Immunocytochemical and flow cytofluorimetric detection of intracellular IL-4, IL-5 and IFN-gamma: applications using blood- and airway-derived cells.

We have compared an immunocytochemical and a flow cytofluorimetric method to detect intracellular IFN-gamma, IL-4 and IL-5 in T-cell clones, peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid (BALF) cells. Intracellular bound cytokine-specific antibodies were visualized either with amino-ethyl carbazole (for immunocytochemistry), or with fluorescent antibodies (for flow cytofluorimetry). The staining was inhibited with recombinant cytokines and corresponded qualitatively and quantitatively to cytokine levels in the supernatants of T-helper-0 (Th0), Th1 and Th2 clones. In analysing in vitro stimulated cells, sufficient signal in the fluorimetric assay was only obtained after the addition of monensin to the cultures. We then observed a good correlation between immunocytochemical (with no monensin added) and the flow cytofluorimetric staining for all three cytokines (PBMC, IFN-gamma and IL-4, rho = 0.9, no IL-5 detectable; clones, IL-5, rho = 0.81, all three p < 0.05). However, compared to flow cytometry, a greater percentage of positively stained cells was frequently observed using immunocytochemistry. In BALF cells, the immunocytochemical method was able to detect significant percentages of positive cells without in vitro stimulation of the cells, in contrast to the flow cytofluorimetric method. In BALF cells from sarcoidosis patients, T-cells were mainly IFN-gamma-positive (immunocytochemically assessed), both with (mean +/- SEM, 39.7 +/- 9.8%), and without (3.5 +/- 1.3%) in vitro stimulation. In BALF cells from allergic subjects, the immunocytochemical method showed lymphocytes positive for IFN-gamma (40.3 +/- 8.3%), IL-4 (19.1 +/- 0.49) and IL-5 (6.1 +/- 3.1). We conclude that both methods can be used to assess the production of IFN-gamma, IL-4 or IL-5 at the single-cell level in T-cell clones, PBMC and cells from the BALF. The high sensitivity and the low number of cells required for the immunocytochemical method indicate that this method can provide detailed information on cytokine production of airway-derived cells in diseases with airway inflammation such as sarcoidosis and asthma.

Repeated allergen challenge as a new research model for studying allergic reactions.

Repeated allergen challenge might be a more relevant model for studying symptomatic disease, because it gives more information on the patient's handling of chronic allergen exposure. Thus, this experimental model has more resemblance to the situation of natural allergen exposure, and the allergen load can be standardized. However, the response to repeated allergen challenge in individual patients can show a large variation, that is from a strongly enhanced response to complete diminution of the response. Successful allergen immunotherapy can change the response pattern of repeated allergen challenge in the skin into down-regulation of the late reaction. Chronic or repeated allergen exposure may result in an enhanced allergen-specific allergic response, involving allergen-specific T-cell activation. Different subsets of T cells can exert either activating or suppressive effects on inflammatory cells involved in subsequent allergic reactions. CD8+ T cells might exert suppressive effects, because they seem to be associated with a subsequent down-regulation of the late skin reaction after repeated allergen challenge (Fig. 4). Further studies are needed to compare the responses to repeated allergen challenge with the response to natural seasonal allergen exposure in the same patients and to explore possible underlying mechanisms using, for example, nasal biopsies.

Effect of local allergen priming on early, late, delayed-phase, and epicutaneous skin reactions.

Allergic disease is reflected in a chronic inflammatory response to an allergen. It is thought that local allergen priming underlies this chronicity. To assess the effect of allergen priming on the amplitude and histologic effect of the allergic reaction, four sequential, intracutaneous skin tests were done with 48-h intervals in 13 patients allergic to the house-dust mite Dermatophagoides pteronyssinus (Dpt). Reactions were measured at 15 min, and at 6, 24, and 48 h. Subsequently, epicutaneous tests were done on Dpt-primed spots (n = 5). At 6, 24, and 48 h, reactions increased after priming (P < 0.006), with unaltered early reactions. Epicutaneous reactions to Dpt on primed spots were larger than in epicutaneous controls on similarly primed skin. Local intradermal priming results in greater inflammatory responses at both intra- and epicutaneous challenge. This mechanism may explain the chronicity of allergic reactions at epithelial surfaces.

Sensitivity to hydrocortisone is a relevant factor in the immunoendocrine relationship. I. The cell-mediated immune response in relation to blood levels and in vitro immunosuppressive effects of hydrocortisone in patients with asthma and healthy control subjects.

