Original Paper Int Arch Allergy Immunol 2013;160:165–172 DOI: 10.1159/000339743
Received: November 14, 2011 Accepted after revision: May 24, 2012 Published online: September 25, 2012
Differences in Systemic and Skin Migrating-Specific CD4+ T Cells in Papular Urticaria by Flea Bite Omar Dominguez-Amorocho a Silvia Duarte d John Mario González e Evelyne Halpert f María Claudia Ortega b Adriana Rodríguez c Elizabeth García g Adriana Cuellar a a
Departamento de Microbiología, Facultad de Ciencias, b Departamento de Pediatría, y c Facultad de Odontología, Pontificia Universidad Javeriana, d Facultad de Medicina, Universidad Militar Nueva Granada, e Facultad de Medicina, Universidad de los Andes, y Departamentos de f Dermatología Pediátrica y g Alergia e Inmunología Pediátrica, Fundación Santa Fe de Bogotá, Bogotá, Colombia
Key Words CD4+ T lymphocytes ⴢ Cytokines ⴢ Flea allergy ⴢ Papular urticaria ⴢ Regulatory cells
Abstract Background: Papular urticaria by flea bite is a chronic allergic condition in which clinical improvement may occur at the age of 7 years, thus representing a natural model of acquired immunologic tolerance in humans. The aim of this study was to characterize regulatory cells and specific responses to flea antigens of CD4+ T lymphocytes expressing cutaneous migration markers in patients with papular urticaria caused by flea bite and with different disease evolution times. Methods: Cell populations were characterized by flow cytometry in samples from patients and healthy controls. Specific cell stimulation was performed with a complete flea body extract. The Mann-Whitney U test was used for comparisons. Results: Total dendritic cells were lower in patients than in healthy controls. No quantitative differences were found in CD4 regulatory T cells. CD4+ T cells from patients produced more IL-4, lL-10, IL-17, and IFN- ␥. Patients who experienced the onset of symptoms within the first 5 years of age showed a greater percentage of local (cutaneous lymphocyte anti-
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gen +) IL-4- and IL-17-producing cells, while patients who experienced the onset of symptoms after the age of 5 years had a higher percentage of systemic (cutaneous lymphocyte antigen –) IL-10-producing cells. Conclusion: Analysis of the cellular immune response against whole flea antigen in patients with papular urticaria by flea bites suggests a possible participation of inflammatory cytokines in the skin reaction (Th17) and a systemic control mechanism (IL-10). This pattern of cytokine production in patients could be a consequence of an impaired dendritic cell population. Copyright © 2012 S. Karger AG, Basel
Introduction
Papular urticaria is a chronic allergic disease clinically diagnosed during the first years of life as a cutaneous hypersensitivity reaction following exposure to ectoparasites, including fleas. Most patients exhibit clinical remission after the age of 5–7 years. Studies of the immune response have shown a differential IgE and IgG antibody response among patients, depending on the time of evolution of the disease [1, 2] and the pattern of cellular infiltration at the lesion site conCorrespondence to: Dr. Adriana Cuellar Departamento de Microbiología, Facultad de Ciencias Pontificia Universidad Javeriana Carrera 7a, No. 43–82, Ed. 52, Oficina 608, Bogotá 11001000 (Colombia) Tel. +57 1 320 8320, ext. 4072, E-Mail acuellar @ javeriana.edu.co
sisting primarily of eosinophils and CD4+ T lymphocytes [2]. Furthermore, a predominantly Th2 response to a polyclonal stimulus [3] and functional differences in monocyte-derived dendritic cells (DC) from patients compared to those originating from healthy donors has also been described [4]. Studies on T lymphocyte responses have used whole circulating cell populations. However, given the compartmentalization of the immune response, evaluating cell subpopulations that express specific homing markers may allow a better approach to the analysis of the local response. Lymphocytes with a skin migration pattern express cutaneous leukocyte antigen (CLA). A selective role of this molecule in cutaneous cell migration has been confirmed by the demonstration that vitamin A and a component derived from vitamin D3 induce reduced expression of CLA in human CD3+ T lymphocytes [5], leading to a lower level of CD4+ T-lymphocyte infiltration and decreased skin inflammation [5, 6]. An important aspect of allergic inflammation control is related to the regulatory potential of cell populations like DC and regulatory T cells [7, 8]. Human DC have been classified into two groups according their expression of different cell markers as myeloid DC (mDC) and plasmacytoid DC (pDC) [9]. The role of these cell populations in PUFB has not been studied in detail. Herein, we report the results of a detailed characterization of circulating cell populations and of the cytokine-specific responses to flea antigens in CD4+ T lymphocytes expressing CLA and in potentially regulatory cells in patients at different disease evolution times and in healthy controls.
