Correct usage of multiple transcription initiation sites and C/EBP-dependent transcription activation of the rat XDH/XO TATA-less promoter requires downstream elements located in the coding region of the gene.

In the present study, we have shown that a downstream element located in the coding region of the TATA-less rat xanthine dehydrogenase/oxidase (XDH/XO) gene (-7 to +42) plays an important role in transcription initiation and C/EBP transcriptional activation. Previous work from our laboratory has shown that the promoter is organized with multiple initiator elements (Inr 1, 2, 3 and 4) which are important for transcription initiation. Additionally, we had identified two C/EBP binding sites upstream of this promoter. Deletional and mutational studies revealed that C/EBP binding was not essential for the basal level of transcriptional initation. However when XO-luciferase constructs include downstream sequence extending to +42 there is development of C/EBP sensitivity as well as a shift in the initiator usage. In the absence of the downstream element, primer extension analyses reveals Inr 3 and 4 to be the major start sites but in the presence of this additional sequence the usage is shifted to Inr 1 and 2. This shift in Inr usage more closely resembles that seen in intact macrophages or liver cells. Gel mobility shift assays indicate the presence of several binding factors located in this downstream region, one of which has been identified as YY-1. We postulate that YY-1 allows DNA bending which permits the upstream C/EBP elements to exhibit a transcriptional activation which is not seen when the downstream element is absent. This study presents a potential model for regulation of the XDH/XO promoter.

Multiple domains for initiator binding proteins TFII-I and YY-1 are present in the initiator and upstream regions of the rat XDH/XO TATA-less promoter.

We previously reported that the TATA-less rat xanthine dehydrogenase/oxidase (XDH/XO) promoter is organized with multiple initiator elements (Inr 1, 2, 3 and 4). Additionally, we identified six factor binding footprints in the upstream region of this promoter (FP 1-FP 6), two of which (FP 2 and FP 4) we showed to be C/EBP binding sites. In this report we continue our characterization of the XDH/XO promoter, detailing other cis elements which comprise the Inr and upstream binding factors. Interestingly, multiple binding domains for known initiator binding proteins, YY-1 and USF-related factor/TFII-I, have been identified which potentially play an important role in transcription initiation.

In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

Multiple initiators and C/EBP binding sites are involved in transcription from the TATA-less rat XDH/XO basal promoter.

In the present study, we have explored further the organization of the TATA-less rat xanthine dehydrogenase/oxidase gene (XDH/XO). A DNase I hypersensitive site has been identified which it colocalizes with the basal promoter reported previously [Chow et al. (1994) Nucleic Acids Res., 22, 1846-1854]. Gel mobility shift assays indicate the presence of multiple binding factors located in the promoter. At least six footprints were detected of which two have been shown to be C/EBP binding sites. Members of the C/EBP-alpha and C/EBP-beta, but not C/EBP-delta, family are able to bind to these two sites. Deletional and mutational studies revealed that C/EBP binding is not essential for the basal level of transcription initiation of this promoter. Much of the transcriptional activity resides in the -102 to -7 DNA fragment, which contains all initiator activity which acts unidirectionally. Within this fragment, four putative initiator elements could be identified; interestingly, the linear integrity of these initiators is important for efficient transcription of the XDH/XO gene. Separation of the initiators leads to a complete loss of transcription activity; however, this loss could be partially restored by the introduction of an Sp1 binding site upstream of the separated initiators. Despite a difference in usage/frequency of initiation at the various initiators, primer extension analyses reveal similar positions for transcription initiations in both XDH/XO reporter constructs and in the endogenous XDH/XO gene. The differential usage of initiators may imply a possible post-transcriptional regulation for the XDH/XO gene.

Nitric oxide inactivates xanthine dehydrogenase and xanthine oxidase in interferon-gamma-stimulated macrophages.

