Studies of meconium-induced lung injury: inflammatory cytokine expression and apoptosis.

To review current literature related to cellular mechanisms of meconium-induced lung injury (MILI). Review of published experimental in vitro and in vivo MAS studies using human and animal lung cells. We found that meconium induces expression of cytokines and angiotensin II (ANG II)-induced apoptotic process in the lung cells. We postulate that inflammatory cytokines induce ANG II expression, which causes apoptotic cell death after binding to its AT1 receptors. We also demonstrated expression of serpins associated with meconium instillation into the lungs. Serpins are proteins that inhibit cellular proteases and elastases. Expression of serpins may be an attempt to recover lung from these injurious effects. In summary our studies show that whereas meconium induces inflammatory cytokines and subsequent cell apoptosis, the lung cells also try to protect themselves by inducing serpins. The balance of these interactions will determine the residual damage. We believe these new findings are very important in understanding of MILI.

Meconium-induced release of nitric oxide in rabbit alveolar cells.

Previous studies have shown meconium-induced lung injury occurs throughout release of inflammatory cytokines. The exact mechanism of cytokine-induced apoptosis is not known. In this study we hypothesized that meconium-induced apoptosis in the lungs is mediated through the production of inducible nitric oxide (NO). We studied two groups of newborn rabbit pups: one group was instilled with meconium and other with normal saline. We measured precursors of NO in lung lavage from both groups of rabbits and NO levels were calculated accordingly. The levels of NO and NO-derivatives increased significantly in both groups. However NO expression in meconium group 2 h after meconium instillation was significantly higher than in saline-instilled group suggesting NO production plays a role in meconium-induced inflammation.

Characterization of serine/cysteine protease inhibitor alpha1-antitripsin from meconium-instilled rabbit lungs.

We have recently purified from meconium-instilled rabbit lungs a novel serine proteinase inhibitor, with an apparent molecular mass of 50 kDa, which we assign to be alpha1-antitripsin. We hypothesize that serpin may attenuate pulmonary inflammation and improve surfactant function after meconium aspiration. Alpha1-antitripsin is a member of the proteinase inhibitor (serpin) superfamily and inhibitor of neutrophil elastase, and it can be identified as a member of the family by its amino acid sequence due to the high degree of conserved residues. Alpha1-antitripsin is synthesized by epithelial cells, macrophages, monocytes, and neutrophils. Deficiency in alpha1-antitripsin leads to exposure of lungs to uncontrolled proteolytic attack from neutrophil elastase or other damaging factors culminating in lung destruction and cell apoptosis. We hypothesize that accumulation of alpha1-antitripsin in the lungs serves as a predisposed protection against meconium-induced lung injury. In this paper, we show how this knowledge can lead to the development of novel therapeutic approaches for treatment of MAS.

Inflammatory response and apoptosis in newborn lungs after meconium aspiration.

An important feature of meconium-instilled newborn lungs is an inflammatory response and apoptotic cell death. It was recently demonstrated by our group and supported by several other investigators in a relatively short period of time. Apoptosis exists also in healthy lungs, but in meconium-instilled lungs its level is usually dramatically higher. Apoptosis is characterized by loss of cell function, decrease in cell size, and its morphology. Apoptosis plays an important role in normal cell life, but increased levels of apoptosis induce great damage for any tissues. Apoptosis in the lungs has been greatly overlooked for the past decade, and meconium-induced apoptosis is a relatively new event and not effectively studied at the present time. This Review summarized current knowledge regarding meconium-induced inflammation and apoptosis in newborn lungs.

Cell death and lung cell histology in meconium aspirated newborn rabbit lung.

UNLABELLED: Meconium aspiration syndrome (MAS) is a major cause of newborn mortality and morbidity. In this study we investigated the inflammatory responses and morphological changes in the newborn lung to debris-free meconium instillation. We developed a model for studies of MAS using 2-week-old rabbit pups. Cell death was assessed by DNA staining and detection of DNA fragmentation by in situ end labeling. Cell death was seen in association with an increase of inflammatory cytokines levels, studied by ELISA. Necrotic cells were detected by staining of lavage cells with ethidium bromide and 4',6'-diamino-2'-phenylidon. Meconium instillation resulted selectively in loss of airway and alveolar epithelial cells followed by cell death, which increased with time. Necrotic cells looked smaller and damaged with maximal counts at 24 h after instillation. CONCLUSION: Meconium instillation into lungs caused massive cell death, possibly by apoptosis, and necrosis that may have been activated by the inflammatory cytokine production.

Apoptosis of lung epithelial cells in response to TNF-alpha requires angiotensin II generation de novo.

