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The Journal of Immunology, 2007, 178: 3767-3776.
Copyright © 2007 by The American Association of Immunologists, Inc.

Monocyte-Dependent Fibroblast CXCL8 Secretion Occurs in Tuberculosis and Limits Survival of Mycobacteria within Macrophages1

Cecilia M. O’Kane*, Joseph J. Boyle{dagger}, Donna E. Horncastle{dagger}, Paul T. Elkington* and Jon S. Friedland2,*

* Department of Infectious Diseases Immunity and {dagger} Department of Histopathology, Imperial College, Hammersmith Campus, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CXCL8 is a chemokine that is implicated in the formation of tuberculous (TB) granulomas and in immunity to Mycobacterium tuberculosis (Mtb). Fibroblast chemokine secretion is important for modulating inflammatory responses in chronic lung disease and inflammatory arthritis but has not been investigated in the pathophysiology of TB. In this study, we used a cellular model to examine monocyte/macrophage-dependent stimulation of fibroblasts by Mtb in the regulation of chemokine secretion, particularly that of CXCL8. Human lung fibroblasts grown in collagen were stimulated with conditioned medium from Mtb-infected monocytes (CoMTb). CoMTb-induced prolonged dose-dependent, p38-mediated expression of stable CXCL8 mRNA by fibroblasts accompanied by a >10-fold increase in CXCL8 secretion (487 ± 88 ng/ml vs 48.6 ± 34 ng/ml in controls) at 120 h. Fibroblasts strongly expressed CXCL8 in vivo in human TB granulomas. Inhibition of TNF-{alpha} or IL-1 in CoMTb abrogated the induction of CXCL8 at a pretranscriptional level. CXCL8 secretion was NF-{kappa}B, C/EBP, and JNK dependent. Sustained NF-{kappa}B activation was demonstrated beyond 24 h in response to CoMTb. Exogenous CXCL8 reduced the survival of Mtb within macrophages, and inhibition of CXCL8 was associated with intracellular mycobacterial proliferation. These data show that fibroblasts have a previously unrecognized role in modulating inflammation in TB by their CXCL8-dependent contribution to cell recruitment and mycobacterial killing within the granuloma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis (TB)3 is characterized by granuloma formation. Regulation of granuloma formation is poorly understood, although TNF-{alpha} has a key role in animal and human studies (1, 2, 3, 4, 5). Chemokines, small (8–10kDa) peptides involved in cell recruitment to sites of inflammation, are implicated in granuloma formation and maintenance (6). The prototypic chemokine CXCL8 has monocyte, lymphocyte, and neutrophil chemoattractant properties (7, 8, 9). CXCL8 is necessary for granuloma formation in the rabbit model of TB (8). In humans a CXCL8 gene polymorphism is associated with susceptibility to TB (10), and decreased CXCL8 secretion occurs in HIV-infected patients with military TB (11).

CXCL8 is not readily detected in normal tissue because of its unstable mRNA but can be induced upon stimulation with pathogens, toxins, or cytokines (12). Regulation is cell and stimulus-specific. All three MAPK pathways may regulate CXCL8 secretion. p38 MAPK stabilizes CXCL8 mRNA (12, 13), JNK acts pretranscriptionally (12, 14), and ERK is occasionally described in CXCL8 regulation (15). Downstream, the transcription factors NF-{kappa}B, C/EBP, and AP-1 interact variably in the activation of the CXCL8 promoter (12).

CXCL8 secretion in TB-infected tissues is largely attributed to leukocytes. Monocytes secrete CXCL8 in response to Mycobacterium tuberculosis (Mtb) or cytokines (16, 17, 18). However, we have previously also described the pulmonary epithelium as a source of CXCL8 in TB (19). The role of lung stromal cells in directing inflammatory responses is increasingly recognized. Fibroblasts and airway smooth muscle cells act as a major source of chemokines in chronic obstructive pulmonary disease, pulmonary fibrosis, and lung infection (20, 21, 22, 23). In extrapulmonary sites such as rheumatoid joints, sustained fibroblast chemokine secretion results in the retention of leukocytes in joints and in persistent inflammation (24). Fibroblast chemokine secretion in TB has not been investigated. Fibroblasts at the periphery of the granuloma are ideally poised to direct cell recruitment into the granuloma.

CXCL8 in TB-infected tissue may have additional immune effects on chemotaxis. It is antiapoptotic (25) and proangiogenic (26). CXCL8 has also been reported to affect intracellular pathogen survival, promoting the survival of Chlamydia pneumoniae within neutrophils (25) but increasing the killing of neutrophil-phagocytosed avirulent mycobacteria (27). However, the neutrophil is not the target cell of Mtb and there are no data suggesting that CXCL8 affects the survival of Mtb.

This study investigated CXCL8 secretion by fibroblasts in TB and its effect on intracellular Mtb survival. We show that fibroblasts are capable of potent CXCL8 secretion in vivo and in vitro. This is dependent on monocyte-derived TNF-{alpha} and IL-1. We also demonstrate that CXCL8 limits the growth of intracellular Mtb, indicating that fibroblasts can modulate the immune response to TB by secreting CXCL8, which both induces chemotaxis and enhances the macrophage killing of Mtb.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

Materials used were LPS free. SB203580, SP600125, and PD98059 were obtained from Cell Signaling Technology. Anti-TNF-{alpha} and IL-1Ra were from Peprotech. Anti-I{kappa}B{alpha} and beta Abs were from Santa Cruz Biotechnology. Cholera and pertussis toxins were from Calbiochem. All other reagents were from Sigma-Aldrich unless stated otherwise.

