Timothy grass (Phleum pratense L.) pollen as allergen carriers and initiators of an allergic response.

Contrary to indoor allergen exposure (e.g. house dust mite), there is no reliable quantitative association between pollen exposure and symptoms of allergic diseases. Therefore we studied localization and release of major allergens from timothy grass (Phleum pratense L.) pollen using different methods and pollen grain sources. Localization of major allergens Phl p 5 and Phl p 1 was visualized by field emission scanning electron microscopy after anhydrous fixation and immunogold silver staining in a three-dimensional reconstruction; Phl p 5 was found in the cytoplasm and on the exine, Phl p 1 in the intine. No allergens were found inside the starch granules. Allergen liberation from pollen grains was studied in vitro under physiological conditions (30 min, 37 degrees C) at pH 6. 0, 7.4 and 9.0. Besides total protein measurements in the supernatant, major allergens were determined by immunoblot, Phl p 5 was quantitated by ELISA. There were striking differences in total protein and major allergen release between freshly collected and commercially available grass pollen grains as well as among freshly collected pollen between rural meadows and areas near high-traffic roads. There was a significantly different release of total protein being lowest in supernatants from commercially available pollen grains (rural/traffic vs. commercial, p<0.001), and of Phl p 5 major allergen (rural>traffic>commercial, p<0.005). Therefore, allergen bioavailability seems to be an important parameter in order to establish reliable dose-response relationships for the outdoor allergen response. Pollen grains incubated in aqueous protein-free buffer solution were also found to secrete significant amounts of eicosanoids namely prostaglandin E2 and leukotriene B4. Pollen grains thus do not act only as allergen carriers but also might have important implications on early events as initiators of allergy.

Air pollution and allergy: experimental studies on modulation of allergen release from pollen by air pollutants.

The fact that allergic diseases increase in prevalence is a generally accepted and worldwide phenomenon. The causes for this increase are not known: only hypothetical concepts exist. Epidemiological studies comparing Eastern and Western European populations have shown a striking difference in the prevalence of respiratory atopic diseases, which is lower in the East. At the same time, different patterns of air pollution have been described, namely 'classical' type I, characterized by SO2 and dust prevailing in the East, and 'modern' type II, characterized by organic compounds, fine particles and ozone, which is more prominent in the West. Type II was associated in multivariate regression analysis with increased prevalence of IgE-mediated allergy. Pollen grains collected from industrial regions with high polyaromatic hydrocarbon load in West Germany, but not in East Germany, were shown to be agglomerated with airborne particles. In vitro exposure of pollen to particles indicated morphological changes and increased allergen release from the pollen. In vitro exposure of pollen to gaseous pollutants (SO2 and NO2) under different conditions of humidity resulted in SO2-induced, but not NO2-induced reduction of allergen release from pollen. It is concluded that the bioavailability of grass pollen allergens may be modulated by air pollutants, supporting the concept of an interaction between pollen and pollutants in the atmosphere outside the organism which in turn may affect allergy-relevant phenomena.

Biomaterial-induced alterations of human neutrophils under fluid shear stress: scanning electron microscopical study in vitro.

Morphological changes of human polymorphonuclear neutrophils (PMN) adhering to hydrophilic (glass) and hydrophobic (FEP-Teflon, polyethylene, polypropylene) surfaces were studied in a parallel-plate flow chamber at the light and scanning electron microscopical levels. The PMN were exposed to a shear stress of 0.19 Pa (1.9 dynes cm-2) or were allowed to adhere without the stress component (static control) during 30 min for all four biomaterials. Observation by light microscopy was performed in situ in the flow chamber at 1, 5, 10, 15, 20, 25 and 30 min. The total number of adherent cells as a function of time and the activation status of the population on the basis of morphological criteria were determined. On the hydrophilic material adhesion of activated PMN was significantly higher (P < 0.05) than on the more hydrophobic surfaces. This effect was most pronounced for the adhesion of neutrophils to glass and polypropylene (PP). Polyethylene (PE) showed only minor adhesion rates. Scanning electron microscopy revealed details of cell shape changes and permitted a more precise classification of populations of neutrophils based on distinctive shapes. As PMN were exposed to shear stress on glass, the majority of cells exhibited surface veils, ridges and ruffles, suggesting a high level of cell migration. In this case, on polymeric surfaces the presence of filopodial networks (FEP-Teflon) and ameoboid cell shapes (PP and PE) was noted. The results suggest that a low shear stress, as well as various chemical and physical properties of biomaterial surfaces, are together responsible for differentiation of PMN populations on solid substrata.

