Cellular Profiles in Peripheral Blood Accompanying Particular Asthmatic Response Types

Background: Patients with allergic bronchial asthma develop various asthmatic response types to bronchial challenge with allergen, such as immediate (IAR), late (LAR), dual late (DLAR) or delayed (DYAR), displaying different clinical, immunologic and pharmacologic features. This study deals with count changes of particular blood cells accompanying the IAR, LAR and DYAR. Methods: In 63 patients developing 22 IAR, 26 LAR and 15 DYAR, the repeated allergen challenges were supplemented with recording of blood cell counts, Th 1 /Th 2 ratio, leukotrines B 4 (LTB 4) and C 4 (LTC 4), eosinophil cationic protein (ECP), myeloperoxidase (MPO), and histamine in blood, and intracellular IFN-γ and IL-4 in peripheral blood mononuclear cells. Results: The IAR was accompanied by increased eosinophil and basophil counts, increased serum concentrations of histamine, LTC 4 and ECP, decreased Th 1 /Th 2 ratio in favour of Th 2 cells, and increased intracellular IL-4. The LAR was associated with increased eosinophil and neutrophil counts, increased serum concentrations of LTC 4 and LTB 4 , unchanged Th 1 /Th 2 ratio, and increased intracellular IL-4. The DYAR was accompanied by increased total leukocyte, neutrophil, monocyte, lymphocyte and thrombocyte counts, increased serum concentrations of LTB 4 and MPO, increased Th 1 /Th 2 ratio in favour of Th 1 cells, and increased intracellular IFN-γ. Conclusions: These results provide evidence for different involvement of particular blood cell types and different hypersen-sitivity mechanisms in IAR, LAR and DYAR. The monitoring of peripheral blood cell counts seems to be an useful supplementary parameter to the bronchial challenge with allergen.

The purpose of this study, being a continuation of our previous pilot studies [16,17] was: 1) To investigate the changes in cellular counts in peripheral blood associated with particular types of asthmatic response to allergen challenge (BPT); 2) To assess the possible involvement of the individual circulating cell types in the hypersensitivity mechanisms underlying the particular types of asthmatic response.

Patients
Sixty-three asthmatics examined at our Department of Allergology & Immunology, Institute Medical Sciences "De Klokkenberg", Breda, The Netherlands) and developing 22 IARs, 26 LARs and 15 DYARs to bronchial challenge with allergen (BPT) (Figures 1-3), volunteered to participate in this study.These patients, 21 -44 years of age, suffered from reversible bronchial obstruction, alternating with symptom-free periods, without any restrictive changes of their pulmonary function [18].They had no airway infections and did not use oral corticosteroids or immunotherapy.They were examined by routine diagnostic procedure, serving also as an inclusion-exclsion criteria, including also 87 BPTs with inhalant allergens and 63 PBS (phosphate buffered saline) control challenges (Tables 1 and 2) All BPTs were performed in a period without manifest bronchial complaints, outside the allergen-relevant season and during hospitalization.
In these 63 patients the positive BPTs with the same allergens (Table 2) and the PBS controls were repeated 2 -3 weeks later (Figures 1-3) and supplemented with recording of cell counts and other factors in venous blood before, and 1, 12, 24, 36, 48, 56 and 72 hours after the challenge (Tables 3-5).The local ethical committee (IBR-MCK) approved this study and an informed consent was obtained from all participants.

Control Subjects
In 17 healthy subjects volunteering to participate as control subjects 17 BPTs with PBS were supplemented with the blood cell counts up to 72 hours after the challenge (Table 1).

Allergens
Dialyzed and lyophilized allergen extracts (Allergopharma, Reinbek, Germany) diluted in PBS were used in concentrations of 100 -500 BU/mL for skin tests and 1000 -3000 BU/mL for BPTs (Table 2).The recommended concentrations by the manufacturer were 500 BU/mL for skin tests and 5000 BU/mL for the BPTs.

Bronchial Provocation Tests (BPT)
The BPTs were performed by means of spirometry (Spirograph D-75 Lode, Groningen, The Netherlands) re-cording the FVC and FEV 1 values.The aerosolized allergen extracts and PBS were inhaled using Wiebadener Doppel-Inhalator at an airflow of 10 L/min.The nebulizer output was 0.12 -0.14 mL/min and the aerosol particles had a median mass diameter of 2.8 -3.6 μ.
The BPTs, being a modification of the European standard [19,20], were performed as follows: 1) Initial (baseline) values recorded at 0, 5 and 10 minutes; 2) PBS control values recorded at 0, 5 and 10 minutes after a 10 minute PBS inhalation; 3) Inhalation of allergen aerosol for 2 × 5 minutes, with inserted spirometry measurement, followed by the recording of the FEV1 and FVC values at 0, 5, 10, 20, 30, 45, 60, 90 and 120 minutes and the every hour up to 12th hour, every second hour during the 22nd and 38th , the 46th and 62nd and at the 72nd hour interval.The PBS control challenge was performed by the same schedule as that of the BPTs with allergens.A 5-day interval has always been inserted between the consecutive tests.
The intra-assay as well as the inter-assay coefficients of variations were less than 5%.