Fibroblast culture

MRC5 cells (human fetal lung fibroblasts; European Collection of Cell Cultures (ECACC) no. 84101801) were cultured in modified Eagle’s medium essential (MEME; Invitrogen Life Technologies) supplemented with 2 mM glutamine, nonessential amino acids, and 10% FCS (BioWest). For stimulation experiments, 1 x 105 cells were seeded in a 0.5 ml type I collagen gel (final concentration of collagen per gel, 2.4 mg/ml; Nutacon). After polymerization at 37°C for 2 h the gels were bathed in MEME with 1% FCS and stimulated.

Monocyte isolation and purification

Monocytes were isolated by density centrifugation and adherence from a single donor buffy coat (U.K. National Blood Transfusion Service, Collindale, U.K.) or peripheral blood from healthy volunteers as previously described (28).

Mtb culture

The virulent Mtb strain H37Rv Pasteur was cultured in 7H9 broth supplemented with ADC enrichment medium as previously described (29).

Generation of conditioned medium from Mtb-infected monocytes (CoMTb)

Monocytes in serum-free RPMI 1640 were infected with Mtb (strain H37Rv) at multiplicity of infection (MOI) of 10 bacilli per monocyte for 24 h. Supernatants were filtered through a 0.2-µm filter (Whatman) before aliquoting and freezing at –20°C for later use. The MOI was confirmed by colony counting in triplicate. Conditioned medium from uninfected monocytes (CoMcon) was used as a control.

ELISA for CXCL8

CXCL8 ELISA from BioSource International was developed according to the manufacturer’s instructions. The lower limit of detection of this assay was 30 pg/ml.

RNase protection assay (RPA)

The human chemokine nonradioactive RPA kit hCK5 (BD Pharmingen) was developed according to manufacturer’s instructions. The chemiluminescent signal was detected by a nucleic acid detection module from Pierce.

Western blotting

Western blotting for I{kappa}B{alpha}/I{kappa}Bbeta and total/phosphorylated p38 detection was conducted as previously described (30).

Transient transfection studies

MRC5 cells were seeded in monolayers in 12-well plates at 6 x 104 cells/well and grown overnight in MEME with 10% FCS. Plasmid DNA (CXCL8 promoter linked to firefly luciferase and the housekeeping gene thymidine kinase promoter linked to Renilla luciferase in a 10:1 ratio) was mixed with Superfect (Qiagen) in additive-free MEME for 15 min before its addition to MRC5 cells dropwise and incubation in the presence of FCS for 1 h at 37°C. Cells were then washed four times with HBSS and the medium was replaced with MEME plus 1% FCS. Cells were then stimulated with CoMTb or CoMcon. Lysates were harvested at the given time points using passive lysis buffer from the dual luciferase assay kit (Promega), and luminescence was detected using this kit as previously described (30).

Nuclear NF-{kappa}B detection

MRC5 cells were grown to confluence in 100-mm dishes in MEME with 10% FCS. Medium was changed to MEME plus 1% FCS before stimulation with CoMTb or CoMcon at 1/10 dilution. Cells were scraped into 1 ml of PBS. Cytoplasmic and nuclear extracts were prepared using a Perbio N-PER nuclear and cytoplasmic extraction kit, and protein content was quantified by a Bradford assay. The kinetics of nuclear NF-{kappa}B translocation were investigated using the TransAM NF-{kappa}B p65 transcription factor assay kit (Active Motif), and subunit identification was made with the TransAM NF-{kappa}B family kit (Active Motif) according to the manufacturer’s instructions.

Intracellular bacilli survival assay

Monocyte-derived macrophages (MDMs) were matured as previously described (28) and infected with virulent Mtb (strain H37Rv) at a MOI of 1. Supernatants were collected at 72 h and cells were lysed with 0.1% saponin. Lysates and supernatants were serially diluted in 7H9 broth and plated onto 7H11 agar for colony counting in triplicate. CFUs were counted on day 14. Experiments were repeated using macrophages derived from five different volunteers.

Immunohistochemistry

Immunohistochemistry for CXCL8 and the mesenchymal marker vimentin (which labels fibroblasts and macrophages) was performed on paraffin-embedded lung biopsies from six patients with culture-proven TB. Sections were probed with primary Abs (CXCL8 (R&D Systems) and vimentin, clone V9 (Sigma-Aldrich)), which were detected with the Biogenex nonbiotinylated kit according to the manufacturer’s instructions. Ethical approval for the use of archived biopsy lung samples was from the Hammersmith Hospital Research Ethics Committee (London, U.K.).

Data analysis

Data were tested for normality using SPSS software, version 12.0, and were normally distributed in all cases except for Fig. 7. For normally distributed data, multiple group comparisons were made using ANOVA with a post hoc Bonferroni correction. Nonparametric testing was used for data presented in Fig. 7. The Mann-Whitney U test was used for comparisons of two groups, while the Kruskall-Wallis H test was used for comparing three or more groups. Unless stated otherwise, data are mean + SEM of experiments conducted in triplicate on at least two occasions.


