Cultured Mast Cells from Patients with Asthma and Controls Respond with Similar Sensitivity to Recombinant Der P2-Induced, IgE-Mediated Activation

Authors


Correspondence to: H. J. Hoffmann, Department of Pulmonary Medicine, Aarhus University Hospital, Building 2b, Nørrebrogade 44, DK-8000 Aarhus C, Denmark. E-mail: hans.jurgen.hoffmann@ki.au.dk

Abstract

The function of cultured mast cells may depend on genetic or environmental influence on the stem cell donor. This study investigates whether asthma or atopy in the donor influenced the growth and sensitivity of mast cells cultured from patients with asthma and healthy controls under identical conditions. Mast cells were cultured from peripheral blood from twelve patients with an objectively confirmed asthma diagnosis and eight healthy subjects. During the last 2 weeks of culture, mast cells were incubated with IL-4 and 80 kU/l recombinant human IgE containing two clones (7% + 7%) specific for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum CD63 median fluorescence intensity was 20 456 ± 1640 (SE) for patients with asthma and 22 275 ± 1971 (SE) for controls (ns). The fraction of CD63 positive cells was 54.4% in patients with asthma and 48.4% in controls (ns). The allergen concentration inducing 50% of the maximal CD63 response was similar in patients with asthma [−0.4795 log ng/ml ± 0.092 (SE)] and controls (−0.6351 log ng/ml ± 0.083, ns) and in atopic and non-atopic subjects. When cultured, sensitized and activated under identical conditions, mast cells from allergic asthmatics and healthy controls respond similar. Activation of cultured mast cells appears to depend on culture conditions (IL-4, IgE) rather than on donor status as atopy and asthma.

Introduction

In allergic reactions, cytokines produced by T-helper 2 lymphocytes, like IL-3, IL-4, IL-5, IL-6 [1] and IL-9 [2] interact with mast cells through specific surface receptors. In vitro, the presence of IL-4, together with stem cell factor (SCF) and IL-6, increases mast cell maturation and induces the expression of the high-affinity IgE receptor FcεRI [3-5] as well as IgE-dependent mediator release [1]. Binding of IgE to FcεRI increases the expression of FcεRI on mast cells [6]. Distinct properties of the IgE repertoire (e.g. affinity of IgE for allergen, clonality of allergen, fraction of specific IgE of total IgE) determine the extent of effector cell degranulation upon exposure to allergen [7]. The functionality of the mast cells in allergy is commonly studied in vitro. Upregulation of CD63 correlates well with histamine release upon degranulation in basophils [8] and mast cells [9]. Importantly, to what extent any differences obtained by this approach depend on properties of the donor and/or the culture environment the cells are differentiating in.

In this study, we tested the reproducibility of our allergen-specific activation model and investigated the influence of donor status (asthma or atopy) on cell number and sensitivity as CD63 upregulation of in vitro differentiated human mast cells cultured from peripheral blood under identical conditions.

Material and methods

Mast cells progenitor cells were isolated from 400 ml peripheral blood. Mast cell lines were generated from CD133+ precursor cells by culture for 8 weeks as previously described [9-11]. The purified cells were resuspended in complete medium (CM); StemSpan medium with IL-6 (50 ng/ml), stem cell factor (100 ng/ml), pen/strep (100 μg/ml) and cultured at 37 °C in a humidified inline image incubator at a density of 5 × 105 cells/ml. The culture medium was supplemented with IL-3 (1 ng/ml) for the first 3 weeks of culture. In week 4–6, the cells were cultured in CM only. From week 7, FBS (10%) and recombinant IL-4 (10 ng/ml, PeproTech Nordic, Stockholm, Sweden) were added to CM, and mast cells were sensitized with 80 kU/l recombinant human IgE-containing IgE specific for the major house dust mite allergen Der p2 (7% clone P4E and 7% clone H12) [7], as previously described [9]. IgE concentration was measured with ImmunoCAP (Thermo Fisher King, Uppsala, Sweden). The total number of cells was recorded weekly.

The sensitivity to IgE-mediated activation of peripheral blood-derived mast cells was investigated as FcεRI-mediated upregulation of the activation marker CD63. Mast cells were activated with recombinant Der p2 allergen in log10 dilutions from 0.0001 to 100 ng/ml for 30 min in a humidified incubator at 37 °C [9]. Two independent non-activated cell suspensions served as negative controls. The cell suspensions and one negative control were then incubated with anti-CD63 FITC (Biolegend, San Diego, CA, USA) for 20 min at 4 °C, washed and acquired on a FACS Canto II.