Recent work from this laboratory demonstrated that apoptosis of pulmonary alveolar epithelial cells (AEC) in response to Fas requires angiotensin II (ANGII) generation de novo and binding to its receptor (Wang et al., 1999b, Am J Physiol Lung Cell Mol Physiol 277:L1245-L1250). These findings led us to hypothesize that a similar mechanism might be involved in the induction of AEC apoptosis by TNF-alpha. Apoptosis was detected by assessment of nuclear and chromatin morphology, increased activity of caspase 3, binding of annexin V, and by net cell loss inhibitable by the caspase inhibitor ZVAD-fmk. Purified human TNF-alpha induced dose-dependent apoptosis in primary type II pneumocytes isolated from rats or in the AEC-derived human lung carcinoma cell line A549. Apoptosis in response to TNF-alpha was inhibited in a dose-dependent manner by the nonselective ANGII receptor antagonist saralasin or by the nonthiol ACE inhibitor lisinopril; the inhibition of TNF-induced apoptosis was maximal at 50 microgram/ml saralasin (101% inhibition) and at 0.5 microgram/ml lisinopril (86% inhibition). In both cell culture models, purified TNF-alpha caused a significant increase in the mRNA for angiotensinogen (ANGEN), which was not expressed in unactivated cells. Transfection of primary cultures of rat AEC with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of TNF-stimulated apoptosis by 72% (P < 0.01). Exposure to TNF-alpha increased the concentration of ANGII in the serum-free extracellular medium by fivefold in A549 cell cultures and by 40-fold in primary AEC preparations; further, exposure to TNF-alpha for 40 h caused a net cell loss of 70%, which was completely abrogated by either the caspase inhibitor ZVAD-fmk, lisinopril, or saralasin. Apoptosis in response to TNF-alpha was also completely inhibited by neutralizing antibodies specific for ANGII (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by TNF-alpha requires a functional renin/angiotensin system (RAS) in the target cell. They also suggest that therapeutic control of AEC apoptosis in response to TNF-alpha is feasible through pharmacologic manipulation of the local RAS.

Human lung myofibroblast-derived inducers of alveolar epithelial apoptosis identified as angiotensin peptides.

Earlier work from this laboratory found that fibroblasts isolated from fibrotic human lung [human interstitial pulmonary fibrosis (HIPF)] secrete a soluble inducer(s) of apoptosis in alveolar epithelial cells (AECs) in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L819-L828, 1995]. The cultured human fibroblast strains most active in producing the apoptotic activity contained high numbers of stellate cells expressing alpha-smooth muscle actin, a myofibroblast marker. The apoptotic activity eluted from gel-filtration columns only in fractions corresponding to proteins. Western blotting of the protein fraction identified immunoreactive angiotensinogen (ANGEN), and two-step RT-PCR revealed expression of ANGEN by HIPF fibroblasts but not by normal human lung fibroblasts. Specific ELISA detected angiotensin II (ANG II) at concentrations sixfold higher in HIPF-conditioned medium than in normal fibroblast-conditioned medium. Pretreatment of the concentrated medium with purified renin plus purified angiotensin-converting enzyme (ACE) further increased the ELISA-detectable ANG II eightfold. Apoptosis of AECs in response to HIPF-conditioned medium was completely abrogated by the ANG II receptor antagonist saralasin (50 microg/ml) or anti-ANG II antibodies. These results identify the protein inducers of AEC apoptosis produced by HIPF fibroblasts as ANGEN and its derivative ANG II. They also suggest a mechanism for AEC death adjacent to HIPF myofibroblasts [B. D. Uhal, I. Joshi, C. Ramos, A. Pardo, and M. Selman. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1192-L1199, 1998].

Fas-induced apoptosis of alveolar epithelial cells requires ANG II generation and receptor interaction.

Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L885-L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 microg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II (P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.

Angiotensin II induces apoptosis in human and rat alveolar epithelial cells.

Recent work from this laboratory demonstrated potent inhibition of apoptosis in human alveolar epithelial cells (AECs) by the angiotensin-converting enzyme inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998]. On this basis, we hypothesized that apoptosis in this cell type might be induced by angiotensin II (ANG II) through its interaction with the ANG II receptor. Purified ANG II induced dose-dependent apoptosis in both the human AEC-derived A549 cell line and in primary type II pneumocytes isolated from adult Wistar rats as detected by nuclear and chromatin morphology, caspase-3 activity, and increased binding of annexin V. Apoptosis also was induced in primary rat AECs by purified angiotensinogen. The nonselective ANG II-receptor antagonist saralasin completely abrogated both ANG II- and angiotensinogen-induced apoptosis at a concentration of 50 microgram/ml. With RT-PCR, both cell types expressed the ANG II-receptor subtypes 1 and 2 and angiotensin-converting enzyme (ACE). The nonthiol ACE inhibitor lisinopril blocked apoptosis induced by angiotensinogen, but not apoptosis induced by purified ANG II. These data demonstrate the presence of a functional ANG II-dependent pathway for apoptosis in human and rat AECs and suggest a role for the ANG II receptor and ACE in the induction of AEC apoptosis in vivo.

