of MAP kinases in M. bovis BCGinduced CXCL8 expression in human epithelial cells.

2. Materials and methods 2.1. Reagents The speciWc inhibitor of p38 MAP kinase pathway, SB203580, and the MEK1/2 inhibitors, PD98059, and U0126 (Calbiochem–Novabiochem, San Diego, CA) were used and dissolved in DMSO (Sigma–Aldrich). Actinomycin D (ActD) was purchased from Sigma– Aldrich (St. Louis, MO). 2.2. Mycobacteria Mycobacterium bovis (ATCC 35733) was obtained from the American Type Culture Collection (Rockville, MD, USA). M. bovis was grown at 37 °C in Sauton medium for 2 weeks. Cultures were harvested by centrifugation and quantiWed by a colony forming unit (CFU) assay. Aliquots of the stock were kept at ¡70 °C. 2.3. Cell culture The human epithelial HEp-2 cell line was originally acquired from the American Type Culture Collection (Rockville, MD, USA). Cells were grown at 37 °C in a humidiWed 5% CO2 incubator using minimum essential medium Eagle with 2 mM of L-glutamine, 1 mM of sodium pyruvate, 0.1 mM of non-essential amino acids, and Earle’s BSS adjusted to contain 1.5 g/L of sodium bicarbonate and 10% heat-inactivated foetal bovine serum (Gibco-BRL, Rockville, MD, USA). The human alveolar epithelial A549 cell line was maintained in a humidiWed 5% CO2 atmosphere in Dulbecco’s modiWed Eagle’s medium. Cells (105/well) were infected with

mycobacteria using an opsonized bacteria-to-cell ratio of 3:1. Control cultures with no mycobacteria were always included. SB203580, PD98059, U0126 or ActD were added to some of the cultures. Each experiment was repeated a minimum of three times with diVerent donors. 2.4. Western blot analysis HEp-2 cells were infected with M. bovis BCG at a bacteria/cell ratio of 3:1 in RPMI-1640 media and incubated at 37 °C in 5% CO2. After 30 min cells were washed three times with cold PBS and cell lysates were prepared using ice-cold lysis buVer (150 mM NaCl, 1 mM phenylmethylsulfonyl Xuoride, 1 g/ml aprotinin, 1 g/ml leupeptin, 1 g/nl pepstein, 1 mM pervanadate, 1 mM EDTA, 1% igepal, 0.25% deoxycholic acid, 1 mM NaF, and 50 mM Tris–HCl (pH 7.4)). Following lysis the cellular suspension was centrifuged, and the clariWed lysates were stored at ¡20 °C. Equal amounts of protein were separated on SDS–polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes (Bio-Rad Laboratories, Florida, USA). After blocking with 5% nonfat milk in TBS-T (Tris-buVered solution, pH 7.6, 0.05% Tween 20), the membranes were incubated overnight with primary antibodies phospho-p38, total p38, phospho ERK1/2, or total ERK1/2 from New England Biolabs (Schwalbach, Germany) as indicated by the manufacturer’s instructions. The primary antibody reaction was followed by 1 h incubation with the appropriate horseradish peroxidaseconjugated goat anti-rabbit immunoglobulin G at room temperature. The peroxidase-positive bands were detected by immersing the blots in a developing solution (73 mM sodium acetate, pH 6.2) containing 0.3% diaminobenzidine tetrahydrochloride and 0.04% H2O2 at room temperature for 5 min. The enzyme reaction was terminated by washing the blots in 0.1 M H2SO4. 2.5. CXCL8 ELISA CXCL8 levels were determined in culture supernatants from control and M. bovis-infected HEp-2 cells by using the kit human CXCL8 enzyme-linked immunosorbent assay (ELISA) system (R&D Systems, Minneapolis, MN, USA). The ELISA assays were carried out according to the manufacturer’s instructions. 2.6. Assessment of CXCL8 gene expression by RT-PCR HEp-2 cells were treated with PD98059 (50 M) or U0126 (30 M) for 30 min, and were then infected with M. bovis BCG at a MOI D 3. Total RNA from cells was isolated by the method of Chomczynski and Sacchi [21]. BrieXy, total RNA was extracted with TRIzol (Life Technologies, Rockville, MD) for 5 min at room temperature, transferred to a microfuge tube, and then 200 l of chloroform was added, vortex mixed, and incubated for

