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- Material and methods
Background: Serum specific IgE, basophil histamine release, and blood eosinophil parameters are associated with allergic rhinitis, but investigations of the relationship to the severity of allergic symptoms are few and conflicting. Our study aimed to investigate the seasonal changes in the following laboratory tests: specific IgE, basophil histamine release, eosinophil counts, and serum and plasma eosinophil cationic protein (ECP) and eosinophil protein X (EPX), and to analyze, in detail, the relationship of each individual test to the severity of symptoms in rhinitis patients allergic to both birch and grass pollen.
Methods: The above tests were performed on blood samples obtained from 49 allergic rhinitis patients during the birch-pollen season, during the grass-pollen season, and after the seasons. Symptom-medication diaries were filled in during both pollen seasons. We used partial least square (PLS) analysis to establish an optimal statistical link between the symptom score and medication and the laboratory tests, in an investigator-independent way.
Results: Increases in specific IgE, basophil histamine release, eosinophil counts, serum ECP and EPX, and plasma EPX were observed from the birch-pollen season to the grass-pollen season, followed by a decrease from the grass-pollen season to after the pollen seasons, except for the specific IgE. No seasonal changes in plasma ECP and total IgE were seen. The PLS analysis found a relationship between symptom score and medication and the aggregate laboratory tests (F-test value 40.2, correlation 0.34 for the cumulative relation). However, the variation in laboratory tests could explain only half of the total variation in symptoms and less than a quarter of the total variation in medication. The symptom score and, to a minor degree, medication were especially correlated with the basophil histamine-release results, with a decreasing relevance of specific IgE, eosinophil counts, total IgE, serum and plasma EPX, and serum ECP. Plasma ECP was not related to the symptom score and medication.
Conclusions: A significant relationship between the severity of allergic rhinitis and various allergic inflammatory markers was found but could account for only a minor part of the variation in the patients' evaluation of their disease.
Abbreviations: ACRI: mean daily capsules of acrivastine; ANTA: mean daily antazoline-naphazoline eye-drops; CAP: specific IgE measured by Pharmacia CAP System; ECP: eosinophil cationic protein; EOS: eosinophil counts; EPX: eosinophil protein X; HR: histamine release; HR15: lowest concentration of extract giving ≥15 ng histamine/ml blood in histamine-release test; LEU: leukocyte counts; MAX: maximum histamine released (ng/ml) at any concentration of extract in histamine-release test; NAL: nasal lavage; PLS: partial least square analysis; PRED: mean daily mg of prednisolone; SYMP: mean daily value of total symptom score.
Seasonal allergic rhinitis, or hay fever, is a prevalent allergic inflammatory disease that seems to be well defined by a coincidental occurrence of pollen exposure and symptoms. Accordingly, symptom scores from patient diaries have been much used in attempts to quantify the severity of the allergic disease. This approach is not without problems, however, and a surrogate marker is needed, since diaries are time-consuming and laborious for the patient. Moreover, symptom scores depend upon medication, and several investigators have combined symptom scores and use of medication into a symptom-medication score. However, the relative weight of symptom scores and of medication has varied from one study to another (1[2-4]–5). Thus, it would be preferable to use a method of weighting symptom and medication scores that is dependent on the actual data, and not on a somewhat arbitrary weight chosen by the investigator.
The allergic inflammation is initiated by allergen cross-linking of specific IgE antibodies, which are attached to mast cells and basophil leukocytes. During the subsequent late phase, the eosinophil leukocyte is the most characteristic cell of the inflammatory reaction in the nasal mucous membrane (6[7-13]–14). Analysis of markers of these cornerstones of the allergic inflammation could prove highly relevant as a practical and objective replacement of or supplement to symptom-medication scores, provided that they are well correlated with the patient's symptoms. The literature gives several examples of seasonal variations in IgE serum levels (15, 16), basophil histamine release (HR) ( 17), and eosinophil activity (18[19, 20]–21), but no clear pattern has emerged.
