Despite advances in medical therapy such as broad-spectrum antibiotics, invasive monitoring, and mechanical ventilation, pneumonia remains a serious problem for hospitalized patients as well as for people in the community at large. In the United States, pneumonia represents the sixth most common cause of death, with estimated health costs of more than $20 billion a year (1). Nosocomial pneumonia remains the leading cause of death from a hospital-acquired infection (2). In ventilated patients, the incidence of pneumonia is roughly 9% to 24% in patients intubated for longer than 48 h, whereas the incidence is even higher in chronically ventilated patients (3). Pneumonia can also be a devastating complicating factor in patients with acute respiratory distress syndrome (ARDS) (4). For surgical patients, pneumonia has become the most common postoperative complication, exceeding wound infection, with an overall incidence of 5% to 8% (5). Mortality associated with the disease ranges from 20% to 70%, increasing the relative risk of death for a patient by 3- to 4-fold (2,3,5).
We have proposed to investigate the role of the protein, lipopolysaccharide (LPS)-binding protein (LBP), in fighting bacterial pneumonia. Since its first isolation from rabbit acute phase serum in 1986 (6), LBP has been widely accepted as an important component of the immune response to gram-negative bacteria (7,8). LBP, a 58- to 60.5-kD acute phase glycoprotein, recognizes the highly conserved lipid A moiety of LPS, which triggers activation of host immune cells by binding to CD14, a glycosyl phosphatidyl inosital (GPI)-linked receptor expressed on monocytic cells and also found in soluble form in serum. When present in high concentrations, LPS aggregates can independently activate monocytic cells by binding directly to CD14. However, in the presence of LBP, this activation occurs at much lower concentrations because LBP facilitates disaggregation of LPS and subsequent binding of monomers to CD14 (9). Intracellular signaling is carried out by downstream Toll-like receptors, TLR4 and TLR2 (10–13). Macrophage activation ultimately leads to release of proinflammatory cytokines, enhanced phagocytosis, heightened oxidative burst capacity, activation of complement, and if severe enough, development of septic pathophysiology. In addition, activated macrophages produce chemokines that recruit neutrophils in response to infection. In humans, the C-X-C chemokine IL-8 has been defined as the primary neutrophil chemoattractant (14). In mice, KC and macrophage inflammatory protein-2α (MIP-2α) are the closest murine homologues to IL-8 (15).
Serum LBP levels rise in the setting of sepsis, and local expression of LBP in the lung has been shown to be upregulated following distant injury and infection (16,17). Whether upregulation of LBP in the setting of sepsis is beneficial or detrimental to the host is unclear. Based on its facilitation of LPS monocyte activation, high LBP levels might be expected to render the host more sensitive to LPS and more prone to develop an overwhelming systemic inflammatory response leading to septic complications of shock, acute respiratory distress syndrome (ARDS), coagulopathy, or end-organ damage. Early studies using antibodies against LBP appeared to support this hypothesis (18,19). Animal studies using LBP knockout mice have yielded conflicting results (20,21), however, and new lines of evidence now suggest that LBP's primary biological role may not be to amplify the proinflammatory cascade in response to LPS.
Although LBP animal studies to date have primarily involved models of systemic infection with i.p. injection of either LPS or bacteria, our study focuses on LBP's local role in the lung. Originally thought to be synthesized only in the liver, LBP has since been shown to be produced at multiple extrahepatic sites, including lung (16,17,22), gut (23), and skin (24). In humans, the presence of LBP in bronchoalveolar lavage (BAL) fluid from patients with asthma and ARDS suggests that this protein may be important in the pathophysiology of these diseases (25,26). Our group has previously demonstrated particularly high expression of LBP within the lungs of animals undergoing an acute phase response (27). Given LBP's ability to markedly enhance phagocytosis and killing of bacteria by alveolar macrophages in vitro (28), we hypothesize that local production of LBP may promote bacterial killing in vivo. In our current studies, we sought to determine the role of LBP in local lung defenses against bacterial challenge using a model of Klebsiella pneumoniae in LBP knockout mice.
MATERIALS AND METHODS
LBP knockout mice were a gift from Douglas Golenbock (Boston University School of Medicine) (21). These animals had been backcrossed into the background C57BL/6 strain at least 12 times before we acquired our colony. They were subsequently housed in a specific pathogen-free environment and allowed to breed. Female LBP knockout mice ranging from 12 to 16 weeks in age and appropriate age- and weight-matched female C57BL/6 control mice (Jackson Laboratory, Bar Harbor, ME) were used for all our experiments. The control mice were housed under specific pathogen-free conditions and were allowed to acclimate to their new surroundings for 1 week prior to being used in experiments. Experiments were performed in accordance with the National Institutes of Health guidelines, and approval was obtained from the University of Michigan Animal Care and Use Committee.