Figure 7
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  		FIGURE 7. CXCL8 reduces survival of Mtb in macrophages in vitro. A, The effect of exogenous CXCL8 on Mtb survival in macrophages. MDMs were obtained by a culture of monocytes in RPMI 1640 containing 10% FCS and 20 ng/ml M-CSF for 5 days. Cells were washed, bathed in serum-free RPMI 1640, and then infected with Mtb at a MOI of 1 in the presence or absence of exogenous CXCL8 (100 ng/ml). Cells were lysed in 0.1% saponin in PBS at 72 h and colony counts were plated in triplicate from the cell lysates. Data are representative of experiments using MDMs derived from 5 separate donors. *, p = 0.03 for CXCL8-treated MDMs compared with control MDMs. B, The effect of exogenous CXCL8 on the number of Mtb bacilli in the supernatants of infected cells. Infections were conducted as in A. At 72 h colony counts were plated in triplicate from the supernatants of the cells infected in A. Note the logarithmic scale on the y-axis. C, CxCL8 inhibition increases Mtb proliferation. MDMs were preincubated for 2 hours with an anti-CxCL8 Ab at 5 mg/ml before infection with Mtb at a MOI of 1. Colony counts were conducted on cell lysates at 72 h. *, p = 0.001 for CXCL8 5 µg/ml compared with control (no anti-CXCL8). D, Chemokine receptor activation and inhibition alters intracellular Mtb survival. Cells were stimulated with cholera or pertussis toxin (each 100 ng/ml) for 2 h before infection with Mtb. Lysates were plated for colony counts in triplicate at 72 h. *, p < 0.001 compared with control. Data for all four subfigures are median, interquartile range, and 95% confidence interval. The y-axis on each figure represents number of CFUs x 106.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Pulmonary fibroblasts secrete CXCL8 in human TB

CXCL8 expression was examined in human lung tuberculous granulomas. Widespread CXCL8 positivity was evident in the granulomas and surrounding alveoli (Fig. 1, Ai and Aii). Both the central granuloma mononuclear cells (Fig. 1Aiii) and the peripheral fibroblasts (Fig. 1Aiv) were strongly immunopositive for CXCL8. We compared CXCL8 secretion by monocytes and fibroblasts in a cellular model of TB infection. In vivo lung fibroblasts are only infrequently infected with Mtb (31), but in the TB granuloma the fibroblasts are stimulated by cytokines and growth factors secreted by Mtb-infected monocytes and macrophages. To mimic this in vivo Mtb-monocyte-fibroblast interaction, monocytes were infected with Mtb at a MOI of 10 and fibroblasts were stimulated with CoMTb for 24 h (Fig. 1B). Basal CXCL8 secretion per 1 x 105 cells was similar for fibroblasts and monocytes. Monocyte CXCL8 secretion increased by a factor of ~26-fold on Mtb infection (from 0.46 ± 0.04 to 12.3 ± 0.68 ng). In contrast, fibroblast CXCL8 secretion increased 1000-fold on stimulation with CoMTb (from 0.24 ± 0.02 to 241 ± 37.54 ng). Thus, fibroblasts are potentially a major source of CXCL8 secretion in the TB granuloma. Of note, fibroblasts did not up-regulate the secretion of CXCL8 in response to direct infection with Mtb (data not shown).


Figure 1
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  		FIGURE 1. Pulmonary fibroblasts express CXCL8 in vivo and in vitro in a cellular model of TB. A, Immunohistochemical staining of the tuberculous granuloma. Lung biopsies of patients with culture-proven TB were immunostained for CXCL8 (red) and the mesenchymal marker vimentin (blue). Ai, Low power view indicating widespread CXCL8 staining in the granuloma and surrounding lung tissue in pulmonary TB. Aii, Concentrated CXCL8 staining in both the center and periphery of the granuloma. Aiii, High power view of the center of the granuloma showing CXCL8 secretion by the mononuclear cells. Aiv, High magnification view of a peripheral granuloma fibroblast showing coexpression of CXCL8 and vimentin. Scale bars are given for each figure. B, Comparison of the effects of direct and indirect infection on CXCL8 secretion by monocytes and fibroblasts. Monocytes were infected with Mtb at a MOI of 10 for 24 h or stimulated with 7H9 broth alone (control, uninfected). CoMTb from 1 x 105 monocytes was used to stimulate 1 x 105 fibroblasts for 24 h. CXCL8 in monocyte conditioned medium and the tissue culture fluid surrounding fibroblasts was assayed by ELISA. Results are expressed as total CXCL8 per 1 x 105 cells of each type. **, p < 0.0001 for Mtb-infected monocytes compared with uninfected monocytes, *, p < 0.01 for CoMTb-stimulated fibroblasts compared with control fibroblasts. Data are mean + SEM.

 
To further investigate fibroblast CXCL8 secretion, we performed kinetic and dose-response experiments. CoMcon caused a small but significant up-regulation in fibroblast CXCL8 compared with unstimulated fibroblasts (data not shown), indicating that crosstalk between unstimulated monocytes and fibroblasts drives some CXCL8 secretion. Over a 120-h period CoMTb induced a sustained increase in fibroblast CXCL8 secretion compared with CoMcon (Fig. 2A), which was statistically significant from 24 h onward. This secretion followed a sustained increase in CXCL8 mRNA, which was first detectable at 2 h (data not shown) and reached a peak at 24 h (Fig. 2B). A marked induction of CXCL8 mRNA was detectable in the CoMTb-stimulated fibroblasts for up to at least 48 h (Fig. 2B). CXCL8 mRNA was only faintly detected in CoMcon-stimulated cells at 48 h.


