Abstract
Rationale: Acetylated Pro-Gly-Pro (Ac-PGP) is an endogenous degradation product of extracellular collagen that binds to leukocyte-expressed chemoattractant receptor CXCR2. Although certain agents that block CXCR2-mediated signaling protect against experimental sepsis, the roles of Ac-PGP and CXCR2 in sepsis are unclear.
Objectives: To investigate the role of Ac-PGP and its receptor, CXCR2, in murine models of cecal ligation and puncture (CLP)–induced polymicrobial sepsis and organ injury.
Methods: The impact of in vivo Ac-PGP treatment on animal survival after induction of experimental sepsis was assessed. Vital organ inflammation and immune cell apoptosis were evaluated by histology, and the modulation of proinflammatory cytokine production and bactericidal activity by Ac-PGP in mouse and human blood leukocytes was measured.
Measurements and Main Results: The activation of CXCR2 by tripeptide agonist Ac-PGP dramatically improved survival in three experimental sepsis models. Ac-PGP elicited bactericidal activity via the generation of hydrogen peroxide, inhibited lung inflammation, and reduced immune cell apoptosis. Fluorescein isothiocyanate–labeled PGP bound directly to CXCR2, and the protective effect of Ac-PGP in sepsis was abolished in CXCR2-deficient mice. Ac-PGP treatment enhanced the production of type 1 cytokines (IFN-γ and IL-12) but inhibited the production of proinflammatory cytokines (tumor necrosis factor [TNF]-α, IL-1β, and IL-6) in vivo. In vitro, Ac-PGP directly increased IFN-γ production and decreased the LPS-stimulated production of TNF-α by mouse splenocytes and human leukocytes. Furthermore, direct treatment of LPS-stimulated splenocytes with IFN-γ resulted in diminished secretion of TNF-α and IL-6.
Conclusions: CXCR2 and Ac-PGP are thus novel target and starting molecules, respectively, for the development of therapeutic agents against sepsis.
The role of acetylated Pro-Gly-Pro (Ac-PGP), an endogenous degradation product of extracellular collagen, in the pathophysiology of sepsis is unclear.
Ac-PGP, an endogenous degradation product of extracellular collagen, is efficacious against polymicrobial sepsis–induced organ injury and lethality by activating CXCR2.
Sepsis is a complex clinical syndrome common in major intensive care units that results from a harmful or damaging host response to infection (1). It is estimated that more than 200,000 deaths have been caused by sepsis in the United States (2). Moreover, the incidence of severe sepsis and septic shock has dramatically increased during the past two decades (3). Because the mortality of severe sepsis and septic shock is so high, the development of new and efficient therapeutic agents against sepsis is critical (1, 3).
Sepsis-induced mortality is closely associated with failed regulation of the inflammatory response, which is caused by substantial impairment of the innate immune system (4–6). It has also been shown that excessive apoptosis of lymphocytes occurs during sepsis, which results in multiorgan failure (7, 8). During the early stage of sepsis, several proinflammatory cytokines including tumor necrosis factor (TNF)-α and IL-1β are dramatically increased, which causes systemic dysfunction of the host immune system (9–11). In addition, the crucial role that invasive bacteria play in the progression and pathogenesis of sepsis is well known. Therefore, it is reasonable to focus on developing an effective way to control bacteria by enhancing phagocyte activity, inhibiting the production of proinflammatory cytokines, and blocking immune cell apoptosis.
CXCR2 is a classic chemoattractant receptor and is found on phagocytic cells such as neutrophils, monocytes, and macrophages (12). CXCL8 binds to CXCR2 in humans (13, 14). Although there is no mouse homolog of CXCL8, its functions are replaced by other mouse chemokines, including CXCL1 (KC, keratinocyte-derived chemokine). Past studies have indicated that the in vitro activation of CXCR2 by CXCL8 or CXCL1 induces leukocyte chemotactic migration in neutrophils and monocytes (15, 16). Clinical data show that the expression of CXCR2, but not of CXCR1, is down-regulated by 50% in the neutrophils of patients with sepsis compared with normal control subjects (17, 18). The down-regulation of CXCR2 leads to impaired neutrophil activity triggered by CXCR2 ligands (17, 18). A previous report showed that the administration of CXCR2-blocking antibodies increased the survival rate in an experimental sepsis model (19). Deficiency of CXCR2 also was shown to increase the survival rate (19). It was also reported that exposure to high systemic levels of CXCL8 causes endothelial dysfunction and loss of the normal anticoagulant state of the endothelium (20). Kuliopulos and colleagues demonstrated that administration of the novel pepducin ligand derived from the CXCR2 intracellular region (x1/2pal-3: pal-RTLFKAHMGQKHR), which blocks CXCR2-mediated signaling by CXCL8 or CXCL1, dramatically increased the survival of cecal ligation and puncture (CLP)–septic mice (21). From these reports, CXCR2 and its cognate ligands are clearly important proinflammatory response–inducing targets.
