Hemorrhagic shock renders patients susceptible to the development of acute lung injury in response to a second inflammatory stimulus by as yet unclear mechanisms. We investigated the role of neutrophils (PMN) in alveolar macrophage (AMφ) priming, specifically, the role in mediating Toll-like receptor (TLR)4 and TLR2 cross talk in AMφ. Using a mouse model of hemorrhagic shock followed by intratracheal administration of LPS, we explored a novel function of shock-activated PMN in the mechanism of TLR2 upregulation induced by LPS-TLR4 signaling in AMφ. We showed that antecedent hemorrhagic shock enhanced LPS-induced TLR2 upregulation in AMφ. In neutropenic mice subjected to shock, the LPS-induced TLR2 expression was significantly reduced, and the response was restored upon repletion with PMN obtained from shock-resuscitated mice but not by PMN from sham-operated mice. These findings were recapitulated in mouse AMφ cocultured with PMN. The enhanced TLR2 upregulation in AMφ augmented the expression of macrophage inflammatory protein-2, TNF-α, and macrophage migration inhibitory factor in the AMφ in response to sequential challenges of LPS and peptidoglycan, a prototypical TLR2 ligand, which physiologically associated with amplified AMφ-induced PMN migration into air pouch and lung alveoli. Thus TLR2 expression in AMφ, signaled by TLR4 and regulated by shock-activated PMN, is an important positive-feedback mechanism responsible for shock-primed PMN infiltration into the lung after primary PMN sequestration.
- acute lung injury
- innate immunity
- Toll-like receptor
the development of acute lung injury and acute respiratory distress syndrome in individuals following major trauma contributes significantly to morbidity and mortality in this patient population (2, 18). Studies have suggested that the global ischemia-reperfusion related to resuscitation from hemorrhagic shock promotes the development of lung injury by priming the innate immunity for an exaggerated inflammatory response to a second, often trivial, inflammatory stimulus, the so-called “two-hit” hypothesis (27). Using a rodent model of hemorrhage/resuscitation, we previously demonstrated that an antecedent shock leads to augmented lung neutrophil (PMN) sequestration and lung injury in response to a small dose of intratracheal lipopolysaccharide (LPS) (14). This effect appeared to be mediated by the increased LPS-stimulated release from alveolar macrophages (AMφ) of proinflammatory cytokines and chemokines, including TNF-α and cytokine-induced neutrophil chemoattractant (CINC) (12, 14). These findings suggest a hemorrhagic shock-primed cellular responsiveness to pathogenic product.
Macrophage activation in response to microbial infection depends on Toll-like receptors (TLRs), a family of pattern recognition receptors and key regulators of both innate and adaptive immunity (23). To date, 13 mammalian TLR paralogs have been identified (10 in humans and 12 in mice) (3). TLR2 is crucial for the propagation of the inflammatory response to components of gram-positive and gram-negative bacteria and mycobacteria such as peptidoglycan (PGN), lipoteichoic acid (LTA), bacterial lipoproteins, lipopeptides, and lipoarabinomannan (19, 22, 34, 36). TLR2 is predominantly expressed in cells involved in the first line of host defense, including monocytes, macrophages, dendritic cells, and PMN (17, 29, 37, 38). TLR4 has been identified as the receptor for LPS and LTA (20, 26). Recently, we reported that TLR4 signaling upregulates TLR2 in LPS-stimulated endothelial cells, and thus the cells' response to TLR2 ligands is significantly amplified (10). These results raised the possibility of a mechanism of inducible cell sensitivity to infection. These observations led us to further investigate the role of TLR4-TLR2 cross talk in AMφ priming in the setting of hemorrhage/resuscitation.
In the present study, using both in vivo hemorrhage/resuscitation mouse model and in vitro PMN-AMφ coculture approaches, we demonstrated that the TLR4-dependent TLR2 upregulation in AMφ is significantly augmented by antecedent shock, and this effect of shock is particularly mediated by shock-activated PMN. The functional relevance of the amplified TLR4-induced TLR2 expression in AMφ was evident by a markedly increased expression of chemokine macrophage inflammatory protein-2 (MIP-2), cytokine migration inhibitory factor (MIF), and TNF-α in the AMφ as well as induction of augmented PMN migration in response to sequential challenges of LPS and PGN. Thus, TLR2 expression in AMφ, signaled by TLR4 and regulated by hemorrhagic shock-activated PMN, could be an important positive-feedback mechanism responsible for shock-primed AMφ activation and alveolar neutrophilia following primary PMN sequestration.
