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Roles of Intracellular Calcium and NF-κB in the Bacillus Calmette- Guérin-Induced Secretion of Interleukin-8 from Human Monocytes
Cellular Immunology 211, 113–122 (2001) doi:10.1006/cimm.2001.1816, available online at http://www.idealibrary.com on
Roles of Intracellular Calcium and NF-B in the Bacillus CalmetteGue´rin-Induced Secretion of Interleukin-8 from Human Monocytes Patricia Me´ndez-Samperio, Janet Palma, and Abraham Va´zquez Departamento de Inmunologı´a, Escuela Nacional de Ciencias Biolo´gicas, IPN, Carpio y Plan de Ayala, 11340 Me´xico, D.F. Me´xico Received December 27, 2000; accepted May 29, 2001
Interleukin (IL)-8 secretion contributes to the early host response against mycobacterial infection by increasing local inflammation and recruiting professional phagocytes. Because the mechanisms through which Mycobacterium bovis Calmette-Gue´rin (BCG) induces IL-8 secretion are unknown, the aim of the present study was to characterize the nature of IL-8 production induced by BCG in human monocytes. In this study, we found that the induction of IL-8 synthesis was dose- and time-dependent after stimulation with BCG. This IL-8 secretion was not attributed to LPS contamination or the presence of TNF-␣. We also determined that BCG-induced IL-8 secretion occurs through a mechanism that requires intracellular calcium and likely involves a calmodulin-sensitive step. Interestingly, BCG-induced secretion of IL-8 from human monocytes resulted from transcriptional up-regulation of the IL-8 gene. Moreover, we present evidence that BCG activates nuclear translocation of the transcription factor NF-B, since pretreatment of monocytes with sulfasalazine, a inhibitor of NF-B activity, blocked the ability of BCG to induce IL-8 secretion in a dose-dependent manner, producing 92.5% inhibition at a concentration of 2 mM. These results were further supported by the fact that treatment of cells with curcumin, another well-described inhibitor of NF-B activity with a different mechanism of action, significantly diminished the effect of BCG on IL-8 secretion. Together, these studies are the first to demonstrate that BCG-induced IL-8 secretion by human monocytes appears to be mediated by intracellular Ca 2ⴙ and is NF-B-dependent and at the same time suggest that production of IL-8 in response to M. bovis BCG can contribute to the initial local and systemic inflammatory response in human tuberculosis. © 2001 Academic Press
INTRODUCTION
to Mycobacterium tuberculosis infection, T cells and macrophages are recruited to the site of infection, resulting in tissue inflammation. The mechanism for recruitment of these cells is involving chemokines. Interleukin (IL)-8, 1 first isolated from monocytes as a neutrophil attractant (3, 4), is a member of the CXC chemokine subfamily that is also chemotactic for T lymphocytes (5) and monocytes (6). IL-8 release from monocyte/macrophages is enhanced after exposure to M.tuberculosis and its cell wall component (7) and following phagocytosis of M.tuberculosis by human monocytic cell lines (8). IL-8, by binding to either of its two specific receptors called CXCR-1 and CXCR-2 (9), induces a variety of cell type-specific responses, including enhanced killing of various microorganisms, such as M. tuberculosis. Circulating levels of IL-8 in plasma of patients with pulmonary tuberculosis have been found to be increased with time as a result of anti-tuberculosis therapy, and high admission plasma levels of IL-8 are of clinical prognostic significance in tuberculosis (10). IL-8 secretion is mainly regulated at the transcriptional level (11). The promoter of the IL-8 gene contains binding sites for a number of important transcription factors, including nuclear factor-B (NF-B) (12–14). NF-B proteins are normally retained in the cytoplasm bound to the IB␣ family of inhibitory molecules (15). Following cellular activation, IB␣ is phosphorylated (16) on specific serine residues and rapidly degraded (17), allowing NF-B to pass into the nucleus, where it can bind the IL-8 B-binding site. Recently, it has been demonstrated that IL-8 secretion induced by bacterial pathogens such as Helicobacter pylori requires activation of the transcription factor NF-B (18). However, whether induction of IL-8 from human monocytes by M. bovis BCG is mediated through NF-B factor has not been reported. In this report, we demonstrated that human monocytes pro1
Tuberculosis is a major health problem which is responsible for almost 3 million deaths annually worldwide (1,2 ). During the course of the immune response
Abbreviations used: BAPTA/AM, 1,2-bis(2-aminophenoxy)ethaneN,N,N⬘,N⬘-tetraacetic acid tetra(acetoxymethyl)esther; BCG, Mycobacterium bovis bacillus Calmette-Gue´rin; IL-8, interleukin-8; M.O.I., multiplicity of infection.
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0008-8749/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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duced significant IL-8 levels following stimulation with M. bovis BCG. This increased IL-8 secretion was mediated through a mechanism requiring intracellular Ca 2⫹ and calmodulin activation. In addition, NF-B inhibitors blocked the ability of M. bovis BCG to induce IL-8 secretion. These findings should increase our understanding of the regulation of IL-8 synthesis in mycobacterial infections, and they suggest that regulated production of IL-8 after stimulation with BCG can contribute to host immune defenses by attracting monocytes, lymphocytes, and neutrophils. MATERIALS AND METHODS Reagents EDTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid tetra(acetoxymethyl) esther (BAPTA/AM), sulfasalazine, curcumin, lipopolysaccharide (LPS) from Escherichia coli serotype 0128:B12, polymyxin B sulfate, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride) were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-TNF-␣ (AB 210-NA) was purchased from R&D Systems (Minneapolis, MN). Live M. bovis BCG, Danish Strain 1331, was kindly supplied by Dr. J. Ruiz-Puente (Instituto Nacional de Higiene, Me´xico). BCG was grown at 37°C in Sauton medium using stationary tissue culture flasks. Mycobacterial viability, as assessed by the number of CFU, was 60 to 70%. Cell Culture Whole heparinized blood was obtained from healthy consenting volunteers. Peripheral blood mononuclear cells (PBMC) were separated out by density gradient centrifugation on Ficoll–Histopaque 1077 (Sigma, St. Louis, MO). The PBMC were enriched for monocytes by adherence on petri dishes at 37°C for 1 h. Then, plates were vortexed and nonadherent cells were removed by vigorous washing with RPMI 1640 (Gibco, BRL). Adherent cells were then cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FCS overnight and monolayers were washed again before infection with BCG. More than 85% of cells purified by this technique were determined to be monocytes. Cells were infected in triplicate with BCG. The multiplicity of infection (m.o.i.) ranged from 0.1 to 3 live M. bovis BCG organisms per cell. After infection, cells were washed twice with warm RPMI to remove extracellular bacteria and
TABLE 1 Inhibition of TNF-␣-Mediated Toxicity by Anti-TNF-␣ Antibody Stimulus TNF-␣ TNF-␣ TNF-␣ TNF-␣ TNF-␣ TNF-␣ TNF-␣
⫹ ⫹ ⫹ ⫹
anti-TNF-␣ anti-TNF-␣ anti-TNF-␣ Control IgG
Concentration
LDH-release units
0.1ng/ml 1.0ng/ml 3.0ng/ml 0.5g/ml 1.0g/ml 5.0g/ml 5.0g/ml
457 926 1783 1702 1050 482 1694
Note. L929 cells (10 4) were cultured for 1 h in culture medium containing either increasing concentrations of anti-TNF-␣ or isotype control IgG and then incubated with TNF-␣ (3 ng/ml) for 24 h. Cytolysis was assessed by measuring LDH release. Similar results were observed in three additional experiments.