Acetylated Pro-Gly-Pro (Ac-PGP) is an endogenous degradation product of the extracellular matrix that binds CXCR2 and stimulates human polymorphonuclear cells (22–24). Here, we demonstrate the therapeutic effects of the CXCR2 agonist Ac-PGP in preventing lethal progression to severe sepsis after microbial infection.
Male wild-type albino Institute of Cancer Research Center (ICR) mice were used for evaluation of the therapeutic effects of Ac-PGP; IFN-γ–deficient mice (C57BL/6 background) were kindly donated by Y. K. Kim (Pohang University of Science and Technology, Pohang, South Korea). CXCR2-deficient mice (C57BL/6 background) were purchased from Jackson Laboratory (Bar Harbor, ME). All experiments involving animals adhered to guidelines and received the approval of the Institutional Review Committee for Animal Care and Use at Dong-A University (Busan, South Korea). For CLP, mice were anesthetized with pentothal sodium (50 mg/kg, intraperitoneal), and a small abdominal midline incision was made to expose the cecum. The cecum was then ligated below the ileocecal valve, punctured twice through both surfaces (or once for the measurement of cytokine production) with a 22-gauge needle, and the abdomen was closed. Sham CLP mice were subjected to the same procedure, but without ligation of the cecum. Survival was monitored once daily for 10 days. For the LPS or Escherichia coli model, E. coli (1 × 109 cells per mouse) or LPS (60 mg/kg) was injected intraperitoneally. Survival was monitored once daily for 10 days.
Unlabeled Ac-PGP and fluorescein isothiocyanate (FITC)–labeled PGP (FITC–6-aminocaproic acid–PGP), with purity greater than 99.6%, were synthesized by Anygen Company (Gwangju, South Korea). The PGP ligand-binding assay was performed as described previously (25). Briefly, RBL-2H3 cells or murine CXCR2 (mCXCR2)–expressing RBL-2H3 cells (gift from R. Pardi, San Raffaele University School of Medicine, San Raffaele, Italy) were seeded at 1 × 105 cells per well in a 24-well plate and cultured overnight. After treating the cells with blocking buffer (33 mM HEPES [pH 7.5], 0.1% bovine serum albumin in RPMI) for 2 hours, 50 μM FITC-labeled PGP was added to the cells in binding buffer (phosphate-buffered saline [PBS] containing 0.1% bovine serum albumin) in the absence or presence of unlabeled Ac-PGP (10 mM). The mix was then incubated for 3 hours at 4°C with continuous agitation. The samples were then washed five times with ice-cold binding buffer, and 300 μl of fixation buffer (PBS containing 5% fetal bovine serum, 0.1% sodium azide, 0.1% paraformaldehyde) was added to each well. Samples were acquired on a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences, San Jose, CA) was used for data analysis.
Mice were subjected to CLP surgery and given PBS or Ac-PGP at a dose of 17.5 mg/kg 2 hours later. The mice were killed 24 hours after surgery, after which their lungs were fixed, sectioned, and stained with hematoxylin and eosin for morphological analysis.
Twenty-four hours after CLP, peritoneal lavage fluids were collected and cultured overnight on blood-agar base plates (Trypticase soy agar deeps; BD Biosciences, San Jose, CA) at 37°C. The number of CFUs was determined as previously described (26).
The extent of pulmonary edema was quantified by evaluating the wet-to-dry (W/D) weight ratio of the lung as previously described (27). Whole harvested wet lungs were weighed and then placed in an oven for 48 hours at 60°C. The dry weight was then measured and the W/D weight ratio was calculated.
The TUNEL (terminal deoxynucleotidyltransferase [TdT]–mediated dUTP nick end labeling) assay was performed with paraffin-embedded tissue sections, which were first deparaffinized according to a standard histological protocol. The sections were then permeabilized with Triton X-100 at 4°C for 2 minutes and flooded with TdT enzyme and digoxigenin-dUTP reaction buffer (TUNEL) reagent for 60 minutes at 37°C. We also performed immunohistochemistry for cleaved caspase-3 in paraffin-embedded tissue sections that were first deparaffinized according to a standard histological protocol as previously described (28).