MATERIALS AND METHODS
Hemorrhagic shock and lung injury.
Male C57BL/6 wild-type (WT) mice and C3H/HeJ mice were purchased from the Jackson Laboratory; TLR2−/− mice were obtained from Dr. Shizuo Akira (Research Institute for Microbial Diseases, Osaka Univ., Osaka, Japan) (35). All experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee of Veterans Affairs Pittsburgh Healthcare System. Mice were 9–10 wk of age at the time of experiments. Animals were anesthetized with 50 mg/kg ketamine and 5 mg/kg xylazine administered intraperitoneally. Femoral arteries were cannulated for monitoring of mean arterial pressure (MAP), blood withdrawal, and resuscitation. Hemorrhagic shock was initiated by blood withdrawal and reduction of the MAP to 40 mmHg within 15 min. Blood was collected into a 1-ml syringe and heparinized to prevent clotting. After a hypotensive period of 60 min, animals were resuscitated by transfusion of the shed blood and Ringer's lactate in a volume equal to that of the shed blood, over a period of 30 min. The catheters were then removed, the femoral artery was ligated, and the incisions were closed. Sham animals underwent the same surgical procedures without hemorrhage and resuscitation. Animals then underwent a tracheotomy using a 20-gauge catheter 1 h after the end of resuscitation (or sham) and received either repurified LPS [Escherichia coli O111:B4; Sigma, St. Louis, MO; LPS repurification was carried out as previously described (19a)] or 30 μg/kg in 50 μl of saline (SAL) or SAL alone intratracheally. The experimental protocol was described as having animals in one of four groups: sham/SAL, shock/SAL, sham/LPS, and shock/LPS. Using this protocol, we previously showed that animals subjected to shock/LPS exhibited increased lung permeability and PMN counts in bronchoalveolar lavage (BAL) fluid compared with all other groups, whereas the sham/LPS group had a small increase in BAL PMN count but no change in permeability (14). At various time points after LPS or SAL administration, AMφ were recovered from BAL fluid for TLR2 detection by RT-PCR and Western blotting.
In vivo PMN depletion and repletion.
PMN repletion in neutropenic mice was performed by tail vein injection of PMN (∼2 × 106 cells) isolated from mouse blood. The immunomagnetic separation system (BD Biosciences Pharmingen, San Diego, CA) (8) was used to isolate PMN. Viability of the isolated PMN was >95%, and PMN population was >95% as assessed by trypan blue exclusion (4) and Wright-Giemsa staining, respectively.
BAL was performed as previously described (14). Normally, the BAL fluid contains ∼91% of AMφ and ∼9% of other cells, including PMN, lymphocytes, and erythrocytes. The immunomagnetic separation system as described above was used to isolate AMφ from BAL fluid. Magnetic nanoparticle-conjugated antibodies (anti-mouse Gr-1, anti-CD4, anti-CD8, and anti-CD45R/B220 antibodies; BD Biosciences) were chosen to label and remove PMN and lymphocytes. The resulting cells consisted of >98% macrophages, and cell viability was >95%.
PMN-AMφ coincubation and air pouch PMN migration assay.
PMN-AMφ coincubation was performed using Transwell. Peripheral PMN were isolated from the mice subjected to either hemorrhage/resuscitation or sham operation, and the AMφ were recovered from BAL fluid using the methodology as described above. The coculture was then stimulated with repurified LPS (1 μg/ml) for 2–8 h in DMEM containing 10% FCS at a concentration of 1 × 106 cells/ml of medium. In some experiments, we first stimulated the cocultures with repurified LPS for 2 h, removed PMN and added PGN (1 μg/ml; Staphylococcus aureus, Sigma), and continued incubation for another 2 h. MIP-2, MIF, and TNF-α expression in the AMφ lysates was measured by Western blotting. In some experiments, the AMφ, which were preincubated with shock-activated PMN and sequentially treated with LPS and PGN (as described above), were collected from the Transwell, resuspended in Ringer's lactate in a concentration of 1 × 106 cells/ml, and injected into a mouse air pouch to induce PMN migration.
