Molecular Medicine

, Volume 14, Issue 7–8, pp 465–475 | Cite as

Epithelial Cell Apoptosis and Neutrophil Recruitment in Acute Lung Injury—A Unifying Hypothesis? What We Have Learned from Small Interfering RNAs

  • Mario Perl
  • Joanne Lomas-Neira
  • Chun-Shiang Chung
  • Alfred Ayala
Open Access
Review Article


In spite of protective ventilatory strategies, Acute Lung Injury (ALI) remains associated with high morbidity and mortality. One reason for the lack of therapeutic options might be that ALI is a co-morbid event associated with a diverse family of diseases and, thus, may be the result of distinct pathological processes. Among them, activated neutrophil- (PMN-) induced tissue injury and epithelial cell apoptosis mediated lung damage represent two potentially important candidate pathomechanisms that have been put forward. Several approaches have been undertaken to test these hypotheses, with substantial success in the treatment of experimental forms of ALI. With this in mind, we will summarize these two current hypotheses of ALI briefly, emphasizing the role of apoptosis in regulating PMN and/or lung epithelial cell responses. In addition, the contribution that Fas-mediated inflammation may play as a potential biological link between lung cell apoptosis and PMN recruitment will be considered, as well as the in vivo application of small interfering RNA (siRNA) as a novel approach to the inhibition of ALI and its therapeutic implications.


ALI and Acute Respiratory Distress Syndrome [ARDS] represent a form of lung dysfunction characterized by hypoxemia and diffuse bilateral infiltrates of the lung often accompanied by pulmonary edema, reduction of lung compliance, and a decrease in the functional residual capacity of the lungs (1,2). In 1967, Ashbaugh et al. described 12 patients with acute respiratory distress, oxygen refracted cyanosis, and diffuse lung infiltrates, and, thus, provided an early description of ALI (3). ALI and ARDS can be of pulmonary (direct) or extra-pulmonary (indirect) origin. Direct ALI (for example, from pneumonia or aspiration) attributes to about 55% of ALI, meanwhile, indirect ALI can be seen in 20% (for example, due to extra-pulmonary sepsis or trauma) (4). In about 21% of instances, there are mixed factors contributing to ALI; in about 4%, no underlying pathophysiology can be distinguished (4). The incidence of ALI and ARDS in the United States of America has been reported to be 79:100,000 and 59:100,000 persons per year, respectively (2,5,6). In the next 25 years, it is anticipated that the incidence will double, based on the exponential growth of the population (5). The highest incidence of ALI is seen during sepsis, accounting for 46% of direct and 33% of indirect ALI (5); in total, around 25% of all ARDS cases stem from a severe sepsis (4). Around 7% of intensive care patients eventually will develop ALI/ARDS (4,7). Recent studies suggest a lethality of around 40% for ALI or ARDS (2,5), partly influenced by premorbid conditions (8) and the underlying pathophysiology (9). Thus, the presence of sepsis represents one of the highest risks factors for mortality from ARDS (up to 50%) (4,10,11). Even when patients are fortunate enough to survive ARDS, they suffer from long term disabilities, such as reduced vital capacity and physical strength and also from cognitive deficits (12, 13, 14).

While advances have been made, the therapeutic interventions to treat ALI or ARDS still remain limited. They include lung protective ventilatory regimens with low tidal volume ventilation (15) and intermittent prone positioning, at least in a selected patient population (2). Several treatment options such as corticosteroids, immuno-nutrition, surfactant, etc. (reviewed in 2) still have to be proven to have beneficial effects on outcome parameters. However, thus far, no pathophysiologic driven therapeutic intervention has yet become available.

The aim of this review is, therefore, to elucidate mechanisms of ALI pathogenesis, focusing on two main theories. The first, that neutrophils (PMN) can play a central role in driving ALI and the second, that lung epithelial cell apoptosis represents an important pathological aspect inducing lung injury. In this regard, we discuss the data in the field as well as some of our own findings using a clinically relevant double-hit mouse model of indirect ALI induced by hemorrhagic shock and subsequent polymicrobial sepsis. In addition, a novel strategy, i.e. the use of siRNA in mouse lungs in vivo, has served us in broadening our understanding of the pathology of ALI.

Pathophysiology of ALI

The pathophysiologic mechanisms of ALI are insufficiently understood. In the common finale of this heterogeneous entity, the alveolo-capillary barrier is compromised allowing consecutive edema formation in the interstitium as well as alveoli, thus compromising gas exchange leading to organ dysfunction and respiratory failure. Histological evaluation of lungs from ALI patients indicated substantial accumulation of activated PMNs, diffuse alveolar damage, loss of epithelial integrity, and increased pulmonary edema (16,17). There are several proposed mechanisms which are believed to account for this phenomenon.

Neutrophil Hypothesis of ALI

Among leukocytes, PMNs are considered the first line of defense against microorganisms. Rapidly recruited to the inflammatory site/organ, they exert a variety of primarily beneficial functions (phagocytosis, production of reactive oxygen species [ROS], and nitric oxide species [NOS], degranulation of lytic enzymes, etc.), that, when well orchestrated, enable clearance of the invading pathogen. However, it is also hypothesized that activated PMN may possess harmful potential when these same functions are directed at otherwise normal host tissue, culminating in injury and organ damage (Figure 1). There is substantial evidence that the lung is particularly susceptible for PMN accumulation. In contrast to most other organs, where PMN sequestration occurs at post-capillary venules, pulmonary PMN retention takes place within the pulmonary capillaries, representing a complex interconnecting network of short capillary segments where the course from arteriole to venule crosses numerous alveolar walls and often includes more than 50 capillary segments. The blood in this complex network contains 50 times more PMN compared with most other vascular beds (reviewed in 18).
Figure 1

Proposed mechanisms of acute lung injury through hemorrhagic shock (HEM) “priming” for inflammation (Infl./red color)/apoptosis (Ao/ gray color)/injury, and “triggered” by a subsequent infectious insult: The resting lung (A) is primed by divergent inflammatory mediators released during an initial event (for example, shock, inflammation, etc.) that acts on a number of cells in the blood (MΦ: monocytes, PMN: neutrophils) and lung (EP: epithelial cells, EC: endothelial cells, AMΦ: alveolar macrophages) (B). These cells, in turn, stimulate, either separately or concomitantly, the pro-inflammatory response and/or the Ao of a small number of EP, both through Fas-FasL activation. The release of chemokines like MCP-1 then primes the AMΦ. When, at a later time, a subsequent inflammatory/infectious (‘trigger’) event takes place (C) the local EC, AMΦ, and/or EP become activated, release chemokines and activating agents that recruit the primed and now activated leukocytes (PMN, MΦ) to the lung (D). These activated leukocytes then transmigrate into the interstitium and alveoli where they perform their effector roles (in the absence of infection, the effector response may be solely injurious). In addition, they may propel the inflammatory/apoptotic response into a vicious cycle by further activating Fas through FasL on their cell surface (E).

PMN accumulation has been observed early in lung tissue (19,20) as well as in bronchoalveolar lavage fluids (BALF) of ARDS patients (16). When developed during neutropenia, ALI rapidly progressed once PMN counts were restored (21,22). Furthermore, the degree of neutrophilia in BALF has been correlated with poor prognosis in septic ARDS (23). In an experimental setting of indirect ALI stemming from hemorrhagic shock (HEM) followed by polymicrobial sepsis, we found that, in response to HEM, circulating PMNs exhibited an ex vivo increase in respiratory burst capacity and a decrease in apoptosis. This is consistent with the concept that shock/injury can produce in vivo “priming,” the significance of which could be seen when HEM then was followed by sepsis, as recruitment of these PMNs into the lung occurred, along with the development of ALI (24). Also, when these HEM-primed PMNs were injected intravenously into PMN-depleted animals, which subsequently underwent cecal ligation and puncture (CLP) to induce sepsis, ALI again resulted (24). Upon this background, using different models of ALI, we (25) and others (26, 27, 28, 29, 30, 31, 32) have found that depletion of PMNs actually may serve to decrease injury associated with ALI. Thus, depletion of PMNs prior to HEM and sepsis markedly reduced the extent of lung inflammation and ameliorated lung protein influx and the severity of ALI (25), which is in line with findings during transfusion-induced ALI (29).

Inflammation—Priming PMNs. Inflammation is closely linked to the pathogenesis of ALI. Levels of Interleukin (IL)-8 and IL-1 have been found increased in the lungs of ALI patients (33,34) and the persistent increase of inflammatory cytokines in the lung correlated with poor outcome in ALI (35, 36, 37). Inflammatory mediators exert several effects on PMNs. For example, complement protein C5a activates PMNs, thus contributing to their pulmonary sequestration and mediating lung tissue injury (38,39). Several inflammatory mediators, including complement, cytokines, chemokines, and lipid mediators also are able to induce the expression of surface molecules on the endothelium, which further promote PMN recruitment (40,41). Tumor necrosis factor (TNF)-α and IL-1β are two important cytokines that are found regularly in the BALF of ARDS patients (36,42,43) at higher concentrations than in their plasma, thus supporting their local origin (35,44). However, anti-TNF and anti-IL-1 therapies have failed to protect septic patients from ALI (44,45). Pro-inflammatory cytokines also are regularly expressed by activated PMNs traveling to the lung (32). They appear to be a relevant source of IL-1β, favoring the subsequent release of other mediators, such as TNF-α, MIP-2, and IL-8 (46, 47, 48). However, an early anti-inflammatory response driven by IL-1-, TNF- and IL-6-receptors, with early peaking IL-10 levels, was also shown in ARDS patients (41). In addition, PMN stimulation with lipopolysaccharide (LPS) or TNF-α showed an activation of nuclear factor kappa B (NF-κB), p38, and AKT (49,50). The degree of nuclear translocation of NF-κB in peripheral PMNs from patients with septic ALI was linked to ventilator time and survival (50).

