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Published online before print May 3, 2004
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,1
* Department of Cell and Molecular Biology, University of Rhode Island, Kingston;
Shock-Trauma Research Laboratories in the Division of Surgical Research, Department of Surgery, Rhode Island Hospital and Brown University School of Medicine, Providence; and
Department of Surgery, New York University and North Shore-Long Island Jewish Hospitals, Manhasset
1Correspondence: Division of Surgical Research, Aldrich 227, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903. E-mail: AAYALA{at}LIFESPAN.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: neutrophils • keratinocyte-derived chemokine • macrophage inflammatory protein-2 • mouse
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The clinical pathology of acute lung injury includes increased microvascular permeability, inflammation, pulmonary edema, and the accumulation of activated neutrophils in lung tissue and bronchoalveolar lavage fluid (BALF). It is the presence of activated neutrophils in the lungs of patients with inflammatory lung disease that has focused much current research on the role of neutrophils in the pathogenesis ALI [2 , 3 , 6 , 9 ]. In preliminary studies, we observed that mice treated with an antibody against a neutrophil cell-surface marker, Gr1, showed a significant reduction in inflammatory mediators and lung injury in our model of Hem priming for acute lung injury [10 ]. In addition, published, adoptive transfer studies infusing neutrophils from Hem mice into neutrophil-depleted mice showed a passive transfer of priming to the neutrophil-depleted mice via the infused neutrophils [4 , 11 ]. The induction of sepsis in these mice produced significant inflammatory lung injury and elevation of inflammatory mediators consistent with the nondepleted, control group [11 , 12 ].
As first responders to sites of inflammation driven by the systemic release of proinflammatory mediators, neutrophils contribute to the containment of pathogens and the resolution of the immune response [13 ]. Yet, the persistence of activated neutrophils in the lungs of trauma patients in the absence of a localized site of infection or injury is believed to directly and/or indirectly interfere with the successful resolution of the innate immune response to inflammation and to contribute to the pathogenesis of ALI [5 , 14 ].
A potent family of chemotactic polypeptides, chemokines, regulate neutrophil migration, activation, and extravasation into sites of inflammation. Chemokines are secreted by immune cells (macrophage, monocytes, and neutrophils) as well as nonimmune cells (endothelial cells) in response to an inflammatory stimuli. Interleukin-8 (IL-8), a human CXC chemokine, has been found elevated in neutrophil-infiltrated lung tissue, organ cavity fluids, and plasma from patients with acute inflammatory diseases [15 ]. Our laboratory and others have found IL-8 homologues, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-2 (MIP-2) elevated in the plasma and lung tissue of mice in response to Hem, which is a common complication in traumatic injury [12 ]. In addition, these chemokines appear to contribute to a state of priming of the phagocyte compartment predisposing the animal to increased susceptibility to organ injury, as we have observed in the lungs, in response to subsequent, infectious challenge [11 , 12 ]. Using neutralizing antibodies, we found anti-MIP-2 but not anti-KC produced a significant reduction in lung tissue and plasma IL-6, with antibody reduction in neutrophil influx and increased lung-tissue IL-10 content when administered immediately following Hem [11 , 12 ]. This has led to further questions regarding the regulatory functions of these two chemokines, as they are believed to share a common receptor, a homologue of the human CXC chemokine receptor 2 (CXCR2).
Based on the above data from our laboratory and others, we hypothesize that inhibition of CXCR2 signaling immediately following Hem will suppress KC and MIP-2 priming of neutrophils, thereby reducing the inflammatory injury observed following the subsequent induction of sepsis. For this purpose, we used antileukinate, a hexapeptide inhibitor of the human CXCR2 receptor, to inhibit CXCR2 signaling in our mouse model of Hem priming for ALI [16 , 17 ].
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice
Male C3H/HeN mice (Charles River Laboratories, Wilmington, MA), 7–9 weeks of age, were used for all experiments. Experiments described in this article were performed in accordance with the National Institutes of Health guidelines and approval from the Animal Use Committee of Rhode Island Hospital (Providence).
