Abstract
Rationale: CD14 is a pattern recognition receptor that can interact with a variety of bacterial ligands. During gram-negative infection, CD14 plays an important role in the induction of a protective immune response by virtue of its capacity to recognize lipopolysaccharide in the bacterial cell wall. Knowledge of the contribution of CD14 to host defense against gram-positive infections is limited.
Objectives: To study the role of CD14 in gram-positive bacterial pneumonia.
Methods: CD14 knockout (KO) and normal wild-type (WT) mice were intranasally infected with Streptococcus pneumoniae.
Measurements and Main Results: CD14 KO mice demonstrated a strongly reduced lethality, which was accompanied by a more than 10-fold lower bacterial load in lung homogenates but not in bronchoalveolar lavage fluid at 48 hours after infection. Strikingly, CD14 KO mice failed to develop positive blood cultures, whereas WT mice had positive blood cultures from 24 hours onward and eventually invariably had evidence of systemic infection. Lung inflammation was attenuated in CD14 KO mice at 48 hours after infection, as evaluated by histopathology and cytokine and chemokine levels. Intrapulmonary delivery of recombinant soluble CD14 to CD14 KO mice rendered them equally susceptible to S. pneumoniae as WT mice, resulting in enhanced bacterial growth in lung homogenates and bacteremia, indicating that the presence of soluble CD14 in the bronchoalveolar compartment is sufficient to cause invasive pneumococcal disease.
Conclusion: These data suggest that S. pneumoniae uses (soluble) CD14 present in the bronchoalveolar space to cause invasive respiratory tract infection.
CD14 has a central role in host defense against gram-negative infection. The contribution of CD14 in gram-positive infection is limited.
CD14, either cell bound or soluble, facilitates invasive pneumococcal disease during respiratory tract infection with Streptococcus pneumoniae.
Investigations of the role of CD14 during inflammation and infection in vivo have almost exclusively focused on LPS and gram-negative bacterial infections (15–21). These studies have established that CD14 plays a pivotal part in systemic and pulmonary inflammation induced by LPS. The recognition of LPS by CD14, resulting in a rapid induction of an innate immune response via TLR4, contributes to an effective host defense against intact gram-negative bacteria. Indeed, elimination or inhibition of CD14 has been found to facilitate the outgrowth of several gram-negative pathogens in vivo (19–21). In this respect, our laboratory recently documented a clear role for CD14 in improving the clearance of clinically relevant pathogens such as Haemophilus influenzae (22) and Acinetobacter baumannii (23) from the mouse respiratory tract. In contrast to this abundant data on the contribution of CD14 in gram-negative infections, knowledge of the role of this receptor in host defense against gram-positive bacteria is limited. In a model of severe sepsis induced by intravenous or intraperitoneal injection of Staphylococcus aureus, CD14 knockout (KO) mice displayed unaltered bacterial loads and survival when compared with normal wild-type (WT) mice (24). More recently, CD14 KO mice were found to be more susceptible to meningitis induced by intrathecal administration of Streptococcus pneumoniae, as reflected by higher disease severity scores and an accelerated mortality (25).
S. pneumoniae is the most prevalent microorganism in community-acquired pneumonia, which is responsible for more than half a million cases each year in the United States alone, and has a fatality rate of 5 to 7% (26, 27). Bacteremia with S. pneumoniae originates from the lungs in almost 90% of cases. In addition, in recent sepsis trials, the pneumococcus was an important causative pathogen, especially in the context of pneumonia (28). We here sought to determine the role of CD14 in the host response to respiratory tract infection caused by S. pneumoniae.
C57BL/6 WT mice were purchased from Charles River (Maastricht, The Netherlands). CD14 KO mice, backcrossed to a C57BL/6 genetic background, were obtained from Jackson Laboratories (Bar Harbor, ME) and bred in the animal facility of the Academic Medical Center in Amsterdam. Sex- and age-matched (10–12 wk) mice were used. All experiments were approved by the Animal Care and Use Committee of the University of Amsterdam.
Pneumonia was induced as described earlier (29–31). Mice were lightly anesthetized by inhalation of isoflurane (Upjohn, Ede, The Netherlands) and 50 μl containing 1–5 × 104 cfu S. pneumoniae serotype 3 (ATCC 6303; American Type Culture Collection, Rockville, MD) were inoculated intranasally. In these experiments, mice were killed at 5, 24, or 48 hours after infection or monitored for 2 weeks. In a separate experiment, mice infected with S. pneumoniae received either saline or recombinant mouse soluble (s) CD14 (1 μg; Biometec, Greifswald, Germany) intranasally at 0 and 24 hours relative to the time of infection; mice were killed 48 hours after infection. In an additional survival experiment, mice received either saline or sCD14 at 0, 24, and 48 hours relative to the time of infection.
