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Pulmonary and Critical Care Division
Departments of Physiology and Surgery, TuftseCNew England Medical Center, Boston, Massachusetts
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut
Department of Pathology, Brown University School of Medicine, Providence, Rhode Island
ABSTRACT
The cascade of cellular and molecular pathways mediating acute lung injury is complex and incompletely defined. Although the Src and Jak family of kinases is upregulated in LPS-induced murine lung injury, their role in the development of lung injury is unknown. Here we report that systemic inhibition of these kinases using specific small molecule inhibitors (PP2, SU6656, tyrphostin A1) significantly attenuated LPS-induced lung injury, as determined by histologic and capillary permeability assays. These inhibitors blocked LPS-dependent cytokine and chemokine production in the lung and in the serum. In contrast, lung-targeted inhibition of these kinases in the airway epithelium via adenoviral-mediated gene transfer of dominant negative Src or of suppressor of cytokine signaling (SOCS-1) disrupted lung cytokine production but had no effect on systemic cytokine production or lung vascular permeability. Mice were significantly protected from lethal LPS challenge by the small molecule inhibitors of Jak and Src kinase. Importantly, this protection was still evident even when the inhibitors were administered 6 hours after LPS challenge. Taken together, these observations suggest that Jak and Src kinases participate in acute lung injury and verify the potential of this class of selective tyrosine kinase inhibitors to serve as novel therapeutic agents for this disease.
Key Words: acute lung injury Jak kinase lipopolysaccharide signal transducers and activators of transcription Src kinase
Acute lung injury (ALI) as manifested by the acute respiratory distress syndrome is a devastating clinical problem, with mortality rates as high as 60% (1, 2). Studies in both humans and animals indicate that a network of proinflammatory cytokines, such as tumor necrosis factor- (TNF-) and interleukin 6 (IL-6), and chemokines, such as IL-8, are critical for initiating, amplifying, and perpetuating lung injury induced by diverse stimuli (3). The production of antiinflammatory cytokines, such as IL-10, is important to promote resolution of the injury process. The persistent elevation of proinflammatory cytokines in the serum of patients with ALI has been associated with increased mortality (4). Conversely, lung-protective ventilation as well as corticosteroids for unresolving cases have been demonstrated to decrease inflammatory cytokines and patient mortality in ALI (5eC8). Pharmacotherapy aimed at blocking a specific cytokine, such as IL-1 or TNF-, has been unsuccessful. These treatment failures point out our lack of understanding of the cellular and molecular mechanisms that underlie ALI and also suggest that a more global inhibition of cytokine signaling may be needed.
Signaling pathways upregulated in animal models of ALI in either the lung or phagocytic cells include the nuclear factor-B (NF-B) pathway, mitogen-activated protein (MAP) kinase and phosphatidylinositol 3eCkinase pathways (9, 10). In addition, we have previously determined that two tyrosine kinases, Src and Jak, are rapidly activated in an LPS model of ALI in the lung (11). The Jak family of kinases is tightly associated with cytokine receptors and plays a critical role in activating multiple downstream signaling pathways. The Src family of kinases has also been shown to participate in cytokine signaling and inflammatory responses (12, 13). Importantly, the Src family kinases have been shown to be critical regulators of cell signaling in immune cells (14). Both of these tyrosine kinases have many downstream signaling effectors, but the signal transducer and activator of transcription (STAT) factors are a well-described common target. The STATs, similar to NF-B, regulate the expression of genes that are critical for inflammatory and immune responses (15). Previously, it has been determined that several STAT proteins are upregulated in animal models of lung injury (11, 16).
Endotoxin or LPS from the outer wall of gram-negative bacteria induces a sepsis syndrome accompanied by key features of ALI, including the recruitment of inflammatory cells into the lung with subsequent increases in capillary permeability and alveolar edema (17). Protein tyrosine kinases, including Src and Jak, are activated by LPS and are required for a number of LPS-mediated responses in macrophages (18eC20). Mice lacking Jak or Src family members are resistant to high-dose LPS challenge, but the LPS effects on lung injury in these animals have not been examined (21, 22).