were then cultured in fresh RPMI at 37°C in 5% CO 2. In some experiments, cultured monocytes were preincubated with medium or various inhibitors for 30 min prior to the addition of BCG. When indicated, LPS was used at a final concentration of 10 g/ml. To some cultures neutralizing antibody to TNF-␣ was added. Culture supernatants for the detection of IL-8 were harvested, centrifuged to remove any debris, and then stored in frozen aliquots at ⫺20°C. Measurement of IL-8 Concentrations of IL-8 in culture supernatants were determined by using the Biotrak human IL-8 enzymelinked immunosorbent assay (ELISA) system (Amersham, Aylesbury, UK) according to the manufacturer’s instructions. The minimum detectable dose of IL-8 is 0.5 pg/well. IL-8 concentrations are expressed as picograms per 10 5 cells. Northern Blot Analysis of IL-8 mRNA Expression Monocytes were infected with BCG at m.o.i. ⫽ 3 in the presence and the absence of the NF-B inhibitor curcumin (20 M). Cells were then lysed in Trizol (Life Technologies), and total RNA was purified according to the manufacturer’s instructions. After quantification on spectrophotometer, 20 g of total RNA from each sample was electrophoresed in a 1.2% agarose gel and transferred to nitrocellulose membranes. The blots were hybridized with a 32P-labeled cDNA probe for human IL-8. A cDNA probe for chicken glyceraldehyde-
FIG. 1. Induction of IL-8 by bacillus Calmette-Gue´rin (BCG) in human monocytes. (A) Human peripheral blood-derived monocytes (10 5/well) were infected with BCG at m.o.i. ⫽ 3 (*), m.o.i. ⫽ 1 (䊐), or m.o.i. ⫽ 0.1 (⌬) or incubated with medium alone (f). Cell culture supernatants were harvested at 0, 2, 8, and 24 h following infection and IL-8 protein was measured by ELISA as described under Materials and Methods. Results are the mean ⫾ SEM of four independent experiments. (B) Human monocytes were infected with BCG (m.o.i. ⫽ 3) or stimulated with LPS (10 g/ml), in the presence or the absence of polymyxin B (5 g/ml). After an 24-h incubation, IL-8 levels were measured in cell culture supernatants by ELISA. Data are expressed as the mean ⫾ SEM of five separate experiments.
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3-phosphate dehydrogenase (GAPDH) mRNA was used as a control to determine loading equivalence. Analysis and Presentation of Data Results of ELISAs are expressed as pg/10 5 cells, means ⫾ SEM of at least three independent experiments. Statistical analysis was performed by using Student’s t test. A P value less than 0.01 was considered statistically significant. RESULTS Human Monocytes Produce IL-8 in Response to BCG Stimulation We first examined the IL-8 production of human monocytes following stimulation with BCG at m.o.i. ⫽ 3, 1, or 0.1. IL-8 secretion was clearly detected in a dose-dependent manner after stimulation with BCG, and a maximal production was reached with m.o.i. ⫽ 3 of BCG (Fig. 1A). No IL-8 secretion was observed in the absence of stimulation. The IL-8 response of BCGinfected cell cultures increased over time to reach a maximum 24 h after infection (Fig. 1A). Parallel experiments were performed to measure IL-8 secretion by human monocytes infected with BCG or cultured with lipopolysaccharide. As shown in Fig. 1B, BCG induced significantly higher production of IL-8 than lipopolysaccharide (P ⬍ 0.01). The IL-8 production experiments were repeated with BCG or LPS in the presence of polymyxin B (5 g/ml). As shown in Fig. 1B, polymyxin B was able to inhibit LPS-induced IL-8 secretion. In contrast, polymyxin B treatment had no significant inhibitory effect on the IL-8 levels stimulated by BCG (Fig. 1B). These data exclude the possibility that LPS contamination is responsible for the effects observed with M. bovis BCG. On the other hand, it is well known that human cells produce TNF-␣ in response to mycobacterial antigens (19, 20). In addition, it has been demonstrated that, in pulmonary epithelial cells, TNF-␣ stimulates IL-8 production (21). Therefore, we evaluated the potential role of TNF-␣ in the capacity of human monocytes infected with BCG to produce IL-8 by using neutralizing antibodies to TNF-␣. In three experiments, anti-TNF-␣ antibodies had no significant effect on IL-8 production (data not shown). In contrast, addition of these antibodies significantly reduced TNF␣-mediated cytotoxicity (Table 1).