The role of endogenous hydrocortisone in the regulation of lymphocyte activity was assayed in patients with asthma (patients with chronic nonspecific lung disease, characterized by attacks of dyspnea, alternating with symptom-free periods) and healthy control subjects. After priming, delayed-type hypersensitivity skin reactions were induced with Helix pomatia hemocyanin (HPH). Hydrocortisone blood levels were measured. The effect of hydrocortisone on HPH-induced lymphocyte proliferation was determined in vitro. The results demonstrate that hydrocortisone in low concentrations (100 ng/ml) inhibited in vitro lymphocyte proliferation equally in patients and control subjects. However, both groups demonstrated a large interindividual variation in hydrocortisone sensitivity. Therefore, in order to determine the immunologic effect of hydrocortisone blood levels in vivo, a hydrocortisone suppression index (HSI) was calculated by use of the information on hydrocortisone concentrations in vivo and the biologic effect of hydrocortisone in vitro. This HSI appeared to be inversely related with the in vivo cell-mediated immune response to HPH. This was reflected in an inverse correlation between HSI and delayed-type hypersensitivity skin reactions to HPH, both in patients (R = -0.64), in control subjects (R = -0.69), and in the total group (R = -0.68; p less than 0.001). No differences were observed between patients and control subjects. It is concluded that endogenous hydrocortisone is likely to play an important role in the regulation of lymphocyte activity in patients with asthma and healthy control subjects. This may have important consequences for the clinical expression of asthmatic symptoms, since the role of lymphocyte activity in the pathogenesis of asthma is increasingly recognized.

Primary cell-mediated immune response in bronchial asthma. Relationship between primary in vitro and in vivo cell-mediated and antibody responses in patients with asthma and healthy controls.

Eleven patients with asthma and ten sex and age matched healthy controls were immunized with the primary immunogen Helix pomatia Haemocyanin (HPH). The amplitude and the kinetics of in vitro cell-mediated immune response were measured by HPH-induced lymphocyte proliferation. Lymphocytes were also challenged in vitro with mitogens and recall antigens. In vivo cell-mediated immunity was determined by inducing delayed type hypersensitivity reactions with HPH. Anti-HPH antibody responses in the IgE, IgG and IgM classes were measured to gain an insight into the relation between cell-mediated and humoral immune responses in patients with asthma and healthy controls. The in vitro and in vivo cell-mediated response and the IgM antibody response did not differ between patients with asthma and controls. The IgE and IgG antibody responses, however, were increased in the patients. IgM antibody response correlated with both the in vitro and in vivo cell-mediated response (R = 0.45, P less than 0.05). IgE and IgG antibody responses however were not correlated with cell-mediated responses. These data suggest that the primary abnormality in immune regulation in patients with asthma concerns the control of the IgE and IgG class antibody responses.

A comparison of salmeterol and formoterol in attenuating airway responses to short-acting beta2-agonists.

In vitro data suggest that salmeterol, contrary to formoterol, can partly antagonise the effect of short-acting beta(2)-agonist rescue medication. To explore whether this occurs in vivo, we compared the effects of increasing doses (200-3200 microg) of fenoterol on the recovery of methacholine induced bronchoconstriction as well as PD(20) methacholine in 23 asthmatic patients, during two-week treatment periods with placebo, and standard doses of salmeterol or formoterol in a double blind, double-dummy, crossover study. Salmeterol showed a slightly higher propensity for the development of bronchodilator tolerance. The recovery of methacholine induced bronchoconstriction was more complete during regular use of formoterol relative to salmeterol. During regular use of both long-acting beta(2)-agonists the bronchoprotective efficacy of fenoterol was attenuated, but this was more pronounced during salmeterol than during formoterol. The mean maximum increase in PD(20) metacholine after the highest dose of fenoterol was 3.97 DD during placebo, 2.47 DD during formoterol (p<0.001) and 1.81 DD during salmeterol treatment (p<0.001). We conclude that in asthmatic patients the efficacy of short-acting beta(2)-adrenoceptor agonists can be significantly attenuated during regular use of long-acting beta(2)-agonists. In this respect, differences were observed between salmeterol and formoterol that may represent the expression of partial antagonism by salmeterol.

Induced sputum and bronchoalveolar lavage as tools for evaluating the effects of inhaled corticosteroids in patients with asthma.

