Detection of activated basophils using flow cytometry for diagnosis in atopic patients.

ORIGINAL ARTICLES

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Detection of activated basophils using flow cytometry for diagnosis in atopic patients

G. J. N. Cozon*^, **, J. Ferrándiz*, D. Peyramond*** and J. L. Brunet***

*UFR Lyon-Nord JE 1947 Université Claude Bernard Lyon 1. France. **Unité d''Immunologie and ***Service des maladies infectieuses, Hôpital de la Croix-Rousse. France.

Correspondence:

Dr. Grégoire Cozon

Unité d''Immunologie, Hôpital de la Croix-Rousse

69317 Lyon Cedex 04 France

E-mail: [e-mail]

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RESUMEN

Resumen: Los basófilos humanos liberan los mediadores de la reacción alérgica después de la fijación de los alergenos en los receptores de la IgE. La activación específica de los basófilos es detectable por citometría de flujo (FCM) utilizando un anticuerpo monoclonal conjugado anti-CD63 marcado con fluresceína.

Objetivos: este estudio evalúa la detección de basófilos por FCM en el diagnóstico de rutina de enfermedades atópicas en comparación a los prick test cutáneos y anticuerpos inmunoglobulina E específicos.

Métodos y resultados: la sangre total de 20 pacientes probablemente atópicos se preincubó con interleucina-3 (IL-3) y a continuación se incubó con alergenos específicos.

Después de marcar con anticuerpos anti-CD63, se detectaron basófilos activados por medio de citometría de flujo.

La preincubación por IL-3 aumentó la expresión espontánea de CD63, incluso a bajas concentraciones (0,1 ng/ml) en los basófilos de dos de los 20 pacientes. La sensibilidad y la especificidad de la citometría de flujo fueron respectivamente 0,56 ± 0,17 (m ± SD) y 1,0 ± 0,0 para la detección de basófilos activados con ácaros sin preincubación con IL-3 y 0,73 ± 0,13 y 1,0 ± 0,0 para la detección de basófilos activados con polen de gramíneas.

La preincubación con IL-3 aumentó la sensibilidad de una forma dosis dependiente, pero redujo la especificidad del FCM en la detección de hipersensibilidad a los ácaros del polvo.

Conclusión: el método permite una detección fácil y rápida de los basófilos activados de sangre total y pudiera resultar interesante para detectar alergia a alergenos no convencionales como medicamentos.

Palabras clave: Basófilos. Alergia. Atópicas. IgE. Citometría del flujo. Polvo-ácaros. Hierba polen. CD63.

SUMMARY

Background: human basophils release mediators of allergy after cross-linking of IgE receptors by allergens. Specific activation of basophils is detectable through flow cytometry (FCM) using an anti-CD63 fluorescein-conjugated monoclonal antibody.

Objectives: this study evaluate the detection of activated basophils by FCM in routine diagnosis of atopic diseases as regard to skin prick tests and specific immunoglobulin E antibodies.

Methods and results: whole blood from twenty patients suspected of atopy was preincubated with interleukin-3 (IL-3), then incubated with specific allergens. After staining using anti-CD63 antibodies, activated basophils were detected through FCM.

IL-3-preincubation increases the spontaneous expression of CD63 even at low concentrations (0.1 ng/ml) on the basophils of 2 patients out of 20. The sensitivity and specificity of FCM were respectively 0.56 ± 0.17 (m ± SD) and 1.0 ± 0.0 for the detection of dust mite-activated basophils without IL-3 preincubation, and 0.73 ± 0.13 and 1.0 ± 0.0 for the detention of grass pollen-activated basophils. IL-3-preincubation increased the sensitivity in a dose-dependent manner but decreased the specificity fo FCM for detecting dust mite hypersensitivity.

Conclusion: this method allow for rapid and easy detection of activated basophils from whole blood, and could be of interest for detecting allergies to non-conventional allergens such as pharmaceutical drugs.

Key words: Basophil. Allergy. Atopy. IgE. Flow cytometry. Dust mite. Grass pollen. CD63.