PMNs on Their Way to Lung— Sequestration, Adhesion, Migration, Activation, and Tissue Injury. First, in response to inflammatory mediators, PMNs migrate to the pulmonary capillaries in preparation for extravasation (Figure 1). Initial changes in the cytoskeleton prevent PMNs from deforming, making it less likely for them to pass through the pulmonary capillaries (51). In this regard, activated PMNs from ARDS patients appeared even more rigid than those from septic patients (52). Second, emigration of PMNs and passage through the endothelium can be regulated through adhesion molecules (51). While their initial sequestration appears to be independent from adhesion molecules, the durability of this process could rely on them. Thus, while L-selectin-deficient mice showed a normal pool of marginated PMNs and cleavage of L-selectin from the PMN surface did not alter this margination (53,54), L-selectin deficient mice exhibited only a very short and transient leukopenia in response to complement activation (53,55). PMNs can adhere to endothelial cells using CD11/CD18 interacting with intercellular adhesion molecule-1 (ICAM-1) on the endothelial side. Thus, PMN emigration in response to Escherichia coli (E.coli), E.coli lipopolysaccharide, Pseudomonas aeruginosa, immunoglobulin (Ig) G immune complexes, and IL-1 was mediated through CD11/CD18, but this did not appear to be the case in response to Streptococcus pneumoniae, Group B Streptococcus, Staphylococcus aureus, hyperoxia, or C5a (56). Even in CD11/CD18-dependent migration, blocking of CD18 reduced PMN emigration only by 60% to 80%, suggesting that other redundant mechanisms mediated these effects (56). Cytokines and chemokines such as IL-8 also have been shown to be involved in increasing β2-integrin avidity (57). It is important to understand that activation of PMNs is associated closely with the endothelial interaction mediated by the adhesion molecules (57,58). Rolling PMNs might well integrate the sum total of inputs received while scanning the endothelium. If an activation threshold is reached, β2-integrins switch to the high-affinity conformation, redistribute on the cell surface, and trigger arrest and adhesion (59,60). However, integrins also are involved in PMN migration, phagocytosis, respiratory burst, and even cytokine production (57,61,62).

As eluded to above, chemokines are critically involved in the activation and recruitment of PMNs to the lung, potentially contributing to their harmful effects on an organ level. In this regard, associations between chemokines, lung neutrophil influx, and alveolo-capillary dysfunction have been reported (63,64). In our experiments, we observed that during hemorrhage-induced septic ALI (24), as well as after experimental blunt chest trauma (65), chemokines are markedly upregulated locally in the lung of the animals, as well as systemically. Blockade of the CXCR (chemokine [CXC motif] receptor) 2 using antileukinate, a hexapeptide inhibitor, following HEM, reduced lung PMN influx in response to subsequent sepsis and additionally decreased lung inflammation and lung protein leak, while pulmonary IL-10 levels were markedly increased (66). We also administered anti-KC or anti-MIP-2 antibody into mice immediately following HEM. The adoptive transfer of PMN isolated from the anti-MIP, but not from the anti-KC-treated donors, exhibited a reduction of lung inflammation and lung PMN influx in the recipients in response to sepsis when compared with control Ig-treated donors (67). These data demonstrated that hemorrhage-induced priming of PMNs not only mediated experimental ALI, but also that this process was differentially effected by MIP-2 and KC, although both signal through CXCR2 in mice. However, as humans utilize IL-8 in place of the two mouse a-chemokines and because receptors for IL-8, i.e. CXCR1 and CXCR2, also are utilized differently, these murine chemokine data may not translate directly to the patient setting of ALI/ARDS (68).

Once activated PMNs have reached the lungs, they possess the potential to induce tissue injury. The release of ROS/NOS has long been thought to be a central effector mechanism of PMN-mediated lung injury (69). In this regard, while the inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase attenuated sepsis-associated ALI (70), surprisingly, NADPH oxidase-deficient animals were not protected from complement-induced ALI (71). On the other hand, inhibition/ deficiency of the nitric oxide synthetase showed protection in complement-/ endotoxin-induced ALI (71,72). However, ROS and NOS also play additional roles as signaling molecules modulating kinases and phosphatases, receptors and transcription factors (reviewed in 73) complicating the interpretation as to whether their net pulmonary effect is one of harm or help.

Proteolytic enzymes also are a part of the PMN pathogen-clearing arsenal. While neutrophil elastase, cathepsin G, and metalloproteinases all have important roles in clearing invading pathogens and function in aspects of tissue remodeling/wound healing, they also are able to mediate neutrophil-induced tissue damage and can degrade extracellular matrix efficiently. Thus, while inhibition of neutrophil elastase improved respiratory function during septic ALI (74), its deficiency also rendered mice more susceptible to gram negative, but not gram positive sepsis (75). In addition, deficiency of elastase and/or cathepsin G was accompanied by increased susceptibility to fungal infections, but decreased susceptibility to endotoxin-mediated inflammatory ALI (76). Activated gelatinase A correlated with protein accumulation in the epithelial lining fluid in ARDS patients and might, therefore, be a potential marker for ARDS (77). The inhibition of matrix metalloproteinases and elastase prevented lung dysfunction and reduced septic ALI in pigs (78). The detailed mechanisms by which PMN mediate ALI via the involvement of proteolytic enzymes are reviewed in detail elsewhere (79,80).

Regulation of PMN Apoptosis. Neutrophils, once mature, exhibit a constitutive form of programmed cell death with a life span between 6 to 12 h in circulation. Under normal circumstances, activated PMNs are eliminated fairly quickly once the invading pathogen has been cleared. Several inflammatory agents, such as LPS, TNF, IL-8, IL-6, IL-1, Granulocyte [macrophage] colony-stimulating factor (G [M] -CSF), etc., can inhibit PMN apoptosis (81, 82, 83, 84). The delayed apoptotic response provides PMNs with a longer life span, which, in turn, allows them to accumulate at local tissue sites of inflammation/infection. In this respect, PMNs obtained from patients following major trauma (85,86), burn injury (87), sepsis (88), and ARDS (89) all showed evidence of decreased apoptosis. The anti-apoptotic effect of ARDS plasma on PMNs appears to be mediated through the GM-CSF receptor (90). In sepsis, the delay in apoptosis was associated with a decrease in caspase-3 and -9 activity and a prolonged maintenance of the mitochondrial membrane potential (88). The delay of PMN apoptosis also involved the active regulation of CXC receptors by PMNs themselves (91). However, controversy persists as to whether this sustained activation actually contributes to organ injury. Activated PMNs have been shown to exert damaging effects in the lungs (68,92). However, following G-CSF treatment in pneumonia patients, no differences in outcome or time of recovery were noted, while it appeared that complications such as ARDS were decreased (93). These data were complemented by studies that indicated that GM-CSF in the air spaces was associated with improved survival in patients with ARDS (94).

In our model of hemorrhage-induced septic ALI, it was evident that prolonging the lifespan of activated PMNs by using transgenic mice that overexpress the anti-apoptotic protein Bcl-2 in a myeloid-restricted fashion (95) did not exacerbate acute lung injury, although a prolonged presence of activated PMNs in the lung was evident (96). In contrast, we observed an initial survival benefit when the life span of PMNs was further prolonged, likely due to an enhanced capacity to clear bacteria as evidenced by the lower bacteria counts in the lung (96). In addition, in an inflammatory/ non-infectious environment, such as SIRS, mimicked by intraperitoneal injection of E. coli-LPS, prolonging the lifespan of activated PMNs was detrimental for the animals’ survival and was associated with an exacerbation of lung injury (96). Additionally, during non-infectious/ inflammatory ALI, failure to clear PMNs from the lungs contributed to increased inflammation and mortality (97). Together, these data imply that the tissue environment (infectious versus inflammatory) that the neutrophil encounters plays an important role in determining whether PMNs mediate organ damage or not. However, the mechanisms which determine whether PMN turn bad or good are not understood well and require further investigation.

Epithelial Cell Hypothesis

The above information makes a strong case for PMNs playing a significant role in ALI. However, it is interesting to note that ARDS has been described as developing in patients with neutropenia (98, 99, 100). In addition, following G-CSF therapy in pneumonia and sepsis patients, the incidence of ARDS was not increased (93,101) and, during experimental ALI or pneumonia, PMNs have been shown to migrate to the lungs without exerting deleterious effects (102,103). Thus, other mechanisms also are likely to be involved in the pathogenesis of ALI.

