Shizu Hayashi

Alveolar macrophage-epithelial cell interaction following exposure to atmospheric particles induces the release of mediators involved in monocyte mobilization and recruitment

BackgroundStudies from our laboratory have shown that human alveolar macrophages (AM) and bronchial epithelial cells (HBEC) exposed to ambient particles (PM10) in vitro increase their production of inflammatory mediators and that supernatants from PM10-exposed cells shorten the transit time of monocytes through the bone marrow and promote their release into the circulation.MethodsThe present study concerns co-culture of AM and HBEC exposed to PM10 (EHC-93) and the production of mediators involved in monocyte kinetics measured at both the mRNA and protein levels. The experiments were also designed to determine the role of the adhesive interaction between these cells via the intercellular adhesion molecule (ICAM)-1 in the production of these mediators.ResultsAM/HBEC co-cultures exposed to 100 μg/ml of PM10 for 2 or 24 h increased their levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, macrophage inflammatory protein (MIP)-1β, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6 and ICAM-1 mRNA, compared to exposed AM or HBEC mono-cultures, or control non-exposed co-cultures. The levels of GM-CSF, M-CSF, MIP-1β and IL-6 increased in co-cultured supernatants collected after 24 h exposure compared to control cells (p < 0.05). There was synergy between AM and HBEC in the production of GM-CSF, MIP-1β and IL-6. But neither pretreatment of HBEC with blocking antibodies against ICAM-1 nor cross-linking of ICAM-1 on HBEC blocked the PM10-induced increase in co-culture mRNA expression.ConclusionWe conclude that an ICAM-1 independent interaction between AM and HBEC, lung cells that process inhaled particles, increases the production and release of mediators that enhance bone marrow turnover of monocytes and their recruitment into tissues. We speculate that this interaction amplifies PM10-induced lung inflammation and contributes to both the pulmonary and systemic morbidity associated with exposure to air pollution.

A gene expression signature of emphysema-related lung destruction and its reversal by the tripeptide GHK

BackgroundChronic obstructive pulmonary disease (COPD) is a heterogeneous disease consisting of emphysema, small airway obstruction, and/or chronic bronchitis that results in significant loss of lung function over time.MethodsIn order to gain insights into the molecular pathways underlying progression of emphysema and explore computational strategies for identifying COPD therapeutics, we profiled gene expression in lung tissue samples obtained from regions within the same lung with varying amounts of emphysematous destruction from smokers with COPD (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified in each tissue sample using the mean linear intercept (Lm) between alveolar walls from micro-CT scans.ResultsWe identified 127 genes whose expression levels were significantly associated with regional emphysema severity while controlling for gene expression differences between individuals. Genes increasing in expression with increasing emphysematous destruction included those involved in inflammation, such as the B-cell receptor signaling pathway, while genes decreasing in expression were enriched in tissue repair processes, including the transforming growth factor beta (TGFβ) pathway, actin organization, and integrin signaling. We found concordant differential expression of these emphysema severity-associated genes in four cross-sectional studies of COPD. Using the Connectivity Map, we identified GHK as a compound that can reverse the gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with TGFβ pathway activation. Treatment of human fibroblasts with GHK recapitulated TGFβ-induced gene-expression patterns, led to the organization of the actin cytoskeleton, and elevated the expression of integrin β1. Furthermore, addition of GHK or TGFβ restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD.ConclusionsThese results demonstrate that gene-expression changes associated with regional emphysema severity within an individuals lung can provide insights into emphysema pathogenesis and identify novel therapeutic opportunities for this deadly disease. They also suggest the need for additional studies to examine the mechanisms by which TGFβ and GHK each reverse the gene-expression signature of emphysematous destruction and the effects of this reversal on disease progression.

