Cytokine balance in the lungs of patients with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) involves an intense inflammatory response in the lungs, with accumulation of both pro- and antiinflammatory cytokines in bronchoalveolar lavage fluid (BALF). Our goal was to determine how the balance between pro- and antiinflammatory mediators in the lungs changes before and after the onset of ARDS. We identified 23 patients at risk for ARDS and 46 with established ARDS and performed serial bronchoalveolar lavage (BAL). We used immunoassays to measure tumor necrosis factor alpha (TNF-alpha) and soluble TNF-alpha receptors I and II; interleukin 1 beta (IL-1 beta), IL-1 beta receptor antagonist, and soluble IL-1 receptor II; IL-6 and soluble IL-6 receptor; and IL-10. We used sensitive bioassays to measure net TNF-alpha, IL-1 beta, and IL-6 activity. Although individual cytokines increased before and after onset of ARDS, greater increases occurred in cognate receptors and/or antagonists, so that molar ratios of agonists/antagonists declined dramatically at the onset of ARDS. The molar ratios remained low for 7 d or longer, limiting the activity of soluble IL-1 beta and TNF-alpha in the lungs at the onset of ARDS. This significant antiinflammatory response early in ARDS may provide a key mechanism for limiting the net inflammatory response in the lungs.

Fas/Fas ligand system mediates epithelial injury, but not pulmonary host defenses, in response to inhaled bacteria.

The Fas/Fas ligand (FasL) system has been implicated in alveolar epithelial cell apoptosis during pulmonary fibrosis and acute respiratory distress syndrome. However, Fas ligation can also lead to cell activation and cytokine production. The goal of this study was to determine the role of the Fas/FasL system in host defenses against Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We administered bacteria by aerosolization into the lungs of Fas-deficient (lpr) mice and wild-type (C57BL/6) mice and measured bacterial clearance at 6 and 12 h. One hour prior to euthanasia, the mice received an intraperitoneal injection of human serum albumin (HSA) for alveolar permeability determinations. At all times after bacterial challenges, the lungs of the lpr mice contained similar or lower numbers of bacteria than those of the C57BL/6 mice. Alveolar permeability changes, as determined by bronchoalveolar lavage fluid HSA concentrations, were less severe in the lpr mice 6 h after the challenges. In response to E. coli, the lpr mice had significantly more polymorphonuclear leukocytes (PMN) and macrophage inflammatory protein 2 in the lungs, whereas histopathologic changes were less severe. In contrast, in response to the gram-positive cocci, the lpr animals had similar or lower numbers of PMN. We conclude that the Fas/FasL system contributes to the development of permeability changes and tissue injury during-gram negative bacterial pneumonia. The Fas/FasL system did not have a major role in the clearance of aerosolized bacteria from the lungs at the bacterial doses tested.

Amniotic fluid lipopolysaccharide-binding protein and soluble CD14 as mediators of the inflammatory response in preterm labor.

OBJECTIVE: Our purpose was to determine the association of lipopolysaccharide-binding protein (LBP) and soluble CD14 (sCD14) with the proinflammatory response among women in preterm labor. The binding of lipopolysaccharide (LPS) with LBP and sCD14 activates macrophages at LPS concentrations up to 1000 times lower than required with LPS alone. LBP and sCD14 in amniotic fluid could explain the high concentrations of cytokines present in the amniotic fluid of culture-positive women and the presence of cytokines in the amniotic fluid of culture-negative women. STUDY DESIGN: A cohort of 169 afebrile women in preterm labor with intact membranes had amniotic fluid collected by transabdominal amniocentesis for culture and for LBP, sCD14, and interleukin 6 (IL-6) determinations by enzyme-linked immunosorbent assay. IL-6 levels of >2 ng/mL were considered elevated. Statistical analyses included analysis of variance, multiple comparisons with Bonferroni correction, and linear regression. RESULTS: All 169 amniotic fluid samples had measurable LBP and sCD14. Subjects were categorized by amniotic fluid culture results and IL-6 concentration into 3 groups: (1) positive amniotic fluid culture, (2) negative amniotic fluid culture, elevated IL-6 concentration, and (3) negative amniotic fluid culture, low IL-6 level. Geometric mean LBP and sCD14 levels differed significantly among groups such that levels were approximately twice as high among pregnancies with positive amniotic fluid culture or elevated IL-6 compared with those without evidence of inflammation (both P <.001). sCD14 was inversely associated with enrollment gestational age independent of amniotic fluid culture results and IL-6 concentrations. Among culture negative, low IL-6 pregnancies, sCD14 decreased 3.5% for each additional week of gestation (95% confidence interval [CI], 0.01%-6.4%; P =.02). LBP levels showed a similar trend in this group (P =.09). One hundred eleven subjects had detectable IL-6 levels. Among these subjects, IL-6 increased by 2.1-fold for every 10-fold increase in LBP (95% CI, 1.1-4.0; P =.02) and by 28.4-fold for every 10-fold increase in sCD14 (95% CI, 10.4-77.4; P <.001) with adjustment for gestational age by linear regression. CONCLUSIONS: LBP and sCD14 are present in amniotic fluid of preterm pregnancies and are linearly associated with amniotic fluid IL-6 concentrations. These molecules may amplify the cytokine response and thereby help explain the presence of cytokines in amniotic fluid when culturable quantities of microbes are absent.

