Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP−/− mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-α, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2−/− mice. Minor effects such as moderately decreased TNF-α and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR−/− animals. In contrast to all other mice studied, only TLR4−/− and PAFR−/− mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.

Gram-positive infections are a worldwide threat and a major cause of mortality. Infections of the respiratory tract are frequently caused by Gram-positive bacteria, and pneumonia is the seventh leading cause of mortality in the United States (1). Although much has been learned about Gram-negative infections and the importance of LPS therein, less is known about the host response to Gram-positive pathogens.

Lipoteichoic acid (LTA)2 is a major constituent of the outer cell wall of Gram-positive bacteria and the predominant mediator of inflammatory responses to these microorganisms (2, 3). LTA shares many pathogenic properties with LPS and is able to induce the production of a variety of proinflammatory cytokines and chemokines by cells of the innate immune system (4, 5, 6, 7). From in vitro studies, it is known that the cellular recognition and signaling receptor for LTA is TLR2 (8, 9, 10, 11, 12, 13). However, there is continuing controversy regarding the possibility of TLR4 being a receptor for LTA (14, 15). Although most investigators who excluded TLR4 as a signaling receptor for LTA studied cells from C3H/HeJ mice (i.e., mice that harbor a mutated TLR4), transfected cell lines, or applied neutralizing anti-TLR4 Abs, Takeuchi and colleagues (14) examined peritoneal macrophages from TLR4−/− mice and thereby found a diminished TNF-α and IL-6 response following stimulation with purified LTA from Staphylococcus aureus (8, 11, 12, 14, 15, 16, 17). A more recent article even disclosed TLR-independent (but CD14-dependent) responses by human polymorphonuclear cells (PMN) that were incubated with LTA (18). Importantly, the roles of TLR2 and TLR4 in the lung inflammatory response to LTA in vivo have not been investigated thus far.

Besides TLR2 and TLR4, several other molecules have been implicated in cellular responsiveness to LTA: in particular, CD14, LPS-binding protein (LBP), and the receptor for platelet-activating factor (PAFR). TLRs such as TLR2 colocalize with CD14 and LTA in lipid rafts of epithelial cells before trafficking to the Golgi apparatus (10) and CD14-dependent responses to diverse LTA preparations from different bacteria have been studied in various cell types in vitro (5, 11, 12, 15, 17, 18, 19). Most of these investigators described some degree of CD14 dependency when studying endpoints like cytokine/chemokine release or the rate at which PMNs undergo apoptosis. Of note, CD14 expression within the pulmonary compartment is not restricted to macrophages because CD14 has been detected on respiratory epithelial cells (20). Moreover, soluble CD14 can be recovered from alveolar lavage fluid (21). LBP is well-known as an acute phase protein that transfers LPS monomers from aggregates to the CD14/TLR4 receptor complex, thereby enhancing the immune response to LPS up to 1000-fold in vitro (22, 23, 24). LBP is also produced by respiratory epithelial cells in vitro and LPS administration results in a dose-dependent increase in bronchoalveolar lavage (BAL) fluid (BALF) LBP in vivo (21, 25, 26). However, LBP does not unequivocally augment inflammation as illustrated by our recent finding that pulmonary LBP inhibits the inflammatory response during LPS-induced lung inflammation in vivo (26). Less is known about the potential role of LBP during LTA-induced inflammation. Although few studies illustrated the requirement for LBP (11, 27), another report showed LBP-independent responses to LTA (19). Another important receptor for LTA, specifically on respiratory epithelial cells, has been identified to be PAFR (28). Lemjabbar et al. (28) disclosed that the interaction of LTA with PAFR induced the induction of NF-κB and mucin production within the lungs, while epithelial TLR2, in contrast to macrophage TLR2, did not play a role. More recent investigations revealed PAFR as an important factor in the LTA-induced NO production in vitro (29).

