Effect of alcohol on bacterial translocation in rats

Bacterial translocation has been proposed to be important in the pathophysiology of sepsis, as well as to be a consequence of sepsis. To study the effect of alcohol on bacterial translocation from the gut, normal Sprague-Dawley rats were administered alcohol by gavage by two regimens: Acute (3.7 g/kg, one dose) or Subacute (1 of 2 doses, 2.4 or 3.7 g/kg/day once daily for 14 days). Mesenteric lymph node cultures were performed, and portal venous blood was assayed for endotoxin. Ileal and cecal permeability studies were performed in the Acute and Subacute groups using fluorescein isothiocyanate-labeled dextrans of either 4,000 or 70,000 kDa size. As an index of the effect of systemic endotoxin, tissues from mesenteric lymph nodes, liver, and intestinal Peyer's patches were assayed for the presence of mRNA for tumor necrosis factor. Additionally, because extrapulmonary sepsis has been shown to suppress pulmonary antibacterial defenses, animals in the Subacute group were challenged by aerosol inoculation with Pseudomonas aeruginosa to determine bacterial clearance and alveolar cellular responses. The results show that neither of the alcohol regimens resulted in bacterial growth from mesenteric lymph nodes or portal blood. Animals in the Subacute group had more endotoxin present in portal blood than did the Control group (92.9 pg/ml vs. 40.2 pg/ml; p < 0.02). None of the animals had demonstrable mRNA for tumor necrosis factor in any of the tissues assayed. There were no demonstrable increases in ileal or cecal permeability for either the small or large molecular weight dextran in either alcohol group. Furthermore, there was no delay in the clearance of P. aeruginosa from the lung in the Subacute group, but these animals recruited fewer neutrophils into the airspaces in response to this challenge than did the Control animals. Thus, alcohol intoxication does not result in bacterial translocation from the gut in this model. Despite higher levels of portal venous endotoxin in the animals in the Subacute alcohol group, no adverse systemic consequences of this phenomenon could be demonstrated.     

Modulation of macrophage function for defence of the lung against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common respiratory tract pathogen in certain groups of compromised hosts, most notably those with cystic fibrosis. The pathogenicity of P. aeruginosa may depend in part upon its capacity to resist normal phagocytic cell clearance. We have recently shown that phagocytosis of P. aeruginosa by macrophages is a unique two-step process; binding is glucose-independent but ingestion occurs only in the presence of D-glucose or D-mannose. P. aeruginosa is the only particle we have found which is ingested by macrophages in a glucose-dependent manner. Since glucose is present in only negligible quantities in the endobronchial space, P. aeruginosa may be pathogenic by virtue of its capacity to exploit the opportunity presented in the lower airway to resist normal nonspecific phagocytic defences. The purpose of the studies reported here is to better understand the glucose-dependent phagocytosis of P. aeruginosa and to design novel therapies to facilitate phagocytic cell clearance of it from the lower respiratory tract. We have shown that phagocytosis of unopsonized P. aeruginosa depends upon facilitated transport of glucose into macrophages via the GLUT1 isoform. After transport into the macrophage, the glucose must be metabolized to trigger phagocytosis of P. aeruginosa; pretreatment with 2-deoxyglucose or 5-thioglucose abrogates glucose-dependent ingestion. We have recently demonstrated that pulmonary alveolar macrophages (as opposed to all other macrophage phenotypes studied) lack the capacity to transport glucose and to phagocytose unopsonized P. aeruginosa; however, after the cells have been cultured in vitro for 48 hours, they are able to perform both functions. Whereas most macrophages (such as peritoneal cells) primarily depend upon glycolysis for metabolic energy, pulmonary alveolar macrophages reside in a high oxygen tension environment and appear to utilize oxidative phosphorylation. Treatment of freshly explanted pulmonary alveolar macrophages with sodium azide (to poison oxidative respiration) dramatically enhances both glucose transport and glucose-dependent phagocytosis of P. aeruginosa. We are currently investigating the compromised phagocytic function of pulmonary alveolar macrophages and the mechanism by which azide enhances glucose transport and phagocytosis of P. aeruginosa. Although physiological measurements have indicated that glucose is removed from the endobronchial space by an active transport process of the lung epithelium, the types of glucose transporters that are expressed in the lung are as yet unknown. Using RT-PCR, we have amplified a product from human and murine lung RNA which has a high degree of homology with members of the sodium-dependent glucose transporter (SGLT) family. The ultimate goal of these studies is to design novel agents for enhancing the phagocytic function of pulmonary alveolar macrophages. Delivery of simple glucose by aerosol would not be effective because (i) it would be exported by sodium-dependent active transport and (ii) pulmonary alveolar macrophages lack the capacity to transport glucose. Various approaches for targeting glucose to alveolar macrophages by receptor-mediated endocytosis are under investigation.     

Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia

To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 x 10(5) CFU of P. aeruginosa. In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 x 10(7) CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P. aeruginosa pneumonia.     

5-Lipoxygenase reaction products modulate alveolar macrophage phagocytosis of Klebsiella pneumoniae

The leukotrienes are potent lipid mediators of inflammation formed by the 5-lipoxygenase-catalyzed oxidation of arachidonic acid. Although the effects of leukotrienes on neutrophil chemotaxis and activation have been established, their role in modulating innate host defense mechanisms is poorly understood. In a previous study (M. Bailie, T. Standiford, L. Laichalk, M. Coffey, R. Strieter, and M. Peters-Golden, J. Immunol. 157:5221-5224, 1996), we used 5-lipoxygenase knockout mice to establish a critical role for endogenous leukotrienes in pulmonary clearance and alveolar macrophage phagocytosis of Klebsiella pneumoniae. In the present study, we investigated the role of specific endogenous leukotrienes in phagocytosis of K. pneumoniae and explored the possibility that exogenous leukotrienes could restore phagocytosis in alveolar macrophages with endogenous leukotriene synthesis inhibition and enhance this process in leukotriene-competent cells. Rat alveolar macrophages produced leukotriene B4 (LTB4), LTC4, and 5-hydoxyeicosatetraenoic acid (5-HETE) during the process of phagocytosis, and the inhibition of endogenous leukotriene synthesis with zileuton and MK-886 dramatically attenuated phagocytosis. We also observed a reduction in phagocytosis when we treated alveolar macrophages with antagonists to the plasma membrane receptors for either LTB4, cysteinyl-leukotrienes, or both. In leukotriene-competent cells, LTC4 augmented phagocytosis to the greatest extent, followed by 5-HETE and LTB4. These 5-lipoxygenase reaction products demonstrated similar relative abilities to reconstitute phagocytosis in zileuton-treated rat alveolar macrophages and in alveolar macrophages from 5-lipoxygenase knockout mice. We conclude that endogenous synthesis of all major 5-lipoxygenase reaction products plays an essential role in phagocytosis. The restorative and pharmacologic effects of LTC4, LTB4, and 5-HETE may provide a basis for their exogenous administration as an adjunctive treatment for patients with gram-negative bacterial pneumonia.     

The locus of tumor necrosis factor-alpha action in lung inflammation

The pulmonary host response to infection and inflammation appears, at least in part, to be compartmentalized from the systemic host response. Tumor necrosis factor-alpha (TNF-alpha) has been implicated in lung inflammation and injury, but its site(s) of action has not been clearly defined. To investigate this, transgenic mice (surfactant apoprotein C promotor/soluble TNF receptor type II-Fc fusion protein ([SPCTNFRIIFc] mice) were generated in which TNF-alpha was selectively antagonized in the distal lung through tissue-specific expression of sTNFRIIFc, a soluble TNF inhibitor. The lung inflammatory response in these mice to pulmonary challenge with Micropolyspora faeni antigen or lipopolysaccharide (LPS) was compared with the response of wild-type mice, wild-type mice treated with recombinant sTNFRIIFc intravenously, and type I TNF-receptor knockout mice. Recruitment of polymorphonuclear leukocytes (PMN) to the lung after challenge with M. faeni antigen was essentially abolished in the TNFRI knockout mice and markedly reduced in the SPCTNFRIIFc mice. Wild-type mice given sTNFRIIFc intravenously in amounts resulting in lung concentrations similar to those in SPCTNFRIIFc mice also showed significantly reduced lung PMN recruitment, whereas those given doses that achieved such concentrations in the blood but low levels in the lung did not. In contrast, PMN recruitment to the lung following aerosol challenge with LPS was reduced significantly in the TNFRI knockout mice and in mice given high-dose sTNFRIIFc intravenously, but was not reduced significantly in SPCTNFRIIFc mice. Thus, inhibition of PMN recruitment in response to M. faeni antigen correlated largely with the extent of intrapulmonary inhibition of TNF-alpha, whereas the response to LPS correlated best with the extent of extrapulmonary inhibition of TNF-alpha. These studies indicate that TNF-alpha may act at different loci to mediate lung inflammation, with the site of action depending in part on the nature of the inflammatory stimulus, and that SPCTNFRIIFc mice provide a tool by which the locus of TNF action can be addressed.     