Mouse hemorrhage/sepsis model for ALI
Hemorrhage
The Hem model we have used for these experiments has been described previously [4
]. In brief, mice were anesthetized with methoxyflurane and restrained in supine position, and catheters were inserted into both femoral arteries. Anesthesia was discontinued, and blood pressure was continuously monitored through one catheter attached to a blood pressure analyzer (MicroMed, Louisville, KY). When fully awake, as determined by a mean blood pressure of 
95 mmHg, the mice were bled over a 5- to 10-min period to a mean blood pressure of 30 mmHg (±5 mmHg) and kept stable for 90 min. Immediately following Hem, mice were resuscitated intravenously with Ringers lactate at four times drawn blood volume. Following resuscitation, arteries were ligated, catheters removed, and catheter sites sutured. Before returning mice to their cages, CXCR inhibitor, antileukinate, in 100 µl phosphate-buffered saline (PBS), was administered at 0 mg, 0.4 mg, or 1.0 mg/mouse subcutaneously (s.c.) [17
]. Sham-Hem was performed as a control, and these mice were anesthetized and restrained, and their femoral arteries were ligated, but no blood was drawn.
Polymicrobial sepsis
Twenty-four hours post-Hem (or Sham-Hem), sepsis was induced as a secondary challenge via cecal ligation and puncture (CLP) as described previously [4
]. To summarize, mice were anesthetized with methoxyflurane and restrained in supine position. A 1-cm midline incision was made; the cecum was ligated with 5–0 silk thread and punctured twice with a 21-gauge needle. The cecum was then replaced, the incision was sutured, and lidocane was applied, abdominal layer then skin. Mice were resuscitated with 1 ml Ringers lactate s.c. and returned to their cages.
Sample collection
Twenty-four hours post-CLP, mice were killed with an overdose of methoxyflurane.
Blood was collected via cardiac puncture into heparinized syringes. Blood samples were centrifuged, and plasma was collected and stored at –70°C for later cytokine analysis.
BALF was collected to assess protein concentration as an index of lung permeability (injury). The trachea was exposed via a midline incision and cannulated with a sterile polypropylene 18-gauge catheter. The lungs were gently lavaged with 0.6 ml saline three times for an average of 1 ml lavage fluid total per lung. Lavage fluid was centrifuged 1000 g for 8 min at 4°C. Protein concentration in lavage fluid was assessed by Bradford assay.
Lung tissue was harvested for assessment of neutrophil influx [myeloperoxidase (MPO)]. As a result of the degradation of tissue architecture observed in lavaged mouse lungs, additional mice were used for histological assessment. For lung histology, the trachea was cannulated, and the lungs were gently inflated to 25 cm pressure with 10% formalin, then excised, and fixed in 10% formalin.
Methods of assessment
Lung MPO activity as an assessment of neutrophil influx was measured according to established protocols [18
]. Briefly, lung tissue was homogenized in 0.5 ml 50 mM potassium phosphate buffer, pH 7.4, and centrifuged at 40,000 g at 4°C for 30 min. The supernatant was reserved for cytokine analysis. The remaining pellet was resuspended in 0.5 ml 50 mM potassium buffer, pH 6.0, with 0.5% hexadecyltrimethylammonium bromide, sonicated on ice, and then centrifuged at 12,000 g at 4°C for 10 min. Supernatants were then assayed at a 1:20 dilution in reaction buffer (530 nmol/L o-dianisidine, 150 nmol/L H2O2 in 50 mM potassium phosphate buffer) and read at 490 nm.
Cytokine ELISAs for mouse were performed on plasma and lung-tissue homogenates and as per the manufacturer’s protocol for indirect ELISA (R&D Systems).