Pulmonary infections due to S. aureus are a serious threat, with LTA being a major immunogenic determinant. Although LBP is produced locally and CD14, TLR2, 4,and PAFR are expressed on alveolar macrophages (AMs) as well as on respiratory epithelial cells (20, 25, 26, 28, 29, 30, 31), knowledge of the precise function of these molecules during pulmonary inflammation induced by Gram-positive cell wall components in vivo is highly limited. Therefore, in the present study, we sought to systematically determine the importance of pulmonary LBP, CD14, TLR2, and TLR4 as well as PAFR in acute lung inflammation induced by highly purified LTA in vivo.

Pathogen-free, 8- to 12-wk-old C57BL/6, wild-type mice were purchased from Harlan Sprague Dawley. LBP−/− mice were generated as described previously and backcrossed to a C57BL/6 background 11 times (24). CD14−/−C57BL/6 mice were obtained from The Jackson Laboratory (32). TLR2−/− (14) and TLR4−/− (33) mice were generated as described and backcrossed 6 times to a C57BL/6 background. PAFR−/− mice were generated as described (34) and backcrossed 7 times to a C57BL/6 background. All mice were bred in the animal facility of the Academic Medical Center (Amsterdam, Netherlands). Age-matched female mice were used in all experiments. The Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands) approved all experiments.

Acute lung inflammation was induced as described previously (6, 26, 35). Briefly, mice were anesthetized by inhalation of isoflurane (Abbott Laboratories) and highly purified LTA (from S. aureus (16), LPS content below 5 pg/mg LTA as assessed by Limulus amebocyte lysate assay), diluted in 50 μl of sterile saline, was instilled intranasally (i.n.). Control mice received sterile saline. LTA purity of 99% was confirmed using magnetic resonance imaging; no lipoprotein contamination could be discerned (36). The optimal LTA dose (100 μg/mouse) was determined in preceding dose-finding experiments (data not shown). After 6 h, mice were anesthetized with hypnorm (Janssen Pharmaceutica) and midazolam (Roche) and sacrificed by bleeding out the vena cava inferior. Blood was collected in EDTA containing tubes and plasma was stored at −20°C until further usage. Based on previous experiments, the 6-h time point was chosen as the ideal time to simultaneously assess cytokine/chemokine alterations and cell influx. In some experiments, relative lung weights were examined by comparing mouse weight/lung weight at t = 6 h.

The trachea was exposed through a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter (Abbott). Bilateral BAL was performed by instilling two 0.5-ml aliquots of sterile saline. Approximately 0.9 ml of BALF was retrieved per mouse. Total cell numbers were counted from each sample using a hemocytometer (Türck chamber); BALF differential cell counts were done on cytospin preparations stained with Giemsa. BALF supernatant was stored at −20°C for cytokine and LBP measurements.

Lungs for histology were harvested at 6 h after infection, fixed in 10% formalin, and embedded in paraffin. Four-micrometer sections were stained with H&E and analyzed blinded for groups. To quantify lung inflammation and damage, the entire lung surface was semiquantitatively scored as described previously (13). In brief, the following parameters were analyzed: interstitial inflammation, intra-alveolar inflammation, endotheliitis, and bronchitis. Each parameter was graded on a scale of 0–3, with 0: absent, 1: mild, 2: moderate, and 3: severe. The total “lung inflammation score” was expressed as the sum of the scores for each parameter, the maximum being 12.

AMs for in vitro assays were harvested from TLR2−/−, TLR4−/−, and wild-type mice by BAL (n = 6–7/strain). Cells were washed and resuspended in RPMI 1640 containing 1 mM pyruvate, 2 mM l-glutamine, penicillin, streptomycin, and 10% FCS. Cells were allowed to adhere for 2 h in 96-well microtiter plates (Greiner) at a final concentration of 1 × 105 cells/well. Adherent monolayer cells were stimulated with LPS (from Escherichia coli O55:B5, 500 ng/ml; Sigma-Aldrich), highly purified LTA (from S. aureus, 40 μg/ml), or RPMI 1640 for 6 h. This specific LTA dose has been chosen based on previous in vitro experiments, as an equivalent to the LPS dose used. Supernatants were collected and stored at −70°C until assayed for TNF-α.