Lung bacterial loads were determined as described earlier (29–31). Briefly, mice were anesthetized with Hypnorm (Janssen Pharmaceutica, Beerse, Belgium) and midazolam (Roche, Meidrecht, The Netherlands), and blood and lungs were collected. Lungs were homogenized at 4°C in 5 volumes of sterile isotonic saline with a tissue homogenizer (Biospect Products, Bartlesville, OK). Serial 10-fold dilutions in sterile isotonic saline were made from these homogenates (and blood), and 50-μl volumes were plated onto sheep blood agar plates and incubated overnight at 37°C and 5% CO2.
Bronchoalveolar lavage fluid (BALF) was obtained as described earlier (32). Briefly, the trachea was exposed through a midline incision and BALF was harvested by instilling and retrieving two 0.5-ml aliquots of sterile isotonic saline. Total cell numbers were counted using a Z2 Coulter particle count and size analyzer (Beckman-Coulter, Inc., Miami, FL). BALF differential cell counts were carried out on cytospin preparations stained with modified Giemsa stain (Diff-Quick; Baxter, McGraw Park, IL).
Lungs for histology were prepared and analyzed as described earlier (30). The parameters for bronchitis, edema, interstitial inflammation, intraalveolar inflammation, pleuritis, and endothelialitis were graded on a scale of 0 to 4, with 0 as “absent” and 4 as “severe.” The total “lung inflammation score” was expressed as the sum of the scores for each parameter, the maximum being 24. Granulocyte staining was performed using fluorescein isothiocyanate–labeled rat anti-mouse Ly-6G mAb (Pharmingen, San Diego, CA), exactly as described (30).
Lung homogenates were prepared as described earlier (30). Tumor necrosis factor (TNF)-α, IL-6, IL-10, and monocyte chemoattractant protein (MCP)-1 were measured by cytometric beads array (CBA) multiplex assay (BD Biosciences, San Jose, CA). IL-1β, macrophage inflammatory protein (MIP)-2, and cytokine-induced neutrophil chemoattractant (KC) were measured by ELISA (R&D Systems, Abingdon, UK). Total protein concentrations were measured in BALF using the BCA protein kit (Pierce, Rockford, IL). sCD14 was measured by ELISA (Biometec).
All data are given as means ± SEM and were analyzed using GraphPad Prism 4 (GraphPad Prism version 4.00 for Windows; GraphPad Software, San Diego, CA). Differences between groups were analyzed using the Mann-Whitney U test or Kruskal-Wallis analysis where appropriate. For survival analyses, Kaplan-Meier analysis followed by log rank test or Cox regression analysis were performed where appropriate. A p value less than 0.05 was considered statistically significant.
To investigate the role of CD14 in the outcome of pneumococcal pneumonia, WT and CD14 KO mice were inoculated with S. pneumoniae (either 1 or 5 × 104 cfu in two independent experiments) and monitored for 14 days (Figures 1A and 1B). After infection with the lower dose, WT mice started dying after 2 days and 93% had died by Day 7. In contrast, the first CD14 KO mice died after 4 days and only 21% had died at the end of the observation period (p < 0.0001 for the difference between groups). After infection with the higher dose, the vast majority of WT mice died shortly after the second day and all animals were dead at Day 6; CD14 KO mice displayed a delayed mortality and 16% survived (p < 0.005 for the difference between groups). These data suggested that CD14 contributes to lethality during S. pneumoniae pneumonia.
Figure 1. CD14 knockout (KO) mice are protected against pneumococcal pneumonia. Survival after intranasal infection with (A) 1 × 104 cfu or (B) 5 × 104 cfu Streptococcus pneumoniae. Mortality was assessed four times daily for 14 days (n = 13–14 mice/group in each experiment).