In the development of ALI/ARDS, the loss of epithelial cells in the lungs with consecutive impairment of the integrity of the alveolo-capillary barrier is commonly noted (19,104). Type I epithelial cells make up approximately 90% of the pulmonary surface, and, while type II epithelial cells account for only 10%, they are critical because they produce surfactant and may differentiate into type I cells (17). Impairment of the alveolar epithelial barrier is, in many ways, important in the development of ALI. On the one hand, the epithelial barrier is, under physiologic conditions, less permeable when compared with the endothelial barrier, thus, destruction of its integrity prompts a progressive influx of protein-rich fluid into the alveoli (17,102). On the other hand, the loss of epithelial integrity represents an impairment of the physiologic trans-epithelial fluid transport and further inhibits the re-absorption of the alveolar edema (105,106). Thus, while an isolated injury to the endothelium still leaves intact the capacity of the epithelium to counteract pulmonary edema formation, the fluid balance becomes rapidly disturbed upon injury of the lung epithelium (107,108). Therefore, it is not surprising that the extent of epithelial damage and impaired edema re-absorption was linked clinically to outcome in ALI (109). Additionally, the accompanying reduction of surfactant production led, via an increase in the surface tension, to an augmented respiratory effort affecting gas exchange (110,111).

Programmed cell death/apoptosis of lung epithelial cells represents a potentially important mechanism contributing to the loss of this cell type in the development of ALI (Figure 1). Bachhofen et al. reported that patients who died from ARDS exhibited excessive apoptotic alterations in the chromatin of their alveolar type II cells (19). Subsequent studies then confirmed these DNA fragmentations (112) as well as an increased expression of the pro-apoptotic protein Bax (Bcl-2 associated X protein) (113). More recent studies also have suggested a role for lung epithelial cell apoptosis in pediatric ARDS (114). Importantly, epithelial cell and PMN apoptosis is differentially regulated during ARDS. While, as mentioned earlier, PMNs show a decreased rate of apoptosis, it is increased markedly in epithelial cells (107). Local mediators in the lungs of these patients may regulate these opposing effects (115,116). Interestingly, the induction of lung epithelial cell apoptosis appears to lead to the development of ALI, which is further aggravated, when apoptosis is additionally induced in macrophages (117). Furthermore, inhibition of apoptosis, using a caspase inhibitor, protected mice from the lethal consequences of endotoxin-associated ALI (118). In addition, Bcl-x(L) treatment reduced albumin leakage and lung tissue damage in LPS-mediated ALI (119).

In the induction of lung apoptosis during ALI, Fas- (an apoptotic death receptor pathway) mediated cell death also appears to play a major role (Figure 2). In this regard, during endotoxin-induced ALI, an increase of Fas expression on epithelial cells, as well as an immigration of Fas-ligand (FasL) expressing cells in(to) the lung, was observed (120). Human lung epithelial cells also expressed Fas and were, particularly in the distal airways, sensitive to Fas-mediated apoptosis (121). Caspase-cleaved cytokeratin-18, a marker for epithelial cell apoptosis, also was increased in BALF during ARDS (122). During ALI and ARDS, an increased concentration of Fas and FasL in patients’ BALF and lung tissue was detected (122,123) and BALF from ARDS patients was shown to induce apoptosis in healthy lung epithelial cells (116). Furthermore, ARDS non-survivors exhibited markedly higher FasL concentrations when compared with survivors (116). Particularly during septic ALI, an infection-severity dependent activation of the Fas-FasL system in the lung was observed (124). FasL concentrations have been reported to be much higher locally than systemically, suggesting its origin is pulmonary (122,123). In this respect, there appear to be several potential sources of FasL: infiltrating monocytes have been implicated (but not alveolar macrophages) (125), PMNs have been implicated (126), and FasL may be cleaved from cell membranes by the activation of matrix metalloproteinases 3 and 7 (127,128). However, anti-apoptotic proteins such as soluble Fas (129) also appear to be increased during ARDS (122,123) and have been described to correlate with clinical features such as the PaO2/FiO2 ratio (122). This may point toward a simultaneous activation of protective anti-apoptotic mechanisms during ALI.
Figure 2

Proposed FasL-Fas signaling in lung epithelial cells and its potential effects. Also, illustrated points at which the pathway(s) has (have) been inhibited. FADD (Fas associated death domain), FLIP (FLICE [FADD like interleukin-1] inhibitory protein), TRAF (Tumor Necrosis Factor Receptor-associated Factor), Raf, ERK (extracellular signal regulated kinase), NF-κB (nuclear factor kappa B), siRNA (small interfering RNA).

Under experimental conditions, the instillation of Fas-activating antibody into murine lungs induced apoptosis of lung epithelial cells, PMN recruitment and impairment of the alveolo-capillary barrier (130). This development of Fas-dependent lung injury was linked to Fas-expression on non-myeloid but not myeloid cells (131). Furthermore, blocking of the Fas-FasL system reduced the development of endotoxin-mediated ALI (120). Our experiments further demonstrated the relevance of Fas-mediated apoptosis in the lung during HEM-induced septic ALI (132). Fas and FasL mutant animals exhibited less pulmonary epithelial cell apoptosis in response to the insult when compared with background animals. In addition, the extent of ALI as assessed histologically and by protein influx was diminished significantly. This was associated with a survival benefit for Fas mutant mice when compared with background animals (132). It also has been reported that Fas-FasL deficiency is associated with a reduction in the degree of injury seen during direct ALI in E. coli, S. aureus, or S. pneumoniae sepsis models (133). Similar results were described for sepsis induced by Legionella pneumonia (134). In contrast, data from pulmonary sepsis following lung infection with P. aeruginosa suggest a reduction in lung apoptosis in the absence of Fas signaling as well, but a decreased survival rate was observed, associated with an increased dissemination of the bacteria (135). It should be noted that Fas-mediated apoptosis in the lung appears to be modulated by several factors such as surfactant protein A, Angiotensin II, transforming growth factor (TGF)-beta, decoy receptor 3, etc. (reviewed in 108), and that other apoptotic pathways, for example, mediated by TNF-α (136) or mitochondria (137), appear to play a role under certain circumstances, too.

Fas-Induced Inflammation—Linking PMN Recruitment and Lung Apoptosis?

Recent studies suggest that activation of Fas serves not only to induce apoptosis, but also to induce the secretion of cytokines and chemokines by a variety of cell types (138) (Figure 2). In murine lungs, activation of Fas initiated a profound inflammatory response with an early generation of chemokines and subsequent recruitment of neutrophils (130,132,139). This response could be reduced by antagonizing Fas ligand (139). Triggering of Fas also led to increased concentrations of TNF-α, MIP-1 α, MIP-2, monocyte chemotactic protein (MCP)-1, and IL-6, and compromised the alveolo-capillary barrier (133). LPS-mediated pulmonary inflammation also appears to be regulated through Fas (140). Mice, genetically altered to express Fas either on myeloid or on non-myeloid cells in the lung, presented no marked increase of inflammatory mediators following Fas activation (131), in contrast to non altered mice lungs (130, 132,139). With respect to cell types involved, it has been indicated that activation of Fas on monocyte cell lines induced production of MIP-2 (141). The activation of an inflammatory program by Fas also was shown for peritoneal macrophages (142), monocytes and macrophages (143), endothelial cells (144), and others (145, 146, 147, 148, 149, 150, 151).

Our own experiments with the Fas and FasL mutant mice showed a marked decrease in inflammation in the lungs of these animals in response to hemorrhage-induced septic ALI, when compared to background animals (132). This phenomenon also was evident following Fas silencing in the mouse lungs using siRNA (152) and was associated, in both cases, with a reduction of PMN immigration into the lungs (132,152). Further experiments revealed that lung epithelial cells were capable of secreting MIP-2, KC, and MCP-1 in vitro in response to Fas activation, through mechanisms involving ERK and, potentially, FLIP (132), complementing the findings of other researchers who demonstrated that a NF-κB dependent mechanism appears to underlie this response (151). Instillation of a Fas-activating antibody into transgenic murine lungs in which lung macrophage numbers are markedly reduced displayed a similar inflammatory response as seen in background animals, further supporting a role for Fas-mediated, epithelial cell-induced pulmonary inflammation in vivo (132). The fact, that inhibition of Fas activation modulated both PMN recruitment into the lung as well as pulmonary epithelial apoptosis, intriguingly points at its value as a potential therapeutic target in ALI.

Using siRNA in the Lung

The use of silencing RNA (siRNA) represents not only a potentially powerful experimental approach to allow us to better understand the evolving pathology of ALI, but may possibly represent a novel therapeutic approach to the treatment of this condition. The history of siRNA, its discovery, development, the mechanisms involved, as well as its successful initial uses in mammals in vivo are described elsewhere (153, 154, 155, 156). With respect to its application in vivo, the lung appears to be a good candidate, as it can be accessed straightforwardly intranasally (i.n.) or intratracheally (i.t.). Nevertheless, nucleic acid transfer efficiency can be diminished substantially by phospholipids and proteins, which are major components of the airway surface liquid (157). Interestingly, unlike systemically, delivery of naked siRNA into the lungs has proven very efficient (152,158,159), thus potentially complex and costly approaches using vector systems or chemical siRNA modifications may not be necessary. In this regard, the delivery of siRNA inhibitors of SARS coronavirus in 5% glucose was superior, even in reducing the severity of the disease, than when given in modified calf pulmonary surface active material (160). As early as 2004, the feasibility of a surfactant-based or naked siRNA approach in the mouse lung targeting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or heme oxygenase-1 during ischemia-reperfusion, respectively, has been demonstrated (161,162). Phase I/II trials for Respiratory Syncytial Virus have been conducted and are now on their way to evaluate the safety, tolerability, and antiviral activity of siRNA treatment in human lungs (155,158,163).