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10 min. After centrifugation for 5 min, the aqueous layer was transferred to a fresh tube, and RNA was precipitated by adding an equal volume of isopropanol. After centrifugation, the RNA pellet was dried and dissolved in 50 l of sterile water. The RNA concentration was determined by optical density measurements. Cellular RNA (2 g) was reversibly transcribed using random hexamer primers (Gibco-BRL, Rockville, MD) and Superscript II reverse transcriptase (Gibco-BRL). The resulting cDNA was ampliWed for a total of 20 cycles in the standard reaction mixture using Taq DNA polymerase and the appropriate CXCL8 primers (Clontech Laboratories, Palo Alto, CA, USA). PCR products were electrophoresed on a 2% agarose gel, visualized by ethidium bromide staining and photographed. Expression of GAPDH was used to check RNA integrity. 2.7. Statistical analysis Results were analyzed by Student’s t test. p 6 0.05 was considered signiWcant.

3. Results 3.1. Mycobacterium bovis BCG activates MAPKs in epithelial cells HEp-2 In a previous study, secretion of CXCL8 in epithelial cells infected with M. bovis was determined [12]. In this study, we examined whether M. bovis stimulates MAPKs in HEp-2 cells by using Western blot analysis with anti-phospho-speciWc Abs against p38 MAPK or ERK1/2 kinases. The data indicate that M. bovis BCG infection induced strong phosphorylation of p38 and ERK(1/2) MAP kinases in HEp-2 cells (Figs. 1A and B). To conWrm that M. bovis BCG activates MAPKs in

Fig. 1. Mycobacterium bovis BCG-induced phosphorylation of p38 and ERK1/2 in HEp-2 cells. HEp-2 cells were exposed to M. bovis BCG (3:1 bacteria/cell), and after a period of 30 min cell lysates were prepared and assessed for p38 (A) and ERK(1/2) (B) MAP kinases activation by Western blot. HEp-2 cells were pretreated with SB203580 (30 M) (A) or PD98059 (50 M) (B) for 30 min and then stimulated with M. bovis. Cell extracts were prepared and subjected to immunoblot analysis. These results are representative of one of three experiments performed independently.

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HEp-2 cells, phosphorylation of p38 MAP kinase by M. bovis BCG in HEp-2 cells was inhibited by the p38 MAP kinase inhibitor, SB203580 (Fig. 1A). A similar inhibitory eVect of an inhibitor of MEK PD98059, was observed on activation of ERK(1/2) MAP kinase by M. bovis BCG in HEp-2 cells (Fig. 1B). 3.2. CXCL8 production by M. bovis BCG-infected HEp-2 cells occurred via the MEK-dependent signal pathway To gain an insight into molecular mechanisms leading to M. bovis BCG-induced CXCL8 production, we studied the eVect of speciWc inhibitors on signal transduction through the p38 MAPK and MEK/ERK pathways in HEp-2 cells. We preincubated HEp-2 cells with diVerent concentrations of SB203580, to speciWcally inhibit p38 MAP kinase activity [22] or with PD98059, an inhibitor of MEK via upstream activator-dependent phosphorylation [23] before M. bovis infection. Results were that M. bovis BCG-induced CXCL8 production was signiWcantly reduced by PD98059 in a dose-dependent manner, beginning at 5 M PD98059 (p < 0.01) (Fig. 2B), whereas SB203580 had no signiWcant eVect (Fig. 2A). To conWrm the regulatory role of ERK pathway, we also evaluated the eVect of a second and more potent MEK inhibitor (U0126, a direct inhibitor of MEK-1 and -2) [24]. Fig. 3 shows that this MEK inhibitor was also capable of signiWcantly blocking (92% inhibition at 30 M) M. bovis BCG-induced CXCL8 secretion in a dose-dependent manner. A similar eVect was observed with other epithelial A549 cell line (Fig. 3). The next step in the investigation led us to examine whether the CXCL8 inhibition reported in Fig. 2B and 3 occurred at the pre- or post-transcriptional level. Therefore, the impact of PD98059 and U0126 on CXCL8 gene expression was monitored. As shown in Fig. 4, inhibition of MEK1/2 reduced M. bovis BCGmediated CXCL8 mRNA expression, a reduction that correlated with the high level of downregulation observed for the CXCL8 protein secretion (Figs. 2B and 3). These results indicate that abrogation of CXCL8 production by blocking MEK1/2-dependent signals is a result of pretranscriptional downregulation of CXCL8. In order to verify or exclude whether transcriptional upregulation of the CXCL8 gene is the predominant mechanism through which M. bovis BCG induces CXCL8 secretion, HEp-2 cells were pretreated with or without various concentrations of the transcriptional inhibitor actinomycin D for 30 min at 37 °C prior to the addition of BCG (MOI D 3) for 48 h at 37 °C. Then the culture supernatants were assayed for CXCL8 content by ELISA. Results from all Wve experiments showed that, addition of actinomycin D signiWcantly reduced in a concentration-dependent manner the ability of M. bovis BCG to induce CXCL8 secretion (Fig. 5).

