We investigated the roles of the potent, chemotactic antimicrobial proteins S100A8, S100A9, and S100A8/A9 in leukocyte migration in a model of streptococcal pneumonia. We first observed differential secretion of S100A8, S100A9, and S100A8/A9 that preceded neutrophil recruitment. This is partially explained by the expression of S100A8 and S100A9 proteins by pneumocytes in the early phase of Streptococcus pneumoniae infection. Pretreatment of mice with anti-S100A8 and anti-S100A9 Abs, alone or in combination had no effect on bacterial load or mice survival, but caused neutrophil and macrophage recruitment to the alveoli to diminish by 70 and 80%, respectively, without modifying leukocyte blood count, transendothelial migration or neutrophil sequestration in the lung vasculature. These decreases were also associated with a 68% increase of phagocyte accumulation in lung tissue and increased expression of the chemokines CXCL1, CXCL2, and CCL2 in lung tissues and bronchoalveolar lavages. These results show that S100A8 and S100A9 play an important role in leukocyte migration and strongly suggest their involvement in the transepithelial migration of macrophages and neutrophils. They also indicate the importance of antimicrobial proteins, as opposed to classical chemotactic factors such as chemokines, in regulating innate immune responses in the lung.
In recent years, the differences between antimicrobial peptides and chemotactic factors have become less distinct. For example, some CC and CXC chemokines such as CCL20 and CXCL10 have been reported to inhibit microbial growth (1, 2). Conversely, defensins, cathepsin G, and CAP37/azurocidin, which are well known for their antimicrobial properties, have recently been shown to be chemotactic for T lymphocytes, monocytes, and neutrophils (3, 4, 5). S100A8 and S100A9 are other antimicrobial proteins that exert characteristic chemotactic and proinflammatory activities.
S100A8 and S100A9 are expressed by neutrophils, monocytes, and activated endothelial and epithelial cells (6, 7, 8, 9, 10). They exist as noncovalently bound homodimers, but also as heterodimers (S100A8/A9) that inhibit bacterial adhesion to mucosal epithelium and bacterial growth through zinc chelation (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). Studies on inflammatory disorders such as cystic fibrosis, gout, and rheumatoid arthritis have shed light upon the association between the secretion of these S100 proteins in biological liquids and pathogenesis (8, 21, 22, 23, 24, 25).
In addition to their antimicrobial properties, S100A8, S100A9, and S100A8/A9 are potently chemotactic for neutrophils and monocytes (26, 27). These proteins are involved in transendothelial migration of leukocytes by inducing neutrophil adhesion to fibrinogen by activation of the β2 integrin Mac-1 (26, 28). They also participate in neutrophil migration to inflammatory sites in response to LPS and monosodium urate crystals (27, 29), which places them in the innate immune response.
Multiple chemotactic and antimicrobial factors are simultaneously present at the site of infection during the immune response; thus, the task of distinguishing their relative roles is rather complicated. In this study, we have further delineated the roles of S100A8 and S100A9 in leukocyte migration by studying bacterial load, leukocyte influx in the lung, and mouse survival that occurs in streptococcal pneumonia. Infection by Streptococcus pneumoniae, a major etiological agent of community-acquired pneumonia, which is the most common cause worldwide of death from infection (30), first takes hold through adhesion of these bacteria to the epithelium within the alveoli. This in turn causes local release of proinflammatory mediators such as cytokines, chemokines, and leukotrienes, which activate the surrounding tissue and further induce inflammatory factors in that tissue. Neutrophils and monocytes/macrophages are among the first cells to migrate to these sites of inflammation where they kill pathogens by phagocytosis, release of oxygen radicals and tissue degrading enzymes, or secretion of antimicrobial peptides.
We report here that S100A8 and S100A9 were released at high levels during the course of infection and that their presence correlated with increased neutrophil migration. More importantly, blockade of S100A8 and S100A9 activity was also associated with an accumulation of neutrophils and monocytes in lung tissue, exemplifying the importance of these antimicrobial proteins in regulating the migration of these cells to the lung alveoli more precisely in the epithelial migration.