A mouse air pouch was prepared as described (9). To perform the PMN migration assay, the pretreated AMφ, as described above, were injected into the air pouch serving as a chemoattractant resource. Air pouch lavage fluid was then collected for PMN counts at 4 h after the injection of the macrophages.
Total RNA from AMφ was isolated using TRI-REAGENT (Molecular Research Center, Cincinnati, OH) following the manufacturer's instructions. Total RNA was then reverse transcribed using a SuperScript Preamplification kit (Invitrogen, Carlsbad, CA). Primers for TLR2 amplification were: position 1008 forward 5′-ATACTAACTTGGCCAGGTTC-3′, position 1590 reverse 5′-AAAGTGTTCCTGCTGATGTC-3′, amplifying 582 bp. Primers for mouse GAPDH were purchased from R&D Systems (Minneapolis, MN). The product of reverse transcription was amplified following the kit's instructions. PCR products were separated using 1.2% agarose gel and identified by ethidium bromide staining. Expression of mRNA was quantitated using Scion Image software (Scion, Frederick, MD) and normalized by the GAPDH signal.
Western blot analysis.
Aliquots of AMφ lysates were separated on a 10% SDS-PAGE under nonreducing conditions. Equivalent loading of the gel was determined by quantitation of protein as well as by reprobing membranes for actin detection. Separated proteins were electroblotted onto polyvinylidene difluoride membranes and blocked for 1 h at room temperature with Tris-buffered saline containing 1% BSA. The membranes were then probed with primary antibody (polyclonal anti-TLR2, -MIP-2, -MIF, or -TNF-α antibody purchased from Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 h. After being washed, primary antibodies associated with the membranes were detected on autoradiographic film by horseradish peroxidase-conjugated secondary antibodies and the ECL plus chemiluminescent system (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. Blots were quantitated using Scion Image software and normalized by actin signal.
The data are presented as means ± SE of n determinations as indicated in the figures. Data were analyzed by one-way analysis of variance; post hoc testing was performed using the Bonferroni modification of the t-test. Where individual studies are demonstrated, these are representative of at least three independent studies.
LPS upregulates TLR2 expression in AMφ through TLR4 signaling.
As shown in Fig. 1A, LPS challenge of AMφ, which were isolated from the BAL fluid of WT mice, was associated with an increased TLR2 mRNA expression at 1 h and more marked increases at 2 and 6 h. The increased TLR2 mRNA trended back to basal level 16 h after LPS stimulation. However, in AMφ derived from C3H/HeJ mice, which are not responsive to LPS because of a point mutation of TLR4 affecting the Toll/IL-1 receptor domain (32, 33), LPS failed to induce TLR2 expression (Fig. 1A). This result suggests that TLR4 signaling mediates the LPS-induced upregulation of TLR2. The changes in protein expression of TLR2 shown in Fig. 1B paralleled the changes in mRNA.
The promoter region of the TLR2 gene contains two NF-κB consensus binding sequences believed to regulate gene transcription (28). To address the role of NF-κB in mediating LPS-induced upregulation of TLR2 in AMφ, we assessed the effects of IKK-NBD, an NF-κB inhibitor (6), on the LPS-induced TLR2 expression in AMφ. As shown in Fig. 1, C and D, the NF-κB inhibitor (100 μM) partially but significantly attenuated the LPS-induced upregulation of TLR2 expression in WT AMφ at 2 h after LPS challenge (Fig. 1, C and D). This result demonstrates a role for NF-κB signaling in mediating TLR4-TLR2 cross talk.
Hemorrhage/resuscitation enhances LPS-induced TLR2 upregulation in AMφ.