Using siRNA to Treat Murine ALI. With respect to the application of siRNA in the in vivo setting of the lung, initially we attempted to extend our observation that blockade of MIP-2 or KC with conventional antibodies effected the development of ALI (67) by using an in vivo approach silencing these same chemokines locally in the lung (164). Subsequently, we initiated studies with anti-apoptotic siRNA against Fas and caspase-8 assessing their capacity to protect the lung from the detrimental effects of hemorrhage-induced septic ALI (152).

However, to establish the initial feasibility of in vivo siRNA administration, mice overexpressing green fluorescent protein (GFP) were chosen to receive a single intratracheal instillation of a GFP-silencing RNA. What we found was that green fluorescence in the lungs of these animals at 18 h post-instillation was reduced by at least 65% when compared with vehicle-treated GFP mice, while no decrease in fluorescence was seen in other organs outside the lungs (152,164). The use of siRNA at these concentrations neither induced substantial activation of type I interferons (165,166) via activation of TLR-3 (167), TLR-7 (168), TLR-8, TLR-9, and protein kinase-R(PKR) pathways (169, 170, 171) nor via more classic proinflammatory processes (169,171). This is in line with reports demonstrating that intravenous delivery of naked siRNA did not induce an interferon response (170). However, the induction of STAT (signal transducers and activator of transcription)-1 in this context appears to be dose dependent (165), and thus it cannot be ruled out that different dosages, delivery, or timing of administration might have provided dissimilar results. To gain some insight into the cellular localization of siRNA in the lung, we followed the intrapulmonary deposition of Cy-5 fluorochrome labeled siRNA uptake by confocal immunofluoresence microscopy. Interestingly, labeled siRNA was found to co-localize only with lung epithelial cells counterstained with anti-cytokeratin-18 at 24 h post instillation, but not alveolar macrophages (152). However, negative results for alveolar macrophages may not preclude the uptake of siRNA by this cell type as one can imagine different kinetics in the various cell types with respect to siRNA uptake and processing. Relative to this, the feasibility of gene silencing in macrophages using siRNA has been described in vitro (172, 173, 174), however, silencing of typically macrophage-derived molecules such as TNF-α and IL-6 during indirect murine lung injury remained unsuccessful in our hands (unpublished observations). Whether this might be attributable to the underlying cell type or perhaps to the degree of up-regulation of a certain gene during pathological conditions remains to be elucidated. Irrespective these and the experiments below represent the first studies to demonstrate the feasibility of in vivo lung siRNA as treatment for developing ALI.

To finally assess the therapeutic capacity of such a siRNA approach, experiments were designed to modulate PMN immigration based on the neutrophil hypothesis using in vivo siRNA constructs against the murine chemokines KC and MIP-2 during the development of indirect septic ALI. To this end, suitable siRNA constructs were instilled into the lungs of animals 2 h following HEM and prior to CLP. ALI was assessed 24 h after the induction of sepsis. Silencing of MIP-2 markedly reduced tissue and plasma IL-6 concentrations, tissue MIP-2, as well as lung PMN influx, interstitial edema, alveolar congestion, and disruption of lung tissue architecture (164). In contrast, KC-siRNA treatment, while reducing plasma KC, tissue KC, and tissue IL-6, produced neither a significant reduction in plasma IL-6 nor lung PMN influx nor lung damage (164). Alternatively, based on the epithelial cell hypothesis, siRNA sequences against Fas and caspase-8 were intratracheally instilled during septic ALI. Interestingly, while these sequences markedly diminished lung Fas and caspase-8 expression in a gene specific fashion, when lung apoptosis was assessed, pulmonary tissue caspase-3 activity was reduced only in response to Fas but not caspase-8 silencing. As silencing here, as commonly observed, did not result in a total depletion of the target protein, but rather in a diminished expression, it is possible that sufficient active caspase-8 was still present to mediate the apoptotic effects either through direct activation of caspase-3 or indirectly through cleavage of the proapoptotic protein Bid (152,175). Our data also indicated that silencing of Fas on lung epithelial cells was associated with a reduction in lung inflammation, PMN influx, and a diminished extent of lung injury (118), thus emphasizing the relevance of epithelial cell integrity during indirect septic ALI.


Keeping in mind the diversity of clinical scenarios, we use the inclusive acronym ALI, one could think that it might be exactly this imprecise global view that prohibits the development of novel therapeutic approaches for ARDS. The diversity of underlying pathologic mechanisms prohibits the formulation of a unified pathophysiology of this clinical entity. Whether different forms of ALI (for example, direct versus indirect) are more of a response to a certain patho-mechanism than another remains to be determined. However, while there may be numerous diverse stimuli that can initiate the pathogenesis of this clinical entity, the final steps in ALI/ARDS, such as compromise of the alveolocapillary barrier function, appear to be somewhat common. Here we have reviewed the data which support the role of the activated PMN in lung injury as well as the contribution of epithelial cell death as independent entities contributing to ALI. Further, we have shown how recent studies looking at epithelial cells and the silencing of Fas-induced apoptosis and/or inflammation in these cells may serve as a lynchpin linking these two pathological processes. Upon early Fas activation, alveolar macrophages and lung epithelial cells might produce chemokines in the lung, thus attracting activated and potentially harmful neutrophils, monocytes, or even T-lymphocytes to the site of injury and potentiating the degree of injury. Finally, we trust we have illustrated how the application of siRNA-induced gene knockdown in the lung not only has produced novel insights into pathogenesis of ALI, but may represent a potential exciting therapeutic approach for its treatment.



This work was supported in part by funds from the University of Ulm Medical School (M.P.) as well as NIH-RO1s GM53209 and HL73525 (A.A.).