Host Response to the Lung Microbiome in Chronic Obstructive Pulmonary Disease

RATIONALEnThe relatively sparse but diverse microbiome in human lungs may become less diverse in chronic obstructive pulmonary disease (COPD). This article examines the relationship of this microbiome to emphysematous tissue destruction, number of terminal bronchioles, infiltrating inflammatory cells, and host gene expression.nnnMETHODSnCulture-independent pyrosequencing microbiome analysis was used to examine the V3-V5 regions of bacterial 16S ribosomal DNA in 40 samples of lung from 5 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 4) and 28 samples from 4 donors (controls). A second protocol based on the V1-V3 regions was used to verify the bacterial microbiome results. Within lung tissue samples the microbiome was compared with results of micro-computed tomography, infiltrating inflammatory cells measured by quantitative histology, and host gene expression.nnnMEASUREMENTS AND MAIN RESULTSnTen operational taxonomic units (OTUs) was found sufficient to discriminate between control and GOLD stage 4 lung tissue, which included known pathogens such as Haemophilus influenzae. We also observed a decline in microbial diversity that was associated with emphysematous destruction, remodeling of the bronchiolar and alveolar tissue, and the infiltration of the tissue by CD4(+) T cells. Specific OTUs were also associated with neutrophils, eosinophils, and B-cell infiltration (Pu2009<u20090.05). The expression profiles of 859 genes and 235 genes were associated with either enrichment or reductions of Firmicutes and Proteobacteria, respectively, at a false discovery rate cutoff of less than 0.1.nnnCONCLUSIONSnThese results support the hypothesis that there is a host immune response to microorganisms within the lung microbiome that appears to contribute to the pathogenesis of COPD.

Chromosomal structure is altered by mutations that suppress or enhance position effect variegation

We examined the genetic, morphological, and molecular effects of position effect variegation inDrosophila, and the effects of mutations that either suppress [Su(var)] or enhance [E(var)] this phenomenon. All eightSu(var) mutations examined strongly suppress the inactivation of variegating alleles of the genes white [In(l) wm4], brown [In (2R)bwVDe2] and Stubble [T(2;3)SbV]. TheE(var) mutation enhances variegation of these loci. The chromosomal region 3C-E (26 bands) which includes the white locus is usually packaged as heterochromatin in salivary glands of the variegating strainwm4. Addition of any of theSu(var) mutations restores a more euchromatic morphology to this region. In situ hybridization to polytene chromosomes and DNA blot analyses of gene copy number demonstrate that the DNA of thew+ gene is less accessible to its probe in the variegatingwm4 strain than it is in the wildtype or variegation-suppressed strains. Blot analysis of larval salivary gland DNA indicates that the white gene copy number does not vary among the strains. Hence, the differences in binding of thew+ gene probe in the variegating and variegation-suppressed strains reflect differences in chromosomal packaging rather than alterations in gene number. The effects of variegation and theSu(var) mutations on chromatin structure were analyzed further by DNAse I digestion and DNA blot hybridization. In contrast to their dramatic effects on chromosomal morphology and gene expression, theSu(var) mutations had negligible effects on nuclease sensitivity of the white gene chromatin. We suggest that the changes in gene expression resulting from position effect variegation and the action of theSu(var) mutations involve alterations in chromosomal packaging.

What Drives the Peripheral Lung–Remodeling Process in Chronic Obstructive Pulmonary Disease?

The smaller airways (<2 mm in diameter) offer little resistance in normal lungs but become the major site of obstruction in chronic obstructive pulmonary disease (COPD). We examined bronchiolar remodeling in COPD by combining quantitative histology, micro-computed tomography (CT), and gene expression studies. Volumes of bronchiolar tissue, total collagen, collagen-1, and collagen-3 were measured in lung tissue from 52 patients with different levels of COPD severity. Micro-CT was used to measure the number and lumen area of terminal bronchioles in four lungs removed before lung transplantation and in four donor lungs that served as controls. Laser capture microdissection provided 136 paired samples of bronchiolar and surrounding lung tissue from 63 patients and the gene expression of a cluster of tissue repair genes was compared. This study shows that total bronchiolar tissue decreased with progression of COPD and was associated with a reduction in total collagen and relative increase in collagen-3 over collagen-1. The micro-CT studies showed a 10-fold reduction in terminal bronchiolar number and a 100-fold reduction in lumen area. Interestingly, most genes associated with tissue accumulation during repair decreased their expression in both airways and in the surrounding lung as FEV(1) declined, but eight genes previously associated with COPD increased expression in the surrounding lung tissue. Our study shows that small airway remodeling is associated with narrowing and obliteration of the terminal bronchioles that begins before emphysematous destruction in COPD and in relation to differential expression of tissue repair genes in the airways and surrounding lung.