Accordingly, our study aimed to determine whether a number of different measurements of allergic inflammatory markers would correlate with the seasonally induced symptoms of patients, and, if so, to identify the analysis having the optimal correlation with symptoms, which would thus be the best suited to supplement/replace symptom scores.
- Top of page
- Material and methods
Our study showed an increase, from the early birch-pollen season (visit 1) to the mid-grass-pollen season (visit 2), in specific IgE, basophil HR, and eosinophil parameters, indicating an activation/upregulation of the IgE synthesis, and the basophil and eosinophil leukocyte, from a low birch-pollen season with few allergic symptoms to a high grass-pollen season with increased allergic symptoms.
An increase in grass-specific IgE from visit 1 to visit 2 was observed, whereas total IgE did not increase. This indicates an antigen-specific activation of the IgE-producing B cell. Basophil leukocytes possess high-affinity receptors for IgE, and mediators are released when the allergen cross-links the specific IgE bound to its high-affinity receptor. We found an increase in both anti-IgE and specific IgE HR from visit 1 to visit 2. Furthermore, there was a clear HR relationship between the anti-IgE response and the allergen-specific response, which could be explained by a high proportion of specific IgE to the total IgE bound to the basophils. The grass parameters increased from visit 1 to visit 2, and, surprisingly, birch-specific IgE and birch HR also increased from visit 1 to visit 2. In our study year, the birch-pollen season started later than usual, and the blood tests at visit 1 were performed at the beginning of the birch-pollen season, when there was only a small amount of pollen. The birch-pollen load increased just after visit 1. A peak response in birch-specific IgE and birch HR after visit 1, which persisted for some time, could explain the observed increases in birch parameters from visit 1 to visit 2. Several studies have concluded that two independent variables control the IgE-mediated response in basophils: the amount of membrane-bound IgE needed to cause measurable HR (basophil sensitivity), and the subcellular factors regulating the degree of HR (basophil reactivity) (29, 30). The HR15 in our study is an expression of cell sensitivity, whereas MAX is an expression of cell reactivity. The birch and grass MAX increased from visit 1 to visit 2, indicating an increase in basophil reactivity. As the MAX calculations are not expressed as “percent release of total cell histamine content”, it cannot be excluded that the observed increase in basophil reactivity reflected an increase in the number of peripheral basophils. However, there was no difference in basophil counts between visit 1 and visit 2 (data not shown). In HR15, only the birch response increased significantly from visit 1 to visit 2, whereas the increase in grass HR15 did not reach statistical significance. The HR15 to grass was high even at visit 1; i.e., the amount of membrane-bound grass-specific IgE was so high that the lowest concentration of the grass extract was sufficient to induce significant HR. If the cutoff (15 ng histamine/ml blood) was increased or lower grass-extract concentrations were used in the HR test, the patient results might have differed more, and the increase in basophil sensitivity have reached significance.
The macrophage-, T-, and B-cell interactions, and the mast-cell/basophil activation induced by an allergen give rise to release of cytokines. This has been associated with the activation and influx of eosinophils into the inflammatory microfocus (10, 31). This is in accordance with a study of in vitro eosinophil release which found a seasonal increase in peripheral eosinophil degranulation, indicating an activation of eosinophils ( 18). In our study, the allergic rhinitis patients showed an increase in blood eosinophil counts and serum ECP and EPX from visit 1 to visit 2, and a postseasonal decrease was found, supporting the concept of eosinophil upregulation and activation. However, the eosinophil counts were significantly correlated with serum ECP and serum EPX, and we did not find an individual increase in the ratios serum ECP/EOS and serum EPX/EOS from visit 1 to visit 2 (paired t-test, data not shown). Therefore, we cannot exclude the possibility that the increase in serum ECP and EPX observed in our study was partly due to an increased number of eosinophils. Several studies have demonstrated that the eosinophil plays a part in the allergic inflammation. A difference in eosinophils, serum ECP, and serum EPX between well-controlled nonallergic asthmatic patients and nonallergic control subjects, as well as between allergic asthmatic patients and nonallergic control subjects after the pollen season, has been reported (18, 19, 21). In another study investigating rhinitis patients vs controls, no difference in serum ECP was observed ( 32). Our study did not find any differences in eosinophils, serum ECP, and serum EPX after the seasons (or at the beginning of the birch-pollen season) between allergic rhinitis patients and healthy controls. The increased levels in asthmatics, but not in rhinitis patients, could have been due to the difference in size of the diseased organ. Fifteen (31%) of the rhinitis patients in our study were also suffering from seasonal asthma. However, no differences were observed either after the season, or in the birch-pollen season between the asthmatic rhinitis patients, the rhinitis patients without asthma, and the control subjects, and no difference was found between the asthmatic rhinitis patients and the rhinitis patients without asthma during the grass-pollen season (data not shown).