Human embryonic kidney (HEK) 293 cells stably expressing CD14, CD14-TLR4, or CD14-TLR2 (37, 38) were cultured overnight at a concentration of 2.5 × 105 cells/ml followed by stimulation with 100 ng/ml LPS (E. coli O55:B5), 10 μg/ml highly purified LTA (S. aureus), or DMEM for 6 h. Supernatants were collected and stored at −70°C until assayed for IL-8.

Murine LBP was measured using a commercially available ELISA (HyCult Biotechnology) according to the manufacturer’s instructions; the detection limit was 0.4 ng/ml. TNF-α, KC, MIP-2 (all R&D Systems), IL-10 (Bender MedSystems), and human IL-8 (Sanquin) were measured using specific ELISAs according to the manufacturer’s instructions. The detection limits were 31 pg/ml for TNF-α, 12 pg/ml for KC, 31 pg/ml for MIP-2, 39 pg/ml for IL-10, and 1 pg/ml for human IL-8. Cytokine and chemokine levels of untreated or NaCl-treated mice were below the detection limit (data not shown). Myeloperoxidase (MPO) concentrations were quantified using a commercially available ELISA (HyCult Biotechnology) according to the manufacturer’s instructions. Total protein in BALF samples was measured using the BCA protein assay kit (Pierce).

Differences between groups were calculated by Mann-Whitney U test. For multiple group comparison, Kruskal-Wallis testing followed by Dunn’s multiple comparison post-hoc test was performed. Values are expressed as mean ± SEM. A p value ≤ 0.05 was considered statistically significant.

We earlier showed that LTA is a potent inducer of chemotactic stimuli (4). Before studying the role of pulmonary LBP in LTA-induced lung inflammation in vivo, we first investigated whether LTA administration influences pulmonary LBP concentrations. As shown in Table I, low levels of alveolar LBP were detectable in saline-treated mice while LTA induced a significant rise in LBP concentrations. Plasma LBP levels were unaffected by pulmonary LTA administration (Table I) and no LBP was detectable in LBP−/− mice. Hence, i.n. administration of LTA induced a rapid increase in pulmonary LBP. Arguing that the local increase in pulmonary LBP might contribute to LTA-induced lung inflammation, we repeated these experiments using wild-type and LBP−/− mice and administered 100 μg of LTA i.n. and enumerated infiltrating PMNs 6 h thereafter. LTA induced a strong influx of cells to the alveolar compartment and differential counts disclosed a predominance of PMNs, although macrophages were also attracted (Fig. 1). However, the LTA-induced acute PMN influx (Fig. 1,A) and number of AMs (22 ± 7 and 19 ± 8 × 103/ml in wild-type and LBP−/− mice, respectively) were not impaired in the absence of LBP. Neither MPO concentrations in BALF (94.3 ± 18 and 78.6 ± 7 μg/ml in wild-type or LBP−/− mice, respectively; n.s., not significant) nor alveolar protein levels (Fig. 1,B) differed between the groups. In addition, an impressive increase in TNF-α, KC, and MIP-2 concentrations in BALF could be measured upon LTA challenge, which again did not depend on the presence of LBP (Fig. 1 C). Hence, these data indicate that the LTA-induced acute pulmonary inflammation does not depend on the presence of LBP in vivo.

Table I.

LBP concentrations in BALF and plasmaa

LBP ConcentrationsBALF (ng/ml)Plasma (μg/ml)
Control 11.7 ± 3.9 5.3 ± 0.9 
6 h post NaCl i.n. 16.8 ± 5.8b 3.5 ± 0.5 
6 h post 100 μg of LTA i.n. 55.7 ± 6.1b,c 4.1 ± 0.3 
LBP ConcentrationsBALF (ng/ml)Plasma (μg/ml)
Control 11.7 ± 3.9 5.3 ± 0.9 
6 h post NaCl i.n. 16.8 ± 5.8b 3.5 ± 0.5 
6 h post 100 μg of LTA i.n. 55.7 ± 6.1b,c 4.1 ± 0.3 
a

Wild-type mice (n = 6–8) were inoculated with saline or 100 μg of LTA i.n. and BAL was performed after 6 h. LBP concentrations were measured with ELISA in BALF and plasma. Samples of untreated mice served as controls (n = 6). Depicted are mean ± SEM.

b

Value of p < 0.05 vs control.

c

Value of p< 0.05 vs NaCl-treated mice.