Immunology of the aging lung

The aging process is associated with multiple deficits in pulmonary immune function. Defense of the airway is impaired in the elderly by decreased mucociliary clearance, alteration in respiratory mechanics and, in some cases, concomitant illnesses that predispose to aspiration. Alveolar defenses can be divided into resident defense mechanisms, inflammatory responses, and specific immune responses. Resident defenses such as macrophage phagocytosis and chemotaxis, although largely intact, may have subtle defects under specific conditions. Inflammatory responses may also be diminished, as evidenced by decreased neutrophil-mediated killing and chemotaxis. Specific immune responses appear to be the most vulnerable to age-associated impairment. Although antigen presentation is well preserved during aging, accessory cell cytokine production may be decreased. T-lymphocyte proliferative responses are markedly decreased, as is the production of and response to intercellular mediators. Age-associated alterations in T cell subpopulations may result in imperfect T cell-B cell interactions leading to expression of abnormal immunoglobulins. These many interacting and compounding impairments may explain the increased susceptibility of the elderly to pulmonary infection and autoimmune diseases.     

Alveolar macrophage release of tumor necrosis factor-alpha in chronic alcoholics without liver disease

Alcohol is an immunosuppressive drug, and chronic abuse has been associated with increased susceptibility to a variety of infections, including bacterial pneumonia and tuberculosis. Alveolar macrophages are the resident phagocytes of the lung and play a central role in lung host defenses against infection ranging from direct antibacterial activity to the release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha). TNFalpha, in particular, plays a key role in the development of the early inflammatory response. In this study, we investigated the effects of chronic alcohol consumption on alveolar macrophage release of TNFalpha in vitro. We prospectively studied lipopolysaccharide (LPS)-stimulated release of TNFalpha from alveolar macrophages obtained from bronchoalveolar lavage fluid (BALF) in 22 alcoholic (18 smokers, 4 nonsmokers) and 7 nondrinking healthy volunteers (3 smokers, 4 nonsmokers). The total number of cells recovered by bronchoalveolar lavage (BAL) and their differential distribution were not significantly different in alcoholics versus controls (43 +/- 8 x 10(6) and 39 +/- 13 x 10(6), respectively). However, the total number of cells recovered from BALF was significantly higher in smokers (51 +/- 8 x 10(6)) than in nonsmokers (19 +/- 5 x 10(6)). Spontaneous (basal) release of TNFalpha by alveolar macrophages was the same in alcoholics and controls. In contrast, LPS-stimulated release of TNFalpha was significantly suppressed in alcoholics compared with that of controls (1343 +/- 271 vs. 3806 +/- 926 U TNF/ml/10(6) cells, respectively, p < 0.015). When controlled for smoking, LPS-stimulated TNFalpha production was suppressed in alcoholic nonsmokers (563 +/- 413 U TNF/ml/10(6)) compared with control nonsmokers (5113 +/- 1264 U TNF/ml/10(6)). LPS-stimulated TNFalpha production was also less in control smokers (2063 +/- 386 U TNF/ml/10(6) cells) than in control nonsmokers (5113 +/- 1264 U TNF/ml/10(6) cells). There was no difference in TNFalpha production between smoking alcoholics and smoking control subjects. We conclude that chronic alcohol consumption significantly suppresses LPS-stimulated alveolar macrophage production of TNFalpha. This effect is obscured if the subject also smokes. Because TNFalpha production is an important element in host defense, this may explain, in part, the susceptibility of chronic alcohol abusers to a variety of infections.     

