Joseph D. Paulauskis

Differential screening identifies genetic markers of monocyte to macrophage maturation.

Maturation of cells of the mononuclear phagocyte lineage from bone marrow precursors to tissue macrophages (MAC) via circulating blood monocytes (MO) is a multistep process only partially understood. Similarly, MAC differentiation can be observed if MO are cultured in vitro. In an attempt to further characterize molecular changes occurring during this process we carried out differential screening of a MO‐derived MAC cDNA library using MO and MAC cDNA. After subcloning and confirmation by a second round of screening, partial sequencing of 41 cDNA clones was performed. In 33 clones the sequences of 7 different previously identified cDNAs were found. The mRNA expression of two of the corresponding genes (apolipoprotein E, ferritin light chain) is already known to be up‐regulated during MAC maturation. For one gene (cathepsin B), a specific up‐regulation of mRNA expression could be shown corresponding to previous protein data. For four genes [human cartilage glycoprotein (HC‐gp39), osteopontin, type IV collagenase, and tryptophanyl‐tRNA synthetase] the specific expression in MAC versus MO was previously unknown but could be confirmed by the use of Northern blot analysis. Of these genes, HC‐gp39 is especially interesting because it is only expressed during the late stages of MAC differentiation. J. Leukoc. Biol. 60: 540–545; 1996.

URBAN AIR PARTICULATE INHALATION ALTERS PULMONARY FUNCTION AND INDUCES PULMONARY INFLAMMATION IN A RODENT MODEL OF CHRONIC BRONCHITIS

Epidemiological studies have reported increased morbidity in human populations following inhalation of elevated levels of urban particulate matter. These responses are especially prevalent in populations with chronic obstructive pulmonary diseases, including chronic bronchitis. Toxicological studies have reported altered pulmonary function and increased pulmonary inflammation following particulate inhalation in the laboratory setting. However, most of these studies have utilized artificial particles that may not accurately mimic outdoor air pollutant conditions. Few studies have utilized actual urban air particle samples in inhalation studies. In the present study, the effects of inhaled concentrated urban air particulates on pulmonary function and pulmonary inflammation are addressed. Normal rats and rats with chronic bronchitis induced by approximately 200 ppm SO(2) for 6 wk were subsequently subjected to filtered air or concentrated air particles (CAPs). Twelve rats per group in 4 groups (48 rats total) were exposed for 5 h/day for 3 consecutive days. The CAPs aerosol levels were 206, 733, and 607 microg/m(3) (MMAD = 0.18 microm, sigma(g) = 2.9) on days 1, 2, and 3, respectively. Following the final day of exposure, pulmonary function parameters, including peak expiratory flow (PEF), tidal volume (TV), respiratory frequency (RF), and minute volume (MV), were measured and compared to preexposure baseline levels. Twenty-four hours following the final day of exposure, bronchoalveolar lavage was performed for total cell counts, differential cell counts, and total lavage protein levels. Pulmonary responses to CAPs in chronic bronchitic animals indicated a significant increase in tidal volume as well as peak expiratory flow. In CAPs-exposed animals without underlying bronchitis, significantly increased tidal volume was observed. Significant pulmonary inflammation was observed in the CAPs-exposed animals, particularly those with chronic bronchitis. Significant increases in neutrophils, lymphocytes, and total lavage protein were observed. These results suggest two distinct mechanistic responses to inhaled particles: a stress-type pulmonary function response marked by increases in flow and volume, that is, deeper breathing; and acute pulmonary inflammation marked by cellular influx, particularly neutrophils. From these data it is concluded that inhaled urban air particles alter pulmonary breathing parameters and increase pulmonary inflammation.

Regulation of macrophage inflammatory protein-1alpha mRNA by oxidative stress.

Accumulation of inflammatory cells within the lung has been implicated in oxidative injury. Recruitment of these cells to a tissue site is a complex process that depends in part upon the local expression of appropriate proinflammatory chemokines. Macrophage inflammatory protein-1α (MIP-1α), a member of the CC subfamily of chemokines, has been shown to contribute to monocyte/macrophage and neutrophil chemotaxis and activation. Our previous work demonstrated that MIP-1α mRNA expression in macrophages is induced by bacterial endotoxin. The objective of this study was to test the hypothesis that an oxidative stress alone may trigger expression of MIP-1α mRNA in macrophages and to determine the mechanism leading to increased expression. A rat alveolar macrophage cell line (NR8383) was exposed to HO or menadione (2-methyl-1,4-naphthoquinone (MQ)), a quinone compound that undergoes redox cycling and generates reactive oxygen species continuously. Steady-state mRNA levels encoding MIP-1α were markedly increased (3-fold) in these cells after 1 h of exposure to 0.5 mM HO, remained higher than control levels after 4 h, and decreased after 6 h. Similarly, MQ (25 or 50 μM) caused a significant increase of MIP-1α mRNA with a maximal induction after 4 h of exposure (5-fold). Both HO and MQ-induced up-regulation of MIP-1α mRNA was suppressed by co-treatment with N-acetylcysteine, a synthetic antioxidant. Co-treatment with actinomycin D reduced the MQ induction of MIP-1α mRNA to a greater extent than the HO-induced increase. Transcription of the MIP-1α gene was increased by exposure to both HO and MQ. HO treatment also induced a marked increase of the MIP-1α mRNA half-life, indicating post-transcriptional stabilization. These observations indicate that an oxidative stress can regulate MIP-1α mRNA expression by two distinct mechanisms: transcriptional activation of the MIP-1α gene and post-transcriptional stabilization of MIP-1α mRNA.