) Intracellular cytokines from activated PBMC cultures
The peripheral blood mononuclear cells (PBMC) were separated by Hypaque-Ficoll density-gradient centrifugation (Pharmacia, Sweden) and cultured at a concentration of 5 × 10 6 cells/mL in presence of 50 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma) and 1 μg/mL ionomycin for 24 hours.After culture, the cells were centrifuged and viability was determined using trypan blue dye exclusion.Supernatants were stored at −80˚C.The concentrations of cytokines were measured in the centrifuged supernatants by means of enzyme-linked immunoassay (ELISA) kits (Quantikine, R & D Systems, utes at 4˚C.The plasma supernatants were removed and processed within 1 hour.The resting aliquots were stored at −70˚C [15].USA), in accordance with the manufacturer's instructions.The minimal detectable limits (pg/mL) were: IFN-γ: 8.0 and IL-4: <10.0.The intra-assay coefficients of variations of these assays were <9% and the inter-assay coefficients of variations were <10% [14,15].
The serum/plasma levels of appropriate factors were measured by using commercially available kits, following manufacturer's recommendations.ECP was estimated in the serum, whereas all other factors were determined in the plasma.All measurements were performed in duplicate by a double-blind schedule.The intra-assay as well as the inter-assay coefficients of variations for all the assay kits employed were <10%.

4) Cellular constituents in the serum/plasma
Venous blood samples (5 mL) were collected into separator tubes (S-Monivette, Sarstedt, Germany), kept at room temperature for 1 hour and then centrifuged at 3000 × g for 10 minutes at 4˚C.The serum supernatants were removed, stored at 2˚C -8˚C and processed within 1 hour.Other venous blood portions were collected in vacutainers containing EDTA, kept for 1 hour at room temperature and then centrifuged at 2500 × g for 10 min-

Statistical Analysis
The asthmatic responses and the PBS controls were statistically analyzed by means of fitting polynomials to the mean curves over time; eight time points within 120 minutes and twenty-five time points up to 72 hours after    the BPT The hypotheses were tested by generalized multivariate analysis of variance model (MANOVA) [21].
The post-challenge cell counts and factors recorded at each of the time points during the asthmatic responses and PBS controls in individual patients were compared with their pre-challenge values and statistically analyzed by Wilcoxon matched-pair signed rank test.The mean post-challenge cell counts and other factors measured at each time point during the asthmatic responses were compared with corresponding PBS values and evaluated by Mann-Whitney U test.A p value < 0.05 was considered to be statistically significant.

Results
The IAR (n = 22) appearing within 120 minutes after the BPT (Figure 1) was statistically significant, both in comparison of the post-challenge with the pre-challenge FEV 1 values and as compared with the PBS controls (p < 0.01, p < 0.001, respectively).
The LAR (n = 26) occurring between 8 -12 after the BPT (Figure 2) was statistically significant both in comparing the post-challenge with the pre-challenge FEV 1 values and in comparison with the PBS controls (p < 0.001, p < 0.001, respectively).
The DYAR (n = 15) appearing between 26 -56 hours after the BPT (Figure 3) was statistically significant, both in comparing the post-with the pre-challenge FEV 1 values and in comparing with the PBS controls (p < 0.001, p < 0.01, respectively).No significant differences were found between the initial and the repeated IAR (p > 0.1), LAR (p > 0.2) or DYAR (p > 0.05).
The IAR was associated with immediate skin response in 73% and positive allergen-specific IgE in the serum in 41% (Table 1).
The LAR was associated with late skin response in 69%, positive specific IgE in the serum in 12%, increased total serum IgG in 38%, IgG 3 in 12%, IgG 4 in 19% (Table 1).
The DYAR was associated with delayed skin response in 60%, and increased total serum IgG in 13% (Table 1).
No significant differences were found in the appearance of the individual asthmatic response types and their cellular profiles in peripheral blood with respect to the particular allergens (p > 0.1).

