Mycobacteria-Induced TNF-α and IL-10 Formation by Human Macrophages Is Differentially Regulated at the Level of Mitogen-Activated Protein Kinase Activity

Norbert Reiling, Antje Blumenthal, Hans-Dieter Flad, Martin Ernst and Stefan Ehlers
J Immunol September 15, 2001, 167 (6) 3339-3345; DOI: https://doi.org/10.4049/jimmunol.167.6.3339

Abstract

The clinical course of mycobacterial infections is linked to the capacity of pathogenic strains to modulate the initial antimycobacterial response of the macrophage. To elucidate some of the mechanisms involved, we studied early signal transduction events leading to cytokine formation by human monocyte-derived macrophages (MDM) in response to clinical isolates of Mycobacterium avium. TNF-α production induced by M. avium was inhibited by anti-CD14 mAbs, but not by Abs against the macrophage mannose receptor. Analysis of mitogen-activated protein (MAP) kinase activation (extracellular signal-regulated kinase 1/2, p38, and c-Jun NH2-terminal kinase) showed a rapid phosphorylation of all three subfamilies in response to M. avium, which was inhibited by anti-CD14 Abs. Using highly specific inhibitors of p38 (SB203580) and MAP kinase kinase-1 (PD98059), we found that activation of the extracellular signal-regulated kinase pathway, but not of p38, was essential for the M. avium-induced TNF-α formation. In contrast, IL-10 production was abrogated by the p38 inhibitor, but not by the MAP kinase kinase-1 inhibitor. In conclusion, M. avium-induced secretion of TNF-α and IL-10 by human macrophages is differentially regulated at the level of MAP kinase activity.

Mycobacterial infections, most prominently tuberculosis and leprosy, remain an important cause of morbidity and mortality worldwide. Mycobacterium avium is one of the most prevalent opportunistic pathogens of immunocompromised patients, particularly those in advanced stages of AIDS, but also infects immunocompetent individuals, such as elderly women, patients with chronic obstructive pulmonary disease, and children, who subsequently develop cervical lymphadenitis (1, 2, 3). Like M. tuberculosis, M. avium is a facultative intracellular bacterium that resides and replicates inside the macrophages of the infected host (4).

Upon initial contact of macrophages with M. avium, a variety of cellular reactions are triggered, including the production of reactive oxygen and nitrogen intermediates and the release of proinflammatory cytokines (e.g., TNF-α and IL-6) (reviewed in Ref. 5) as well as anti-inflammatory cytokines (IL-10 and TGF-β) (6, 7). In vivo, mycobacteria-induced production of TNF-α correlates with accelerated granuloma formation, resulting in rapid containment of the infectious focus (8). In contrast, endogenous IL-10 may down-modulate the antimycobacterial response, as treatment with anti-IL-10 Abs or infection in IL-10-deficient mice leads to increased clearance of some mycobacterial strains (6, 9).

Intracellular pathogens such as mycobacteria select specific host cell receptors to facilitate both adherence and entry, including complement receptor types 1, 3, and 4, surfactant receptors, scavenger receptors, and some integrins (reviewed in Ref. 10). In addition, the mannose receptor plays a prominent part in early internalization and processing of mycobacteria (11), although signaling via the macrophage mannose receptor (MMR)3 has not been conclusively demonstrated. Similarly, CD14 is clearly involved in the response to lipoarabinomannan derived from mycobacteria (12). Whether intact mycobacterial cells use CD14 to initiate cytokine production by macrophages has not been determined to date.

