Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counter-regulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages-cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS. Copyright © American Society for Investigative Pathology.

Gut ischemia/reperfusion induced acute lung injury is an alveolar macrophage dependent event

Background:. Although the role of the lung alveolar macrophage (AM) as a mediator of acute lung injury (ALI) after lung ischemia/reperfusion (I/R) has been suggested by animal experiments, it has not been determined whether AMs mediate ALI after intestinal I/R. The objective of this study was to determine the effect of AM elimination on ALI after intestinal I/R in rats. Mwthods: Male Wistar rats (n = 90) were randomly divided into three groups: the clodronate-liposomes (CLOD-LIP) group received intratracheal treatment with CLOD-LIP; the liposomes (LIP) group received intratracheal treatment with LIP; and the nontreated (UNTREAT) group received no treatment. Twenty-four hours later each group was randomly divided into three subgroups: the intestinal I/R subgroup was subjected to 45-minute intestinal ischemia and 2-hour reperfusion; the laparotomy (LAP) subgroup was subjected to LAP and sham procedures; the control (CTR) subgroup received no treatment. At the end of reperfusion, ALI was quantitated in all the animals by the Evans blue dye (EBD) method. Results: ALI values are expressed as EBD lung leakage (mu g EBD/g dry lung weight). EBD lung leakage values in the CLOD-LIP group were 32.59 +/- 12.74 for I/R, 27.74 +/- 7.99 for LAP, and 33.52 +/- 10.17 for CTR. In the LIP group, lung leakage values were 58.02 +/- 18.04 for I/R, 31.90 +/- 8.72 for LAP, and 27.17 +/- 11.48 for CTR. In the UNTREAT group, lung leakage values were 55.60 +/- 10.96 for I/R, 35.99 +/- 6.89 for LAP, and 30.83 +/- 8.41 for CTR. Within each group, LAP values did not differ from CTR values. However, in the LIP and UNTREAT groups, values for both the LAP and CTR subgroups were lower than values for the I/R subgroup (p < 0.001). The CLOD-LIP I/R subgroup value was less (p < 0.001) than the I/R subgroup values in the LIP and UNTREAT groups. These results indicated that I/R provokes ALI that can be prevented by CLOD-LIP treatment, and further suggested that AMs are essential for ALI occurrence induced by intestinal I/R in rats.

Effect of annexin-A1 peptide treatment during lung inflammation induced by lipopolysaccharide

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Endogenous annexin A1 counter-regulates bleomycin-induced lung fibrosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

Multifaceted roles of the lysosome in the establishment and expansion of tuberculous infection

Thesis (Ph.D.)--University of Washington, 2016-04

Effect of remifentanil hydrochloride administered via constant rate infusion on the minimum alveolar concentration of isoflurane in cats

Objective - To evaluate the effects of increasing doses of remifentanil hydrochloride administered via constant rate infusion (CRI) on the minimum alveolar concentration (MAC) of isoflurane in cats. Animals - 6 healthy adult cats. Procedures - For each cat, 2 experiments were performed (2-week interval). On each study day, anesthesia was induced and maintained with isoflurane; a catheter was placed in a cephalic vein for the administration of lactated Ringer's solution or remifentanil CRIs, and a catheter was placed in the jugular vein for collection of blood samples for blood gas analyses. On the first study day, individual basal MAC (MAC Basal) was determined for each cat. On the second study day, 3 remifentanil CRIs (0.25, 0.5, and 1.0 μg/kg/min) were administered (in ascending order); for each infusion, at least 30 minutes elapsed before determination of MAC (designated as MAC R0.25, MAC R0.5, and MAC R1.0, respectively). A 15-minute washout period was allowed between CRIs. A control MAC (MAC Control) was determined after the last remifentanil infusion. Results - Mean ± SD MAC Basal and MAC Control values at sea level did not differ significantly (1.66 ± 0.08% and 1.52 ± 0.21%, respectively). The MAC values determined for each remifentanil CRI did not differ significantly. However, MAC R0.25, MAC R0.5, and MAC R1.0, were significantly decreased, compared with MAC Basal, by 23.4 ± 79%, 29.8 ± 8.3%, and 26.0 ± 9.4%, respectively. Conclusions and Clinical Relevance - The 3 doses of remifentanil administered via CRI resulted in a similar degree of isoflurane MAC reduction in adult cats, indicating that a ceiling effect was achieved following administration of the lowest dose.