Anti-interleukin 8 autoantibody: interleukin 8 complexes in the acute respiratory distress syndrome. Relationship between the complexes and clinical disease activity.

Increased levels of interleukin 8 (IL-8) are found in bronchoalveolar lavage (BAL) fluids from patients with the acute respiratory distress syndrome (ARDS). However, IL-8 is not an efficient predictor of the course of ARDS. Our prior studies demonstrated that IL-8 present in lung fluids from patients with ARDS is associated with anti-IL-8 autoantibodies (anti-IL-8:IL-8 complexes). These data led us to hypothesize that the complexes might better predict the development of acute lung injury. Accordingly, we measured concentrations of free and complexed IL-8 in BAL fluids from 19 patients at risk and 45 with established ARDS on Days 1, 3, 7, 14, and 21 after the onset of ARDS. The concentrations of anti-IL-8:IL-8 complexes in patients with ARDS on Day 1 were significantly higher than in patients at risk (p < 0.05). There was a significant association between anti-IL-8:IL-8 complex concentrations and the onset of ARDS (p = 0.03). Similarly, anti-IL-8:IL-8 complex concentrations were significantly higher in patients on Day 1 of ARDS who later died (p < 0.05), and the association between high anti-IL-8: IL-8 complex concentrations and the probability of dying was significant (p = 0.03). The presence of anti-IL-8:IL-8 complexes in BAL fluids of patients with ARDS is an important prognostic indicator for the development and outcome of ARDS.

Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome.

Nitric oxide (NO) end-products (nitrate and nitrite) are present in bronchoalveolar lavage (BAL) fluid of patients with inflammatory lung diseases. Reactive oxygen-nitrogen intermediates damage macromolecules by oxidation or nitration of critical residues in proteins. The goal of this study was to measure NO end-products (nitrate+ nitrite), in BAL fluid before and after the onset of acute respiratory distress syndrome (ARDS) and to determine if these products are associated with expression of inducible nitric oxide synthase enzyme (iNOS) in BAL cells and nitration of BAL proteins. We performed bronchoalveolar lavage (BAL) in patients at risk for ARDS (n = 19), or with ARDS (n = 41) on Days 1, 3, 7, 14, and 21 after onset, and measured total nitrite (after reducing nitrate to nitrite) and protein-associated nitrotyrosine concentration in each BAL fluid sample. Cytospin preparations of BAL cells were analyzed by immunocytochemistry for iNOS and nitrotyrosine. Nitrate+nitrite were detected in BAL fluid from patients at risk for ARDS, and for as long as 21 d after the onset of ARDS. Nitrotyrosine was detectable in all BAL fluid samples for as long as 14 d after the onset of ARDS (range, 38.8 to 278.5 pmol/mg of protein), but not in BAL of normal volunteers. Alveolar macrophages of patients with ARDS were positive for iNOS and nitrotyrosine, and remained positive for as long as 14 d after onset of ARDS. The BAL nitrate+nitrite did not predict the onset of ARDS, but the concentration was significantly higher on Days 3 and 7 of ARDS in patients who died. Thus, NO end products accumulate in the lungs before and after onset of ARDS; iNOS is expressed at high levels in AM during ARDS; and nitration of intracellular and extracellular proteins occurs in the lungs in ARDS. The data support the concept that NO-dependent pathways are important in the lungs of patients before and after the onset of ARDS.

Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome.