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.     

Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the delta F508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.     

Interleukin-4 enhances pulmonary clearance of Pseudomonas aeruginosa

To determine the effects of interleukin-4 (IL-4) on bacterial clearance from the mouse lung, transgenic mice expressing IL-4 in respiratory epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice) were infected intratracheally with Pseudomonas aeruginosa. Survival of CCSP-IL-4 mice following bacterial administration was markedly improved compared with that of control mice. While bacteria proliferated in lungs of wild-type mice, a rapid reduction in the number of bacteria was observed in the IL-4 mice as early as 6 h postinfection. Similarly, intranasal administration of IL-4 enhanced bacterial clearance from the lungs of wild-type mice. While acute and chronic IL-4 increased the numbers of neutrophils in bronchoalveolar lavage fluid, bacterial infection was associated with acute neutrophilic pulmonary infiltration, and this response was similar in the presence or absence of IL-4. Local administration or expression of IL-4 in the mouse lung enhanced pulmonary clearance of P. aeruginosa in vivo and decreased mortality following infection.     

Estimation of relative economic value for herd life of dairy cattle from profile equations

The phagocytic capability afforded by neutrophil influx into the lungs is essential to ward off invading bacteria. The objective of this study was to evaluate the effect of prior neutrophil recruitment induced by alveolar instillation of endotoxin (LPS, 200 micrograms/kg) 16 h before a pulmonary infection caused by instillation of live Pseudomonas aeruginosa ([PYO]: 1.5 x 10(8) colony-forming units [cfu]/kg) in rats. A first series of experiments showed that lipopolysaccharide (LPS) instillation induced recruitment of alveolar neutrophils that were capable, ex vivo, of elastase exocytosis, reactive oxygen species secretion, and PYO killing. In a second set of experiments, LPS followed by PYO was compared with PYO alone (n = 11 surviving rats in each group). Parameters were studied 24 h after the bacterial challenge. As compared with PYO alone, pretreatment with LPS followed by PYO was associated with decreased mortality (0% versus 54%, p < 0.05), decreased protein leakage into bronchoalveolar lavage (BAL) fluid (1.8 +/- 0.4 versus 13.5 +/- 2.2 mg/ml, p < 0.001), and improved bacterial clearance from BAL (4.0 +/- 1.4 x 10(2) versus 1.2 +/- 0.5 x 10(4) cfu/ml, p < 0.05) and from pulmonary parenchyma (8.5 +/- 6.4 x 10(5) versus 1.9 +/- 0.8 x 10(7) cfu/ml, p < 0.05). We conclude that prior alveolar endotoxin instillation induces local recruitment of functionally active neutrophils, and that this is associated with resistance to subsequent experimental pneumonia.     

TNF and IL-6 mediate MIP-1alpha expression in bleomycin-induced lung injury

Previously, macrophage inflammatory protein-1alpha (MIP-1alpha), a member of the C-C chemokine family, has been implicated in bleomycin-induced pulmonary fibrosis, a model of the human disease idiopathic pulmonary fibrosis. Neutralization of MIP-1alpha protein with anti-MIP-1alpha antibodies significantly attenuated both mononuclear phagocyte recruitment and pulmonary fibrosis in bleomycin-challenged CBA/J mice. However, the specific stimuli for MIP-1alpha expression in the bleomycin-induced lesion have not been characterized. In this report, two mediators of the inflammatory response to bleomycin, tumor necrosis factor (TNF) and interleukin-6 (IL-6), were evaluated as putative stimuli for MIP-1alpha expression after bleomycin challenge in CBA/J mice. Elevated levels of bioactive TNF and IL-6 were detected in bronchoalveolar lavage (BAL) fluid and lung homogenates from bleomycin-treated CBA/J mice at time points post-bleomycin challenge, which precede MIP-1alpha protein expression. Treatment of bleomycin-challenged mice with soluble TNF receptor (sTNFr) or anti-IL-6 antibodies significantly decreased MIP-1alpha protein expression in the lungs. Furthermore, normal alveolar macrophages secreted elevated levels of MIP-1alpha protein in response to treatment with TNF plus IL-6 or bleomycin plus IL-6, but not TNF, bleomycin, or IL-6 alone. Finally, leukocytes recovered from the BAL fluid of bleomycin-challenged mice secreted higher levels of MIP-1alpha protein, compared to controls, when treated with TNF alone. Based on the data presented here, we propose that TNF and IL-6 are part of a cytokine network that modulates MIP-1alpha protein expression in the profibrotic inflammatory lesion during the response to intratracheal bleomycin challenge.     