Figure 2
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  		FIGURE 2. CoMTb induces prolonged gene expression and secretion of CXCL8 by fibroblasts. A, Kinetics of fibroblast CXCL8 secretion in response to CoMTb. Fibroblasts were seeded in collagen gels in 24-well plates and stimulated with CoMTb (black line) or CoMcon (light gray line) at a dilution of 1/10. Cell culture supernatants were analyzed by ELISA for CXCL8 at 24, 48, 72, 96, and 120 h. CoMTb induced significant up-regulation at all the of time points checked. Data are mean + SEM. *, p < 0.05 for CoMTb-stimulated cells compared with CoMcon cells at all time points. B, CXCL8 mRNA expression in CoMTb- and CoMcon-stimulated fibroblasts. Fibroblasts were seeded in 100-mm dishes and total RNA was extracted at the given time points and analyzed by chemiluminescent RNase protection assay. The housekeeping genes GAPDH and L32 are also shown. C, Dose-response curve for fibroblasts stimulated with CoMTb and CoMcon. Fibroblasts seeded in collagen gels in 24-well plates were stimulated with increasing concentrations of CoMTb and CXCL8 concentrations were measured at 24 h by ELISA. Data are mean + SEM. *, p < 0.05 for all dilutions listed compared with control unstimulated fibroblasts.

 
The induction of CXCL8 secretion occurred in response to very low concentrations of CoMTb (Fig. 2C), with dilutions of 1/500 being capable of driving a >1000-fold increase in CXCL8 secretion at 24 h (37.6 ± 10.1 ng/ml in CoMTb-stimulated cells compared with 315 ± 29 pg/ml in CoMcon-stimulated cells, p = 0.02). The dose-response plateaued at a concentration of 1/50 and thereafter showed a nonsignificant tendency to decline.

CoMTb-induced fibroblast CXCL8 secretion is TNF-{alpha} and IL-1 dependent

Mtb-stimulated monocytes secrete both TNF-{alpha} and IL-1beta (18, 32). Both of these proinflammatory cytokines are recognized stimuli for CXCL8 secretion in fibroblasts and other cells (12, 33). However, TNF-{alpha}- and IL-1beta-independent induction of CXCL8 has been described in both fibroblasts and other cell types (12, 16, 34). We investigated their role in this model of TB. TNF-{alpha} inhibition reduced fibroblast CXCL8 secretion in response to CoMTb to that of CoMcon-stimulated cells (309 ± 38 ng/ml in CoMTb group vs 128 ± 32 ng/ml with anti-TNF-{alpha} Ab at 5 µg/ml, p = 0.008; Fig. 3A). IL-1 inhibition with 100 ng/ml IL-1Ra significantly reduced CXCL8 secretion to 126 ± 21 ng/ml (p < 0.008). A combination of both anti-TNF-{alpha} and IL-1Ra had no greater effect than either alone. Preincubation of the cells with anti-TNF-{alpha} 5 µg/ml or IL-1Ra 100 ng/ml markedly reduced detectable CXCL8 mRNA at 24 h compared with CoMTb stimulation, demonstrating that both of these cytokines act at least in part pretranscriptionally (Fig. 3B). Doses of the inhibitors used were chosen based on the neutralizing dose required to inhibit the quantity of TNF-{alpha} or IL-1beta in CoMTb, as we have previously published (29). CoMTb also induced CCL2 and CXCL10 gene expression in fibroblasts. CCL2 mRNA was reduced by TNF-{alpha} and IL-1 inhibition, whereas CXCL10 was inhibited by anti-TNF preincubation but not by IL-1Ra (data not shown).


Figure 3
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  		FIGURE 3. CXCL8 up-regulation is TNF-{alpha} and IL-1beta dependent. A, The effect of cytokine inhibition on CoMTb-induced CXCL8 secretion by fibroblasts. For TNF-{alpha} inhibition CoMTb was incubated with anti-TNF-{alpha} Ab (µg/ml) for 2 h at 37°C before fibroblast stimulation. For IL-1 inhibition, fibroblasts were seeded in gels and mixed with IL-1Ra (ng/ml). Cells were incubated at 37°C for 2 h before stimulation with CoMTb. CXLC8 was measured at 72 h. Final concentration per well is given. Data are mean + SEM. *, p < 0.01 compared with CoMTb. B, Anti-TNF-{alpha} and IL-1Ra both reduce CXCL8 mRNA in response to CoMTb. Fibroblasts or CoMTb were preincubated with IL-1Ra or anti-TNF as described above. mRNA was extracted at 24 h and analyzed by RPA. C, Synergistic induction of CXCL8 secretion by TNF-{alpha} and IL-1beta. Pulmonary fibroblasts were stimulated directly with either TNF-{alpha} (ng/ml) or IL-1beta (ng/ml) alone or in combination. Data are mean + SEM. *, p < 0.001 for TNF or IL-1 alone vs control; **, p < 0.0005 for TNF + IL-1 vs control.

 
Direct stimulation of MRC5 fibroblasts with either TNF-{alpha} or IL-1beta induced a significant increase in CXCL8 secretion (p < 0.001; Fig. 3C). The combination of the two cytokines had a synergistic effect. This synergistic interaction is consistent with the finding that very low concentrations of CoMTb are sufficient to drive maximal fibroblast CXCL8 secretion.