Methods Study Population We enrolled 20 children aged 2–10 years with a clinical diagnosis of papular urticaria induced by flea bite (PUFB). Study participants were recruited from the Pediatric Dermatology and Allergy clinics at the Fundación Santa Fe de Bogotá, Bogotá, Colombia. Disease evolution time was defined as the time elapsed between the first episode (onset of symptoms) and the date of recruitment into the study. The patients were subdivided into two groups: 10 having less than 5 years since the first onset of symptoms (mean time of evolution, 2.8 8 0.9) and 10 having more than 5 years (mean time of evolution, 7.1 8 1.1). As a control group, we included 10 children within the same age group, who attended the same hospital for minor surgical procedures related to noninflammatory pathologies. None of the controls had a history of PUFB. The informed consent form was signed by the parents or guardians of the children. The study was approved by the institutional review boards of each of the par-
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ticipating institutions. The age distribution in both groups did not show any significant difference. Diagnosis of the Disease The diagnosis of PUFB was conducted clinically. Most patients had lesions appearing as grouped papules, often pruritic. Papules were usually excoriated or crusted, appearing intermittently in a persistent chronic manner and leaving behind hypo- or hyperpigmented macules. They were most frequently located in areas where clothing would fit snugly, such as the sock line and waistband. However, exposed areas of the extremities were also affected in some patients. Obtention of Flea Antigens Aqueous extracts (20% weight/volume) of complete fleas (Ctenocephalides felis felis) (Greer Labs, Lenoir, N.C., USA) were prepared. The fleas were stored at –70 ° C until the time of processing; they were macerated in PBS and constantly shaken for 1 h. The flea extract was centrifuged at 15,000 rpm for 15 min at 4 ° C and passed through a 0.22-m filter to ensure sterility. Protein concentration was determined using a Bradford protein assay. A flea extract containing 20 g of protein/ml was used based on previous antigen titration curves.
Characterization of Cell Populations by Flow Cytometry Peripheral blood mononuclear cells (PBMCs) were obtained by gradient density centrifugation using Ficoll-Hypaque (SigmaAldrich, St. Louis, Mo., USA). A total of 1 ! 106 PBMCs were stained for myeloid and pDC surface markers with a lineage 1 (Lin-1) cocktail of FITC conjugated antibodies (CD3, CD14, CD16, CD19, CD20, and CD56 – dump channel), HLA-DR-PECy7, CD11c-APC, and CD123-PerCP-Cy5,5 (BD Biosciences, San José, Calif., USA). For natural regulatory T cells, 1 ! 106 PBMC were stained with CD3-FITC, CD4-PerCP, CD25-PE-Cy7, and CD127-Biotin (BD Biosciences), followed by incubation with streptavidin SAV APC-Cy7 (BD Biosciences). For Tregs, cells were fixed and permeabilized using a human regulatory T cell staining kit (eBioscience, San Diego, Calif., USA). Intracellular staining was done for the transcription factor FOXP3 using antiFOXP3-PE antibodies. The CD3+ CD4+ CD25+ population was selected based on CD25 expression on the CD3+ CD4+ cells in each sample. Cells were analyzed in a FACSAria flow cytometer and with FACSDiva software (BD Biosciences). At least 8 ! 105 cells were analyzed in each experiment. Intracellular Detection of Cytokines Cytokine production by CD4+ T cells was assessed in cultured PBMCs with the presence of flea extract (20 g/ml) for 12 h, and stimulated with anti-CD28 (0.5 g/ml) and anti-CD49d (0.5 g/ ml). After 3 h of stimulation, 10 g/ml of brefeldin A (BD Golgi Stop; BD Biosciences) was added to block cytokine secretion from the cells. Cells were recovered in cold PBS-EDTA 2 mM and stained with CD3 Pacific Blue, CD4 PerCP, CLA FITC (BD Pharmingen), and a viability marker (LIVE/DEAD쏐 Fixable Aqua Dead Cell Stain; Invitrogen). The cells were permeabilized as described above prior to intracellular detection of interleukin (IL)-10 (anti-IL-10 APC; BD Pharmingen), IL-4 (anti-IL-4 PE; BD Biosciences), interferon gamma (IFN-␥) (anti-IFN-␥ Alexa 700; BD Pharmingen), and IL-17A (anti-IL-17A Alexa Fluor 647; eBioscience). Staphylococcal enterotoxin B (SEB) was used as a posi-
Dominguez-Amorocho et al.