Effect of dexmedetomidine on the minimum alveolar concentration of isoflurane in cats

This study reports the effects of dexmedetomidine on the minimum alveolar concentration of isoflurane (MAC iso) in cats. Six healthy adult female cats were used. MAC iso and dexmedetomidine pharmacokinetics had previously been determined in each individual. Cats were anesthetized with isoflurane in oxygen. Dexmedetomidine was administered intravenously using target-controlled infusions to maintain plasma concentrations of 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, and 20ng/mL. MAC iso was determined in triplicate at each target plasma dexmedetomidine concentration. Blood samples were collected and analyzed for dexmedetomidine concentration. The following model was fitted to the concentration-effect data: where MAC iso.c is MAC iso at plasma dexmedetomidine concentration C, MAC iso.0 is MAC iso in the absence of dexmedetomidine, I max is the maximum possible reduction in MAC iso, and IC 50 is the plasma dexmedetomidine concentration producing 50% of I max. Mean±SE MAC iso.0, determined in a previous study conducted under conditions identical to those in this study, was 2.07±0.04. Weighted mean±SE I max, and IC 50 estimated by the model were 1.76±0.07%, and 1.05±0.08ng/mL, respectively. Dexmedetomidine decreased MAC iso in a concentration-dependent manner. The lowest MAC iso predicted by the model was 0.38±0.08%, illustrating that dexmedetomidine alone is not expected to result in immobility in response to noxious stimulation in cats at any plasma concentration. © 2011 Blackwell Publishing Ltd.

Lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed TNF-related apoptosis-inducing ligand

Mononuclear phagocytes have been attributed a crucial role in the host defense toward influenza virus (IV), but their contribution to influenza-induced lung failure is incompletely understood. We demonstrate for the first time that lung-recruited "exudate" macrophages significantly contribute to alveolar epithelial cell (AEC) apoptosis by the release of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a murine model of influenza-induced pneumonia. Using CC-chemokine receptor 2-deficient (CCR2(-/-)) mice characterized by defective inflammatory macrophage recruitment, and blocking anti-CCR2 antibodies, we show that exudate macrophage accumulation in the lungs of influenza-infected mice is associated with pronounced AEC apoptosis and increased lung leakage and mortality. Among several proapoptotic mediators analyzed, TRAIL messenger RNA was found to be markedly up-regulated in alveolar exudate macrophages as compared with peripheral blood monocytes. Moreover, among the different alveolar-recruited leukocyte subsets, TRAIL protein was predominantly expressed on macrophages. Finally, abrogation of TRAIL signaling in exudate macrophages resulted in significantly reduced AEC apoptosis, attenuated lung leakage, and increased survival upon IV infection. Collectively, these findings demonstrate a key role for exudate macrophages in the induction of alveolar leakage and mortality in IV pneumonia. Epithelial cell apoptosis induced by TRAIL-expressing macrophages is identified as a major underlying mechanism.

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5.

Gene targeting was used to create mice with a null mutation of the gene encoding the common beta subunit (beta C) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3; multi-CSF), and interleukin 5 (IL-5) receptor complexes (beta C-/- mice). High-affinity binding of GM-CSF was abolished in beta C-/- bone marrow cells, while cells from heterozygous animals (beta C+/- mice) showed an intermediate number of high-affinity receptors. Binding of IL-3 was unaffected, confirming that the IL-3-specific beta chain remained intact. Eosinophil numbers in peripheral blood and bone marrow of beta C-/- animals were reduced, while other hematological parameters were normal. In clonal cultures of beta C-/- bone marrow cells, even high concentrations of GM-CSF and IL-5 failed to stimulate colony formation, but the cells exhibited normal quantitative responsiveness to stimulation by IL-3 and other growth factors. beta C-/- mice exhibited normal development and survived to young adult life, although they developed pulmonary peribronchovascular lymphoid infiltrates and areas resembling alveolar proteinosis. There was no detectable difference in the systemic clearance and distribution of GM-CSF between beta C-/- and wild-type littermates. The data establish that beta C is normally limiting for high-affinity binding of GM-CSF and demonstrate that systemic clearance of GM-CSF is not mediated via such high-affinity receptor complexes.