[More] [Minimize]To obtain insight in the involvement of CD14 during the early phase of host defense against pneumococcal pneumonia, bacterial loads were determined in lung homogenates and blood obtained from WT and CD14 KO mice 5, 24, or 48 hours after infection (i.e., at time points before the first WT mice started dying). Although, at the first two time points, the number of S. pneumoniae colony-forming units that were recovered from lung homogenates was similar in WT and CD14 KO mice, at 48 hours after infection the bacterial load in lungs of CD14 KO mice was more than 10-fold lower than in the lungs of WT mice (p < 0.001, Figure 2A). Strikingly, WT mice had positive blood cultures from 24 hours onward (24 h: 4 out of 7 mice; 48 h: 8 out of 8 mice), whereas no CD14 KO mouse had a positive blood culture at 24 hours and S. pneumoniae could be cultured from blood at 48 hours from only one of eight CD14 KO mice (Figure 2A). This latter finding, which was reproduced in three independent experiments, suggested that CD14 contributes to the invasion of pneumococci from the alveolar compartment (the primary site of the infection) into the blood stream. This prompted us to perform the next series of experiments to obtain insight into the bacterial loads in the bronchoalveolar compartment of WT and CD14 KO mice after intranasal instillation of S. pneumoniae. For this, the alveolar space was gently lavaged 5, 24, or 48 hours after infection, and the number of S. pneumoniae colony-forming units was counted in the BALF obtained. In contrast to the differences in bacterial burdens in whole lung homogenates (and blood), BALF of WT and CD14 KO mice contained equal numbers of S. pneumoniae at all time points (Figure 2B). To obtain insight into the location of bacteria in the lungs of CD14 KO and WT mice, we performed Gram stains on lung tissue slides. S. pneumoniae was visualized primarily extracellularly throughout infected areas in the lungs without apparent differences between the two mouse strains (see Figure E1 of the online supplement). Together, these data suggested that CD14 contributes to the invasion of S. pneumoniae from the alveolar space into lung tissue and the circulation.
Figure 2. CD14 KO mice demonstrate reduced invasiveness and dissemination of infection. Bacterial loads in (A) lungs and (B) bronchoalveolar lavage fluid (BALF) at 5, 24, and 48 hours after infection with 5 × 104 cfu S. pneumoniae. BC+ indicates the number of positive blood cultures. Data are means ± SEM (n = 7–8 mice/group at each time point). *p < 0.001 versus WT mice.
[More] [Minimize]To determine the role of CD14 in the induction of pulmonary inflammation in response to S. pneumoniae infection, lung tissue slides were prepared from WT and CD14 KO mice at 5, 24, or 48 hours after infection. Although at 5 hours the extent of lung inflammation did not differ between the two mouse strains, pulmonary inflammation was clearly less pronounced in CD14 KO mice, as determined by the semiquantitative scoring system described in Methods, at both 24 hours (p < 0.05) and 48 hours (p < 0.005) after inoculation (Table 1). Representative slides of lung tissue from WT and CD14 KO mice 24 and 48 hours after inoculation of S. pneumoniae are presented in Figure 3. In addition, CD14 KO mice demonstrated a reduced accumulation of neutrophils in lung tissue at 24 and 48 hours after infection, as visualized by Ly-6G staining (not shown). The reduced lung inflammation in CD14 KO mice was accompanied by an attenuated leak of protein into BALF at 48 hours (p < 0.05 vs. WT mice, Table 1). Of note, uninfected CD14 KO and WT mice displayed similar leukocyte differentiation in peripheral blood (data not shown).
Figure 3. CD14 KO mice display reduced lung inflammation. Representative lung slides of (A and B) WT and (C and D) CD14 KO mice 24 hours (A and C) and 48 hours (B and D) after infection with 5 × 104 cfu S. pneumoniae. Hematoxylin-and-eosin staining; original magnification, ×4.
[More] [Minimize]T = 5 h | T = 24 h | T = 48 h | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| WT | CD14 KO | WT | CD14 KO | WT | CD14 KO | |||||||
| Total lung score, AU | n.d. | n.d. | 7.0 ± 1.5 | 3.0 ± 0.8* | 12.8 ± 1.9 | 5.3 ± 1.6† | ||||||
| Total protein level BAL, μg/ml | 249 ± 8 | 254 ± 12 | 278 ± 24 | 308 ± 25 | 743 ± 123 | 362 ± 47* | ||||||
| Neutrophil count BAL, ×103/ml | 5 ± 2 | 1 ± 0.2* | 48 ± 12 | 16 ± 3* | 100 ± 14 | 97 ± 24 | ||||||
| Cytokine and Chemokine Production in Lung Homogenate (pg/ml) | ||||||||||||
