Acute lung injury (ALI) is a life-threatening complication associated with sepsis, pneumonia, aspiration, multiple blood transfusions, trauma, and acute pancreatitis. Despite improvements in management of critically ill patients, mortality remains about 40%, and survivors often do not return to a normal life (1). Neutrophils are the first immune cells to accumulate in the lung during ALI, recruited by a complex network of cytokines and chemokines produced by tissue macrophages (2). Evidence supporting a causative role for neutrophils in lung injury includes a correlation between the degree of neutrophil accumulation in bronchoalveolar lavage fluid (BALF) and the severity of ALI in humans and a reduction in the severity of ALI in animal models following neutrophil depletion or inhibition of neutrophil accumulation in the lungs (3–7). The major systems by which neutrophils induce lung injury include the release of granule components, formation of neutrophil extracellular traps, and generation of reactive oxygen species (2, 8–10). A number of neutrophil granule components, including serine proteases, matrix metalloproteinases, lactoferrin, LL-37, azurocidin, and defensins, have been shown to contribute to ALI (2), and direct evidence for a role of neutrophil exocytosis was provided by studies in which injection of granule products produced ALI (8).
Although a role for neutrophils in ALI is well established, strategies to control neutrophil activation to prevent tissue and organ injury have not followed. We recently developed a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23) that inhibited secretory vesicle, gelatinase granule, and specific granule exocytosis in human neutrophils (11). Using that TAT-fusion protein, we showed that inhibition of neutrophil exocytosis reduced phagocytosis-stimulated hydrogen peroxide production by 60% and inhibited tumor necrosis factor α (TNF-α) and platelet-activating factor–induced priming of the respiratory burst by up to 80% (11). To determine if administration of TAT-SNAP-23 was able to inhibit inflammatory injury in vivo, the present study used a model of ALI induced by immune complex (IC) deposition in rats. That model is dependent on complement activation, cytokine production, and chemokine-induced neutrophil infiltration (12, 13). Our results show that intravenous administration of TAT-SNAP-23 inhibited the development of increased vascular permeability without affecting neutrophil accumulation in the lungs, and that effect was associated with reduced accumulation of complement components.
MATERIALS AND METHODS
Rodent model of ALI
Acute lung injury was induced in pathogen-free male Long-Evans rats (275–300 g) as previously described (3–7). Briefly, rabbit polyclonal immunoglobulin G (IgG) anti–bovine serum albumin (BSA; 2.5 mg, MP-Biomedics, Solon, Ohio) was injected intratracheally in 0.3 mL of phosphate-buffered saline (PBS), pH 7.4. Immediately thereafter, 10 mg of BSA (Sigma-Aldrich, St Louis, Mo) with 250 μg of fluorescein isothiocyanate (FITC)–BSA (Invitrogen, Carlsbad, Calif) in 0.5 mL of PBS was injected intravenously. Two hours later, 0.05 mg/kg of TAT-SNAP-23 or a control TAT-fusion protein of similar molecular size (TAT-control) (11) was injected intravenously. Animals were killed 4 h after initiation of injury. In separate studies, intravenous administration of TAT-SNAP-23 or TAT-control showed no adverse effects after 24 h. Animal care was performed following the National Institutes of Health guidelines. Animal studies were approved by the University of Louisville Institutional Animal Care and Use Committee.