Mitogen-activated protein (MAP) kinases are ubiquitous serine/threonine protein kinases involved in signal transduction in eukaryotic organisms. Their activity is important for a panoply of cellular functions, ranging from proliferative to differentiative events and programmed cell death (13). At least three distinct families of MAP kinases exist in mammalian cells: the p42/44 extracellular signal-regulated kinase (ERK) MAP kinase, c-Jun NH2-terminal kinases (JNKs), and p38 MAP kinase. All three can be activated independently and simultaneously. Whereas ERK1 and -2 are mainly activated by growth factors or phorbol esters, JNK and p38 are activated by inflammatory cytokines or cellular stresses such as osmotic changes and UV light (14). Bacterial LPS leads to a rapid activation of all three MAP kinase families (15, 16, 17). Whereas p38 and ERK are of critical importance for the release of proinflammatory cytokines such as TNF-α and IL-1 (18), only activated p38, but not ERK, is necessary for IL-10 formation in response to LPS (19).

Whether MAP kinases are involved in TNF-α and IL-10 secretion by human macrophages in response to viable mycobacteria has not been explored in any detail. In this in vitro study we investigated the role of two putative surface receptors for mycobacteria, CD14 and MMR, and made use of selective MAP kinase inhibitors to determine the relative importance of MAP kinases p38 and ERK-1 in signaling TNF-α and IL-10 secretion in response to infection with M. avium.

Materials and Methods

Bacteria

M. avium strains SE01 and 2151 SmO were initially derived from AIDS patients (20, 21). Both strains were cultured in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose, and catalase (BD Biosciences, Heidelberg, Germany) to the mid logarithmic phase. Aliquots were frozen at −70°C until needed. For stimulation experiments M. avium strains were thawed at room temperature and spun down with 2000 rpm (830 × g for 10 min). Pellets were resuspended in PBS and added to the cells for the times indicated. To rule out the presence of LPS in the assays, both strains were tested in a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The effective LPS concentration in the experiments when ratios of 10:1 were used was <2 pg/ml.

Bacterial LPS of Salmonella enterica, serotype friedenau H909, was provided by H. Brade (Research Center Borstel, Borstel, Germany). It was prepared by the hot phenol-water procedure, purified by repeated ultracentrifugation, and converted into the sodium salt form by electrodialysis (22).

Chemicals

SB203580 and PD98059 were purchased from Calbiochem (Schwalbach, Germany). Abs against MAP kinases (p38, phospho-p38, p42/44 (ERK 1/2), phospho-p42/44 (ERK 1/2), JNK, and phospho-JNK) were purchased form New England Biolabs (Schwalbach, Germany). Anti-CD14 (MEM 18) was a gift from V. Horejsi (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic). Isotype control Abs used in culture experiments were obtained from BD PharMingen (Heidelberg, Germany).

Cell preparation and differentiation

PBMC were prepared from venous blood from healthy volunteer donors using density gradient centrifugation (23). Monocytes were isolated from PBMC by counterflow centrifugation as previously described (24). The purity of the monocyte fraction was determined using flow cytometric analysis of CD14 expression (consistently >95%) and positivity for α-naphthyl-esterase staining (>90%). For differentiation into macrophages, monocytes were cultured for 7 days in Teflon bags (Süd Laborbedarf, Gauting, Germany) containing RPMI 1640 supplemented with 2% (v/v) heat-inactivated human serum, 100 U/ml penicillin G (Biochrom, Berlin, Germany), 100 μg/ml streptomycin (Biochrom), 2 mmol/L l-glutamine (Biochrom), and 2 ng/ml M-CSF (R&D Systems, Wiesbaden, Germany). After differentiation cells were washed and cultured (4 × 105/well at 1 × 106/ml) in 24-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark) in RPMI 1640 medium containing 10% (v/v heat-inactivated FCS (Biochrom) and 2 mmol/L l-glutamine (Biochrom). Cell viability in the presence of inhibitors was measured by trypan blue exclusion and by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was 95% of the viability of control (untreated) cultures.

