Hillel S. Koren

Modulation of human alveolar macrophage properties by ozone exposure in vitro

We have investigated changes in human alveolar macrophage (HAM) function after exposure in vitro to ozone (O3) (0.1-1.0 ppm for 2-4 hr). The functions studied reflect concern that O3 is detrimental to host defense mechanisms in the bronchoalveolar spaces. Exposure of HAM to O3 caused a concentration-dependent increase in release of prostaglandin E2 (PGE2), an important modulator of inflammation, phagocytosis, and oxidative burst. Although phagocytosis of particulate immune complexes was decreased by O3, we found no change in the quantity of Fc receptors and complement receptors on the HAM surface. Superoxide (O2-) production in response to phorbol ester was reduced after exposure of HAM to O3 while the basal O2- release in response to plastic adherence was not affected. Growth inhibition of the opportunistic yeast Cryptococcus neoformans by HAM was not affected by O3 exposure. The production of inflammatory mediators and immune modulators such as tumor necrosis factor-alpha, interleukin 1, and interleukin 6 were not induced by exposure to O3. However, compared to controls, O3- exposed HAM produced significantly lower levels of these cytokines when stimulated with bacterial lipopolysaccharide (LPS). Two-dimensional gel electrophoretic analysis of proteins made by HAM following in vitro exposure to O3 identified 11 proteins whose rate of synthesis was significantly altered. Thus, these studies show that exposure to O3 alters the functional competence of HAM. While there is a minimal effect on protein expression or synthesis, the responses of HAM to particulate immune complexes, to bacterial LPS, and to PMA are impaired. The release of arachidonic acid and PGE2 suggest that the effect of O3 is primarily targeted to the HAM cell membrane. These changes may ultimately result in increased susceptibility to inhaled infectious agents in the O3-exposed individual.

UVA II Exposure of Human Skin Results in Decreased Immunization Capacity, Increased Induction of Tolerance and a Unique Pattern of Epidermal Antigen-Presenting Cell Alteration

Abstract— The risks incurred from increased exposure to UVA II (320‐340 nm) (i.e. during sunscreen use and extended outdoor exposure, tanning parlors) are not well understood. Therefore, we explored the effects of UVA II on skin immune responses in humans. After a single local exposure (4 minimum erythemal dose [MED]) using a xenon arc lamp filtered with a narrow bandpass filter (335 ± 5 nm full width at half maximum), individuals were contact‐sensitized with dinitrochlorobenzene (DNCB) through a UVA II exposure site or through normal skin. UVA II induced a marked decrease in the magnitude of skin immune responses (P < 0.0001). The UVA II group had only 29% successful sensitizations, as compared to 83% in the control group. The percentage of individuals who remained tolerant to DNCB after two sensitizations was 23.6% for the UVA II‐exposed group, as compared to 3.8% in the controls (P= 0.006). UVA II also uniquely altered the type of antigen‐presenting cells present in the epidermis. Human leukocyte antigen (HLA)‐DR+ cells in control epidermal cell suspensions (C‐EC) comprised a single, homogeneous population of Langerhans cells (LC) with the phenotype: CD1ahi DRmid CD11b− CD36− (1.5 ± 0.3% of EC). UVA II irradiation reduced the number of such LC to 0.6 ± 0.2% of EC. Although cells expressing the macrophage phenotype: CD1a DRhi CD11b+ CD36+ were increased in UVA II skin, relative to C‐EC, these comprised only 10.1 ± 6.1% of the DR+ cells, which is less than that after UVB exposure. Also distinct from UVB, a third population was found in UVA II‐EC, which exhibited a novel phenotype: CD1a+ DR+ CD36+ CDllb+; these comprised 11.1 ± 6.9% of the DR+ UVA II‐EC.

