M. W. Sielczak

Effects of chemical mediators of anaphylaxis on ciliary function

We assessed the effects of selected chemical mediators of anaphylaxis on CBF in vitro. Ciliated epithelial cells were obtained from the trachea of conscious sheep with a cytology brush and suspended in a perfusion chamber containing KH. Ciliary activity was viewed microscopically and recorded on videotape for subsequent slow-motion analysis of CBF. Prostaglandin E1 (10(-8) M to 10(-6) M), prostaglandin E2 (10(-10) M to 10(-6) M), and leukotriene-C4 (10(-8) M) increased CBF between 7% and 33%. Histamine caused ciliostimulation only at the relatively high concentrations above 10(-5) M (7% increase in CBF), whereas prostaglandin F2 alpha (10(-10) M and 10(-6) M) was without effect. In no preparation was ciliary discoordination observed. These findings indicate that several chemical mediators of anaphylaxis stimulate CBF and that the previously described impairment of mucociliary transport in stable allergic asthma or antigen-induced bronchoconstriction is probably not caused by a primary alteration of ciliary function.

The 5-lipoxygenase inhibitor zileuton blocks antigen-induced late airway responses, inflammation and airway hyperresponsiveness in allergic sheep

Leukotrienes are thought to be involved in allergen-induced airway responses. To test this hypothesis we used a newly described 5-lipoxygenase inhibitor, zileuton, and examined its effect on antigen-induced early and late bronchial responses, airway inflammation and airway hyperresponsiveness in allergic sheep. Early and late responses were determined by measuring specific lung resistance (SRL) before and serially for 8 h after antigen challenge. Airway inflammation was assessed by bronchoalveolar lavage performed before, 8 h after and 24 h after antigen challenge. Airway responsiveness was measured before and 24 h after challenge by determining the dose of inhaled carbachol that caused a 400% increase in SRL (PD400%). The sheep (n = 8) were challenged with Ascaris suum antigen once after vehicle treatment (methylcellulose) and once after treatment with zileuton (10 mg/kg in methylcellulose, p.o.) given 2 h before antigen challenge. Trials were separated by at least 21 days. Zileuton had no effect on the early bronchoconstrictor response to antigen but the drug inhibited the late bronchial response by 55% (P less than 0.05). Unlike the control trial, there was no significant increase in bronchoalveolar lavage eosinophils at 8 h post challenge in the zileuton-treated sheep. Furthermore, zileuton treatment blocked (P less than 0.05) the airway hyperresponsiveness seen 24 h after challenge. Ex vivo formation of leukotriene B4 was inhibited over several hours after a single oral dose of zileuton, indicating that the compound was acting as a 5-lipoxygenase inhibitor in vivo. These results suggest that 5-lipoxygenase metabolites contribute to allergen-induced late responses, airway inflammation and airway hyperresponsiveness in this animal model of asthma.

Airway cell changes in tracheal lavage of sheep after ozone exposure

We were interested in whether ozone (O3) could stimulate the migration of mast cells into the tracheal lumen. To test this we determined the effect of an acute O3 exposure on the types and relative numbers of cells recovered by tracheal lavage. Seven conscious sheep were intubated with an elongated nasotracheal tube. The trachea between the larynx and the cuff of the tracheal tube (15-20 cm) was lavaged repeatedly with 10-15-ml aliquots (total 350 ml) of 0.9% buffered (pH 7.4 saline, which contained the mast cell-stabilizer disodium cromoglycate (10 micrograms/ml). One hour after a baseline lavage, the sheep were exposed on separate occasions to either air (control) or 0.5 ppm O3 for 2 h. Lavages were repeated 24 h later. Cells were recovered from the lavage effluent by centrifugation across a saline/Ficoll Paque gradient. From part of this material we estimated total cells and total viable cells (with Trypan blue). The rest of the material was recentrifuged at 400 X g for 5 min, and cytological slides were made from the cell pellet. Slides were stained with Polichrome and Wright-Giemsa, and were analyzed by light microscopy. The percentages of epithelial cells, macrophages, lymphocytes, and mast cells were determined from a total count of 500 cells/slide. Differences in cell percentages between pre- and postexposure were calculated both for air and O3 exposures, and these differences were compared. Exposure to O3 resulted in an increased number of mast cells and lymphocytes when compared to the changes observed with air. It seems likely that the increase in number of luminal mast cells and lymphocytes following O3 exposure signals an enhanced inflammatory response and that these changes could contribute to O3-induced increased nonspecific airway hyperresponsiveness and susceptibility to allergic IgE-mediated airway reactions.