FIGURE 1.

LTA induces acute pulmonary inflammation is independent of LBP. A, PMN counts; B, total protein; C, TNF-α, KC, and MIP-2 concentrations obtained from BALF (eight mice per strain) 6 h after i.n. administration of 100 μg of LTA. ▪, Wild-type mice (Wt); □, LBP−/− mice. Data are mean ± SEM.

FIGURE 1.

LTA induces acute pulmonary inflammation is independent of LBP. A, PMN counts; B, total protein; C, TNF-α, KC, and MIP-2 concentrations obtained from BALF (eight mice per strain) 6 h after i.n. administration of 100 μg of LTA. ▪, Wild-type mice (Wt); □, LBP−/− mice. Data are mean ± SEM.

Close modal

Next, we were interested in whether the reported importance of CD14 in the inflammatory response to LTA can be reproduced in vivo (5, 11, 12, 15, 17, 18, 19). Wild-type and CD14−/− mice were inoculated i.n. with LTA and the inflammatory response was evaluated. Although pulmonary TNF-α and MIP-2 concentrations were reduced in CD14−/− animals, KC levels, PMN influx, the number of AMs (26 ± 4 and 24 ± 4 × 103/ml in wild-type and CD14−/− mice, respectively; n.s.) as well as alveolar protein concentrations and BALF MPO levels (94 ± 21 and 62 ± 28 μg/ml in wild-type and CD14−/− mice, respectively; n.s.) did not differ between wild-type and gene-deficient mice (Fig. 2). Therefore, CD14 has only a moderate effect on pulmonary inflammation induced by LTA.

FIGURE 2.

LTA-induced pulmonary inflammation partially depends on CD14. A, PMN counts; B, total protein; C, TNF-α, KC, and MIP-2 concentrations obtained from BALF (n = 6/strain) 6 h after i.n. administration of 100 μg of LTA. ▪, Wild-type mice (Wt); □, CD14−/− mice. Data are mean ± SEM; ∗, p < 0.05 vs wild-type mice.

FIGURE 2.

LTA-induced pulmonary inflammation partially depends on CD14. A, PMN counts; B, total protein; C, TNF-α, KC, and MIP-2 concentrations obtained from BALF (n = 6/strain) 6 h after i.n. administration of 100 μg of LTA. ▪, Wild-type mice (Wt); □, CD14−/− mice. Data are mean ± SEM; ∗, p < 0.05 vs wild-type mice.

Close modal

TLR2 has been repeatedly shown to be the most important receptor mediating the response to LTA in vitro (8, 9, 10, 11, 12, 13). In accordance, we found mice lacking TLR2 to be nonresponsive to i.n. LTA. Although in wild-type mice LTA induced brisk responses, the increase in the number of PMN (Fig. 3,A) and AMs (23 ± 3 and 8 ± 3 × 103/m in wild-type and TLR2−/− mice, respectively, p < 0.05), as well as local TNF-α, KC, or MIP-2 release, was abolished in TLR2−/− mice (Fig. 3,B). MPO was barely detectable in BALF from TLR2−/− mice (63 ± 21 and 0.7 ± 0.2 μg/ml in wild-type and TLR2−/− mice, respectively; p < 0.05) and greatly reduced in TLR2−/− lungs (30.5 ± 5.1 and 4.4 ± 1.1 mg/ml in wild-type and TLR2−/− mice, respectively). Furthermore, leakage was significantly reduced in the absence of TLR2 as illustrated by differences in alveolar protein concentrations (Fig. 3,C) and relative lung weights (Fig. 3,D). Histological workup of lung specimens finally confirmed the importance for TLR2 in LTA-induced lung inflammation in vivo (Fig. 3 E). The extent of lung inflammation was more severe in wild-type mice than TLR2−/− animals (inflammation score: 4.6 ± 0.8 in wild-type and 0.6 ± 0.2 in TLR2−/− mice, p < 0.05).