OBJECTIVE: To determine whether bronchoalveolar lavage fluid (BALF) from patients either at risk for the acute respiratory distress syndrome (ARDS) or with sustained ARDS modulates neutrophil apoptosis; to measure the BALF concentrations of the apoptosis inhibitors granulocyte colony-stimulating factor (G-CSF) and granulocyte/macrophage colony-stimulating factor (GM-CSF) before and after the onset of ARDS; and to determine whether the BALF concentrations of G-CSF and/or GM-CSF are associated with clinical outcome. DESIGN: Prospective cohort study. SETTING: Tertiary university hospital. PATIENTS: Twenty patients at risk for ARDS and 45 patients with established ARDS. INTERVENTIONS: Patients at risk for ARDS underwent bronchoalveolar lavage within 24 hrs of being identified, then again 72 hrs later. Patients with ARDS underwent bronchoalveolar lavage within 24 hrs of meeting ARDS criteria, then again on days 3, 7, and 14 of the disease. MEASUREMENTS AND MAIN RESULTS: Normal peripheral blood neutrophil were incubated overnight in BALF from normal volunteers, from patients at risk for ARDS, or from patients with ARDS. neutrophil apoptosis was determined by flow cytometric analysis of annexin V binding. G-CSF and GM-CSF were measured in BALF by immunoassays. Compared with normal BALF, BALF from patients on days 1 and 3 of ARDS inhibited neutrophil apoptosis, but BALF from patients at later stages of ARDS, or from patients at risk for ARDS, did not. The BALF concentrations of both G-CSF and GM-CSF were elevated early in ARDS and decreased toward later stages. Patients who lived had significantly higher concentrations of GM-CSF in the BALF than those who died. CONCLUSIONS: We conclude that the antiapoptotic effect of ARDS BALF on normal neutrophil is highest during early ARDS, and decreases during late ARDS. G-CSF and GM-CSF are present in BALF from patients with ARDS, and their concentrations parallel the antiapoptotic effect of ARDS BALF. These data support the concept that the life-span of neutrophil in the air spaces is modulated during acute inflammation. GM-CSF in the air spaces is associated with improved survival in patients with ARDS.

Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS.

The goal of this study was to determine the changes that occur in surfactant-associated proteins in bronchoalveolar lavage fluid (BAL) and serum of patients at risk for ARDS and during the course of ARDS. We found that the concentrations of SP-A and SP-B were low in the BAL of patients at risk for ARDS before the onset of clinically defined lung injury, whereas the concentration of SP-D was normal. In patients with established ARDS, BAL SP-A and SP-B concentrations were low during the entire 14-d observation period, but the median SP-D concentrations remained in the normal range. Immunoreactive SP-A and SP-D were not increased in the serum of patients at risk for ARDS, but both increased after the onset of ARDS to a maximum on Day 3 and remained elevated for as long as 14 d. The BAL SP-A concentrations were significantly lower in at-risk patients who developed ARDS, and no patient with a BAL SP-A concentration greater than 1.2 microg/ml developed ARDS. On Days 1 and 3 of ARDS, the BAL SP-D concentration was significantly lower in patients who died, and the BAL SP-D concentration was significantly related to the PI(O(2))/FI(O(2)) ratio. Thus, surfactant protein abnormalities occur before and after the onset of ARDS, and the responses of SP-A, SP-B, and SP-D differ in important ways. The BAL SP-A and SP-D measurements can be used to classify patients as high or low risk for progression to ARDS and/or death after the onset of ARDS. Strategies to increase these surfactant proteins in the lungs of patients with ARDS could be useful to modify the onset or the course of ARDS.

Lipopolysaccharide binding protein and soluble CD14 receptor protein in amniotic fluid and cord blood in patients at term.