Abs and flow cytometry

The following Abs were used for immunophenotyping of cells: anti-CD14 (RMO52; Coulter Diagnostics, Krefeld, Germany), anti-carboxypeptidase M (MAX1, provided by R. Andreesen, Department of Hematology and Oncology, University of Regensburg, Regensburg, Germany) (25), and anti-MMR (provided by S. Sozzani, Instituto di Ricerche Farmacologiche “Mario Negri,” Milan, Italy) as well as the appropriate IgG1 and IgG2a isotype control Abs. Binding of the primary Abs was detected by the use of Cy5-labeled goat anti-mouse antiserum (Dianova, Hamburg, Germany). Briefly, 2 × 105 cells were stained at 4°C with saturating Ab concentrations as stipulated by the manufacturer. After staining the cells were washed, resuspended in PBS, and fixed with 3% paraformaldehyde until analysis in a FACSCalibur flow cytometer (BD Biosciences).

Determination of monokine release

For the detection of monokines culture supernatants were harvested at the times indicated and frozen at −20°C until analysis. The concentrations of TNF-α in supernatants were determined using quantitative ELISA provided by H. Gallati (Intex, Muttenz, Switzerland). IL-10 ELISA was purchased from R&D Systems. The assays were conducted as recommended by the manufacturer and as previously described (26).

Analysis of MAP kinase phosphorylation

Cells (4 × 105) were lysed in 100 μl 2× sample buffer (125 mM Tris (pH 6.80, 4% SDS, 20% glycerol, 100 μM DTT, and 0.05% bromophenol blue), loaded onto a 12% SDS-PAGE gel, and run at 100 mA for 1.5 h. Cell proteins were transferred to nitrocellulose membrane (Sartorius, Gottingen, Germany) for 1.5 h at 75 V by wet blot at 4°C (Mini Protean 2, Bio-Rad, Munich, Germany). The nitrocellulose was then blocked with 5% nonfat dry milk (Glücksklee, Nestle, Frankfurt) in TBS with 0.1% Tween 20 for 2 h, washed and incubated with the primary Ab at 4°C overnight as recommended by the manufacturer. The blots were washed four times with TBS with 0.1% Tween 20 and incubated for 1 h with HRP-conjugated goat anti-rabbit IgG Ab (Dianova; p38, phospho-p38, ERK 1/2, phospho-ERK 1/2, JNK) or HRP-conjugated goat anti-mouse IgG Ab (Dianova; phospho-JNK). Immunoreactive bands were developed using a chemiluminescent substrate (ECL, Pharmacia-Amersham, Freiburg, Germany). Autoradiography was performed with exposure times of 15 s to 5 min, whichever were adequate for visualization.

Statistical analysis

Data obtained from independent experiments are presented as the mean ± SD and were compared by Student’s t test or for multiple comparisons by ANOVA. Differences were considered significant for p < 0.05.

Results

M. avium leads to rapid TNF-α production in human MDM

Human MDM were incubated with increasing concentrations of M. avium strain SE01. Analysis after 12 h of culture showed a dose-dependent increase in TNF-α formation (Fig. 1⇓A), with 4–12 ng/ml TNF-α at a ratio of 10 mycobacteria/macrophage. Differing absolute amounts in TNF-α produced are due to donor variability, which can be quite substantial. All experiments were performed at least five times using cells from different donors, and the qualitative effects described here were reproduced in all individuals. The highest peak of TNF-α production in response to M. avium was always detected 8–24 h after stimulation (Fig. 1⇓B). For additional experiments a mycobacteria-macrophage ratio of 3:1 or 10:1 was used, and supernatants were harvested at 18 h.

FIGURE 1.
FIGURE 1.

M. avium-induced TNF-α production in human MDM. A, Human MDMs were incubated with an increasing ratio of M. avium SE01 or LPS (10 ng/ml). Supernatants were harvested after 12 h, and TNF-α formation was measured by ELISA. The means of duplicates ± SD are shown. Data illustrated are representative of four separate experiments with cells from different donors. B, Human MDMs were incubated with increasing ratios of M. avium SE01. Supernatants were harvested after the times indicated, and TNF-α formation was measured by ELISA. The means of duplicates ± SD are shown.

