Ulf Pipkorn

The cellular response of the human allergic mucosa to natural allergen exposure.

It has been suggested that the IgE-dependent late-phase reaction to allergen exposure, with the features of an inflammatory cellular infiltration and airway hyperreactivity, is a link between anaphylaxis and continuous allergic airway disease. Our main knowledge of the cellular response to allergen in sensitized individuals has been derived from allergen-challenge models. To explore the dynamics of the cellular response during the actual disease, patients with a strictly seasonal allergic rhinitis were studied during natural allergen exposure. Ten patients suffering from an isolated birch-pollen allergy were followed from a symptom-free state before, during, and to the height of the birch-pollen season. Repeated parallel cell samplings from the nasal mucosa were performed with cytologic imprints on plastic strips, nasal lavages with the recovery of the cells in the lavage fluid with cytocentrifugation on object slides for cytologic study, and scrapings from the nasal surface with a curette for histologic and ultrastructural evaluation. The histamine content was determined in lavage fluid and cell pellets. The tosyl-alpha-tosyl-L-arginine methyl esterase activity of the nasal lavage fluid was also determined as a biochemical marker of the allergic inflammatory reaction. The birch-pollen season was moderate in terms of pollen counts, and this resulted in mild to moderate nasal symptoms that ran parallel to the birch-pollen counts. The total number of cells recovered in the lavage fluid was 1.2 +/- 0.4 (SEM) x 10(6) before and 3.2 +/- 2.0 per 10(6) cells (not significant) during pollen exposure. Most cells were neutrophils and mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraepithelial Migration of Nasal Mucosal Mast Cells in Hay Fever

Mast cells were studied by light microscopy in mucosal imprints and in biopsies of nasal mucosa of 12 birch pollen allergic individuals before and during the pollen season, using techniques optimized for the demonstration of mucosal mast cells. We also measured the histamine content of nasal mucosa, whole blood and plasma, and counted the numbers of circulating blood basophils. Before the pollen season the nasal mucosa was found to contain many mast cells located in the mucosal connective tissue stroma, and very few cells with basophilic and metachromatic granules were found in mucosal imprints. During the pollen season there was a redistribution of mast cells into the epithelium, many such cells now being recovered in mucosal imprints. The total number of mucosal mast cells counted in tissue sections did not change significantly with the onset of the pollen season, suggesting a redistribution of mucosal mast cells by migration. Judged by morphologic appearance and naphthol-AS-D chloroacetate esterase activity, the intraepithelial mast cells found in tissue sections had rather the properties of tissue mast cells than of blood basophils, and only a few of the basophilic cells of the imprints had a morphology compatible with blood basophils. The histamine content of the mucosa, as well as histamine levels of whole blood and plasma, and circulating blood basophil numbers did not change significantly in relation to the pollen season. These findings suggest that an intraepithelial migration of mucosal mast cells is part of the allergic mucosal response.(ABSTRACT TRUNCATED AT 250 WORDS)

A brush method to harvest cells from the nasal mucosa for microscopic and biochemical analysis

A method is described for the sampling of epithelial cells and other effector cells from the human airway mucosa for structural and biochemical analysis. The cell samples are obtained from the nasal mucosa using a small nylon brush which is rotated over the epithelium and soaked and shaken in a small volume of a balanced salt solution. Morphological evaluation using light microscopy and transmission electron microscopy revealed excellently preserved cytological detail. In asymptomatic individuals the cells harvested were as follows: 45 +/- 5.9% (mean +/- SEM) epithelial cells, 38 +/- 7.1% granulocytes, 16 +/- 2.3% large mononuclear cells (monocytes), and 1.3 +/- 2.3% eosinophils. Repeated measurements in the same individual revealed a coefficient of variation of the order of 40% for the proportions of cells harvested. In comparison with nasal airway lavage, a higher proportion of epithelial cells and monocytes were obtained with the brush method. The cells harvested could also be used for biochemical analysis. The histamine content of the cell pellets was found to be strongly correlated with the mast cell count (r = 0.93) and was estimated to about 10 pg/cell, which is higher than previously reported for mast cells obtained from human lung tissue dispersed by an enzymatic method. The present method appears to be appropriate for the study of cellular events in the nasal mucosal epithelium.