The effects of a cysteinyl leukotriene antagonist (ONO-1078) on antigen-induced responses in allergic sheep

The cysteinyl leukotrienes (LTC4/D4/E4) are putative mediators of asthma. In this study we used sheep allergic to Ascaris suum antigen to examine the effects of a novel orally active cysteinyl LT antagonist, ONO-1078, on antigen-induced early and late responses, airway inflammation, post challenge (24 h) airway hyperresponsiveness (AHR) and mucociliary clearance. Airway responses to antigen were determined by measuring specific lung resistance (SRL) before and for 8 h after challenge, bronchoalveolar lavage (BAL) was used to estimate airway inflammation, and airway responsiveness was measured by determining the carbachol dose that increased SRL by 400% (PC400). We also used a radiographic technique to measure the antigen-induced change in tracheal mucus velocity (TMV), a marker of mucociliary clearance. In two trials separated by at least 21 days, sheep were treated once with ONO-1078 (30 mg/kg, p.o.) and once with placebo (0.5% methylcellulose), 2 h before and 4 h after antigen challenge. Treatment with ONO-1078 (n = 7) provided 40% protection (p < 0.10) against the peak early increase in SRL, resulted in a more rapid reversal of the early response, and provided 96% protection against the peak late (6-8 h) increase in SRL. ONO-1078 also inhibited the AHR 24 h after challenge. In the drug trial, PC400 was unchanged as compared to pre-challenge, whereas in the placebo trial, PC400 was decreased 1.4-fold (p < 0.05). Treatment however, did not affect BAL cell numbers or differential.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast Cells are Important Effector Cells Modulating Hyperoxic Pulmonary Fibrosis. • 2017

Mast cells (M.C) were identified over one century ago. They have been implicated in allergic, as well as various fibrotic processes. (Jr Am Acad of Derm 1990;23:615) Pulmonary Fibrosis (PF) induced by Bleomycin and other agents appears to be modulated by M.C(Am Rev Resp Dis 1984;130:797). Our laboratory has previously shown an association between M.C number and indices of Pulmonary Fibrosis induced by hyperoxic exposure in newborn mice (Ped Research 1994;35:343A). The purpose of this investigation was to directly test the hypothesis that M.C. deficiency reduces or protects against Hyperoxic Pulmonary Fibrosis (HPF). Adult mice heterozygous for the Wv allele were bred. One fourth of their offspring were homozygous and thus mast cell deficient. These mast cell deficient mice (MCD) served as the experimental group for this study, while offspring from breeding normal mice served as control (C). Both groups were exposed to either 60% FiO2 (O2) or room air (RA) under controlled temperature and barometric pressure. At 30days the lungs were prepared for histological examination, determination of collagen (CL) and hydroxyproline (OHPR) content. Data are expressed as X± (SD). NA= not applicable, n.s= not significant a=P< 0.01 Control RA vs O2, b=p< 0.01 MCD RA vs MCD O2, c=p<0.01 Control RA vs MCD RA, d=p<0.001 Control O2 vs MCD O2. These results directly implicate Mast Cells as an important effector cell in Hyperoxia induced pulmonary fibrosis. We speculate that MC products may in part mediate the pulmonary fibrosis response to oxygen. Table

Hyperoxia Affects Somaticovisceral Growth. 363

Exposure to oxygen during the neonatal period is fraught with deleterious effects on lung growth and morphology(Ped Pulm 1993;16:81). Investigators have shown reduced body weight in various species subjected to oxygen exposure. The purpose of this investigation was to evaluate the effects of Hyperoxia (85% FiO2) on somaticovisceral growth in mice. Mice were bred and their offspring were randomized at 2 days of age to receive 85%FiO2(O2) or room air (RA) under controlled temperature, humidity barometric pressure and diurnal cycles Mothers were exposed to oxygen for no longer than 24 hours and alternated to minimize oxygen toxicity. The mice were offered mice chow and water ad lib. Both RA and O2 exposed mice received equivalent nutrient intake. Each group was exposed to 7, 15, or 30days of experimental or control conditions. We measured body weight, and wet to dry weights of selected organs. Data for 30 days exposure is expressed as X± (SD) (mg) Table There were significant differences in final bodyweight between O2 exposed and RA (11.08(1.2) vs 17.2(0.87)gms)p< 0.001 at 30 days. Hyperoxia exposure affected organ growth of heart, lung and brain as early as 7 days of exposure while the most pronounced effect on all organs studied occurred at 30 days. At 30 days heart, lungs, liver and spleen had significant differences in wet/dry ratios suggestive of edema. We conclude that hyperoxia adversely affects somaticovisceral growth. Whether these differences in organ growth are related to impaired protein synthesis and /or impaired growth factors remains to be elucidated.