Mouse neutrophils were isolated from peripheral blood, using Histopaque-1077 solution (Sigma-Aldrich, St. Louis, MO) as previously described (29). Freshly isolated neutrophils from normal mice were stimulated with various concentrations of Ac-PGP for 10 minutes in the presence of cytochalasin B (5 μM). To investigate the role of CXCR2, neutrophils were preincubated with several concentrations of SB225002 or vehicle (dimethyl sulfoxide) for 30 minutes before addition of Ac-PGP (20 μM) for 10 minutes. H2O2 in the supernatant was measured with an H2O2 assay kit (Molecular Probes, Eugene, OR).
The Institutional Review Board at Ajou University Hospital (Suwon, South Korea) approved all human subject protocols, and informed consent was obtained for all blood donations. Peripheral blood was collected from healthy donors, and human peripheral mononuclear cells were separated on a Histopaque-1077 gradient. Human neutrophils were isolated according to the standard procedures of dextran sedimentation, hypotonic lysis of erythrocytes, and use of a lymphocyte separation medium gradient as described previously (29). The isolated human leukocytes were used promptly.
Neutrophil bactericidal activity was measured according to the method of Yan and colleagues (26). Neutrophils were incubated at 37°C on 13-mm plastic coverslips in 60-mm plastic culture dishes (1 × 106 neutrophils per coverslip) for 1 hour. Nonadherent cells were removed with PBS. Adherent neutrophils were incubated with 106 opsonized E. coli for 1 hour. After washing away the unengulfed E. coli, the number of viable bacteria in the neutrophils was determined before and after incubation with several concentrations of Ac-PGP or vehicle for 1 hour. The percentage of bacteria killed was calculated as 100 × (1 – number of CFU after Ac-PGP stimulation/number of CFU before Ac-PGP stimulation).
To measure the production of CLP-induced cytokines in peritoneal lavage fluids, mice were given Ac-PGP 2, 14, 26, and 38 hours after CLP. The peritoneal lavage fluids were collected at various times between 4 and 72 hours after CLP, and the cytokines present in the peritoneal fluid were measured by ELISA (e-Bioscience, San Diego, CA).
Mouse splenocytes (3 × 106 cells/0.3 ml) were placed in RPMI 1640 medium containing 5% fetal bovine serum in 96-well plates and kept in a 5% CO2 incubator at 37°C. The splenocytes were then incubated with LPS (100 ng/ml) for 24 hours in the presence or absence of Ac-PGP, respectively. LPS (100 ng/ml) was added to the cells 30 minutes later, after which the cell-free supernatants were collected, centrifuged, and measured for TNF-α, IL-6, and IFN-γ by ELISA (BD Biosciences Pharmingen) according to the manufacturer's instructions. Regarding the effects of IFN-γ on the production of proinflammatory cytokines by LPS, mouse splenocytes (5 × 105 cells/ml) were stimulated with LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO) with or without several concentrations (1, 10, 100, and 1,000 IU/ml) of IFN-γ (R&D Systems, Minneapolis, MN). TNF-α and IL-6 levels in the supernatants were measured by ELISA (BD Biosciences Pharmingen) 20 hours after the incubation.
Survival data were analyzed by log-rank test. All other data were evaluated by analysis of variance. The Bonferroni test was used for post hoc comparisons and statistical significance was set a priori at P < 0.05.
The therapeutic effect of Ac-PGP on experimental sepsis was investigated in CLP models, using ICR mice. The survival of mice was monitored for 10 days after CLP. Two days after CLP, only 20% of untreated wild-type mice survived (Figure 1A). However, subcutaneous administration of Ac-PGP 2 hours after CLP dramatically increased the survival rate in a dose-dependent manner (Figure 1A). Injection of Ac-PGP (7.5, 12.5, or 17.5 mg/kg) dramatically increased mouse survival compared with the PBS-injected controls (Figure 1A). In terms of injection frequency, survival was greatly improved when Ac-PGP (17.5 mg/kg) was injected 2 hours post-CLP and at 12-hour intervals three or four additional times (Figure 1B). Given these results, our subsequent experiments were performed in CLP mice, using Ac-PGP (17.5 mg/kg) beginning 2 hours after CLP and at 12-hour intervals three additional times. When Ac-PGP was injected 10 hours post-CLP, the therapeutic effect was still shown (Figure 1C).