Intraepithelial migration of mucosal mast cells in hay fever: ultrastructural observations

Evidence has been presented suggesting that a migration of nasal mast cells from the mucosal connective tissue stroma into the epithelium is part of the mucosal response in birch pollen allergy. In a previous study, the identification of these intraepithelial cells as tissue mast cells rather than blood basophils was based on light microscopical morphology and histochemistry. We have now studied the ultrastructure of these cells in mucosal biopsies taken before and during the birch pollen season. Intraepithelial cells with basophil or metachromatic granules were only observed in biopsies taken during the season. Some of these cells had the ultrastructural appearance of tissue mast cells, including cytoplasmic lipid droplets and a granular substructure composed of multilamellar arrays and scrolls, serving to distinguish human mast cells from blood basophils. The ultrastructural traits of the remaining cells were heterogeneous, some reminiscent of human blood basophils, others of globule leucocytes of other species, but entirely typical blood basophils could not be identified. The results thus support our previous suggestion that a migration of mucosal mast cells from the connective tissue stroma into the epithelium is part of the human allergic mucosal response. It cannot be determined whether the ultrastructural heterogeneity of these cells is the result of an adaptation to the intraepithelial environment of one single mast cell type or to the existence of an ultrastructurally distinct mucosal mast cell.

Budesonide and Nasal Allergen Challenge Testing in Man

In a nasal allergen challenge test, the effect of the glucocorticosteroid, Budesonide, on the pollen allergic type 1 reaction has been investigated, Placebo and two different dosages of budesonide were administered intranasally for 1 week before the challenge was performed, The study was designed as a double‐blind cross‐over trial.

Effect of Topical Glucocorticoid Treatment on Nasal Mucosal Mast Cells in Allergic Rhinitis

Glococorticoids were previously considered not to affect the immediate allergic reaction. However, in a nasal allergen challenge, an inhibitory effect on the nasal symptoms induced at the challenge has been shown to occur in patients treated with a recently developed Glucocorticoid, budesonide, for 1 week prior to the challenge. This treatment was also found to reduce tissue histamine levels in the nasal mucosa. Mast cells in the mucosa were therefore studied with a view to finding out whether this reduction could be due to a reduction of mast cells. A double‐blind study was performed in 14 asymptomatic patients. Nasal biopsies were made before and after I weeks treatment with either budesonide or placebo. The number of mast cells was counted in two Epon sections after the specimens has undergone specific staining with toluidine blue. No quantitative or qualitative morphological changes in the mast cells were found as a result of treatment.

Nasal mucosal mast cells and histamine in hay fever, effect of topical glucocorticoid treatment

Symptomatic seasonal allergic rhinitis has previously been found to be associated with a redistribution of mast cells from the subepithelial stroma to the epithelial lining and the surface of the nasal mucosa. The present study was designed in order to elucidate the interaction between topical glucocorticosteroids, effective in the treatment of allergic rhinitis, and the migration of mast cells described earlier. Six patients treated prophylactically in the nose with budesonide were studied. Imprints and biopsies from the nasal mucosa were taken 2-3 weeks before and 2-3 weeks into the birch pollen season. The biopsies were used for light microscopy and tissue histamine determination. The morphologic studies showed, also in the actively treated patients, an increased number of metachromatically stained cells on the nasal mucosal surface of the same order of magnitude as previously reported for untreated patients. We did, however, find a decrease in the histamine content of the nasal mucosa, which was not associated with a decrease in the number of mast cells. Together with similar previous findings in the unstimulated allergic nasal mucosa these results suggest that glucocorticosteroids induce a decrease in the mast cell histamine pool, possibly due to an inhibition of the intracellular synthesis of histamine. This effect might contribute to the clinically beneficial effect of topical glucocorticosteroids in the treatment of hay fever.

Effect of a Topical Glucocorticoid, Budesonide, on Nasal Mucosal Blood Flow as Measured with 133 Xe Wash‐Out Technique

The “vasoconstrictor” effect of dermally applicated steroids is a widely used parameter when determining the potency of glucocorticoids. A similar effect on the nasal mucosa has been suggested as providing an explanation for the clinical effect of topically administered glucocorticoids in the treatment of nasal disorders such as allergic and vasomotor rhinitis. The recently described 133 Xe wash‐out method was used for the purpose of blood flow determination in 11 healthy subjects. In a randomized double blind cross over study, the effect of topically administered budesonide, a non halogenated glucocorticoid, for 1 week was compared with that of placebo. No diference was found to occur in the mucosal blood flow after the administration of budesonide, as compared with placebo. It seems likely that a more complex activity than vasoconstriction is responsible for the clinical effect in the treatment of nasal disorders, and further research is required in order to clarify this issue.