2.0
*
1.5
Total DC (cells/ml)
HLA-DR
Total DC (%)
R1 0.51
40
*
1.0 0.5
LIN
0 PUFB 5
M
c
Fig. 1. DC in PBMCs from healthy controls (HC) and PUFB pa-
tients according to symptoms’ initial onset (less than 5 years: PUFB !5, more than 5 years: PUFB 15). a Dot plot showing total DC (DC) without any lineage marker and high HLA-DR expres-
tive control and nonstimulated cells were used as a negative control in each experiment. Cells were acquired in a FACSAria cytometer and data was analyzed with FACSDiva software (BD Biosciences). Integrated mean fluorescence intensity (iMFI) was calculated by multiplying the frequency of a specific cytokine-producing cell and the respective mean fluorescence intensity. This value was used as an indicator of the quality of a cell population’s functional response [10, 11]. Statistical Analysis A descriptive analysis was made on cell populations, using percentages, means, and standard deviations. Differences among groups were determined with the Mann-Whitney U test using GraphPad Prism 5.0 software. Differences were considered statistically significant when p ! 0.05.
Results
P
HC
M
HC
DC subpopulation (cells/ml)
HC
75 13.02
*
0
b
6.91
*
P
PUFB 5
PUFB 5
*
10
5
0 M
P
HC
M
P
PUFB 5
sion (R1). CD11c+ mDC (R2) and CD123+ pDC (R3). b Frequency (%) and absolute value (cells/ml) for total DC. c Frequency (%) and absolute count (cells/ml) of mDC (M) and pDC (P). Results are expressed as medians 8 ranges. * p ! 0.05. LIN = Lineage.
Total DC were gated based of the absence of lineage marker expression (T, B, and NK lymphocytes; monocytes and granulocytes) and high levels of HLA-DR, a class II major histocompatibility complex (MHC) molecule. In this population (Lin– and HLA-DR+), the expression of the myeloid marker CD11c for mDC and CD123high (IL-3R) as a marker for pDC (fig. 1a) were analyzed. The results showed a significantly lower number (both in percentages and absolute counts) of total DC in patients with PUFB compared with healthy controls (fig. 1b); DC analysis revealed a significant reduction of both subpopulations only in the group of patients with less than 5 years of disease evolution compared to healthy controls (fig. 1c). For Treg characterization, cells were gated based on expression of CD3+ CD4+ CD25+ FoxP3+ CD127low/– (fig. 2a). The results showed no differences in percentages or absolute counts when controls and patients where compared (fig. 2b).
DC and Natural Regulatory T Cells The percentage and absolute counts of DC and natural regulatory T cells (Treg) were evaluated in fresh nonstimulated PBMC from healthy controls and patients suffering from PUFB.
Antigen-Specific CD4+ T Cells Considering the role of effector T cells in the amplification, or control, of allergic inflammation, we analyzed
T Cells in PUFB
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Fig. 2. Natural regulatory T cells from healthy controls (HC) and PUFB patients according to initial onset of symptoms (less than 5 years: PUFB !5, more than 5 years: PUFB 15). a Dot plot showing CD4+ T cells (R1) with CD25 expression (R2). This population was used for evaluating cells with FOXP3 transcription factor intracellular expression and absence or low expression of CD127 (R3) corresponding to natural regulatory T cells. b Frequency (%) and absolute value (cells/ml) of natural Treg. Results are expressed as medians 8 ranges.