Other cytokines identified in CoMTb by bead array and ELISA included oncostatin M, IL-6, and the chemokines CXCL8, CCL2, CCL3, and CCL5 (29, 35). Inhibition of oncostatin M and IL-6 with neutralizing Abs did not alter fibroblast CXCL8 secretion, and broad-spectrum chemokine inhibition with pertussis had no effect (data not shown).

CoMTb induces NF-{kappa}B activation in fibroblasts

NF-{kappa}B, which is normally held in the cytoplasm bound to its inhibitors (I{kappa}B), is implicated in a number of inflammatory responses to TB infection and is required for CXCL8 secretion in response to some but not all stimuli (12, 36). I{kappa}B{alpha} was detectable in the cytoplasm of CoMcon-stimulated fibroblasts. Upon CoMTb stimulation I{kappa}B{alpha} levels decreased within 15 min, remained undetectable at 30 min, and started to rise by 1 h (Fig. 4A). However, total cytoplasmic I{kappa}B{alpha} in CoMTb-stimulated cells remained suppressed for over 4 h compared with CoMcon-stimulated fibroblasts. I{kappa}Bbeta, an inhibitor whose suppression is implicated in prolonged NF-{kappa}B activation (37), was also suppressed by CoMTb and remained undetectable 8 h after stimulation (Fig. 4A).


Figure 4
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  		FIGURE 4. CoMTb drives NF-{kappa}B activation in fibroblasts. A, Both I{kappa}B{alpha} and I{kappa}Bbeta are degraded in the cytoplasm upon CoMTb stimulation. Cytoplasmic extracts were taken at the given time points after stimulation. Cytoplasmic protein extracts (25 µg) were subjected to SDS-PAGE followed by probing for I{kappa}B{alpha} and I{kappa}Bbeta. B, CoMTb stimulation causes nuclear translocation of NF-{kappa}B. Nuclear extracts from CoMTb (black line)- and CoMcon (gray line)-stimulated cells were extracted at given time points and 10 µg analyzed using the TransAM p65 subunit NF-{kappa}B transcription factor assay kit. Results are expressed as spectrophotometric readings per well at OD450. Data are representative of three separate experiments. C, Competition experiments to confirm the specificity of binding with the TransAM p65 subunit NF-{kappa}B assay kit. Reactions were incubated with an excess of wild type (WT) p65 binding oligo (WT comp DNA) or mutated p65 (mut comp DNA). In the case of the WT oligo, p65 in the nuclear extract binds predominantly to the nonfixed excess oligo and not to the fixed oligo at the bottom of the well. p65 does not bind to mutated oligo but to the fixed oligo at the bottom of the well and, thus, the signal is similar to that of nuclear extract with no form of competitor DNA added. D, Identification of NF-{kappa}B subunit up-regulation in response to CoMTb. Both the p65 and p50 NF-{kappa}B subunits are up-regulated in the nuclei of CoMTb- stimulated cells. Nuclear extracts taken at a half-hour were analyzed by the TransAM NF-{kappa}B family assay. Data are representative of three separate experiments and are expressed as ratio of OD450 in CoMcon (light gray boxes): OD450 in CoMTb-stimulated (black boxes) cell nuclear extracts.

 
CoMTb caused a rapid rise in nuclear NF-{kappa}B concentration, which peaked for 2 h before slowly declining. At 24 h there was persistent elevation in nuclear NF-{kappa}B compared with controls. CoMcon caused a small transient rise in detectable nuclear NF-{kappa}B, returning to baseline in under 1 h. Confirming the specificity of the findings, excess competitor but not mutated DNA prevented nuclear binding of NF-{kappa}B (Fig. 4C). The p52, Rel B, and Rel C subunits were detectable in equal amounts in the nuclear extracts from both the CoMcon and CoMTb-stimulated cells. The p50 subunit showed a small rise on CoMTb stimulation, whereas the most marked effect of CoMTb was nuclear translocation of the p65 subunit (Fig. 4D).

CoMTb activates the CXCL8 promoter in pulmonary fibroblasts

To further investigate the transcriptional control of CoMTb-induced fibroblast CXCL8 gene expression, promoter-reporter studies were performed. MRC5 cells were transfected with wild-type or mutant constructs of the CXCL8 promoter linked to a luciferase gene (gift of Dr. W. Reed, University of North Carolina, Chapel Hill, NC). Promoter activation in response to CoMTb was detectable at 4 h with marked activation at 8 h (>100-fold compared with control; p < 0.05). Promoter activity peaked at 24 h and thereafter declined (data not shown). Mutation of the NF-{kappa}B binding site prevented CXCL8 promoter activation in response to CoMTb (Fig. 5C). The CXCL8 promoter also contains a C/EBP binding site that is required for maximal CXCL8 induction in response to some stimuli in many cell types (19, 38). In CoMTb-stimulated fibroblasts, mutation of the C/EBP binding site was associated with complete abrogation of CXCL8 promoter activation (p < 0.001).