Twenty microliters of EDTA-anticoagulated mouse tail vein blood was taken from each animal prior to bacterial inoculation and analyzed by the Hemavet Mascot machine (CDC Laboratories, Oxford, CT). On the day of sacrifice, tail blood was again drawn and analyzed to assess for changes in blood differential with infection.
Klebsiella pneumonia, strain 43816, serotype 2 (American Type Culture Collection, Manassas, VA) was provided by the laboratory of Theodore Standiford (Pulmonary Medicine, University of Michigan). This strain was grown up in trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ) with reinoculation the following morning into fresh tryptic soy broth to bring the bacteria out of stationary phase. The bacteria were then centrifuged at 2000 rpm at 4°C for 10 min (Sorvall RT 6000D, Kendro, Newtown, CT), washed with sterile 0.9% normal saline, centrifuged again, and then resuspended in sterile saline. Optical density was read on the spectrophotometer (Milton Roy, Rochester, NY) at a wavelength of 600 nm. Appropriate serial dilutions were subsequently made to achieve a concentration of 1000 bacteria per 30 μL of inoculum, the LD50 for C57BL/6 using this strain of Klebsiella.
Mice were anesthetized with an i.p. injection of pentobarbital. After the cervical region was prepped with 95% ethanol, dissection was carried out down to the level of the trachea. A 27-gauge needle on a Hamilton syringe was used to deliver 30 μL of bacterial inoculum to each animal. The animal was kept upright for roughly 30 s postintratracheal injection of the bacteria in order to ensure delivery of the bacteria to the most dependent portions of the lung. Metal clips were used for skin closure. The animals were resuscitated with 1 mL of 0.9% normal saline subcutaneously and allowed to recover in a 37°C warmer. Postoperatively, animals were given standard mouse chow and water ad libitum and they were housed in accordance with University of Michigan's Unit for Laboratory Animal Medicine regulations until the time of sacrifice.
At 6, 18, or 24 h post-inoculation, mice were anesthetized with an i.p. pentobarbital injection as above. The abdomen, anterior thorax, and cervical region up to the level of the mandible were prepped with 95% ethanol. The skin overlying the anterior thorax was sharply dissected away, exposing the underlying musculature. The abdomen was then entered sharply just below the xiphoid. The diaphragms were taken down bilaterally, and the anterior rib cage was removed. Blood was obtained via cardiac puncture using a sterile 18-gauge needle and was plated undiluted, as well as diluted 1:100, on 5% sheep blood agar plates (Becton Dickinson) to assess for bacteremia. One milliliter of sterile 0.9% normal saline with 0.05 M EDTA was then injected into the right ventricle to flush out the pulmonary vasculature. The lungs and heart were then dissected free en bloc, with later removal of heart and lymph node tissue. In some experiments, whole lungs were dedicated to one assay; in others, the four lobes of the right lung and single-lobed left lung were used in different analyses.
Myeloperoxidase (MPO) assay
Whole-lung specimens were weighed, homogenized (Polytron Kinematica, Brinkmann Instruments, Westbury, NY), and sonicated (Branson Sonifier 250, Danbury, CT) in 1.5 mL of MPO homogenization buffer containing monobasic and dibasic potassium phosphate, hexadecyltrimethylammonium bromide, and EDTA. The samples were then centrifuged at 16,000 g at 4°C for 15 min. Supernatants were aspirated into fresh tubes and stored on ice until the assay was performed. Samples were run in triplicate with 20 μL of sample and 200 μL of substrate buffer (containing 0.3% hydrogen peroxide and o-dianisidine HCL) per well. The plate reader was programmed to read every 10 s at 465 nm for 90 s with linear function analysis performed of the data obtained. The assay assessed for enzyme oxidative function over time.