Acute ozone-induced lung injury in neutrophil-depleted rats☆

To test the hypothesis that neutrophils contribute to acute, ozone-induced epithelial damage in the lung, rats were depleted of their circulating neutrophils by intraperitoneal injection of a rabbit anti-rat neutrophil serum (ANS) 12 hr prior to an 8-hr exposure to 1.0 ppm ozone. Additional rats were given an injection of normal rabbit serum (NRS) prior to ozone exposure. Exposures were followed by postexposure periods in filtered air for 0, 4, or 16 hr. Control rats were given either ANS or NRS and then exposed only to filtered air. Analysis of bronchoalveolar lavage fluid (BALF) from NRS-treated rats revealed a significant increase in total neutrophils above that of controls at the 4- and 16-hr postexposure times, with a peak increase at 4 hr postexposure. In contrast, there was almost total ablation of the BALF neutrophil response in the ANS-treated rats at all times. Ozone caused an increase in BALF protein, fibronectin, and interleukin-6 above those in controls in both the NRS- and ANS-treated rats, but the only significant difference between the two groups was a level of fibronectin in the neutrophil-depleted animals higher than that in the neutrophil-sufficient animals at the 0-hr postexposure time. Electron microscopic morphometry on lungs fixed by intravascular perfusion demonstrated no significant differences in the volume per surface area epithelial basal lamina (Vs) of necrotic and degenerating epithelial cells in central acini between the neutrophil-depleted and neutrophil-sufficient animals. From these results, we concluded that neutrophils do not play a detectable role in contributing to the early epithelial damage in the lung caused by an acute exposure to ozone.

Human upper respiratory tract responses to inhaled pollutants with emphasis on nasal lavage.

A set of symptoms has been described during the past two decades. These symptoms, which have been called the sick building syndrome, include eye, nose, and throat irritation; headache; mental fatigue; and respiratory distress. It is likely that VOCs present in synthetic materials used in homes and office buildings contribute to these symptoms. There have been few studies, however, in which humans have been exposed to known amounts of VOCs under carefully controlled conditions. In this study, 14 subjects have been exposed to a mixture of VOCs (25 mg/m3 total hydrocarbon) representative of what is found in new homes and office buildings. Because irritation of the nose and throat are symptoms often associated with the upper respiratory tract and may result from an inflammatory response in the upper airways, we have used NAL to monitor PMN influx into the nasal passages following exposure to VOCs. We report statistically significant increases in PMNs both immediately after a 4-hr exposure to VOCs, as well as 18 hr later.

ATTENUATION AND RECOVERY OF PULMONARY INJURY IN RATS FOLLOWING SHORT-TERM, REPEATED DAILY EXPOSURE TO OZONE

Controlled human and epidemiology studies have demonstrated that during repeated exposure to ozone (O 3) attenuation of lung function responses may occur. It is yet unknown whether inflammatory and biochemical effects in lower airways of humans, as observed upon single O 3 exposure, also show a diminutive response following repeated exposure to O 3. The aim of this study was to investigate inflammatory, permeability, and histopathological responses in lungs of rats following repeated daily O 3 exposure and to study the time course of attenuation and recovery of these effects using single O 3 challenges at various postexposure times. To aid in animal-to-human extrapolation, this study and a previously reported human study (Devlin et al., 1997) were designed with similar protocols. Wistar rats were exposed for 5 consecutive days to 0.4 ppm O 3 for 12 h/night. Subsequently, the time course of postexposure recovery was determined by a single challenge of 12 h to 0.4 ppm O 3 after a 5-, 10-, 15-, or 20-day recovery period. Broncho-alveolar lavage (BAL) examination and histopathology were performed 12 h after this O 3 challenge. To quantify the magnitude of the O 3 response, results were compared with a group exposed only once for 12 h to 0.4 ppm O 3 and sacrificed simultaneously. The results demonstrate that a single exposure of 0.4 ppm O 3 causes marked permeability and inflammatory responses in lower airways of rats, as evidenced by enhanced BAL fluid levels of proteins, fibronectin, interleukin (IL)-6, and inflammatory cells. However, 5 days of exposure to 0.4 ppm O 3 for 12 h/night resulted in a complete disappearance of these responses, resulting in BAL fluid values that were not different from those observed in unexposed controls. Postexposure analyses of pulmonary response to O 3 challenges demonstrated that these attenuated responses show a gradual recovery. The data indicate that with respect to BAL fluid levels of albumin, IL-6, and number of macrophages and neutrophils, the period for lung tissue to regain its full susceptibility and responsiveness to O 3 following a 5-day preexposure period is approximately 15-20 days. Remarkably, the total protein and fibronectin responses in BAL fluid still exhibited an attenuated response to an O 3 challenge at 20 days postexposure. Morphometry (number of BrdU-labeled cells in terminal bronchiolar epithelium, and number of alveolar macrophages) showed that after a recovery of 5-10 days following a 5-day preexposure the response to a challenge was identical to that after a single exposure. These results suggest that complete repair from lower airway inflammation caused by short-term, repeated exposure to O 3 may take longer than previously assumed.