Figure 1. Therapeutic effects of acetylated Pro-Gly-Pro (Ac-PGP) in sepsis models. (A) Various doses of Ac-PGP were injected subcutaneously four times into cecal ligation and puncture (CLP)–induced septic mice 2, 14, 26, and 38 hours after CLP. (B) CLP mice were given zero, one, two, three, four, or five injections of Ac-PGP (17.5 mg/kg, subcutaneous) 2, 14, 26, 38, and 50 hours after CLP. (C) Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into CLP mice 10, 22, 34, and 48 hours after CLP. (D and E) Phosphate-buffered saline (PBS) or Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into mice 2, 14, 26, and 38 hours after intraperitoneal injection of either (D) 1 × 109 Escherichia coli or (E) LPS (60 mg/kg). (F) SB225002 (17.5 mg/kg) or vehicle (dimethyl sulfoxide) was injected subcutaneously 2 hours before CLP. Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into mice 2, 14, 26, and 38 hours after CLP. (G) Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into CLP mice 2, 14, 26, and 38 hours after CLP in wild-type or CXCR2-deficient mice. (H) Fluorescein isothiocyanate (FITC)–labeled PGP (50 μM) was incubated for 3 hours at 4°C with vector- or murine CXCR2 (mCXCR2)–expressing RBL-2H3 cells in the absence or presence of excess unlabeled Ac-PGP (10 mM). Binding of FITC-labeled PGP was determined by fluorescence-activated cell-sorting analysis. KO = knockout; NT = no treatment. Results are representative of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, comparing Ac-PGP versus PBS controls (A–F). Sample size: n = 16–24 mice per group (A–G).
[More] [Minimize]The effects of Ac-PGP in other sepsis mouse models were also evaluated. Therapeutic administration of Ac-PGP significantly enhanced survival in mice inoculated with E. coli (1 × 109 cells per mouse) compared with vehicle-treated controls (Figure 1D). Ac-PGP also reduced the incidence of fatal sepsis in mice injected intraperitoneally with LPS at 60 mg/kg (Figure 1E).
We next asked whether the antiseptic activity of Ac-PGP acts via its reported receptor, CXCR2. In mice pretreated with a CXCR2-selective antagonist (SB225002) (30), Ac-PGP failed to improve survival in the CLP model (Figure 1F). Treatment with Ac-PGP also failed to improve the survival of CXCR2-deficient septic mice (Figure 1G).
To examine direct binding of Ac-PGP to mCXCR2, we synthesized FITC-labeled PGP (FITC–6-aminocaproic acid–PGP) and confirmed its bioactivity by neutrophil chemotaxis (data not shown). Labeled PGP bound specifically to mCXCR2+ RBL-2H3 transfectants but not to empty vector controls (Figure 1H). Addition of 200-fold excess unlabeled Ac-PGP competitively inhibited the binding of FITC-labeled PGP to mCXCR2+ cells. We also found that Ac-PGP induced chemotactic migration of mCXCR2+ RBL-2H3 transfectants but not of empty vector controls (data not shown). Together, these results indicate that Ac-PGP acts specifically through CXCR2 to improve survival in experimental sepsis.
Dysfunction of vital organs such as lung and liver is associated with mortality after sepsis (31). Hematoxylin and eosin staining was performed to examine morphological changes. As shown in Figure 2A, CLP caused dramatic inflammation of the liver and lungs. Liver tissues in CLP mice showed more apoptosis, which was reduced by the administration of Ac-PGP. Lungs of CLP mice showed severe alveolar congestion and displayed extensive formation of thrombotic lesions, which were also dramatically inhibited by Ac-PGP (Figure 2B). CLP caused an increase in the lung W/D weight ratio, an additional indicator of acute lung inflammation, which was significantly decreased by administration of Ac-PGP (Figure 2C). Immune cell apoptosis is also accompanied by sepsis (1, 31). As predicted, CLP caused thymic and splenic cell apoptosis, which was inhibited by administration of Ac-PGP (Figures 2A and 2D). The majority of apoptosis in the thymus or spleen of CLP mice occurred in the cortex, which is populated by immature cells. An important morphological change to the apoptotic cells of CLP mice versus those of sham or Ac-PGP mice was the presence of small compacted nuclei (pyknosis) with multiple nuclear fragments (apoptotic bodies) in apoptotic lymphocytes (Figure 2A). DNA fragmentation analysis (TUNEL) confirmed that Ac-PGP inhibited the apoptosis of splenocytes and thymocytes induced by CLP (Figure 2D).