| TNF-α | 34 ± 9 | 29 ± 8 | 108 ± 54 | 267 ± 75 | 421 ± 82 | 528 ± 229 | ||||||
| IL-1β | b.d. | b.d. | 2,960 ± 1,390 | 4,772 ± 1,609 | 12,596 ± 865 | 4275 ± 2872* | ||||||
| IL-6 | 20 ± 8 | 30 ± 11 | 368 ± 142 | 546 ± 181 | 2,972 ± 542 | 479 ± 192‡ | ||||||
| IL-10 | 218 ± 91 | 199 ± 26 | 675 ± 55 | 1,067 ± 127* | 2,038 ± 423 | 566 ± 17† | ||||||
| MCP-1 | 226 ± 35 | 230 ± 34 | 1,219 ± 409 | 776 ± 200 | 7,499 ± 955 | 1,568 ± 459‡ | ||||||
| MIP-2 | 272 ± 30 | 238 ± 29 | 3,112 ± 355 | 3,991 ± 416 | 19,131 ± 4,928 | 7,591 ± 3166* | ||||||
| KC | 179 ± 18 | 320 ± 61 | 2,346 ± 515 | 2,391 ± 575 | 5,922 ± 748 | 2,352 ± 459† | ||||||
| Cytokine and Chemokine Production in Plasma (pg/ml) | ||||||||||||
| TNF-α | 20 ± 8 | 30 ± 11 | 368 ± 142 | 546 ± 181 | 2,972 ± 542 | 479 ± 192‡ | ||||||
| IL-6 | 4 ± 3 | 1 ± 0.3 | 196 ± 71 | 36 ± 9* | 6,281 ± 1,789 | 86 ± 16† | ||||||
| MCP-1 | 15 ± 3 | 11 ± 1 | 49 ± 14 | 14 ± 3† | 1,224 ± 406 | 31 ± 5† | ||||||
The histologic analyses indicated that CD14 deficiency resulted in a reduced lung inflammatory response to S. pneumoniae, including a diminished influx of neutrophils into lung tissue. Considering that neutrophils play an important role in the immune response to bacterial pneumonia (33), we next sought to evaluate the extent of neutrophil recruitment into the bronchoalveolar space. At 5 and 24 hours after infection, the number of neutrophils in BALF of CD14 KO mice was less than that in BALF of WT mice (p = 0.05 and p < 0.05, respectively; Table 1). Forty-eight hours after infection, neutrophil counts were equal in BALF of both mouse strains.
Cytokines and chemokines play an important role in host defense against bacterial pneumonia (34). Thus, we determined the concentrations of TNF-α, IL-1β, IL-6, IL-10, MCP-1, MIP-2, and KC in lung homogenates obtained 5, 24, and 48 hours after infection (Table 1). Except for increased IL-10 production in lungs of CD14 KO mice 24 hours after inoculation (p < 0.05), the levels of these mediators did not differ between the two mouse strains at 5 and 24 hours after inoculation. At 48 hours, CD14 KO mice demonstrated reduced concentrations of all mediators except TNF-α (Table 1). Twenty-four hours after inoculation, IL-6 and MCP-1 levels in plasma were lower compared with those in WT mice; TNF-α, MCP-1, and IL-6 levels were also lower in CD14 KO mice 48 hours after inoculation (Table 1).
We next hoped to determine whether sCD14 could compensate for CD14 gene deficiency during S. pneumoniae pneumonia. First, we measured sCD14 concentrations in BALF harvested from WT mice before and 5, 24, or 48 hours after infection. sCD14 was readily detectable in normal BALF and significantly increased during the course of pneumonia (p < 0.05, Figure 4A). Intranasal administration of recombinant mouse sCD14 to CD14 KO mice changed this mouse strain into a WT phenotype during pneumonia. Indeed, whereas CD14 KO mice were protected against lethality when compared with WT mice (confirming the data presented in Figure 1), CD14 KO mice treated with sCD14 showed accelerated and increased lethality similar to WT mice (Figure 4B). In addition, whereas CD14 KO mice displayed more than 10-fold lower bacterial loads in lung homogenates than WT mice at 48 hours after infection and whereas only one of eight CD14 KO mice had a positive blood culture at this time point versus eight of eight WT mice (confirming the data presented in Figure 2A), CD14 KO mice that had received sCD14 demonstrated similar bacterial loads when compared with WT mice, and seven of eight of CD14 KO mice treated with sCD14 had positive blood cultures (Figure 4C). Moreover, administration of sCD14 to CD14 KO mice enhanced the pulmonary inflammatory response that again was clearly reduced in CD14 KO mice not receiving sCD14, to an extent observed in WT mice, as indicated by the semiquantitative scoring system described in Methods (Table 2). Moreover, CD14 KO mice treated with sCD14 demonstrated an increased inflammation and accumulation of neutrophils in lung tissue slides compared with CD14 KO mice as visualized by hematoxylin and eosin and, respectively, Ly-6G staining (Figure 5). In line with these findings, the lung and plasma concentrations of cytokines and chemokines were increased by sCD14 administration to CD14 KO mice (Table 2). Together, these data indicate that the presence of sCD14 in the bronchoalveolar compartment of CD14 KO mice can fully compensate for CD14 gene deficiency.