After induction of pentobarbital anesthesia, tracheal cutdown was performed. The trachea was isolated and an anterior tracheostomy performed to allow cannulation with PE50 tubing mated to a 23-gauge needle attached to a three-way stopcock/syringe apparatus. The lungs were then lavaged with 2 × 1 mL of warmed Hank's balanced salt solution without Ca2+ or Mg2+ (Invitrogen, Carlsbad, CA formerly Gibco). The lavages were performed in 350-μL increments to minimize traumatic injury to the lungs. The lavage fluid was spun down, with the first milliliter of supernatant saved and quick frozen for later cytokine analysis. The two cell pellets were then resuspended in RPMI (Invitrogen) with 1% fetal calf serum and pooled. After pretreatment with Zap-Oglobin (Coulter, Miami, FL), cell counts were performed using a Coulter Counter (Coulter Electronics, Hialeah, FL). Based on cell counts, 5 × 105 cells were delivered to each slide and cytospins were performed on the samples. Slides were fixed with Diff-Quik (Baxter, Detroit, MI) and cell differentials were performed counting 300 white blood cells (WBC) per slide.
KC, MIP-2α, and TNF-α levels were measured by enzyme-linked immunoabsorbant assay (ELISA) in plasma, BAL fluid, and supernatants from whole-lung homogenates. Sandwich ELISAs were performed using rabbit polyclonal capture antibodies raised against murine recombinant KC and MIP-2α; a portion of these antibodies were biotinylated and used as detection antibodies. A commercially obtained antibody pair (R&D Systems, Minneapolis, MN) was used to assay for TNF-α. Briefly, plates (Immunoplate Maxisorb; Nunc, Neptune, NJ) were coated with anti-mouse cytokine capture antibody and incubated overnight at 4°C. The plates were then washed with buffer solution and nonspecific binding sites were blocked with casein. All subsequent steps were conducted at room temperature. Samples were added in duplicate with plasma diluted 1:10 in diluent buffer, BAL fluid diluted 1:1, and lung homogenates diluted 1:1 and 1:50. Standard curves were prepared using appropriate recombinant murine cytokine or chemokine (R&D Systems). After a 1-h incubation, plates were washed, and biotinylated anti-mouse cytokine detection antibody was added. After a 1-h incubation, plates were again washed and then exposed to horseradish peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. 3,3´5,5´-tetramethylbenzidine (TMB; Genzyme Diagnostics, San Carlos, CA) was then added and the plates were allowed to change color away from light. H2S04 was used to stop the reaction. The plates were then read at 465 and 590 nm on the Biotek plate reader (Bio-Tek Instruments, Winooski, VT) and cytokine concentrations were calculated from the standard curves.
Determination of plasma and lung CFU
Blood samples obtained via sterile cardiac puncture were plated both undiluted and diluted 1:100 on 5% BSA plates and allowed to incubate at 37°C overnight. Left lung lobes were weighed and then homogenized in 1.5 mL of sterile saline with three serial 1:100 dilutions subsequently performed. One hundred-microliter aliquots of each of the three dilutions were plated in duplicate on 5% blood agar plates and allowed to incubate overnight at 37°C. Plates were counted the following day in a blinded fashion and CFUs per gram of lung tissue was calculated for each sample. Representative colonies were swabbed and sent to microbiology for identification.
Analysis was performed using Statview software (SAS Institute, Cary, NC). Unless indicated otherwise, data is expressed as the mean ± SE and was compared using a two-tailed Student's t test. Statistical significance was assigned at P values <0.05. Chi-square analysis was performed on the bacteremia data; Kaplan-Meier analysis with log rank testing was performed on the survival data.
Decreased survival after intratracheal injection of Klebsiella pneumonia
LBP knockout mice and C57BL/6 wild-type mice were each given a 30-μL intratracheal inoculation containing 103Klebsiella pneumoniae, the predicted LD50 dose for the wild-type. There was 100% mortality in the LBP knockout mice by day 3 after bacterial challenge as compared with the 42% mortality in the control mice established by day 4 (Fig. 1; n = 30, 12 wild-type controls and 18 LBP knockouts; log rank test, P < 0.0001). The control animals that survived past day 4 remained alive during the 3-week observation period following the bacterial challenge.
Incidence of bacteremia and pulmonary bacterial load
In order to delineate the mechanism by which LBP affected survival in bacterial pneumonia, we analyzed the bacterial load in the blood and lung homogenates using quantitative bacterial cultures. At 18 h, 25% of the knockouts (2 of 8) were bacteremic vs. none of the control mice (0 of 7). At 24 h, this difference had reached statistical significance with 72% (10 of 14) of the knockout mice bacteremic, whereas only 23% (3 of 13) of control mice demonstrated bacteremia (Fig. 2A;P = 0.012, chi-square test). Figure 2B compares quantitative bacterial counts on supernatants from knockout vs. control lung homogenates taken at 24 h after Klebsiella inoculation. Knockout mice had significantly (P = 0.025) higher numbers of viable bacteria in their lungs at the time of sacrifice compared with controls (log CFU/g lung tissue: 7.40 ± 0.28 vs. 6.35 ± 0.36, respectively, n = 36).