CD 14, and not the MMR, is involved in macrophage activation by M. avium

To analyze which receptor mediates the activation by M. avium SE01, we measured TNF-α formation in the presence or the absence of inhibitory Abs against CD14 (27) or against the MMR (28). Preincubation with the anti-CD14-Ab resulted in a reduction of up to 80% of the M. avium-induced TNF-α formation (SE01; Fig. 2⇓A), whereas anti-MMR Ab did not show a significant effect (Fig. 2⇓B). An IgG1 isotype control Ab used at the same concentration did not affect TNF-α secretion. Therefore, CD14, but not the MMR, is involved in M. avium-induced TNF-α formation of human macrophages. Similar results were obtained when M. avium-induced IL-10 formation was measured (data not shown).

FIGURE 2.
FIGURE 2.

CD14, and not the MMR, is involved in macrophage activation by M. avium. Human macrophages were preincubated with anti-CD14 Abs (A), anti-MMR Abs (B), or isotype control Abs and subsequently stimulated with M. avium strain SE01 (ratio of 3:1). Supernatants were harvested after 18 h, and TNF-α formation was measured by ELISA. The means of duplicates ± SD are shown. A, Data shown are the mean ± SD of three independent experiments; B, data from one representative experiment of three are shown; ∗, p < 0.05; n.s., not significant.

M. avium leads to the phosphorylation of MAP kinases

Because MAP kinases are critical factors mediating cellular responses to many external stimuli, we next examined MAP kinase activation in response to M. avium infection. Macrophages were stimulated with mycobacteria at a ratio of 3:1, and phosphorylation of p38, JNK, and ERK1/2 was analyzed (Fig. 3⇓). M. avium SE01 led to a strong phosphorylation of p38 and JNK and ERK1/2 30–60 min after stimulation. Control cells stimulated with LPS (10 ng/ml) showed a signal of comparable intensity. The observed time courses of all three MAP kinase families were similar.

FIGURE 3.
FIGURE 3.

M. avium readily induces phosphorylation of MAP kinases. Human MDMs were incubated with M. avium strain SE01 at a ratio of 3:1 and LPS (10 ng/ml) for the times indicated. Cells were lysed, and aliquots of total cell lysates were separated by SDS-PAGE and immunoblotted as described. Blots were incubated overnight with specific anti-phospho-JNK (p-JNK), anti-phospho-p38 (p-p38), or anti-phospho-ERK1/2 (p-ERK) as well as specific control Abs for the unphosphorylated forms all three kinases, followed by appropriate peroxidase-coupled secondary reagents and were visualized by ECL. Similar data were obtained in two independent experiments.

CD14 is critical for MAP kinase phosphorylation by M. avium

Because treatment with anti-CD14 mAbs blocked TNF-α formation, we next investigated whether it would also interfere with MAP kinase phosphorylation, a critical prerequisite of MAP kinase activation. Macrophages were incubated in the absence or presence of an anti-CD14 Ab (5 μg/ml) or isotype control Ab for 45 min, subsequently stimulated with mycobacteria for 30 min, and analyzed for MAP kinase phosphorylation. Fig. 4⇓ illustrates that preincubation with anti-CD14 almost completely abrogated MAP kinase phosphorylation (ERK1/2 and p38) of MDMs in response to M. avium SE01. Preincubation with the same concentration of isotype control Ab had no effect. Thus, the availability of CD14 is essential for MAP kinase activation by M. avium.

FIGURE 4.
FIGURE 4.

CD14 is critical for MAP kinase phosphorylation by M. avium. Human MDMs were preincubated with anti-CD14 monoclonal (5 μg/ml) or isotype control Abs (5 μg/ml) for 45 min. Subsequently, M. avium strain SE01 at a ratio of 3:1 and LPS (10 ng/ml) were added for 30 min. Cells were lysed, and aliquots of total cell lysates were separated by SDS-PAGE and immunoblotted as described. Blots were incubated overnight with specific anti-phospho-p38 (p-p38) or anti-phospho-ERK (p-ERK), followed by appropriate peroxidase-coupled secondary reagents and were visualized by ECL. As controls the total amounts of ERK and p38 as detected by anti-ERK and anti-p38 Abs are shown. Similar data were obtained in two independent experiments.