Secretory Activity of Nasal Mucosal Mast Cells and Histamine Release in Hay Fever

Although theoretical considerations and experimental evidence implicate the mast cells in the pathophysiology of the immediate type hypersensitivity reaction, the evidence of their active participation in human allergic disease is still fragmentary. We have therefore sought evidence of mast cell activation in allergic mucosal disease using strictly seasonal allergic rhinitis as a model. Twelve patients with birch pollen-induced hay fever were examined before and well into the birch pollen season. Allergen exposure was monitored by pollen counts and the degree of symptoms registered daily. Small surgical biopsies and mucosal imprints were obtained from each patient before and during the season. Mast cells were analysed by light and electron microscopy and mucosal histamine was measured using a sensitive HPLC assay. We found a reduction in the number of mast cells in the nasal mucosa during pollen exposure (p less than 0.05) but no significant reduction of the histamine content. There was a correlation between the nasal mucosal mast cell density and histamine content before the pollen season (r = 0.76; p less than 0.01), but no such correlation was found during the period of pollen exposure (r = 0.19; n.s.). This finding points to secretory activity by the mast cells during the pollen season and to the appearance of a non-mast cell pool of tissue histamine. Evidence for a secretory activity of the mast cells during the pollen season was also confirmed by electron microscopy. In addition, we found a strong correlation (r = 0.77; p less than 0.01) between the histamine content of the nasal mucosa during the pollen season and the degree of nasal symptoms. The number of epithelium-associated mast cells found on mucosal imprints prior to the pollen season showed a strong correlation with the symptoms experienced later during the period of pollen exposure (r = 0.83; p less than 0.01). Taken together these observations indicate that the mast cell has a pathogenetic role in continuous allergic airway disease and re-emphasizes the role of histamine in the induction of the symptoms of allergic rhinitis.

Phenotypic expression of proteoglycan in mast cells of the human nasal mucosa

SummaryThe phenotypic expression of the proteoglycan of human mast cells in the nasal mucosa and normal skin was analysed using histochemical techniques. Nasal mucosa was obtained from normal subjects, from patients with seasonal allergic rhinitis before and during the pollen season and from patients with nasal polyps. In the latter groups, specimens were taken from both polyp tissue and adjacent nasal mucosa. Formaldehyde treatment blocked the cationic dye binding in 75–84% of the mast cells located in the nasal mucosa, as compared to the optimum fixation with IFAA (iso-osmotic formaldehyde-acetic acid). A significantly lower degree of blocking of dye binding was obtained in the human skin where 45% of the mast cells were susceptible to formaldehyde treatment (P<0.01). The mast cells of the polyp tissue also showed a relatively low degree of blocking (54%), which was significantly lower than the blocking of mast cells of the nasal mucosa taken from the same individuals (P<0.05). Staining of serial tissue sections in Alcian Blue containing graded concentrations of MgCl2 was used to determine the critical electrolyte concentration (CEC) of the dye binding, defined as the salt concentration at which the staining of 50% of the mast cells is extinguished. The CEC of the skin mast cells was 0.64m MgCl2 which is significantly higher than that of the mast cells of the nasal mucosa of normal subjects [0.49m (P<0.05)], allergic subjects [0.52m (P<0.01)], patients with polyp disease [0.52m (P<0.01)] and the polyp tissue proper [0.57m (P<0.05)]. This implies that mast cells of the nasal mucosa contain glycosaminoglycans of a relatively lower charge density and/or molecular size than the connective tissue mast cells found in the human skin. A similar difference has been observed between rat mucosal mast cells, containing a chondroitin suphate proteoglycan, and rat connective tissue mast cells which contain a heparin proteoglycan. However, unlike the rat mucosal cells, the mast cells of the human nasal mucosa showed a weakly fluorescent Berberine binding and, like the rat connective tissue mast cells, entirely lost the ability to bind Toluidine Blue after treatment with nitrous acid. Such treatment results in a deaminative cleavage of heparin and heparan sulphate, but does not degrade chondroitin sulphate. These results provide further evidence of the existence of a distinctive mucosal mast cell phenotype also in man. It is suggested that the lower CEC of the mucosal mast cells is an expression of a content of haparan sulphate, while the relatively higher CEC of the skin mast cells is compatible with a content of heparin.