OBJECTIVES: Our purpose was to examine whether lipopolysaccharide binding protein and soluble CD14 are present in amniotic fluid and to determine whether the lipopolysaccharide binding protein and soluble CD14 concentrations are associated with indicators of infection or labor at term. A lipopolysaccharide-lipopolysaccharide binding protein complex activates macrophages through soluble CD14 at lipopolysaccharide concentrations up to 100 times lower than required with lipopolysaccharide alone. Thus lipopolysaccharide binding protein and soluble CD14 in amniotic fluid could explain the high concentrations of cytokines found in amniotic fluid of culture-positive patients and may even explain the presence of cytokines in some culture-negative patients. STUDY DESIGN: Healthy women at term undergoing cesarean section had amniotic fluid, chorioamnion, decidua, and cord blood obtained. Lipopolysaccharide binding protein was measured by enzyme-linked immunosorbent assay. Amniotic fluid was cultured and assayed for cytokines, and the chorioamnion and decidua were cultured and examined histologically. RESULTS: Lipopolysaccharide binding protein and soluble CD14 were present in all amniotic fluids and fetal cord blood. An elevated level of lipopolysaccharide binding protein (270 ng/ml/mg of protein) was present in the amniotic fluid of 12 (36%) of the 33 patients. An elevated level was associated with microorganisms in the chorioamnion and decidua, cytokines (tumor necrosis factor-alpha, interleukin-6, and interleukin-8) in amniotic fluid, histologic chorioamnionitis, and labor. Among patients in labor, the concentration of lipopolysaccharide binding protein appeared independent of microorganisms in the amniotic fluid. CONCLUSIONS: Lipopolysaccharide binding protein and soluble CD14 are present in amniotic fluid, and concentrations of lipopolysaccharide binding protein are elevated in patients in labor with and without evidence of infection. Lipopolysaccharide binding protein and soluble CD14 may mediate intrauterine inflammatory responses at term.

Neutrophil apoptosis in the acute respiratory distress syndrome.

Little is known about neutrophil (PMN) apoptosis in the acute respiratory distress syndrome (ARDS). We uses morphologic criteria to count apoptotic PMN in bronchoalveolar lavage fluid (BAL) of 35 patients on Days 1, 3, 7, 14, and 21 of ARDS and 13 patients on Days 1 and 3 of risk for ARDS. We found that the proportion of apoptotic PMN in BAL was low throughout the course of ARDS. There was no significant difference between the percentage of apoptotic PMN in patients at risk and patients with established ARDS or between patients who lived (2.4%) and patients who died (1.8%). When normal human PMN were incubated in ARDS BAL, a significantly lower proportion became apoptotic (50 +/- 4%), as compared with PMN incubated in lavage fluid from normal volunteers (76 +/- 7%, p < 0.05). This antiapoptotic effect of ARDS BAL was blocked by immunodepleting BAL of G-CSF and GM-CSF. We conclude that the proportion of apoptotic PMN recovered from the lungs of patients with ARDS is low throughout the course of ARD S. Furthermore, BAL from patients with ARDS prolongs survival of normal human PMN in vitro, and this effect is partially mediated by G-CSF and GM-CSF.

Pulmonary and systemic inflammatory responses in rabbits with gram-negative pneumonia.

The major goals of this study were to define the relationships between intrapulmonary and systemic inflammatory responses in animals with gram-negative pneumonia. We treated rabbits with intrapulmonary Escherichia coli (1 x 10(7) to 1 x 10(10) cfu/ml), and then measured physiologic, cellular, and molecular events in the lungs and systemic circulation for 24 h. The treatment protocols resulted in groups of animals that mimicked the stages of the septic inflammatory response in humans. Animals treated with low inocula had systemic changes consistent with systemic inflammatory response syndrome and cleared the bacteria and inflammatory products from the lungs. Animals treated with high inocula failed to clear bacteria from the lungs, had severe intrapulmonary inflammatory responses, and developed septic shock. Intrapulmonary leukocyte recruitment was directly related to the size of the bacterial inoculum, but lung protein accumulation was not. Tumor neurosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), and GRO were detectable in lung lavage fluid at 4 h and declined by 24 h in animals that cleared intrapulmonary E. coli. In contrast, lavage TNF-alpha, IL-8, and GRO increased over 24 h in animals that failed to clear intrapulmonary bacteria. MCP-1 increased between 4 h and 24 h in the lungs of all of the animals as the histologic response evolved from neutrophilic to mononuclear cell predominance. Thus, the intensity of systemic inflammatory and physiologic responses to intrapulmonary gram-negative infection depends on the inoculum size and whether the bacteria are cleared from or proliferate in the lungs. The results provide experimental support for the recently proposed classification of septic responses in humans.

Relationship between soluble CD14, lipopolysaccharide binding protein, and the alveolar inflammatory response in patients with acute respiratory distress syndrome.