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BCG-Induced IL-8 Secretion Requires Mobilization of Intracellular Ca 2⫹ and Activation of Calmodulin Because Ca 2⫹ ionophores have been shown to elicit IL-8 secretion from monocytes (22), and an intracellular Ca 2⫹ flux is associated with induction of IL-8 secretion by certain signals, such as IL-1 (23), we next sought to determine whether the effect of BCG on IL-8 secretion required mobilization of intracellular Ca 2⫹. We used EDTA to deplete extracellular Ca 2⫹ and used the cell-permeable agent BAPTA/AM to chelate releasable intracellular stores of Ca 2⫹. As evident in Fig. 2A, pretreatment of monocytes with EDTA effectively inhibited in a concentration-dependent manner, first evident at 0.1 mM and maximal at 3 mM (42.5% inhibition), the level of BCG-induced IL-8 secretion. When monocytes were incubated in EDTA and BAPTA/AM, the ability of BCG to activate IL-8 secretion was completely inhibited, in a dose-dependent manner. Maximal inhibition was reached with 30 M BAPTA/AM ⫹ 3 mM EDTA (96.8% inhibition) and 32.5% inhibition was obtained with 0.3 M BAPTA/AM ⫹ 3 mM EDTA (Fig. 2B). Because a major effect of intracellular Ca 2⫹ flux is the activation of calmodulin, we next examined whether the inhibition of calmodulin could diminish BCG-induced secretion of IL-8. W-7 is a potent and specific inhibitor of calmodulin activity and we observed that treatment with this agent abrogated BCGmediated IL-8 secretion by ⬃75% at 20 M compared with control cultures (Fig. 3). These results indicate that both intracellular Ca 2⫹ and calmodulin are required for BCG-induced IL-8 secretion in human monocytes. BCG-Induced Secretion of IL-8 from Human Monocytes Results from Transcriptional Up-regulation of the IL-8 Gene Next, we determined whether transcriptional upregulation of the IL-8 gene is the predominant mechanism through which BCG induces IL-8 secretion in human monocytes. As evident in Fig. 4, pretreatment of monocytes with the transcriptional inhibitor actinomycin D effectively inhibited in a concentration-dependent manner the ability of BCG to induce IL-8 secretion. These results indicate that BCG up-regulates IL-8 secretion primarily through transcriptional upregulation of the IL-8 gene in monocytes. To investigate the mechanism underlying IL-8 gene activation in
FIG. 2. BCG-induced IL-8 secretion is dependent upon intracellular Ca 2⫹. (A) Human monocytes (10 5/well) were incubated with medium alone or with various concentrations of EDTA for 30 min prior to the addition of BCG (m.o.i. ⫽ 3) for 24 h at 37°C. Culture supernatants were assayed for IL-8 content as above. The results represent the mean ⫾ SEM for four donors. (B) Human monocytes were incubated with medium alone or with 3 mM EDTA and various concentrations of BAPTA/AM for 30 min at 37°C. BCG was added and the cells were incubated for an additional 24 h at 37°C. Cell culture supernatants were collected and analyzed for IL-8 measurement by ELISA. Data are expressed as the mean ⫾ SEM of five separate experiments. Reduction of BCG-induced IL-8 secretion after the addition of 10 and 30 M BAPTA/AM plus 3 mM/ml EDTA is statistically significant (*P ⬍ 0.01).
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FIG. 3. Effect of W-7 on IL-8 production by BCG-infected human monocytes. Human monocytes (10 5/well) were incubated with medium alone or increasing concentrations of W-7 for 30 min at 37°C. After this incubaction, BCG was added and the cells were incubated for an additional 24 h at 37°C. Cell culture supernatants were collected and analyzed for IL-8 measurement by ELISA. Shown are the mean ⫾ SEM of five independent experiments. Results in parentheses indicate the percentage of inhibition by W-7 with respect to BCG cultures.
response to BCG, we analyzed the role of the transcriptional regulator NF-B in the control of IL-8 gene expression in human monocytes. In these experiments we pretreated monocytes with sulfasalazine, a well-described inhibitor of NF-B activity. As shown in Fig. 5A, the effect of sulfasalazine was dose-dependent, first evident at 0.2 mM, and maximal at 2 mM (92.5% inhibition). In order to confirm these results, in this work we used another specific inhibitor of NF-B activity (curcumin) with a different mechanism of action. Our results indicate that curcumin significantly diminished the effect of BCG as well (Fig. 5B), suggesting that activation of NF-B is required for the BCG-induced up-regulation of IL-8 expression. To investigate whether the effects of curcumin were exerted at the level of gene expression, total RNA was extracted from monocytes cultured with medium (control), stimulated with BCG, alone or in combination with curcumin, and Northern blot analysis was performed. As depicted in Fig. 6, control monocytes ex-
pressed undetectable or very low constitutive levels of IL-8 mRNA that was markedly up-regulated by infection with BCG. Addition of curcumin significantly diminished IL-8 mRNA expression in BCG-infected monocytes. Curcumin alone did not affect the mRNA levels of the housekeeping gene GAPDH. DISCUSSION An increasing body of evidence suggests that production of IL-8 in response to mycobacterial antigens can contribute to the initial neutrophilic inflammatory response in human tuberculosis. However, despite the evidence of its important role in the pathophysiology of tuberculosis, the intracellular mechanisms leading to mycobacteria-induced IL-8 secretion have not been identified. The goal of this investigation was to characterize part of the molecular mechanism of M. bovis BCG-induced IL-8 secretion. We found a significant release of IL-8 by human monocytes infected with M.
REGULATION OF IL-8 SECRETION IN BCG-INFECTED HUMAN MONOCYTES
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FIG. 4. BCG-induced IL-8 secretion results from transcriptional regulation of the IL-8 gene. Human monocytes (10 5/well) were incubated with medium alone or with various concentrations of actinomycin D for 30 min at 37°C prior to the addition of BCG (m.o.i. ⫽ 3) for 24 h at 37°C. The culture supernatants were assayed for IL-8 content by ELISA. Results are represented as the mean ⫾ SEM of eight separate experiments. The percentage in parentheses indicates inhibition in the presence of actinomycin D compared with BCG cultures which did not receive actinomycin D.