Materials and Methods
S. pneumoniae infection
S. pneumoniae serotype 3 (isolated clinical strain) was grown in 100 ml of brain-heart infusion medium (Difco) at 37°C in 5% CO2 to the midexponential phase (0.4 OD600). Forty milliliters of bacteria culture were pelleted, washed twice, and resuspended in 10 ml of cold endotoxin-free PBS. An OD at 600 nm was read, and an adequate dilution was made to obtain an inoculum of 4 × 105 CFU/50 μl. The bacterial concentration was confirmed by serial dilutions of the inoculum plated on blood-agar. Female CD1 mice, 6–8 wk old (Charles River) were lightly anesthetized by isoflurane and intranasally instilled with 4 × 105 CFU of S. pneumoniae or endotoxin-free PBS. In some experiments, mice were injected i.p. 16 h before infection with 2 mg of purified neutralizing rabbit IgG anti-S100A8 and/or anti-S100A9, or with nonspecific rabbit IgG (27). At various times postinfection, the mice were sacrificed by carbon dioxide asphyxiation, and bronchoalveolar lavage (BAL)3 and lungs were collected as described (31). For survival studies, the morbidity and mortality of infected mice were carefully observed for up to 6 days. Bacterial clearance in anti-S100A8- and/or anti-S100A9-treated mice was determined by serial dilution of BAL and lung homogenates on blood-agar. Animal studies have been reviewed and approved by the Laval University animal protection committee.
Analyses of leukocyte recruitment and quantification of S100A8 and S100A9
BAL was centrifuged at 1200 rpm for 10 min, and the pelleted cells were resuspended in 600 μl of PBS. Total leukocytes were stained with acetic blue and counted with a hematocytometer. Leukocyte subpopulations were determined by Wright-Giemsa stainings of cytospins. Lungs were homogenized in 2 ml of 50 mM phosphate buffer, pH 6, and then diluted by addition of an equal volume of 50 mM phosphate buffer, pH 6, containing 1% hexadecyltrimethylammonium bromide. Diluted homogenates were sonicated for 30 s and centrifuged at 3000 × g for 30 min. The supernatants were used to evaluate neutrophil recruitment in the tissue by measuring endogenous myeloperoxidase. Briefly, in a 96-well plates, 15 μl of myeloperoxidase standards (Sigma-Aldrich), and samples were mixed with 100 μl of the reaction solution containing 0.01 mg/ml o-dianisidine and 0.004% H2O2 in phosphate buffer. After 10 min, 100 μl of 1% sodium azide were added to stop the reaction (31). The OD was read at 450 nm. Quantification of S100A8, S100A9, and S100A8/A9 was done using by ELISA as previously described (27).
Immunohistochemistry, immunofluorescence, and electron microscopy
Lungs were perfused with 4% paraformaldehyde in PBS, fixed for 48 h at 4°C, dehydrated, and embedded in paraffin. For immunohistochemical detection of murine S100A8 and S100A9, 5-μm tissue sections were rehydrated by serial immersion in 100% toluene; then 100, 95, 70, and 50% ethanol; and rinsed in PBS. After inhibition of endogenous peroxidase with 3% H2O2 in PBS for 20 min, nonspecific binding sites were blocked with 10% normal donkey serum (Sigma-Aldrich), 2% BSA in PBS. After three washings with PBS, tissue sections were incubated for 1 h at room temperature with purified anti-murine S100A8 or S100A9 rabbit polyclonal IgG Abs (10 μg/ml) diluted in PBS, 2% normal donkey serum, 2% BSA. Tissue sections were rinsed three times with PBS and incubated for 1 h at room temperature with peroxidase-conjugated donkey anti-rabbit IgG (100 μg/ml). After a washing, the peroxidase activity was revealed with a 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich), 0.024% H2O2 solution for 5 min. Tissue sections were counterstained with H&E.