Interferon-gamma (IFN-gamma) has been reported to up-regulate transcription of the xanthine dehydrogenase (XDH) gene and to regulate XDH and xanthine oxidase (XO) activity in endothelial cells and liver tissue. Macrophages are a source of XDH/XO activity at inflammatory sites and are functionally regulated by IFN-gamma. We studied the effect of IFN-gamma on XDH and XO in rat bone marrow macrophages, rat alveolar macrophages, and murine RAW cells. Instead of an induction of enzyme activity, XDH/XO activity was almost totally lost after incubation with 100 to 1,000 U/ml of IFN-gamma for 24 h in all three cell types. The loss of cell-associated XDH/XO activity was not correlated with the appearance of XDH/XO activity in the media. In addition, the loss of XDH/XO activity could not be accounted for by transcriptional repression, since there was an increase in steady-state levels of XDH mRNA. To determine whether XDH/XO activity might be lost through nitric oxide-mediated inactivation of XDH/XO, we compared the time course and dose response for XDH/XO inactivation with that of nitric oxide production and found them similar. Treatment with the nitric oxide inhibitor N-monomethyl arginine appeared to totally block inactivation of XDH/XO by IFN-gamma. We conclude that upon stimulation with IFN-gamma, inducible nitric oxide in macrophages leads to post-transcriptional inhibition of XDH/XO, possibly minimizing the potential for tissue injury from XO released from macrophages into the inflammatory milieu. Inactivation of XDH may represent yet another "protective" role for nitric oxide at sites of inflammation.

Endotoxin induces the expression of macrophage inflammatory protein 1 alpha mRNA by rat alveolar and bone marrow-derived macrophages.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a newly described cytokine that is present in large amounts in the culture supernatant of an endotoxin-stimulated murine macrophage-like cell line (RAW 264.7). There is increasing information that suggests that this cytokine mediates acute neutrophilic inflammation, although the mechanism of mediation is unknown. Data examining the production and regulation of MIP-1 alpha by primary rat macrophages are lacking, and MIP-1 alpha has not been studied previously in an animal model of endotoxin-induced neutrophilic alveolitis. In this study, we performed Northern analysis of steady-state rat MIP-1 alpha mRNA using an oligonucleotide probe complementary to amino acids 4-13 of murine MIP-1 alpha. Our data demonstrate that rat alveolar and bone marrow-derived macrophages can be induced by in vitro endotoxin treatment to express a 1.1-kb MIP-1 alpha mRNA. Expression of the mRNA could be elicited by treatment with 0.1 to 10.0 micrograms/ml of endotoxin in vitro with peak steady-state levels detectable up to 9 h after adding endotoxin to the media. Alveolar macrophages recovered by whole lung lavage from endotoxin-treated rats expressed increased amounts of the mRNA homologous to MIP-1 alpha mRNA when treated in vitro with endotoxin. We also found that rat neutrophils could be induced by endotoxin in vitro to express the MIP-1 alpha mRNA. We were able to identify MIP-1 alpha in culture supernatant from endotoxin-stimulated rat alveolar and bone marrow-derived macrophages by immunoprecipitation with a specific goat anti-murine MIP-1 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of alveolar macrophage production of chemoattractants by leukotriene B4 and prostaglandin E2.

Alveolar macrophages (AM) appear to influence the recruitment of neutrophils into the lung by the elaboration of both lipid and peptide chemotactic molecules for neutrophils. Little is known about the mechanisms that regulate production or release of chemotactic molecules by AM or the interaction between these classes of chemotactic molecules. We investigated the hypothesis that the lipid mediator leukotriene B4 (LTB4) has an in vitro regulatory action on the production of chemotactic proteins by AM. In these experiments, the chemotactic activity in AM culture supernatants was measured in a modified Boyden chamber. LTB4 treatment increased AM production of chemotactic activity in excess of what might be attributed to the amount of LTB4 measured in the culture supernatant after the incubation period. This effect was magnified by in vivo administration of endotoxin prior to AM harvesting. Pretreatment with LTB4 caused a sustained 250% increase in AM production of chemotactic activity, yet only negligible amounts of LTB4 were measured by gas chromatography/mass spectrometry in the LTB4-pretreated AM culture supernatants, indicating that LTB4 alone did not account for the chemotactic activity observed in our studies. A chemotactic peptide in LTB4-treated AM culture supernatant could be isolated and separated from LTB4 by molecular sieve chromatography. Purified column fractions contained 80% of the chemotactic activity of endotoxin-stimulated AM culture supernatant and had a molecular mass of 10,000 D. In contrast to LTB4, prostaglandin E2 (PGE2) suppressed chemotactic activity production by endotoxin-stimulated AM by 70%. Pretreatment with PGE2 was not effective; PGE2 had to be present in the AM culture medium during endotoxin exposure in order to exert a suppressive effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Paradoxical regulation by PGE-2 on release of neutrophil chemoattractants by rat bone marrow macrophages.