Humoral immunity and regulation of intrapulmonary growth of Legionella pneumophila in the immunocompetent host

The potential role of humoral immunity in regulating intrapulmonary growth of Legionella pneumophila in the immunocompetent host was investigated using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with a virulent strain of L. pneumophila (10(6) bacteria per mouse) resulted in the recruitment of B lymphocytes into the lung and the development of anti-L. pneumophila Ab. Opsonization of L. pneumophila in vitro with anti-L. pneumophila-specific mAb resulted in a significant decrease in intrapulmonary growth of the bacteria at 24 to 72 h postinfection. Transmission electron microscopic analysis of lung tissue from L. pneumophila- infected mice demonstrated that while there was no significant difference between phagocytosis of the unopsonized and opsonized L. pneumophila by alveolar macrophages at 24 h postinfection, phagocytosis of opsonized bacteria by alveolar mononuclear phagocytic cells was significantly enhanced at 48 h postinfection. Depletion of A/J mice of complement before intratracheal inoculation of opsonized L. pneumophila (10(6) bacteria per mouse) did not significantly alter intrapulmonary growth of L. pneumophila. These results suggest that anti-L. pneumophila Ab, produced during replicative L. pneumophila lung infections, may regulate intrapulmonary growth of L. pneumophila in the immunocompetent host by decreasing the viability of extracellular L. pneumophila and by enhancing phagocytosis of the bacteria by alveolar mononuclear phagocytic cells by a complement-independent mechanism.     

Surfactant protein-A binds group B streptococcus enhancing phagocytosis and clearance from lungs of surfactant protein-A-deficient mice

Surfactant protein-A (SP-A) gene-targeted mice clear group B streptococcus (GBS) from the lungs at a slower rate than wild-type mice. To determine mechanisms by which SP-A enhances pulmonary clearance of GBS, the role of SP-A in binding and phagocytosis of GBS was assessed in SP-A (-/-) mice infected with GBS in the presence and absence of exogenous SP-A. Coadministration of GBS with exogenous SP-A decreased GBS colony counts in lung homogenates of SP-A (-/-) mice. SP-A bound to GBS in a calcium-dependent manner. Although pulmonary infiltration with macrophages was not altered in SP-A (-/-) versus wild-type mice after GBS infection, the number of alveolar macrophages with phagocytosed bacteria was lower in the SP-A (-/-) mice than in the wild-type mice. When SP-A was coadministered with GBS, phagocytosis was significantly increased. Oxygen radical production by alveolar macrophages from SP-A (-/-) mice infected with GBS was decreased compared with wild-type controls and was increased when SP-A (-/-) mice were infected in the presence of exogenous SP-A. Superoxide (SO) radical generation was deficient in macrophages from SP-A (-/-) mice. SP-A plays an important role in GBS clearance in vivo, mediated in part by binding to and enhancing GBS phagocytosis and by increasing SO production by alveolar macrophages.     

