Novel Mechanisms of Ozone-Induced Pulmonary Inflammation and Resolution, and the Potential Protective Role of Scavenger Receptor BI.

INTRODUCTION: Increases in ambient levels of ozone (O3), a criteria air pollutant, have been associated with increased susceptibility and exacerbations of chronic pulmonary diseases through lung injury and inflammation. O3 induces pulmonary inflammation, in part by generating damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and scavenger receptors (SRs). This inflammatory response is mediated in part by alveolar macrophages (AMs), which highly express PRRs, including scavenger receptor BI (SR-BI). Once pulmonary inflammation has been induced, an active process of resolution occurs in order to prevent secondary necrosis and to restore tissue homeostasis. The processes known to promote the resolution of inflammation include the clearance by macrophages of apoptotic cells, known as efferocytosis, and the production of specialized pro-resolving mediators (SPMs). Impaired efferocytosis and production of SPMs have been associated with the pathogenesis of chronic lung diseases; however, these impairments have yet to be linked with exposure to air pollutants. SPECIFIC AIMS: The primary goals of this study were: Aim 1 - to define the role of SR-BI in O3-derived pulmonary inflammation and resolution of injury; and Aim 2 - to determine if O3 exposure alters pulmonary production of SPMs and processes known to promote the resolution of pulmonary inflammation and injury. METHODS: To address Aim 1, female wild-type (WT) and SR-BI-deficient, or knock-out (SR-BI KO), mice were exposed to either O3 or filtered air. In one set of experiments mice were instilled with an oxidized phospholipid (oxPL). Bronchoalveolar lavage fluid (BALF) and lung tissue were collected for the analyses of inflammatory and injury markers and oxPL. To estimate efferocytosis, mice were administered apoptotic cells (derived from the Jurkat T cell line) after O3 or filtered air exposure.To address Aim 2, male WT mice were exposed to either O3 or filtered air, and levels of SPMs were assessed in the lung, as well as markers of inflammation and injury in BALF. In some experiments SPMs were administered before exposure to O3or filtered air, to determine whether SPMs could mitigate inflammatory or resolution responses. Efferocytosis was measured as in Aim 1. RESULTS: For Aim 1, SR-BI protein levels increased in the lung tissue of mice exposed to O3, compared with mice exposed to filtered air. Compared with WT controls, SR-BI KO mice had a significant increase in the number of neutrophils in their airspace 24 hours post O3 exposure. The oxPL levels increased in the airspace of both WT and SR-BI KO mice after O3 exposure, compared with filtered air controls. Four hours after instillation of an oxPL, SR-BI KO mice had an increase in BALF neutrophils and total protein, and a nonsignificant increase in macrophages compared with WT controls. O3 exposure decreased efferocytosis in both WT and SR-BI KO female mice.For Aim 2, mice given SPM supplementation before O3 exposure showed significantly increased AM efferocytosis when compared with the O3exposure control mice and also showed some mitigation of the effects of O3 on inflammation and injury. Several SPMs and their precursors were measured in lung tissue using reverse-phase high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS). At 24 hours after O3 exposure 14R-hydroxydocosahexaenoic acid (HDHA) and 10,17-dihydroxydocosahexaenoic acid (diHDoHE) were significantly decreased in lung tissue, but at 6 hours after exposure, levels of these SPMs increased. CONCLUSIONS: Our findings identify novel mechanisms by which O3 may induce pulmonary inflammation and also increase susceptibility to and exacerbations of chronic lung diseases.

Evaluation and application of a plasma ashing method for STEM fiber analysis in human lung tissue.

A preparation technique for fiber analysis in human lung tissue has been developed that involves freeze-drying and low-temperature ashing. Analysis is made in the analytical scanning transmission electron microscope at a magnification of 10,000 x. With the use of a special counting method, the sensitivity is approximately 80,000 fibers per gram dry tissue with lengths less than 5 microns and 40,000 fibers per gram dry tissue with lengths greater than or equal to 5 microns. In spite of the leaching and contamination effects described in the literature--even for chrysotile fibrils taken from the lung, elemental spectra do not differ essentially from the asbestos standard. In order to carry out a complete check of the preparation method, a suspension of standard crocidolite fibers in water was gelatinated. No changes in length, diameter or aspect ratio distribution occurred. After preparation, at least 40% of the fibers were recovered. Compared to the wide range of fiber concentrations observed in human lung tissue, these recovery rates appear adequate for fiber analysis in lung dust for medical or legal purposes. To date, 70 lung dust specimens have been analyzed. These are discussed in the paper.

Potential health risks from the use of fibrous mineral absorption granulates.

Attapulgite (palygorskite) and sepiolite are fibrous clay minerals used commercially as components in a wide variety of products including oil and grease adsorbents, carriers for pharmaceuticals, cosmetics, and pesticides. They are also components of drilling muds and animal litter and they are used as paint thickeners. The current annual worldwide production of these minerals exceeds one million tons. Although fibrous in nature, the fibre length may vary greatly depending on the location of the geological deposits. American attapulgite is short (0.1-2.5 micron in length, median of 0.4 micron) but palygorskite from other parts of the world is much longer (30% longer than 5 micron). Several samples of these materials have been submitted to scanning transmission electron microscopy (STEM). This paper reports the results of microscopic evaluations and makes a comparison with the data from experimental carcinogenicity studies and it is concluded that fibre length is a most important carcinogenic property.

Asbestos dust exposure during brake repair.

About 10,000 tons of chrysotile per year are used in the Federal Republic of Germany for the production of friction materials. During brake repair an unknown number of approximately 300,000 mechanics in automobile service stations are exposed to asbestos dust. In a field study, asbestos fiber concentrations during brake repair were measured. Occupational histories and chest X-rays of brake service mechanics are being examined. Ninety dust measurements in 76 service stations were made by phase contrast microscopy and by scanning transmission electron microscopy. By electron microscopy, extremely fine chrysotile fibers with lengths less than 5 microns were identified in brake drum dust. Fibers with lengths greater than or equal to 5 microns constituted less than 1% of all chrysotile fibers counted in brake drum dust. Short-term asbestos dust exposures were measured by light microscopy in 101 personal samples during blowing out of brakes, and grinding and turning of brake linings. During blowing out of car brakes, as well as during grinding of brake linings, the product of fiber concentration with length greater than 5 microns and sampling time amounted to about 4-5 fibers/ml X min corresponding to a concentration of 10(6) fibers/m3 over 4-5 min. For trucks and buses higher amounts of 5-10 X 10(6) fibers/m3 X min were observed during these operations. From occupational histories of 210 vehicle mechanics, an average duration of employment of mean +/- s = 21 +/- 10 years and a mean cumulative fiber dose of mean +/- s = (0.54 +/- 1.1) X 10(6) fibers/m3 X years were calculated.