FIGURE 3.

LTA-induced pulmonary inflammation entirely depends on TLR2. Mice (n = 6–7/strain) were inoculated i.n. with 100 μg of LTA and (A) PMN counts, (B) TNF-α, KC, and MIP-2 concentrations, as well as (C) total protein levels and (D) relative lung weights (lung weight (g)/mouse weight (g)) were evaluated after 6 h. ▪/Filled symbols, wild-type mice (Wt); □/open symbols, TLR2−/− mice. Data are mean ± SEM (A–C); ∗, p < 0.05 vs wild-type mice. E, Paraffin-embedded lungs, stained with H&E, were scored as described in Materials and Methods. Representative slides of n = 7/group are presented; magnification ×40.

FIGURE 3.

LTA-induced pulmonary inflammation entirely depends on TLR2. Mice (n = 6–7/strain) were inoculated i.n. with 100 μg of LTA and (A) PMN counts, (B) TNF-α, KC, and MIP-2 concentrations, as well as (C) total protein levels and (D) relative lung weights (lung weight (g)/mouse weight (g)) were evaluated after 6 h. ▪/Filled symbols, wild-type mice (Wt); □/open symbols, TLR2−/− mice. Data are mean ± SEM (A–C); ∗, p < 0.05 vs wild-type mice. E, Paraffin-embedded lungs, stained with H&E, were scored as described in Materials and Methods. Representative slides of n = 7/group are presented; magnification ×40.

Close modal

The role of TLR4 in cellular responsiveness to LTA has been debated (8, 11, 12, 14, 15, 17), whereas PAFR has been recognized as the most important epithelial LTA-recognition receptor within the pulmonary compartment in vitro (28). To explore these findings in vivo, we inoculated PAFR−/−, TLR4−/−, and wild-type mice with LTA and evaluated the inflammatory response (Fig. 4). To exclude the possibility of LPS contamination, we performed these experiments with an entirely new LTA batch, obtained from a separate purification procedure. PAFR and TLR4 had a comparable impact on pulmonary inflammation, indicated by strongly diminished cell influx and local TNF, KC, MIP-2, and alveolar protein concentrations in mice lacking either receptor (Fig. 4). Although PMN influx was significantly reduced in PAFR−/− and TLR4−/− mice (Fig. 4,A), the number of AMs was highest in PAFR−/− animals (17 ± 5 × 103/ml in wild-type, 32 ± 6 × 103/ml in PAFR−/−, and 15 ± 3 × 103/ml in TLR4−/− mice, respectively; p < 0.05, when comparing PAFR−/− and TLR4−/− mice). In line with this, MPO concentrations in BALF were lowest in TLR4−/− animals (35.5 ± 9 μg/ml in wild-type, 15 ± 4 μg/ml in PAFR−/−, and 5 ± 2 μg/ml in TLR4−/− mice; p < 0.05, when comparing wild-type and TLR4−/− mice). Of note, the overall inflammatory response in this specific experiment was less pronounced (as compared with Fig. 3), which might be due to batch-to-batch variations in the biological activity of LTA. Hence, both TLR4 and PAFR importantly contribute to pulmonary inflammation induced by LTA in vivo.

FIGURE 4.

TLR4 and PAFR equally contribute to LTA-induced pulmonary inflammation. Indicated mice (n = 5–6/strain) were treated i.n. with 100 μg of LTA and (A) PMN counts, (B) total protein, and (C) TNF-α, KC, and MIP-2 concentrations were assessed after 6 h. ▪, Wild-type mice (Wt); □, TLR4−/− mice; ▦, PAFR−/− mice. Data are mean ± SEM; ∗, p < 0.05 vs wild-type mice.