bovis BCG. These results are in agreement with earlier studies demonstrating IL-8 secretion by human alveolar cells in response to M. bovis (24, 25) and, at the same time, are consistent with the findings that such IL-8 secretion from infected monocytes varied with m.o.i. and in a time-dependent manner (26). In addition, the effect of M. bovis BCG on IL-8 secretion cannot be attributed to the presence of bacterial LPS contamination because polymyxin B treatment failed to inhibit the BCG-induced IL-8 secretion. Recent studies have indicated that phagocytosis of mycobacteria by human monocytes results in secretion of TNF-␣ (19, 20). In addition, it has been shown that in LPS-stimulated human cells, IL-8 secretion was reduced with anti-TNF-␣ Ab (27). Our results, however, showed that in human monocytes infected with M. bovis BCG, production of IL-8 was not dependent on production of TNF-␣, suggesting that IL-8 is part of the initial inflammatory response rather than a component
of the secondary cytokine cascade. The results presented here correlate well with an earlier study reporting that TNF-␣ did not affect IL-8 production by human monocyte– endothelial cell interactions (28). Moreover, our data demonstrated that BCG-induced IL-8 production is mediated by stimulating transcription of the IL-8 gene, as evidenced by the inhibition of IL-8 secretion in the presence of actinomycin D. These data are in agreement with the finding that secretion of IL-8 from monocytes results from transcriptional upregulation of the IL-8 gene (29). Given that intracellular Ca 2⫹ flux is an important second messenger that mediates enhancement of IL-8 secretion in human neutrophils (23) and endothelial cells (30, 31), it is possible that in our system, signaling through Ca 2⫹ mediates BCG-induced secretion of IL-8. In support of this premise, our results demonstrated that complete inhibition of the effects of M. bovis BCG on IL-8 synthesis required both EDTA and BAPTA/
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FIG. 5. Inhibitors of the transcription factor NF-B abrogate the BCG-mediated activation of IL-8 secretion. Human monocytes (10 5/well) were incubated with medium alone or increasing concentrations of sulfasalazine (Sulfasal) (A) or curcumin (Cur) (B) for 30 min at 37°C. BCG was added and the cells were incubated for an additional 24 h. Media samples were collected and analyzed for IL-8 content by ELISA. Results are represented as the mean ⫾ SEM of eight separate experiments. An asterisk indicates P ⬍ 0.01 compared with the value obtained with BCG alone.
REGULATION OF IL-8 SECRETION IN BCG-INFECTED HUMAN MONOCYTES
FIG. 6. Curcumin inhibits IL-8 mRNA expression in BCG-infected monocytes. Total RNA was extracted from monocytes cultured with medium alone (lane 1) or infected with BCG at a m.o.i. of 3 alone (lane 2) or in combination with 20 M curcumin (Cur) (lane 3) and analyzed by Northern blotting. Equal loading of total RNA was ensured by reprobing the same Northern blots for the housekeeping gene GAPDH. The blot shown is representative of three different experiments.
AM. These results indicate that mobilization of intracellular Ca 2⫹and influx of extracellular Ca 2⫹are each required for effective activation of IL-8 gene transcription. These results were further confirmed as the specific calmodulin inhibitor W-7 abrogated IL-8 secretion from human monocytes, suggesting that the effect of M. bovis BCG on IL-8 synthesis requires the activation of calmodulin. Taken together, these data strongly implicate the involvement of an intracellular Ca 2⫹/calmodulin-dependent signaling pathway in M. bovis BCG-induced secretion of IL-8. On the other hand, expression of the IL-8 gene has been reported to be dependent upon activation of NF-B by bacterial pathogens such as H. pylori (18). Our study, however, is the first to demonstrate that M. bovis BCG-mediated IL-8 secretion is NF-B-dependent, since this secretion was blocked by using sulfasalazine, a well-described inhibitor of NF-B activity. We confirmed the activation of this important proinflammatory transcription factor as evidenced by the inhibition of NF-B activation in the presence of the antioxidant curcumin, which has a different mechanism of action. Together, these results suggest that BCG-induced IL-8 secretion appears to be mediated, at least in part, by NF-B. Our data are consistent with the observation that mycobacterial antigens activate NF-B in human cells (32–34). NF-B has been shown to exhibit synergistic and cooperative binding with NF-IL-6 (35, 36) and AP-1 (37) to induce IL-8 secretion. Therefore, our data do not allow us to exclude the possibility that NF-B synergizes with other nuclear factors to regulate IL-8 synthesis during stimulation with BCG. We are currently investigating this possibility. In conclusion, we demonstrated that human monocytes infected in vitro with M. bovis BCG produce IL-8 through up-regulation of its gene. Furthermore, our results demonstrate that intracellular Ca 2⫹, calmodulin, and NF-B are critical for the induction of IL-8 by human monocytes stimulated with M. bovis BCG.
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These findings suggest that NF-B activation and mobilization of Ca 2⫹ have a central role in regulating the production of IL-8 by M. bovis BCG. Further experimental work is needed to know whether the effect of intracellular Ca 2⫹, calmodulin, and NF-B on M. bovis BCG–induced IL-8 production may represent a significant regulatory mechanism in vivo. However, these data may represent an important regulatory mechanism during the immune response to BCG, since IL-8 is a known chemotactic agent for immune cells in humans and hence may be of central importance in granuloma formation. ACKNOWLEDGMENTS We thank Dr. J. Ruiz-Puente (Instituto Nacional de Higiene, Me´xico) for providing the M. bovis BCG; P.M.S. is an EDI, COFAA, and SNI fellow. This work was financed in part by Grant CGPI 200479 to P.M.S.