Figure 2. Effect of acetylated Pro-Gly-Pro (Ac-PGP) on cecal ligation and puncture (CLP)–induced inflammation of vital organs and immune cell apoptosis. (A and B) Phosphate-buffered saline (PBS) or Ac-PGP (17.5 mg/kg, subcutaneous) was administered 2 and 14 hours after CLP. The mice were killed 24 hours after surgery. The livers, lungs, spleens, and thymus were stained with hematoxylin and eosin (A, original magnifications, ×100 and ×400). The data are representative of eight mice per group. (B) Acute lung injury score and thrombi and hyaline membrane score were measured. (C) Ac-PGP (17.5 mg/kg, subcutaneous) was administered 2 and 14 hours after CLP, and the lungs were used to measure the wet-to-dry (W/D) weight ratio 24 hours after CLP in ICR mice. (D) Ac-PGP (17.5 mg/kg) was injected subcutaneously two times into CLP mice 2 and 14 hours post-CLP. The spleen and thymus, which were collected 24 hours after sham CLP, CLP plus PBS, or CLP plus Ac-PGP administration, were subjected to DNA fragmentation analysis (terminal deoxynucleotidyltransferase [TdT]–mediated dUTP nick end labeling; TUNEL) (left) (original magnification, ×400). TUNEL-positive cells were counted (right). Data are expressed as means ± standard error (B–D, n = 8). *P < 0.05; **P < 0.01; ***P < 0.001.
[More] [Minimize]Past studies have shown that CLP-induced lethality is correlated with bacterial colony counts in the peritoneal fluid (1, 31). Here, we tested the effect of Ac-PGP on bacterial clearance from peritoneal fluid. Administration of Ac-PGP dramatically reduced the intraperitoneal bacterial colony count by 82.1% at 24 hours after CLP (Figure 3A). The effect of Ac-PGP on bactericidal activity in vitro was tested with mouse neutrophils. Mouse neutrophils were allowed to ingest E. coli for 1 hour, followed by stimulation with various concentrations (0.1–50 μM) of Ac-PGP for 20 minutes. Stimulation of mouse neutrophils with Ac-PGP markedly enhanced bactericidal activity in a dose-dependent manner (Figure 3B). Inactive control peptide Ac-PGG (acetylated Pro-Gly-Gly) failed to enhance bactericidal activity (Figure 3B). It is well known that reactive oxygen species such as hydrogen peroxide are major molecules involved in bactericidal activity (32). Here, we tested the effect of Ac-PGP on the production of hydrogen peroxide in mouse neutrophils. Stimulation of mouse neutrophils with various concentrations of Ac-PGP strongly enhanced the production of hydrogen peroxide in a concentration-dependent manner (Figure 3C). To test the role of CXCR2 in Ac-PGP–induced hydrogen peroxide production, neutrophils were pretreated with a CXCR2 antagonist (SB225002) before Ac-PGP treatment. Ac-PGP-stimulated hydrogen peroxide production was almost completely inhibited by the CXCR2 antagonist (Figure 3D).
Figure 3. Acetylated Pro-Gly-Pro (Ac-PGP) stimulates bactericidal activity in vivo and in vitro. (A) Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into cecal ligation and puncture (CLP)–induced septic mice 2 and 14 hours after CLP. Peritoneal lavage fluids collected 24 hours after sham CLP, CLP plus phosphate-buffered saline (PBS), or CLP plus Ac-PGP administration were cultured overnight on blood-agar base plates at 37°C; the number of CFUs was then counted. (B) Adherent neutrophils were incubated with 106 opsonized Escherichia coli for 1 hour, and several concentrations of Ac-PGP or inactive control Ac-PGG (acetylated Pro-Gly-Gly) were added for 1 hour. The number of viable bacteria in the neutrophils was then determined. (C) Mouse neutrophils were stimulated with vehicle (PBS) or Ac-PGP (0.1–50 μM) for 15 minutes. (D) Several concentrations of SB225002 were added 5 minutes before the addition of Ac-PGP (20 μM). The amount of H2O2 produced was measured from the supernatant, using an H2O2 assay kit (Molecular Probes). Data are expressed as means ± standard error (A, n = 4; B–D, n = 8). *P < 0.05; **P < 0.01; ***P < 0.001. PMNs = polymorphonuclear cells.
[More] [Minimize]The effect of Ac-PGP on CLP-induced proinflammatory cytokines TNF-α, IL-1β, and IL-6 in peritoneal fluid was evaluated 4 to 72 hours after CLP. CLP induced dramatic increases in TNF-α, IL-1β, and IL-6 within 24 hours (Figures 4A–4C). Ac-PGP injection blunted the proinflammatory cytokine response in the CLP model, and resulted in a significant decrease in TNF-α, IL-1β, and IL-6 levels (Figures 4A–4C). To evaluate the effects of Ac-PGP on proinflammatory cytokine production induced by LPS, the in vitro production of TNF-α and IL-6 by mouse splenocytes was measured. The direct release of TNF-α and IL-6 from mouse splenocytes triggered by LPS was inhibited by in vitro Ac-PGP treatment (Figures 4D and 4E). Interestingly, CXCL1, the mouse counterpart to CXCL8, failed to inhibit LPS-stimulated TNF-α and IL-6 production (Figures 4D and 4E).
