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Keywords:

  • basophil;
  • FACS;
  • IgE;
  • immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Background:  Allergen-specific immunotherapy (ASIT) in allergic rhinitis and asthma is the only treatment that effects the long-term development of these diseases. Basophil allergen threshold sensitivity, CD-sens, which is a valuable complement to resource-demanding clinical challenge tests, was used to monitor the initiation of ASIT induced allergen ‘blocking activity’.

Methods:  Patients IgE-sensitized to timothy (n = 14) or birch (n = 19) pollen were started on conventional (8–16 weeks) or ultra rush ASIT, respectively, and followed by measurements of CD-sens, allergen binding activity (ABA) and serum IgG4- and IgE-antibody concentrations.

Results:  CD-sens decreased during the early phase of ASIT-treatment. In parallel, ABA increased and correlated significantly with the increasing levels of IgG4 antibody concentrations. High dosages of allergen were more effective while mode of dosing up did not seem to matter. No change was seen in basophil reactivity.

Conclusion:  CD-sens and ABA, in contrast to basophil reactivity, seem to be promising tools to monitor protective immune responses initiated by ASIT.

Allergen-specific immunotherapy (ASIT) (1) is commonly used to treat patients with allergic rhino-conjunctivitis and asthma. ASIT decreases symptoms, reduces the need for conventional medication and prevents the onset of asthma and new sensitizations (2). Disadvantages include a minor risk for systemic reactions after the injections (3) and lack of information on optimal dosage of each allergen.

Various approaches have been selected to reach the allergen threshold and maintenance dose of ASIT from a slow increase of the allergen dose over weeks to months to accelerated build-up schedules (4) such as ultra-rush ASIT when incremental doses of allergen are given over days (5).

The intention with ASIT is to induce a clinical tolerance by stimulating the synthesis of ‘blocking antibodies’, supposedly of the IgG4 subclass, and partly interfering with the allergen-specific IgE-sensitization by immune tolerance committed lymphocytes (6, 7). There are at present no good and easy usable methods to monitor the immune response to ASIT. Clinical measures, such as decreased skin test reactivity, assessments of the patient’s clinical symptoms and the amount of medication required are no accurate measures of the immunological changes aimed at by ASIT.

The aim of this study was to compare, during the initiation phase of ASIT with birch or timothy pollen allergen, changes in basophil allergen threshold sensitivity, CD-sens (8, 9), representing effector cell allergen sensitivity and the immune response represented by serum concentrations of allergen-specific IgE and IgG4 antibodies.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients

Patients with allergic rhino-conjunctivitis with positive skin prick tests and IgE antibodies to birch and/or timothy grass pollen referred to routine ASIT were invited to participate in the study.

Birch ASIT (Aquagen® SQ; ALK-Abelló, Hørsholm, Denmark), was initiated as ultra rush regime with daily incremental doses of allergen reaching the maintenance dose on day three (5). Conventional timothy ASIT (Aquagen® SQ) was initiated by weekly injections during 8 or 16 weeks. CD-sens and allergen-specific serum antibody levels were measured before ASIT start and after reaching the maintenance dose (birch n = 19, timothy n = 14). In a number of patients these measures were repeated after an average of 34 (birch; 14 patients) and 57 (timothy; eight patients) weeks of maintenance treatment, that is, before pollen season, which represent a total of 44 and 66 weeks of treatment, respectively.

The study was accepted by the Local Ethics Committee at Lund University, Sweden.

Basophil activation and CD-sens

CD-sens analyses were performed as basophil allergen threshold stimulation (Aquagen® SQ, allergen concentrations: 0.01–10 000 SQU/ml) as previously described in detail (8). The higher the CD-sens, the higher is the basophil allergen sensitivity. The intra assay CV was 13.1% and the inter assay CV 5.4% (8). Percentage CD63 up-regulation after stimulation with one or a few high doses of the allergen in question was taken as a measure of basophil reactivity (9).

Calculation of allergen binding activity

The CD-sens from plasma-containing (untreated) whole blood was compared to the CD-sens in blood samples where the plasma had been removed before allergen stimulation. The ratio CD-sens after/before washing was calculated and termed ‘allergen binding activity’ (ABA) (10).

Immunoglobulin analyses

The serum concentrations of IgE (kU/l) were determined by ImmunoCAP® Total IgE (Phadia AB, Uppsala, Sweden) and of IgE- (kUA/l) and IgG4 antibodies (mg/l) to birch (t3) and timothy grass (g6) by ImmunoCAP® Specific IgE (Phadia AB) according to the manufacturer’s instructions.

Statistical analyses

Paired comparisons over time within patients were performed using Wilcoxon signed-rank test. Correlations between the different parameters were sought by calculating the Spearman correlation coefficient. Differences were considered statistically significant at P < 0.05. Data is presented as median values.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Basophil analyses

When comparing CD-sens in untreated blood before start of ASIT and when the maintenance dose of allergen was reached a significant drop in CD-sens was seen for both timothy (from 39.0 to 3.4; n = 14; P < 0.005) and birch (from 0.8 to 0.1; n = 19; P < 0.0005) (Fig. 1A) while CD-sens of washed basophils did not differ significantly (data not shown). The difference in untreated blood was still significant for both timothy (CD-sens 2.2; n = 8; P < 0.01) and birch (CD-sens 0.2; n = 14; P < 0.0005) after 66 and 44 weeks of treatment, respectively, that is, in a sample drawn just prior to pollen season (pre-season sample). When comparing ABA before treatment (timothy 1.2 and birch 1.3) and after reaching the maintenance dose (timothy 9.6 and birch 9.0) a significant (P < 0.0005) increase was seen for both timothy and birch (Fig. 1B) and the increase was still significant in the pre-season sample (timothy 7.6 and birch 8.3) (P < 0.05). In contrast, basophil reactivity remained unchanged for both allergens at both occasions.

image

Figure 1.  A significant P < 0.0005 drop in timothy (bsl00084) and birch (○) CD-sens in untreated blood was seen after maintenance allergen dose was reached (A). During the same period ABA (B) and allergen-specific IgG4 antibodies (C) increased significantly for both timothy (bsl00084) (P < 0.0005 and P < 0.001, respectively) and birch (○) (P < 0.0005 and P < 0.001, respectively). Basophil cell reactivity did not change.