Figure 4. Treatment with recombinant soluble CD14 results in invasive infection in CD14 KO mice. (A) Soluble CD14 concentrations in BALF of WT mice infected with 5 × 104 cfu S. pneumoniae. Data are means ± SEM (n = 7–8 mice/group at each time point); *p < 0.05 versus naive mice. (B) Survival of WT and CD14 KO mice that received either saline or soluble (s)CD14 intranasally (1 μg; 0, 24, and 48 h). *p < 0.05 versus WT (n = 11–12 mice/group). (C) Bacterial loads in lungs 48 hours after infection with 5 × 104 cfu S. pneumoniae. BC+ indicates the number of positive blood cultures. CD14 KO + sCD14 mice were treated with sCD14 (1 μg) at 0 and 24 hours. Data are means ± SEM (n = 7–8 mice/group at each time point). *p < 0.05 versus CD14 KO mice.
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Figure 5. Treatment with recombinant sCD14 enhances lung inflammation in CD14 KO mice. Representative lung slides of (A) WT and (B) CD14 KO mice treated with saline or (C) sCD14 mice. Mice were killed 48 hours after infection with 5 × 104 cfu S. pneumoniae. Hematoxylin-and-eosin staining; original magnification, ×4. Insets show Ly-6G staining; original magnification, ×4.
[More] [Minimize]WT | CD14 KO | CD14 KO + sCD14 | ||||
|---|---|---|---|---|---|---|
| Total lung score, AU | 15 ± 2 | 4 ± 2*† | 17 ± 2 | |||
| Cytokine and Chemokine Production in Lung Homogenate (pg/ml) | ||||||
| TNF-α | 794 ± 193 | 523 ± 222† | 1,816 ± 322* | |||
| IL-6 | 2,270 ± 898 | 465 ± 119*† | 1,447 ± 310 | |||
| MCP-1 | 6,296 ± 1654 | 941 ± 407*† | 4,576 ± 829 | |||
| Cytokine and Chemokine Production in Plasma (pg/ml) | ||||||
| TNF-α | 623 ± 394 | 92 ± 10 | 310 ± 149 | |||
| IL-6 | 8,675 ± 5,063 | 78 ± 16‡† | 7,291 ± 6,998 | |||
| MCP-1 | 892 ± 361 | 143 ± 8‡ | 827 ± 635 | |||
CD14 is a pattern recognition receptor that has been shown to interact with a variety of bacterial components, including LPS, peptidoglycan, and lipoteichoic acid (4–6). Several studies have indicated that the early recognition of LPS by CD14 is important for mounting an effective innate immune response against gram-negative infections (19, 35). We here report that, much unlike the protective role of CD14 during gram-negative respiratory tract infection (22, 23), CD14 facilitates the outgrowth and, in particular, the dissemination of bacteria during pneumonia caused by S. pneumoniae. Experiments in which sCD14 was administered into the airways of CD14 KO mice established that sCD14 present in the bronchoalveolar compartment is sufficient to render the host more susceptible to pneumococcal pneumonia.
To our knowledge, only two previous studies examined the role of CD14 in host defense against a gram-positive infection. In a model of gram-positive septic shock induced by S. aureus, Haziot and colleagues did not detect a significant part for CD14 in survival or bacterial clearance (24). More recently, Echchannaoui and coworkers reported that CD14 KO mice showed more rapid and more severe signs of disease together with an accelerated lethality in a model of S. pneumoniae meningitis induced by direct intrathecal injection of live bacteria (25). The adverse outcome of CD14 KO mice was accompanied by an enhanced inflammatory response in the central nervous system. In addition, CD14 KO mice demonstrated a transiently enhanced outgrowth of pneumococci in their brains, as reflected by elevated bacterial loads at 24 hours but not at 48 hours. These two earlier studies contrast with our present findings in pneumonia caused by S. pneumoniae. The results of Haziot and colleagues do not necessarily conflict with our current data considering that these authors used a different gram-positive pathogen and a model that, due to its direct systemic nature, likely relies less on local antibacterial effector mechanisms (24). The model of S. pneumoniae central nervous system infection used by Echchannaoui and coworkers differs significantly from the model of S. pneumoniae pneumonia used here. Indeed, in the former study, pneumococci were injected directly into the brain, thereby circumventing normal anatomic barriers, in particular the blood–brain barrier (25). In our pneumonia model, the number of S. pneumoniae colony-forming units remained similar in BALF of CD14 KO and WT mice throughout the course of infection, but the bacterial loads in whole lung homogenates were more than 10-fold lower in the former strain at 48 hours after infection. More strikingly, blood cultures remained negative in CD14 KO mice with a single exception, whereas WT mice developed positive blood cultures from 24 hours onward and invariably had systemic infection at 48 hours. Treatment of CD14 KO mice with recombinant mouse sCD14 via the airways made them fully susceptible to invasive pneumococcal disease, not only confirming that the phenotype of CD14 KO mice in this model is CD14 dependent but also demonstrating that sCD14 is sufficient to reproduce the WT phenotype. Hence, in the bronchoaveolar compartment, (soluble) CD14 is used by S. pneumoniae to cause a full-blown and invasive infection.