Here we have investigated the impact of inhibiting the Src and Jak kinases on LPS-induced ALI in mice by both small molecule inhibitors and adenoviral-mediated gene transfer. Targeted inhibition of these kinases in the airway epithelium prevented LPS-induced cytokine production in the lung, but did not impact systemic cytokine production or ALI. However, the systemic inhibition of Src and Jak kinases resulted in decreased LPS-mediated ALI, systemic cytokine and chemokine production, and mortality even when administered 6 hours after LPS challenge.
Some of the results of these studies have been previously reported in the form of an abstract (23eC25).
METHODS
For a detailed description of the methods, see the online supplement.
Reagents
AG1478, SU6656, and PP2 and were purchased from Calbiochem (La Jolla, CA), Tyrphostin A1 (TyrA1) was obtained from LC Laboratories (Woburn, MA). LPS from Escherichia coli (serotype 055:B5) and all other chemicals, unless indicated, were purchased from Sigma-Aldrich (St. Louis, MO). Phospho-STAT3 (Y705) and STAT3 antibodies, phospho-Jak2, and phospho-Src (Tyr416) were purchased from Cell Signaling (Beverly, MA); Src (pp60) and Jak2 antibodies were obtained from Upstate Biotechnology (Lake Placid, NY).
Lung Injury Model
BALB/c mice were injected intraperitoneally with either 6 mg/kg or 20 mg/kg (lethal challenge) of LPS (11). Animals received tyrosine kinase inhibitors either before or after LPS injection as indicated at doses of 1 mg/kg for TyrA1 and PP2, 8 mg/kg for SU6656, and 0.5 mg/kg of AG1478. For lethal challenge studies, mice were treated with two doses of inhibitors each day (8:00 A.M. and 2:00 P.M.) for 7 days, and followed daily out to 14 days for mortality. All experiments were conducted under a protocol approved by the institutional animal care and research committee.
Histologic Examination
The lungs were embedded in paraffin and processed for immunohistochemistry as previously described (11). Lung sections stained with hematoxylin and eosin were graded for lung injury using previously published criteria by a board-certified, blinded pathologist (26).
Western Blot Analysis
Whole lungs were homogenized in lysis buffer and processed for Western blot analysis as previously described (11).
Serum or Lung Tissue Quantification of Cytokines and Chemokines
Serum and lung tissue homogenates were prepared as previously described (11). The levels of TNF-, IL-6, and macrophage inflammatory protein 2 (MIP-2) in the serum and lung tissue were quantitated by ELISA kits (R&D Systems, Minneapolis, MN).
Adenoviral Infection
Mice were infected intranasally with adenovirus containing dominant negative Src, suppressor of cytokine signaling, or a green flourescent protein (GFP) control adenovirus on Day 1 and 3 as previously described (11). On Day 5, LPS was administered. The replication-deficient adenovirus SOCS-1 and the GFP control were provided by Dr. Daniel Frantz and Steve Shoelson (Harvard University, Boston, MA) (27). The adenovirus expressing the dominant negative c-Src was constructed in our laboratory in collaboration with the Gastroenterology and Research on Absorption and Secretory Processes (GRASP) viral core facility center using replication-deficient adenovirus. Expression of adenovirus was confirmed by examining lung sections using the Zeiss Axiovert 10 fluorescent microscope. (See the online supplement for details.)
Assessment of Lung Capillary Leakage
Evans blue dye (EBD; 20 mg/kg) was injected intravenously. Animals were killed, and the right lung was homogenized with formamide and incubated for 18 hours at 37°C. After centrifugation, the supernatant was collected and the optical density was determined spectrophotometrically at 620 nm. EBD concentration in lung homogenate was calculated against a standard curve and was expressed as micrograms of EBD/gram of tissue.
Statistical Analysis
Data are expressed as means ± SEM. An analysis of variance was performed with Statview statistical analysis software (SAS Institute, Cary, NC), and a difference was accepted as significant if the p value was 0.05 or less as verified by the Bonferroni-Dunn post hoc test. All experiments were repeated at least two times and represent data on 3 to 10 mice per data point. A Student's t test was performed on the means of two sets of sample data and considered significant if the p value was 0.05 or less.