Figure 4. Role of acetylated Pro-Gly-Pro (Ac-PGP) in the production of proinflammatory cytokines. (A–C) Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into cecal ligation and puncture (CLP)–induced septic mice, after which the levels of cytokines in the peritoneal fluids were measured at various time points after CLP. (A) Tumor necrosis factor (TNF)-α; (B) IL-1β; (C) IL-6. Data are expressed as means ± standard error (n = 8). *P < 0.05; **P < 0.01 compared with CLP plus phosphate-buffered saline (PBS). (D and E) Mouse splenocytes were preincubated with PBS, Ac-PGP (1, 5, 10, and 20 μM), or CXCL1 (50 and 100 ng/ml) for 30 minutes and then stimulated with PBS or LPS (100 ng/ml) for 3 hours. The levels of (D) TNF-α and (E) IL-6 were measured by ELISA. Data are presented as means ± standard error (D and E, n = 4). *P < 0.05; **P < 0.01; ***P < 0.001.
[More] [Minimize]The effect of Ac-PGP on CLP-induced helper T-cell type 1 cytokine levels in peritoneal fluid was also measured from 4 to 72 hours after CLP. The levels of helper T-cell type 1 cytokines (IFN-γ, IL-12p70, and IL-2) were significantly increased on injection of Ac-PGP (Figures 5A–5C). Because we found that administration of Ac-PGP enhanced IFN-γ production in vivo, we tested the direct effect of Ac-PGP on the production of IFN-γ by splenocytes. As shown in Figure 5D, activation of splenocytes with Ac-PGP significantly enhanced the production of IFN-γ stimulated by LPS. CXCL1 had little effect on LPS-stimulated IFN-γ production (Figure 5D). We next asked whether IFN-γ itself suppressed production of TNF-α and IL-6 by LPS-stimulated mouse splenocytes. Interestingly, IFN-γ significantly attenuated the LPS-induced production of TNF-α and IL-6 by splenocytes (Figures 5E and 5F).
Figure 5. Acetylated Pro-Gly-Pro (Ac-PGP)–induced antiseptic activity is mediated by IFN-γ. (A–C) Ac-PGP (17.5 mg/kg) was injected subcutaneously four times into cecal ligation and puncture (CLP)–induced septic mice, after which cytokine levels in the peritoneal fluids were measured at various time points after CLP. (A) IFN-γ; (B) IL-12; (C) IL-2. Data are expressed as means ± standard error (n = 8). *P < 0.05; **P < 0.01 compared with CLP plus phosphate-buffered saline (PBS). (D) Mouse splenocytes were preincubated with PBS, Ac-PGP (1, 5, 10, and 20 μM), or CXCL1 (50 and 100 ng/ml) for 30 minutes and then stimulated with PBS or LPS (100 ng/ml) for 3 hours. The levels of IFN-γ were measured by ELISA. (E and F) Mouse splenocytes were stimulated with LPS (100 ng/ml) in the absence or presence of IFN-γ (1, 10, 100, and 1,000 U/ml) for 3 hours. The levels of (E) TNF-α or (F) IL-6 were measured by ELISA. Data are presented as means ± standard error (D–F, n = 4). **P < 0.01; ***P < 0.001. (G and H) Wild-type C57BL/6 or IFN-γ−deficient mice were subcutaneously injected with Ac-PGP (17.5 mg/kg) or PBS two times 2 and 14 hours after CLP. (G) The spleen, which was collected 24 hours after sham CLP, CLP plus PBS, or CLP plus Ac-PGP administration, was used for the TUNEL assay (left) (original magnification, ×400). TUNEL-positive cells were counted (right). Data are expressed as means ± standard error (G, n = 8). *P < 0.05; **P < 0.01; ***P < 0.001. (H) The spleen from the mice was used for immunofluorescence staining with fluorescein isothiocyanate (FITC)–conjugated cleaved caspase-3 antibody (original magnification, ×100). The data are representative of eight mice per group (H). KO = knockout.
[More] [Minimize]We next asked whether IFN-γ plays a role in Ac-PGP–mediated protection against immune cell apoptosis, which is associated with mortality during sepsis (1, 31). CLP triggered substantial splenocyte apoptosis by DNA fragmentation analysis (TUNEL) (Figure 5G). Whereas administration of Ac-PGP dramatically inhibited CLP-induced apoptosis in wild-type mice, Ac-PGP failed to inhibit CLP-induced immune cell apoptosis in IFN-γ–deficient mice (Figure 5G). Similar results were observed when caspase-3 activation was used as a marker of splenocyte apoptosis (Figure 5H). CLP caused substantial caspase-3 activation in splenocytes that was inhibited by in vivo treatment with Ac-PGP (Figure 5H). In IFN-γ knockout mice, however, treatment with Ac-PGP had no effect (Figure 5H). Thus, our data implicate IFN-γ as a key cytokine contributor to the protective effects of Ac-PGP in experimental sepsis.