Prostaglandin E2 (PGE2) was shown to cause up to a 110% increase in the release into media of soluble chemoattractants for neutrophils by cultured rat bone marrow macrophages (RBMM) during a 16 hour incubation period. Coincubating with concentrations of PGE2 of 10 nM and below did not stimulate release of chemoattractants while concentrations between 10(2) and 10(4) nM increased the chemotactic activity of conditioned medium by 40% to 110% (p less than 0.05). In contrast to the effect of coincubating, pre-treatment with PGE2 for 2 and 4 hours was ineffective in stimulating the release of chemoattractants by RBMM. We also assessed whether PGE-2 modulated the release of chemoattractants by RBMM stimulated with endotoxin (LPS). LPS caused a four fold increase in the production of chemoattractants with a peak effect found at an LPS concentration of 1 microgram/ml. Coincubating with PGE2 in concentrations between 10(2) and 10(4) nM paradoxically decreased LPS-stimulated production of chemoattractants by up to 40% (p less than 0.05). Pre-treatment with PGE2 for 4 hours partially blocked LPS-stimulated release of chemotactic activity. These data indicate that PGE-2 has paradoxical effects on the production of chemoattractants by RBMM: being independently stimulatory but down regulating the effects of LPS. These findings suggest the possibility that the activation state of the RBMM may determine the effect of PGE2: quiescent RBMM can be stimulated by PGE2 but LPS-activated RBMM may be suppressed.

Mechanisms and mediators of the adult respiratory distress syndrome.

In 1990, we are much less certain that we understand ARDS than we were in 1982, and we have yet to identify specific therapy. It is tempting to conclude that we have made no progress, but this conclusion would be unwarranted. In those 8 years, important advances have been made. The complement hypothesis has survived, with significant modifications. Recognition of the importance of infection in clinical outcome and of endotoxin in augmentation of neutrophil-mediated injury has evolved in concert and meshes well. A new class of peptide mediators, cytokines, has assumed a central role. Lipid mediators now appear as modulators of cytokine-induced effects by priming, amplification, and regulation of gene expression rather than as unifactorial "causes" of the physiologic manifestations of ARDS. These interdigitating mechanisms have been recognized as pansystemic, resulting in overt multiple organ dysfunction and ultimately in death if amplification mechanisms go unchecked. Technologies in molecular genetics, generally unknown to the pulmonary community in 1982, have had a significant impact. Recombinant cDNA technology has permitted identification of the existence, structure, and functions of novel cytokines; made them available in sufficient quantity for detailed study; and prompted interest in the regulation of gene expression in the evolution and resolution of inflammation. Proteins modified by genetic engineering, as well as monoclonal antibodies and receptor antagonists for specific cytokines, are promising future approaches to therapy. At present, the complexity of the redundant networks by which inflammation is regulated seems bewildering in relation to ARDS. Bewildering or not, the age of the "mediator" of ARDS, and of the corresponding therapeutic "magic bullet," is over. The complexity of the system of regulatory checks and balances must be addressed at the molecular level.

Protection by deferoxamine from endothelial injury: a possible link with inhibition of intracellular xanthine oxidase.

Hydroxyl radical scavengers and xanthine oxidase inhibitors protect cultured bovine pulmonary endothelial cells (BPAEC) from lytic injury by the endotoxin lipopolysaccharide (LPS). We hypothesized that exposure of BPAEC to cytotoxic concentrations of LPS activated intracellular xanthine oxidase, and that intracellular iron-dependent hydroxyl radical formation (a Fenton reaction) ensued, resulting in cell lysis. To test this, the protective effects of deferoxamine against H2O2 and LPS-induced cytotoxicity to BPAEC was assessed by 51Cr release. Preincubation with 0.4 mM deferoxamine conferred 67 +/- 15% (mean +/- SE) protection from LPS-induced cytotoxicity but 48 h of preincubation were required to induce significant protection. Significant protection form a classical Fenton reaction model, injury by 50 microM H2O2, could be induced by a 1-h preincubation with a 0.4 mM deferoxamine. The dissociated time course suggested that deferoxamine might work by different mechanisms in these models. The effects of LPS and deferoxamine on BPAEC-associated xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity were assessed using a spectrofluorophotometric measurement of the conversion of pterin to isoxanthopterin. BPAEC had 106 +/- 7 microU/mg XD+XO activity; XO activity constituted 48 +/- 1% of total XO+XD activity. LPS at a cytotoxic concentration did not alter XO, XD, or percent XO. Deferoxamine had striking proportional inhibitory effects on XO and XD in intact cells. XO+XD activity fell to 6 +/- 1% of control levels during a 48-h exposure of BPAEC to deferoxamine. Deferoxamine did not inhibit XO+XD ex vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of endotoxin-induced cell injury on 70-kD heat shock proteins in bovine lung endothelial cells.