RESULTS
LPS-induced Src and Jak2 Activation Is Blocked in Mouse Lung by Small Molecule Inhibitors
TyrA1 and SU6656 have been found to be specific and effective inhibitors of Jak and Src kinases in vitro, respectively (28eC30). Our prior work indicated that LPS activates the Jak and Src kinases in the lung after 30 minutes, with maximal activation occurring at 2 hours for both kinases (11). Both of these kinases were subsequently downregulated at 12 hours followed by a second wave of activation at 24 to 48 hours. To determine whether these small molecule inhibitors can block the activation of these kinases in vivo, mice were treated with LPS and total lung lysates were assayed for Src and Jak activation with phospho-specific antibodies. These antibodies only recognize the activated forms of both of these kinases. LPS induced a threefold increase in Src phosphorylation in the lung that was completely inhibited by SU6656 (Figure 1A). TyrA1 also inhibited Src phosphorylation (twofold decrease), but to a lesser extent than SU6656. LPS induced a fourfold increase in Jak2 activation that was attenuated by both TyrA1 (twofold decrease) and SU6656 (fourfold decrease; Figure 1B). These results suggest that both Src and Jak kinases can be effectively inhibited in vivo by TyrA1 and SU6656, and that these kinases may mediate in part some of the lung's response to LPS.
Src and Jak Kinase Inhibitors Block STAT3 Activation In Vivo
The STAT3 transcription factor is a downstream effector of both Jak and Src kinases and is activated by tyrosine phosphorylation by these kinases. To further confirm that the previously described kinase inhibitors were effective in vivo, we evaluated their ability to inhibit LPS-induced STAT3 activation in lung. We have previously demonstrated that LPS rapidly activates STAT3 in the lung with kinetics similar to that seen with the Jak and Src kinases (11). STAT3 activation was assayed by evaluating the phosphorylation state of STAT3 with anti-STAT3 (Y705) phospho-specific antibody. The phosphorylation of this conserved C-terminal tyrosine is required for STAT activation. In addition to SU6656, we used the small molecule inhibitor PP2 to determine whether a structurally distinct Src family tyrosine kinase inhibitor would yield similar results (31). Pretreatment of mice with TyrA1 or PP2 blocked the early STAT3 tyrosine phosphorylation in the lung induced 1 hour after LPS injection as determined by Western blot analysis (Figure 2A). The mechanism of STAT activation by LPS is likely to change over time as different cytokines and inflammatory mediators are elaborated. To determine whether these inhibitors were effective at preventing STAT3 activation that occurred at more delayed time points after LPS administration, mice were injected with LPS and then killed after 24 hours. These inhibitors were also effective at preventing LPS-induced STAT3 activation that occurred 24 hours after LPS treatment (Figure 2B). Finally, to evaluate whether the inhibitors were still effective when administered after LPS administration, both PP2 and TyrA1 were given 2 hours after LPS treatment. STAT3 activation by LPS was dramatically reduced at 6 hours after LPS treatment when the inhibitors were administered in this manner (Figure 2C). The administration of inhibitors alone to the mice was well tolerated and did not result in STAT activation. In addition, SU6656 administration had a similar effect on LPS-induced STAT3 activation as PP2 (Figure 2D). Thus, we conclude that these Jak and Src kinase inhibitors prevented LPS-induced STAT activation and thus function effectively in vivo. In addition, these data suggest that STAT3 activation by LPS in the lung requires both Jak and Src kinase activity.
To determine where in the lung the inhibitors were functioning, immunohistochemistry was performed using phospho-specific STAT3 antibody. Our prior work demonstrated that LPS-induced STAT3 activation in the lung occurred most intensely in the airway epithelium, but also was found in the endothelium of multiple vascular beds, as well as in the recruited inflammatory cells (11). When STATs are activated, they translocate to the nucleus; therefore, nuclear staining indicates the site of STAT activation. Mice treated with either inhibitor had less intense LPS-induced airway epithelial and endothelial staining for STAT3 in the lungs than control untreated animals (Figure 3). Thus, these inhibitors were effective in blocking STAT3 activation in the airway epithelium and endothelium of the lung.