Endotoxin-specific NF-κB activation in pulmonary epithelial cells harboring adenovirus E1A

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-κB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-κB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c- jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-κB induction in these cells.

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes.

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila salivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC; tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5–7, 72F1–2, 83F-84A; tRNA3Met, 46A1–2, 61D1–2, 70F1–2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84D3–4, 92B1–9; tRNA4Val, 56D3–7, 70BC.

Functional Changes in Aging Polymorphonuclear Leukocytes

BACKGROUNDnPrevious studies from our laboratory have shown that the expression of L-selectin on polymorphonuclear neutrophils (PMN) decreases as the cell ages in the circulation and that these older PMN have more fragmented DNA and show morphological features of apoptosis.nnnMETHODS AND RESULTSnThe present study was designed to compare the functional capabilities of PMN expressing low levels of L-selectin (L-selectin[low]) and the total population of PMN they were isolated from (L-selectin[mixed]). The results show no difference of the baseline filamentous actin (F-actin) content between PMN expressing low and high levels of L-selectin. However, the ability of L-selectin(low) PMN to assemble F-actin was impaired after stimulation by n-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (1 nmol/L fMLP: P<.02, 10 nmol/L fMLP: P<.01). The ability of L-selectin(low) PMN to change shape when stimulated (10 nmol/L fMLP) was also decreased (P<.05). Filtration studies showed no difference in baseline deformability between L-selectin(low) and L-selectin(mixed) leukocytes, but the L-selectin(low) cells showed a decreased ability to stiffen after fMLP stimulation (P<.05). L-selectin(low) cells demonstrated a decreased ability to migrate toward a chemoattractant (1, 3, and 10 nmol/L fMLP) (P<.004) but have an enhanced ability to upregulate CD18 (P<.00002) and produce hydrogen peroxide (P<.00004).nnnCONCLUSIONSnWe conclude that PMN undergo substantial functional changes as they age in the circulation.

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.

A Lung Tissue Bank for Gene Expression Studies in Chronic Obstructive Pulmonary Disease

A bank of surgically resected human lung tissues frozen at − 70°C after being inflated with support medium for cutting frozen tissue and a separate group inflated with fixative and embedded in paraffin has been established for studies of chronic obstructive pulmonary disease. The present report concerns the quality of RNA that can be extracted from these frozen and fixed tissue samples and from cells obtained from them by laser capture microdissection. The results show that the RNA yield was 257 ± 183 ng/mg and 77 ± 56 ng/mg from randomly selected frozen and paraffin‐embedded tissue, respectively. Intact 18S and 28S rRNA subunits were present in 11/23 frozen and 2/6 paraffin‐embedded specimens. The 375‐bp actin and 296‐bp glyceraldehdye 3‐phosphate dehydrogenase targets were amplified by reverse transcription‐PCR from both sources and the 983‐bp glyceraldehdye 3‐phosphate dehydrogenase and 499‐bp nonhousekeeping integrin‐linked kinase targets from frozen tissue. The minimal amount of RNA required for reverse transcription‐PCR of 296‐bp glyceraldehdye 3‐phosphate dehydrogenase target was 29 pg from frozen tissue when RNA subunits were present and 144 pg when these subunits were absent compared to 0.8 ng from paraffin‐embedded tissue. Ten laser pulses were required to laser capture sufficient cells from frozen tissue to detect amplification of the 375‐bp actin target while more pulses were required for equivalent amplification from paraffin‐embedded tissue. Storage time had no detectable effect on RNA quality. We conclude that both frozen and paraffin‐embedded tissues as well as laser‐captured cells are suitable for gene expression studies but frozen tissue offered greater sensitivity.