For immunofluorescence, paraffin-embedded lung sections were rehydrated and immersed for 1 h in 0.025% NH3, 70% ethanol to decrease autofluorescence. Lung sections were then blocked for 2 h in PBS, 10% normal goat serum (Sigma-Aldrich), 2% BSA, 0.2% Triton before being incubated overnight with a mix of 1/1000 rabbit anti-human prosurfactant protein C (proSP-C; Chemicon) and 1 μg/ml rat anti-S100A8 or rat anti-S100A9 in PBS, 2% normal goat serum, 2% BSA. Equal amounts of isotype control IgG and purified preimmune serum were used as control. After six washes, tissue sections were incubated for 1 h with a mix of ALEXA-488-conjugated goat anti-rabbit IgG (1/200), ALEXA-555-conjugated goat anti-rat IgG (1/200), and the nuclear marker TOPRO-3 (1 μM). Fluorescence was then examined using an Olympus Fluoview FV300 confocal microscope.
For electron microscopy analyses, lungs were fixed in 2.5% glutaraldehyde in phosphate buffer and post fixed in 1% osmium tetroxide. They were embedded in Epon and sectioned for transmission electron microscopy. The samples were then analyzed using a JEOL JEM-1230 transmission electron microscope at 80 kV.
Evaluation of neutrophils sequestration in the lung tissue using flow cytometry
The number of sequestered neutrophil following S. pneumoniae infection was measured following a variant of a protocol described in Ref. 32 . Briefly, at 48 h postinfection, mice were injected i.v. with 10 μg of FITC-labeled anti-Gr1 (to label sequestered neutrophils; Leinco Technologies) or FITC-labeled isotype control (IgG2b; Ebioscience). Ten minutes after injection, the mice were sacrificed by carbon dioxide asphyxia, and the blood, lungs, and BAL were harvested. The lungs were minced and digested using a solution of dispase 2.4 U/ml (BD Biosciences) and collagenase, 124 U/ml (Sigma-Aldrich), in the presence of excess unlabeled anti-Gr1 (to prevent labeling of transendothelial migrated neutrophils by residual labeled anti-Gr1; Leinco Technologies) or isotype control in PBS for 60–90 min at 37°C. After digestion, the lungs were passed through a 70-μm cell strainer (BD Biosciences), centrifuged, and resuspended at 10 × 106 cells/ml in PBS, 2% FBS.
Separate aliquots of 1 × 106 cells from the blood, BAL, and lung were first incubated for 30 min with 0.5 μg of murine Fc block (Ebioscience) in PBS-2% FBS (Wisent) to block Fc receptors. After three washes, the cells were incubated with 1 μg of PE-labeled anti-7/4 (1/50) (to label all neutrophils; Serotec) or PE-labeled IgG2a (Ebioscience) for 30 min. After incubation, the cells were washed three times and fixed with PBS, 2% formaldehyde. Flow cytometry analysis was done on an EPICS XL Coulter flow cytometer. Sequestered neutrophils were determined by defining the Gr-1+ cells present in the 7/4+ (total neutrophil) population per million of 7/4− cells (lung cells).
CXCL2, CXCL1, and CCL2 ELISA
For determination of CXCL2, CXCL1, and CCL2 protein levels, 96-well microtiter plates (NUNC) were coated overnight with goat Abs against CCL2, CXCL2, or CXCL1 in 0.1 M NaHCO3, pH 9.6. The plates were washed three times with PBS, 0.05% Tween 20 (PBST) and blocked for 2 h with PBST, 2% BSA. Murine recombinant chemokine standards or the samples diluted in PBST, 2% BSA were then added and incubated for 2 h. After washing, biotinylated goat anti-CCL2, CXCL1, or CXCL2 were added for 1 h. Poly-HRP streptavidin (1/10,000) (Pierce Biotechnology) was incubated for 45 min, and the plates were revealed with 3,3′,5,5′-tetramethylbenzidine substrate. The reaction was stopped with 0.18 N H2SO4. The OD was read at 450 nm. The lower limit of quantification of these assays was 10 pg/ml. All Abs and recombinant murine chemokines were purchased from R & D Systems.
Experimental data are expressed as mean ± SEM. One-way ANOVA with Dunnett’s post test was performed using GraphPad InStat version 3.05 for Windows (GraphPad Software). Differences were considered statistically significant when p ≤ 0.05.