In vitro assays
Bronchoalveolar lavage fluid was collected by instilling and withdrawing 5 mL of sterile PBS three times via an intratracheal cannula. Lung injury was quantified by measuring leakage of FITC-BSA into lung parenchyma by fluorometry and by measuring the BALF albumin concentration. Cells were recovered from BALF by centrifugation and Wright-stained following a cytospin. A cell differential count was performed, and the total number of neutrophils calculated. Bronchoalveolar lavage fluid was stored at −80°C for mass spectrometry analysis and measurement of cytokine/chemokine levels (Multi-Analyte ELISA array; SA Biosciences, Frederick, Md). For immunoblot analysis, BALF cells were lysed in 40 μL of ice-cold lysis buffer as previously described (11, 14). Lysate or BALF proteins were separated by 4% to 12% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblot analysis for His-tag (1:1,000; Abcam, Cambridge, Mass), C-reactive protein (CRP, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, Calif), or complement C3 (1:1,000, Pierce, Rockford, Ill). Lung homogenates were separated by 4% to 12% gradient SDS-PAGE, followed by immunoblot analysis for caspase 3 (1:500; Cell Signaling Technology, Danvers, Mass). Protein signal was visualized by chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Plasma membrane expression of CD18 was measured in BAL cells by FITC-conjugated monoclonal anti-CD18 (Abcam); secretory vesicle and specific granule exocytosis by isolated human neutrophils was determined by measuring the increase in plasma membrane expression of FITC-conjugated monoclonal anti-CD35 (secretory vesicles, clone E11; Pharmingen, San Diego, Calif) and FITC-conjugated monoclonal anti-CD66b (specific granule, clone CLB-B 13.9; Accurate Chemical, Westbury, NY) in human neutrophils. All the plasma membrane markers were analyzed by flow cytometry as previously described (11, 14).
Human neutrophils were isolated from healthy donors using plasma-Percoll gradients as previously described (11, 14, 16). Wright staining performed on cytospins of isolated cells confirmed that more than 95% of the cells were neutrophils. Trypan blue exclusion staining confirmed more than 97% of cellswere viable. The institutional review boards of the University of Louisville approved the use of human donors for the study.
In a separate group of rats, lungs were fixed by intratracheal instillation of 10% buffered formaldehyde. Sections of embedded lung were stained by hematoxylin and eosin. The extent and location of neutrophils in the lungs were determined by immunohistochemistry for myeloperoxidase (MPO) using a polyclonal anti-rat MPO antibody (1:500; Abcam).
Mass spectrometry analysis of BALF
To achieve greater sensitivity for identification of low-abundance proteins, BALF supernatants were immunodepleted of seven high-abundance serum proteins (albumin, IgG, transferrin, fibrinogen, IgA, α-2 macroglobulin, IgM) using a ProteomeLab IgY R7 LC-2 column according to manufacturer guidelines. Proteins in the flow-through fractions were reduced, alkylated, and digested with trypsin, as previously described (14, 15). Tryptic peptides were separated by two-dimensional liquid chromatography and tandem mass spectrometry data generated by nanospray ionization into an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, Mass). The acquired mass spectrometry data were searched against a rat RefSeq protein database modified to include BSA using the SEQUEST (version 27 revision 11) algorithm, as described previously (14, 15). The database analysis was performed with SequestSorcerer (Sage-N Research, San Jose, Calif), and high-probability peptide and protein identifications were assigned from the SEQUEST results using the ProteinProphet (tools.proteomecenter.org/software.php) and SageN Sorcerer statistical platforms. Scaffold 3 proteomic analysis software (ProteomeSoftware, Inc, Portland, Ore) was used for quantitative comparison using a label-free spectral counting method (14, 15). Qualitative comparison of protein expression patterns was performed by Ingenuity Pathways Analysis software (http://ingenuity.com) (16).
All data are expressed as mean ± SEM. Statistical analysis was performed using analysis of variance with the Tukey-Kramer multiple-comparisons test. Differences were considered statistically significant when P < 0.05.
Administration of TAT-SNAP-23 ameliorates ALI
To determine an effective dose of TAT-SNAP-23, a preliminary study was performed in which 0.01, 0.05, or 0.1 mg/kg was injected intravenously 2 h after IC deposition in the lung. Bronchoalveolar lavage fluid was collected at 4 h, and vascular permeability was measured by the amount of FITC-BSA fluorescence. Induction of ALI resulted in a 5-fold increase in fluorescence in BALF that was reduced by 54% following administration of 0.01 mg/kg of TAT-SNAP-23, by 57% with 0.05 mg/kg, and 35% with 0.1 mg/kg (data not shown). Based on those results, 0.05 mg/kg TAT-SNAP-23 was used in all subsequent experiments.