Fluorescent staining of neutrophils in frozen lung-tissue sections was performed as per the manufacturer’s protocol (Molecular Probes, Eugene, OR). Following rinsing to remove mounting media and blocking with PBS plus 5% serum for 30 min at 37°C, slides were incubated with purified goat polyclonal antibody against a peptide within an internal region of MPO of mouse origin (MPO L-20:SC-16129, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature in a humidity chamber. Slides were rinsed and incubated with a biotinylated rabbit anti-goat immunoglobulin G2b antibody for 30 min at room temperature and then rinsed. Slides were then incubated with a streptavidin Alexa Fluor® 594 conjugate (Molecular Probes) for 30 min, rinsed, and compared with negative-control slides (without primary). Slides were then cover-slipped with a mounting medium for fluorescence microscopy with 6-diamidine-2'-phenylindole dihydrochloride (DAPI; Vector Laboratories, Burlingame, CA), a DNA stain. To establish the total number (%) of cells (per field) that were neutrophils (MPO+) present in the sample, tissue sections were randomly screened (seven to eight fields/slide) at 400x (25 µm2/field). The number of MPO-positive cells (Alexa Fluor® 594) was divided by the number of DAPI-positive cells and then multiplied times 100 to derive the percent MPO-positive cells.
Immunohistochemical staining for assessment of neutrophil influx and tissue architecture
Staining for leukocyte-specific esterase, Naphthol AS-D chloroacetate esterase (Sigma Diagnostics, St. Louis, MO), was performed on frozen tissue sections fixed in citrate-acetone-formaldehyde. Slides were incubated in a solution of sodium nitrate, Fast Red Violet BL base solution, TRIZMAL 6.3 buffer, and Naphthol AS-D chloroacetate solution in deionized water for 15 min at 37°C. Following rinsing, slides were counterstained with Gills hemotoxylin solution and cover-slipped. Stained lung sections were examined microscopically for morphology and positively stained cells. To establish the total number (%) of cells (per field) that were neutrophils (esterase +) present in the sample, tissue sections were randomly screened (seven to eight fields/slide) at 400x (25 µm2/field).
Statistical analysis
Data was expressed as means ± SEM. Statistical error was determined using one-way ANOVA; the post-hoc test was Tukey’s. Calculations were performed using SigmaStat for Windows Version 2.03. P values <0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antileukinate differentially abrogates Hem-induced, systemic inflammatory response subsequent to septic challenge
The systemic inflammatory response is mediated in part by the secretion of cytokine/chemokines into the blood by local tissue cells in the peritoneal cavity [19
, 20
]. In the PBS vehicle group, Hem followed by septic challenge, produced increased levels of plasma IL-6 and a suppression of IL-10 in the blood. Antileukinate treatments significantly reduced levels of IL-6 and increased systemic levels of IL-10 in a dose-dependent manner (Fig. 2A
and 2B
).
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Antileukinate treatment significantly reduced Hem-induced neutrophil recruitment to lungs following subsequent septic challenge
The influx of neutrophils to the lungs of Hem septic mice was assessed by MPO activity. Antileukinate treatment reduced MPO activity to near Sham-Hem/CLP levels (Fig. 3A
). Immunofluorescent staining of lung-tissue sections with antibody against MPO confirmed MPO enzymatic activity data with a decrease in MPO-positive staining cells from 22% ± 6 in the PBS vehicle Hem controls (in keeping with results observed following infectious challenge and inflammatory lung injury, Holt et al. [21
] and Moore et al. [22
]) to 6% ± 2 in the 1.0-mg antileukinate-treated mice. The percent of MPO-positive staining cells in lung tissue from Sham-Hem/CLP mice was 7% ± 3.