Changes in airway inflammation can be studied with bronchoalveolar lavage, but the widespread use of this procedure is limited by its invasiveness. The aim of this study was to evaluate the usefulness of induced sputum as a non-invasive alternative to bronchoalveolar lavage for studying changes in airway inflammation in patients with asthma. Thirty patients were treated for 12 weeks with an inhaled corticosteroid (fluticasone propionate (FP), 250 microg twice daily) or a short-acting beta-agonist (salbutamol (Sb), 400 microg twice daily) in a double-blind, double-dummy, randomized parallel group study. Sputum induction with hypertonic saline solution was performed twice before treatment and after 4, 8, 10, and 11 weeks of treatment. Bronchoalveolar lavage fluid divided into two pools (first 60 mL portion as bronchoalveolar lavage/bronchial wash (BAL/BW) and subsequent 80 mL as bronchoalveoalar lavage (BAL)) was obtained before and after 12 weeks of treatment. Changes in cell differentials and plasma-protein leakage (alpha2-macroglobulin, albumin, and their ratio (relative coefficient of excretion, RCE)) were analyzed in induced sputum and were compared with changes in BAL/BW and BAL. During treatment with FP, the PC20histamine (interpolated concentration of histamine that caused a fall in FEV1 of 20% of the baseline value) increased (P < .0001), and the percentage of eosinophils (P = .004), levels of (alpha2-macroglobulin (P = .09) and RCE (P = .007) decreased in sputum. These changes were different from those in the Sb group (PC20histamine P< .0001, eosinophils P= .004, alpha2-macroglobulin P= .003, RCE P = .01), in which alpha2-macroglobulin showed a significant increase (P = .015). Changes in the percentage of eosinophils and in the levels of alpha2-macroglobulin in sputum were associated with changes in the PC20histamine (Rs = -0.59, P = .007 and Rs = -0.47, P = .03, respectively). These correlations did not reach significance in BAL/BW and BAL fluid. The statistical power to detect changes in induced sputum was higher for the percentage of eosinophils and similar for plasma protein leakage as compared with analysis of BAL/BW and BAL fluid. We conclude that the analysis of induced sputum is a useful, non-invasive alternative to bronchoalveolar lavage for assessing the effects of antiinflammatory drugs in asthma.

HLA-DR expression is induced on keratinocytes in delayed hypersensitivity but not in allergen induced late-phase reactions.

In view of increasing evidence suggesting an active immunoregulatory role of the skin keratinocytes and the observation that the differentiation of allergen specific T lymphocytes is critical in the development of allergy, we evaluated epidermal expression of HLA-DR antigen in skin reactions induced with an atopen (house dust mite) and with an non-atopic antigen (Hemocyanin). Two groups of patients with house dust mite (Dermatophagoides pteronyssinus [Der p]) allergy were compared, one group was skin tested with Der p, the other group was immunized and subsequently skin tested with Helix pomatia Hemocyanin (HPH). Biopsy specimens taken at 48 h after the HPH (n = 11) and Der p (n = 11) tests were analysed immunohistologically. Reactions in both groups were comparable in size. Immunohistological analysis showed domination by CD4+ lymphocytes. Expression of HLA-DR antigen by epidermal keratinocytes was observed in six out of 11 of the HPH induced reactions, but in none of the Der p induced reactions. Eosinophils were spotted only throughout the Der p induced reactions, showing a good correlation with the number of CD4 positive lymphocytes. The lack of HLA-DR expression by keratinocytes during the allergen-induced reaction, compared with the Hemocyanin induced reaction can be the result of a difference in cytokine profile of the lymphocytes dominating the dermal infiltrate. On the other hand evidence exists that defective HLA-DR expression by keratinocytes enhances antigen induced lymphocyte activation, and may thus contribute to the development of allergen-specific T-lymphocytes.

Allergen-induced changes in airway responsiveness are related to baseline airway responsiveness.

In the literature, bronchial allergen challenge is usually reported to result in an increase in histamine-induced airway responsiveness (AR). The present study investigated the relation between baseline AR and allergen-induced changes in AR. The effect of allergen challenge on AR was investigated in 21 atopic asthmatic patients. Allergen challenge resulted in a significant decrease in PC20 histamine after 24 h. When the group was divided into three subgroups according to baseline PC20 histamine, a significant decrease in PC20 histamine was found only in patients with relatively high baseline PC20 histamine (groups 1 and 2). A significant inverse correlation was found between baseline PC20 and allergen-induced PC20 histamine. The effect of repeated allergen challenge on AR was studied in eight patients. The first allergen challenge resulted in a significant decrease in PC20 histamine; no further decrease in mean PC20 histamine was seen after the second allergen challenge. These results suggest that allergen-induced changes in AR occur mainly in patients with relatively high baseline PC20 values. Once an increase in AR is induced, further allergen challenge does not always result in further increase in AR.