To address the role of antecedent hemorrhagic shock in the regulation of TLR2 in AMφ, animals were subjected to hemorrhage/resuscitation (shock) or sham operation and then given LPS or SAL intratracheally at 1 h after resuscitation. AMφ were then recovered from BAL fluid at 1–4 h after LPS or SAL for TLR2 mRNA detection. As shown in Fig. 2A, AMφ from sham animals demonstrated a low level of TLR2 mRNA expression. Shock alone induced a slight increase in TLR2 in AMφ by 4 h. Administration of LPS to sham animals resulted in an increase in TLR2 mRNA in AMφ by 2 h, which increased further by 4 h. However, animals subjected to shock before LPS exhibited a marked increase in TLR2 mRNA by 2 and 4 h (1.6- and 2.3-fold increase vs. sham/LPS, respectively). Considered together, these data support the notion of a priming role for hemorrhagic shock in the LPS-induced TLR2 upregulation in AMφ.
To address the role of TLR4 signaling in shock-modulated TLR2 expression, C3H/HeJ mice were subjected to shock followed by intratracheal LPS. By 4 h after LPS administration, LPS failed to upregulate TLR2 mRNA and protein expression in the AMφ derived from C3H/HeJ mice (Fig. 2, B and C). These results suggest that modulation of LPS-induced TLR2 upregulation by hemorrhagic shock occurs through a TLR4-dependent mechanism.
Shock-activated PMN contribute to shock-enhanced TLR2 upregulation in AMφ.
We have previously shown that PMN and PMN-derived oxidants play an important role in mediating the upregulation of TLR2 in lung endothelial cells induced by TLR4 signaling (10). In this study, we hypothesized that PMN initially sequestered into alveoli after shock and LPS stimulation would contribute to the augmented TLR4-induced TLR2 expression in AMφ through PMN-AMφ interaction. We used the PMN-AMφ coculture system to test the hypothesis. AMφ from WT mice were cocultured with PMN that were isolated from either sham-operated or shock-resuscitated WT mice in the presence or absence of LPS for up to 6 h. The alterations in TLR2 mRNA and protein expression in the AMφ are shown in Fig. 3, A and B. Constitutive expression of TLR2 in the AMφ could be detected in the absence of LPS treatment (time 0), and this expression was unaltered when the AMφ were cocultured with PMN derived from mice subjected to either sham or shock (lanes 1–3). Coculture of AMφ with PMN derived from mice subjected to hemorrhagic shock caused a significantly higher level of TLR2 expression in the AMφ in response to LPS at the 6-h time point (lane 10) compared with the group cocultured with PMN isolated from sham animals (lane 9). These results suggest a dominant role of shock-activated PMN in the response.
To address the role of PMN-derived oxidants in signaling LPS-induced TLR2 expression in AMφ, antioxidant membrane-permeable polyethylene glycol (PEG)-catalase (1,000 U/ml) was applied to the coculture system. As shown in Fig. 3, A and B, PEG-catalase attenuated the effect of shock-activated PMN in increasing TLR2 expression in LPS-stimulated AMφ. Because the coculture experiments were performed using the Transwell approach, the results suggested that the interaction between AMφ and PMN might not be through a direct coupling.
To confirm the role of PMN, we next evaluated the response to shock/LPS challenge in AMφ of WT mice after the depletion of circulating PMN. In some cases, we replenished the PMN in the mice made neutropenic, to address the causal role of PMN in mediating the response. As shown in Fig. 4A, at 4 h after LPS challenge, neutropenia induced in mice subject to hemorrhagic shock was associated with a ∼58% reduction in TLR2 expression in AMφ (lane 6) compared with mice subjected to shock/LPS with no PMN depletion (lane 5). In contrast, depletion of PMN did not alter TLR2 expression in sham/LPS-treated AMφ (lane 2). Repletion with WT PMN isolated from animals subjected to hemorrhagic shock restored the expression of TLR2 in AMφ in response to shock/LPS (lane 9). However, repletion with PMN derived from sham-operated animals failed to restore TLR2 expression (lane 7). Interestingly, replenishing the neutropenic sham/LPS mice with PMN that were obtained from mice subjected to hemorrhagic shock resulted in a ∼3.3-fold increase in TLR2 expression in AMφ (lane 4) compared with non-neutropenic sham/LPS mice (lane 1). Together, these results demonstrate the critical role of shock-activated PMN in the augmented TLR4-induced TLR2 expression in AMφ. The changes in protein level of TLR2 shown in Fig. 4B paralleled the changes in mRNA (Fig. 4A).