Because we observed that Ac-PGP strongly stimulated the antibacterial activity of mouse neutrophils, we tested the bactericidal effects of Ac-PGP on human neutrophils. Ac-PGP had a dose-dependent effect on bacterial killing by human neutrophils: as little as 100 nM Ac-PGP significantly enhanced the bactericidal activity, and treatment with 20 μM Ac-PGP resulted in nearly complete killing of added opsonized E. coli (Figure 6A).
Figure 6. Acetylated Pro-Gly-Pro (Ac-PGP) enhances bactericidal activity, decreases tumor necrosis factor (TNF)-α production, and increases IFN-γ release from human neutrophils and peripheral blood mononuclear cells (PBMCs). (A) Adherent human neutrophils were incubated with 106 opsonized Escherichia coli for 1 hour, and several concentrations of Ac-PGP were added for 1 hour. The number of viable bacteria in the neutrophils was then determined. (B and D) Human neutrophils or (C and E) PBMCs were preincubated with phosphate-buffered saline (PBS), Ac-PGP (1, 5, 10, and 20 μM), or CXCL8 (50 and 100 ng/ml) for 30 minutes and then stimulated with PBS or LPS (100 ng/ml) for 3 hours. The levels of (B and C) TNF-α and (D and E) IFN-γ were measured by ELISA. Data are presented as means ± standard error (A–E, n = 4). *P < 0.05; **P < 0.01; ***P < 0.001. PMN = polymorphonuclear cells.
[More] [Minimize]We next examined the effects of Ac-PGP on cytokine production by human neutrophils and peripheral blood mononuclear cells (PBMCs). Similar to mouse splenocytes, the production of TNF-α by LPS-stimulated human neutrophils or PBMCs was significantly inhibited by in vitro treatment with Ac-PGP (Figures 6B and 6C), whereas the production of IFN-γ was significantly enhanced (Figures 6D and 6E). Interestingly, the human CXCR2 chemokine agonist CXCL8 had little effect on LPS-stimulated TNF-α or IFN-γ production (Figures 6B–6E). Thus, Ac-PGP improved the antiseptic activities of both human and mouse leukocytes.
Despite aggressive treatment, mortality due to sepsis remains high (1, 31). An important approach to controlling sepsis is the neutralization of endotoxin, although its effectiveness is controversial (31). Other approaches to treating sepsis include targeting proinflammatory mediators such as TNF-α and IL-1β (1, 27). In this study, we demonstrate that the administration of Ac-PGP after induction of sepsis by CLP effectively prevented CLP-induced lethality in mice via multiple therapeutic pathways: (1) enhanced bactericidal activity mediated in part by increased secretion of IFN-γ; (2) an antiinflammatory effect via diminished release of proinflammatory TNF-α, IL-1β, and IL-6; and (3) an antiapoptotic effect on immune cells. We also clearly demonstrate that the therapeutic effect of Ac-PGP requires tripeptide engagement with CXCR2, because pretreatment of mice with CXCR2-specific antagonist (SB225002) abolished the efficacy of Ac-PGP in CLP-induced sepsis. Furthermore, the therapeutic effect of Ac-PGP against experimental sepsis was also completely absent in CXCR2-deficient mice, thus indicating that Ac-PGP acts via CXCR2 to prevent septic mortality. We also demonstrated that FITC-labeled PGP binds specifically to mCXCR2+ cells (Figure 1H). Together, these results indicate that Ac-PGP acts through CXCR2 to elicit antiseptic activity.
The bacterial colony count dramatically increases during the progression of sepsis, and controlling bacterial load is critical to its treatment. Ac-PGP significantly enhanced bactericidal activity in vivo (Figure 3A) and strongly stimulated mouse and human neutrophil bactericidal activity in vitro (Figures 3B and 6A), likely through enhanced production of the toxic intracellular mediator hydrogen peroxide (Figure 3C). Elevated systemic levels of proinflammatory cytokines cause generalized vasodilation and vascular leakiness, reduced tissue oxygenation, and leukocyte extravasation and associated tissue destruction, all of which contribute to lethal vital organ inflammation and failure in patients with severe sepsis (1). Here, we show that Ac-PGP inhibits the production of proinflammatory cytokines TNF-α, IL-1β, and IL-6 in peritoneal fluid after experimental sepsis (Figures 4A–4C), thus linking Ac-PGP–dependent enhanced survival with a reduction in deleterious proseptic cytokines.