Dose Response for UV‐induced Immune Suppression in People of Color: Differences Based on Erythemal Reactivity Rather than Skin Pigmentation¶†

Ultraviolet radiation (UVR) is known to suppress immune responses in human subjects. The purpose of this study was to develop dose responses across a broad range of skin pigmentation in order to facilitate risk assessment. UVR was administered using FS 20 bulbs. Skin pigmentation and UVR sensitivity were evaluated using Fitzpatrick classifications, minimal erythemal dose (MED), slope of the erythemal dose response curve (sED), baseline pigmentation and tanning response. To assess immune responses dinitrochlorobenzene (DNCB) was applied to irradiated buttock skin 72 h after irradiation. Two weeks later DNCB was applied to the inside upper arm. Skin thickness was measured before and after challenge. Dose response was modeled (to obtain a regression line) for the entire group of 185 subjects. With the exception of sED none of the above‐mentioned pigmentation indicators contributed significantly to variability around the regression line. Thus, differences in sensitivity for multiple skin types based on Fitzpatrick classification or MED were not observed. However, differences in immune sensitivity to UVR were detected between subjects with steep erythemal dose response curves and those with moderate or flat responses. For subjects with steep erythemal responses the dose calculated to suppress the immune response by 50% was 114 mJ/cm2. This group included individuals with Fitzpatrick skin types I–V, MED for these subjects ranged from 30 to 80 mJ/cm2. The 50% suppression dose for subjects with weak or no erythemal response could not be computed (the dose response was flat). This resistant group included subjects with skin types IV–VI and MED for these subjects ranged from 41 to >105 mJ/cm2. This study provides a human dose response for UVR suppression of contact sensitivity that will be useful in risk assessment. It is the first study to provide this information using the FS sun lamp and is the first study to include people of color. The sED appears to be a new variable for identifying sensitive subjects at risk of UVR‐induced immune suppression.

Breakage and binding of DNA by reaction products of hypochlorous acid with aniline, 1-naphthylamine, or 1-naphthol

Hypochlorous acid (HOCl) is a chemically reactive oxidant and a potent microbicidal agent that is synthesized in phagosomes of inflammatory neutrophils and released into extracellular spaces. Besides reducing pathogenicity by reacting with phagocytized infectious agents, HOCl may damage tissues and yield toxic products upon reaction with various other molecules, including xenobiotics. As model xenobiotics, the substituted aryl compounds aniline, 1-naphthylamine, and 1-naphthol (1-NOH) were investigated herein for their potential to react with HOCl and the transformed into genotoxic products. The compounds were first exposed to HOCl (25-150 microM) in phosphate buffer and afterward used to treat human fibroblasts or purified DNA. DNA single-strand breaks in cells and the binding of HOCl-reacted 1-[14C]NOH to purified DNA were assessed by DNA alkaline elution and scintillation spectrometry, respectively. It was found that neither HOCl nor compounds alone could break cellular DNA. But HOCl-reacted compounds produced up to 400 rad equivalents of DNA breaks. HOCl reaction products of aniline and the model bicyclic aryl compounds differed in their DNA-breaking characteristics. HOCl-reacted 1-[14C]NOH was stable and bound to DNA at up to 124 pmol/mg DNA. Sodium thiosulfate, glutathione, and taurine inhibited the transformation reactions; but only the former two blocked binding of HOCl-reacted 1-NOH to DNA. Ultraviolet spectra showed that HOCl reacted rapidly (less than 1 min) and equally well with 1-NOH at pH 7.2 or at an intraphagosomal pH of 5.0. Reaction concentrations of HOCl in this study were 2- to 11-fold lower than levels generated in vitro by stimulated neutrophils. These results show that certain aryl compounds can react readily with approximated physiological levels of HOCl (-OCl) to form relatively long-lived products that bind DNA and are genotoxic to human cells.