Functional characterization of rat chemokine macrophage inflammatory protein-2

Expression of mRNA for the C-X-C chemokine, macrophage inflammatory protein-2 (MIP-2), is induced during acute inflammation in rat models of disease. We have characterized the phlogistic potential of rat recombinant MIP-2 (rMIP-2) protein in vitro and in vivo. Recombinant MIP-2 caused marked PMN chemotaxis in vitro, with peak chemotactic activity at 10 nM. Incubation of whole blood with rMIP-2 caused a significant loss of L-selectin and a significant increase in Mac-1 expression on the PMN surface. Under similar conditions rMIP-2 also caused a modest respiratory burst in PMNs. The intratracheal instillation of 10 and 50μg of rMIP-2 caused a significant influx of PMNs into the airspace of the lungs. Rat MIP-2 is a potent neutrophil chemotactic factor capable of causing neutrophil activation and is likely to function in PMN recruitment during acute inflammation in rat disease models.

Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts

We have isolated and sequenced overlapping cDNA clones for rat KC*. The 0.93 kb cDNA has a single open reading frame of 288 nucleotides, and substantial sequence identity with the platelet-factor 4 family members mouse KC, hamster gro, and human gro. Using cloned cDNA as a probe, expression of KC mRNA in lavaged rat alveolar macrophages (AMs) increased after lipopolysaccharide (LPS) treatment. We also studied expression in vitro by a rat fetal lung fibroblast cell line, RFL-6. Expression of KC mRNA in RFL-6 cells increased after treatment with interleukin 1 or with conditioned medium from rat AMs treated with LPS.

In vitro efficacy of morpholino-modified antisense oligomers directed against tumor necrosis factor-alpha mRNA.

Chemical modification of antisense oligonucleotides to increase nuclease resistance may improve their efficacy within enzyme-rich cellular targets (e.g. macrophages). We evaluated a panel of morpholino antisense oligomers (M-AS) for their ability to inhibit macrophage tumor necrosis factor-α (TNF-α) release and compared them to phosphodiester (O-AS) and phosphorothioate (S-AS) types of oligonucleotides. M-AS inhibited translation in vitro (rabbit reticulocyte lysate) of target mRNA at concentrations as low as 200 nM (e.g. percent inhibition by M-AS 2 at 0.2, 1.0, and 2.0 μM was 40.9 ± 5.3%, 50.2 ± 4.6%, and 57.7 ± 3.6%, respectively, n = 4, p ≤ 0.002 versus control). Similarly, M-AS 2 effectively, albeit partially, inhibited TNF-α production by LPS-stimulated macrophages (RAW 264.7 cells). Incubation of cells with 25 μM M-AS 2 resulted in 32.6 ± 2.6% (n = 3, p = 0.002 versus control) decrease in TNF-α release. In contrast, S-AS inhibited translation of the target mRNA in the rabbit reticulocyte lysate assay, but not in the cell-based assay. In fact, S-AS nonspecifically augmented TNF-α release. O-AS were without effect in either system. Uptake studies with fluorescent M-AS revealed that inhibitory effects were seen despite relatively low cellular uptake (intracellular concentration 30.5 ± 6.7 nM; efficiency of uptake 0.1%). In contrast, flow cytometric and confocal analysis revealed that S-AS were avidly taken up by RAW 264.7 cells, confirming that their lack of efficacy was not due to lack of uptake. With improved methods of delivery, M-AS may represent an important therapeutic modality.

Rat alveolar macrophages express preprotachykinin gene-I mRNA-encoding tachykinins

Although the tachykinins substance P (SP) and neurokinin A have been largely localized to neurons, eosinophils have also been shown to express these peptides. Our aim was to determine whether rat alveolar macrophages (AM) express preprotachykinin gene-I (PPT-I) mRNA that encodes these tachykinins and to examine expression during inflammation. PPT-I mRNA was detected by reverse transcription (RT)-polymerase chain reaction (PCR) in AM and brain (control) but not in peritoneal macrophages. Northern analysis showed that PPT-I mRNA was induced two- to fourfold by in vivo treatment of rats with intratracheal lipopolysaccharide (LPS) and in vitro after 4 h of exposure to LPS. This increase was inhibited by dexamethasone. In situ RT-PCR and immunocytochemistry further confirmed that AM express PPT-I mRNA and SP-like immunoreactivity, respectively, which was enhanced by LPS treatment. A 1.3-kb transcript consistent with PPT-I mRNA was detected by Northern analysis of bronchoalveolar lavage neutrophils. Therefore, rat AM express PPT-I mRNA that is upregulated in AM by LPS and is attenuated by dexamethasone. PPT-I mRNA was also detected in lung neutrophils.