CD14 KO mice demonstrated less lung inflammation, in particular at 48 hours after infection, as reflected by histopathology and cytokine and chemokine levels. Of note, whereas granulocyte staining of lung sections revealed a reduced neutrophil recruitment into lung tissues of CD14 KO mice at 48 hours, BALF of CD14 KO and WT mice contained equal neutrophil numbers at this time point. This finding is possibly related to the reduced bacterial load in lung tissue but not in BALF of CD14 KO mice (i.e., the reduced bacterial load in lung tissue may provide a less potent stimulus for the influx of neutrophils). CD14 KO mice were previously reported to have elevated TNF-α and IL-6 levels in blood during S. aureus–induced sepsis (24) and elevated TNF-α and MIP-2 levels in brain homogenates during S. pneumoniae–induced meningitis (25). In the present study, TNF-α in lungs was the only cytokine that was not affected by CD14 deficiency; all other mediators measured in lungs, including IL-6 and MIP-2, were lower in CD14 KO mice. This finding could be explained in two mutually nonexclusive ways. First, CD14 could play a direct role in the responsiveness of cytokine-producing cells to S. pneumoniae. In support of this possibility, we found that alveolar macrophages obtained from CD14 KO mice produced less TNF-α and IL-6 on stimulation with heat-killed S. pneumoniae (data not shown). Second, CD14 KO mice had a lower bacterial load in their lungs at 48 hours after infection, and thus were exposed to a less potent proinflammatory stimulus. In line with these findings, earlier studies have demonstrated a clear correlation between the pulmonary bacterial load and the extent of lung inflammation, including cytokine levels during experimentally induced S. pneumoniae pneumonia (34).
The pneumococcal cell wall contains phosphoryl choline that can specifically bind the platelet activating factor receptor (PAFR), an interaction that facilitates bacterial entry into these cells (36–38). Furthermore, the capacity of S. pneumoniae to transcytose to the basal surface of rat and human endothelial cells is dependent on the PAFR. Our laboratory recently provided evidence that this mechanism is important for the virulence of S. pneumoniae during murine respiratory tract infection in vivo (29). Using PAFR KO mice, we demonstrated that the PAFR is used by S. pneumoniae to induce lethal pneumonia, as reflected by a strongly reduced mortality, an attenuated bacterial outgrowth in the lungs, and a diminished dissemination of the infection in PAFR KO mice. As such, the phenotype of PAFR KO mice strongly resembles the phenotype of CD14 KO mice in this model. It is tempting to speculate that (soluble or surface) CD14 is involved in the presentation (of components) of S. pneumoniae to the PAFR so that the phosphoryl–PAFR-mediated invasion is facilitated. The possibility exists that (soluble or surface) CD14 serves as a chaperone that facilitates internalization and thus invasiveness of S. pneumoniae. CD14 itself is known to bind LPS and rapidly traffic between the cell membrane and intracellular compartments (39, 40). The more recent observation that binding and internalization of polyinosine-polycytidylic acid (pIpC) depends on CD14 illustrates that this property of CD14 is not restricted to LPS (41). Although we were not able to verify direct binding of CD14 to S. pneumoniae using in vitro binding assays or fluorescence microscopy (data not shown), we certainly cannot exclude the possibility of CD14-mediated internalization of bacteria in vivo.
An interaction between CD14 and TLRs is unlikely to explain our observations. Indeed, although CD14 can facilitate the presentation of several bacterial components to either TLR2 or TLR4, the presence of neither of these pattern recognition receptors facilitates invasive pneumococcal infection: both TLR2 and TLR4 have no/little contribution to host defense against pneumococcal pneumonia (30, 31, 42). Moreover, mice deficient for the common TLR adaptor protein MyD88 displayed a strongly reduced resistance against nasopharyngeal infection with S. pneumoniae (43). Thus, if the role of CD14 observed here would rely on TLRs, one would expect that CD14 KO mice would have been more susceptible to rather than protected against pneumococcal pneumonia.
Our study is the first to identify a detrimental role for CD14 in host defense against a common bacterial infection. We show that (soluble) CD14 is required for the development of severe invasive pneumonia on infection of the lower airways by S. pneumoniae. Our current data strongly suggest that S. pneumoniae specifically uses (soluble) CD14 in the bronchoalveolar compartment to cause invasive disease by a TLR-independent mechanism.