The median maximum activation [CD63 median fluorescence intensity (MFI) and CD63%] and sensitivity measured as the effective allergen concentration inducing 50% of the maximum response (EC50Der p2) of the mast cell lines by allergen-mediated activation were evaluated by fitting nonlinear curves to activation data in Prism 5.0d (Graph Pad). The negative control was incubated with PBS, and a gate-defining activated cell was set at 2% CD63 positive cells. Patients with asthma and healthy controls were each considered as one group. Individual measurements of asthmatics and controls were compared with nonparametric statistics due to the small sample size.

We determined the reproducibility of our allergen-specific activation model by splitting stem cell cultures of three donors (one woman, one allergic asthmatic, median age 48 years) into three evenly sized cultures immediately after isolation of CD133+ precursor cells. At maturity, sensitized IL4-matured mast cell reactivity (%CD63+) and sensitivity (EC50 for CD63 activation) of the cultures differentiated from each donor were compared with each other.

The second study included twelve patients (Table 1) with an objectively confirmed asthma diagnosis according to GINA guidelines [12]. Rhinitis was diagnosed according to ARIA guidelines [13]. Inclusion criteria were age >18 years, no current smoking and a smoking history of <10 pack-years, no previous pharmacological treatment for asthma except for short acting beta2-agonists as needed, no current airway infection and otherwise healthy. Eight healthy subjects fulfilling the same inclusion criteria but without asthma were included as controls. The local ethics committee in Copenhagen, Denmark, approved the study (Nr H-2-2010-130). All subjects gave written informed consent.

Table 1. Characteristics of subjects
 Controls (n = 8)Patients with asthma (n = 12)P-value
  1. M, male; F, female; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; NS, not significant.

  2. a

    Data are given as median and range.

Agea, (years)23.0 (22.5–23.25)26.0 (21.0–33.0)NS
Gender (M/F, n)4/47/5NS
Former smoker22NS
FEV1a Litre4.14 (3.75–4.73)3.45 (3.14–4.26)NS
FVCa Litre5.17 (4.69–5.92)4.76 (3.90–5.88)NS
GINA asthma severitya 1 (= 4), 2 (= 7), 3 (= 1) 
ARIA rhinitis score1 (= 2)1 (= 3), 3 (= 4), 4 (= 3) 
Positive skin prick test28 

Ten participants were atopic and had a positive skin prick test (>3 mm) to at least one of ten common allergens (birch, grass, mugwort, horse, dog, cat, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Alternaria alternata and Cladosporium herbarum, manufactured by ALK-Abello, Hørsholm, Denmark) (Table 1).

Results

In the reproducibility study, the median maximal CD63% of the parallel cultures from the each donor were measured for three donors after activation with Der p2 allergen. The median CD63 span and log EC50 for CD63% varied by 14% and 0.52 log ng/ml, respectively (Fig. 1).

Figure 1.

Analysis and reproducibility. (A) Analysis of mast cell activation by measurement of CD63 expression. Mast cells were gated as a coherent population in the left pane, and a marker was set at 2% of resting cells (light grey peak) in the right panel. Activation with 1 ng/ml Der p2 (46%, black stippled line) and 100 ng Der p2 (87.8%, dark grey histogram) are shown. (B) The percentage activated cells (CD63%) measured after allergen-specific activation of mast cells differentiated in parallel cultures (A–C) from one representative donor.

There was no difference in stem- or mast cell yields between patients with asthma and controls, and between all atopic- and all non-atopic subjects, or between the atopics and non-atopics in the asthma group (Table 2).

Table 2. Mast cell numbers obtained from asthmatics and control persons (median and range)
 Controls (= 8)Patients with asthma (= 12)P-value
  1. NS, not significant.

  2. a

    Data are given as median and range.

Number of CD133+ cells isolated per ml blooda1758 (1313–2439)2506 (2025–3325)NS
Total number of isolated CD133+ cells at baseline (week 0)a0.74 (0.59–1.08) × 1061.01 (0.86–1.19) × 106NS
Total number of mast cells (week 7)a6.9 (4.47–10,12) × 10611.0 (5.14–15.68) × 106NS
Nr of cell doublings (mast cells/progenitor cells)9.03 (6.22–11.8)9.50 (6.0–14.3)NS

After activation, the median maximal CD63MFI was 20 456 ± 1640 [standard error (SE)] for patients with asthma and 22 275 ± 1971 (SE) for controls (ns) (Fig. 2A), and the median percentage of CD63+ mast cells was 54.4% for patients with asthma and 48.4% for controls (ns) (Fig. 2B). The median sensitivity, EC50Der p2, based on the percentage CD63+ mast cells of all mast cell cultures differentiated from patients with asthma and controls was similar [−0.4795 log ng/ml ± 0.092 (SE)] in patients with asthma and −0.6351 log ng/ml ± 0.083 controls, ns). There was no difference in sensitivity between atopic subjects and non-atopic controls, or between the atopic and non-atopic patients within the asthma group (> 0.05).