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The reduction in BALF protein and MPO activity in the antileukinate-treatment groups is further supported by lung-tissue histology. Lung-tissue sections from Hem/septic mice showed significant septal thickening and cellular infiltrate (Fig. 3C) ; antileukinate treatment attenuated septal thickening and cellular infiltrate seen in the lungs of Hem/septic mice (Fig. 3D) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our murine model of ALI, we have described a predisposition/priming for inflammatory lung injury in animals that are subjected to an initial sublethal challenge (hypotensive shock/Hem) before the induction of sepsis. This priming stimulus was significantly yet differentially blunted when antibody against neutrophil chemotactic proteins KC and MIP-2 was administered immediately following Hem [12 ]
To further elucidate the contribution of neutrophils in the pathogenesis of ALI via the chemokines KC and MIP-2, disruption of their chemokine/receptor-signaling pathway was achieved using antileukinate, a hexapetide inhibitor of Glu-Leu-Arg+ (ELR+) CXCRs. Antileukinate has been demonstrated to significantly attenuate pulmonary fibrosis in a Bleomycin model of lung injury in mice [17 ]. Additionally, Maus et al. [24 ] blocked neutrophil chemotaxis to the lungs of lipopolysaccharide-challenged and CC chemokine ligand 2-treated mice following treatment with antileukinate.
In this study, we have demonstrated that disruption the chemokine/receptor migratory signaling cascade with antileukinate immediately following an initial challenge, Hem, significantly reduces inflammatory lung injury observed subsequent to the induction of sepsis.
Lung-tissue levels of proinflammatory cytokine IL-6 and chemokines KC and MIP-2 were considerably reduced in mice that received antileukinate following Hem and septic challenge. This is in agreement with histological MPO activity and BALF protein findings. Lung-tissue histology showed a marked reduction in septal thickening/congestion as well as decreased cellular infiltrate. Antileukinate inhibition of neutrophil migration to the lungs was observed in the significant reduction in MPO activity and the number of fluorescent MPO+ cells detected in lung tissue from treated mice. In addition, BALF protein concentration, a measure of lung leak/injury, was also reduced significantly.
Systemically, inhibition of ELR+ CXCRs underscores the complex and dynamic cellular and molecular relationships/interactions associated with the evolution of sepsis. Plasma levels of IL-6 decreased in a dose-dependent manner, suggesting that antileukinate inhibition of neutrophil priming for activation and migration suppresses the secretion of IL-6 into the blood by local tissue and immune cells. Alternatively, suppression of an early neutrophil response serves to limit the intensity of the inflammatory signal and by doing so, reduces the production of IL-6. As mortality studies have not been performed on the antileukinate-treated mice, the beneficial or deleterious effects of suppressed, systemic IL-6 levels in this model are not known.
It is interesting that systemic levels of KC and MIP-2 did not reflect the overall decrease in cytokine levels observed in lung tissue. Systemic levels of MIP-2 were significantly elevated above the PBS control for both doses of antileukinate. It is possible that this divergent effect is a consequence of a lack of neutrophil-mediated/signaling interactions with cells from the inflammatory environment as a result of CXCR blockade. In this respect, it is suggested that neutrophils migrate along gradients of chemoattractants produced by cells (including endothelial/epithelial, tissue macrophage, neutrophils, fibroblasts) responding to inflammatory stimuli such as tumor necrosis factor 
(TNF-), endotoxin, IL-1, and complement [23
, 25
, 26
]. Additionally, cell-to-cell interactions involved in the inflammatory signaling cascade contribute to the production of cytokines/chemokines [27
], facilitate the diapedesis of neutrophils into the lung [28
], and serve to stimulate production of anti-inflammatory mediators (i.e., IL-10) [30
]. Neutrophil interactions with endothelial cells along vessel walls at sites of inflammation stimulate the adherence and transmigration of neutrophils from the circulation. Miotla et al. [28
] found that mice deficient in P-selectins and antibody blockade of P-selectins (neutrophil-associated adhesion molecule) on endothelial cells in the pleural cavity showed significantly (90–70%, respectively) reduced neutrophil emigration into the inflamed/infected tissue. Aujuebor et al. [30
] showed a strong corollary between neutrophil influx into the lung in response to chemokine signaling and an up-regulation, ostensibly by tissue macrophages, of endogenous IL-10, which is an important anti-inflammatory mediator shown to suppress expression of proinflamatory cytokines, TNF-, and IL-1 as well as CXC and CC chemokines [31
]. With that said, the inhibition of neutrophil targeting to the lung may produce an up-regulation of systemic MIP-2 in an attempt to compensate for the absence of chemotaxing cells.