The ERK pathway is critical for M. avium-induced TNF-α formation

ERK1/2 and p38 have both been shown to be pivotal in cytokine formation induced by LPS (29). To further understand the functional role of these kinases in the activation of macrophages induced by M. avium, we used highly specific inhibitors of either kinase and measured TNF-α formation. Both inhibitors, SB203580 (p38) (18) and PD98059 (MAP kinase kinase, MEK) (30), dose dependently reduced LPS-induced TNF-α formation of human macrophages by 85 and 80%, respectively (Fig. 5⇓, A and C). In contrast, TNF-α formation of macrophages in response to M. avium was completely unaffected by the p38 inhibitor SB203580 (Fig. 5⇓B). However, the MEK inhibitor PD98059 did reduce TNF-α secretion of MDMs by up to 85% (Fig. 5⇓D). The latter result was confirmed by the use of a second and more potent MEK inhibitor U0126 (data not shown).

FIGURE 5.
FIGURE 5.

The MEK inhibitor PD98059, but not the p38 inhibitor SB203580, reduces the M. avium-induced TNF-α formation of human MDMs. Human MDMs were preincubated with medium containing 0.1% DMSO as solvent control, 1 and 3 μM SB203580, or 3 and 10 μM PD98059 for 45 min. Subsequently, LPS (10 ng/ml; A and C) and M. avium strain SE01 (B and D) were added at a ratio of 3:1. Supernatants were harvested after 18 h, and TNF-α formation was measured by ELISA. Data shown are the mean ± SD of three independent experiments performed in duplicates; ∗, p < 0.05; n.s., not significant.

To verify that reduced TNF-α production in the presence of PD98059 was indeed a consequence of inhibited ERK phosphorylation, MDMs were cultured in the presence or the absence of 10 μM of the MEK inhibitor PD98059 for 45 min. Cells were stimulated for 30 min with M. avium SE01, 10 ng/ml LPS, and 10 ng/ml PMA, and ERK phosphorylation was analyzed. PD98059 inhibited M. avium SE01-induced ERK phosphorylation (Fig. 6⇓A). LPS-induced phosphorylation of ERK was affected to a similar extent, as described previously (31), whereas PMA-mediated activation, which is known to stimulate cells via different and independent mechanisms, was almost unaltered. The p38 phosphorylation in response to LPS and M. avium SE01 was unaffected in the presence of PD98059 (Fig. 6⇓B).

FIGURE 6.
FIGURE 6.

The MEK inhibitor PD98059 abrogates M. avium-induced phosphorylation of ERK, but not p38, in human MDMs. Human MDMs were preincubated in the presence of 10 μM PD98059 for 45 min or with medium containing 0.1% DMSO as solvent control. Cells were then incubated with M. avium strain SE01 at a ratio of 3:1 and LPS (10 ng/ml) for 30 min. Cells were lysed, and aliquots of total cell lysates were separated by SDS-PAGE and immunoblotted as described. A, Blots were incubated overnight with specific anti-phospho-ERK (p-ERK), anti-ERK (ERK); B, anti-phospho-p38 (p-p38) and anti-p38 (p38), followed by appropriate peroxidase-coupled secondary reagents and visualized by ECL.