Heat shock proteins (HSPs) have been remarkably conserved throughout evolution. It has been assumed that induction of HSPs remains a stereotypic response to injury, important for survival of eukaryotic cells during euthermic injury. However, there are few studies of this phenomenon in endothelial cells, and none in pulmonary endothelial cells. We studied the induction of synthesis of 70-kD proteins in bovine pulmonary artery endothelial cells (BPAECs) in response to heat shock and to euthermic injury induced by bacterial endotoxin. First, in response to heat, BPAECs showed rapid and reversible heat-induced synthesis of 70-kD proteins, readily detectable by one-dimensional SDS-PAGE of [35S]methionine-labeled BPAECs. Heat shock at 42 degrees C for 3 h or 43 degrees C for 2 h suppressed total protein synthesis by 30% (P less than 0.001) but an increased rate of synthesis of 70-kD protein continued, representing an increasing fraction of total protein synthesis. Heat-induced synthesis of 70-kD protein returned to baseline levels 8 h after heat shock. Northern analysis showed that mRNA for a protein homologous to a conserved amino acid sequence in the family of species-homologous 70-kD heat shock proteins (HSP 70) was induced by a 15-min incubation at 42 degrees C and remained detectably increased for 6 h. We next assessed whether euthermic injury by bacterial endotoxin (LPS) generated a similar response. LPS was cytotoxic by BPAECs as assessed morphologically, by release of 51Cr from prelabeled cells, and by a significant suppression of total protein synthesis (range, 35 to 70%; P less than 0.001). Despite cytotoxicity, LPS did not induce 70-kD protein at a level that could be detected by SDS-PAGE, and no increase in mRNA for HSP 70 was detected by Northern analysis. LPS-injured BPAECs remained "competent" to induce both 70-kD proteins and mRNA for HSP 70 in response to heat shock. We conclude that at least quantitatively, induction of HSP 70 by BPAECs is not a stereotypic response to injury but rather is at least relatively injury-specific. However, competence to induce HSP 70 appears to be extremely resilient: it is retained in dysfunctional BPAECs in the face of profound inhibition of global protein synthesis, suggesting an important homeostatic role.

Effects of D-galactosamine-induced acute liver injury on mortality and pulmonary responses to Escherichia coli lipopolysaccharide. Modulation by arachidonic acid metabolites.

Multiple extrapulmonary organ system failures increase mortality, permeability edema, and alveolar inflammation during gram-negative sepsis because of abnormal regulation of host inflammatory responses. We tested the hypothesis that acute hepatocytic injury induced by the selective hepatotoxin, D-galactosamine (GalN), augments mortality and amplifies pulmonary microvascular permeability to albumin and neutrophilic influx after administering Escherichia coli lipopolysaccharide (LPS) 24 h later by impairing the metabolism of endogenously synthesized products of arachidonic acid. We determined the lung extravascular leak of 125I-human serum albumin measured at multiple time points after LPS and enumerated polymorphonuclear leukocytes (PMNs) in bronchoalveolar lavage fluid (BALF). Because the liver is important in prostaglandin (PG) and leukotriene (LT) metabolism, we measured plasma concentrations of 6-keto-PGF1 alpha and thromboxane B2 (TxB2) in addition to paired plasma BALF concentrations of LTB4 and BALF LTC4 60 min and 24 h after LPS. We further assessed the protective effects of a single 20-mg/kg injection given intraperitoneally (i.p.) of the LTA4 synthetase inhibitor, diethylcarbamazine (DEC). After 400 mg/kg GalN, LPS at 2.5 or 1.25 mg/kg i.p. increased mortality (p less than 0.001), albumin leak 60 and 90 min after LPS (p less than 0.05), plasma 6-keto-PGF1 alpha, TxB2, and LTB4 levels and BALF LTC4 within 60 min (p less than 0.05). LTB4 and LTC4 levels in BALF 24 h later were similarly increased (p less than 0.05) as were bronchoalveolar PMNs (p less than 0.001). DEC improved mortality and albumin leak (p less than 0.001), reduced lung influx of PMNs and peripheral leukocytosis (p less than 0.05), attenuated plasma LTB4 and BALF LTC4 levels 60 min after LPS (p less than 0.05), and decreased BALF LTB4 and LTC4 at 24 h (p less than 0.05), but was associated with higher plasma 6-keto-PGF1 alpha and TxB2 values at 60 min. Changes in eicosanoid levels and modulation of responses by DEC in this model suggest that impaired metabolism of endogenously synthesized leukotriences by the damaged liver underlies these phenomena. We conclude that this mechanism may enhance septic lung injury during acute liver dysfunction.