The effects of bacterial endotoxin (lipopolysaccharide, LPS) are amplified by lipopolysaccharide binding protein (LBP) and CD14, resulting in cellular activation at very low concentrations of LPS. To investigate the importance of this pathway in acute lung injury, we measured LPS, LBP, and soluble CD14 (sCD14) in the bronchoalveolar lavage fluid (BAL) of 82 patients with acute respiratory distress syndrome (ARDS). LBP and sCD14 increased markedly in BAL of patients with ARDS. sCD14 and LBP each were strongly related to BAL total protein and polymorphonuclear neutrophil (PMN) concentration, whereas LPS concentration was not. Multivariate analyses showed sCD14 to be strongly related to BAL total protein, even after controlling for LPS and LBP concentrations. sCD14 was strongly and independently related to PMN concentration, after controlling for BAL LPS, LBP, and interleukin-8 (IL-8). The BAL LPS concentration was not strongly related to either BAL total protein or BAL PMN. The BAL sCD14 and LBP values were similar in all subgroups of patients with ARDS, and were not related to survival. The serum LBP and sCD14 were elevated in ARDS, but were not related to BAL total protein, LBP, sCD14, PMN, or clinical outcome. Thus, LBP and sCD14 reach high concentrations in the lungs of patients with ARDS, and BAL sCD14 is strongly related to two major indices of lung inflammation: total protein and PMN concentration. CD14-dependent mechanisms may contribute to lung inflammation in ARDS.

Deposition of particles in the lungs of infant and adult rats after direct intratracheal administration.

The study of neonatal pulmonary defense mechanisms has been limited by difficulty in administering foreign material into the lungs of newborn animals. We have developed and standardized a simple method for the intratracheal instillation of particles into the distal airways of newborn rats. We used this method to compare intrapulmonary particle deposition in neonatal and adult rats. We instilled 51Cr-labeled microspheres (11 microns diameter) by direct intratracheal inoculation into the lungs of neonatal (< 2- or 19-h-old) and adult (6-week-old) Sprague-Dawley rats. Immediately after microsphere instillation, the lobar distribution of the particles was analyzed by scintillation counting. The anatomic location of the particles was determined by autoradiography. The instilled microspheres reached all lobes in both lungs of neonatal and adult rats. The pattern of distribution in the right lung was nearly identical in the neonatal and adult rats. In the left lung, however, particles deposited preferentially in the cranial lobe in neonates but in the caudal lobe of adult rats. Analysis of the location of particle deposition in the lungs indicated that 64% +/- 6.85 (n = 4) reached the distal airways in the neonates vs. 85% +/- 3.3 (n = 6) in the adults, whereas the remainder deposited in the conducting airways. This method provides an effective means of delivering particles into distal airspaces of neonatal rats and can be used to study pulmonary defense mechanisms in newborn animals.

Effects of endotoxin in the lungs of neonatal rats: age-dependent impairment of the inflammatory response.

Age-dependent maturation of the intrapulmonary inflammatory responses to bacterial lipopolysaccharide (LPS) was studied because nosocomial gram-negative infections cause morbidity in newborn infants. Escherichia coli LPS or live E. coli were injected into the airways of neonatal or adult rats; intrapulmonary recruitment of leukocytes was measured 6 h later. Neonates showed age- and dose-dependent impairment of intrapulmonary neutrophil recruitment after intratracheal administration of LPS or live E. coli that persisted for the first 28 days of life. Neonatal and adult alveolar macrophages released similar amounts of neutrophil chemotactic activity and tumor necrosis factor in response to incubation with LPS in vitro. Treatment of neonates with intratracheal or systemic interferon-gamma did not augment the response to LPS. Thus, intrapulmonary inflammatory responses to LPS and gram-negative bacteria are impaired early in life and do not approach adult levels until approximately 4 weeks of age.

The effect of type-specific polysaccharide capsule on the clearance of group B streptococci from the lungs of infant and adult rats.

To study the importance of the type-specific polysaccharide capsule of group B streptococci (GBS) in the pathogenesis of lung infections, bacterial clearance rates and lung cellular responses to encapsulated and unencapsulated variants of types III and Ia GBS strains were investigated in rats. Bacteria were instilled by direct intratracheal inoculation to simulate aspiration during parturition. Neonates failed to eliminate encapsulated or unencapsulated GBS strains within 6 h of inoculation, whereas adults cleared greater than 90% of each strain within 6 h. Neutrophils accumulated rapidly in the lungs of neonates and adults in response to all GBS strains. Immediately after inoculation, neonatal alveolar macrophages contained fewer encapsulated GBS than did adult alveolar macrophages and fewer encapsulated than unencapsulated GBS, suggesting that the capsule impairs the initial phagocytosis of GBS in the lungs of neonates. Animal age was a more important determinant of bacterial elimination from the lung than the type-specific GBS capsule. However, both age and the bacterial capsule were important determinants of systemic dissemination.