Secretion of S100A8 and S100A8/A9 precedes leukocyte recruitment during S. pneumoniae infection
To study the involvement of S100A8 and S100A9 in leukocyte recruitment, we used a murine model of S. pneumoniae lung infection. Mice were inoculated by nasal instillation with 4 × 105 CFU of S. pneumoniae to mimic the human infection. Lung infection was validated by the observation of progressive neutrophil and macrophage recruitment to the lung tissue and airspace. Neutrophil recruitment in lung tissue was first detected at 6 h, peaked by 24 h (Fig. 1,A), and then remained constant until the death of the mouse. In contrast, neutrophil accumulation in the alveolar space was slower, with maximal infiltration of neutrophils (∼8 × 105 cells) occurring at 48 h. Macrophage migration steadily increased during the infection, reaching a maximum of 1.8 × 106 cells at 72 h (Fig. 1 B).
To determine the role of S100A8 and S100A9 in leukocyte migration during infection in the lungs, S100A8, S100A9, and S100A8/A9 were quantified in lung tissue and BAL of mice infected with S. pneumoniae. As shown in Fig. 1,C, S100A8 rapidly peaked in lung tissue at close to 200 μg/lung, appearing at 12 h before declining by 36 h postinjection. S100A8 preceded S100A9 and S100A8/A9 release, with maximum presence of these proteins detected at 48 h at ∼40 and 107 μg/lung, respectively. In contrast, concentrations of S100A8/A9 and S100A8 in the BAL peaked at 24 and 36 h (70 μg/ml, both) and remained relatively constant until the death of the mouse (Fig. 1 D). Interestingly, compared with the other S100 proteins, little S100A9 was released in these conditions. Thus secretion of S100 proteins preceded infiltration of neutrophils in the lung and alveolar space. Moreover, the differential release of these proteins suggested distinct roles for these proteins in the host primary responses to S. pneumoniae.
To further characterize the release of S100 proteins, we examined the localization of these proteins in the lung during infection. Immunostaining of lung tissue sections showed that S100A8 and S100A9 proteins were localized mainly in pneumocytes, neutrophils, and macrophages at early stages of infection (Fig. 2, A–C). Confocal imaging of lung tissue sections indicated that type II pneumocytes (proSPC+) expressed S100A8 and S100A9 (Fig. 3). Neutrophils and macrophages (data not shown) were also strongly positive for S100A8 and S100A9 proteins.
At later times postinfection, the lung tissue did not show diffused distribution of S100 proteins but rather defined localization around the inflamed bronchi and bronchioli. Their high levels at the inflammatory site were associated with a loss of integrity of the leukocyte-infiltrated lung (Fig. 2, D–F). These results suggested that the antimicrobial proteins S100A8 and S100A9 could play important but distinct roles in the pathogenesis of S. pneumoniae infection.
Blockade of S100A8 and S100A9 activities significantly inhibits neutrophil and macrophage recruitment in the airspace
S100 proteins have both intracellular and extracellular activities. Conclusions from studies using knockout mice defective in S100A8 or S100A9 gene are therefore difficult to reach, as both intra- and extracellular activities are inhibited in these animals. In addition, S100A8-deficient mice die during embryogenesis (33). It is thus almost impossible to use knockout mice for the study of the extracellular activities of S100A8 and S100A9. To circumvent this problem, we used blocking mAbs to determine the importance of extracellular S100A8/A9 in leukocyte migration and disease genesis (27, 29). To examine the roles of S100A8 and S100A9 in innate immune responses to S. pneumoniae, their activities were blocked by i.p. injection of purified IgG directed against S100A8 and/or S100A9 16 h before infection. Interestingly, no significant changes were observed in S. pneumoniae clearance (Fig. 4) or the survival rates between treated and untreated infected mice (Fig. 5). Anti-S100A8 and anti-S100A9 slightly increased mouse survival rates (but not significantly), whereas the combination of both Abs had no effect. As expected, passive immunization with nonspecific Abs (preimmune serum) protected mice from early death associated with pneumonia (34).