The authors thank Joost Daalhuisen, Ingvild Kop, and Marieke ten Brink for technical assistance during the animal experiments and Anita de Boer and Regina de Beer for assistance during pathology lung slide preparations. They thank Michael Tanck for statistical advice.
| 1. | Stefanova I, Horejsi V, Ansotegui IJ, Knapp W, Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 1991;254:1016–1019. |
| 2. | Haziot A, Tsuberi BZ, Goyert SM. Neutrophil CD14: biochemical properties and role in the secretion of tumor necrosis factor-alpha in response to lipopolysaccharide. J Immunol 1993;150:5556–5565. |
| 3. | Landmann R, Muller B, Zimmerli W. CD14, new aspects of ligand and signal diversity. Microbes Infect 2000;2:295–304. |
| 4. | Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 1990;249:1431–1433. |
| 5. | Kusunoki T, Hailman E, Juan TS, Lichenstein HS, Wright SD. Molecules from Staphylococcus aureus that bind CD14 and stimulate innate immune responses. J Exp Med 1995;182:1673–1682. |
| 6. | Cleveland MG, Gorham JD, Murphy TL, Tuomanen E, Murphy KM. Lipoteichoic acid preparations of gram-positive bacteria induce interleukin-12 through a CD14-dependent pathway. Infect Immun 1996;64:1906–1912. |
| 7. | Pugin J, Schurer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ, Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 1993;90:2744–2748. |
| 8. | Miyake K. Innate recognition of lipopolysaccharide by Toll-like receptor 4-MD-2. Trends Microbiol 2004;12:186–192. |
| 9. | Miller SI, Ernst RK, Bader MW. LPS, TLR4 and infectious disease diversity. Nat Rev Microbiol 2005;3:36–46. |
| 10. | Bazil V, Strominger JL. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes. J Immunol 1991;147:1567–1574. |
| 11. | Haziot A, Chen S, Ferrero E, Low MG, Silber R, Goyert SM. The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol 1988;141:547–552. |
| 12. | Labeta MO, Durieux JJ, Fernandez N, Herrmann R, Ferrara P. Release from a human monocyte-like cell line of two different soluble forms of the lipopolysaccharide receptor, CD14. Eur J Immunol 1993;23:2144–2151. |
| 13. | Landmann R, Zimmerli W, Sansano S, Link S, Hahn A, Glauser MP, Calandra T. Increased circulating soluble CD14 is associated with high mortality in gram-negative septic shock. J Infect Dis 1995;171:639–644. |
| 14. | Bufler P, Stiegler G, Schuchmann M, Hess S, Kruger C, Stelter F, Eckerskorn C, Schutt C, Engelmann H. Soluble lipopolysaccharide receptor (CD14) is released via two different mechanisms from human monocytes and CD14 transfectants. Eur J Immunol 1995;25:604–610. |
| 15. | Leturcq DJ, Moriarty AM, Talbott G, Winn RK, Martin TR, Ulevitch RJ. Antibodies against CD14 protect primates from endotoxin-induced shock. J Clin Invest 1996;98:1533–1538. |
| 16. | Schimke J, Mathison J, Morgiewicz J, Ulevitch RJ. Anti-CD14 mAb treatment provides therapeutic benefit after in vivo exposure to endotoxin. Proc Natl Acad Sci USA 1998;95:13875–13880. |
| 17. | Spek CA, Verbon A, Aberson H, Pribble JP, McElgunn CJ, Turner T, Axtelle T, Schouten J, Van Der Poll T, Reitsma PH. Treatment with an anti-CD14 monoclonal antibody delays and inhibits lipopolysaccharide-induced gene expression in humans in vivo. J Clin Immunol 2003;23:132–140. |
| 18. | Tasaka S, Ishizaka A, Yamada W, Shimizu M, Koh H, Hasegawa N, Adachi Y, Yamaguchi K. Effect of CD14 blockade on endotoxin-induced acute lung injury in mice. Am J Respir Cell Mol Biol 2003;29:252–258. |
| 19. | Le Roy D, Di Padova F, Adachi Y, Glauser MP, Calandra T, Heumann D. Critical role of lipopolysaccharide-binding protein and CD14 in immune responses against gram-negative bacteria. J Immunol 2001;167:2759–2765. |
| 20. | Frevert CW, Matute-Bello G, Skerrett SJ, Goodman RB, Kajikawa O, Sittipunt C, Martin TR. Effect of CD14 blockade in rabbits with Escherichia coli pneumonia and sepsis. J Immunol 2000;164:5439–5445. |
| 21. | Opal SM, Palardy JE, Parejo N, Jasman RL. Effect of anti-CD14 monoclonal antibody on clearance of Escherichia coli bacteremia and endotoxemia. Crit Care Med 2003;31:929–932. |
| 22. | Wieland CW, Florquin S, Maris NA, Hoebe K, Beutler B, Takeda K, Akira S, van der Poll T. The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable Haemophilus influenzae from the mouse lung. J Immunol 2005;175:6042–6049. |
