Gunilla Halldén

Markers of inflammation and bronchial reactivity in children with asthma, exposed to animal dander in school dust

Several studies have confirmed the presence of animal dander allergens in school dust but the effect of this indirect animal exposure on health has not been evaluated. In this study we investigated bronchial reactivity and markers of eosinophil activity and inflammation during two separate weeks of school in 10 children with mild asthma and a positive skin prick test to cat and dog. At the beginning and the end of the first week the children underwent bronchial challenges with methacholine, and at the beginning and the end of the second week they underwent nasal lavages (NAL) and induced sputum samplings. Blood and urine samples for analysis of inflammatory markers were obtained before and after both school weeks. Peak expiratory flow (PEF) and symptoms of asthma and allergy were recorded daily, and spirometry was performed on each visit. The exposure to animal dander allergens was estimated from dust samples obtained in the subjects’ schools and homes. Bronchial sensitivity to methacholine increased in the week when this was measured. The proportion of eosinophils in peripheral blood, and urinary eosinophil protein X (EPX), decreased in both weeks. There was a trend towards an increase of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) in sputum in the week when these proteins were measured. The concentrations of cat (Fel d1) and dog (Can f1) allergens were higher in dust collected in schools than in homes. Our results show that in children with mild asthma and animal dander allergy, there is a significantly increased bronchial sensitivity to methacholine after one school week. There is also a significant decrease in the number of circulating eosinophils and a trend towards an increase of sputum EPO, which could correlate with the early phase of eosinophil recruitment to the lungs. These effects may be related to the continuous exposure to animal allergens in school dust.

Difference in the Blood Monocyte Phenotype Between Uremic Patients and Healthy Controls: Its Relation to Monocyte Differentiation into Macrophages in the Peritoneal Cavity

The phenotypic alterations between blood monocytes from 11 patients with end-stage renal disease, who had been on peritoneal dialysis for less than one week, and blood monocytes from 10 healthy controls, were analyzed. In addition, peritoneal macrophages in the dialysate effluent were enclosed. Analysis of functional receptor density was performed using immunostaining and flow cytometry. The phenotypic characterization was selected to represent various biological functions such as adhesion, phagocytosis (CD11b/CD18, CD11c/CD18, CD16), antigen-presentation (HLA-DR, ICAM-1), differentiation (transferrin receptor, CD71), receptor for LPS (CD14) and initiation of the coagulation cascade (Tissue factor, CD142). The proportion of CD16-positive blood monocytes and the quantitative level of ICAM-1 were higher in the patient group, compared to healthy controls. A significant increase in the quantitative level of CD11b/CD18, CD11c/CD18, HLA-DR and ICAM-1, transferrin receptor, CD 14 and CD 16, was found on peritoneal macrophages, compared to monocytes, harvested both from the corresponding patients, as well as from healthy donors. In contrast, we did not find any significant differences in the expression of tissue factor between monocytes and peritoneal macrophages. In conclusion, phenotypic differences exist between monocyte populations in the blood circulation of CAPD patients, and healthy individuals. We also show that transmigration of monocytes into the peritoneal cavity implies a selective up-regulation of functional receptors, preferentially related to adhesion, and antigen-presentation in a steady-state situation in non-infected CAPD patients.

Eotaxin Increases the Expression of CD11b/CD18 and Adhesion Properties in IL5, But Not fMLP-Prestimulated Human Peripheral Blood Eosinophils

A selective recruitment of eosinophils to sites of inflammation is claimed to be controlled by regulation of cytokines, chemokines and adhesion molecules. In animal models, eotaxin has been suggested to be a potent chemokine since it in cooperation with interleukin-5 induce selective chemotaxis and infiltration of eosinophils to lung tissue after an allergen provocation. We have investigated the in vitro effect of eotaxin on human peripheral blood eosinophils with respect to CD11b/CD18 expression and adhesion properties to the matrix protein fibronectin. We did not find any effect of eotaxin per se on CD11b/CD18 expression, neither on eosinophils from healthy subjects nor from patients with asymptomatic pollen related asthma. However, eotaxin significantly upregulated the quantitative level of CD11b/CD18 and increased the adhesion to fibronectin in eosinophils from healthy subjects preincubated in vitro with interleukin-5, but not in eosinophils preincubated with fMLP. Moreover, eosinophils harvested 24 hours after an in vivo allergen inhalation provocation in asthmatics, upregulated CD11b/CD18 after in vitro incubation with eotaxin alone.