However, as seen in Fig. 6,A, blockade of S100A8 and S100A9 activities led to a 45% decrease of neutrophils recruitment in the BAL 24 h postinfection (p < 0.01 and p < 0.05 compared with control mice, Dunnett multiple comparison test). Moreover, at 48 h postinfection, pretreatment with anti-S100A8 and anti-S100A9 inhibited neutrophil migration to the alveolar space by 70% (Fig. 6,B; p < 0.05, Dunnett multiple comparison test). These results indicated that S100A8 and S100A9 are required for neutrophil recruitment in the alveolar space. This requirement was even more important for macrophage recruitment, with a reduction of 82% in mice treated with anti-S100A8 and anti-S100A9 24 h postinfection (Fig. 6,C; p < 0.05, Dunnett multiple comparison test). This result was confirmed at 48 h postinfection, where the blockade of S100A8 and S100A9, alone or in combination, led to a reduction of ∼80% of macrophage infiltration in the alveolar space (Fig. 6 D; p < 0.01, Dunnett multiple comparison test). These results indicated that S100A8 and S100A9 play an important role in the migration of leukocytes, and particularly macrophages, in the alveolar space during S. pneumoniae infection.
To determine at which stage of this migration cascade S100 proteins played a role, phagocyte migration to the lung tissue was evaluated by measuring endogenous myeloperoxidase. At 48 h postinfection, pretreating infected mice with anti-S100A8 and anti-S100A9 led to a 68% increase of endogenous myeloperoxidase in the lung tissue compared with the control mice (Fig. 7; p < 0.05, Dunnett multiple comparison test). This accumulation was associated with a decrease in leukocytes in the airspace, suggesting that S100A8 and S100A9 play a role in the transmigration of phagocytes from the lung tissue to the alveolar space.
To test this hypothesis, FITC-labeled anti-Gr1 (anti-neutrophil) Abs were injected i.v. 10 min before the mice were euthanized to stain sequestered neutrophils. The lungs were then harvested and single cell suspensions were analyzed by flow cytometry following labeling of all neutrophils with the neutrophil-specific Ab 7/4. Under these conditions, sequestered neutrophils were positively labeled with both FITC-labeled Gr1 and PE-labeled 7/4, whereas neutrophils that have already migrated in the tissue were labeled only with the Ab 7/4. As shown in Fig. 8,A, the number of neutrophils present in the lung remained unchanged in anti-S100A8 and anti-S100A9-treated mice. Moreover, no significant changes in the number of neutrophils sequestered in the lung vasculature were observed by blocking S100A8 and S100A9 proteins compared with control mice (Fig. 8 B). In addition, the number of circulating neutrophils remained unaffected by the injection of anti-S100 proteins (data not shown).
Similarly, electron microscopy analyses of lung tissues show neutrophils and macrophages in alveoli of infected animals (Fig. 9, A and B), whereas most of the neutrophils and macrophages were found in lung tissue, but not alveoli of anti-S100A8 and anti-S100A9-treated animals (Fig. 9, C and D). These results confirmed the hypothesis that S100A8 and S100A9 are important for neutrophil transepithelial, but not transendothelial migration in the lung.
To further decipher the mechanism by which anti-S100A8 and anti-S100A9 caused the accumulation of phagocytes in lung tissue, the expression of the chemokines CXCL1, CXCL2, and CCL2 was evaluated by ELISA in BAL and lung tissues. Treatment of mice with anti-S100A9 alone or in combination with anti-S100A8 led to 50 and 100% increases in the lungs of CCL2 and CXCL2 levels, respectively, compared with control mice. (Fig. 10, A, C, and E; p < 0.01 for CXCL2 and p < 0.05 for CCL2, Dunnett multiple comparison test). However, a 2.5-fold increase of CCL2 levels was also observed in the BAL of mice treated with anti-S100A9 or anti-S100A9 in combination with S100A8. (Fig. 10, B, D, and F; p < 0.01, Dunnett multiple comparison test). Similarly, a 20% increase of CXCL1 levels was observed after the blockade of S100A8 and S100A9. The augmented presence of chemokines attracting both neutrophils and monocyte/macrophages in the BAL and lung tissues of anti-S100A8 and anti-S100A9-treated mice was convincing evidence against an indirect effect of S100A8 and S100A9 in directing phagocyte migration in the lung.