Inhibition of Src and Jak Kinase Activity Decreases LPS-induced Lung Injury
To examine whether the Jak or Src family of kinases mediates ALI, we evaluated the effect of inhibiting these kinases on LPS-induced ALI as determined histologically. Mice were pretreated with PP2, SU6656, TyrA1, or a combination of both SU6656 and TyrA1 before LPS administration, and lung tissue was obtained for pathologic examination. Light microscopic evaluation of the lung sections indicated neutrophil infiltration begins at 6 hours after LPS injection, whereas hemorrhage and endothelial and epithelial damage begins at 16 hours and further increased by 24 hours (data not shown and Figure 4). Twenty-four hours after LPS treatment the mice were lethargic, with evidence of respiratory distress and ocular edema. In contrast, mice receiving LPS plus either of the inhibitors were grossly indistinguishable from the untreated control mice. Histologic examination of the lungs of mice that received inhibitors in addition to LPS injection revealed a significant decrease in multiple features of ALI, including inflammation (as indicated by inflammatory cell number), edema, hemorrhage, and atelectasis compared with mice that received LPS alone as determined by a previously published lung injury score (Figure 4 and Table 1) (26).
Inhibition of Src and Jak Tyrosine Kinase Activity Decreases LPS-induced Lung Permeability
A hallmark of ALI is alveolar-capillary membrane damage with subsequent increases in microvascular permeability. To determine whether LPS-induced changes in vascular permeability could be attenuated by blocking Src and Jak kinase activity, the EBD assay was performed (32). As shown in Figure 5A, lung EBD concentration increased more than twofold in mice treated with LPS for 20 hours (p < 0.001). The administration of TyrA1, SU6656, or both together resulted in a significant decrease in EBD concentration in each of the experimental groups (p < 0.05 for all inhibitors). Because pretreatment with inhibitors does not reflect what happens most often clinically, we examined the effect on vascular permeability when inhibitors were administered after LPS treatment (Figure 5A). LPS-mediated changes in vascular permeability as determined by EBD analysis were likewise inhibited when animals were first treated with inhibitors 6 hours after LPS administration (p < 0.05 for all inhibitors). Thus, systemically inhibiting Src and Jak kinase activity protected against LPS-induced lung injury as determined by both histologic and capillary permeability analyses.
Inhibition of Src and Jak Kinases Decreases LPS-induced Cytokine Production in the Lung and Serum
TNF- and IL-6 are proximal proinflammatory cytokines upregulated after LPS administration. In addition, the prolonged generation of IL-6 in humans with ALI has been associated with increased mortality (4). To determine whether inhibition of Src and Jak kinases attenuated cytokine production, LPS-induced TNF- and IL-6 levels were evaluated by ELISA after treatment with TyrA1 and SU6656. As shown in Figure 5B, 6 hours after LPS administration, TNF- levels increased more than fourfold in both the serum and the lung as determined by ELISA (p 0.001). This LPS-induced TNF- production in both the lungs and serum was significantly decreased by treatment with TyrA1 and SU6656 alone or in combination when administered 2 hours after LPS injection (p 0.05 for all treatments). LPS treatment also resulted in a greater than 20-fold increase in IL-6 levels in both the serum and the lungs (p 0.0001). Similarly, both inhibitors alone or in combination were effective at inhibiting IL-6 production in both the lungs and serum (p 0.05 for all treatments; Figure 5C). Similar inhibition of LPS-induced TNF- and IL-6 generation was seen at 24 hours when the inhibitors were administered up to 6 hours after LPS treatment (data not shown).
Inhibition of Src and Jak Kinases Decreases LPS-induced Chemokine Generation
Neutrophil sequestration into the lung is critical for host defense but also contributes to the development of ALI. In rodent models of lung injury, the chemokine MIP-2 has been shown to be essential for neutrophil recruitment into the lung because rodents do not have IL-8 (33, 34). We determined that LPS treatment resulted in a greater than 250-fold increase in serum MIP-2 levels compared with that in control animals (Figure 5D). However, animals that received either TyrA1 or SU6656 in addition to LPS injection had a significant decrease in serum MIP-2 compared with control animals. MIP-2 levels have also been demonstrated to be elevated in neutrophil-infiltrated lung tissue in response to inflammatory stimuli; therefore, we evaluated the ability of these inhibitors to diminish lung MIP-2 generation in response to LPS. Similar to that seen with serum, the inhibitors significantly decreased LPS-induced MIP-2 production in the lung (Figure 5D). These results together with the histologic evaluation suggest that the Src and Jak kinase inhibitors may exert a beneficial effect on ALI by decreasing the numbers of neutrophils migrating into the lung.