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Lin, Y., Zhang, M., and Barnes, P. F., Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis. Infect. Immun. 66, 1121–1126, 1998. Standiford, T. J., Kunkel, S. L., Basha, M. A., Chensue, S. W., Lynch, J. P., Toews, G. B., Westwick, J., and Strieter, R. M., Interleukin-8 gene expression by a pulmonary epithelial cell line: A model for cytokine networks in the lung. J. Clin. Invest. 86, 1945–1951, 1990. Lukacs, N. W., Strieter, R. M., Ellner, V., Evanoff, H. L., Burdick, M. D., and Kunkel, S. L., Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte:endothelial cell interactions. Blood 86, 2767–2773, 1995. Linevsky, K. J., Pothoulakis, C., Keates, S., Warny, M., Keates, A. C., LaMont, J. T., and Kelly, C. P., IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes. Am. J. Physiol. 273, G1333–1339, 1997. Volk, T., Hensel, M., Mading, K., Egerer, K., and Kox W. J., Intracellular Ca 2⫹ dependence of nitric oxide mediated enhancement of interleukin-8 secretion in human endothelial cells. FEBS Lett. 415, 169 –171, 1997. Dolmetsch, R. E., Xu, K., and Lewis, R. S., Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392, 933–936, 1998. Wickremasinghe, M. I., Thomas, L. H., and Friedland, J. S., Pulmonary epithelial cells are a source of IL-8 in the response to Mycobacterium tuberculosis: Essential role of IL-1 from infected monocytes in a NF-B-dependent network. J. Immunol. 163, 3936 –3947, 1999. Toossi, Z., Xia, L., Wu, M., and Salvekar, A., Transcriptional activation of HIV by Mycobacterium tuberculosis in human monocytes. Clin. Exp. Immunol. 117, 324 –330, 1999. Ott, M., Lovett, J. L., Mueller, L., and Verdin, E., Superinduction of IL-8 in T cells by HIV-1 tat protein is mediated through NF-B factors. J. Immunol. 160, 2872–2880, 1998. Mastronarde, J. G., Monick, M. M., and Hunninghake, G. W., Oxidant tone regulates IL-8 production in epithelium. Am. J. Respir. Cell Mol. Biol. 13, 237–244, 1995. Mastronarde, J. G., He, B., Monick, M. M., Mukaida, N., Matsushima K., and Hunninghake, G. W., Induction of interleukin (IL)-8 gene expression by respiratory syncytial virus involves activation of nuclear factor (NF)-B and NF-IL6. J. Infect. Dis. 174, 262–267, 1996. Yasumoto, K., Okamoto, S., Mukaida, N., Mai, M., and Matsushima, K., Tumor necrosis factor ␣ and interferon ␥ synergistically induce interleukin-8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-B-like binding sites of the interleukin 8 gene. J. Biol. Chem. 267, 22506 –22511, 1992.
Roles of Intracellular Calcium and NF-B in the Bacillus CalmetteGue´rin-Induced Secretion of Interleukin-8 from Human Monocytes Patricia Me´ndez-Samperio, Janet Palma, and Abraham Va´zquez Departamento de Inmunologı´a, Escuela Nacional de Ciencias Biolo´gicas, IPN, Carpio y Plan de Ayala, 11340 Me´xico, D.F. Me´xico Received December 27, 2000; accepted May 29, 2001
Interleukin (IL)-8 secretion contributes to the early host response against mycobacterial infection by increasing local inflammation and recruiting professional phagocytes. Because the mechanisms through which Mycobacterium bovis Calmette-Gue´rin (BCG) induces IL-8 secretion are unknown, the aim of the present study was to characterize the nature of IL-8 production induced by BCG in human monocytes. In this study, we found that the induction of IL-8 synthesis was dose- and time-dependent after stimulation with BCG. This IL-8 secretion was not attributed to LPS contamination or the presence of TNF-␣. We also determined that BCG-induced IL-8 secretion occurs through a mechanism that requires intracellular calcium and likely involves a calmodulin-sensitive step. Interestingly, BCG-induced secretion of IL-8 from human monocytes resulted from transcriptional up-regulation of the IL-8 gene. Moreover, we present evidence that BCG activates nuclear translocation of the transcription factor NF-B, since pretreatment of monocytes with sulfasalazine, a inhibitor of NF-B activity, blocked the ability of BCG to induce IL-8 secretion in a dose-dependent manner, producing 92.5% inhibition at a concentration of 2 mM. These results were further supported by the fact that treatment of cells with curcumin, another well-described inhibitor of NF-B activity with a different mechanism of action, significantly diminished the effect of BCG on IL-8 secretion. Together, these studies are the first to demonstrate that BCG-induced IL-8 secretion by human monocytes appears to be mediated by intracellular Ca 2ⴙ and is NF-B-dependent and at the same time suggest that production of IL-8 in response to M. bovis BCG can contribute to the initial local and systemic inflammatory response in human tuberculosis. © 2001 Academic Press
INTRODUCTION
to Mycobacterium tuberculosis infection, T cells and macrophages are recruited to the site of infection, resulting in tissue inflammation. The mechanism for recruitment of these cells is involving chemokines. Interleukin (IL)-8, 1 first isolated from monocytes as a neutrophil attractant (3, 4), is a member of the CXC chemokine subfamily that is also chemotactic for T lymphocytes (5) and monocytes (6). IL-8 release from monocyte/macrophages is enhanced after exposure to M.tuberculosis and its cell wall component (7) and following phagocytosis of M.tuberculosis by human monocytic cell lines (8). IL-8, by binding to either of its two specific receptors called CXCR-1 and CXCR-2 (9), induces a variety of cell type-specific responses, including enhanced killing of various microorganisms, such as M. tuberculosis. Circulating levels of IL-8 in plasma of patients with pulmonary tuberculosis have been found to be increased with time as a result of anti-tuberculosis therapy, and high admission plasma levels of IL-8 are of clinical prognostic significance in tuberculosis (10). IL-8 secretion is mainly regulated at the transcriptional level (11). The promoter of the IL-8 gene contains binding sites for a number of important transcription factors, including nuclear factor-B (NF-B) (12–14). NF-B proteins are normally retained in the cytoplasm bound to the IB␣ family of inhibitory molecules (15). Following cellular activation, IB␣ is phosphorylated (16) on specific serine residues and rapidly degraded (17), allowing NF-B to pass into the nucleus, where it can bind the IL-8 B-binding site. Recently, it has been demonstrated that IL-8 secretion induced by bacterial pathogens such as Helicobacter pylori requires activation of the transcription factor NF-B (18). However, whether induction of IL-8 from human monocytes by M. bovis BCG is mediated through NF-B factor has not been reported. In this report, we demonstrated that human monocytes pro1
Tuberculosis is a major health problem which is responsible for almost 3 million deaths annually worldwide (1,2 ). During the course of the immune response
Abbreviations used: BAPTA/AM, 1,2-bis(2-aminophenoxy)ethaneN,N,N⬘,N⬘-tetraacetic acid tetra(acetoxymethyl)esther; BCG, Mycobacterium bovis bacillus Calmette-Gue´rin; IL-8, interleukin-8; M.O.I., multiplicity of infection.