| 23. | Knapp S, Wieland CW, Florquin S, Pantophlet R, Dijkshoorn L, Tshimbalanga N, Akira S, van der Poll T. Differential roles of CD14 and Toll-like receptors 4 and 2 in murine acinetobacter pneumonia. Am J Respir Crit Care Med 2006;173:122–129. |
| 24. | Haziot A, Hijiya N, Schultz K, Zhang F, Gangloff SC, Goyert SM. CD14 plays no major role in shock induced by Staphylococcus aureus but down-regulates TNF-alpha production. J Immunol 1999;162:4801–4805. |
| 25. | Echchannaoui H, Frei K, Letiembre M, Strieter RM, Adachi Y, Landmann R. CD14 deficiency leads to increased MIP-2 production, CXCR2 expression, neutrophil transmigration, and early death in pneumococcal infection. J Leukoc Biol 2005;78:705–715. |
| 26. | Campbell GD Jr. Commentary on the 1993 American Thoracic Society guidelines for the treatment of community-acquired pneumonia. Chest 1999;115:14S–18S. |
| 27. | Bernstein JM. Treatment of community-acquired pneumonia: IDSA guidelines. Infectious Diseases Society of America. Chest 1999;115:9S–13S. |
| 28. | Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely EW, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699–709. |
| 29. | Rijneveld AW, Weijer S, Florquin S, Speelman P, Shimizu T, Ishii S, van der Poll T. Improved host defense against pneumococcal pneumonia in platelet-activating factor receptor-deficient mice. J Infect Dis 2004;189:711–716. |
| 30. | Knapp S, Wieland CW, van't Veer C, Takeuchi O, Akira S, Florquin S, van der Poll T. Toll-like receptor 2 plays a role in the early inflammatory response to murine pneumococcal pneumonia but does not contribute to antibacterial defense. J Immunol 2004;172:3132–3138. |
| 31. | Branger J, Knapp S, Weijer S, Leemans JC, Pater JM, Speelman P, Florquin S, van der Poll T. Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice. Infect Immun 2004;72:788–794. |
| 32. | Rijneveld AW, Florquin S, Branger J, Speelman P, Van Deventer SJ, van der Poll T. TNF-alpha compensates for the impaired host defense of IL-1 type I receptor-deficient mice during pneumococcal pneumonia. J Immunol 2001;167:5240–5246. |
| 33. | Knapp S, Schultz MJ, Poll TV. Pneumonia models and innate immunity to respiratory bacterial pathogens. Shock 2005;24:12–18. |
| 34. | Moore TA, Standiford TJ. Cytokine immunotherapy during bacterial pneumonia: from benchtop to bedside. Semin Respir Infect 2001;16:27–37. |
| 35. | Haziot A, Ferrero E, Kontgen F, Hijiya N, Yamamoto S, Silver J, Stewart CL, Goyert SM. Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity 1996;4:407–414. |
| 36. | Wissner A, Schaub RE, Sum PE, Kohler CA, Goldstein BM. Analogues of platelet activating factor. 4. Some modifications of the phosphocholine moiety. J Med Chem 1986;29:328–333. |
| 37. | Cabellos C, MacIntyre DE, Forrest M, Burroughs M, Prasad S, Tuomanen E. Differing roles for platelet-activating factor during inflammation of the lung and subarachnoid space: the special case of Streptococcus pneumoniae. J Clin Invest 1992;90:612–618. |
| 38. | Cundell DR, Gerard NP, Gerard C, Idanpaan-Heikkila I, Tuomanen EI. Streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor. Nature 1995;377:435–438. |
| 39. | Thieblemont N, Wright SD. Transport of bacterial lipopolysaccharide to the Golgi apparatus. J Exp Med 1999;190:523–534. |
| 40. | Latz E, Visintin A, Lien E, Fitzgerald KA, Monks BG, Kurt-Jones EA, Golenbock DT, Espevik T. Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the Toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. J Biol Chem 2002;277:47834–47843. |
| 41. | Lee HK, Dunzendorfer S, Soldau K, Tobias PS. Double-stranded RNA-mediated TLR3 activation is enhanced by CD14. Immunity 2006;24:153–163. |
| 42. | Albiger B, Dahlberg S, Sandgren A, Wartha F, Beiter K, Katsuragi H, Akira S, Normark S, Henriques-Normark B. Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection. Cell Microbiol 2006 Sep 27; [Epub ahead of print]. |
| 43. | Albiger B, Sandgren A, Katsuragi H, Meyer-Hoffert U, Beiter K, Wartha F, Hornef M, Normark S, Normark BH. Myeloid differentiation factor 88-dependent signalling controls bacterial growth during colonization and systemic pneumococcal disease in mice. Cell Microbiol 2005;7:1603–1615. |