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INTRODUCTION

Human basophils and mast cells are central to the pathogenesis of chronic allergic diseases such as allergic conjunctivitis or asthma. They are the only cells that synthesize histamine and express plasma membrane receptors that bind the Fc* portion of IgE with high affinity (Fc*RI). In atopic patients, allergens such as dust mite or grass pollen cross-link specific IgE. Activation of basophils by IgE cross-linking induces fusion of cytoplasmic granules with the plasma membrane and the subsequent release of mediators. Immediate hypersensitivity is also involved in some drug allergies. Clinicians need simple biological tests to confirme atypical clinical symptoms, especially in drug allergies where drug-specific IgE assays are not available. Basophil activation can be detected and quantified via measurement of released mediators, such as histamine or leukotriene C4, or via measurement of the expression of activation markers on the basophil surface (reviewed in 1). Flow cytometric detection of activated basophils has already been described showing either detectable alterations in light scatter characteristics or in actin polymerization (reviewed in 2), or modification of staining with specific monoclonal antibodies (mab) to CD63 (3) or to CD45 (4, 5). Routine use of flow cytometry (FCM) to detect activated basophils after specific activation has been described (6) but all the methods involved large amounts of blood for preparing basophil-enriched cell suspensions. In addition, activated basophils are not always detected after non-specific activation by anti-IgE (7) or N formyl-methionyl-leucyl-phenylalanine of blood cells from non-atopic subjects even after interleukin-3 (IL-3) preactivation (Cozon et al unpublished data). Preactivation of basophils with IL-3 is used in many research studies and may sensitize the basophil response to stimuli (1, 8-11). The present study was initiated to evaluate a simple flow cytometric method for routine detection of activated basophils in whole blood from atopic patients after allergen-specific stimulation. The findings show that FCM would be very useful for rapid detection of specific and immediate hypersensitivity.

MATERIAL AND METHODS

Patients

Twenty patients routinely referred to our department for suspected atopy were included in the study. Patients taking corticosteroids or antihistaminics in the prior month were excluded from the study. Allergies to dust mite Dermatophagoides pteronyssimus (Der p) or grass pollen Dactylis glomerate (Dac g) were suspected upon clinical history, and diagnosed upon the presence of either a positive skin prick test to these allergens (Stallergenes Fresnes, France) or allergen-specific IgE antibodies assayed by cap test (Pharmacia Diagnostics, Uppsala, Sweden). The surface of definite skin reaction was measured using Powercadd software (Engineered Software, Greensboro, NC). Immediate skin reaction was defined as positive if the surface of the skin reaction was greater than 20 mm² (12), and equivocal when measured between 10 and 20 mm². Allergen-specific IgE antibodies were positive when higher than 0.35 International Units (IU).

Blood specimens were collected from patients with their informed consent, using Vacutainer™ tubes (Becton Dickinson, Meylan, France) containing lithium heparin anticoagulant.

Cell activation

Optimal incubation time for IL-3 preactivation and exposure to allergen were established in preliminary experiments. Within two hours of blood withdrawing, aliquots of hundred µl of whole blood were incubated for 30 minutes at 37° C in 5% CO₂ with 10 µl of IL-3 (Sigma, Saint Quentin Fallavier, France) at different dilutions (final dilutions: 0 ng/ml, 0.1 ng/ml, 1 ng/ml, and 10 ng/ml) in order to preactivate the basophils. Freeze-dried commercial allergenic preparations were reconstituted with distilled water at a concentration of 100 cutaneous reactivity index/ml, according to manufacturer''s instructions, then diluted 1:10 and 1:100 in phosphate buffer saline (PBS) (Sigma). Preactivated blood samples were incubated at 37° C with 100 µl of PBS in duplicate, or FMLP (Sigma) diluted in PBS at 0.5 10^­6M and 0.5 10^­7M, or else with allergen extracts. After 15 minutes of incubation, red cells were lysed with 155 mmol/l of NH₄Cl, 10 mmol/l of KHCO₃ and 0.1 mmol/l of EDTA. The leukocytes were recovered by centrifugation and the pellets stained with a combination of two monoclonal antibodies labeled with distinguishable fluorochromes for 15 minutes, in the dark, at +4° C. To estimate activated basophils, a combination was used of polyclonal fluorescein isothiocyanate (FITC)-conjugated goat antibodies to human IgE (Sigma) with either phycoerythrin (PE)-conjugated anti-human CD63 (Caltag Laboratories, San Francisco, CA) or PE-conjugated murine IgG1 isotype control antibodies (Caltag Laboratories). The cells were then washed and resuspended in PBS-0.1% Bovine Serum Albumin (BSA)-5 mM EDTA and analyzed the same day by two-color analysis on a FaCScan™ flow cytometer (Becton Dickinson). With Lysis II software (Becton Dickinson), green fluorescence triggering in the fl1 channel and side scatter (SSC) were used to gate on basophils that expressed a high density of IgE (IgE^bright) (Fig. 1A). Double staining using FITC-conjugated goat antibodies to human IgE and PE-conjugated anti-human CD14 showed that 2.6% to 13% of the cells in this gate were CD14-positive. All the patients included in the present study had clearly-identifiable basophils in flow cytometry. Data from 1,000 basophils were then displayed as two-color dot plots [green vs. orange (fl2) fluorescence] over the IgE-positive cells to measure the proportion of activated basophils that express CD63 (Fig. 1B-C). Percentages of activated basophils were estimated by taking the values obtained with irrelevant-control antibodies having identical isotypes, and subtracting them from those obtained with anti-CD63 antibodies. It is noteworthy that less than 1% of the basophils were stained with the irrelevant-control antibodies.