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0008-8749/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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duced significant IL-8 levels following stimulation with M. bovis BCG. This increased IL-8 secretion was mediated through a mechanism requiring intracellular Ca 2⫹ and calmodulin activation. In addition, NF-B inhibitors blocked the ability of M. bovis BCG to induce IL-8 secretion. These findings should increase our understanding of the regulation of IL-8 synthesis in mycobacterial infections, and they suggest that regulated production of IL-8 after stimulation with BCG can contribute to host immune defenses by attracting monocytes, lymphocytes, and neutrophils. MATERIALS AND METHODS Reagents EDTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid tetra(acetoxymethyl) esther (BAPTA/AM), sulfasalazine, curcumin, lipopolysaccharide (LPS) from Escherichia coli serotype 0128:B12, polymyxin B sulfate, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride) were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-TNF-␣ (AB 210-NA) was purchased from R&D Systems (Minneapolis, MN). Live M. bovis BCG, Danish Strain 1331, was kindly supplied by Dr. J. Ruiz-Puente (Instituto Nacional de Higiene, Me´xico). BCG was grown at 37°C in Sauton medium using stationary tissue culture flasks. Mycobacterial viability, as assessed by the number of CFU, was 60 to 70%. Cell Culture Whole heparinized blood was obtained from healthy consenting volunteers. Peripheral blood mononuclear cells (PBMC) were separated out by density gradient centrifugation on Ficoll–Histopaque 1077 (Sigma, St. Louis, MO). The PBMC were enriched for monocytes by adherence on petri dishes at 37°C for 1 h. Then, plates were vortexed and nonadherent cells were removed by vigorous washing with RPMI 1640 (Gibco, BRL). Adherent cells were then cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FCS overnight and monolayers were washed again before infection with BCG. More than 85% of cells purified by this technique were determined to be monocytes. Cells were infected in triplicate with BCG. The multiplicity of infection (m.o.i.) ranged from 0.1 to 3 live M. bovis BCG organisms per cell. After infection, cells were washed twice with warm RPMI to remove extracellular bacteria and
TABLE 1 Inhibition of TNF-␣-Mediated Toxicity by Anti-TNF-␣ Antibody Stimulus TNF-␣ TNF-␣ TNF-␣ TNF-␣ TNF-␣ TNF-␣ TNF-␣
⫹ ⫹ ⫹ ⫹
anti-TNF-␣ anti-TNF-␣ anti-TNF-␣ Control IgG
Concentration
LDH-release units
0.1ng/ml 1.0ng/ml 3.0ng/ml 0.5g/ml 1.0g/ml 5.0g/ml 5.0g/ml
457 926 1783 1702 1050 482 1694
Note. L929 cells (10 4) were cultured for 1 h in culture medium containing either increasing concentrations of anti-TNF-␣ or isotype control IgG and then incubated with TNF-␣ (3 ng/ml) for 24 h. Cytolysis was assessed by measuring LDH release. Similar results were observed in three additional experiments.
were then cultured in fresh RPMI at 37°C in 5% CO 2. In some experiments, cultured monocytes were preincubated with medium or various inhibitors for 30 min prior to the addition of BCG. When indicated, LPS was used at a final concentration of 10 g/ml. To some cultures neutralizing antibody to TNF-␣ was added. Culture supernatants for the detection of IL-8 were harvested, centrifuged to remove any debris, and then stored in frozen aliquots at ⫺20°C. Measurement of IL-8 Concentrations of IL-8 in culture supernatants were determined by using the Biotrak human IL-8 enzymelinked immunosorbent assay (ELISA) system (Amersham, Aylesbury, UK) according to the manufacturer’s instructions. The minimum detectable dose of IL-8 is 0.5 pg/well. IL-8 concentrations are expressed as picograms per 10 5 cells. Northern Blot Analysis of IL-8 mRNA Expression Monocytes were infected with BCG at m.o.i. ⫽ 3 in the presence and the absence of the NF-B inhibitor curcumin (20 M). Cells were then lysed in Trizol (Life Technologies), and total RNA was purified according to the manufacturer’s instructions. After quantification on spectrophotometer, 20 g of total RNA from each sample was electrophoresed in a 1.2% agarose gel and transferred to nitrocellulose membranes. The blots were hybridized with a 32P-labeled cDNA probe for human IL-8. A cDNA probe for chicken glyceraldehyde-
FIG. 1. Induction of IL-8 by bacillus Calmette-Gue´rin (BCG) in human monocytes. (A) Human peripheral blood-derived monocytes (10 5/well) were infected with BCG at m.o.i. ⫽ 3 (*), m.o.i. ⫽ 1 (䊐), or m.o.i. ⫽ 0.1 (⌬) or incubated with medium alone (f). Cell culture supernatants were harvested at 0, 2, 8, and 24 h following infection and IL-8 protein was measured by ELISA as described under Materials and Methods. Results are the mean ⫾ SEM of four independent experiments. (B) Human monocytes were infected with BCG (m.o.i. ⫽ 3) or stimulated with LPS (10 g/ml), in the presence or the absence of polymyxin B (5 g/ml). After an 24-h incubation, IL-8 levels were measured in cell culture supernatants by ELISA. Data are expressed as the mean ⫾ SEM of five separate experiments.
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3-phosphate dehydrogenase (GAPDH) mRNA was used as a control to determine loading equivalence. Analysis and Presentation of Data Results of ELISAs are expressed as pg/10 5 cells, means ⫾ SEM of at least three independent experiments. Statistical analysis was performed by using Student’s t test. A P value less than 0.01 was considered statistically significant. RESULTS Human Monocytes Produce IL-8 in Response to BCG Stimulation We first examined the IL-8 production of human monocytes following stimulation with BCG at m.o.i. ⫽ 3, 1, or 0.1. IL-8 secretion was clearly detected in a dose-dependent manner after stimulation with BCG, and a maximal production was reached with m.o.i. ⫽ 3 of BCG (Fig. 1A). No IL-8 secretion was observed in the absence of stimulation. The IL-8 response of BCGinfected cell cultures increased over time to reach a maximum 24 h after infection (Fig. 1A). Parallel experiments were performed to measure IL-8 secretion by human monocytes infected with BCG or cultured with lipopolysaccharide. As shown in Fig. 1B, BCG induced significantly higher production of IL-8 than lipopolysaccharide (P ⬍ 0.01). The IL-8 production experiments were repeated with BCG or LPS in the presence of polymyxin B (5 g/ml). As shown in Fig. 1B, polymyxin B was able to inhibit LPS-induced IL-8 secretion. In contrast, polymyxin B treatment had no significant inhibitory effect on the IL-8 levels stimulated by BCG (Fig. 1B). These data exclude the possibility that LPS contamination is responsible for the effects observed with M. bovis BCG. On the other hand, it is well known that human cells produce TNF-␣ in response to mycobacterial antigens (19, 20). In addition, it has been demonstrated that, in pulmonary epithelial cells, TNF-␣ stimulates IL-8 production (21). Therefore, we evaluated the potential role of TNF-␣ in the capacity of human monocytes infected with BCG to produce IL-8 by using neutralizing antibodies to TNF-␣. In three experiments, anti-TNF-␣ antibodies had no significant effect on IL-8 production (data not shown). In contrast, addition of these antibodies significantly reduced TNF␣-mediated cytotoxicity (Table 1).