The Bacteroides fragilis BtgA mobilization protein binds to the oriT region of pBFTM10.

The Bacteroides fragilis conjugal plasmid pBFTM10 contains two genes, btgA and btgB, and a putative oriT region necessary for transfer in Bacteroides fragilis and Escherichia coli. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of btgA revealed three sets of inverted repeats, potentially locating the oriT region. A 304-bp DNA fragment comprising this putative oriT region was cloned and confirmed to be the functional pBFTM10 oriT by bacterial conjugation experiments using E. coli and B. fragilis. btgA was cloned and overexpressed in E. coli, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognate oriT. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to the oriT-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.

Tumor necrosis factor alpha gene regulation: enhancement of C/EBPbeta-induced activation by c-Jun.

Tumor necrosis factor alpha (TNF alpha) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPbeta played an important role in the regulation of the TNF alpha gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPbeta and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPbeta, expression of c-Jun by itself had relatively little effect on TNF alpha promoter activity. However, the combination of C/EBPbeta and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNF alpha promoter. To determine if C/EBPbeta and c-Jun might cooperate to regulate the cellular TNF alpha gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPbeta and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNF alpha than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/-99-bp AP-1 binding site cooperated with endogenous C/EBPbeta in the activation of the -120 TNF alpha promoter-reporter. DNA binding assays using oligonucleotides derived from the TNF alpha promoter suggested that C/EBPbeta and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNF alpha gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPbeta and that this interaction contributes to the expression of the cellular TNF alpha gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNF alpha gene.

Mechanisms of stabilizing nucleosome structure. Study of dissociation of histone octamer from DNA.

The influence of ionic strength on DNA-histone and histone-histone interactions in reconstituted nucleosomes was studied by measuring the parameters of histone tyrosine fluorescence: fluorescence intensity and lambda(max) position. The first parameter is sensitive to histone-DNA interactions. The changes of the second one accrue due to hydrogen bond formation/disruption between tyrosines in the histone H2A-H2B dimer and the (H3-H4)2 tetramer. The simultaneous measurement of these parameters permits the recording of both the dissociation of histone complexes from DNA, as well as changes in histone-histone interactions. As ionic strength is increased, the H2A-H2B histone dimer dissociated first, followed by dissociation of the (H3-H4)2 tetramer [Yager, T.G., McMurray, C.T. and Van Holde, K.E. (1989) Biochemistry 28, 2271-2276]. The H2A-H2B dimer is dissociated in two stages: first, the ionic bonds with DNA were disrupted, followed by the dissociation of the histone dimer from the tetramer. And secondly, the disruption of dimer-tetramer specific H-bonds. It was established that the energy of electrostatic interactions of the histone dimer with DNA within the nucleosome is much less than the energy of interaction of the histone dimer with the tetramer.

The influence of antibiotics and antitumor agents on the relaxation activity of Pisum sativum leaf chloroplast topoisomerase I.

DNA topoisomerase was isolated from pea leaf chloroplasts. The relaxation activity of this topoisomerase was Mg2+ dependent and sensitive to ethidium bromide and novobiocin, a gyrase inhibitor. Chloroplast topoisomerase (Topo I) was ATP independent, as shown by the characteristic gel distribution of topoisomers. Topoisomerase, compared with the known eucaryotic topoisomerase I, was not stimulated by polyamines as are spermidine, spermine, and cadaverine. Ethidium bromide, DAPI, heparin, nalidixic acid, and m-AMSA (but not camptothecin) were able to inhibit the relaxation activity of chloroplast topo I. Nalidixic acid, novobiocin, m-AMSA, camptothecin, and amiloride were tested for their effects on the topoisomerase-catalyzed "cleavage complex" between DNA and chloroplast DNA topoisomerase I.

Rapid method for the fractionation of nuclear proteins and their complexes by batch elution from hydroxyapatite.

A new procedure for the separation and purification of nuclear proteins and their complexes by batch elution from hydroxyapatite is presented. This method allows to isolate such proteins with different basic character faster and more efficiently than procedures using column chromatography, while showing high selectivity, sensitivity, simplicity, mild conditions of purification, reproducibility and protein stability.