Application of X-ray microanalysis to study of the expression of endothelial adhesion molecules on human umbilical vein endothelial cells in vitro.

A semi-quantitative procedure is described, which allows the evaluation of expression levels of endothelial adhesion molecules on cultured human umbilical vein endothelial cells (HUVEC) using energy dispersive X-ray microanalysis (EDX). As a model two adhesion molecules, E-selection (CD62E; ELAM-1/endothelial leukocyte adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1; CD54), were localized by the use of the silver-enhancement colloidal gold method after stimulation of HUVEC with endotoxin lipopolysaccharide (LPS), tumour necrosis factor (TNF) or a phorbol ester (PMA). The analysis was performed in a scanning electron microscope (SEM) at an accelerating voltage of 15 kV with scanned areas of 200 x 400 microns. The semi-quantitative data indicated that in LPS-treated groups both adhesion molecules were expressed at a significantly higher level than in all other groups (P < 0.01). In addition, after a 4 h treatment the expression levels of E-selectin in all groups were higher compared to ICAM-1. The experimental data from X-ray microanalysis were compared with data obtained from an enzyme-linked immunosorbent assay (ELISA) and similar values were found for both types of preparation. This microanalytical method is relatively simple and seems to be suitable for immunogold labelling studies on different types of endothelial cells in vitro.

Application of immunogold labelling for light and electron microscopic localization of endothelial leukocyte adhesion molecule 1 (ELAM-1) on cultured human endothelial cells.

This study describes the expression characteristics of E-selectin molecules using immunogold histochemical techniques on cultured human umbilical vein endothelial cells (HUVEC). The expression of E-selectin was induced by tumour necrosis factor-alpha (TNF-alpha, 300 U/ml), phorbol ester (PMA, 10 ng/ml) and bacterial lipopolysaccharide (LPS, 4 micrograms/ml). No expression was demonstrated on control cells. Using the silver-enhanced colloidal gold-labelling technique, at the light microscopical level, HUVEC could be distinctively subdivided into three staining types. The cell labelling index, expressed as the number of 'positively' stained cells as a proportion of all viewed cells was the highest in the LPS group. For transmission electron microscopy (TEM) the preembedding immunocytochemical staining method and embedding in epoxy resin (Agar 100) according to standard procedures was used. In TEM gold particles were localized in close association with the apical plasma membrane, as well as on the surface of microvillus-like projections (the latter by TNF-alpha group). For high resolution scanning electron microscopy (HR-SEM) the secondary (SEI) and the backscattered electron imaging (BEI) modes were used. Gold particles were randomly distributed over the whole cell surface, although they appeared to be denser in the perinuclear zone. The quantitative evaluation on SE and BE viewing (the number of gold particles per cell area in microns 2) demonstrated the highest density of labelling in the LPS-treated group, but there was only a significant difference between LPS and TNF-alpha groups (P < 0.01, t-test). Furthermore, the ultrastructural studies indicated that treatment with substances which up-regulate E-selectin expression was not related to toxic cell damage or significant alterations of cellular ultrastructure.

Effect of acute lead intoxication on the ultrastructure of neutrophils in the peripheral blood of the rat.