We found that Ac-PGP, but not CXCL1, an alternate CXCR2 ligand, inhibited the LPS-stimulated production of several proinflammatory cytokines such as TNF-α and IL-6 by mouse splenocytes (Figures 4D and 4E). We also observed that Ac-PGP, but not CXCL8, inhibited LPS-stimulated TNF-α production by human neutrophils and PBMCs (Figures 6B and 6C). These results suggest that CXCR2 can discriminate between different agonists, and that different agonists can trigger different cellular effector functions. Indeed, in a previous report we showed that the chemoattractant receptor formyl peptide receptor-like 1 can be differentially activated by two different agonists, resulting in different cellular responses (33). On the basis of the differential regulation of CXCR2 by alternate agonist ligands, we suggest that CXCR2 may mediate antiinflammatory as well as proinflammatory responses, depending on the agonist.
The role of IFN-γ in sepsis is controversial. Whereas IFN-γ is down-regulated in patients with severe sepsis, and treatment with IFN-γ significantly improved monocyte immune function and survival in immune-depressed patients with sepsis (4, 34), others showed paradoxically that IFN-γ inhibition led to improved survival in endotoxin shock or rat CLP models (35, 36). Dighe and colleagues previously hypothesized that a therapeutic approach designed to augment IFN-γ production may be beneficial for the treatment of sepsis, given that it stimulates phagocytic bactericidal activity (37). Even though a clinical study demonstrated that septic patients who have been treated with IFN-γ show improved clinical outcomes (34), the underlying mechanism remains unclear. In our study, we show that the administration of Ac-PGP enhanced IFN-γ production in a CLP model and in vitro, and that the antiseptic activity of Ac-PGP was decreased in IFN-γ–deficient mice (Figures 5G and 5H), suggesting that the therapeutic effect of Ac-PGP was mediated by IFN-γ. To uncover the mechanism of action of IFN-γ on Ac-PGP–stimulated antiseptic activity, we tested the direct effect of IFN-γ on the production of proinflammatory cytokines TNF-α and IL-6 in LPS-stimulated splenocytes. Interestingly, IFN-γ significantly diminished the production of TNF-α and IL-6 by LPS (Figures 5E and 5F). This result strongly indicates that IFN-γ can stimulate antiseptic activity not only by enhancing bactericidal activity but also by inhibiting the LPS-induced release of proinflammatory cytokines TNF-α and IL-6.
Elevated bronchoalveolar lavage fluid levels of Ac-PGP were reported in mice after exposure to aerosolized LPS, as well as in human patients with chronic obstructive pulmonary disease (24). Furthermore, chronic airway exposure to Ac-PGP over 12 weeks caused alveolar enlargement and right ventricular hypertrophy in mice, similar to the pathology observed in patients with chronic obstructive pulmonary disease (24). Thus, whereas Weathington and colleagues demonstrated pulmonary pathology after Ac-PGP treatment in otherwise healthy mice, we show here that Ac-PGP is efficacious in treating experimental sepsis. Because similar doses were used in both studies (∼10–20 mg/kg), the in vivo effects of Ac-PGP may therefore depend on the route of administration (aerosolized vs. subcutaneous), frequency of dosing (chronic, twice per week for 12 wk; vs. acute, one to five doses over a 50-h period), or the health of the animal. In sepsis, Ac-PGP likely exerts its effects through altering systemic cytokine levels (enhancing IFN-γ secretion and dampening TNF-α and IL-1β release) and enhancing bacterial killing, whereas airway-specific administration of Ac-PGP in healthy mice may serve primarily to recruit neutrophils to the lung, where they damage alveolar tissue. Thus, although initially characterized as a proinflammatory mediator of lung pathology, our data suggest that Ac-PGP may be a useful therapeutic agent with which to treat sepsis.
In conclusion, CXCR2 activation by Ac-PGP effectively prevented the progression to severe sepsis after microbial infection via enhanced bactericidal activity, increased IFN-γ production, and decreased proinflammatory cytokine release. Thus, therapeutics based on Ac-PGP–mediated activation of CXCR2 might represent a novel approach for the treatment of sepsis.
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* Present address: Cancer Research Institute, College of Medicine, Seoul National University, Seoul 110-744, South Korea.
Supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare, and Family Affairs, Republic of Korea (A090110), and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. 2009 0093198).
Contribution of the authors: Conception and design: S.K., Y.Y., Y.B.; analysis and interpretation: S.K., J.S., H.L., H.K., J.P.; drafting the manuscript for important intellectual content: B.Z., S.B., Y.B.
This article has an online supplement, which is available from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201101-0004OC on April 21, 2011
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