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Fig. 3. The MEK inhibitor U0126 reduces the M. bovis BCG-induced CXCL8 secretion in HEp-2 cells or A549 cells. Cells were treated with medium containing 0.075% (vol/vol) DMSO as solvent control, or increasing doses of U0126 for 30 min prior to infection with M. bovis BCG (MOI D 3). After 48 h incubation, supernatants were collected and CXCL8 was measured by ELISA. Data shown are means § SD of four independent experiments. The percentage in parentheses indicates inhibition in the presence of U0126 compared with M. bovis BCG cultures which did not receive U0126.

Fig. 2. PD98059, but not a p38 MAPK inhibitor, reduced M. bovis BCG-induced CXCL8 release from HEp-2 cells. HEp-2 cells were preincubated with increasing concentrations of SB203580 (A) or PD98059 (B) for 30 min before being treated with M. bovis BCG (MOI D 3) for 48 h at 37 °C. The amount of CXCL8 secreted was measured by ELISA. All data are presented as means § SEM of Wve independent experiments. Reduction of M. bovis BCG-induced CXCL8 secretion after addition of 5 and 50 M PD98059 is statistically signiWcant (*p < 0.01).

4. Discussion The pivotal roles of MAPKs in CXCL8 production have been documented [25,26]. However, depending on the stimuli and cell types, three MAPKs have been reported to diVerentially mediate CXCL8 expression [27–29]. In this study, we show that M. bovis BCG indeed can rapidly stimulate p38 MAPK and ERK1/2 phosphorylation in HEp-2 cells. Despite this, we provide evidence leading to the observation that M. bovis BCGinduced CXCL8 production depends on MEK, but not on p38 MAPK. First, M. bovis BCG-stimulated CXCL8 protein secretion was reduced by a MEK inhibitor (PD98059), but not by a p38 MAPK (SB203580) inhibitor. Second, these results were conWrmed by using a more speciWc MEK inhibitor (U0126) with diVerent

Fig. 4. EVect of speciWc inhibitors of MAPK kinases 1 and 2 (MEK1/2) on M. bovis BCG-induced CXCL8 gene expression. HEp-2 cells were treated with PD98059 (50 M) or U0126 (30 M) for 30 min at 37 °C prior to stimulation with M. bovis BCG. After incubation total RNA was isolated and RT-PCR was performed using 1 g of RNA. PCR products were run on a 2% agarose gel containing ethidium bromide. The results depicted are representative of three independent experiments. GAPDH, glyceraldehyde 3-phosphato dehydrogene probe used to conWrm equal RNA loading.

mechanism of action [24]. Third, both inhibitors signiWcantly reduced CXCL8 mRNA expression. Although previous reports have demonstrated mycobacterial-induced CXCL8 production [7,8], the upstream signaling mechanism involved in this M. bovis BCGinducible epithelial cell function has not been fully elucidated. Therefore, in this study, we for the Wrst time have demonstrated the involvement of the MEK1/2 MAPK cascade in the regulation of CXCL8 in M. bovis BCGinfected epithelial cells.