Figure 2.

The CD63 MFI and the % CD63+ mast cells cultured from patients with asthma and controls were similar (median; IQR). A; The maximal CD63 MFI measured on allergen-specific activated mast cells from patients with asthma (red) and controls (dotted blue). B; The median % CD63+ mast cells measured on allergen-specific activated mast cells from patients with asthma (red) and controls (dotted blue).

Discussion

In the normal human lung, the majority of the mast cells reside in the peripheral regions and express very little FcεRI [14]. In contrast, most mast cells in the bronchial mucosa express high levels of FcεRI [14]. Mast cells act as airway sentinels that detect environmental hazards. If they bind allergen-specific IgE on cell surface FcεRI, exposure to the allergen triggers an IgE-dependent activation [15]. In patients with atopic uncontrolled asthma, the infiltration of mast cells into the alveolar parenchyma is increased, and the expression of FcεRI is dramatically upregulated compared with healthy controls [16]. A similar but less pronounced shift of alveolar mast cells to a high FcεRI-expressing phenotype also takes place in mild atopic asthma [17].

CD117+ progenitors are primarily found in the CD133+ CD34+ haematopoietic stem cell subset [18]. Atopic patients have more CD34+ progenitor cells than non-atopics [19], but the frequency of CD133+ progenitors and mast cell numbers resulting from them have not yet been characterized. In this study, we found a tendency for more CD133+ progenitor- and mast cells in patients with asthma compared with the healthy control persons (Table 2), but no significant difference. Neither could we detect any difference between atopic patients and non-atopics. We could thus not confirm the previously reported results (the CD133+ and CD34+ subsets overlap to a large extend), but this may be due to lack of statistical power or differences among the patient cohorts. As we have not used the same methods as Shemi et al. [19], the data are not directly comparable.

In this study, mast cells were cultured with IL-4 as this has been found to enhance the expression of FcεRI [3] and lead to terminal maturation [5]. They were then sensitized with 80 kU/l total IgE containing 7% of two species of IgE specific for Der p2 [3, 7]. In physiological terms, the present IgE composition and concentration were thus not unusually high. In fact, the used IgE levels correspond to what can be found in the 75–90th percentile of an adult reference population [20] or in allergic patients with moderate to high total IgE [21].

We reasoned that three triplicate repeats would give a good indication of the reproducibility of the assay we developed for measuring mast cell sensitivity. The triplicate cultures for each donor had been separated for 8 weeks, simulating maturation of mast cells from the same donor at three separate occasions. The reproducibility for the maximal activity was very good at 14%, and the sensitivity measurement was good as well, with 0.5 log units as the median variation of triplicates of three donors.

We have recently shown that mast cells sensitized with 8–800 kU/l of the same composition of Der p2 IgE used in the present study respond by not only CD63 upregulation but also histamine release and prostaglandin D2 synthesis after allergen exposure [9] and that this activation is comparable with activation of human basophils [8]. At very low (<1 kU/l) and very high (>800 kU/l) IgE concentrations, the four mast cell lines that we reported on previously, responded with greater variation than more moderate levels such as 80 kU/l [9]. Here, we chose an IgE concentration that was elevated, but not atopic, that is, below 100 kU/l, and at which mast cell lines would behave similarly.

Both mast cell maximal expression and sensitivity of CD63 were similar in mast cell lines cultured from patients with asthma and healthy controls, and in atopic subjects and non-atopic controls. Importantly, our results thus suggest that factors like IL-4 and IgE in the local micro-environment in the lung, rather than genetic disposition, play a major role in the maturation of mast cells [5].

In summary, the present study reveals that cultured mast cells from patients with asthma and healthy controls respond surprisingly similar when cultured under identical conditions and stimulated with a high level of IgE to mimic an allergen-induced activation. Whether or not any differences may occur in response to lower concentrations of total and specific IgE during culture, or measuring other parameters of activation, emerges as an important area for further investigations.

Acknowledgment

The authors would like to thank Helle Skjøth and Ellen-Margrethe Raaby for excellent technical assistance.

Author contribution

AS and VB recruited patients and collected clinical data. IKK did all cell culture work. HJH, AS and VB designed the study. IKK wrote the first draft of the study. All authors contributed to writing.

Conflict of interest

IKK, AS, RD, JSE, VB, and HJH have no conflict of interest to report. GL is an employee of ALK-Abello.

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