The Acute-Phase Response in the Liver Is Attenuated by Src and Jak Kinase Inhibitors
To determine whether the systemic administration of the Src and Jak kinase inhibitors was also downregulating signaling pathway outside the lung, we assessed STAT3 activation in the liver. Acute-phase response genes are induced in the liver in response to LPS (35). STAT3 is required for the induction of all genes downstream of IL-6 and most genes downstream of LPS in the liver (36). As seen in Figure 5E, STAT3 phosphorylation was robustly induced at 6 hours and only weakly present at 24 hours. Administering SU6656 was effective at attenuating STAT3 activation at the earlier time point (6-hour), whereas TyrA1 was effective only at the later (24-hour) time point. Thus, our data indicate that the systemic administration of the Src and Jak kinase inhibitors attenuates the acute-phase inflammatory response in the liver as evidenced by decreased LPS-mediated STAT3 activation. Collectively, these data indicate that preventing Src and Jak kinase activation may afford protection against LPS-induced ALI by attenuating the inflammatory response in organs outside the lung.
Adenoviral-mediated Gene Transfer of SOCS-1 and DN-Src Inhibits LPS-induced STAT3 Activation in Mouse Lung
Small molecule inhibitors, such as SU6656 and TyrA1, when administered intraperitoneally affect multiple organs and cell types and could have nonspecific effects on other kinases. Therefore, to more precisely target inhibition of the Src and Jak kinases, adenoviral-mediated gene transfer was used to overexpress SOCS-1 (AdSOCS-1), a naturally occurring Jak kinase inhibitor, or a dominant negative form of Src (AdDN-Src), which is kinase inactive, in the lung. Intranasal administration of adenovirus has been shown to be an effective method to overexpress genes in the airway epithelium of mice (11, 37). As shown in Figure 6A, LPS-induced STAT3 activation in the lung was diminished by infection with AdSOCS-1 or AdDN-Src, but not by the control AdGFP virus. The extent of adenoviral infection was determined by examining the fluorescence of lung sections from mice infected with adenovirus containing a GFP tag. As seen in Figure 6B, there was marked and extensive fluorescence along the airway epithelium in the GFP-infected animals, with only minimal background fluorescence in animals infected with a virus lacking GFP. Thus, these results indicate that LPS-mediated STAT3 activation in mouse lung requires Src and Jak kinase activation in the lung itself. In addition, it demonstrates that adenoviral-mediated gene transfer is an effective way to inhibit Src and Jak kinases locally in the lung epithelium.
Overexpression of AdSOCS-1 and AdDN-Src Inhibits Lung Cytokine Production, but Has No Effect on Lung Vascular Permeability
The lung epithelium is a major source of cytokine generation in the lung (38, 39). We therefore examined the effect of overexpressing AdSOCS-1 and AdDN-Src on LPS-mediated cytokine production in the lung. LPS-mediated cytokine production in mice infected with AdGFP resulted in a greater than fivefold increase in TNF- production in both serum (p < 0.0001) and in lung (p < 0.0001; Figure 6C), which was similar in magnitude to the results obtained from uninfected mice (Figure 5). Lung TNF- production was significantly inhibited by adenoviral-mediated gene transfer of both DN-Src (p 0.0001) and SOCS-1 (p 0.005). In contrast, serum TNF- levels were unchanged by adenoviral-mediated gene transfer of DN-Src and SOCS-1. These results suggest that LPS-mediated TNF- generation in the lung is dependent on the airway epithelium, because this is the cell type where overexpression of these genes is occurring. Furthermore, these data demonstrate that specific inhibition of either Src or Jak kinases locally in the lung attenuates some of the lung's inflammatory response as evidenced by decreased TNF- generation, while not altering the systemic LPS response, as evidenced by the unchanged serum TNF- levels.