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Serological analyses

Low levels of IgG4 antibodies to both timothy (1.7 mg/l) and birch (3.6 mg/l) were detected in most of the patients before ASIT treatment. The levels of IgG4 antibodies to both timothy and birch were significantly increased at maintenance dose (39.2 mg/l; n = 14; P < 0.0001 and 20.7 mg/l; n = 19; P < 0.0001, respectively) (Fig.1C) and even more in the pre-season samples (timothy 167 mg/l, P < 0.01; and birch 57 mg/l, P < 0.001).

Correlations between basophil activation and serological parameters

A significant correlation was found between ABA and concentration of IgG4 antibodies, for birch (rS = 0.67; n = 19; P < 0.01) already in the maintenance dose samples (data not shown). This correlation was even more pronounced in the pre-season samples for both birch (rS = 0.80; n = 14; P < 0.001) and timothy (rS = 0.83; n = 8; P < 0.05) (Fig. 2).

image

Figure 2.  ABA representing the ratio of CD-sens of plasma containing whole blood to CD-sens of blood samples after plasma removal, for birch (rS = 0.80; P < 0.001) and timothy (rS = 0.83; P < 0.05) correlated significantly to the level of IgG4 antibodies in the pre-season sample. Both allergens are plotted together.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

It is generally agreed that ASIT (1) is able to modulate the immune response of the IgE antibody initiated inflammation responsible for the allergic symptoms. An IgE-sensitization, as well as allergic symptoms, is initiated by extremely small, μg per season, amounts of allergen (11). However, by stimulating the patient’s immune system with increasing allergen dosages, up to mg, a state of allergen-specific immune tolerance, presumably based on ‘blocking’ antibodies, preferentially IgG4, and allergen-specific lymphocytes, is developed (6, 7, 12).

Basophil allergen threshold sensitivity, CD-sens, is an established way to quantify allergen sensitivity (8–10) and could be useful to monitor changes in allergen sensitivity of patients undergoing ASIT. The patients in this study were diagnosed as having allergic rhino-conjunctivitis due to IgE-sensitization to birch or timothy pollen. The birch allergic patients were treated with an ultra rush initiation phase and the timothy allergic with conventional dosing up regimes of eight or 16 weeks. Since one effect of ASIT is presumed to be stimulation of the production of allergen blocking factors in serum the ABA (10) was measured. If ABA is present more allergen is needed to trigger the basophils and the CD-sens, and presumably allergy expression, will be lower.

Before start of ASIT all patients had a high CD-sens to the allergen in question but essentially no ABA. However, already when the patients had reached the maintenance dose the median ABA value had increased approx. ten fold. No significant difference was seen between the rush and conventional initiation mode. At pre-season, the effects of ASIT were more prominent in the group treated with timothy allergen as compared to birch pollen, that is, drop in median CD-sens (timothy 18 times, birch four times), increase in median ABA (timothy six times, birch six times) and increase in median IgG4-anibody levels (timothy 98 times, birch 16 times). The explanation is most likely differences in time for maintenance dose; timothy was given for almost twice as long time as birch, 58 and 34 weeks, respectively.

The effect of ASIT and its optimal design has long been debated (13) and it would be of great importance and interest to use the objective and risk-free CD-sens as a complement to organ sensitization during significant modifications of the conventional ASIT scheme, for example, the low dose sublingual immunotherapy (14) and a rapid and high dosing up using anti-IgE to cover for side effect (15).

Allergen-specific IgG, and especially IgG4, antibodies are thought to represent allergen blocking factors in plasma (12) and to achieve a good IgG4 antibody response is one main purpose with ASIT (6). Very low levels were found before ASIT, but the allergen injections of ASIT stimulated a strong increase. Thus, already at maintenance dosage the levels had increased more than 20 times and continued to increase during further treatment. The correlation with ABA strongly supports the hypothesis that IgG4 antibodies are ‘blocking’ allergen access to inflammatory cells and thus symptoms. However, ASIT most likely has additional effects on the basic mechanisms of allergy (16), and these effects should be included in ABA.

Interestingly, CD-sens in washed basophils did not change significantly during the course of treatment. In contrast basophils from patients receiving six years of anti-IgE treatment expressed a decrease in allergen sensitivity most likely dependent on cellular changes (10). It would be most interesting to see whether this phenomenon will appear also after many years of ASIT treatment.

In conclusion, already the early phase of ASIT resulted in a decrease in patient allergen sensitivity, as measured by CD-sens, combined with a dramatic increase in IgG4 antibodies and ABA, while no effect of ASIT was seen on the basophil reactivity. CD-sens and ABA, in contrast to basophil reactivity, seem to be promising tools to monitor presumably protective immune responses initiated by ASIT. A higher total dose of allergen given over time resulted in more allergen blocking activity. If a mixture of noncross-reacting allergens are given in the same injection the allergen to which the patient has highest IgE antibody levels will set the roof for the amounts of the mixture given. As a consequence optimal stimulation of the other allergens in the mixture will be difficult to reach.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We would like to thank Ingegerd Ågren-Andersson for performing the ImmunoCAP® analyses and Åse Olerud for helping with the basophil analyses.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References