We found no differences in eosinophil parameters and basophil HR (except birch HR) of the allergic patients between visit 1 (birch season) and visit 3 (after the seasons). The birch-pollen load was low before visit 1, and the patients had few symptoms. On the other hand, the eosinophil and basophil HR parameters were increased during the peak grass-pollen season, with a high pollen load and with symptoms in all patients. These results could indicate that the severity of the allergic rhinitis is influenced by the eosinophil and basophil leukocyte.
The production of specific IgE and subsequent sensitization of mast cells and basophils are important factors involved in the pathophysiology of allergic rhinitis, and eosinophils participate in the local allergic inflammation, but it is presently not known to what extent the different parameters can explain disease severity. The partial least square PLS analysis has previously been used to describe the relationship between air-pollution parameters and plant-growth parameters ( 28). In our study, the PLS analysis may possibly explain to what extent the different in vitro parameters express the severity of allergic rhinitis.
The accuracy of the symptom and medication scores predicted by the PLS analysis was high, showing a means to combine a symptom and medication score in an investigator-independent way. We found a correlation among most of the different in vitro parameters; therefore, the PLS analysis seems a good choice, as this multivariable model solves the collinearity problem (intercorrelation between variables). The model showed a significant relationship between clinical symptoms and the blood-measurement markers of immunoinflammation, with an indication of different importance among the individual tests. When looking at symptom score and medication, the variance explained was higher for the symptoms than the medication. This could be explained by the patients' attitude toward symptoms and medication. In another study, we followed patients during both pollen seasons and found that they tended to use as little medication as possible in order to control symptoms to a certain individual level (data to be published). The prednisolone seemed to be of no importance, perhaps because only 10 patients in our study used prednisolone, and the mean daily prednisolone consumption was low. We did not find differences in any blood-test parameters between the allergic patients with or without prednisolone treatment at the three visits, again, presumably, because of the minimal prednisolone medication. However, several studies have shown that glucocorticoids affect the allergic inflammation directly by cell receptors, or indirectly by disruption of the cytokine network; e.g., the eosinophil generation, survival, and function ( 33). The PLS-analysis results of the relationship between symptom and medication scores and the different laboratory tests showed that a few selected tests tell as much as all the tests put together. The HR test seemed to be the most relevant parameter of clinical symptoms during the pollen season. However, although a significant relationship was found between in vitro tests and the symptom and medication scores, the PLS model explained only less than half of the total variation in symptoms and less than one-quarter of the total variation in medication. This indicates that blood parameters do not necessarily reflect the actual disease status of the target organ. The measurements of number of eosinophils, concentration of ECP/EPX in the tissue, and the releasibility of tissue basophil/mast cells might have increased the percentage of the total variation in disease severity explained by these parameters. Other tissue immunologic cells such as CD4+ T-helper cells ( 7), Langerhans' cells ( 34), and endothelial cells (35, 36) have been shown to be related to allergic inflammation, indicating that allergic inflammation and its relation to the severity of the allergic disease is very complex.
In conclusion, a seasonal increase in the blood measurements of basophil HR, eosinophils, serum ECP and EPX, and specific IgE was observed in allergic rhinitis patients. A relationship was observed between the severity of allergic rhinitis and these laboratory tests, and the basophil HR test proved to be the most relevant test. However, the present study indicates that other factors contribute to the severity of allergic rhinitis.