To further address the role of TLR4 in PMN activation, we replenished neutropenic shock/LPS WT mice with C3H/HeJ PMN. As shown in Fig. 4, A and B, PMN collected from C3H/HeJ mice subjected to either sham or shock failed to restore TLR2 expression in mice subjected to shock/LPS (lanes 8 and 10), suggesting an important role for TLR4 in the activation of PMN by shock/LPS.
Increased TLR2 expression in AMφ results in enhanced cytokine expression and PMN migration.
To address the effect of shock-enhanced LPS/TLR4 activation of TLR2 in AMφ on inflammatory cytokines, we assessed MIP-2, MIF, and TNF expression in AMφ using sequential challenges of LPS and PGN, a ligand for TLR2. In the AMφ-PMN coculture system, LPS was added to the cocultures at time 0, followed by removal of the PMN and addition of PGN to the AMφ at 2 h (the time point at which TLR2 was upregulated in the AMφ as described above). MIP-2, MIF, and TNF protein levels in the AMφ were then assessed at 4 h by Western blotting. PMN-derived cytokines were excluded by removing PMN from the system before addition of PGN. LPS or PGN alone induced a slight increase in MIP-2, MIF, and TNF in AMφ that were preincubated with PMN isolated from the mice subjected to either sham or shock (Fig. 5, A–C). Whereas the sequential challenges of LPS and PGN caused small increases in the expression of inflammatory cytokines in AMφ that were preincubated with PMN isolated from sham-operated animals, AMφ that were preincubated with shock-activated PMN exhibited marked increases in MIP-2, MIF, and TNF expression in response to the sequential challenges of LPS and PGN (Fig. 5, A–C). These results suggest a priming role vis-à-vis cytokine expression of the upregulated TLR2 in enhancing AMφ response to bacterial components.
To establish a linkage between the increased expression of the cytokines in AMφ and PMN migration, we recovered AMφ from the AMφ-PMN coculture system in which the AMφ were preincubated with shock-activated PMN and sequentially stimulated with LPS and PGN, and we injected the AMφ into the mouse air pouch to induce PMN migration. PMN counts in the air pouch lavage fluid at 4 h after the injection of AMφ are shown in Fig. 6A. AMφ that were preincubated with shock-activated PMN and challenged with LPS and PGN induced a significantly higher number of air pouch PMN, which was 2.7- and 2.4-fold greater than that in the groups preincubated with no PMN or the PMN isolated from sham-operated mice, respectively. These data paralleled with the changes in MIP-2, MIF, and TNF expression in the AMφ as shown in Fig. 5.
As chemokine-dependent PMN migration is an important determinant of lung PMN infiltration, we next addressed the role of shock-enhanced LPS upregulation of TLR2 in the formation of alveolar neutrophilia using the in vivo hemorrhage-resuscitation model in WT, C3H/HeJ, and TLR2−/− mice. We first challenged the mice with hemorrhage/resuscitation, and 1 h after resuscitation, we administered LPS plus PGN intratracheally. We counted PMN in BAL at 2- and 6-h points after LPS/PGN administration to represent PMN infiltration at the initial phase and later phase, respectively. As shown in Fig. 6B, at the 2-h time point, either LPS alone or LPS/PGN caused an ∼8.3-fold increase in BAL PMN in WT sham-operated animals (groups 2 and 4) compared with SAL control (group 1). In contrast, LPS alone and LPS/PGN caused a 31.2- and 30.0-fold increase in alveolar PMN in WT mice subjected to shock (groups 6 and 8) compared with saline control (group 5), respectively. PGN alone caused a 4.1- and an 11.5-fold increase in BAL PMN in animals subjected to sham operation (group 3) and shock (group 7) at 2 h compared with SAL controls (groups 1 and 5), respectively. At the 6-h time point, PGN alone did not alter BAL PMN in either sham-operated or shocked animals (groups 3 and 7). In contrast, LPS alone caused a significant further increase in BAL PMN at 6 h in both sham-operated and shocked mice compared with that observed at 2 h (groups 2 and 6). However, whereas LPS/PGN caused a 2.8-fold further increase in BAL PMN in WT animals subjected to sham operation at 6 h (group 4), the treatment of LPS/PGN induced a 5.2-fold further increase in BAL PMN in WT mice subjected to shock at 6 h compared with the 2-h point (group 8), but LPS/PGN only caused a 2.0-fold further increase in TLR2−/− mice (group 10). In C3H/HeJ shocked mice, BAL PMN levels at 2 and 6 h were markedly attenuated in response to LPS/PGN challenge (group 9) compared with WT shocked mice.