To determine the effect of inhibiting exocytosis with TAT-SNAP-23 on ALI, four groups of rats were used. A control group received intratracheal and intravenous saline, whereas the other three groups received anti-BSA intratracheally and BSA and FITC-labeled BSA intravenously. One group also received an intravenous injection of 0.05 mg/kg TAT-SNAP-23, and a second received 0.05 mg/kg TAT-control 2 h after IC deposition. At 4 h, rats were killed, and bronchoalveolar lavage was performed, or lung tissue obtained for histology. Figure 1A shows the BALF fluorescence resulting from leakage of FITC-labeled BSA from blood into the lungs. As seen in the preliminary study, ALI was associated with a significant increase in BALF fluorescence that was significantly reduced following administration of TAT-SNAP-23, but not TAT-control. Figure 1B shows total protein in BALF from rats with ALI with and without treatment with TAT-control or TAT-SNAP-23. Those results confirm that treatment with TAT-SNAP-23, but not TAT-control, significantly reduced the increased vascular permeability associated with ALI. A differential cell count was performed in the BALF cells recovered from the four groups. The average number of neutrophils was very low in the control animals (0.07 × 106 ± 0.02 × 106, n = 8 animals). About 95% of the cells recovered from rats with ALI were neutrophils, as previously described (12, 13). Acute lung injury was associated with a significant increase in total neutrophil count (5.2 × 106 ± 2 × 106, n = 8 animals) that was not affected by treatment with TAT-SNAP-23 (8 × 106 ± 2.8 × 106, n = 8 animals) or TAT-control (9 × 106 ± 2.2 × 106, n = 8 animals). No statistical difference was found between the numbers of cells recovered from BALF of ALI, or TAT-SNAP-23–, or TAT-control–treated animals. Thus, TAT-SNAP-23 treatment significantly reduced the increased vascular permeability and total protein levels following IC deposition, whereas neutrophil infiltration was not affected.
Figure 1C shows representative lung histology from four groups. Rats administered saline showed a normal lung structure, whereas rats with untreated ALI demonstrated intra-alveolar edema and hemorrhage and neutrophil infiltration. Treatment with TAT-SNAP-23 resulted in a reduction in lung edema and hemorrhage, although infiltrating neutrophils were still observed. Lung histology in rats treated with TAT-control was similar to that seen in rats with untreated ALI. To confirm the observation that neutrophil infiltration was not impaired in TAT-SNAP-23–treated animals, immunohistochemistry for MPO was performed. Figure 1D shows that the number and location of neutrophils were similar with or without TAT-SNAP-23 treatment. Thus, histology supported the BALF findings that TAT-SNAP-23, but not TAT-control, inhibited the increased vascular permeability produced by IC deposition, whereas neutrophil migration into lungs was not affected.
To determine if neutrophils infiltrating the lungs contained TAT-fusion proteins, immunoblot analysis for the His-tag present in the TAT-fusion proteins was performed in cells contained in BALF. Figure 2 shows that cell lysates from rats treated with TAT-SNAP-23 and TAT-control contained proteins at the appropriate molecular size that stained for the His-tag (11). As the majority of cells contained in BALF were neutrophils, we interpret those results to indicate that TAT-fusion proteins entered circulating neutrophils that subsequently migrated into the lungs.
Effect of TAT-SNAP-23 on neutrophil exocytosis and generation of inflammatory stimuli
To determine if intravenous administration of TAT-SNAP-23 inhibited neutrophil exocytosis, plasma membrane expression of a granule marker CD18 (β2 integrin) by BALF neutrophils was analyzed by flow cytometry. Table 1 shows that neutrophils from rats with ALI showed a significant increase in the plasma membrane expression of CD18, indicating that exocytosis had occurred in infiltrating neutrophils. Bronchoalveolar lavage fluid neutrophils obtained from animals with ALI that were also treated with TAT-SNAP-23, but not TAT-control, showed a small but significant reduction in the plasma membrane expression of CD18 (Table 1).