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BCG-Induced IL-8 Secretion Requires Mobilization of Intracellular Ca 2⫹ and Activation of Calmodulin Because Ca 2⫹ ionophores have been shown to elicit IL-8 secretion from monocytes (22), and an intracellular Ca 2⫹ flux is associated with induction of IL-8 secretion by certain signals, such as IL-1 (23), we next sought to determine whether the effect of BCG on IL-8 secretion required mobilization of intracellular Ca 2⫹. We used EDTA to deplete extracellular Ca 2⫹ and used the cell-permeable agent BAPTA/AM to chelate releasable intracellular stores of Ca 2⫹. As evident in Fig. 2A, pretreatment of monocytes with EDTA effectively inhibited in a concentration-dependent manner, first evident at 0.1 mM and maximal at 3 mM (42.5% inhibition), the level of BCG-induced IL-8 secretion. When monocytes were incubated in EDTA and BAPTA/AM, the ability of BCG to activate IL-8 secretion was completely inhibited, in a dose-dependent manner. Maximal inhibition was reached with 30 M BAPTA/AM ⫹ 3 mM EDTA (96.8% inhibition) and 32.5% inhibition was obtained with 0.3 M BAPTA/AM ⫹ 3 mM EDTA (Fig. 2B). Because a major effect of intracellular Ca 2⫹ flux is the activation of calmodulin, we next examined whether the inhibition of calmodulin could diminish BCG-induced secretion of IL-8. W-7 is a potent and specific inhibitor of calmodulin activity and we observed that treatment with this agent abrogated BCGmediated IL-8 secretion by ⬃75% at 20 M compared with control cultures (Fig. 3). These results indicate that both intracellular Ca 2⫹ and calmodulin are required for BCG-induced IL-8 secretion in human monocytes. BCG-Induced Secretion of IL-8 from Human Monocytes Results from Transcriptional Up-regulation of the IL-8 Gene Next, we determined whether transcriptional upregulation of the IL-8 gene is the predominant mechanism through which BCG induces IL-8 secretion in human monocytes. As evident in Fig. 4, pretreatment of monocytes with the transcriptional inhibitor actinomycin D effectively inhibited in a concentration-dependent manner the ability of BCG to induce IL-8 secretion. These results indicate that BCG up-regulates IL-8 secretion primarily through transcriptional upregulation of the IL-8 gene in monocytes. To investigate the mechanism underlying IL-8 gene activation in
FIG. 2. BCG-induced IL-8 secretion is dependent upon intracellular Ca 2⫹. (A) Human monocytes (10 5/well) were incubated with medium alone or with various concentrations of EDTA for 30 min prior to the addition of BCG (m.o.i. ⫽ 3) for 24 h at 37°C. Culture supernatants were assayed for IL-8 content as above. The results represent the mean ⫾ SEM for four donors. (B) Human monocytes were incubated with medium alone or with 3 mM EDTA and various concentrations of BAPTA/AM for 30 min at 37°C. BCG was added and the cells were incubated for an additional 24 h at 37°C. Cell culture supernatants were collected and analyzed for IL-8 measurement by ELISA. Data are expressed as the mean ⫾ SEM of five separate experiments. Reduction of BCG-induced IL-8 secretion after the addition of 10 and 30 M BAPTA/AM plus 3 mM/ml EDTA is statistically significant (*P ⬍ 0.01).
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FIG. 3. Effect of W-7 on IL-8 production by BCG-infected human monocytes. Human monocytes (10 5/well) were incubated with medium alone or increasing concentrations of W-7 for 30 min at 37°C. After this incubaction, BCG was added and the cells were incubated for an additional 24 h at 37°C. Cell culture supernatants were collected and analyzed for IL-8 measurement by ELISA. Shown are the mean ⫾ SEM of five independent experiments. Results in parentheses indicate the percentage of inhibition by W-7 with respect to BCG cultures.
response to BCG, we analyzed the role of the transcriptional regulator NF-B in the control of IL-8 gene expression in human monocytes. In these experiments we pretreated monocytes with sulfasalazine, a well-described inhibitor of NF-B activity. As shown in Fig. 5A, the effect of sulfasalazine was dose-dependent, first evident at 0.2 mM, and maximal at 2 mM (92.5% inhibition). In order to confirm these results, in this work we used another specific inhibitor of NF-B activity (curcumin) with a different mechanism of action. Our results indicate that curcumin significantly diminished the effect of BCG as well (Fig. 5B), suggesting that activation of NF-B is required for the BCG-induced up-regulation of IL-8 expression. To investigate whether the effects of curcumin were exerted at the level of gene expression, total RNA was extracted from monocytes cultured with medium (control), stimulated with BCG, alone or in combination with curcumin, and Northern blot analysis was performed. As depicted in Fig. 6, control monocytes ex-
pressed undetectable or very low constitutive levels of IL-8 mRNA that was markedly up-regulated by infection with BCG. Addition of curcumin significantly diminished IL-8 mRNA expression in BCG-infected monocytes. Curcumin alone did not affect the mRNA levels of the housekeeping gene GAPDH. DISCUSSION An increasing body of evidence suggests that production of IL-8 in response to mycobacterial antigens can contribute to the initial neutrophilic inflammatory response in human tuberculosis. However, despite the evidence of its important role in the pathophysiology of tuberculosis, the intracellular mechanisms leading to mycobacteria-induced IL-8 secretion have not been identified. The goal of this investigation was to characterize part of the molecular mechanism of M. bovis BCG-induced IL-8 secretion. We found a significant release of IL-8 by human monocytes infected with M.