Recent studies have supported a two-hit hypothesis in the pathogenesis of lung injury in trauma patients, namely that an initial stimulus may prime for subsequent organ damage in response to a second, often minor, insult (12, 14, 27). One possible mechanism underlying this phenomenon may be that circulating PMN are primed for increased lung sequestration and cytotoxic activity (5). PMN sequestration in the lung is a result of a cascade of cellular events in which PMN, endothelia, epithelia, and AMφ act in concert. The role of AMφ in the regulation of PMN sequestration is thought to be critical, since AMφ initiate PMN migration through direct interactions with PMN mediated by the expression of chemokines and cytokines. We have previously reported that priming of AMφ after hemorrhage and resuscitation contributes to augmented lung injury, through increased release of chemokines and proinflammatory cytokines, and subsequent PMN sequestration (12, 14). However, questions remain regarding how AMφ are primed and whether or not interactions between PMN and AMφ can contribute to, and thus prime, AMφ for amplified pulmonary inflammation in response to a trivial infection. In the present study, we observed that LPS/TLR4 signaling upregulated TLR2 expression in AMφ, and shock-activated PMN played a critical role in the mechanism of TLR2 upregulation. This cross talk between TLR4 and TLR2 in AMφ resulted in the amplification of expression of cytokines and chemokines in response to the bacterial products LPS and PGN and subsequently led to enhanced PMN sequestration in the lung. Thus the present study demonstrates a novel mechanism underlying hemorrhagic shock-primed lung injury, namely that hemorrhagic shock-activated PMN that were initially sequestered in alveoli can instruct AMφ to upregulate TLR2, thereby sensitizing AMφ to TLR2 ligands and promoting enhanced lung inflammation (Fig. 7).
The role of TLR4 signaling in regulating TLR2 expression was clearly delineated in the studies using C3H/HeJ mice as shown in Fig. 1. This result is consistent with previous reports showing that LPS, through TLR4, upregulates TLR2 expression in macrophages (23, 28). However, we further observed that TLR4 signaling is also required for the shock-enhanced TLR2 expression, since LPS challenge of C3H/HeJ mice failed to induce enhanced TLR2 expression in a setting of hemorrhage/resuscitation. Since we used repurified LPS in all experiments to eliminate any possible effects of non-LPS contaminations, the observed TLR2 upregulation was secondary to the activation of TLR4 signaling rather than the direct LPS activation of TLR2. In in vitro studies, we found a reduced expression of TLR2 in AMφ in which NF-κB was inhibited by IKK-NBD, indicating a role of NF-κB in mediating the TLR4-TLR2 cross talk. However, since the inhibitory effect of IKK-NBD seemed partial, the role of other transcript factors, such as CCAAT/enhancer-binding protein, cAMP response element binding protein, and signal transducers and activators of transcription (28) may need to be investigated.
Our previous study demonstrated that antecedent shock prevents the LPS-induced reduction in TLR4 expression, thereby breaking the negative feedback mechanism of TLR4 regulation in response to LPS (13). This might be one of the mechanisms underlying cell priming for increased susceptibility to LPS after resuscitated hemorrhagic shock. However, in clinical scenarios, a variety of pathogens are involved in posttrauma lung infection. TLR2 is capable of recognizing a much broader range of pathogen components than TLR4 can. For example, TLR2 can recognize the components derived from gram-positive and gram-negative bacteria, mycobacteria, mycoplasma, and fungi (7, 16, 19, 22, 25, 30, 34, 35). Thus upregulated TLR2 may contribute to increased response in the lung to a variety of pathogens after trauma. In the present study, we showed that upregulation of TLR2 significantly amplified chemokine and cytokine expression in AMφ in response to PGN and functionally enhanced PMN migration. Thus the present study suggests an important physiological significance of the TLR4-TLR2 cross talk in activating a positive-feedback signal leading to amplified AMφ activation and local inflammation in response to invading pathogens.