FIGURE 4.

TLR4 and PAFR equally contribute to LTA-induced pulmonary inflammation. Indicated mice (n = 5–6/strain) were treated i.n. with 100 μg of LTA and (A) PMN counts, (B) total protein, and (C) TNF-α, KC, and MIP-2 concentrations were assessed after 6 h. ▪, Wild-type mice (Wt); □, TLR4−/− mice; ▦, PAFR−/− mice. Data are mean ± SEM; ∗, p < 0.05 vs wild-type mice.

Close modal

After having investigated the requirement for LBP, CD14, TLR2, TLR4, and PAFR for an appropriate proinflammatory response, we asked whether these receptors also affect the anti-inflammatory response. Neither LBP nor CD14 or TLR2 deficiency resulted in altered IL-10 concentrations (Fig. 5). Of great interest, however, PAFR−/− and TLR4−/− mice displayed significantly higher alveolar IL-10 levels when compared with wild-type mice (Fig. 5).

FIGURE 5.

Enhanced anti-inflammatory IL-10 response in TLR4−/− and PAFR−/− mice. IL-10 concentrations were measured in BALF of indicated mice (n = 5–8/strain) 6 h after i.n. administration of 100 μg of LTA. ▪, Wild-type mice (Wt); □, LBP, CD14, TLR2, or TLR4−/− mice; ▦, PAFR−/− mice. Date are mean ± SEM; ∗, p < 0.05 vs wild-type mice.

FIGURE 5.

Enhanced anti-inflammatory IL-10 response in TLR4−/− and PAFR−/− mice. IL-10 concentrations were measured in BALF of indicated mice (n = 5–8/strain) 6 h after i.n. administration of 100 μg of LTA. ▪, Wild-type mice (Wt); □, LBP, CD14, TLR2, or TLR4−/− mice; ▦, PAFR−/− mice. Date are mean ± SEM; ∗, p < 0.05 vs wild-type mice.

Close modal

Given the rather unexpected finding of decreased pulmonary inflammation in TLR4−/− mice treated with LTA, we then were interested in the role of primary AMs herein. For this purpose, isolated AMs from wild-type, TLR4−/−, and TLR2−/− mice were stimulated with LTA or LPS in vitro and released TNF-α, MIP-2, KC, and IL-10 was quantified. Fig. 6,A illustrates that the in vitro TNF-α response of murine AMs to LTA depended exclusively on TLR2. In addition, measurements of LTA-induced MIP-2 and KC release also revealed the requirement for TLR2 but not TLR4 (data not shown), whereas IL-10 levels were below the detection limit. Moreover, human HEK cells, transfected with either CD14 or combinations of CD14 and TLR2 or TLR4, only responded to LTA in the presence of TLR2 (Fig. 6 B). Together, the in vitro response to LTA specifically requires TLR2.

FIGURE 6.

In vitro response to LTA depended on TLR2. A, Stably transfected HEK cells were incubated in quadruplicates with DMEM medium, LPS (100 ng/ml), or LTA (10 μg/ml) for 6 h and IL-8 release was measured in supernatants using an ELISA. B, Adherent primary AMs, isolated from wild-type, TLR4−/−, or TLR2−/− mice (n = 6–7 mice/strain), were stimulated with LPS (500 ng/ml), LTA (40 μg/ml), or were left untreated (medium). TNF-α release was evaluated in supernatants. Data are mean ± SEM; ∗, p < 0.05 vs wild type. Representative data of two independent experiments are shown.

FIGURE 6.

In vitro response to LTA depended on TLR2. A, Stably transfected HEK cells were incubated in quadruplicates with DMEM medium, LPS (100 ng/ml), or LTA (10 μg/ml) for 6 h and IL-8 release was measured in supernatants using an ELISA. B, Adherent primary AMs, isolated from wild-type, TLR4−/−, or TLR2−/− mice (n = 6–7 mice/strain), were stimulated with LPS (500 ng/ml), LTA (40 μg/ml), or were left untreated (medium). TNF-α release was evaluated in supernatants. Data are mean ± SEM; ∗, p < 0.05 vs wild type. Representative data of two independent experiments are shown.