Figure 5
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  		FIGURE 5. CoMTb induces CXCL8 promoter activation in fibroblasts. A, Kinetics of fibroblast CXCL8 promoter activation in response to CoMTb. MRC5 cells were transiently transfected with plasmids containing the CXCL8 promoter linked to firefly luciferase and the housekeeping gene thymidine kinase promoter linked to beetle luciferase. Cells were stimulated with CoMTb (black line) or CoMcon (gray line) and lysed at the given time points, and luciferase was measured by the Dual luciferase assay. Results are expressed as relative luminescence for firefly/beetle luciferase (the ratio of CXCL8 promoter activation to thymidine kinase promoter activation). Data are mean + SEM. *, p < 0.05 for CoMTb-stimulated cells compared with CoMcon. B, Schematic diagram of the CXCL8 promoter showing the NF-{kappa}B and C/EBP binding sites; details of the mutations are listed on the right. C, The effect of mutation of NF-{kappa}B or C/EBP binding sites. Fibroblasts were transiently transfected with plasmids containing the wild type (WT) promoter or the promoter with a mutated NF-{kappa}B ({Delta}NF-{kappa}B) or mutated C/EBP ({Delta}C/EBP) binding site and stimulated with CoMTb. Luciferase activity was measured at 24 h by the Dual luciferase assay. Data are mean + SEM. *, p < 0.001 compared with WT promoter.

 
CoMTb-induced CXCL8 secretion is dependent on the p38 and JNK MAPK pathways

All three MAPK subfamilies have been variably implicated in the induction of CXCL8 (12), depending on the specific stimulus and cell type. For example, CXCL8 was secreted in response to cytokines by primary bronchial epithelial via an ERK- but not a p38- or JNK-dependent mechanism (39). In contrast in CoMTb-stimulated fibroblasts, inhibition of the p38 MAP kinase pathway resulted in a dose-dependent reduction in CXCL8 secretion at 72 h (p < 0.05 for SB203580 at 20 µM compared with CoMTb alone; Fig. 6A). CoMTb stimulation of fibroblasts resulted in a rapid phosphorylation of p38, within 15 min, that slowly declined (Fig. 3B). p38 remained phosphorylated at 24 h, indicating a prolonged activation. Because fibroblast CXCL8 mRNA remains elevated for >48 h in response to CoMTb (Fig. 2B), we investigated whether p38 caused mRNA stabilization. In Fig. 6C, lane 1 shows baseline CXCL8 mRNA before stimulation with CoMTb. At 2 h there was little detectable CXCL8 mRNA in response to CoMcon (Fig. 6C, lane 2) but marked up-regulation in response to CoMTb (lane 3), which persisted at 24 h as before (lane 6). Preincubation of cells with SB203580 to inhibit p38 phosphorylation did not reduce CoMTb-induced CXCL8 at 2 (Fig. 6C, lane 4) or 24 h (lane 8). Actinomycin D, which inhibits new gene transcription, was added to cells 2 h after stimulation with CoMTb but was not associated with a reduction in CXCL8 mRNA at 24 h after stimulation (Fig. 6C, lane 7), indicating that the CXCL8 mRNA induced by CoMTb in the first 2 h was stable (Fig. 6C). However, in cells preincubated with p38 inhibitor before stimulation with CoMTb and treated with actinomycin D after 2 h, no CXCL8 mRNA was detectable at 24 h (Fig. 6C, lane 9), indicating that mRNA stabilization requires p38 activity (6C).


Figure 6
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  		FIGURE 6. p38 and JNK MAP kinase pathways are required for fibroblast CXCL8 induction in response to CoMTb. A, p38 inhibition. Cells in collagen gels were preincubated with SB203580 (concentration in µM), a p38 inhibitor, for 2 h before stimulation with CoMTb. CXCL8 concentrations were measured at 72 h. Data are mean + SEM. *, p < 0.05 compared with CoMTb-stimulated cells. B, Phosphorylation of p38 in response to CoMTb. Cells were stimulated with CoMTb or CoMcon and lysed at the given time points for Western blot analysis. CoMTb induced a rapid marked phosphorylation followed by a prolonged decline, with persistent elevation of phosphorylated p38 at 24 h. Data are representative of two separate experiments. C, p38 activation mediates CXCL8 mRNA stability. Fibroblasts were preincubated for 2 h in the presence or absence of SB203580 (10 µM) before stimulation with either CoMcon or CoMTb. Two hours after stimulation, actinomycin (10 µg/ml) was added to CoMTb-stimulated cells to prevent new gene transcription. mRNA was collected at the baseline 2 and 24 h after stimulation with CoMcon or CoMTb and analyzed by RPA. Data are representative of three separate experiments. D, JNK inhibition. Cells in collagen gels were preincubated with SP600125 (concentration in µM), a JNK inhibitor, for 2 h before stimulation with CoMTb. CXCL8 was measured in the surrounding tissue culture fluid at 72 h. Data are mean + SEM. *, p < 0.05 compared with CoMTb-stimulation alone. E, The effect of JNK inhibition on CXCL8 mRNA. Cells were preincubated for 2 h in presence or absence of SP600125 (µM) before stimulation with CoMTb. mRNA expression at 24 h was analyzed by RPA.

 
To further investigate the role of the MAPKs, we inhibited the JNK and ERK pathways. Inhibition of ERK with PD98059 did not significantly affect CXCL8 secretion, whereas JNK inhibition caused a dose-dependent decrease in CXCL8 (Fig. 6D). JNK phosphorylation was also induced rapidly upon CoMTb stimulation of fibroblasts, and although levels of phosphorylated JNK declined within 1 h there was sustained up-regulation of JNK phosphorylation at 24 h compared with CoMcon-stimulated cells (data not shown). Like p38, JNK affects CoMTb-induced CXCL8 at a posttranscriptional level because although secretion is reduced with SP600125, CXCL8 mRNA accumulation was not inhibited (Fig. 6E).