Control Subjects
Neither significant changes in the counts of any cell type nor significant changes in the concentrations of the other factors in blood were found in the control subjects after the PBS control challenges (p > 0.05).
The cellular changes in blood during the individual asthmatic response types have been investigated intensively in animals [28,29].However, papers concerning repeated counting of the blood cell, especially in con-junction with their constituents in the blood, during the particular asthmatic response types in humans are not numerous [9,11,12,16,17,[22][23][24][25][26][27].Moreover, in most of these studies only some of the cell types were counted.
The studies concerning the cellular changes on the materials collected directly from the asthmatic lungs, such as (induced) sputum, BAL fluid or lung biopsies, provide important data related to the site of the immunologic processes, which is the bronchial tissue and bronchial lumen [6][7][8][9]30,31].However, the bronchial tissue also has relationships with other organs, especially with the vascular system and blood, and immunologic events occurring in the bronchial tree may display reciprocal influence with the capillary network and circulating blood cells [1,5,6,10,13,26,[30][31][32][33].The inflammatory cells participating in the immunologic processes in the bronchial tissue are either cells recruited from the circulation into the airway tissues or the cells already resident in those tissues.Vice versa, some of the inflammatory cells after participating in the bronchial immunologic processes can re-migrate into the circulation, and various factors released in the bronchial tissue can penetrate into the circulation and affect there the circulating cells [1-3, 5,6,9,10,13,22-27].
The role of a particular blood cell type in a certain immunologic process may be defined by a its morphologic and functional properties and by its temporary stage and location [6,8,9,26,30,[34][35][36]. The cells can be involved in an immunologic process either as its participant or as its target.This role can be evaluated by various criteria: 1) The cell count (density) and its changes in time, related to a certain event, e.g.allergen exposure/challenge, representing the dynamic aspect of such involvement; 2) The activation degree of the cells, associated often with intracellular granule changes; 3) The ability of the cells to generate and release typical constituents during a certain period of the immunologic process; 4) the localization and kinetics of that cell type [1][2][3][7][8][9][10]13,23,26,27,30].The satisfactory evaluation of the role of a certain cell type requires measurement of the representative parameters before and repeatedly after a well-defined event, such as bronchial challenge with an allergen [3,[10][11][12][13][14][15][16][17].
The blood cell counting cannot be fully compared with other techniques, such as measurements of cells in the (induced) sputum, BAL fluid or bronchial biopsies, having also their advantages and disadvantages [10,24,[31][32][33][34][35][36].Their important advantage is the generation of highly specific data related directly to the immunologic event in the bronchial tree and mucosa.Their disadvantage includes relatively high variations in the cell counts and in amounts of the recovered fluid (e.g.BAL), the necessity of special facilities and personal skill, and some standardization problems [30][31][32]35].Moreover, the BAL and lung biopsies represent also a certain burden and risk for the patient, and cannot be therefore repeated frequently within a short period of time [32][33][34][35].
Conversely, the blood cell counts can also be influenced by various extra-pulmonary factors and these data may therefore be less specific for the bronchial events than the cell counts in the sputum or BAL fluid.On the other hand, the blood cell counting is an easy, non-burdening, method, requiring no special facilities, which can be repeated without any limitation.
Our results demonstrating different kinetics of the individual cell types in peripheral blood during the particular asthmatic response types suggest involvement of different immunologic mechanisms in these types.Our results confirm also the existence of the so-called noneosinophilic or neutrophilic asthma phenotypes [4,[6][7][8]27].They also suggest the need to refine the classical interpretation of the blood eosinophilia as one of the most important indicator for the allergic bronchial asthma.
During the IAR, the eosinophil counts decreased after the BPT, followed by their increase, together with increased serum concentrations of ECP and LTC 4 .The LAR was accompanied by increased eosinophil counts and increased concentrations of LTC 4 , but not by changes of ECP.These findings suggest different involvement of eosinophils in the IAR and in the LAR.In contrast, no changes in eosinophil count were recorded during the DYAR [14][15][16]37,38].
Another interesting finding were increased neutrophil counts accompanied by concentration changes of LTB 4 but not of MPO during the LAR, whereas increased neutrophil counts during the DYAR were associated with concentration changes both of LTB 4 and MPO [13-17, 37,38].
The changes in Th 1 /Th 2 ratio in favour of Th 2 and increased intracellular concentrations of IL-4, but not that of IFN-γ, during the IAR would suggest an active involvement of Th 2 -cells in immunologic mechanisms underlying this asthmatic response type.
The changes in Th 1 /Th 2 ratio in favour of Th 1 and increased intracellular concentrations of IFN-γ, but not that of IL-4, during the DYAR would indicate an important role of Th 1 -cells in the immunologic processes leading to the DYAR.The balanced Th 1 /Th 2 ratio, upon increased IL-4 concentration, would point to an almost simultaneous involvement of Th 1 and Th 2 cells in mechanisms underlying the LAR [13][14][15][16][17]37,38].
The serial monitoring of the blood cell counts during the BPTs can be helpful to discriminate the particular asthmatic response types and can act as an additional confirmation of the recorded asthmatic response type.This technique can also be combined with other techniques, if necessary.