M. avium-induced TNF-α formation is p38 independent

In contrast to its prominent effect on LPS-induced TNF-α formation, the p38 inhibitor SB203580 did not influence M. avium SE01-induced TNF-α secretion (Fig. 5⇑B). To exclude that this effect was specific for a particular strain of M. avium, we performed p38 inhibition experiments with another clinical isolate of M. avium, the smooth opaque variant of strain 2151. Human MDMs were preincubated with SB203580 and subsequently stimulated with increasing concentrations of M. avium 2151 SmO (Fig. 7⇓B). Corroborating the results obtained with M. avium SE01, no inhibition was observed. Rather, there was a tendency to an increased TNF-α formation in the presence of SB203580, although this did not attain statistical significance in all experiments (see Fig. 7⇓B vs Fig. 5⇑B). Because TNF-α production was always lower when using M. avium than when using LPS as a stimulus, it appeared possible that the inhibitory effect of SB203580 was mostly quantitative, rather than qualitative. To test this possibility, a wide range of LPS concentrations (0.01 pg/ml to 10 ng/ml) were used to induce both high and low amounts of TNF-α in MDMs. Indeed, TNF-α formation was sensitive to inhibition by SB203580 at all given LPS concentrations (Fig. 7⇓A). Therefore, whereas p38 MAP kinase activity is essential for TNF-α production induced by LPS, it is not necessary for TNF-α production induced by M. avium.

FIGURE 7.
FIGURE 7.

Stimulation of human macrophages by LPS and M. avium: influence of the p38 inhibitor SB203580. Human MDMs were preincubated with medium containing 0.1% DMSO as solvent control (▦) and 3 μM SB203580 (▪) for 45 min and were subsequently incubated with increasing concentrations of LPS (A) and M. avium strain 2151 SmO (B). Supernatants were harvested after 18 h, and TNF-α formation was measured by ELISA; ∗, p < 0.05.

M. avium-induced IL-10 production is p38 dependent, but ERK independent

The activity of p38 is also important for the production of IL-10 by human monocytes (19). Therefore, we analyzed M. avium-induced IL-10 production in the absence or presence of SB203580. Fig. 8⇓B shows that the M. avium-induced IL-10 production of MDM was blocked by 70% when using SB203580, thus demonstrating that the inhibitor is active in M. avium-infected macrophages. Similar to results with LPS stimulation, the ERK inhibitor PD 98059 did not affect IL-10 production by MDM infected with M. avium (Fig. 8⇓D), whereas LPS-induced IL-10 production was enhanced. In conclusion, p38 MAP kinase activity is necessary, but ERK activity is dispensable, for the IL-10 production of human macrophages stimulated with M. avium.

FIGURE 8.
FIGURE 8.

The p38 inhibitor SB203580, but not the MEK inhibitor PD98059, reduces the M. avium-induced IL-10 formation of human MDMs. Human MDMs were preincubated with medium containing 0.1% DMSO as a solvent control or 3 and 10 μM SB203580 or PD98059 for 45 min. Subsequently, LPS (10 ng/ml; A and C) and M. avium were added at a ratio of 3:1 (B and D). Supernatants were harvested after 48 h, and IL-10 formation was measured by ELISA. Data shown are the mean ± SD of three independent experiments performed in duplicates; ∗, p < 0.05; n.s., not significant.

Discussion

We studied receptor usage and early signal transduction events involved in M. avium-induced activation of human macrophages. We found that CD14, but not the MMR, was critically involved in TNF-α and IL-10 secretion. M. avium led to the rapid phosphorylation of all three MAP kinase families in a CD14-dependent manner. In contrast to LPS-induced signaling, activation of ERK, but not p38 activity, was essential for the secretion of TNF-α in response to M. avium, whereas p38 activity, but not ERK activity, was critical for IL-10 secretion induced by M. avium. Therefore, macrophage-dependent orchestration of pro- vs anti-inflammatory events is regulated very early during infection with M. avium at the level of MAP kinase activity.