Figure 1.--Dot plot analysis of blood leukocytes from a grass pollen-atopic patient stained with fluorescein-conjugated anti-IgE and phycoerythrin-conjugated anti-CD63 after lysis of red blood cells. A: correlative display of side-light scatter (SSC) on X axis vs. IgE green fluorescence (FL1) on Y axis. IgE^bright cells are gated in region R1. B-C: red fluorescence (FL2) on X axis shows the expression of CD63 on IgE^bright cells (Y axis) after incubation of IL-3-primed whole blood in either phosphate buffer saline (B) or a specific allergen (C).

To calculate the sensitivity and specificity of the detection of activated basophils in FCM after specific activation, positive and negative status of the 20 pacientes were established for both allergens as specified above using the skin prick test and RAST results. The detection of activated basophils in FCM was considered as positive for an allergen when the specific-activated basophil percentage was 10% more than the PBS control.

RESULTS

As regards the results of specific IgE antibodies, five patients were neither allergic to dust mite nor grass pollen (group 1), four were allergic to Der. p (group 2), six to Dac g (group 3), and five to both allergens (group 4) (table I). The Der p-specific skin prick test and IgE were discordant in 3 cases out of 20 when considering a positive skin reaction and 5 out of 20 with an equivocal skin reaction, respectively (table I). The Dac g-specific skin test and IgE were discordant in 1 case out of 20 when considering a positive skin reaction and 2 out of 20 with an equivocal skin reaction (table I).

 

Table I Results of total IgE and specific IgE in international units (IU) and specific skin tests to dust mite Dermatophagoides pteronyssimus (Der p) or grass pollen Dactylis glomerate (Dac g) antigens in 20 atopic patients*
Patients	Group	Total IgE  (IU)	Der p- specific  IgE (IU)	Der g- specific  IgE (IU)	Der p- specific prick  test mm^2	Der g- specific prick  test mm^2
1	1	1,276	0	0	12	0
2	1	211	0	0	0	14
3	1	9	0	0	13	15
4	1	16	0	0	0	0
5	1	249	0	0	0	0
6	2	143	2.46	0	226	0
7	2	755	78	0	42	0
8	2	254	26	0	50	0
9	2	206	124	0	100	0
10	3	59	0	21.46	10	61
11	3	202	0	29.19	0	52
12	3	23	0	8	0	231
13	3	45	0	27	0	52
14	3	74	0	5.42	0	80
15	3	623	0	10	0	27
16	4	1,963	3.16	608	0	15
17	4	753	21.52	5.3	39	25
18	4	283	165	2.68	494	186
19	4	1,341	6	321	0	100
20	4	83	0.42	19.62	15	183
*Patients were classified in 4 groups:
group 1: patients non-allergic to dust mite or grass pollen;
group 2: patients allergic to Der p;
group 3: patients allergic to Dac g;
group 4: patients allergic to both allergens.