Induction of High Mobility Group-I(Y) Protein by Endotoxin and Interleukin-1β in Vascular Smooth Muscle Cells ROLE IN ACTIVATION OF INDUCIBLE NITRIC OXIDE SYNTHASE

Nonhistone chromosomal proteins of the high mobility group (HMG) affect the transcriptional regulation of certain mammalian genes. For example, HMG-I(Y) controls cytokine-mediated promoters that require transcription factors, such as nuclear factor-κB, for maximal expression. Even though a great deal is known about how HMG-I(Y) facilitates expression of other genes, less is known about the regulation of HMG-I(Y) itself, especially in cells in primary culture. Therefore we investigated the effect of endotoxin and the cytokine interleukin-1β on HMG-I(Y) expression in vascular smooth muscle cells. Induction of HMG-I(Y) peaked after 48 h of interleukin-1β stimulation (6.2-fold) in cells in primary culture, and this increase in mRNA corresponded to an increase in HMG-I(Y) protein. Moreover, immunohistochemical staining revealed a dramatic increase in HMG-I(Y) protein expression in vascular smooth muscle cells after endotoxin stimulation in vivo. This increase in HMG-I(Y) expression (both in vitro and in vivo) mirrored an up-regulation of inducible nitric oxide synthase, a cytokine-responsive gene. The functional significance of this coinduction is underscored by our finding that HMG-I(Y) potentiated the response of inducible nitric oxide synthase to nuclear factor-κB transactivation. Taken together, these studies suggest that induction of HMG-I(Y), and subsequent transactivation of iNOS, may contribute to a reduction in vascular tone during endotoxemia and other systemic inflammatory processes.

16S rRNA Restriction Fragment Length Polymorphism Analysis of Bacterial Diversity as a Biomarker of Ecological Health in Polluted Sediments from New Bedford Harbor, Massachusetts, USA

Abstract A polymerase chain reaction (PCR)-based method was developed to compare bacterial diversity among environmental sites with varying degrees of anthropogenic impact. New Bedford Harbor, MA, a US Environmental Protection Agency-designated Superfund hazardous waste site, was studied to assess changes in bacterial diversity resulting from long-term inputs of organic and inorganic pollutants. Total DNA was extracted from surficial sediments sampled from four sites along a transect of decreasing contamination (Upper and Lower Acushnet Estuary, New Bedford Harbor, and Buzzards Bay, respectively). Oligonnucleotide primers specific to conserved regions of the 16S rRNA gene were used to PCR-amplify sequences from DNA extracts. Restriction fragment length polymorphism (RFLP) analysis resulted in generation of a number of unique operational taxonomic units (OTUs). Cluster analysis of fragment pattern data using the computer program RESTSITE ® allowed for bacterial diversity estimations, which, in agreement with standard culture techniques, showed higher bacterial diversity in New Bedford Harbor sediments, relative to Buzzards Bay. By employing bacterial diversity as a sensitive indicator of environmental stress, the method has wide applicability to many environments for the assessment of anthropogenic impact on aquatic ecosystems.

Contamination of water supplies with Cryptosporidium parvum and Giardia lamblia and diarrheal illness in selected Russian cities

Cryptosporidium parvum and Giardia lamblia are important agents of waterborne diarrheal illness worldwide. While giardiasis is routinely diagnosed in Russia with a chemical staining technique, data on the prevalence of cryptosporidiosis are scarce. Monitoring of the respective parasites in water supplies in Russia is very limited. A health survey conducted in the city of Cherepovets and three other cities in the European part of Russia using enzyme-linked immunosorbent assays (ELISA) demonstrated that 6.9% of diarrheal patients tested had C. parvum antigens in their fecal samples; 9.4% had G. lamblia antigens. A survey of occurrence of these parasites in water supplies in Cherepovets and seven other cities demonstrated that source and finished water samples from several of these cities were contaminated with either C. parvum oocysts or G. lamblia cysts. The surveys were not designed to assess associations between presence or concentrations of C. parvum and G. lamblia in water and related gastrointestinal diseases in exposed populations. Rather, the goals were to demonstrate the presence of disinfection-resistant protozoan parasites in untreated and treated waters, and the importance of these pathogens as causative agents of diarrheal illnesses in a number of Russian cities.