To determine whether inhibiting these kinases locally in airway epithelium also resulted in protection from lung injury, the effect of intranasal administration of AdSOCS-1 and AdDN-Src on vascular permeability was determined. As seen in Figure 6D, the overexpression of AdSOCS-1 and AdDN-Src via intranasal administration did not protect against the LPS-induced increases in vascular permeability as determined by EBD assay. These results indicate that inhibition of these kinases in only the airway epithelium, while diminishing cytokine generation in the lung, is not sufficient to prevent the development of ALI in response to systemic LPS administration. This finding is not unexpected because systemic cytokines remain elevated and Src and Jak kinases remain active in other cell types throughout the lung.
Src and Jak Tyrosine Kinase Inhibitors Prevent Mortality after Lethal LPS Challenge
Nonspecific tyrosine kinase inhibitors have been shown to be protective against endotoxic shock in animals, although the signaling pathways that have exerted this protective effect have not been well characterized (40eC42). To examine whether the Src and Jak tyrosine kinase inhibitors would attenuate LPS-induced mortality, mice were given high-dose LPS challenge (20 mg/kg). As seen in Figure 7A, mice treated with LPS alone had 100% mortality within 30 hours of LPS administration. In contrast, mice pretreated with TyrA1 or SU6656 before LPS administration and twice daily thereafter had a significantly decreased mortality rate to 30% (p 0.0001) at 2 weeks. Moreover, mice treated with a combination of the two inhibitors had an even greater survival advantage with no mortality seen at 2 weeks (p 0.0001). Although there is a trend in improved survival with both inhibitors, this effect was not statistically significant compared with inhibitors given alone as determined by log-rank analysis (p = 0.3).
We subsequently evaluated the ability of the inhibitors to prevent mortality when administered after LPS challenge (Figure 7B). Although the result was less dramatic than that seen with animals pretreated with inhibitors, there was still a highly significant improvement (p < 0.005) in survival in the animals that received the inhibitors 6 hours after LPS treatment compared with control animals (survival: LPS, 0%; TyrA1, 57%; SU6656, 57%; and both TyrA1 and SU6656, 67%). In addition, the time to death was delayed in all of the treatment groups compared with control animals. Treatment of the mice with the epidermal growth factor (EGF) receptor tyrosine kinase inhibitor AG1478 had no effect on LPS-mediated STAT activation (data not shown) or LPS-mediated mortality (Figure 7C). Thus, the effects of SU6656 and TyrA1 are specific. Importantly, we have determined that inhibiting the Jak and Src kinases systemically through the administration of SU6656 and TyrA1 is protective against lethal LPS challenge, even when administered post-LPS.
To determine whether inhibition of the Jak and Src kinases in the lung epithelium could prevent LPS-induced lethality, we intranasally administered adenovirus overexpressing inhibitors of Src and Jak kinases or GFP as a control. As seen in Figure 7D, the targeted inhibition of these kinases had no effect on LPS-induced mortality. This result is not surprising as we demonstrated that these viruses did not prevent LPS-mediated increases in lung vascular permeability or systemic cytokine production.
DISCUSSION
Although both the Jak and Src family of kinases have been shown to be important for the systemic response to LPS, the role of these kinases in the lung's inflammatory response is not well characterized (21, 22, 43). Our data indicate that both the Src and Jak kinases mediate LPS-induced ALI. This was demonstrated histologically as well as functionally because there was significantly less capillary leak in mice treated systemically with Src and Jak kinase inhibitors. Importantly, this protective effect was still evident even when these kinases were inhibited 6 hours after LPS treatment. A significant survival advantage was also seen in mice that had received Jak and Src kinase inhibitors after high-dose LPS challenge.
The mechanism of LPS-induced Src and Jak activation in the lung is likely to be multifactorial and change over time as different cytokines and inflammatory mediators are elaborated. LPS has been shown to activate these kinases in macrophages. In addition, LPS can have direct effects on multiple other cell types throughout the body, including the lung endothelium, to produce inflammatory cytokines and reactive oxygen species (44). In the past, we have determined that STAT3 activation by LPS requires reactive oxygen species and it is likely that Src and Jak activation by LPS may also be in part redox-dependent (45). Both Jak and Src kinases have been shown to be redox-regulated as has LPS-induced signaling in some instances (46, 47).