REGULATION OF IL-8 SECRETION IN BCG-INFECTED HUMAN MONOCYTES
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FIG. 4. BCG-induced IL-8 secretion results from transcriptional regulation of the IL-8 gene. Human monocytes (10 5/well) were incubated with medium alone or with various concentrations of actinomycin D for 30 min at 37°C prior to the addition of BCG (m.o.i. ⫽ 3) for 24 h at 37°C. The culture supernatants were assayed for IL-8 content by ELISA. Results are represented as the mean ⫾ SEM of eight separate experiments. The percentage in parentheses indicates inhibition in the presence of actinomycin D compared with BCG cultures which did not receive actinomycin D.
bovis BCG. These results are in agreement with earlier studies demonstrating IL-8 secretion by human alveolar cells in response to M. bovis (24, 25) and, at the same time, are consistent with the findings that such IL-8 secretion from infected monocytes varied with m.o.i. and in a time-dependent manner (26). In addition, the effect of M. bovis BCG on IL-8 secretion cannot be attributed to the presence of bacterial LPS contamination because polymyxin B treatment failed to inhibit the BCG-induced IL-8 secretion. Recent studies have indicated that phagocytosis of mycobacteria by human monocytes results in secretion of TNF-␣ (19, 20). In addition, it has been shown that in LPS-stimulated human cells, IL-8 secretion was reduced with anti-TNF-␣ Ab (27). Our results, however, showed that in human monocytes infected with M. bovis BCG, production of IL-8 was not dependent on production of TNF-␣, suggesting that IL-8 is part of the initial inflammatory response rather than a component
of the secondary cytokine cascade. The results presented here correlate well with an earlier study reporting that TNF-␣ did not affect IL-8 production by human monocyte– endothelial cell interactions (28). Moreover, our data demonstrated that BCG-induced IL-8 production is mediated by stimulating transcription of the IL-8 gene, as evidenced by the inhibition of IL-8 secretion in the presence of actinomycin D. These data are in agreement with the finding that secretion of IL-8 from monocytes results from transcriptional upregulation of the IL-8 gene (29). Given that intracellular Ca 2⫹ flux is an important second messenger that mediates enhancement of IL-8 secretion in human neutrophils (23) and endothelial cells (30, 31), it is possible that in our system, signaling through Ca 2⫹ mediates BCG-induced secretion of IL-8. In support of this premise, our results demonstrated that complete inhibition of the effects of M. bovis BCG on IL-8 synthesis required both EDTA and BAPTA/
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FIG. 5. Inhibitors of the transcription factor NF-B abrogate the BCG-mediated activation of IL-8 secretion. Human monocytes (10 5/well) were incubated with medium alone or increasing concentrations of sulfasalazine (Sulfasal) (A) or curcumin (Cur) (B) for 30 min at 37°C. BCG was added and the cells were incubated for an additional 24 h. Media samples were collected and analyzed for IL-8 content by ELISA. Results are represented as the mean ⫾ SEM of eight separate experiments. An asterisk indicates P ⬍ 0.01 compared with the value obtained with BCG alone.
REGULATION OF IL-8 SECRETION IN BCG-INFECTED HUMAN MONOCYTES
FIG. 6. Curcumin inhibits IL-8 mRNA expression in BCG-infected monocytes. Total RNA was extracted from monocytes cultured with medium alone (lane 1) or infected with BCG at a m.o.i. of 3 alone (lane 2) or in combination with 20 M curcumin (Cur) (lane 3) and analyzed by Northern blotting. Equal loading of total RNA was ensured by reprobing the same Northern blots for the housekeeping gene GAPDH. The blot shown is representative of three different experiments.
AM. These results indicate that mobilization of intracellular Ca 2⫹and influx of extracellular Ca 2⫹are each required for effective activation of IL-8 gene transcription. These results were further confirmed as the specific calmodulin inhibitor W-7 abrogated IL-8 secretion from human monocytes, suggesting that the effect of M. bovis BCG on IL-8 synthesis requires the activation of calmodulin. Taken together, these data strongly implicate the involvement of an intracellular Ca 2⫹/calmodulin-dependent signaling pathway in M. bovis BCG-induced secretion of IL-8. On the other hand, expression of the IL-8 gene has been reported to be dependent upon activation of NF-B by bacterial pathogens such as H. pylori (18). Our study, however, is the first to demonstrate that M. bovis BCG-mediated IL-8 secretion is NF-B-dependent, since this secretion was blocked by using sulfasalazine, a well-described inhibitor of NF-B activity. We confirmed the activation of this important proinflammatory transcription factor as evidenced by the inhibition of NF-B activation in the presence of the antioxidant curcumin, which has a different mechanism of action. Together, these results suggest that BCG-induced IL-8 secretion appears to be mediated, at least in part, by NF-B. Our data are consistent with the observation that mycobacterial antigens activate NF-B in human cells (32–34). NF-B has been shown to exhibit synergistic and cooperative binding with NF-IL-6 (35, 36) and AP-1 (37) to induce IL-8 secretion. Therefore, our data do not allow us to exclude the possibility that NF-B synergizes with other nuclear factors to regulate IL-8 synthesis during stimulation with BCG. We are currently investigating this possibility. In conclusion, we demonstrated that human monocytes infected in vitro with M. bovis BCG produce IL-8 through up-regulation of its gene. Furthermore, our results demonstrate that intracellular Ca 2⫹, calmodulin, and NF-B are critical for the induction of IL-8 by human monocytes stimulated with M. bovis BCG.
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These findings suggest that NF-B activation and mobilization of Ca 2⫹ have a central role in regulating the production of IL-8 by M. bovis BCG. Further experimental work is needed to know whether the effect of intracellular Ca 2⫹, calmodulin, and NF-B on M. bovis BCG–induced IL-8 production may represent a significant regulatory mechanism in vivo. However, these data may represent an important regulatory mechanism during the immune response to BCG, since IL-8 is a known chemotactic agent for immune cells in humans and hence may be of central importance in granuloma formation. ACKNOWLEDGMENTS We thank Dr. J. Ruiz-Puente (Instituto Nacional de Higiene, Me´xico) for providing the M. bovis BCG; P.M.S. is an EDI, COFAA, and SNI fellow. This work was financed in part by Grant CGPI 200479 to P.M.S.
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