CXCL8 limits the intracellular growth of Mtb in macrophages in vitro

Finally, we investigated whether fibroblast-derived CXCL8 could alter the survival of Mtb in macrophages. MDMs were infected with Mtb for 72 h in the presence or absence of exogenous CXCL8. We chose this time point on the basis of preliminary data confirming that at 72 h we could demonstrate significant proliferation of bacilli in MDMs without excessive macrophage cytotoxicity. The addition of CXCL8 to infected macrophages caused a significant reduction in the survival of intracellular or cell-associated Mtb (Fig. 7A; p = 0.03), although it had no effect on extracellular Mtb in the culture supernatant (Fig. 7B). Total Mtb (cell-associated and extracellular Mtb) was significantly reduced in the presence of exogenous CXCL8 (median 1.55 x 106, interquartile range 0.97–2.04 x 106 vs 2.81 x 106, range 2.11–3.17 x 106; p < 0.03). Data suggest that CXCL8 does not merely alter the uptake of bacilli by MDMs but reduces the survival of the cell-associated mycobacteria. Because macrophages infected with Mtb themselves secrete CXCL8, we hypothesized that the inhibition of CXCL8 in Mtb-infected cultures would be associated with increased bacillary proliferation. MDMs were pretreated for 2 h with anti-CXCL8 Ab and then infected for 72 h with Mtb (Fig. 7C). Anti-CXCL8 Ab resulted in an ~2-fold increase in Mtb growth within cells (p < 0.05). Finally, we reproduced the effect of CXCL8 on Mtb survival with cholera toxin, which activates G{alpha}s protein coupled receptors (p < 0.001; Fig. 7D). In addition, the inhibition of chemokine receptors with pertussis toxin (thereby blocking the effect of Mtb-induced CXCL8) was associated with an increase in intracellular Mtb, which just failed to reach statistical significance (p = 0.055; Fig. 7D). To ascertain whether this effect was specific, we tested the effect of CXCL12, another CXC chemokine secreted in high quantities by activated fibroblasts (40), and CCL5, which is also secreted by pulmonary fibroblasts (41) and is known to be up-regulated in the host response to Mtb (30), on the intracellular growth of mycobacteria. Neither had any significant effect (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
These data suggest a key role for fibroblasts in host defense to TB. We have shown that in an in vitro model of TB, which mimics the monocyte-fibroblast cellular interactions of the granuloma, fibroblasts are capable of very high-level CXCL8 secretion. On a cell for cell basis, fibroblasts stimulated with CoMTb secreted ~20 times more CXCL8 than Mtb-infected monocytes. This comparison has limitations in that the fibroblasts and monocytes were not derived from the same donor and the stimulus to CXCL8 secretion is different for each cell type. However, the observation is repeatable on multiple occasions and underlines the principle that fibroblasts are a major potential source of CXCL8 in TB. Immunohistochemical staining of the TB granuloma confirms that fibroblasts secrete CXCL8 in TB. CXCL8 staining has been identified previously in TB granulomas but not localized to fibroblasts (42). These data are consistent with observations in nonpulmonary sites and in nontuberculous disease where the fibroblast has a key role orchestrating and perpetuating the chronic inflammatory response (43, 44). Furthermore, the sustained and potent secretion of CXCL8, a mediator that we demonstrate reduces mycobacterial survival, suggests a key role for fibroblasts in the immune response to Mtb infection.

The secretion of CXCL8 by fibroblasts in vitro is a sustained response associated with prolonged expression of stable mRNA, suggesting that fibroblast-induced leukocyte recruitment may be a an ongoing feature of TB as long as fibroblasts are exposed to inflammatory cytokines secreted by Mtb-infected mononuclear cells. CXCL8 is secreted in response to very low concentrations of CoMTb, indicating that only small numbers of infected monocytes in the granuloma may drive significant fibroblast CXCL8 secretion. As bacilli are killed and cleared, the monocyte secretory profile will approach that of CoMcon, resulting in decreased fibroblast CXCL8 secretion. Thus, fibroblasts that tend to be found at the periphery of the granuloma may function as key regulators of the size and turnover of the granuloma.

We have demonstrated that CoMTb-induced CXCL8 secretion is dependent on both IL-1 and TNF-{alpha}, two prominent cytokines in the host response to Mtb. Both of these stimuli are known to induce CXCL8 from pulmonary fibroblasts (45, 46, 47), but the synergistic interaction we found is not well-recognized in the lung mesenchyme, and their role in driving CXCL8 secretion by fibroblasts in TB is not previously described. TNF-{alpha} and IL-1beta-independent induction of CXCL8 has been shown in fibroblasts and other cells (12, 16, 34). Because this interaction is synergistic, the inhibition of one factor alone will lead to a marked reduction in CXCL8 concentrations. Clinically, TNF-{alpha} inhibition leads to a breakdown of granuloma in patients already infected with Mtb or results in poor granuloma formation in response to new infection (5). This may be explained in part by a subsequent failure to secrete CXCL8 in the granuloma periphery by fibroblasts, leading to reduced cell recruitment to, and failure to retain cells within, the granuloma.