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor.

Confluent monolayers of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells (HUVE) inhibited by 80 to 90% the production of O2- by added human neutrophils (PMNs) stimulated by plasma membrane receptor-mediated activators (formylmethionylleucylphenylalanine [fMLP], opsonized zymosan, heat-killed Staphylococci), but not by non-plasma membrane receptor-mediated activators (phorbol myristate acetate and delta-hexachlorocyclohexane). Degranulation induced by fMLP was also inhibited by BPAE. Inhibition was not affected by eicosatetraynoic acid (ETYA) or indomethacin. To assess the role of cell-cell contact, 0.45-microns-pore culture plate inserts were employed to prevent PMN-endothelial cell contact during incubation. A similar amount of inhibition of stimulated PMNs superoxide production was seen as compared to PMN-endothelial incubations where contact occurred. A soluble component released by BPAE monolayers, when added to PMNs, duplicated the inhibition seen by BPAE-PMN co-incubation. Incubation of BPAE with adenosine deaminase did not reduce inhibition of O2- production compared to controls without adenosine deaminase. There was no evidence of endothelial scavenging of O2- generated by hypoxanthine-xanthine oxidase, and inhibition of endothelial superoxide dismutase did not diminish the inhibitory effort. We conclude that cell contact is not required for BPAE inhibition of fMLP-stimulated O2- production by PMN, and that scavenging of superoxide anion is not the mechanism. The inhibitor appears to be a polypeptide with an apparent molecular weight between 1,000 and 10,000 D and does not appear to be adenosine, an arachidonate metabolite, or superoxide dismutase. The mechanism may involve down-regulation of plasma membrane receptor-mediated activation of PMNs.

Lung microvascular transport properties measured by multiple indicator dilution methods in patients with adult respiratory distress syndrome. A comparison between patients reversing respiratory failure and those failing to reverse.

We conducted indicator dilution studies on the lungs of patients in the early phases of adult respiratory distress syndrome (ARDS) to test the hypothesis that capillary permeability was increased in patients with respiratory failure. Indicator dilution studies were performed using 51Cr-erythrocytes, 125I-albumin, 14C-urea, and 3H-water as tracers. The injectate was infused as a bolus into a central venous line. Peripheral arterial blood was collected and counted for radioactivity. Mathematical analysis of the indicator curves yielded cardiac output, measures of the product of capillary permeability and surface area for urea (PS and D1/2S), the intravascular lung volume (Vv), and the extravascular lung water volume (Ve). Permeability was separated from surface area by normalizing PS and D1/2S to Vv. Patients could be divided into 16 in whom blood gas determinations and radiologic criteria for ARDS were reversed and 23 in whom they were not. We examined indicator dilution and other measures of lung function in the two groups to determine whether significant differences in microvascular function existed. PS and PS/Vv were significantly higher in the nonreversal patients. Ve was above normal, but not different between groups. Linear regression analysis showed significant correlations for all of the following in the nonreversal group: Ve and all measures of permeability, pulmonary vascular resistance (PVR), and the inverse of permeability-surface area measures and AaDO2 and PVR. Only measures of Ve and PS correlated in the reversal group. These results support the hypothesis that capillary permeability is increased in patients with early ARDS and continuing respiratory failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased intrapulmonary retention of radiolabeled neutrophils in early oxygen toxicity.