Quantitative, rather than qualitative, differences in CD69 upregulation in human blood eosinophils upon activation with selected stimuli

Background: The study aimed to investigate whether CD69 expression on granulocytes is subject to specific regulation by inflammatory mediators, and, if so, to identify these factors in relation to eosinophil activity markers such as the EG2 epitope and ECP release.

The degree of natural allergen exposure modifies eosinophil activity markers in the circulation of patients with mild asthma

We have previously found that CD9, CD11b, and intracellular ECP (EG2) may be used as activity markers for eosinophils in vitro. The main object of the present study was to determine whether these markers can reflect eosinophil activation in vivo in relation to allergen exposure. For this purpose, six patients with a history of allergic rhinitis and occasional asthma symptoms during the pollen season participated. Blood donors served as controls. Peripheral blood eosinophils were analyzed according to the FOG method and flow cytometry, before and during one birch pollen season with high pollen load (HPL) and one with low pollen load (LPL). The CD9 expression on peripheral eosinophils from the patients was significantly increased both before (P<0.05) and during (P<0.01) HPL season, and CD11b expression solely during HPL season (P = 0.01) as compared to controls. The intracellular expression of the EG2 epitope was increased before (P<0.01) and during (P<0.05) HPL season, and increased significantly (P<0.05) during season as compared to before. No changes were observed before and during LPL season. The proportion of eosinophils was increased both before (P<0.05) and during (P<0.001) the HPL season as compared to controls. The markers CD9, EG2, and, to a lesser extent, CD11b seem to detect activated eosinophils in the circulation, whereas EG2 may also reflect increased antigen exposure during season.

Allergen-induced inflammation in the nose : a comparison of acute and repeated low-dose allergen exposure

To investigate allergic rhinitis induced by two experimental provocation models, we compared local inflammation with markers of eosinophil activity in peripheral blood. Patients with strictly seasonal allergic rhinitis were selected and investigated outside the pollen season. An acute challenge with increasing doses of allergen every 15 min until symptoms occurred was performed in nine patients. Nasal lavage and blood samples were taken before and 4 and 24 h after challenge. After a 6‐week washout period, 10 patients were submitted to 7 days of repeated low allergen exposures. One small dose (≅/100 of the acute dose) was given each day. Blood and lavage samples were taken prior to and after the period. As control four patients were challenged with diluent only. The acute challenge resulted in sneezing and nasal discharge and blockage and was accompanied by a rise in histamine and eosinophil cationic protein (ECP) in lavage fluid after 4 h and continuing after 24 h, when there also was a rise in the number of eosinophils and ECP in peripheral blood. The repeated low‐dose exposures caused very few symptoms but produced increased ECP in the lavage Auid and a trend toward increased histamine concentration. There were no changes in ECP, intracellular EG2 binding, or number of eosinophils in the blood. No changes were seen in the control group. Our findings show that changes in eosinophil mediator release in nasal lavage can be seen after very low, but repeated, allergen exposures despite no, or minimal, clinical symptoms.

No signs of activity markers in peripheral blood despite increased bronchial reactivity after repeated low-dose allergen exposure.

The allergen inhalation test can be used as an experimental model to study pathophysiological events in allergic asthma. Repeated low‐dose inhalations of allergen induce increased bronchial hyperresponsiveness (BHR) and resemble natural allergen exposure. The objective of the present study was to investigate whether eosinophil recruitment and activation in peripheral blood, differences in expression of lymphocyte surface antigens and increased bronchial responsiveness to histamine occur during and after repeated low‐dose bronchial allergen challenge. Fourteen atopic asthmatic patients were challenged in a randomized double‐blind manner for 7 days with either allergen in very low doses or placebo. We measured the concentration of eosinophils, eosinophil cationic protein (ECP) and the expression of the EG2‐epitope on intracellular ECP in eosinophils and the expression of lymphocyte surface antigen markers in peripheral blood. The challenge period started and ended with a histamine provocation. The repeated low‐dose allergen exposure resulted in a significant increase in BHR. No changes were seen in the placebo group. Concerning the inflammatory parameters in peripheral blood, no significant changes were seen during or after the week of low‐dose allergen inhalations. Our results show that very low, repeated doses of allergen induce increased airway reactivity despite lack of evident clinical symptoms or signs of activation of inflammatory cells in peripheral blood.