In this study, we examined the functions of S100A8 and S100A9 by observing leukocyte migration in the mouse lung during streptococcal pneumonia. Our results indicate that S100A8 and S100A9 are released differentially during the course of infection. At an early phase of infection, the pneumocytes might be one of the sources of these two proteins. This presence is correlated with increased neutrophil and macrophage migration at latter stages of infection. More importantly, blockade of S100A8 and S100A9 activity strongly inhibited phagocyte recruitment to the alveoli without affecting the sequestration of neutrophils within the vasculature, or the number of circulating neutrophils or their migration in lung tissue. The reduced migration of phagocytes in the alveoli was not caused by altered secretion of chemokines in the lung tissue or alveoli. What emerges from the results is the importance of these antimicrobial proteins in promoting leukocyte migration to the lung alveoli.
A possible explanation for these observations would be that S100A8 and S100A9 act by preventing neutrophil apoptosis. The observed reduced numbers of neutrophils in the alveolar space could therefore be explained by an enhanced apoptosis of neutrophils in the presence of anti-S100A8 and anti-S100A9. However, this is unlikely to be the case as a similar diminution of neutrophils numbers in Ab-treated animals was not seen in lung tissue. In addition, we observed very few apoptosing neutrophils in BALs of untreated or Ab-treated animals, either by flow cytometry, optical or electron microscopy.
S100A8 and S100A9 did not induce the transepithelial migration of phagocytes indirectly by inducing the secretion of chemokines in the alveolar lumen, given that alveolar secretion of CXCL1 and CCL2 was down-regulated by S100A8 and S100A9. This is intriguing considering that S100A8 and S100A9 induce the expression and secretion of CXCL8 in epithelial cells (35) and of proinflammatory cytokines such as IL-6, CXCL8, IL-1β, and TNF-α in monocytes (36). The increased presence of chemokines in the BAL of anti-S100A8- and anti-S100A9-treated animals was associated with a diminished presence of phagocytes. It is thus possible that the reduced uptake of chemokines via chemokine receptors present on (fewer) leukocytes could contribute to this augmentation, although the increased presence of chemokines and phagocytes (as observed by myeloperoxidase) in lung tissue provide evidence against this hypothesis. Alternatively, the secretion of an inhibitory molecule by phagocytes that down-regulates the secretion of CCL2 and CXCL1 could also explain this effect. Further experimentation will be necessary to test these hypotheses.
Antimicrobial peptides and chemokines are simultaneously present in the lung during streptococcal infection and the task of distinguishing their relative functions is complex. Recently, studies have shown that antimicrobial and chemotactic peptides have certain activities in common; for example, defensins and CAP37, both antimicrobial peptides, induce weak to moderate inflammatory reaction when injected in mice (3). Moreover, β-defensins bind and activate the chemokine receptor CCR6 (37). Conversely, antimicrobial activities have been attributed to certain chemokines (1, 2). These duplicated activities make it difficult to ascertain the relative roles of these cytokines. Studies by Fillion and others, using a combination of three neutralizing Abs (anti-CCL2, anti-CCL3, and anti-CCL5) in S. pneumoniae-infected mice showed only a 33% a reduction in macrophage infiltration and no significant decrease in neutrophils migration in the alveolar space (38). Similarly, blockade of CXCL2 activities led to only a 20% decrease of neutrophil recruitment in the airspace in a pneumolysin-induced lung inflammation (39). These results suggest that these chemokines could have a relatively minor role in neutrophil migration to the lung during streptococcal infection. This is in contrast to the drastic effect of inhibition of S100A8 and S100A9, which emphasizes the importance of these proteins in the innate response.