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Fig. 5. Mycobacterium bovis BCG-induced CXCL8 secretion results from transcriptional regulation of the CXCL8 gene. HEp-2 cells were treated with medium alone or with various concentrations of actinomycin D (ActD) for 30 min at 37 °C prior to the addition of M. bovis BCG (MOI D 3) for 48 h at 37 °C. The culture supernatants were assayed for CXCL8 content by ELISA. Results are represented as means § SEM of Wve independent experiments.

In this study, further experiments were conducted to determine whether this cellular regulation was taking place at the pre- and/or post-transcriptional level(s). We found that MEK1/2 inhibitors exerted their down-regulatory eVect on CXCL8 expression by M. bovis BCGinfected HEp-2 cells at the pre-transcriptional level. Our observation is in agreement with those of Linevsky et al. [30], who reported that secretion of CXCL8 from monocytes results from transcriptional upregulation of the CXCL8 gene. Moreover, our data, indicating a key role for transcriptional activation in epithelial cells in response to mycobacterial antigen stimulation, are perfectly in agreement with a previous study which has demonstrated that the induction of chemokine secretion from epithelial cells by M. tuberculosis occurs at the transcriptional level [31]. On the other hand, several reports have indicated evidence for cross-talk between MAPKs and NF-B signaling pathways [32,33]. To assess this possibility, experiments to determine whether CXCL8 expression attenuating eVects of speciWc MEK inhibitors, PD98059 and U0126, are probably caused by a reduction in activation of the CXCL8 promoter and/or by any component of the NF-B signaling cascade are in progress. We have demonstrated the noninvolvement of p38 MAPK in M. bovis BCG-induced CXCL8 production in epithelial cells. According to previous study [34], p38 MAPK mediates the stimulating action of CXCL8 synthesis by lung epithelial cells. In this regard, activation of p38 MAP kinase has been shown to be indispensable for interleukin-1-induced CXCL8 expression in epithelial cells [35]. With respect to these diVerential and contradictory results several reasons possibly exist to explain

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these data. First, p38
and p38 isoforms might exert distinct action in regulating CXCL8 synthesis. Second, that the upstream signal transduction for CXCL8 protein expression varies among diVerent activating inducers. Third, SB203580 might have p38 MAPK-independent nonspeciWc action, which possibly can inXuence CXCL8 protein translation and/or stability [36]. On the other hand, Toll-like receptor 2 (TLR2) has been implicated in the recognition of M. bovis BCG particulate or soluble antigens leading to macrophage proinXammatory cytokine production [37]. However, a recent report has demonstrated that the proinXammatory response to live M. bovis BCG is independent of TLR2 and TLR4 [38]. Since abundant expression of TLR2 mRNA in human lung epithelial cells has been documented [39], and TLR2 mediated responses involve p38 MAPkinase activation, it may be a valuable undertaking to carry out an analysis of the dependency on TLR2 and TLR4 for M. bovis-induced CXCL8 production by human epithelial cells. Collectively, the Wndings reported in the present study suggest that MEK1/2-dependent signaling events play pivotal roles in M. bovis BCG-induced CXCL8 expression in human epithelial cells. Further experimental work is needed to know whether the eVect of MEK1/2 on M. bovis BCG-induced CXCL8 secretion may represent a signiWcant regulatory mechanism in vivo. However, these data may represent an important regulatory mechanism during the immune response to BCG, since CXCL8 induction during mycobacterial infection is a major neutrophil-activating factor and chemotactic.

Acknowledgments This research was supported by a grant from the Coordinación General de Posgrado e Investigación (CGPI) No. 20050019. P.M.S. is a COFAA, EDI, and SNI fellow.

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