Intra-assay reproducibility of the detection of activated basophils in flow cytometry was estimated by replicate (n = 5) determination on blood from a Der p-allergic patient. Means of CD63-positive basophils (± standard deviation) were 0.8% (± 0.8%) and 60% (± 7.3%) for untreated and Der p-activated basophils, respectively, without IL-3 preincubation, and 1.1% (± 0.3%) and 68% (± 7.5) for untreated and Der p-activated basophils after IL-3 preincubation (1 ng/ml).

Spontaneous expression of CD63 on untreated basophils in whole blood varied from 0 to 9.9% of IgE+ basophils; mean = 3.9% ± 2.9 (standard deviation).

The figure 2 shows that preincubation of whole blood with IL-3 increased spontaneous expression of CD63 even at low concentrations (0.1 ng/ml) on the basophils of 2 patients (nº 1 and 18) out of 20.

Figure 2.--Percentage of CD63-positive basophils from atopic patients (n = 20) after incubation of whole blood with increasing amounts of interleukin-3 (IL-3). Aliquots of whole blood were incubated for 30 min. at 37° C in 5% CO₂ with IL-3 at different dilutions (final dilutions: 0 ng/ml, 0.1 ng/ml, 1 ng/ml, and 10 ng/ml). After red blood cell lysis, leukocytes were incubated with a combination of polyclonal fluorescein-conjugated goat antibodies to human IgE and monoclonal phycoerythrin-conjugated anti-human CD63 antibodies. Percentages of activated basophils were measured through flow cytometry as described in Methods.

Figure 3 shows that the difference of percentages between specifically-activated basophils and control basophils can occur or increase with IL-3 preactivation. The percentage of activated basophils was identical for both tested dilutions of allergens except for one case (see below).

Figure 3.--Percentage of CD63-positive basophils from atopic patients'' basophils (n = 20) after incubation of IL-3-primed whole blood in dust mite Dermatophagoides pteronyssimus (Der p) or grass pollen Dactylis glomerate (Dac g) antigens. Aliquots of whole blood were primed with increasing concentrations of IL-3 (0, 0.1, 1 and 10 ng/ml), and then incubated in PBS, Der p or Dac g antigens for 15 min. After red blood cell lysis and incubation in a combination of polyclonal fluorescenin-conjugated anti-IgE antibodies and monoclonal phycoertythrin-conjugated anti-CD63 antibodies, cells were analyzed through flow cytometry as described in Methods.

One patients (nº 4) who had a negative prick test to dust mites and no specific IgE had a positive reaction to the 1:10 dilution of Der p. in the presence of IL-3. There was no basophil activation with the 1:100 dilution of the allergen. The specific reactivity of IL-3-primed basophils from this patient to only one dilution of dust mite antigen was confirmed two months later.

A difference of 10% between negative controls and specific-activated basophil percentages was chosen in order to affirm specific activation. Using this cut-off value, FCM sensitivity and specificity of FCM were, respectively, 0.56 ± 0.17 (m ± SD) and 1.0 ± 0.0 for the detection of Der p-specific activated basophils in atopic patients without IL-3 pre-incubation and 0.73 ± 0.13 and 1.0 ± 0.0 for the detection of Dac g-specific activated basophils (table II). Preincubation of whole blood with IL-3 increased sensitivity in a dose-dependent manner but slightly decreased FCM specificity for detecting dust mite hypersensitivity (table II).

 

Table IISensitivity and specificity of the flow cytometric detection of activated basophils in IL-3-primed whole blood in the presence of specific antigens according to the concentration of IL-3 and the atopic status of 20 patients as detailed in Methods*
	Dermatophagoides pteronissimus		Dactylis glomerate
Interleukin-3	Sensitivity	Specificity	Sensitivity	Specificity
ng/ml	m ± SD	m ± SD	m ± SD	m ± SD
0	0.56 ± 0.17	1.0 ± 0.0	0.73 ± 0.13	1.0 ± 0.0
0.1	0.67 ± 0.16	0.91 ± 0.09	0.91 ± 0.09	1.0 ± 0.0
1	0.67 ± 0.16	0.91 ± 0.09	1.0 ± 0.0	1.0 ± 0.0
10	0.78 ± 0.14	0.91 ± 0.09	1.0 ± 0.0	1.0 ± 0.0
*Results are expressed as means (m) ± standard deviation (SD).