17.3
98.3
0.53
CD4
CD4
R1
Treg cells (%)
15
36.8
R2
10 5 0
CD25 R3
90 Treg (cells/ml)
CD3
FOXP3
5.36
60 30 0
a
NS
SEB
14.4
b
CD127
HC
PUFB 5
0.10 0.05 0 0.01
2.81
0.05
0.20 IL-10
CD4
0.15
**
0.10 0.05 0 1.03
0.12
**
0.20 0.15
IL-4
0.00
0.10 0.05 0 2.14
0.08
0.20
*
0.15
IL-17
0.01
0.10 0.05 0
a
b
HC
PUFB
Fig. 3. Detection of intracellular cytokines in nonstimulated cells (NS) and cells stimulated with SEB such as polyclonal stimuli or flea extract (FE). a Dot plot showing IL-10, IL-4, IL-17, and IFN-␥ detection. b Analysis
of comparison of cells obtained from healthy controls (HC) and the total number of patients (PUFB). Results are expressed as medians 8 ranges. * p ! 0.05, ** p ! 0.01.
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Dominguez-Amorocho et al.
CD3+ CD4+ T cells (%)
Cytokine iMFI
3.0
5,000
IFN-␥+
1.5
*
*
0.2
3,750
*
*
0.1
2,500
0.005
1,250
0
0
*
5,000
3.0
IL-10+
* *
1.5 1.5 1.0 0.5
3,750
*
2,500
0.05
1,250
0
0
3.00
*
2.25
5,000
*
IL-4+
0.75
2,500
0.1
1,250
0
0
3.00
*
CD4 T-cell frequency in response to flea extract in CLA– and CLA+ populations in patients with different disease evolution times (less than 5 years: PUFB !5, more than 5 years: PUFB 15). a Frequency of populations producing different cytokines. b Analysis of iMFI. Results are expressed as medians 8 ranges. * p ! 0.05.
IL-17+
2.25
+
*
*
*
*
3,750
1.50
Fig. 4. Cytokine-producing CD3+ and
*
60,000
*
40,000
*
1.50
20,000
0.75
5,000
0.10
2,500
0.05 0
0 CLA–
a
CLA+
PUFB 5
CLA–
b
CLA+
PUFB 5
the production of IL-10, IL-17, IL-4, and IFN-␥ by CD3+ CD4+ cells specific for flea antigens. As a negative control for cytokine secretion we used nonstimulated cells, and as a positive control we used cells exposed to a polyclonal stimulus (fig. 3). The results indicate that PUFB patients have a significantly higher proportion of CD4+ T cells producing the evaluated cytokines compared to healthy controls (fig. 3). No differences were found when patient groups were subdivided by disease evolution time. To dissect the specificity for homing, or systemic function of those cells, the cytokine-secreting popula-
tion was further divided into CLA+ and CLA–, and according to the time elapsed from the initial onset of symptoms. A significantly higher percentage of CD4+ CLA+ T lymphocytes producing IL-4, IL-10, and IL-17 was found in patients with a shorter disease duration. In this group, we also found higher levels of IFN-␥-producing cells in CLA– cells (fig. 4a). Those results were mirrored by the cytokine iMFI analysis (fig. 4b) In patients with more than 5 years of disease evolution, a similar percentage of IL-4-producing CD4+ T cells in
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CLA+ or CLA– was found. In this group, IL-10- and IFN␥-producing CD4+ T cells were higher in CLA– and IL-17 was higher in the CLA+ compartment (fig. 4a). In the cytokine iMFI analysis, we found a significant difference in the production of IL-10 and IFN-␥ in CLA– (fig. 4b). Within the CLA+ population, significantly higher percentages of IL-4- and IL-17-producing cells were observed in patients with less than 5 years of disease evolution compared with patients with a longer disease history (fig. 4a). The same pattern was observed by cytokine iMFI analysis (fig. 4b). Within the CLA population, there were more IL-10- and less IFN-␥-producing cells in patients with a longer time of disease evolution (fig. 4a).