Diminished neutrophil response to infection
Neutrophil recruitment is a critical component of the immune response to bacterial challenge, and differences in neutrophil response may lead to differences in bacterial load. Therefore, we performed peripheral blood counts to evaluate neutrophil response, as well as MPO assays and BAL cell counts to evaluate local neutrophil recruitment into the lung. The results from peripheral blood analysis are depicted in Figure 3. The black arrow indicates inoculation with Klebsiella after the first blood draw. There are several differences in baseline bloodwork between the knockout and control mice. Overall, knockouts have higher total white blood cell counts (Fig. 3A), which appears to be primarily due to greater numbers of circulating lymphocytes (Fig. 3C) and monocytes (Fig. 3D). Interestingly, not only do LBP knockout mice have lower numbers of circulating neutrophils at baseline, but they actually display lower circulating neutrophil counts at all time points (0, 18, and 24 h post-bacterial challenge) when compared with controls (Fig. 3B).
Analysis of local pulmonary recruitment of neutrophils demonstrated a statistically significant reduction in MPO activity (84 ± 7 vs. 141 ± 24, respectively) in whole-lung homogenates from knockout mice compared with controls at 18 h following bacterial challenge (Fig. 4A; n = 22, P = 0.022). Analysis of BAL fluid, reflecting inflammatory cell recruitment into the alveolar space, demonstrated significantly reduced numbers of neutrophils (PMN/mL: 1.6 × 105 ± 3.0 × 104 vs. 3.3 × 105 ± 5.7 × 104) 18 h after bacterial inoculation in the alveoli of knockout mice vs. controls (Fig. 4B; n = 21, P = 0.012). Comparison of numbers of recruited macrophages did not reveal any significant differences between the two strains of mice.
Measurement of chemokine and cytokine levels in serum and lung
Chemokine levels were measured in plasma, BAL fluid, and supernatants from lung homogenates. We looked specifically at two C-X-C chemokines, KC and MIP-2α, murine analogues for IL-8 instrumental in neutrophil recruitment. As expected, chemokine expression was mainly a localized event with no appreciable systemic levels detectable in plasma (data not shown). MIP-2α studies failed to show any statistically significant differences between groups (data not shown). There were, however, significant reductions in levels of KC in BAL fluid at 6 h (Fig. 5A;P = 0.0048) and in lung parenchyma at 18 h (Fig. 5B;P = 0.028) from knockout mice compared with controls. TNF-α was found to be significantly elevated (P = 0.0206) in plasma from knockout mice 18 h post-bacterial inoculation compared with controls (Fig. 5C). Levels of TNF-α in the lung, however, demonstrated no significant differences between knockouts and controls (data not shown).
We have known for some time that LBP is locally produced in the lung and is upregulated in various disease states such as ARDS, asthma, acute lung injury, and bacterial infection (22,25,29,30). The implications of this, however, were uncertain. Early LBP studies seemed to suggest that it sensitized the host to gram-negative endotoxemia, increasing the likelihood of the development of overwhelming sepsis. Preliminary work in relation to ARDS and LBP initially led investigators to view LBP as a deleterious rather than protective element in the setting of infection. However, these early studies tended to use LPS or endotoxin as the challenge rather than live bacteria. These models are necessarily of limited clinical relevance as they fail to recreate the condition where the host must deal with a rapidly replicating organism, not only a set amount of offending antigen.
Recent in vitro studies have shown that LBP may be protective, enhancing alveolar macrophage phagocytosis and bacterial killing (28,31,32) as well as at times acting as a lipid-transfer protein, transferring LPS to high density lipoprotein (HDL) thereby neutralizing it (33). There is some evidence that the divergent properties of LBP may in fact be concentration-dependent: at low concentrations, LBP may act to potentiate macrophage activation by LPS, whereas at higher acute-phase concentrations, LBP may actually inhibit LPS-induced cell activation (34,35). The role of LBP in infection and inflammatory disease, however, is still not fully understood.