Effects of oxygen exposure on it vitro function of pulmonary alveolar macrophages

Bacterial infection may complicate pulmonary oxygen (O2) toxicity, and animals exposed to high O2 concentrations show depressed in vivo pulmonary bacterial inactivation. Therefore, in vitro studies were undertaken to define the mechanism by which O2 alters pulmonary antibacterial activity. Normal and BCG pretreated rabbits were exposed to 100% O2 for 24, 48, and 72-h periods. Pulmonary alveolar macrophages (PAM) were obtained from the experimental animals and from nonoxygen exposed controls by bronchopulmonary lavage. O2 exposure did not alter cell yield or morphology. PAMs were suspended in 10% serum-buffer, and phagocytosis of (14C)Staphylococcus aureus 502A and (14C)Pseudomonas aeruginosa was measured. Comparison of the precent uptake of the 14C-labeled S. aureus after a 60-min incubation period demonstrated that normal PAMs exposed to O2 for 48 h showed a statistically significant increase in phagocytosis when compared to their controls (43.5 vs. 29.2%). A similar, but smaller increase was seen after 24-h O2 exposures. 48 and 72-h O2 exposures produced no significant changes in phagocytosis in PAMs from BCG-stimulated rabbits. Normal PAMs also showed an increased phagocytosis of Ps. aeruginosa after 48-h oxygen exposure. No impairment of in vitro bactericidal activity against either S. aureus 502A or Ps. aeruginosa could be demonstrated in PAMs from normal rabbits exposed to O2 for 48 h. These results indicate that the in vitrophagocytic and bactericidal capacity of the rabbit PAM is relatively resistant to the toxic effects of oxygen, and that imparied in vivo activity may possibly be mediated by effects other than irreversible metabolic damage to these cells. The mechanism for the observed stimulation of phagocytosis remains to be determined.     

Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells

Mycobacterium tuberculosis infects one-third of the world's human population. This widespread infection depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. DNA sequences of M. tuberculosis have been cloned; these confer on a nonpathogenic Escherichia coli strain an ability to invade HeLa cells, augment macrophage phagocytosis, and survive for at least 24 hours inside the human macrophage. This capacity to gain entry into mammalian cells and survive inside the macrophage was localized to two distinct loci on the cloned M. tuberculosis DNA fragment.     

Effect of alveolar lining material on phagocytic and bactericidal activity of lung macrophages against Staphylococcus aureau

Unstimulated rabbits were sacrificed and their lungs washed with heparinized saline. After alveolar macrophages were harvested, the cell-free lavage fluid was centrifuged at 47,000 X g to recover a small, whitish, surface-active pellet (F fraction.) The supernatant was concentrated 15-fold by vacuum dialysis (P fraction). Alveolar macrophages in a serum-free system were challenged with radiolabeled (32P) Staphylococcus aureus preincubated in either balanced salt solution or F or P fraction. A small increase in alveolar macrophage bacterial uptake occurred with P fraction-treated staphylococci. P fraction from locally immunized animals further enhanced phagocytosis. In bactericidial experiments, alveolar macrophages were allowed to phagocytize staphylococci preincubated in either balanced salt solution or F fraction. Intracellular bactericidal activity of alveolar macrophages was quantitated by lysotaphin lysis of extracellular bacteria and quantitation of viable intracellular bacteria. Enhanced lung macrophage bactericidal activity against F fraction-incubated staphylococci was noted.     

IL-10 is a major mediator of sepsis-induced impairment in lung antibacterial host defense

To explore the mechanism of immunosuppression associated with sepsis, we developed a murine model of sepsis-induced Pseudomonas aeruginosa pneumonia. CD-1 mice underwent either cecal ligation and 26-gauge needle puncture (CLP) or sham surgery, followed by the intratracheal (i.t.) administration of P. aeruginosa or saline. Survival in mice undergoing CLP followed 24 h later by the i.t. administration of saline or P. aeruginosa was 58% and 10%, respectively, whereas 95% of animals undergoing sham surgery followed by P. aeruginosa administration survived. Increased mortality in the CLP/P. aeruginosa group was attributable to markedly impaired lung bacterial clearance and the early development of P. aeruginosa bacteremia. The i.t. administration of bacteria to CLP-, but not sham-, operated mice resulted in an impressive intrapulmonary accumulation of neutrophils. Furthermore, P. aeruginosa challenge in septic mice resulted in a relative shift toward enhanced lung IL-10 production concomitant with a trend toward decreased IL-12. The i.p., but not i.t., administration of IL-10 Abs given just before P. aeruginosa challenge in septic mice significantly improved both survival and clearance of bacteria from the lungs of septic animals administered P. aeruginosa. Finally, alveolar macrophages isolated from animals undergoing CLP displayed a marked impairment in the ability to ingest and kill P. aeruginosa ex vivo, and this defect was partially reversed by the in vivo neutralization of IL-10. Collectively, these observations indicate that the septic response substantially impairs lung innate immunity to P. aeruginosa, and this effect is mediated primarily by endogenously produced IL-10.     