Discussion
DC influence lymphocyte activation and polarization towards effector phenotypes [12]. It has been established that allergic patients skew their T helper response toward Th2; however, this polarization depends on the previous encounter with professional antigen-presenting cells, such as DC [13]. It has been reported that mDC-secreted products involved in attracting CD4+ Th2 cells, neutrophils, and eosinophils amplify the allergic inflammation [14]. The pDC role in immune allergic response amplification or inhibition is not yet clear since increases [15] and reductions [16] have been reported in affected tissues. In a murine model, pDC have been associated with protection from allergic inflammation in the airways [17]. Studies in infants have shown that a decrease in circulating pDC, but not mDC, is associated with the appearance of clinical symptoms of allergic asthma [18, 19]. Our findings of decreased numbers of peripheral DCs in PUFB patients might reflect a recruitment of this cell population to inflammatory sites, as seen in asthmatic patients [20, 21]. However, it should be noted that there was a reduction of both mDC and pDC, but only in patients having a shorter time of progression of the disease. This shift in DC appears to be associated with the acquisition of an immune stage conducive to clinical improvement, as most PUFB patients experience an improvement at the age of 5–7 years after disease onset. In a previous study, we reported functional differences in monocytederived DCs from PUFB patients who had a higher expression of co-stimulating molecules such as CD86 and HLA-DR, which might indicate a greater ability to stimulate T cells. Likewise, reduced regulatory activity was observed, with lower IL-10 and IL-6 secretion compared to healthy controls [4]. 170
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Natural Treg were identified as: CD3+ CD4+ CD25+ FoxP3+ CD127low. Most human CD4+ T regulatory Treg cells express low levels of IL-7 alpha receptor chain (CD127) because FoxP3 can be upregulated by activation, even on cells without regulatory activity [22]. Reports concerning natural Treg circulating levels have shown an increase in these cells in asthmatic children, albeit with a lack of functional activity compared with healthy controls [23]. Children with clinically active allergy to cow’s milk have lower circulating CD4+ CD25+ cells than asymptomatic children [24]. Contrasting with previous studies, the results of our work suggest no differences in frequency for this population between affected individuals and healthy controls. Within the population of Treg cells, there appears to be a subpopulation that is generated after antigenic stimulation (flea bite) and it is characterized by the production of IL-10 [25]. Our results suggest that individuals with PUFB have a greater percentage of systemic IL-10producing cells compared to healthy controls, mainly in the group with a longer history of the disease. The association of IL-10-secreting cells and allergy is controversial [26], but PBMCs from grass-allergic patients have an IL-10 response not found in healthy individuals [27]. In vitro, neutralization of IL-10 increased proinflammatory cytokine secretion [28] and allergenpulsed DCs in the presence of IL-10-induced regulatory cells [29]. Interestingly, successful allergen-specific immunotherapy has been associated with IL-10 production in allergic patients treated for allergy to birch pollen [30] and dust mites [31]. According to the importance of IL-4 and its proinflammatory role, our results show a higher frequency of IL-4-producing CD4+ T cells in cells that are involved in the local immune response in the skin, as was observed for CLA+ compared to CLA– CD4+ T cell populations. In addition, this difference was not observed in patients with a longer evolution time of the disease. Bearing in mind that clinical observation suggests that PUFB patients show clinical improvement with time and repeated exposure to flea antigens, these results may indicate that the most severe symptoms, present in the early stages of the disease, could be associated with the predominant TH2 response from specific cutaneous CD4+ T cells in these patients. The same behavior is observed in IL-17-producing CD4+ T cells and in patients with longer disease evolution times. This premise could indicate that TH17 response regulation occurs late in the process of desensitization to flea antigens.
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Although the same behavior is observed in IL-10-producing CD4+ T cells, it is possible that this response is related to an increase in regulatory mechanisms associated with the IL-4- and IL-17-mediated inflammatory response. Interestingly, we observed that PUFB patients have a predominant frequency of producing IFN-␥ CD4+ T cells associated with the systemic response (CLA– CD4+ T cells), with no difference related to the disease evolution time, which supports our conclusions. We have shown that patients with papular urticaria compared with healthy controls have a stronger response in terms of cytokine production after stimulation with flea antigen and this response is different in patients depending on the time of disease evolution.
Acknowledgments The authors want to thank doctors Armando Rojas, Clara Ines Ortiz, Manuel Forero, and Mariela Tavera for their collaboration with the volunteers participating in this study. Many thanks also go to Luis Miguel Franco, Baylor College of Medicine, Houston, Tex., USA for his constructive suggestions for the manuscript. This work was supported by Colciencias research project No. 120340820416 and Universidad Militar Nueva Granada’s research project No. MED-155. Researchers’ participation was supported by Pontificia Universidad Javeriana, Universidad Militar Nueva Granada, Fundación Santa Fe de Bogotá, and Universidad de los Andes (Bogotá, Colombia).
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