Our data represents the first instance where LBP's role has been studied in the setting of a mucosal bacterial challenge, here specifically in the lungs. We clearly demonstrate a dramatic difference in mortality between LBP knockout mice and control mice in this pneumonia model with 100% mortality in the LBP knockout mice vs. only 42% mortality in the controls. This impressive difference between the two groups spurred our investigation into its possible mechanisms. With the finding of earlier onset of bacteremia in knockout mice, we postulated that there was some deficiency in the knockout mouse innate immune system at the level of the lung, resulting in a reduced ability to contain infection at a local level, hence leading to early bacteremia and sepsis. The significantly higher bacterial load detected in lung samples from knockout mice also suggested that bacterial killing at the local level was compromised in the absence of LBP. We had previously shown that LBP significantly enhances alveolar macrophage phagocytosis and killing of bacteria (28). The question was whether this alone could account for the dramatic differences in immunity or whether neutrophil recruitment and/or function might also be compromised in the absence of LBP.
In our study, the MPO assay assessed for sequestered neutrophils in the lungs after bacterial inoculation. Reduced MPO levels in the lungs from knockout mice vs. controls at 18 h after bacterial challenge confirmed impaired neutrophil recruitment in the absence of LBP. Cytospin differentials and cell counts on BAL fluid were in keeping with the MPO results, demonstrating statistically significant reductions in the numbers of neutrophils in the alveolar spaces of knockout vs. control mice. Significantly lower levels of the neutrophil chemokine, KC, found in knockout BAL fluid at 6 h and in knockout lung parenchyma at 18 h may partially explain why there is diminished neutrophil recruitment to the infection site.
Therefore, we postulate that LBP affects local pulmonary immune defenses not only by enhancing macrophage effector functions such as phagocytosis and bacterial killing, but also by altering macrophage regulatory functions such as chemokine production. In the absence of LBP, there appears to be impaired LPS activation of alveolar macrophages, possibly leading to less effective phagocytosis and killing of bacteria by the macrophages as well as decreased synthesis of C-X-C chemokines necessary for effective neutrophil recruitment into the lung. Neutrophil recruitment is essential for the ultimate eradication of rapidly proliferating bacteria; it is felt that alveolar macrophages are generally unable to accomplish this alone. In vivo studies using murine pneumonia models have demonstrated the importance of C-X-C chemokines in bacterial clearance and survival (36,37).
In addition, LBP-deficient animals may suffer not only from suppressed induction of mediators of neutrophil recruitment, but also from impairment of immunity due to baseline hematologic deficiencies. The neutropenia that we identified in these knockout mice has not been reported previously in the literature. Lower circulating neutrophil counts in knockout mice both before and after bacterial challenge cannot be explained by neutrophil sequestration in areas of infection. Therefore, this diminished neutrophil response to infection may reflect hematopoietic events at the level of the bone marrow, namely deficient granulocyte production. In fact, there have been other reports of pronounced hematologic abnormalities seen in knockout mice; for example, CD18-deficient mice display dramatically increased numbers of circulating neutrophils (38).
The high systemic TNF-α levels seen in knockout mice at 24 h postinfection, although at first surprising, may simply reflect a severe degree of sepsis in these immunocompromised mice. By 24 h, we know that the knockout mice, unlike controls, are largely bacteremic. At high concentrations of LPS, macrophage activation with production of proinflammatory cytokines such as TNF-α occurs in the absence of LBP. Circulating monocytes brought into contact with large amounts of LPS and gram negatives in the bloodstream may be the source of this cytokine.
In conclusion, our experiments represent the first time that LBP has been shown to be essential in host defenses against a mucosal bacterial challenge. Given the marked differences in survival, bacteremia, efficiency of bacterial killing, chemokine levels, and neutrophil recruitment between knockout mice and controls, we assert that LBP is essential in innate immune defenses against bacterial infection in the lung. Recent demonstration of local production of LBP by type II pneumocytes with upregulation of protein expression by these cells in response to glucocorticoids and proinflammatory cytokines IL-1β, IL-6, and TNF-α (16) lends further support to a possible central role for LBP in pulmonary host defenses. Neonatal alveolar macrophages have also been recently shown to produce LBP at physiologically relevant concentrations with further inducible expression in response to LPS challenge (17). Increased LBP production in the neonatal lung could represent a compensatory response by the innate immune system until adequate adaptive immunity develops. Given our findings and those of others, the presence of LBP within the lung is clearly important and should no longer be considered an incidental finding. Further work must be done to determine whether dysregulation of LBP expression or function may play a role in various pathologic disease states in the lung.
We would like to thank Dr. Douglas Golenbock of Boston University School of Medicine for graciously donating the LBP knockout mice for our experiments. We also thank Dr. Theodore Standiford and his laboratory in the Department of Pulmonary Medicine at University of Michigan for assistance with the Klebsiella pneumonia model. We wish to express our gratitude to Dr. Nancy Gong and Mita Ghosh for their assistance with experiments.
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