Close modal

Pneumonia is associated with a profound inflammatory response within the pulmonary compartment. Inflammation induced by Gram-positive pathogens is predominantly elicited by bacterial cell wall components such as LTA and peptidoglycan (3, 6). Although many investigators studied the requirement of pattern recognition receptors that sense the presence of LTA in vitro, little is known about these pathways in vivo. We here for the first time investigated the recognition machinery for LTA during pulmonary inflammation in vivo using mice lacking LBP, CD14, TLR2, TLR4, or PAFR. In accordance with earlier in vitro studies, we revealed in particular TLR2, and to a lesser extent PAFR, to contribute to pulmonary inflammation caused by LTA in vivo. Quite surprisingly, we discerned an important role for TLR4 in LTA-induced lung inflammation in vivo, while CD14 played a moderate role and LBP was not required.

Pulmonary host defense against invading pathogens invariably involves the activation of macrophages and epithelial cells, the release of proinflammatory cytokines and chemokines, and the recruitment of PMNs. Our data underline the crucial importance of TLR2 during LTA-induced lung inflammation in vivo because the inflammatory response was abolished in the absence of TLR2. However, we observed that despite the presence of TLR2, other surface receptors such as PAFR and TLR4 also contributed—albeit to a lesser degree—to the pulmonary immune response. Because the absence of these receptors did not abolish, but rather diminished, pulmonary inflammation, we hypothesize two potential scenarios: first, LTA might be an actual ligand for TLR4 and/or PAFR; second, the LTA-TLR2-induced inflammation instigates the generation of endogenous mediators that serve as ligands for PAFR and/or TLR4 and thus synergize with LTA in the inflammatory response. Potential endogenous mediators that are in particular generated during pulmonary inflammation, such as PAF, fragmented hyaluronan, oxidation products, biglycans, or heat shock proteins have been identified as PAFR and/or TLR4 ligands (39, 40, 41, 42, 43, 44, 45). In addition, LTA might form complexes with endogenous molecules that in turn synergize and enhance the inflammatory response. This synergistic effect has been shown for hemoglobin and LTA (46) and more recent data disclosed a role for TLR4 herein (47). However, due to the potential leakage of hemoglobin during BAL, we were not able to reliably measure hemoglobin levels in this model. Of note, BALF did not contain visible erythrocytes after the lavage procedure.

Performing in vitro assays using primary AMs and transfected HEK cells, we were able to clearly confirm that TLR2 but not TLR4 is the LTA-recognizing receptor. The finding that TLR4 played a role during LTA-induced lung inflammation in vivo certainly warranted us to exclude the possibility of LPS contamination. We believe that LPS contamination cannot explain our findings because the LPS content of the LTA preparation was very low (<5 pg of LPS/mg of LTA), which results in i.n. inoculation of a maximum of 0.5 pg of LPS; this amount is not sufficient to induce a detectable inflammatory response in the mouse lung (26, 48). In addition, contaminating LPS would have elicited an inflammatory response in TLR2−/− mice, which clearly did not occur. To our knowledge, this is the first study investigating the function of TLR4 in LTA-induced inflammation in vivo. Earlier reports that studied and excluded the role of TLR4 in vitro used either transfected cell lines, neutralizing Abs, or cells from C3H/HeJ mice (8, 11, 12, 15, 16, 17). In fact, Takeuchi et al. (14) were the only ones that used cells from the same mouse strain described here and found TLR4−/− peritoneal macrophages to be hyporesponsive to LTA. Importantly, by stimulating TLR4−/− macrophages with various Gram-positive cell wall components including peptidoglycan, Takeuchi et al. (14) clearly demonstrated an appropriate responsiveness comparable to wild-type animals. In addition, our group recently showed that TLR4−/− mice bred in our institution mount an unaltered (and strong) inflammatory response in their lungs upon intrapulmonary delivery of Burkholderia pseudomallei (49). Together, these data make the possibility of an overall impaired inflammatory response in the absence of TLR4 unlikely. However, our results from in vitro stimulations of primary AMs obtained from wild-type, TLR2−/−, and TLR4−/− mice definitely point toward TLR2 as the LTA-signaling cell receptor (Fig. 6). Hence, the possibility of LTA being a direct ligand for TLR4 seems unlikely, whereas the in vivo effects described here might be related to the multifaceted interplay between different cell types and mediators within the lungs that cannot be revealed using in vitro techniques.