In contrast to MIP-2, KC in the plasma was reduced significantly at the lower dose of antileukinate yet not at the higher dose. The meaning of these divergent effects is not immediately clear; however, distinctions between MIP-2, a homologue of human IL-8, and KC, which shares a greater sequence homology with human growth-related oncogene- (Gro-)/melanoma growth-stimulating activity (MGSA), have been reported. Hachicha et al. [32
] and Brown et al. [33
] found increased levels of KC but not MIP-2 in inflammatory synovitis and an experimental lyme disease model, respectively. In addition, Wang et al. [34
] has reported that overexpression of MGSA/Gro- up-regulated murine oncogene M-Ras protein expression. Recently, Armstrong et al. [36
] found differential expression of KC and MIP-2 in a tissue-, temporal-, and stimuli-dependent manner. These examples highlight the apparent divergence in these two CXC chemokines despite evidence that they share a common receptor. In previous ALI studies, we have reported that antibody depletion of peripheral blood neutrophils produced a significant elevation in plasma levels of MIP-2 compared with controls, whereas KC plasma levels were depressed [10
]. These data taken together suggest a more active/specific role for MIP-2 than KC in neutrophil chemotaxis in the acute, inflammatory process, where neutrophil depletion [10
] and neutrophil chemokine receptor inhibition in a sepsis-induced inflammatory environment stimulate significant MIP-2 secretion but not KC.
An explanation for the dose-dependent increase in plasma IL-10 has proven confounding. IL-10 is an anti-inflammatory cytokine produced by T helper cell type 2 (Th2) lymphocytes, monocytes, and epithelial cells. It is associated with immunosuppression and the down-regulation of interferon-
and other Th1/proinflammatory cytokines. In septic patients, elevated levels of IL-10 have proven to be indicative of a poor outcome (high mortality) [36
, 37
]. It is possible that the elevated, systemic levels of IL-10 reflect a contemporaneous response to septic bacterial challenge not specifically associated with the inhibition of neutrophil chemotaxis. Although not assessed here, it could be speculated that although neutrophils from antileukinate-treated mice do not home to the lung, they may transit from circulation to the liver. Once in the liver, their uptake by Kuppfer cells could serve as a stimulus for IL-10 release [38
], thereby elevating systemic levels of IL-10. However, a clear understanding of this augmentation of systemic IL-10 release remains to be established.
The recruitment of inflammatory cells to sites of infection or inflammation is a complex and dynamic biological process. In the pathogenesis of ALI, activated neutrophils become dysregulated, trafficking to the lungs, where they are sequestered and continue to release tissue-damaging mediators/enzymes after clearance of infectious agents. Chemokines secreted by cells in the inflammatory environment target neutrophils to these sites. KC and MIP-2 are two murine neutrophil chemokines found elevated in the plasma and lung tissue of Hem/septic mice. Signaling for neutrophil recruitment occurs when KC or MIP-2 binds to their receptor, a homologue of the human IL-8 chemokine receptor, CXCR2. We hypothesized that inhibition of neutrophil chemotaxis via CXCR2 signal inhibition would reduce inflammatory lung injury in our model of Hem-induced neutrophil priming for ALI. Our data support this hypothesis and with further research, present a potential, therapeutic approach to the treatment of ALI.
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ACKNOWLEDGEMENTS |
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Received November 5, 2003; revised February 26, 2004; accepted March 26, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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J. Immunol. 159,3595-3602[Abstract]
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