Eosinophil activity markers in peripheral blood have high predictive value for bronchial hyperreactivity in patients with suspected mild asthma.

The present study aimed to evaluate the predictive value of eosinophils and markers of their activity for bronchial hyperreactivity (BHR) in a population of patients with recently developed clinical symptoms of asthma. The activation of eosinophils was estimated by measuring eosinophil cationic protein (ECP) in serum. In addition, flow cytometry was used to measure the expression of the EG2-epitope on intracellular ECP in eosinophils from peripheral blood. Twenty-eight consecutive patients with clinical history of asthma were studied. Of the 28 patients, 18 had a positive bronchial challenge test measured as PD20 < or = 1600 micrograms histamine. A significantly higher concentration of eosinophils and a trend to higher ECP in the peripheral blood was found in the hyperreactive group than in the nonreactive group. However, the intracellular expression of ECP did not correlate with the PD20 value, and no significant difference between the groups was found. With one eosinophil activity marker, either serum ECP or EG2, BHR could be predicted in 70% of the patients. If we combined any two of the activity markers (serum ECP, EG2, or the percentage of eosinophils), the predictive value increased to 100%. We conclude that the blood eosinophil concentration, as well as, to some extent, serum ECP, has a high specificity for BHR in patients with recently developed clinical symptoms of asthma. Despite normal bronchial reactivity, some patients had signs of activated eosinophils, i.e., high serum ECP and increased EG2 expression. Thus, these markers may reflect early stages in the development of BHR. Our results also indicate that a combined evaluation of percentage of eosinophils and of eosinophil activity markers is of clinical value to predict BHR.

T‐cell subsets in adenoids and peripheral blood related to age, otitis media with effusion and allergy

Adenoids and peripheral blood samples from 29 children (20–120 months of age) undergoing adenoidectomy for long‐standing otitis media with effusion (OME) (n=16) or obstructive adenoid hyperplasia (n=13) were investigated by flow cytometry for their T‐lymphocyte profile. Eleven of the enrolled children were allergic to inhalant and/or food allergens. For the whole group, the percentage of helper T cells belonging to the memory phenotype (CD4+/CD45RO+ cells) was significantly higher in adenoids than in blood (p<0.0001), while the same cell category increased with age in peripheral blood (p<0.01). A highly significant negative regression (p<0.001) was found between age and the percentage ratio of CD4+ cells that were CD45RO+ in adenoids and blood. Allergic children had a higher CD4+/CD8+ ratio for cells expressing CD45RO+ (p<0.05) in adenoids. The results of this study indicate that adenoids participate in the development of an immunological memory. Our findings support a relationship between allergy and memory cells in adenoids.

Eosinophil activity markers in peripheral blood have high predictive value for bronchial hyper-reactivity in patients with

The present study aimed to evaluate the predictive value of eosinophils and markers of their activity for bronchial hyper‐reactivity (BHR) in a population of patients with recently developed clinical symptoms of asthma. The activation of eosinophils was estimated by measuring eosinophil cationic protein (ECP) in serum. In addition, flow cytometry was used to measure the expression of the EG2‐epitope on intracellular ECP in eosinophils from peripheral blood. Twenty‐eight consecutive patients with clinical history of asthma were studied. Of the 28 patients, 18 had a positive bronchial challenge test measured as PD20 1600 μg histamine. A significantly higher concentration of eosinophils and a trend to higher ECP in the peripheral blood was found in the hyper‐reactive group than in the nonreactive group. However, the intracellular expression of ECP did not correlate with the PD, value, and no significant difference between the groups was found. With one eosinophil activity marker, either serum ECP or EG2, BHR could be predicted in 70% of the patients. If we combined any two of the activity markers (serum ECP, EG2, or the percentage of eosinophils), the predictive value increased to 100%. We conclude that the blood eosinophil concentration, as well as, to some extent, serum ECP, has a high specificity for BHR in patients with recently developed clinical symptoms of asthma. Despite normal bronchial reactivity, some patients had signs of activated eosinophils, i.e., high serum ECP and increased EG2 expression. Thus, these markers may reflect early stages in the development of BHR. Our results also indicate that a combined evaluation of percentage of eosinophils and of eosinophil activity markers is of clinical value to predict BHR.