  1. 1.
    Bernard GR et al. (1994) The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am. J. Respir. Crit. Care Med. 149:818–24.PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Wheeler AP, Bernard GR. (2007) Acute lung injury and the acute respiratory distress syndrome: a clinical review. Lancet. 369:1553–64.PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Ashbaugh DG, Bigelow DB, Petty TL, Levine BE. (1967) Acute respiratory distress in adults. Lancet. 2:319–23.CrossRefGoogle Scholar
  4. 4.
    Brun-Buisson C et al. (2004) Epidemiology and outcome of acute lung injury in European intensive care units. Results from the ALIVE study. Intensive Care Med. 30:51–61.PubMedCrossRefPubMedCentralGoogle Scholar
  5. 5.
    Rubenfeld GD et al. (2005) Incidence and outcomes of acute lung injury. N Engl. J. Med. 353:1685–93.PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Herridge MS, Angus DC. (2005) Acute lung injury—affecting many lives. N. Engl. J. Med. 353:1736–8.PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Goss CH, Brower RG, Hudson LD, Rubenfeld GD. (2003) Incidence of acute lung injury in the United States. Crit. Care Med. 31:1607–11.PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Yilmaz M et al. (2007) Six-month survival of patients with acute lung injury: prospective cohort study. Crit. Care Med. 35:2303–7.PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Calfee CS et al. (2007) Trauma-associated lung injury differs clinically and biologically from acute lung injury due to other clinical disorders. Crit. Care Med. 35:2243–50.PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Vincent JL, Zambon M. (2006) Why do patients who have acute lung injury/acute respiratory distress syndrome die from multiple organ dysfunction syndrome? Implications for management. Clin. Chest Med. 27:725–31.PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Monchi M et al. (1998) Early predictive factors of survival in the acute respiratory distress syndrome. A multivariate analysis. Am. J. Respir. Crit. Care Med. 158:1076–81.PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Hopkins RO, Herridge MS. (2006) Quality of life, emotional abnormalities, and cognitive dysfunction in survivors of acute lung injury/acute respiratory distress syndrome. Clin. Chest Med. 27:679–89.PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Herridge MS et al. (2003) One-year outcomes in survivors of the acute respiratory distress syndrome. N. Engl. J. Med. 348:683–93.PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Suratt BT, Parsons PE. (2006) Mechanisms of acute lung injury/acute respiratory distress syndrome. Clin. Chest Med. 27:579–89.PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    ARDS Network. (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N. Engl. J. Med. 342:1301–8.CrossRefGoogle Scholar
  16. 16.
    Pittet JF, Mackersie RC, Martin TR, Matthay MA. (1997) Biological markers of acute lung injury: prognostic and pathogenetic significance. Am. J. Respir. Crit. Care Med. 155:1187–205.PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Ware LB, Matthay MA. (2000) The acute respiratory distress syndrome. N. Engl. J. Med. 342:1334–49.PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Burns AR, Smith CW, Walker DC. (2003) Unique structural features that influence neutrophil emigration into the lung. Physiol. Rev. 83:309–36.PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Bachofen M, Weibel ER. (1982) Structural alterations of lung parenchyma in the adult respiratory distress syndrome. Clin. Chest Med. 3:35–56.PubMedPubMedCentralGoogle Scholar
  20. 20.
    Bachofen M, Weibel ER. (1977) Alterations of the gas exchange apparatus in adult respiratory insufficiency associated with septicemia. Am. Rev. Respir. Dis. 116:589–615.PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Rinaldo JE, Borovetz H. (1985) Deterioration of oxygenation and abnormal lung microvascular permeability during resolution of leukopenia in patients with diffuse lung injury. Am. Rev. Respir. Dis. 131:579–83.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Azoulay E et al. (2002) Deterioration of previous acute lung injury during neutropenia recovery. Crit. Care Med. 30:781–6.PubMedCrossRefPubMedCentralGoogle Scholar
  23. 23.
    Steinberg KP et al. (1994) Evolution of broncho-alveolar cell populations in the adult respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 150(1):113–122.PubMedCrossRefPubMedCentralGoogle Scholar
  24. 24.
    Ayala A et al. (2002) Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency. Am. J. Pathol. 161:2283–94.PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Lomas-Neira J, Chung CS, Perl M, Gregory S, Biffl W, Ayala A. (2006) Role of alveolar macrophage and migrating neutrophils in hemorrhage-induced priming for ALI subsequent to septic challenge. Am. J. Physiol. Lung Cell. Mol. Physiol. 290:L51–8.PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Stephens KE, Ishizaka A, Wu ZH, Larrick JW, Raffin TA. (1988) Granulocyte depletion prevents tumor necrosis factor-mediated acute lung injury in guinea pigs. Am. Rev. Respir. Dis. 138:1300–7.PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Inoue S et al. (1995) Anti-neutrophil antibody attenuates the severity of acute lung injury in rats with experimental acute pancreatitis. Arch. Surg. 130:93–8.PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Clark SC, Rao JN, Flecknell PA, Dark JH. (2003) Pentoxifylline is as effective as leukocyte depletion for modulating pulmonary reperfusion injury. J. Thorac. Cardiovasc. Surg. 126:2052–7.PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Looney MR, Su X, Van Ziffle JA, Lowell CA, Matthay MA. (2006) Neutrophils and their Fc gamma receptors are essential in a mouse model of transfusion-related acute lung injury. J. Clin. Invest. 116:1615–23.PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Heflin JAC, Brigham KL. (1981) Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia. J. Clin. Invest. 68:1253–60.PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Till GO, Johnson KJ, Kunkel R, Ward PA. (1982) Intravascular activation of complement and acute lung injury. J. Clin. Invest. 69:1126–35.PubMedCrossRefPubMedCentralGoogle Scholar
  32. 32.
    Abraham E, Carmody A, Shenkar R, Arcaroli J. (2000) Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am. J. Physiol Lung Cell. Mol. Physiol. 279:L1137–45.PubMedCrossRefGoogle Scholar
  33. 33.
    Donnelly SC et al. (1993) Interleukin-8 and development of adult respiratory distress syndrome in at-risk patient groups. Lancet. 341:643–7.PubMedCrossRefGoogle Scholar
  34. 34.
    Pugin J, Ricou B, Steinberg KP, Suter PM, Martin TR. (1996) Proinflammatory activity in bron-choalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am. J. Respir. Crit. Care Med. 153:1850–6.PubMedCrossRefGoogle Scholar
  35. 35.
    Meduri GU et al. (1995) Inflammatory cytokines in the BAL of patients with ARDS. Persistent elevation over time predicts poor outcome. Chest. 108:1303–14.PubMedCrossRefGoogle Scholar
  36. 36.
    Siler TM, Swierkosz JE, Hyers TM, Fowler AA, Webster RO. (1989) Immunoreactive interleukin-1 in bronchoalveolar lavage fluid of high-risk patients and patients with the adult respiratory distress syndrome. Exp. Lung Res. 15:881–94.PubMedCrossRefGoogle Scholar
  37. 37.
    Meduri GU et al. (1995) Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 beta and IL-6 levels are consistent and efficient predictors of outcome over time. Chest. 107:1062–73.PubMedCrossRefGoogle Scholar
  38. 38.
    Hammerschmidt DE, Weaver LJ, Hudson LD, Craddock PR, Jacob HS. (1980) Association of complement activation and elevated plasma-C5a with adult respiratory distress syndrome. Patho-physiological relevance and possible prognostic value. Lancet. 1:947–9.PubMedCrossRefGoogle Scholar
  39. 39.
    Jacob HS, Craddock PR, Hammerschmidt DE, Moldow CF. (1980) Complement-induced granu-locyte aggregation: an unsuspected mechanism of disease. N. Engl. J. Med. 302:789–94.PubMedCrossRefGoogle Scholar
  40. 40.
    Zimmerman GA et al. (1999) Endothelial activation in ARDS. Chest. 116:18S–24S.PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Park WY et al. (2001) Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 164:1896–903.PubMedCrossRefPubMedCentralGoogle Scholar
  42. 42.
    Schutte H et al. (1996) Bronchoalveolar and systemic cytokine profiles in patients with ARDS, severe pneumonia and cardiogenic pulmonary edema. Eur. Respir. J. 9:1858–67.PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Suter PM et al. (1992) High bronchoalveolar levels of tumor necrosis factor and its inhibitors, in-terleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am. Rev. Respir. Dis. 145:1016–22.PubMedCrossRefPubMedCentralGoogle Scholar
  44. 44.
    Goodman RB et al. (1996) Inflammatory cyto-kines in patients with persistence of the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 154:602–11.PubMedCrossRefPubMedCentralGoogle Scholar