Although the inhibitory effect of TAT-SNAP-23 on CD18 expression suggests that intravenous administration of TAT-SNAP-23 partially inhibited neutrophil exocytosis, other possibilities are that TAT-SNAP-23 treatment reduced expression of neutrophil stimuli in BALF. To determine if BALF from TAT-SNAP-23–treated animals contained stimuli of neutrophil exocytosis, in vitro degranulation assays were performed with isolated human neutrophils incubated with BALF from the four different animal groups. Secretory vesicle and specific granule exocytosis was determined by an increased plasma membrane expression of CD35 and CD66b, respectively. Figure 3, A and B, shows that BALF obtained from rats with ALI with or without treatment with TAT-SNAP-23 or TAT-control stimulated exocytosis equivalent to that stimulated by N-formylmethionine-leucine-phenylalanine (fMLF), whereas human neutrophils incubated with BALF from control animals showed no increase in CD35 and CD66b expression compared with basal levels. These data indicate that there are sufficient stimuli in the BALF of TAT-SNAP-23–treated animals to activate neutrophils; however, those animals showed less severe tissue injury and reduced upregulation of CD18. In vitro degranulation assays using human neutrophils pretreated with TAT-SNAP-23 followed by incubation with the different BALF showed inhibition of secretory vesicle and specific granule exocytosis (data not shown). No differences in TNF-α and IL-6 levels were observed in BALF from rats with ALI with or without treatment with TAT-SNAP-23 or TAT-control (data not shown). No change in caspase 3 levels was observed in lung homogenates from rats with ALI with or without treatment with TAT-SNAP-23 or TAT-control (data not shown). Taken together, the results suggest that administration of TAT-SNAP-23 resulted in amelioration of ALI by reducing neutrophil exocytosis, whereas generation of proinflammatory stimuli, apoptosis, and infiltration of neutrophils into the lungs were not affected.
Proteomic analysis of BALF
To examine potential mechanisms by which inhibition of neutrophil exocytosis reduced ALI, identification and quantification of proteins contained in BALF from rats with untreated ALI or ALI treated with TAT-SNAP-23 or TAT-control were performed by mass spectrometry. A total of 117 proteins were identified from the three groups that had spectral counts greater than 1 (see Table, Supplemental Digital Content 1, at https://links.lww.com/SHK/A153, which contains the accession number and spectral counts for all the proteins identified in the BALF). Those proteins were subjected to Ingenuity Pathways Knowledge Base analysis to assign functional and canonical pathways. Table 2 shows the seven functional and canonical pathways containing the largest number of identified proteins. This analysis indicates that a number of proteins present in BALF from rats with ALI are associated with pathways related to an immune or inflammatory response. Functional pathways included antigen presentation (23 proteins), humoral immune response (nine proteins), inflammatory response (30 proteins), and immune cell trafficking (23 proteins). Canonical pathways included the acute-phase response (24 proteins), the complement system (12 proteins), and the coagulation system (six proteins).
To determine if TAT-SNAP-23 treatment altered protein expression in BALF, the spectral counts of all 117 proteins were normalized to the total protein concentration. Of the 117 proteins, 78 proteins (67%) were reduced by at least 50% in the TAT-SNAP-23–treated rats, 32 proteins (28%) had similar spectral counts among the three groups, and six proteins (5%) showed at least a 50% increase in the spectral counts in TAT-SNAP-23–treated animals (see Table, Supplemental Digital Content 1, at https://links.lww.com/SHK/A153). Of the 78 proteins reduced in BALF from TAT-SNAP-23–treated rats, 12 proteins were components of the complement cascade, three proteins were components of the coagulation pathway, four were neutrophil granule proteins, and one was an acute-phase protein (Table 3). Validation of the proteomic data was performed by immunoblot analysis for two identified proteins, the acute-phase protein CRP and the complement protein C3. As shown in Figure 4, a reduction of CRP (Fig. 4A) and C3 (Fig. 4B) was observed in BALF from TAT-SNAP-23–treated animals compared with TAT-control or untreated ALI.