The influence of lead acetate on the ultrastructure of peripheral neutrophils was investigated in adult male rats. It was found that a single intraperitoneal administration of lead at dose 150 mg Pb/kg b.w. led to ultrastructural changes in the neutrophils at 3 and 6 h post injection. At the time of testing the exposed population had a mean (+/- SD) blood lead concentration from 206.2 +/- 24.8 micrograms/100 ml to 75.2 +/- 9.9 micrograms/100 ml compared with a mean value of 4.0 +/- 0.7 micrograms/100 ml for the control groups. The ultrastructural alterations such as irregular nuclei with deep invaginations, plasma membrane pockets, the presence of vacuoles with a heterogeneous material and an increasing amount of RER cisternae, were most clearly expressed 6 h after lead administration. In addition there appeared sometimes nuclear pockets, and a prominent crystalline inclusion placed in the cytoplasmic matrix of some neutrophils. No differences in the mitochondrial morphology and cytoplasmic granule pattern between exposed and control rats were observed.

The small intestinal enterocytes of rats during lead poisoning: the application of the Timm sulphide silver method and an ultrastructural study.

Young male rats were divided into 3 groups which received approximately 100 mg of a lead acetate/kg b.w. per day in their drinking water during 2, 30 and 60 d, respectively. Samples from the jejunum were processed for transmission electron microscopy (TEM), and after 60 d of exposure blocks of tissue were also processed to evaluating the Timm sulphide silver reaction sites in the epithelial absorptive cells in TEM according to Dancher and Zimmer (5). The ultrastructure of enterocytes in poisoned rats at 2 days was similar to the controls. A marked feature of about one third of the rat enterocytes exposed to lead for 30 d was the presence of numerous, small rough-membraned vesicles and prominent, dilated Golgi complexes in their cytoplasm. Most of the enterocytes at the 60th d of lead-exposed rats had a vacuolated cytoplasm associated with the prominent Golgi complexes and vacuoles of various size. They also had pleomorphic rough-membraned vesicles and dilated cisternae of the RER. The presence of the Timm reaction deposits has been observed on the microvillar surface of the brush border in connection with the enterocyte plasma membrane, and in the extracellular space between epithelial cells. Furthermore, Timm precipitates were found in extravascular spaces surrounding capillaries, between endothelial cells, and in the capillary lumen.

Electron microscopical localization of the lead in peripheral blood neutrophils of the rat with timm sulphide silver method and X-ray probe microanalysis.

The distribution of lead ions in rat peripheral neutrophils was investigated using Timm sulphide silver method. In the neutrophils of adult male rats the Pb-precipitates were electron microscopically localized at 1.5, 3.0 and 6.0 hours after a single intraperitoneal injection of lead at dose 150 mgPb kg b. w. After increased lead ions input to cell matrix a formation of cytoplasmic metal deposits as well as an aggregation of Pb-complexes within deep invaginations of nuclear membrane was detectable. Furthermore, the neutrophils were prepared to examination by scanning electron microscope according to Domagala et al. (1979). In neither case there was a clear difference of neutrophil surface morphology between exposed and control populations. The same preparations were then used to X-ray probe microanalysis, and correlation between the presence of Timm reaction product and lead indication was obtained.

Application on Timm sulphide silver method for electron microscope localization of lead ions in blood cells.

The application of the Timm sulphide silver method for the demonstration of lead ions in peripheral blood cells is investigated, using male white adult Wistar rats treated with a single dose of lead acetate, range 150 mg/kg b.w. To improve the Timm reaction for electron microscopy, fixation of whole cells with glutaraldehyde fixative solution saturated with H2S, an agarose embedding and physical development of thick sections without prior cryostat sectioning are presented. At 6.0 hours after injection both erythrocytes as well as white cells reveal the positive Timm reaction. All types of blood cells contain numerous cytoplasmic precipitates illustrating the intracellular lead accumulation. It is shown that the invaginations of white cell nuclear membrane possess a storing function as areas of lead depots. As a rule, the neutrophils display a highest amount of cytoplasmic precipitates and exclusively a low amount of reaction products in basophils is observed. At 14 days after injection, precipitates are present only in erythrocytes and monocytes. A suggestion of a possible functional significance of changes in the Timm staining pattern in blood cell types is discussed in this paper.