Our data indicate that both the Src and Jak kinases are required for LPS-induced signaling in the lung. This was demonstrated by the finding that both the small molecule inhibitors SU6656 and TyrA1 prevented LPS-induced STAT activation in the lung. In addition, PP2, which is known to preferentially target Src kinase over Jak2, had the same effect (48). More specific inhibition of Src and Jak kinases by overexpressing DN-Src and SOCS-1 also significantly attenuated LPS-induced STAT activation in the lung. Our data in the lung demonstrating that SU6656 blocked both Src and Jak activation by LPS also suggest that these kinases may reside in a common pathway downstream of LPS, with Src upstream of Jak kinase. This is consistent with in vitro work by others and us (29, 49).
Tyrosine kinases are required for the activation of multiple signaling pathways downstream of LPS in phagocytic cells (50). In addition, the subsequent cytokine cascades induced by LPS in both macrophages and other cell types exert their cellular effects through tyrosine phosphorylation-dependent pathways. Consequently, systemically targeting tyrosine kinases should be an efficient way to dampen multiple aspects of LPS-mediated inflammation in different cell types and organs. Others have shown a beneficial effect of tyrosine kinase inhibitors on LPS- or zymosan-mediated ALI or multiorgan failure using genistein, a nonspecific tyrosine kinase inhibitor, or tyrphostins, such as TyrA1 or TyrAG126 (40, 42, 51, 52). These studies have suggested that the protective effects of these agents were caused by inhibition of either the MAPK or NF-B pathways. Our data suggest another possible mechanism of how these inhibitors afford protection from ALI. Namely, we demonstrate that TyrA1, PP2, and SU6656 also inhibit Src and Jak tyrosine kinases. These results are consistent with work by others demonstrating that mice deficient in Src and Jak kinase family members are protected from LPS-induced lethality (21, 22). Conversely, animals with constitutive activation of the Src family kinase Hck were found to have an exaggerated inflammatory response to LPS in the lungs, which included increased production of cytokines, such as TNF- (43).
The persistent elevation of proinflammatory cytokines in humans with ALI or sepsis has been associated with a worse outcome (4). Therefore, one mechanism by which inhibition of Src and Jak kinases may protect against LPS-induced ALI is by decreasing the generation of proinflammatory cytokines arising from phagocytic cells, the lung, or other organs. Supporting this idea are our data demonstrating that inhibiting these kinases results in significantly decreased LPS-mediated production of the proximal proinflammatory cytokines TNF- and IL-6 in both the serum and lungs of animals. In addition, these inhibitors dampened LPS-induced signaling in organs, such as the liver, as evidenced by their ability to decrease LPS-induced STAT3 activation (36).
Src and Jak kinases are important for neutrophil and macrophage effector function; therefore, inhibiting Src and Jak may be protective through decreasing inflammatory cell migration and/or function (19, 21). Our histologic data support this contention because there were fewer inflammatory cells in the lungs of mice treated with either inhibitor of Src or Jak kinases. The levels of the chemokine MIP-2 correlate with neutrophil sequestration and lung injury in animal models of ALI (53). Conversely, neutralizing MIP-2 has been shown to decrease neutrophil recruitment in the lung (33). Our data, which demonstrate a significant reduction in LPS-induced MIP-2 generation in both the serum and lungs of mice receiving the inhibitors, suggest that another mechanism of protection of these inhibitors is to decrease neutrophil sequestration in the lungs.