Sequential lung injuries, such as oxygen toxicity followed by septicemia, are common during the adult respiratory distress syndrome (ARDS). As these forms of vascular injury may be mediated in part by polymorphonuclear leukocytes (PMN), aberrant interactions between PMN and previously injured pulmonary endothelium are of both theoretical interest and clinical importance. The present study was undertaken to test the hypothesis that early oxygen toxicity at a dose that injuries pulmonary endothelium relatively selectively alters intrapulmonary neutrophil kinetics. Unanesthetized rats breathing 1.0 atmospheres oxygen for 36 h showed ultrastructural endothelial damage but no edema, injury, or neutrophilic inflammation by histologic criteria. However, in these oxygen-toxic animals, whereas initial accumulation of radiolabeled PMN in lungs was normal, washout of PMN was abnormal at 120 min after infusion, at which point the pulmonary retention of radiolabeled PMN in the lungs of oxygen-treated animals was significantly higher than in control animals (139% of control, p less than 0.0096). Features of our methodology, including avoidance of osmotic stress and use of paired control animals, appear to have greatly enhanced the sensitivity of radiolabeled neutrophils for detecting a subtle abnormality of neutrophil-endothelial interactions. Our studies in the oxygen toxicity model provide the first demonstration in vivo of abnormal intrapulmonary neutrophil kinetics in early oxygen toxicity prior to the onset of histologic evidence of lung injury or inflammation.

High-dose corticosteroids in patients with the adult respiratory distress syndrome.

Corticosteroids are widely used as therapy for the adult respiratory distress syndrome (ARDS) without proof of efficacy. We conducted a prospective, randomized, double-blind, placebo-controlled trial of methylprednisolone therapy in 99 patients with refractory hypoxemia, diffuse bilateral infiltrates on chest radiography and absence of congestive heart failure documented by pulmonary-artery catheterization. The causes of ARDS included sepsis (27 percent), aspiration pneumonia (18 percent), pancreatitis (4 percent), shock (2 percent), fat emboli (1 percent), and miscellaneous causes or more than one cause (42 percent). Fifty patients received methylprednisolone (30 mg per kilogram of body weight every six hours for 24 hours), and 49 received placebo according to the same schedule. Serial measurements were made of pulmonary shunting, the ratio of partial pressure of arterial oxygen to partial pressure of alveolar oxygen, the chest radiograph severity score, total thoracic compliance, and pulmonary-artery pressure. We observed no statistical differences between groups in these characteristics upon entry or during the five days after entry. Forty-five days after entry there were no differences between the methylprednisolone and placebo groups in mortality (respectively, 30 of 50 [60 percent; 95 percent confidence interval, 46 to 74] and 31 of 49 [63 percent; 95 percent confidence interval, 49 to 77]; P = 0.74) or in the reversal of ARDS (18 of 50 [36 percent] vs. 19 of 49 [39 percent]; P = 0.77). However, the relatively wide confidence intervals in the mortality data make it impossible to exclude a small effect of treatment. Infectious complications were similar in the methylprednisolone group (8 of 50 [16 percent]) and the placebo group (5 of 49 [10 percent]; P = 0.60). Our data suggest that in patients with established ARDS due to sepsis, aspiration, or a mixed cause, high-dose methylprednisolone does not affect outcome.

Shift in subfractions of rat alveolar macrophages in vivo during endotoxin-induced alveolitis.

To elucidate changes in alveolar macrophages that accompany sepsis-induced lung injury, this study analyzed the subfractions of alveolar macrophages (AM) recovered by lung lavage during the onset of endotoxin-induced acute neutrophilic alveolar inflammation in the rat model. Centrifugation on continuous self-generated density gradients of Percoll was used to fractionate AM into subpopulations between density limits 1.012 and 1.130. Two-thirds of AM recovered from pathogen-free control rats (group C) were in a fraction with a density range of 1.058-1.078 ["normal" density fraction, (ND)]. Only 6% were located in a very low density (VLD) fraction 1.037-1.048. Neutrophils accounted for less than 1% of recovered cells and usually were found in the fraction with density range of 1.079-1.130. By contrast, if rats underwent lung lavage 15 hours after the administration of endotoxin (group E), only 38% of macrophages were recovered in the "normal" density fraction, whereas 26% of the AM recovered were in the VLD fraction. This shift in the relative sizes of the density based subpopulations coincided with the onset of acute bronchoalveolar inflammation as indicated by the recovery of neutrophils by bronchoalveolar lavage (PMN = 7 X 10(4) in C, vs. 9.4 X 10(5) in E, p less than .001). The macrophages on the low density subfractions showed functional impairment: they were less viable in culture and migrated poorly in response to endotoxin-activated serum compared to macrophages in the "normal" density fraction from the endotoxin-treated animals. The rapid emergence of the low density population after endotoxin could represent an influx of new cells, but more likely indicates that injury to or previous activation of resident macrophages has caused their density to decrease. We speculate that the emergence of a population of AM in airspaces with low density and impaired function could weaken pulmonary host defence following endotoxemia.