  45. 45.
    Opal SM, Cross AS. (1999) Clinical trials for severe sepsis. Past failures, and future hopes. Infect. Dis. Clin. North Am. 13:285–97, vii.PubMedCrossRefPubMedCentralGoogle Scholar
  46. 46.
    Abraham E, Kaneko DJ, Shenkar R. (1999) Effects of endogenous and exogenous catecholamines on LPS-induced neutrophil trafficking and activation. Am. J. Physiol. 276:L1–8.PubMedPubMedCentralGoogle Scholar
  47. 47.
    Shenkar R, Abraham E. (1999) Mechanisms of lung neutrophil activation after hemorrhage or endotoxemia: roles of reactive oxygen intermediates, NF-kappa B, and cyclic AMP response element binding protein. J. Immunol. 163:954–62.PubMedPubMedCentralGoogle Scholar
  48. 48.
    Parsey MV, Tuder RM, Abraham E. (1998) Neu- trophils are major contributors to intraparenchy-mal lung IL-1 beta expression after hemorrhage and endotoxemia. J. Immunol. 160:1007–13.PubMedPubMedCentralGoogle Scholar
  49. 49.
    Miskolci V et al. (2007) NFkappaB is persistently activated in continuously stimulated human neu- trophils. Mol. Med. 13:134–42.PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Yang KY, Arcaroli JJ, Abraham E. (2003) Early alterations in neutrophil activation are associated with outcome in acute lung injury. Am. J. Respir. Crit. Care Med. 167:1567–74.PubMedCrossRefPubMedCentralGoogle Scholar
  51. 51.
    Doerschuk CM. (2001) Mechanisms of leukocyte sequestration in inflamed lungs. Microcirculation. 8:71–88.PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Skoutelis AT et al. (2000) Neutrophil deformabil- ity in patients with sepsis, septic shock, and adult respiratory distress syndrome. Crit. Care Med. 28:2355–9.PubMedCrossRefPubMedCentralGoogle Scholar
  53. 53.
    Doyle NA et al. (1997) Neutrophil margination, sequestration, and emigration in the lungs of L-selectin-deficient mice. J. Clin. Invest. 99:526–33.PubMedCrossRefPubMedCentralGoogle Scholar
  54. 54.
    Kubo H et al. (1999) L- and P-selectin and CD11/CD18 in intracapillary neutrophil sequestration in rabbit lungs. Am. J. Respir. Crit. Care Med. 159:267–74.PubMedCrossRefPubMedCentralGoogle Scholar
  55. 55.
    Simon SI et al. (1999) Signaling functions of L-se-lectin in neutrophils: alterations in the cytoskele-ton and colocalization with CD18. J. Immunol. 163:2891–901.PubMedPubMedCentralGoogle Scholar
  56. 56.
    Doerschuk CM, Tasaka S, Wang Q. (2000) CD11/CD18-dependent and -independent neu-trophil emigration in the lungs: how do neu-trophils know which route to take? Am. J. Respir. Cell Mol. Biol. 23:133–6.PubMedCrossRefPubMedCentralGoogle Scholar
  57. 57.
    Williams MA, Solomkin JS. (1999) Integrin- mediated signaling in human neutrophil functioning. J. Leukoc. Biol. 65:725–36.PubMedCrossRefPubMedCentralGoogle Scholar
  58. 58.
    Lowell CA, Berton G. (1999) Integrin signal transduction in myeloid leukocytes. J. Leukoc. Biol. 65:313–20.PubMedCrossRefPubMedCentralGoogle Scholar
  59. 59.
    Schymeinsky J, Mocsai A, Walzog B. (2007) Neu-trophil activation via beta2 integrins (CD11/CD18): molecular mechanisms and clinical implications. Thromb. Haemost. 98:262–73.PubMedCrossRefPubMedCentralGoogle Scholar
  60. 60.
    Ley K. (2002) Integration of inflammatory signals by rolling neutrophils. Immunol. Rev. 186:8–18.PubMedCrossRefPubMedCentralGoogle Scholar
  61. 61.
    Nathan C et al. (1989) Cytokine-induced respiratory burst of human neutrophils: Dependence on extracellular matrix proteins and CD11/CD18 in- tegrins. J. Cell. Biol. 109:1341–9.PubMedCrossRefPubMedCentralGoogle Scholar
  62. 62.
    Walzog B et al. (1999) A role for beta(2) integrins (CD11/CD18) in the regulation of cytokine gene expression of polymorphonuclear neutrophils during the inflammatory response. FASEB J. 13:1855–65.PubMedCrossRefPubMedCentralGoogle Scholar
  63. 63.
    Perkins GD, Nathani N, McAuley DF, Gao F, Thickett DR. (2007) In vitro and in vivo effects of salbutamol on neutrophil function in acute lung injury. Thorax. 62:36–42.PubMedCrossRefPubMedCentralGoogle Scholar
  64. 64.
    Belperio JA et al. (2002) Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J. Clin. Invest. 110:1703–16.PubMedCrossRefPubMedCentralGoogle Scholar
  65. 65.
    Perl M et al. (2006) The pulmonary and hepatic immune microenvironment and its contribution to the early systemic inflammation following blunt chest trauma. Crit. Care Med. 34:1152–9.PubMedCrossRefPubMedCentralGoogle Scholar
  66. 66.
    Lomas-Neira JL, Chung CS, Grutkoski PS, Miller EJ, Ayala A. (2004) CXCR2 inhibition suppresses hemorrhage-induced priming for acute lung injury in mice. J. Leukoc. Biol. 76:58–64.PubMedCrossRefPubMedCentralGoogle Scholar
  67. 67.
    Lomas JL et al. (2003) Differential effects of macrophage inflammatory chemokine-2 and ker-atinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice. Shock. 19:358–65.PubMedCrossRefPubMedCentralGoogle Scholar
  68. 68.
    Martin TR. (2002) Neutrophils and lung injury: getting it right. J. Clin. Invest. 110:1603–5.PubMedCrossRefPubMedCentralGoogle Scholar
  69. 69.
    Shasby DM et al. (1982) Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals. Am. Rev. Respir. Dis. 125:443–7.PubMedPubMedCentralGoogle Scholar
  70. 70.
    Wang W, Suzuki Y, Tanigaki T, Rank DR, Raffin TA. (1994) Effect of the NADPH oxidase inhibitor apocynin on septic lung injury in guinea pigs. Am. J. Respir. Crit. Care Med. 150:1449–52.PubMedCrossRefPubMedCentralGoogle Scholar
  71. 71.
    Kubo H et al. (1996) Preservation of complement-induced lung injury in mice with deficiency of NADPH oxidase. J. Clin. Invest. 97:2680–4.PubMedCrossRefPubMedCentralGoogle Scholar
  72. 72.
    Kristof AS, Goldberg P, Laubach V, Hussain SN. (1998) Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury. Am. J. Respir. Crit. Care Med. 158:1883–9.PubMedCrossRefPubMedCentralGoogle Scholar
  73. 73.
    Fialkow L, Wang Y, Downey GP. (2007) Reactive oxygen and nitrogen species as signaling molecules regulating neutrophil function. Free Radic. Biol. Med. 42:153–64.PubMedCrossRefPubMedCentralGoogle Scholar
  74. 74.
    Endo S et al. (2006) Sivelestat sodium hydrate improves septic acute lung injury by reducing alveolar dysfunction. Res. Commun. Mol. Pathol. Pharmacol. 119:53–65.PubMedPubMedCentralGoogle Scholar
  75. 75.
    Belaaouaj A et al. (1998) Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat. Med. 4:615–8.PubMedCrossRefPubMedCentralGoogle Scholar
  76. 76.
    Tkalcevic J et al. (2000) Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity. 12:201–10.PubMedCrossRefPubMedCentralGoogle Scholar
  77. 77.
    Delclaux C et al. (1997) Gelatinases in epithelial lining fluid of patients with adult respiratory distress syndrome. Am. J. Physiol. 272:L442–51.PubMedPubMedCentralGoogle Scholar
  78. 78.
    Carney DE et al. (1999) Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass. Circulation. 100:400–6.PubMedCrossRefPubMedCentralGoogle Scholar
  79. 79.
    Kawabata K, Hagio T, Matsuoka S. (2002) The role of neutrophil elastase in acute lung injury. Eur. J. Pharmacol. 451:1–10.PubMedCrossRefPubMedCentralGoogle Scholar
  80. 80.
    Moraes TJ, Zurawska JH, Downey GP. (2006) Neutrophil granule contents in the pathogenesis of lung injury. Curr. Opin. Hematol 13:21–7.PubMedCrossRefPubMedCentralGoogle Scholar
  81. 81.
    Dunican AL, Leuenroth SJ, Grutkoski P, Ayala A, Simms HH. (2000) TNFalpha-induced suppression of PMN apoptosis is mediated through interleukin-8 production. Shock. 14:284–8.PubMedCrossRefPubMedCentralGoogle Scholar
  82. 82.
    Dunican AL, Leuenroth SJ, Ayala A, Simms HH. (2000) CXC chemokine suppression of polymor-phonuclear leukocytes apoptosis and preservation of function is oxidative stress independent. Shock. 13:244–50.PubMedCrossRefPubMedCentralGoogle Scholar
  83. 83.
    Jimenez MF et al. (1997) Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch. Surg. 132:1263–9.PubMedCrossRefPubMedCentralGoogle Scholar
  84. 84.
    Ertel W et al. (1999) Granulocyte colony-stimulating factor inhibits neutrophil apoptosis at the local site after severe head and thoracic injury. J. Trauma. 46:784–92.PubMedCrossRefPubMedCentralGoogle Scholar
  85. 85.
    Biffl WL et al. (1999) Neutrophils are primed for cytotoxicity and resist apoptosis in injured patients at risk for multiple organ failure. Surgery. 126:198–202.PubMedCrossRefPubMedCentralGoogle Scholar
  86. 86.
    Biffl WL et al. (2001) Neutrophil apoptosis is delayed by trauma patients’ plasma via a mechanism involving proinflammatory phospholipids and protein kinase C. Surg. Infect. (Larchmt). 2:289–93.PubMedCrossRefPubMedCentralGoogle Scholar
  87. 87.
    Chitnis D, Dickerson C, Munster AM, Winchurch RA. (1996) Inhibition of apoptosis in polymor-phonuclear neutrophils from burn patients. J. Leukoc. Biol. 59:835–9.PubMedCrossRefPubMedCentralGoogle Scholar
  88. 88.
    Taneja R et al. (2004) Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity. Crit. Care Med. 32:1460–9.PubMedCrossRefPubMedCentralGoogle Scholar