The major finding of the present study is that in vivo administration of a novel fusion protein containing a SNARE domain from SNAP-23 resulted in amelioration of injury in a neutrophil-dependent ALI rat model. Using a well-characterized rat model of neutrophil-dependent ALI induced by alveolar deposition of IC (3–7, 12, 13, 17–19), intravenous administration of that fusion protein inhibited the development of lung injury, as measured by a reduction in albumin leakage and by reduced alveolar hemorrhage and edema on histologic examination. In addition, proteomic analysis of BALF suggested that one mechanism by which inhibition of neutrophil exocytosis reduced ALI is via reduced activation of the complement and coagulation cascades. The data suggest that targeting SNARE protein-mediated exocytosis may be an effective therapeutic strategy for neutrophil-mediated diseases.
SNARE proteins mediate exocytosis in a variety of cell types, including neuronal cells, leukocytes, endothelial and epithelial cells (20, 21). We recently showed that incubation of isolated human neutrophils with a novel TAT-fusion protein containing the SNARE domain of SNAP-23 significantly inhibited stimulated exocytosis of secretory vesicles, gelatinase granules, and specific granules, but not azurophilic granules (11). That inhibition of exocytosis was associated with a reduction in phagocytosis-stimulated respiratory burst activity and with inhibition of TNF-α– and platelet-activating factor–induced priming of fMLF-stimulated superoxide release. Based on the ability of TAT-SNAP-23 to bind SNARE protein complexes in vitro, the mechanism of inhibition of exocytosis was proposed to be interference with SNARE protein coiled-coil formation (11). The current study provides evidence that TAT-SNAP-23 inhibited neutrophil exocytosis in vivo. Neutrophils obtained from BALF of TAT-SNAP-23–treated rats contained TAT-SNAP-23 and showed reduced plasma membrane expression of CD18. The increase in plasma membrane expression of CD18 in stimulated neutrophils results from exocytosis of gelatinase and specific granules (22, 23). In addition, quantitative proteomic analysis of BALF found reduced levels of several neutrophil granule proteins, including MPO, collagenase, and lysozyme.
As SNARE proteins are involved in protein trafficking in all cells, the possibility that TAT-SNAP-23 inhibited ALI through an action on cells other than neutrophils was considered. N-ethylmaleimide-sensitive factor, like SNAP-23, is another molecule involved in vesicular trafficking (21). Morrell et al. (24) showed that intravenous administration of a TAT-fusion protein containing an inhibitory peptide derived from N-ethylmaleimide-sensitive factor inhibited vascular endothelial cell exocytosis and blocked neutrophil infiltration into the peritoneum in a mouse model of inflammatory peritonitis. If TAT-SNAP-23 acted similarly, reduced neutrophil recruitment into the lungs would be predicted. The presence of similar numbers of neutrophils in the lungs and BALF of TAT-SNAP-23–treated and untreated animals in our study argues against an effect of TAT-SNAP-23 on vascular endothelial cell exocytosis. In addition, the presence of similar levels of cytokines, including TNF-α and IL-6, in BALF of all three groups of rats with alveolar IC deposition indicates that administration of TAT-SNAP-23 had no effect on cytokine production and release by resident macrophages, lung parenchymal cells, and infiltrating neutrophils. In vitro studies performed with isolated human neutrophils exposed to BALF from all three groups of rats showed that the reduced plasma membrane expression of CD18 in rat neutrophils derived from BALF was not due to the absence of adequate proinflammatory stimuli. Taken together, our data suggest that TAT-SNAP-23 inhibits development of ALI by blocking exocytosis of neutrophils that have infiltrated into the lung in response to the inflammatory response initiated by IC deposition.