To date, most investigators have focused on the role of NF-κB in mycobacteria-induced macrophage activation (32, 33). This is the first study reporting that all three MAP kinase families (p38, ERK1/2, and JNK) are rapidly phosphorylated in response to intact viable M. avium. Our next goal was to understand in which way M. avium-induced MAP kinase activation was causally related to TNF-α formation. p38 activity is of critical importance for the release of TNF-α and IL-1 in monocytes stimulated by LPS (34). In brief, p38 activates MAP kinase-activated protein kinase 2, which then phosphorylates heat shock protein 27, further leading to the initiation of TNF-α mRNA translation. The pyridinyl imidazole SB203580, a cell-permeable, highly specific p38 inhibitor, binds to the p38 ATP binding site (35), therefore inhibiting the activity, but not the phosphorylation, of the enzyme. Although SB203580 clearly reduced LPS-mediated TNF-α formation, M. avium-induced TNF-α secretion, as shown for two different clinical isolates, was not affected, even when 3 μM inhibitor was used. This concentration is well above the IC50 of 0.15 μM described by other investigators (36). Even when the ratio of mycobacteria per macrophage was raised to 10:1 and 30:1, no inhibitory effect of SB203580 on M. avium-induced TNF-α formation was seen. This indicates that the p38-independent TNF-α formation is not strain or dose specific, but may represent a general reaction pattern of the macrophage responding to M. avium and, possibly, other mycobacteria. That there is a qualitative, rather than quantitative, difference in cellular activation in response to mycobacteria compared with LPS is further supported by our finding that the LPS-induced TNF-α was always sensitive to inhibition by the p38 inhibitor, even when very low concentrations of LPS, e.g., 50 pg/ml, were used (Fig. 7⇑A).

Only T cells were previously shown to release TNF-α independently of p38 activity (36). This is the first demonstration that in macrophages also p38 activity may not be per se involved in TNF-α formation, but is instead closely linked to the stimulus used. Whereas LPS from Gram-negative bacteria requires p38 activity for TNF-α production, mycobacteria such as M. avium do not. Our findings suggesting a distinct role of p38 in mycobacterial infections are corroborated by a very recent report by Chan et al. (37), who show that SB203580 does not block, but rather enhances, lipoarabinomannan-induced nitrite production in murine RAW264 macrophages. Other investigators demonstrated an inhibition of LPS-induced nitrite formation by the same inhibitor (38).

In contrast to TNF-α, we found IL-10 production to be significantly reduced in the presence of SB203580. IL-10 is an important regulator of myeloid cells, which plays an overall anti-inflammatory role, potently inhibiting the capacity of monocytes/macrophages to secrete inflammatory cytokines as well as down-regulating their capacity to serve as accessory cells (39). Previous studies demonstrated that p38 activation is a key signaling step in the IL-10 biosynthesis induced by LPS (19). We now show that this also holds true for the IL-10 production by macrophage induced by mycobacteria. Because myeloid IL-10 production is regulated in an autocrine/paracrine network by stimulatory cytokines such as TNF-α or IL-1 (40), one may argue that in our system the M. avium-induced TNF-α ultimately might lead to IL-10 formation by inducing p38 activity. Although we have not formally ruled out this possibility, we consider it unlikely, because blocking of M. avium- induced TNF-α formation by the MEK inhibitor PD98059 had no influence on IL-10 formation.

The involvement of the ERK pathway was analyzed by the use of PD98059, a selective inhibitor of MEK1, the dual specificity kinase that activates ERK via phosphorylation on both threonine and serine residues (41). We found that both the M. avium- and the LPS-induced TNF-α formation was drastically reduced in the presence of PD98059, which effectively inhibited ERK1/2 phosphorylation to either stimulus. Therefore, in contrast to p38, the ERK pathway is critically important in the M. avium-induced activation and subsequent TNF-α formation of human MDMs.

Our data indicate that there might be an early branching point in the mycobacteria-induced activation cascade of the macrophage, at least with respect to cytokine induction, that fundamentally differs from that induced by LPS. Therefore, we analyzed whether this discrepancy would also be apparent at the level of receptor usage.