Effects of Phosgene Exposure on Lung Arachidonic Acid Metabolism

AbstractPhosgene can acylate macromolecules and may react with and affect enzymes involved in arachidonic acid metabolism. We examined the effects of an acute phosgene exposure in vivo and in vitro on lung arachidonic acid metabolism. Fischer 344 rats were exposed either to air or to phosgene (0.05–7.0 ppm) for 4 h and the lungs were lavaged at 0, 4, 20, and 44 h postexposure. Leukotriene B, (LTBJ, peptide leuko-trienes C4 D4 and E4 (LTC4/D/E4, and prostaglandin E2 (PCE2) were measured in la-vage fluid by radioimmunoassay Phosgene exposure in vivo (0.7–7.0 ppm for 4 b) produced significant decreases in concentrations of PGE, (maximal decrease of 74%), LTB, (maximal decrease of 59%), and LTC4/D4/E4 (maximal decrease of 97%) measured in rat lavage fluid immediately postexposure. Associated with this decrease in eicosa-noid production was a decrease in the number of alveolar macrophages and an increased number of neutrophils recovered in the lavage fluid of phosgene-exposed rats. Lung lavage eicosanoid conce...

Inhibition of Human Natural Killer Cell Activity Following in Vitro Exposure to Ozone

AbstractPrevious findings have revealed impairment of several host defense mechanisms in animals and humans resulting from ozone exposure. In this study we have examined the effect of in vitro ozone exposure on human peripheral blood natural killer (NK) cell activity measured against K562 tumor target cells. The data show that NK activity is inhibited in a time-dependent manner with marked suppression observed after 6 h of ozone exposure. Significant inhibition occurred at three different levels of ozone exposure (1.0, 0.5, and 0.78 ppm) and effector cell:target cell (E:T) ratios (50:1; 259 and 72.5:1) compared to air controls (p < .05). The capacity of ozone exposed NK cells to kill tumor cells decreased in a linear fashion as the level of ozone increased from 0.78 to 7.0 ppm (p - .006 at 50:l; .004 at 25:1). The ozone effect was not merely due to a nonspecific toxicity; as NK activity recovered within 78 h following exposure either spontaneously or by stimulation with IL 2. Moreover, unexposed cells tre...

The Natural Killer Cell‐Like Lytic Activity Expressed by Cytolytic T Lymphocytes is Associated with the Expression of a Novel Function‐Associated Molecule

Experiments were performed to analyse the natural killer cell (NK)‐like cytotoxicity frequently expressed by human antigen‐specific cytolytic T lymphocytes (CTL). To this end, several monoclonal antibodies (MoAbs) previously shown to identify a novel function‐associated molecule (FAM) involved in human NK function were utilized. Flow cytometry revealed that these MoAbs reacted with the majority of human NK, but only with a subpopulation of CTL isolated from primary mixed lymphocyte cultures. Preincubation of CTL with the MoAbs inhibited the NK‐like lysis of K562 targets. Experiments with anti‐CD3 MoAb demonstrated that neither the NK‐like cytotoxicity of CTL nor the lytic activity of NK were mediated by the CD3 complex. Expression of the novel FAM was found to develop in T‐cell cultures at the time that NK‐like cytotoxicity was observed. Repeated in vitro antigenic stimulation of CTL was shown to result in loss of NK‐like cytotoxicity, as well as loss of the FAM on the CTL surface. Thus, NK‐like cytotoxicity displayed by antigen‐specific CTL appears to be mediated by a novel FAM that is identical to that structure found on NK.