Amplified AMφ activation and PMN infiltration induced by PGN are dependent on augmented upregulation of TLR2 that resulted from the interaction of PMN and AMφ. The primarily sequestered PMN seem to be important in priming for consequent enhanced PMN infiltration. This is evident by the BAL PMN counts in in vivo hemorrhage-resuscitation model as shown in Fig. 6B. In the early phase, PMN infiltration is dependent on TLR4 signaling, since BAL PMN increased at the 2-h time point in WT and TLR2−/− mice, but not in TLR4 mutant mice, in response to the treatment of shock/LPS/PGN. LPS alone increased the BAL PMN counts, whereas PGN alone did not. However, in the later phase (by 6 h), the amplified PMN infiltration is secondary to upregulated TLR2 expression as evident by the facts that 1) BAL PMN was markedly elevated in WT animals, but not in TLR2-deficient or TLR4 mutant animals, which were subjected to shock/LPS/PGN challenges; 2) either LPS or PGN alone failed to induce an amplified PMN infiltration in WT shocked mice; and 3) LPS plus PGN failed to increase the PMN counts in WT sham-operated animals. Thus the findings explored an important role of the shock-activated PMN and TLR4 signaling in activating a positive-feedback signal leading to exaggerated lung inflammation through the upregulation of TLR2 expression in AMφ (Fig. 7).
TLR4 per se seems play a role in shock activation of PMN as shown in Fig. 4. Repletion of neutropenic WT shocked animals with PMN derived from C3H/HeJ shocked mice did not restore AMφ expression of TLR2 in response to LPS. One possible mechanism underlying this phenomenon may be that shock activates PMN through releasing endogenous “danger signals” (24) and activating TLR4. Recent studies have shown that endogenous danger molecules, such as high mobility group box 1 protein (HMGB1), are potent activators of TLR4 (31). HMGB1 expression, for example, in the lung increased within 4 h after hemorrhage and is believed to contribute to the development of posthemorrhage acute lung injury (21). Another plausible explanation is that other than TLR4 playing a role in activating PMN by shock, TLR4 is also required as an LPS receptor to “fully” activate PMN in response to the following LPS challenge. However, for the PMN activation, the role of TLR4 in mediating shock-derived signals seems more important than that in mediating LPS signal, since repletion of neutropenic shocked animals with PMN from WT sham-operated mice, which possess functional TLR4, was not able to restore TLR2 expression in AMφ. Further investigation is required to define the precise nature of TLR4-dependent activation of PMN by shock.
Oxidants appear to participate in the regulation of TLR2 gene expression as demonstrated in Fig. 3. The most probable explanation is that oxidants enhance signaling through the TLR4-NF-κB cascade and thus promote augmented LPS-induced TLR2 gene transcription. Evidence supporting an oxidant effect on this signaling pathway is derived from two observations made in our prior reports (12, 14). First, in animal experiments, antioxidant N-acetylcysteine (NAC) addition during resuscitation prevented the augmented NF-κB translocation in AMφ after LPS treatment. Second, NAC supplementation reversed the enhanced LPS-induced gene transcription of the NF-κB-dependent genes, such as CINC and TNF, in AMφ from the animals exposed to antecedent shock. It was found in our previous studies that PMN NAD(P)H oxidase served as an important source of oxidants for mediating PMN-endothelial cell cross talk (10, 11). Further studies are required to define the source(s) of oxidants released from shock-activated PMN.
In summary, the present study demonstrates a function of shock-activated PMN in mediating TLR4-TLR2 cross talk in AMφ and AMφ priming, which acts in a positive-feedback manner to amplify pulmonary neutropenia and inflammation.
This work was supported by a research grant from the Department of Surgery, University of Pittsburgh, and National Heart, Lung, and Blood Institute Grant HL-079669.
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- Copyright © 2006 the American Physiological Society