The MMR is a C-type lectin that mediates attachment to glycoconjugates terminating in mannose, fucose, and N-acetylglucosamine. The MMR has been shown to mediate phagocytosis of various mycobacterial species (11, 42) and has been reported to trigger the production of reactive oxygen intermediates by macrophages (43), but other investigators failed to detect a role of MMR in generation of O2− or in the change in phosphorylation of protein tyrosine residues (42). Our experiments using inhibitory Abs against the MMR (28) did not reveal an important role for this receptor in the M. avium-induced TNF-α formation of human MDMs.

A very prominent player in the macrophage activation cascade after contact with bacterial compounds as well as mycobacterial components such as liporarabinomannan is CD14, a cell surface, glycosylphosphatidyl inositol-linked, 55-kDa glycoprotein expressed predominantly on myelomonocytic cells (including monocytes and macrophages) (12, 44, 45). We found MAP kinase activation and cytokine formation induced by viable M. avium to be dependent on CD14. The molecular mechanism by which CD14 mediates the activation of MDM by intact mycobacteria is not clear. Due to the lack of an intracellular signaling tail, CD14 on its own is not able to transmit a signal into a cell. Recent discoveries show that CD14 interacts with Toll-like receptors (TLRs) to transmit a signal (46). TLRs comprise a family of innate immune signaling receptors that are capable of discriminating between bacterial components originating from different sources (47). Recent evidence suggests that TLR4 is essential for the recognition of Gram-negative bacteria, whereas TLR2 is a key signaling factor in cell responsiveness to isolated components of Gram-positive bacteria (48) and of mycobacteria (49). With respect to intact viable mycobacteria the situation might be even more complex. Means et al. (50) reported that intact M. tuberculosis may use both TLR2 and TLR4 to activate NF-κB, and Lien et al. (51) demonstrated that M. avium-mediated TNF-α formation by human PBMC could be only partially inhibited by an anti-TLR2 Ab. This inhibition was reflected at the level of NF-κB signaling, as demonstrated in a panel of TLR-transfected Chinese hamster ovary cells. It will be interesting to investigate whether and to what extent TLR2-mediated signaling contributes to MAP kinase activation and cytokine formation by primary macrophages in response to intact M. avium organisms.

Regardless of whether CD14 or TLR2 is the first step in the activation process, CD14 is critically involved in both M. avium-induced ERK and p38 activation. Our data suggest that the branching point in M. avium-induced signal transduction is downstream of CD14 but upstream of the MAP kinase level. Most signals transmitted via TLRs make use of an adapter molecule, MyD88, although MyD88-independent pathways are also being identified (52). Whether differential MAP kinase usage is directly related to differential TLR and/or MyD88 usage remains to be determined.

In summary, the requirement for activated MAP kinases clearly differs between M. avium and LPS-triggered cytokine secretion. Whereas p38, and not ERK, activity is necessary for IL-10 secretion, ERK activity, and not p38, is indispensable for the generation of TNF-α. This distinct usage of MAP kinase pathways in the production of regulatory cytokines offers an intriguing novel target for therapeutic intervention in mycobacteria-induced inflammation.

Acknowledgments

We thank V. Horesji (Prague) for the kind gift of the anti-CD14 (MEM-18) mAb, and A. J. Ulmer (Research Center Borstel) for helpful discussions. We gratefully acknowledge the expert technical assistance of R. Bergmann, E. Kaltenhäuser, S. Kutsch, and S. Kröger-Albrecht.

Footnotes

  • ↵1 This work was supported in part by the Deutsche Forschungsgemeinschaft (Re1228/3-1, to N.R. and S.E.).

  • ↵2 Address correspondence and reprint requests to Dr. Norbert Reiling, Division of Molecular Infection Biology, Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, D-23845 Borstel, Germany. E-mail address: nreiling{at}fz-borstel.de

  • ↵3 Abbreviations used in this paper: MMR, macrophage mannose receptor; MAP, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAP kinase kinase; ERK, extracellular signal-regulated kinase; MDM, monocyte-derived macrophage; TLR, Toll-like receptor.

  • Received July 17, 2000.
  • Accepted July 9, 2001.

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