In addition, we have demonstrated a role for CXCL8 in reducing the survival of Mtb within infected macrophages. CXCL8 is unlikely to be directly toxic to M. tuberculosis given that it had no effect on extracellular bacilli but appears to modulate macrophage activity to improve their killing capacity. The fact that mycobacterial counts in the supernatants of infected cells at 72 h were unchanged in response to CXCL8 suggests that CXCL8 does not affect the uptake of the bacilli but alters their survival within the cells. Total (supernatant plus cell-associated) mycobacterial counts were reduced in the presence of CXCL8, so this is not simply a reflection of differential distribution of the bacilli over the time course of the experiment. The effects of CXCL8 were reproduced by treating macrophages with cholera toxin, a nonspecific Gs protein-coupled receptor activator. However, the capacity of CXCL8 to control mycobacterial proliferation is not a broad-spectrum effect of GPCR activation because it was not reproduced by other chemokines. Treatment of cells with pertussis toxin was associated with an increased bacillary proliferation within macrophages that just failed to reach statistical significance. The data also demonstrate that autocrine stimulation of Mtb-infected macrophages by chemokines plays a role in controlling TB proliferation. Specifically, inhibition of autocrine CXCL8 mimicked the effect of pertussis on bacilli numbers. Although Mtb-infected macrophages themselves secrete CXCL8, we showed that exogenous CXCL8 caused an increase in bacterial killing so that within the granuloma fibroblast CXCL8 secretion is likely to further control the rate of proliferation of Mtb. The mechanism by which CXCL8 reduces the survival of intracellular Mtb is not yet known but may be related to its effect on apoptosis (25) or reactive oxygen or nitrogen intermediates (48). This is the subject of ongoing further work, because defining this may help design new therapeutic targets to aid in the killing of mycobacteria.

The signaling pathways regulating CXCL8 secretion in lung fibroblasts in response to CoMTb include NF-{kappa}B and p38 and JNK MAPKs. The requirement for NF-{kappa}B is well described although not universal in the induction of CXCL8 (12, 36). I{kappa}Bbeta degradation in the cytoplasm is usually more prolonged than that of I{kappa}B{alpha} (37), and this finding in fibroblasts is consistent with the sustained p38 phosphorylation and CXCL8 secretion seen in response to CoMTb. I{kappa}Bbeta is thought to mediate more prolonged immune responses, and not all of the inflammatory stimuli that activate NF-{kappa}B involve I{kappa}Bbeta degradation (37). The NF-{kappa}B subunits that primarily translocated to the nucleus were p65 and p50, although there was constitutive nuclear expression of p52, Rel C, and Rel B. p65 up-regulation was considerably more marked than that of p50, possibly indicating a role for signaling by p65 homodimers (49, 50). Unlike p50 homodimers, which are usually transcriptionally repressive, p65 dimers are generally activators. The nuclear subunits identified are distinct from our findings in respiratory epithelial cells, where CoMTb induced the nuclear translocation of Rel C in addition to p65 and p50 (19), and demonstrates that the responses are cell specific. The importance of NF-{kappa}B in this case is emphasized by the fact that mutation of the NF-{kappa}B binding site is associated with the abrogation of CoMTb-induced CXCL8 promoter activation. Interestingly, mutation of the C/EBP binding site also completely abrogated CXCL8 activation in response to CoMTb in fibroblasts, whereas in pulmonary epithelial cells such mutation only partly reduced CXCL8 promoter activity (19). An absolute requirement for C/EBP binding for CXCL8 promoter activation has been described in some studies, although in most studies C/EBP was required only for maximal induction (12, 51).

Prolonged p38 phosphorylation occurred in fibroblasts in response to CoMTb and mediated mRNA stability. Our group has recently demonstrated p38 phosphorylation in the tuberculous granuloma and the surrounding lung parenchyma (29), confirming the importance of this pathway in vivo. In addition, intact JNK signaling was essential for CXCL8 secretion, which is consistent with published data and is associated downstream with AP-1 activation (12). JNK activity regulating CXCL8 at a posttranslational level has not been described, although JNK-induced posttranslational modification of other proteins is recognized (52).

In summary, we propose on the basis of our data that fibroblasts stimulated by Mtb-infected monocytes have a role in the regulation of cell recruitment to and cell retention within the granuloma by secreting CXCL8. CXCL8 induction is sustained and TNF-{alpha} and IL-1 dependent, and it occurs in response to prolonged NF-{kappa}B activation, p38-induced mRNA stabilization, and JNK phosphorylation. Fibroblasts exert negative feedback control on this by secretion of CXCL8 that, as we show for the first time, limits the proliferation of intracellular Mtb and, hence, further monocyte-dependent fibroblast stimulation.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was funded by the Wellcome Trust (to C.M.O., P.T.E., and J.S.F.). Back

2 Address correspondence and reprint requests to Prof. J. S. Friedland, Department of Infectious Diseases, Imperial College, Hammersmith Campus, Du Cane Road, London, U.K. E-mail address: j.friedland{at}imperial.ac.uk Back

3 Abbreviations used in this paper: TB, tuberculosis; Mtb, Mycobacterium tuberculosis; CoMcon, conditioned medium from uninfected monocytes; CoMTb, conditioned medium from Mtb-infected monocytes; MDM, monocyte-derived macrophage; MOI, multiplicity of infection; RPA, RNase protection assay. Back

Received for publication January 18, 2006. Accepted for publication December 28, 2006.


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 Introduction
 Materials and Methods
 Results
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 Disclosures
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