STAT3 was identified as an acute-phase response gene in the liver and thus has a pivotal role in orchestrating inflammatory responses by upregulating the expression of cytokines, chemokines, and inflammatory mediators (54, 55). STAT3 has also been shown to be critical for antiinflammatory responses orchestrated by IL-10 (56). Therefore, predicting the effect of STAT3 blockade is difficult. We and others have recently demonstrated that STAT3 is activated very early in the lungs in multiple different models of ALI, suggesting that it might play a role in the initiation of lung injury (11, 57). Although some prior studies suggest that STAT3 plays a largely antiinflammatory role in LPS-mediated responses, elucidation of the role of STAT3 has been hindered by the fact that deletion of the STAT3 gene in mice results in embryonic lethality (58). As a result, these studies have relied on the targeted disruption of STAT3 in mice in one or a couple of cell types (myeloid, endothelial, and epithelial cells) and have found enhanced inflammation in response to endotoxin (55, 59, 60). Mice with a deletion of the alternately spliced STAT3- also manifest enhanced inflammatory responses to LPS, suggesting that this STAT3 isoform plays a largely antiinflammatory role (61). It is likely that the net effect of STAT3 deletion is dependent on when and from what cell types it is ablated. Supporting this notion are data demonstrating that STAT3 deletion in T cells is protective in an experimental enterocolitis model (62). Our data suggest that the global inhibition of STAT3 in multiple cell types could have an overall beneficial effect against LPS-induced inflammation and suggest another mechanism by which Src and Jak kinase inhibition is beneficial in protecting against LPS-mediated ALI and sepsis. Interestingly, use of AG1478, an inhibitor of the EGF receptor tyrosine kinase, had no effect on LPS-induced STAT3 activation or mortality, suggesting that the beneficial effect was correlated with Src and Jak kinase as well as STAT3 inhibition.
It is possible that the kinase inhibitors used in these studies are also preventing the activation of other STATs, such as STAT4 or STAT6, which have been shown to be proinflammatory in other models of sepsis (63). We have data that the kinase inhibitors used here also prevented LPS-induced STAT1 activation (data not shown). In addition, tyrosine kinase activity is required for maximal activation of other signaling pathways, such as the NF-B and MAPK pathways. Therefore, these kinase inhibitors may also attenuate ALI through the disruption of other proinflammatory signaling pathways. Interestingly, recent genetic evidence from Drosophila indicates that the STAT and NF-B pathways cooperate in mediating the immune response to endotoxin (64).
The fact that targeted disruption of these kinases by adenoviral-mediated gene transfer of SOCS and DN-Src diminished lung cytokine production in response to LPS, but was unable to protect against capillary permeability, suggests that this therapeutic approach is unlikely to be beneficial for patients with ALI. The failure of this targeted approach is likely because it does not impact on the endothelial injury or the systemic inflammation that accompanies ALI. Recent data suggest that lung epithelial cell STAT3 may be important in limiting lung injury in response to hyperoxia (60, 65). Therefore, it is possible that inhibiting LPS-induced STAT3 activation in just the lung epithelium, as was the case in our adenoviral experiments, is deleterious. Unlike the studies with hyperoxia, however, in our studies with LPS we did not see enhanced pulmonary capillary permeability in animals overexpressing DN-Src or SOCS-1, but rather no significant improvement. Whether STAT3 plays a similar role in the lung in response to LPS-induced ALI in the epithelium is unclear and will be the focus of future investigation.
Although some recent advances have been made in the treatment of ALI, it remains a significant clinical problem with substantial patient morbidity and mortality. Understanding the molecular and cellular mechanisms that mediate ALI is crucial for the development of more effective treatment strategies. Our study suggests that Jak and Src family kinases participate in ALI and verify the potential of this class of selective tyrosine kinase inhibitors as novel therapeutic agents for this disease. Tyrosine kinase inhibitors have already been used successfully in humans for the treatment of malignancies (66). Finally, the fact that both TyrA1 and SU6656 were highly effective at reducing lung injury and mortality when administered 6 hours after LPS administration has important clinical ramifications because most patients with ALI and sepsis present hours after the inciting event.
Acknowledgments
The authors thank Dr. Barry Fanburg and Dr. Nicholas Hill for their assistance in reviewing this article. In addition, the authors thank Kathleen Riley for her technical assistance with the fluorescent microscope.
A.R.S. is supported by National Heart, Lung, and Blood Institute grant R01HL-68753-01A1, the American Lung Association of Middlesex County, the American Lung Association of Western Massachusetts, TuftseCNew England Medical Center Research Fund, and Gastroenterology and Research on Absorption and Secretory Processes Digestive Disease Center grant P30-DK-34928. B.H.C. is supported by NIH GM51551.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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