The various chemotactic signals present at the inflammatory site probably act in sequence (40), and some chemotactic factors probably stimulate leukocyte firm adhesion to the endothelium to promote transendothelial migration. Other chemotactic factors then guide leukocytes within the tissue to the general area of inflammation where pathogen- and innate immunity-derived factors (such as formyl peptides and C5a) override these signals and lead phagocytes to the pathogen. The importance of this sequential action of chemotactic factors emerged in a study by Coates and McColl (41), who showed that the activities of complement-derived factors dominate over chemokines during leukocyte migration in response to bacterial infection. That inhibition of S100A8 and S100A9 blocks phagocyte migration to the alveoli, but not lung tissue, and supports that these proteins play a role in the last steps of phagocyte migration.
Leukocyte migration to the lung is tightly regulated. To gain access to the luminal space, leukocytes must first extravasate, migrate within the lung tissue, and then cross the basolateral to the luminal side of the epithelial cells. S100A9 and S100A8/A9 were previously shown to promote monocyte transendothelial migration (42). In addition, S100A9 stimulates neutrophil adhesion to the extracellular matrix protein fibronectin (N. Anceriz and P. A. Tessier, unpublished observations). Thus, the assumption that S100A9 could facilitate leukocyte extravasation and movement within the tissue is valid; however, in our experiments phagocytes accumulated within the tissue in mice treated with anti-S100A8 and anti-S100A9, which favors that S100A8 and S100A9 play important roles in neutrophil migration across epithelial cells, but not in lung transendothelial migration. This is intriguing as it implies a specialized role for S100A8 and S100A9 in transepithelial migration. Interestingly, such an activity has been recently described for hepoxilin A3, a lipid mediator (43).
S100A8 and S100A9 regulate the passage of leukocytes through the endothelium by enhancing the expression of adhesion molecules such as ICAM-1 and VCAM-1 on endothelial cells (44) and by activating their counterreceptor Mac-1 on neutrophils (26, 45). In addition, S100A8/A9 has recently been shown to down-regulate tight junction proteins on endothelial cells (44). Thus, they could facilitate leukocyte transendothelial migration by enhancing both leukocyte adhesion and endothelial cell permeability. Although S100A8 and S100A9 are not critical to neutrophil transendothelial migration in the lung, they could play a similar role by increasing the permeability of the alveolar epithelium by regulating tight junction genes. Additional experiments are needed to test this hypothesis.
It is becoming increasingly clear that the release of intracellular contents of any cell into the extracellular space activates the immune system. As the first cells to reach inflammatory sites, neutrophils are well positioned to signal impending harm. S100A8/A9 accounts for almost 40% of neutrophil cytosolic proteins (46). In addition, S100A8 and S100A9 induce NFκB activation in mononuclear cells, activate lymphocyte, and assist neutrophil and monocyte migration (27, 36, 42, 47). As such, they are powerful danger signals that activate both the innate and adaptive immune responses. Consequently, the death of few cells, particularly neutrophils, or the active secretion of their intracellular content, could send a powerful danger signal alerting the innate immune system of damage caused by a pathogen. That S100A8 was more important in neutrophil migration to the lung in response to S. pneumoniae infection than LPS-induced inflammation (48) supports this idea. This might reflect a role for S100A8 and S100A9 as danger signals for the immune system, as unlike a true bacterial infection, LPS inflammation would not result in the death of cells.
In conclusion, in these murine studies, we report convincing evidence of the critical role of S100A8 and S100A9 proteins of regulating macrophage and neutrophil transepithelial migration from the lung tissue to the alveolar space in the host response to S. pneumoniae infection. These results indicate the importance of antimicrobial peptides, as opposed to classic chemotactic factors, in regulating inflammatory reactions. In addition, they suggest that S100 proteins could act as potent danger signals for the immune system.
We thank Susanne Richardson for reviewing this manuscript and the technicians from the Université Laval electron micoscopy service for their help in preparation and analysis of lung sections.
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.
This work was supported by Canadian Institutes of Health Research Grant 57777 and the Fonds de la Recherche en Santé du Québec (to P.A.T.).
Abbreviations used in this paper: BAL, bronchoalveolar lavage; PBST, PBS 0.05% Tween 20.