Early and late allergic reaction in the nose assessed by whole body plethysmography.

Physiological changes during late phase nasal responses after allergen challenge are difficult to establish and different criteria are used for the definition of a positive late phase nasal reaction. The objective of this study was to assess the value of whole body plethysmography in detecting changes in nasal airway resistance after allergen challenge and to suggest criteria for the definition of early and late phase nasal reactions. Nasal challenge with allergen was performed in 15 allergic patients. Nasal resistance was followed until 10 h after allergen challenge and on a control day using whole body plethysmography. The mean percentage changes in the inspiratory nasal resistance during the early phase period (0.25-2 h) and the late phase period (4-10 h) were significantly higher on the allergen challenge day than on the control day (p=0.001 and p=0.01, respectively). The mean percentage change in the inspiratory nasal resistance during the early and late phase period on the control day plus 2 times the standard deviation served as cut-off point for a positive reaction. Using this definition, all patients had early reactions and 7 of the 15 patients (47%) also had late reactions. We conclude that whole body plethysmography is a useful, noninvasive method for the measurement of the physiological changes in the nose following allergen challenge.

Influx of neutrophils into the airway lumen at 4 h after segmental allergen challenge in asthma.

BACKGROUND: Segmental allergen challenge is a powerful tool to study inflammatory reactions in asthmatic airways. There is little information on the early events at 5 min and 4 h after allergen challenge with respect to the cell influx and the chemokine interleukin-8 (IL-8). METHODS: Seven mild to moderate allergic asthmatics (AA group), 5 allergic nonasthmatics (ANA group) and 5 nonallergic controls underwent segmental allergen challenge, with allergen doses based upon skin reactivity. Bronchoalveolar lavage (BAL) samples were obtained before, 5 min and 4 h postchallenge, and were analyzed for cell numbers and differential counts, eosinophil and neutrophil chemotactic activity, and levels of IL-8. RESULTS: At 5 min postchallenge, no changes were observed compared to baseline. At 4 h postchallenge, an increase was found in the number of neutrophils and the levels of IL-8, which was dependent on the dose of allergen in the AA and ANA group. At the same allergen dose, the increases in neutrophils and levels of IL-8 were calculated to be 91 and 67 times higher, respectively, in AA than in ANA. Levels of IL-8 correlated with the number of neutrophils and with the in vitro neutrophil chemotactic activities in BAL fluid. CONCLUSIONS: Neutrophil chemotactic activity is increased in BAL fluid at 4 h after segmental allergen challenge. We suggest that apart from IgE-mediated mast cell degranulation, additional local factors in the airways determine the degree of IL-8 increase and neutrophil influx.

A double-blind study on the effect of inhaled corticosteroids on plasma protein exudation in asthma.

Plasma protein exudation into the airways is an important pathophysiological event in asthma. The effect of 12 wk of treatment with inhaled fluticasone propionate (FP; 250 microgram twice a day) or salbutamol (Sb; 400 microgram twice a day) on plasma protein leakage was compared in a double-blind, randomized parallel-group study of 30 patients with asthma. Primary outcomes were plasma protein leakage and size selectivity of the blood-airway lumen barrier, cell differentials in BAL fluid, and bronchial responsiveness to histamine (PC20histamine). Two independent procedures to account for the effect of variable dilution of BAL on the levels of albumin (Alb) and alpha2-macroglobulin (A2M) in BAL fluid consisted of correction based on urea levels and on the application of the relative coefficient of excretion [RCE = ([A2M] in BAL fluid/[A2M] in serum)/([Alb] in BAL fluid/[Alb] in serum)]. In the FP group a significant decrease was found in the A2M level and the RCE, and in the percentage of eosinophils in BAL fluid. The PC20histamine increased significantly (mean increase, 2.4 doubling doses), whereas PC20histamine decreased in the Sb group. Differences between groups were significant except for the decrease in eosinophils. We conclude that 12 wk of FP (250 microgram twice a day) decreased the permeability of the blood-airway lumen barrier, in particular for high molecular weight proteins.

Segmental allergen challenge induces plasma protein leakage into the airways of asthmatic subjects at 4 hours but not at 5 minutes after challenge.