PAFR has been reported to recognize LTA. Lemjabbar et al. (28) demonstrated that respiratory epithelial cells signal the presence of LTA via PAFR and that this process occurs, in contrast to macrophages, irrespective of TLR2. However, the precise participation of PAFR in the inflammatory response to LTA is difficult to discern. Although LTA has been proposed as a direct ligand for PAFR by some investigators, other reports indicate that LTA may (possibly via TLR2) induce the release of PAF that in turn acts via PAFR (28, 29). Our studies do not allow for distinguishing between these two pathways, and PAF concentrations are hard to determine in diluted samples such as BALF due to the low sensitivity of available assays and the fact that PAF largely exists in a cell-associated form (29, 50). However, our data clearly indicate an important role for PAFR during LTA-induced acute lung injury, irrespective of the mode of activation.

Of great interest, we observed increased IL-10 levels in BALF from both TLR4−/− and PAFR−/− mice challenged with LTA. It is known that under normal circumstances, anti-inflammatory pathways are activated within the pulmonary compartment, most likely to prevent perpetual inflammation due to inhalation of minute amounts of, e.g., polluted air in daily life (51). Bronchial epithelial cells have been identified as the source of constitutive pulmonary IL-10 in humans and mice (51, 52). Once the lung is exposed to potentially harmful substances, such as bacteria, the initiation of an inflammatory response is crucial for the elimination of the infection and to prevent further damage to the host (53). Enhanced IL-10 levels have been repeatedly shown to worsen the host defense during bacterial pneumonia in vivo (53, 54, 55, 56). LTA-triggered activation via TLR2 induces an inflammatory response, while TLR4 and/or PAF-mediated signaling might also counteract the constitutive anti-inflammatory milieu. Counteracting anti-inflammatory pathways might be a direct result of LTA-induced TLR4/PAF involvement or an indirect consequence of above-mentioned endogenous mediators that are generated in the course of TLR2-triggered inflammation. The lack of inflammation and consecutive absence of endogenously generated mediators would explain our observation of unaltered IL-10 levels in TLR2−/− mice. Another explanation, not excluding the one suggested above, could be that the presumed immunosuppressive effects mediated by TLR2 (57) may become more visible in the absence of receptors mediating inflammatory responses (such as TLR4 and PAFR).

Together, our data illustrate the principal importance of pulmonary TLR2 as the signaling receptor for LTA in vivo and in vitro. Additionally, we discovered that PAFR and TLR4 importantly contribute to the pulmonary inflammation induced by LTA in vivo. Although LTA might be a direct ligand for PAFR, the possibility exists that pulmonary TLR4 and/or PAFR signal the presence of TLR2-triggered endogenous, proinflammatory mediators in vivo.

We are grateful to Dr. Satoshi Ishii and Prof. Takao Shimizu for providing PAFR−/− mice and Prof. Shizuo Akira for providing TLR2−/− and TLR4−/− mice. The expert technical assistance of Joost Daalhuisen and Ingvild Kop was highly appreciated.

The authors have no financial conflict of interest.

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.

2

Abbreviations used in this paper: LTA, lipoteichoic acid; PMN, polymorphonuclear cell; LBP, LPS-binding protein; PAFR, platelet-activating factor receptor; BAL, bronchoalveolar lavage; BALF, BAL fluid; AM, alveolar macrophage; HEK, human embryonic kidney; i.n., intranasal(ly); KC, keratinocyte-derived chemokine; MPO, myeloperoxidase; n.s., not significant.

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