  89. 89.
    Fialkow L et al. (2006) Neutrophil apoptosis: a marker of disease severity in sepsis and sepsis-induced acute respiratory distress syndrome. Crit. Care. 10:R155.PubMedCrossRefPubMedCentralGoogle Scholar
  90. 90.
    Goodman ER et al. (1999) Role of granulocyte-macrophage colony-stimulating factor and its receptor in the genesis of acute respiratory distress syndrome through an effect on neutrophil apoptosis. Arch. Surg. 134:1049–54.PubMedCrossRefPubMedCentralGoogle Scholar
  91. 91.
    Dunican A, Grutkoski P, Leuenroth S, Ayala A, Simms HH. (2000) Neutrophils regulate their own apoptosis via preservation of CXC receptors. JSurg. Res. 90:32–8.CrossRefGoogle Scholar
  92. 92.
    Abraham E. (2003) Neutrophils and acute lung injury. Crit. Care Med. 31:S195–9.PubMedCrossRefPubMedCentralGoogle Scholar
  93. 93.
    Nelson S et al. (1998) A randomized controlled trial of filgrastim as an adjunct to antibiotics for treatment of hospitalized patients with community-acquired pneumonia. CAP Study Group. J. Infect. Dis. 178:1075–80.PubMedCrossRefPubMedCentralGoogle Scholar
  94. 94.
    Matute-Bello G et al. (2000) Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome. Crit. Care Med. 28:1–7.PubMedCrossRefPubMedCentralGoogle Scholar
  95. 95.
    Lagasse E, Weissman IL. (1994) bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages. J. Exp. Med. 179:1047–52.CrossRefGoogle Scholar
  96. 96.
    Perl M et al. (2007) Beneficial versus detrimental effects of neutrophils are determined by the nature of the insult. J. Am. Coll. Surg. 204:840–52.PubMedCrossRefPubMedCentralGoogle Scholar
  97. 97.
    Teder P et al. (2002) Resolution of lung inflammation by CD44. Science. 296:155–8.PubMedCrossRefPubMedCentralGoogle Scholar
  98. 98.
    Laufe MD, Simon RH, Flint A, Keller JB. (1986) Adult respiratory distress syndrome in neutropenic patients. Am. J. Med. 80:1022–6.PubMedCrossRefPubMedCentralGoogle Scholar
  99. 99.
    Vansteenkiste JF, Boogaerts MA. (1989) Adult respiratory distress syndrome in neutropenic leukemia patients. Blut. 58:287–90.PubMedCrossRefPubMedCentralGoogle Scholar
  100. 100.
    Ognibene FP et al. (1986) Adult respiratory distress syndrome in patients with severe neutropenia. N. Engl. J. Med. 315:547–51.PubMedCrossRefPubMedCentralGoogle Scholar
  101. 101.
    Wunderink R et al. (2001) Filgrastim in patients with pneumonia and severe sepsis or septic shock. Chest. 119:523–9.PubMedCrossRefPubMedCentralGoogle Scholar
  102. 102.
    Wiener-Kronish JP, Albertine KH, Matthay MA. (1991) Differential responses of the endothelial and epithelial barriers of the lung in sheep to Escherichia coli endotoxin. J. Clin. Invest. 88:864–75.PubMedCrossRefPubMedCentralGoogle Scholar
  103. 103.
    Walker DC, Behzad AR, Chu F. (1995) Neutrophil migration through preexisting holes in the basal laminae of alveolar capillaries and epithelium during streptococcal pneumonia. Microvasc. Res. 50:397–416.PubMedCrossRefPubMedCentralGoogle Scholar
  104. 104.
    Ware LB, Matthay MA. (2001) Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 163:1376–83.PubMedCrossRefPubMedCentralGoogle Scholar
  105. 105.
    Modelska K, Pittet JF, Folkesson HG, Courtney Broaddus V, Matthay MA. (1999) Acid-induced lung injury. Protective effect of anti-interleukin-8 pretreatment on alveolar epithelial barrier function in rabbits. Am. J. Respir. Crit. Care Med. 160:1450–6.PubMedCrossRefPubMedCentralGoogle Scholar
  106. 106.
    Sznajder JI. (1999) Strategies to increase alveolar epithelial fluid removal in the injured lung. Am. J. Respir. Crit. Care Med. 160:1441–2.PubMedCrossRefPubMedCentralGoogle Scholar
  107. 107.
    Martin TR, Hagimoto N, Nakamura M, Matute-Bello G. (2005) Apoptosis and epithelial injury in the lungs. Proc. Am. Thorac. Soc. 2:214–20.PubMedCrossRefPubMedCentralGoogle Scholar
  108. 108.
    Martin TR, Nakamura M, Matute-Bello G. (2003) The role of apoptosis in acute lung injury. Crit. Care Med. 31:S184–8.PubMedCrossRefPubMedCentralGoogle Scholar
  109. 109.
    Matthay MA, Wiener-Kronish JP. (1990) Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am. Rev. Respir. Dis. 142:1250–7.PubMedCrossRefPubMedCentralGoogle Scholar
  110. 110.
    Strohmaier W et al. (2005) Bilateral lavage with diluted surfactant improves lung function after unilateral lung contusion in pigs. Crit. Care Med. 33:2286–93.PubMedCrossRefPubMedCentralGoogle Scholar
  111. 111.
    Greene KE et al. (1999) Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS. Am. J. Respir. Crit. Care Med. 160:1843–50.PubMedCrossRefPubMedCentralGoogle Scholar
  112. 112.
    Bardales RH, Xie SS, Schaefer RF, Hsu SM. (1996) Apoptosis is a major pathway responsible for the resolution of type II pneumocytes in acute lung injury. Am. J. Pathol. 149:845–52.PubMedPubMedCentralGoogle Scholar
  113. 113.
    Guinee D Jr et al. (1997) The potential role of BAX and BCL-2 expression in diffuse alveolar damage. Am. J. Pathol. 151:999–1007.PubMedPubMedCentralGoogle Scholar
  114. 114.
    Bem RA, Bos AP, Matute-Bello G, van TM, van Woensel JB. (2007) Lung epithelial cell apoptosis during acute lung injury in infancy. Pediatr. Crit. Care Med. 8:132–7.PubMedCrossRefPubMedCentralGoogle Scholar
  115. 115.
    Matute-Bello G et al. (1997) Neutrophil apoptosis in the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 156:1969–77.PubMedCrossRefPubMedCentralGoogle Scholar
  116. 116.
    Matute-Bello G et al. (1999) Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS). J. Immunol. 163:2217–25.PubMedPubMedCentralGoogle Scholar
  117. 117.
    Miyake Y et al. (2007) Protective role of macrophages in noninflammatory lung injury caused by selective ablation of alveolar epithelial type II Cells. J. Immunol. 178:5001–9.PubMedCrossRefPubMedCentralGoogle Scholar
  118. 118.
    Kawasaki M et al. (2000) Protection from lethal apoptosis in lipopolysaccharide-induced acute lung injury in mice by a caspase inhibitor. Am. J. Pathol. 157:597–603.PubMedCrossRefPubMedCentralGoogle Scholar
  119. 119.
    Chen H et al. (2007) Anti-apoptotic PTD-FNK protein suppresses lipopolysaccharide-induced acute lung injury in rats. Exp. Mol. Pathol. 83:377–84.PubMedCrossRefPubMedCentralGoogle Scholar
  120. 120.
    Kitamura Y et al. (2001) Fas/FasL-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice. Am. J. Respir. Crit. Care Med. 163:762–9.PubMedCrossRefPubMedCentralGoogle Scholar
  121. 121.
    Nakamura M et al. (2004) Differential response of human lung epithelial cells to Fas-induced apoptosis. Am. J. Pathol. 164:1949–58.PubMedCrossRefPubMedCentralGoogle Scholar
  122. 122.
    Lee KS et al. (2007) Evaluation of bronchoalveolar lavage fluid from ARDS patients with regard to apoptosis. Respir. Med. 102:464–9.PubMedCrossRefPubMedCentralGoogle Scholar
  123. 123.
    Albertine KH et al. (2002) Fas and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome. Am. J. Pathol. 161:1783–96.PubMedCrossRefPubMedCentralGoogle Scholar
  124. 124.
    Hashimoto S et al. (2000) Upregulation of two death pathways of perforin/granzyme and FasL/Fas in septic acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 161:237–43.PubMedCrossRefPubMedCentralGoogle Scholar
  125. 125.
    Kiener PA et al. (1997) Human monocytic cells contain high levels of intracellular Fas ligand: rapid release following cellular activation. J. Immunol. 159:1594–8.PubMedPubMedCentralGoogle Scholar
  126. 126.
    Serrao KL, Fortenberry JD, Owens ML, Harris FL, Brown LA. (2001) Neutrophils induce apoptosis of lung epithelial cells via release of soluble Fas ligand. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L298–305.PubMedCrossRefPubMedCentralGoogle Scholar
  127. 127.
    Powell WC, Fingleton B, Wilson CL, Boothby M, Matrisian LM. (1999) The metalloproteinase matrilysin proteolytically generates active soluble Fas ligand and potentiates epithelial cell apoptosis. Curr. Biol. 9:1441–7.PubMedCrossRefPubMedCentralGoogle Scholar
  128. 128.
    Matsuno H et al. (2001) Stromelysin-1 (MMP-3) in synovial fluid of patients with rheumatoid arthritis has potential to cleave membrane bound Fas ligand. J. Rheumatol. 28:22–8.PubMedPubMedCentralGoogle Scholar
  129. 129.
    Cheng J et al. (1994) Protection from Fas-mediated apoptosis by a soluble form of the Fas molecule. Science. 263:1759–62.PubMedCrossRefPubMedCentralGoogle Scholar
  130. 130.
    Matute-Bello G et al. (2001) Fas (CD95) induces alveolar epithelial cell apoptosis in vivo: implications for acute pulmonary inflammation. Am. J. Pathol. 158:153–61.PubMedCrossRefPubMedCentralGoogle Scholar
  131. 131.
    Matute-Bello G et al. (2005) Fas-mediated acute lung injury requires fas expression on non-myeloid cells of the lung. J. Immunol. 175:4069–75.PubMedCrossRefPubMedCentralGoogle Scholar
  132. 132.
    Perl M et al. (2007) Fas-induced pulmonary apoptosis and inflammation during indirect acute lung injury. Am. J. Respir. Crit. Care Med. 176:591–601.CrossRefGoogle Scholar