DISCUSSION

Activation of basophils and mast cells by IgE cross-linking is the main mechanism of the pathogeny of atopy. Interaction of allergens with IgE-sensitized mast cells and basophils induces cell degranulation and the release of inflammation mediators and leads to the clinical manifestations of allergy. Detection of specific IgE antibodies in atopic patients is routinely used for diagnosis of specific type-1 hypersensitivity to a large array of allergens. But in some cases there is discrepancy between specific IgE assay and skin tests, and in other cases no specific IgE assays are available. Others tests, such as histamine release or leukotriene C3 production have been described for detecting specific allergies, but as these tests require radioactivity detection equipment, they are unsuitable for routine use in many laboratories. Flow cytometric detection of activated basophils has been described using the cell surface adhesion molecule CD11b (13), detection of CD63 (3), or variations in CD45 staining (5). Staining with anti-CD63 antibodies seems to be one of the easiest tools for routine diagnosis because of the clear-cut appearance of the molecule after basophil activation (3). The present study shows clearly that CD63 staining is very useful in detecting activated basophils from atopic patients. All the tested patients had IgE^bright cells that can be mainly considered as basophils because only 2.6% to 13% of the cells in this gate were CD14-positive monocytes. Nevertheless, when testing non-atopic subjects, IgE^bright cells were sometime absent and the detection of the CD63 molecule on basophils was impossible (data not shown). High expression of Fc*R1 on the basophil surface has already been correlated to serum IgE concentrations (14, 15). It can explain the absence of IgE^bright cells in some non-atopic subjects.

Six patients have equivocal results of prick test to one or both allergens (table I). Among them, after in vitro activation with the incriminate allergen, CD63-positive basophils were present in whole blood from one patient (nº 20) only after IL-3 preincubation at 10 ng/ml. In this case, a low level of Der p-specific IgE antibodies (O.42 IU) was also detected by cap test.

IL-3 has already been used to sensitize basophils in many routine and research studies (6, 8-11). The present study shows that IL-3 sensitizes the specific activation of basophils in most cases, increasing the sensitivity of the test at low concentrations for pollen grass allergies, and in a dose-dependent manner for dust mite hypersensitivity. The specificity was only slightly decreased by preincubation of basophils with increasing concentrations of IL-3. Nevertheless, in some cases, even at low concentrations (i.e. 0.1 ng/ml), IL-3 activated the basophils to express CD63 without any other stimulus (Fig. 2). Patient nº 1 with a dose-dependent response to IL-3 had a high level of total IgE and was allergic to hymenopter venom but not to grass pollen or dust mites. In this case there was no difference between PBS and allergen-incubated basophils even when IL-3 preactivation increased the number of CD63-positive basophils. In the case of patient nº 4, IL-3 preincubation sensitized the in vitro activation of basophils with the 1:10 dilution of dust mite allergen, giving false positive results as regards specific IgE detection. Nevertheless, in this case we cannot rule out allergies because of the clinical background. On the other hand, priming the basophils with IL-3 is necessary for detecting the specific activation of basophils to some drugs such as antibiotics (Cozon et al. unpublished data).

In conclusion, the present study describes a simple method for detecting the specific activation of basophils in allergic patients. Although this method was not compared with histamine release which requires radioactivity detection equipment, the sensitivity and the specificity of this flow cytometric method mean that it can be proposed routine studies. It is a rapid method which uses only small amounts of whole blood. It can be applied in routine laboratories equipped with a flow cytometer, thereby sidestepping the use of radioactive agents. The sensitivity of the assay can be increased in some cases using IL-3 preincubation to prime the basophils. This method could be of interest for the biological detection of allergies to certain antigens such as pharmaceutical drugs in atopic patients.

ACKNOWLEDGMENTS

We thank S. Adrian for critically reading and revising the text. This work was supported by grants from the Hospices Civils de Lyon AO95 France and Laboratories Boiron Ste Foy les Lyon France.

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