Liver as a focus of impaired oxygenation and cytokine production in a porcine model of endotoxicosis

OBJECTIVES: To determine whether the liver is a focus of insufficient oxygenation and whether liver is a source of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in a porcine model of endotoxicosis. DESIGN: In vivo, prospective, controlled, repeated-measures, experimental study. SETTING: Experimental physiology laboratory in a university. SUBJECTS: Juvenile pigs, weighing 22 to 35 kg. INTERVENTIONS: Catheters for blood sampling were inserted into the carotid artery, portal vein, hepatic vein, and pulmonary artery of anesthetized animals. Ultrasonic flow probes were placed on the portal vein and the hepatic artery. During surgery, normal saline was infused intravenously at 25 mL/kg/hr. Following stabilization, animals were allocated randomly to one of two groups. The endotoxemic group (n = 6) received 50 mg/kg of purified Escherichia coli lipopolysaccharide infused into the external jugular vein over 1 hr. The control group (n = 6) received a sham saline infusion infused over 1 hr. Once the endotoxin or sham infusion was initiated, the rate of the intravenous saline infusion was increased to 48 mL/kg/hr for the remainder of the experiment. Measurements were obtained before the endotoxin or sham infusion, immediately after the infusion, and every 30 mins thereafter for 4 hrs. MEASUREMENTS AND MAIN RESULTS: Blood gases, lactate, and bioactive TNF and IL-6 concentrations were measured from the carotid artery, portal vein, hepatic vein, and pulmonary artery. The porcine model is characterized by systemic hypotension, pulmonary hypertension, and maintenance of cardiac output. Despite decreased hepatic oxygen delivery in endotoxemic animals (p < .02), there was no change in hepatic oxygen consumption compared with controls. Throughout the experiment, there was net hepatic consumption of lactate in both groups. There was no significant hepatic production (or consumption) of TNF or IL-6 in either group. CONCLUSIONS: In this porcine model of endotoxicosis, there is a reduction of hepatic oxygen delivery but dysoxia is not present. The liver is not a source of TNF or IL-6 in this model of endotoxicosis.     

Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism

To study the rate and regulation of alveolar fluid clearance in acute pneumonia, we created a model of Pseudomonas aeruginosa pneumonia in rats. To measure alveolar liquid and protein clearance, we instilled into the airspaces a 5% bovine albumin solution with 1.5 microCi of 125I-human albumin, 24 h after intratracheal instillation of bacteria. The concentration of unlabeled and labeled protein in the distal airspaces over 1 h was used as an index of net alveolar fluid clearance. Since there was histologic evidence of alveolar epithelial injury, several methods were used to measure alveolar fluid clearance, including the use of experiments in rats with blood flow and the use of experiments in rats without blood flow, so that movement across the epithelial barrier would be minimized in the latter group. The results with each method were identical. We found that P. aeruginosa pneumonia increased alveolar liquid clearance over 1 h by 48% in studies with blood flow, and by 43% in rats without blood flow, compared with respective controls (P < 0.05). In both studies, this increase was inhibited with amiloride. However, propranolol had no inhibitory effect, thus ruling out a catecholamine-dependent mechanism to explain the increase in alveolar fluid clearance. An antitumor necrosis factor-alpha neutralizing antibody, instilled into the lung 5 min before bacteria, prevented the increase in alveolar liquid clearance in rats with pneumonia (P < 0.05). Also, TNFalpha (5 microg) instilled in normal rats increased alveolar liquid clearance by 43% over 1 h compared with control rats (P < 0.05). In normal rats instilled with TNFalpha, propranolol had no inhibitory effect. In conclusion, gram-negative pneumonia markedly upregulates net alveolar epithelial fluid clearance, in part by a TNFalpha-dependent mechanism. This finding provides a novel mechanism for the upregulation of alveolar epithelial sodium and fluid transport from the distal airspaces of the lung.