Relationship between induced sputum cytology and inflammatory status with lung structural and functional abnormalities in asbestosis

Background Asbestosis is associated with lung cellular and immunological abnormalities. Induced sputum cytology and local and systemic markers of inflammation may be helpful to characterize disease status and progression in these patients. Methods Thirty-nine ex-workers with asbestosis on high-resolution CT (HRCT) and 21 non-exposed controls were evaluated. Sputum cytology and IL-8 in serum and sputum were related to lung function impairment. Results Subjects with asbestosis had reduced sputum cellularity but higher macrophagel neutrophil ratio and % macrophage as compared with controls. Sputum and serum IL-8 were also higher in patients with asbestosis (P < 0.05). In addition, evidence of lung architectural distorption on HRCT was associated with increased levels of serum IL-8. Interestingly, absolute macrophage number was negatively correlated with total lung capacity (r = -0.40; P = 0.04) and serum IL-8 to lung diffiusing capacity (r = -0.45; P = 0.01). Conclusions Occupationally exposed subjects with asbestosis on HRCT have cytologic abnormalities in induced sputum and increased local and systemic pro-inflammatory status which are correlated to functional impairment.

Temporal evolution of epithelial, vascular and interstitial lung injury in an experimental model of idiopathic pulmonary fibrosis induced by butyl-hydroxytoluene

This study was undertaken to test whether the structural remodelling of pulmonary parenchyma can be sequentially altered in a model and method that demonstrate the progression of the disease and result in remodelling within the lungs that is typical of idiopathic pulmonary fibrosis. Three groups of mice were studied: (i) animals that received 3-5-di-tert-butyl-4-hydroxytoluene (BHT) and were killed after 2 weeks (early BHT = 9); (ii) animals that received BHT and were killed after 4 weeks (late BHT = 11); (iii) animals that received corn oil solution (control = 10). The mice were placed in a ventilated Plexiglas chamber with a mixture of pure humidified oxygen and compressed air. Lung histological sections underwent haematoxylin-eosin, immunohistochemistry (epithelial, endothelial and immune cells) and specific staining (collagen/elastic fibres) methods for morphometric analysis. When compared with the control group, early BHT and late BHT groups showed significant decrease of type II pneumocytes, lower vascular density in both and higher endothelial activity. CD4 was increased in late BHT compared with early and control groups, while CD8, macrophage and neutrophil cells were more prominent only in early BHT. The collagenous fibre density were significantly higher only in late BHT, whereas elastic fibre content in late BHT was lower than that in control group. We conclude that the BHT experimental model is pathologically very similar to human usual interstitial pneumonia. This feature is important in the identification of animal models of idiopathic pulmonary fibrosis that can accurately reflect the pathogenesis and progression of the human disease.

G551D CF mice display an abnormal host response and have impaired clearance of Pseudomonas lung disease

Several cystic fibrosis (CF) mouse models demonstrate an increased susceptibility to Pseudomonas aeruginosa lung infection, characterized by excessive inflammation and high rates of mortality. Here we developed a model of chronic P. aeruginosa lung disease in mice homozygous for the murine CF transmembrane conductance regulator G551D mutation that provides an excellent model for CF lung disease. After 3 days of infection with mucoid P. aeruginosa entrapped in agar beads, the G551D animals lost substantially more body weight than non-CF control animals and were less able to control the infection, harboring over 40-fold more bacteria in the lung. The airways of infected G551D animals contained altered concentrations of the inflammatory mediators tumor necrosis factor-alpha, KC/N51, and macrophage inflammatory protein-2 during the first 2 days of infection, suggesting that an ineffective inflammatory response is partly responsible for the clearance defect.

T cell derived cytokines in lung-phase immunity to Schistosoma mansoni

In C57Bl/6 strain mice vaccinated with radiation-attenuated cercariae of Schistosoma mansoni immune elimination of challenge parasites occurs in the lungs. Leococytes were recovered from the lungs of such mice by bronchoalveolar lavage and cultured in vitro with larval antigen; the profile of cytokines released was then analyzed. From 14 days after vaccination, BAL cultures contained infiltrating lymphocytes wich produced abundant quantitties of IFN-g and IL-3. Challenge of vaccinated mice resulted in a second influx of IFN-g nd IL-3- producing cells, earlier than after vaccination or in the appropriate contropls. Ablation studies revealed that CD4+ T cells were the source of IFN-g. The timing of cytokine production after vaccination, and challenge was coincident with the phases of macrophage activation previously reported. At no time could lymphocytes in BAL cultures to stimulated to proliferate with either larval Ag or mitogen, in contrast to splenocytes from the same mice. Furthermore, T cell growth factor activity was not detected in BAL cultures stimulated with Ag. We suggest that the lymphocytes recruited to the lungs are memory/effector cells, When Ag. released challenge schistosomula is presented to these cells, they respond by secreting cytokines wich mediate the formation of cellular aggregates around the parasites, blocking their onward migration.