We have investigated whether increased plasma protein leakage is present early after segmental allergen challenge in allergic asthma. Seven asthmatic subjects with mild allergy (AA group) and 5 non-asthmatic subjects with allergy (ANA group) were challenged with allergen doses based on similar early skin reactions; 5 healthy control subjects without allergy (C group) were challenged with the highest dose applied in the subjects with allergy. Bronchoalveolar lavage (BAL) fluid was obtained before, at 5 minutes after, and at 4 hours after challenge from different segments. Levels of albumin (Alb) and alpha2-macroglobulin (A2M) were measured in BAL fluid and serum. In addition, we calculated the relative coefficient of excretion as follows: RCE = ((A2M in BAL fluid)/(A2M in serum))/((Alb in BAL fluid)/(Alb in serum)). Also, levels of tryptase as a marker of mast cell activation and tumor necrosis factor-alpha (TNF-alpha), a possible inducer of plasma protein leakage, were determined. At 5 minutes after challenge, in none of the groups was a significant change found in the parameters for protein leakage. Levels of tryptase were increased in the subjects with allergy at 5 minutes after challenge only (P = .004). At 4 hours after challenge, levels of Alb (P = .03) and A2M (P = .04) and the RCE (P = .04) were increased in the AA group only. At 4 hours, levels of TNF-alpha were increased, with no significant differences among the three groups. In the asthmatic subjects with allergy, levels of TNF-alpha correlated with levels of Alb (r = 0.85, P = .02). In conclusion, at 4 hours after segmental allergen challenge, plasma protein leakage was increased in the asthmatic subjects only. The increase in levels of TNF-alpha in all groups indicates that the presence of TNF-alpha alone was not sufficient to cause plasma protein leakage within 4 hours after allergen challenge. Our results confirm the concept that plasma exudation after allergen exposure is a pathophysiologic event associated with asthma.

The primary immune response in bronchial asthma. I. A kinetic study of Helix pomatia hemocyanin-specific IgE, IgG, IgA, and IgM antibody responses in patients with asthma and in matched controls.

Eleven asthmatic patients with allergy and 10 age- and sex-matched controls were immunized subcutaneously with 1 mg of the primary test immunogen Helix pomatia hemocyanin (HPH). The HPH-specific IgE, IgG, IgA, and IgM antibody response was measured by ELISA. After immunization, not only the IgE but also the IgG antibody response of the patients exceeded that of the controls (p less than 0.01). IgA antibody response also tended to be higher in the asthmatic group (NS). Except for an earlier rise after immunization in the asthmatic group (p less than 0.05), IgM peak responses did not differ significantly between the two groups. There was a high correlation (r = 0.9) between the magnitude of the IgE and the IgG antibody responses. IgM antibody response did not correlate with the response in any of the other antibody classes measured. The total serum immunoglobulin concentration was determined before immunization. Only the IgE level was significantly higher in the asthmatic group (p less than 0.01). No correlation was found between serum immunoglobulin concentrations and the magnitude of the HPH-specific antibody response in the same class. We conclude that the increased humoral responsiveness of asthmatic patients with allergy is not restricted to the IgE class nor to a limited number of commonly encountered antigens (allergens).

Allergen immunotherapy inhibits airway eosinophilia and hyperresponsiveness associated with decreased IL-4 production by lymphocytes in a murine model of allergic asthma.

In the present study, we investigated whether allergen immunotherapy is effective in a murine model with immunologic and pathophysiologic features reminiscent of allergic asthma. Ovalbumin-sensitized mice received increasing (1 microgram to 1 mg) subcutaneous doses of ovalbumin twice a week for 8 wk according to a semirush immunotherapy protocol as used in allergic patients. During immunotherapy, an initial rise in serum levels of ovalbumin-specific antibodies (immunoglobulin [Ig]G1, IgE, IgG2a) occurred, after which IgE levels decreased sharply concomitant with an increase in IgG2a levels. The increase in IgG2a levels, with the decline in IgE levels, suggests that during immunotherapy interferon-gamma production is increased or interleukin (IL)-4 production is decreased. After immunotherapy, inhalation challenge of the mice with ovalbumin revealed almost complete inhibition (98%, P < 0.01) of eosinophil infiltration into bronchoalveolar lavage and airway hyperresponsiveness (100% at 320 microgram/kg methacholine, P < 0.05) compared with sham-treated animals. In addition, IL-4 production of thoracic lymph node cells stimulated with ovalbumin in vitro was largely reduced (60%, P < 0.05) after immunotherapy. Thus, effective immunotherapy in this animal model appears to be due to modulation of antigen-specific T cells. Similar effects on airway symptoms and IL-4 production can be obtained within 1 wk by three injections of the highest dose of ovalbumin (1 mg). This animal model will be used as a preclinical model to improve allergen immunotherapy and to gain more insight into the mechanisms involved.