  133. 133.
    Matute-Bello G et al. (2001) Fas/Fas ligand system mediates epithelial injury, but not pulmonary host defenses, in response to inhaled bacteria. Infect. Immun. 69:5768–76.PubMedCrossRefPubMedCentralGoogle Scholar
  134. 134.
    Tateda K et al. (2003) Hyperoxia mediates acute lung injury and increased lethality in murine Legionella pneumonia: the role of apoptosis. J. Immunol. 170:4209–16.PubMedCrossRefPubMedCentralGoogle Scholar
  135. 135.
    Grassme H et al. (2000) CD95/CD95 ligand interactions on epithelial cells in host defense to Pseudomonas aeruginosa. Science. 290:527–30.PubMedCrossRefPubMedCentralGoogle Scholar
  136. 136.
    Liu AN et al. (1999) Perforin-independent CD8(+) T-cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-alpha: relative insensitivity to Fas ligand. Am. J. Respir. Cell Mol. Biol. 20:849–58.PubMedCrossRefPubMedCentralGoogle Scholar
  137. 137.
    Buccellato LJ, Tso M, Akinci OI, Chandel NS, Budinger GR. (2004) Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells. J. Biol. Chem. 279:6753–60.PubMedCrossRefPubMedCentralGoogle Scholar
  138. 138.
    Arai H, Gordon D, Nabel EG, Nabel GJ. (1997) Gene transfer of Fas ligand induces tumor regression in vivo. Proc. Natl. Acad. Sci. U. S. A. 94:13862–7.PubMedCrossRefPubMedCentralGoogle Scholar
  139. 139.
    Wortinger MA et al. (2003) Fas ligand-induced murine pulmonary inflammation is reduced by a stable decoy receptor 3 analogue. Immunology. 110:225–33.PubMedCrossRefPubMedCentralGoogle Scholar
  140. 140.
    Matute-Bello G, Winn RK, Martin TR, Liles WC. (2004) Sustained lipopolysaccharide-induced lung inflammation in mice is attenuated by functional deficiency of the Fas/Fas ligand system. Clin. Diagn. Lab. Immunol. 11:358–61.PubMedPubMedCentralGoogle Scholar
  141. 141.
    Neff TA et al. (2005) Relationship of acute lung inflammatory injury to Fas/FasL system. Am. J. Pathol. 166:685–94.PubMedCrossRefPubMedCentralGoogle Scholar
  142. 142.
    Hohlbaum AM, Gregory MS, Ju ST, Marshak Rothstein A. (2001) Fas ligand engagement of resident peritoneal macrophages in vivo induces apoptosis and the production of neutrophil chemotactic factors. J. Immunol. 167:6217–24.PubMedCrossRefPubMedCentralGoogle Scholar
  143. 143.
    Park DR et al. (2003) Fas (CD95) induces pro-inflammatory cytokine responses by human monocytes and monocyte-derived macrophages. J. Immunol. 170:6209–16.PubMedCrossRefPubMedCentralGoogle Scholar
  144. 144.
    Yamaoka-Tojo M et al. (2003) Dual response to Fas ligation in human endothelial cells: apoptosis and induction of chemokines, interleukin-8 and monocyte chemoattractant protein-1. Coron. Artery Dis. 14:89–94.PubMedCrossRefPubMedCentralGoogle Scholar
  145. 145.
    Guo Z, Zhang M, Tang H, Cao X. (2005) Fas signal links innate and adaptive immunity by promoting dendritic-cell secretion of CC and CXC chemokines. Blood. 106:2033–41.PubMedCrossRefPubMedCentralGoogle Scholar
  146. 146.
    Guo Z et al. (2003) Fas ligation induces IL-1beta-dependent maturation and IL-1beta-independent survival of dendritic cells: different roles of ERK and NF-kappaB signaling pathways. Blood. 102:4441–7.PubMedCrossRefPubMedCentralGoogle Scholar
  147. 147.
    Rescigno M et al. (2000) Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1beta, and the production of interferon gamma in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses. J. Exp. Med. 192:1661–8.PubMedCrossRefPubMedCentralGoogle Scholar
  148. 148.
    Choi C et al. (2001) Fas-induced expression of chemokines in human glioma cells: involvement of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Cancer Res. 61:3084–91.PubMedPubMedCentralGoogle Scholar
  149. 149.
    Choi C, Gillespie GY, Van Wagoner NJ, Benveniste EN. (2002) Fas engagement increases expression of interleukin-6 in human glioma cells. J. Neurooncol. 56:13–9.PubMedCrossRefPubMedCentralGoogle Scholar
  150. 150.
    Schaub FJ et al. (2000) Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells. Nat. Med. 6:790–6.PubMedCrossRefPubMedCentralGoogle Scholar
  151. 151.
    Hagimoto N et al. (1999) Induction of interleukin-8 secretion and apoptosis in bronchiolar epithelial cells by Fas ligation. Am. J. Respir. Cell Mol. Biol. 21:436–45.PubMedCrossRefPubMedCentralGoogle Scholar
  152. 152.
    Perl M et al. (2005) Silencing of fas, but not cas-pase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis. Am. J. Pathol. 167:1545–59.PubMedCrossRefPubMedCentralGoogle Scholar
  153. 153.
    Kumar LD, Clarke AR. (2007) Gene manipulation through the use of small interfering RNA (siRNA): from in vitro to in vivo applications. Adv. Drug Deliv. Rev. 59:87–100.PubMedCrossRefGoogle Scholar
  154. 154.
    Aigner A. (2007) Nonviral in vivo delivery of therapeutic small interfering RNAs. Curr. Opin. Mol. Ther. 9:345–52.PubMedGoogle Scholar
  155. 155.
    de FA, Vornlocher HP, Maraganore J, Lieberman J. (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat. Rev. Drug Discov. 6:443–53.CrossRefGoogle Scholar
  156. 156.
    Martin SE, Caplen NJ. (2007) Applications of RNA interference in mammalian systems. Annu. Rev. Genomics Hum. Genet. 8:81–108.PubMedCrossRefGoogle Scholar
  157. 157.
    Ernst N et al. (1999) Interaction of liposomal and polycationic transfection complexes with pulmonary surfactant. J. Gene Med. 1:331–40.PubMedCrossRefGoogle Scholar
  158. 158.
    Thomas M et al. (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc. Natl. Acad. Sci. U. S. A. 102: 5679–84.PubMedCrossRefPubMedCentralGoogle Scholar
  159. 159.
    Thomas M, Lu JJ, Chen J, Klibanov AM. (2007) Non-viral siRNA delivery to the lung. Adv. Drug Deliv. Rev. 59:124–33.PubMedCrossRefGoogle Scholar
  160. 160.
    Li BJ et al. (2005) Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat. Med. 11:944–51.PubMedCrossRefGoogle Scholar
  161. 161.
    Massaro D, Massaro GD, Clerch LB. (2004) Noninvasive delivery of small inhibitory RNA and other reagents to pulmonary alveoli in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 287(5):L1066–L1070.PubMedCrossRefGoogle Scholar
  162. 162.
    Zhang X et al. (2004) Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis. J. Biol. Chem. 279:10677–84.PubMedCrossRefGoogle Scholar
  163. 163.
    Akhtar S, Benter IF. (2007) Nonviral delivery of synthetic siRNAs in vivo. J. Clin. Invest. 117:3623–32.PubMedCrossRefGoogle Scholar
  164. 164.
    Lomas-Neira JL, Chung CS, Wesche DE, Perl M, Ayala A. (2005) In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury. J. Leukoc. Biol. 77:846–53.PubMedCrossRefPubMedCentralGoogle Scholar
  165. 165.
    Sledz CA, Holko M, de Veer MJ, Silverman RH, Williams BR. (2003) Activation of the interferon system by short-interfering RNAs. Nat. Cell Biol. 5:834–9.PubMedCrossRefPubMedCentralGoogle Scholar
  166. 166.
    Moss EG, Taylor JM. (2003) Small-interfering RNAs in the radar of the interferon system. Nat. Cell Biol. 5:771–2.PubMedCrossRefPubMedCentralGoogle Scholar
  167. 167.
    Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. (2001) Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 413:732–8.PubMedCrossRefPubMedCentralGoogle Scholar
  168. 168.
    Hornung V et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11:263–70.PubMedCrossRefGoogle Scholar
  169. 169.
    Robbins MA, Rossi JJ. (2005) Sensing the danger in RNA. Nat. Med. 11:250–1.PubMedCrossRefPubMedCentralGoogle Scholar
  170. 170.
    Heidel JD, Hu S, Liu XF, Triche TJ, Davis ME. (2004) Lack of interferon response in animals to naked siRNAs. Nat. Biotechnol. 22:1579–82.PubMedCrossRefPubMedCentralGoogle Scholar
  171. 171.
    Akira S. (2003) Toll-like receptor signaling. J. Biol. Chem. 278:38105–8.PubMedCrossRefGoogle Scholar
  172. 172.
    Wang XM, Kim HP, Song R, Choi AM. (2006) Caveolin-1 confers antiinflammatory effects in murine macrophages via the MKK3/p38 MAPK pathway. Am. J. Respir. Cell Mol. Biol. 34:434–42.PubMedCrossRefGoogle Scholar
  173. 173.
    Ulanova M et al. (2007) Involvement of Syk protein tyrosine kinase in LPS-induced responses in macrophages. J. Endotoxin Res. 13:117–25.PubMedCrossRefGoogle Scholar
  174. 174.
    Tephly LA, Carter AB. (2007) Constitutive NADPH oxidase and increased mitochondrial respiratory chain activity regulate chemokine gene expression. Am. J. Physiol. Lung Cell. Mol. Physiol. 293:L1143–55.PubMedCrossRefGoogle Scholar
  175. 175.
    Perl M, Chung CS, Ayala A. (2005) Apoptosis. Crit. Care Med. 33:S526–9.PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Feinstein Institute for Medical Research 2008

Authors and Affiliations

  • Mario Perl
    • 1
  • Joanne Lomas-Neira
    • 2
  • Chun-Shiang Chung
    • 2
  • Alfred Ayala
    • 2
  1. 1.Department of Traumatology, Hand- and Reconstructive SurgeryUniversity of Ulm Medical SchoolUlmGermany
  2. 2.Division of Surgical Research, Department of SurgeryRhode Island Hospital and Brown UniversityProvidenceUSA

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