The proteomic analysis of BALF identified complement and coagulation proteins, as well as CRP, and the neutrophil-derived proteins MPO, collagenase, gelatinase, and vitamin D–binding protein in rats with ALI. Quantitative analysis found that a number of proteins in the complement and coagulation systems were reduced in BALF from TAT-SNAP-23–treated animals. Complement activation is necessary for neutrophil recruitment and IC-induced ALI in the rodent model used in this study (3–7, 17–19, 25). Complement activation and generation of C5a result in activation of alveolar macrophages stimulating the release of proinflammatory cytokines such as TNF-α, IL-1, and chemokines such as macrophage inflammatory protein-2 and mouse keratinocyte chemoattractant (12, 13, 19). In addition, C5a and the membrane attack complex (C5b-9) are involved in upregulation of endothelial cell adhesion molecules, which participate in neutrophil migration into the lung parenchyma (17). This proinflammatory environment results in enhanced neutrophil recruitment and activation leading to tissue damage. It has been shown that intratracheal administration of a neutralizing antibody to C5a or inducing the IC-ALI in a C5 knockout mice resulted in attenuation of neutrophil infiltration and amelioration of lung injury (26–28). Our data show that blocking neutrophil exocytosis by administration of TAT-SNAP-23 results in reduction of complement components and amelioration of lung injury. Previous reports showed that intratracheal injection of neutrophil granule components produced ALI (8) and that specific granule contents, including serine proteases, matrix metalloproteinases, LL-37, lactoferrin, defensins, and azurocidin, may contribute to ALI (2). An interaction of those granule components with the complement cascade has not been examined. Our findings suggest the novel hypothesis that neutrophil exocytosis participates in ALI through release of granule components that, among other actions, regulate complement activation. Neutrophils and alveolar macrophages cleave C5 to generate active C5a, and serine proteases participate in C5a generation (29, 30). Our findings suggest that inhibition of neutrophil exocytosis not only limits the release of granule components capable of directly inducing tissue injury, but also prevents activation of an amplification loop, which results from activation of the complement cascade.
The ability of BALF from both treated and untreated groups to stimulate equal neutrophil exocytosis in vitro indicates IC deposition results in the generation of equivalent amounts of neutrophil stimuli. We have reported previously that TNF-α is a potent stimulus of neutrophil exocytosis (11). The presence of substantial amounts of TNF-α in TAT-SNAP-23–treated animals supports the conclusion that TAT-SNAP-23 inhibited ALI by blocking neutrophil exocytosis. Although the mechanism by which exocytosis contributed to complement activation was not examined in our study, Camous et al. (31) recently described a novel mechanism by which the alternative complement pathway is activated on the surface of neutrophils. Neutrophils primed by TNF-α and stimulated by fMLF demonstrated increased plasma membrane expression of properdin released from granules, which activated the alternative complement pathway resulting in plasma membrane deposition of C3. We reported previously that properdin is a component of neutrophil-specific granules (22). Ritis et al. (32) reported that activation of neutrophil C5a receptors triggers activation of the extrinsic coagulation cascade via release of tissue factor. It has been shown that high levels of C5a result in upregulation of factor H, a key complement inhibitor, suggesting that C5a controls its own generation to maintain homeostasis (33). Flierl et al. (34) reported that neutralization of C5a via systemic infusion of anti-C5a resulted in reduction of factor H and amelioration of trauma-induced ALI. Similarly, in the present study, inhibition of neutrophil exocytosis resulted in a reduction in both C5 and factor H and amelioration of IC-induced ALI. Thus, neutrophil degranulation might contribute to complement activation leading to tissue damage through a mechanism that remains to be determined.
A proteomic study performed on BALF of patients with acute respiratory distress syndrome (ARDS) found higher levels of complement components C3, C4, and C9 compared with normal subjects (35). In the same study, BALF from ARDS patients showed increasing expression of C3 as the disease progressed (35). In addition, this study showed increased S100 proteins (S100 A8 and S100 A9) at day 1 in BALF of ARDS patients compared with control subjects. We reported previously that S100 A8 and S100 A9 proteins are components of neutrophil-specific and gelatinase granules (22). Those reports and our data support a pathophysiologically relevant link between neutrophil exocytosis and activation of the complement and coagulation cascade following IC deposition. Understanding the mechanisms involved in regulation of neutrophil exocytosis may lead to new therapeutic strategies to improve the outcome of ALI.
The authors thank Karen Brinkley, Junyi Le, Shweta Tandon, Timmothy M. Creed, Terri Manning, and Daniel Wilkey for their expert technical help.
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