Correlation Between the Extent of In Vitro Complement Activation by Allergens and the Clinical Symptoms in the Same Patients

Recently we published several findings indicating that complement activation occurs on the allergen-exposed mucosa of the sensitized patients, contribute to the development of allergic inflammation and may determine the severity of symptoms. Ragweed (RW) allergy was selected as a model of pollen allergy since it is the most common inhalation allergen in Hungary and in the United States, too.

In early August 1992 (that is during the RW blooming season) blood samples were taken from 40 RW allergic patients (8). These sera were incubated with ragweed allergen extract (RWA) (20 AU/ml) and the formation of complement activation products was determined. After taking blood samples the allergic patients were asked to fill diaries about their symptoms during the subsequent 4 weeks. This diary included subjective scores to be given to nasal-, eye-, throat-, pulmonary-, and ear symptoms (0 = no symptom, 1 = mild symptom, 2 = medium symptom, 3 = severe symptom, disturbing daily life). Symptoms' scores were summed. 26 of the diaries could be evaluated, and compared to RWA induced AP activation (C3bBbP formation). We found a significant positive correlation between eye-, and throat symptoms and the activation of the AP (p=0.03) and p=0.006, respectively). The correlation between nasal symptoms and C3bBbP formation was of marginal significance (p=0.07).

When the 26 allergic subjects were divided into two groups according to the extent of C3bBbP formation (>120 %, n=15 or <120 %, n=ll compared to buffer control) upon incubation with 20 AU/ml RWA, we found that the sum of eye and nasal symptoms' scores that developed in a 4-week period subsequent to blood sampling obtained in different patients were significantly higher in patients with more pronounced allergen-induced AP activation (128 ± 22 vs. 28 ± 6, p=0.0009, and 149 ± 22 vs. 57 ± 13, p=0.0032, respectively)

An other study was performed in twenty-two 15-17 year old RW allergic adolescents (34). Serum samples taken during the RW blooming season were incubated with 100 M-g/ml RWA and the generation of different complement activation products were measured by ELISA or RIA tests. Symptom scores were registered for 4 weeks during the RW blooming season. The patients were divided according to the extent (low or high) of generation of the complement activation products and symptom scores registered in the two groups were compared by using the two-way ANOVA method. Significantly higher symptom scores were obtained in the high than in the low complement activation group (p values: 0.049 for C1rC1sC1inh, 0.022 for C3bBbP, 0.015 for C5b-9, 0.0001 for C3a, 0.0008 for C5a). Similar results were obtained at the measurement performed in the sera obtained from the same patients half a year before the season (p values: 0.022 for C3bBbP, 0.005 for C5b-9) (Figure 2).

Next, we analyzed the relationship between the results of the usual allergologic tests (IgE estimation, titration skin prick test) and the extent of in vitro ragweed allergen extract (RWA)-induced complement activation in the sera of the same 48 patients suffering from late-summer allergy (35). For obtaining estimation about skin reactivity to RWA of a patient, skin prick testing was performed by titration. The aqueous extract of RWA was applied in three different concentrations (103, 104, and 105 biological units [BU]/ml). The results were evaluated after 15 minutes. The lowest concentration which gave rise to a histamine-equivalent positive reaction was considered as the measure of skin reactivity and was expressed in arbitrary units. Skin reactivity was designated as one arbitrary skin reactivity unit (SRU) if 105 BU/ml RWA produced a wheal with a diameter equal to the histamine control at a given patient, while 103 and 104 BU/ml RWA induced less or no reaction.

Figure 2. Symptoms scores registered during the ragweed blooming seasons in 22 ragweed allergic adolescents with high (above median of the whole group) and low (below median) C3a and C5a generation in their serum samples incubated with 100 ng/ml RWA

Skin reactivity was designated as 10 or 100 SRU if histamine-equivalent positive reaction was obtained already with 104 and 103 BU/ml RWA, respectively. Sera of these patients were incubated with 20, 100, and 400 U/ml RWA and generation of two complement activation products, alternative pathway C3-convertase (C3bBbP) and terminal pathway activation complex (C5b-9) was measured by ELISA methods. A strong positive correlation (Spearman correlation coefficient r = 0.495, p=0.0004, and r = 0.454, p=0.0012, respectively) was found between individual skin reactivity to RWA and C3bBbP generation induced by 20 and 100 allergological units/ml (U/ml) RWA (Figure 3).

20 100 400

final serum concentration of RWA extract, U/ml

20 100 400

final serum concentration of RWA extract, U/ml

Figure 3. Correlation between the skin reactivity to ragweed as determined by a dilution prick test and the extent of C3bBbP formation in the sera of ragweed allergic patients incubated with different amounts of ragweed allergen extract. *Compared to 1SRU, MannWhitney test

Figure 3. Correlation between the skin reactivity to ragweed as determined by a dilution prick test and the extent of C3bBbP formation in the sera of ragweed allergic patients incubated with different amounts of ragweed allergen extract. *Compared to 1SRU, MannWhitney test

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