Atopic dermatitis (AD) is a chronic, pruritic, relapsing skin disorder that is often associated with a personal or a family history of asthma, allergic rhinitis, or conjunctivitis. It negatively impacts the quality of life of those affected and that of their families 1.
AD is a multifactorial disease comprising genetic factors, skin barrier dysfunction, and immunological disturbance. Two hypotheses of the mechanism of AD have been proposed; one hypothesis holds that the primary defect is induced by an immunologic disturbance that causes IgE-mediated sensitization, with epithelial-barrier dysfunction considered as a consequence of the local inflammation. The other hypothesis proposes that an intrinsic defect in the epithelial cells leads to the barrier dysfunction; the immunologic aspects are considered to be an epiphenomenon 2.
The skin immune system can be divided into early innate immune responses, which promote cutaneous inflammation, and adaptive immune responses, which promote memory responses against foreign antigens 3.
Antigen-specific IgE is the major recognition structure for allergens on mast cells and basophils. It may also be instrumental for the induction of allergen-specific tolerance or in anti-inflammatory mechanisms 4.
Toll-like receptors (TLRs) are a recently identified group of pattern recognition receptors that are involved in mechanisms of host defense against a wide range of pathogenic microorganisms 5. TLRs play an essential role in the activation of innate immune response to microbial pathogens including bacteria, parasites, and fungi. TLRs also interact with a variety of endogenous human ligands 6.
In the epidermis, keratinocytes have been shown to express functional TLRs. Trafficking immune system cells in the skin that express TLRs include Langerhans cells, monocytes/macrophages, dendritic cells, melanocytes, granulocytes, T and B lymphocytes, and mast cells. Endothelial cells of the microvasculature, stromal cells (fibroblasts and adipocytes), and airway epithelium also express TLRs. Each of these cell types has distinct TLR expression patterns and likely contributes toward cutaneous immune responses 7.
As TLRs are influencing adaptive immunity, the regulation of TLR expression at the sites of some skin diseases may be important in their pathophysiology. This has been shown in some studies on AD, acne vulgaris, leprosy, psoriasis, and Lyme disease 8. TLRs also play a role in asthma, acute respiratory distress syndrome, cardiac ischemia, coronary artery disease, inflammatory bowel disease, rheumatoid arthritis, acute tubular necrosis, fertility, and others 9.
Aim of the work
The aim of this work is to study the clinical features of AD and to evaluate the expression of TLR2 on peripheral blood monocytes of AD patients and correlate it with the serum level of IgE. It also aims to evaluate the relation between the expression of TLR2 and the severity of disease.
Participants and methods
The study included 30 patients with AD, 14 males (46.7%) and 16 females (53.3%) between 6 months and 26 years of age (mean±SD 8.52±7.21). They were selected randomly from those attending the Dermatology Outpatient Clinic at Al Zahraa University Hospital and the Medical Service Unit at the National Research Centre during the period from October 2009 to April 2010. The controls included 25 age-matched and sex-matched healthy volunteers, 15 males (60%) and 10 females (40%), between 3 months and 25 years of age (mean±SD 6.30±6.70). Patients with various degrees of severity of AD and variable durations of illness were selected. All patients with AD fulfilled the diagnostic criteria of Hanifin and Rajka 10. Patients with psoriasis, acne vulgaris, any autoimmune or endocrinal disease, or those taking any medication that affects the immune system were excluded. Patients had not been treated with UV light or systemic immunomodulatory agents for at least 4 weeks before vein puncture. Patients or their legal guardians provided informed written consent, and the study was approved by the local ethical committee. All patients included in the study were subjected to full history taking, a general examination, and a dermatological examination.
Two venous blood samples were collected from each AD patient and healthy donor. The blood samples obtained first were centrifuged and serum probes were stored at −20°C until collection of all samples to determine the total IgE levels according to the product manual. The samples obtained the second time were whole blood on EDTA for TLR2 assessment without storage.
Determination of total IgE
- The serum total IgE concentration was measured using a Modular E170 automated analyzer (Roche, Basel, Switzerland).
- A liquid-phase immunoassay was used for the accurate determination of serum total IgE. A fully automated, quantitative IgE assay was used that has a reverse sandwich architecture using monoclonal mouse anti-human IgE antibody covalently bound to paramagnetic particles in the solid phase and capturing the sample IgE. Bound sample IgE reacts with liquid biotin-labeled allergen, which is detected by chemiluminescence using acridiniumester-labeled streptavidin 11.
- Total IgE levels were quantified in protein units designated as kU/l with a lower detection limit of 0.35 kU/l.
- Total serum IgE levels less than 100 kU/l are in the normal or borderline range and IgE levels of at least 100 kU/l were classified as pathological.
- Patients with serum total IgE less than 100 kU/l were categorized into an intrinsic AD group, whereas the other patients were included in the extrinsic AD group.
Determination of toll-like receptor 2
The kits used were supplied from BD Pharmingen (New Jersey, USA) (catalog number: 558318, size: 100 tests, volume per test: 20 μl, clone: 11G7, immunogen: human TLR2-transfected cell line, isotype: mouse IgG1 kappa, storage buffer: aqueous buffered solution containing BSA and 0.09% sodium azide and reactivity: QC testing: human) according to the following steps:
- Leukocytes in 100 µl whole blood were labeled with 10 µg/ml of anti-CD14 mAb 26ic, which is a monocyte marker (a kind gift from Dr Jos A.G. van Strijp and Dr Kok P.M. van Kessel from the Eijkman-Winkler Institute, Utrecht University, Utrecht, the Netherlands), anti-TLR2 mAb (Becton-Dickinson, Mountain View, California, USA), and proper isotype-matched control mAbs to detect nonspecific staining (IgG1 for CD180, IgG2a for TLR2, and IgG2b for CD14; Serotec, Denmark) and then with a phycoerythrin-labeled goat anti-mouse IgG conjugate (Dako, Glostrup, Denmark).
- The erythrocytes were lysed and the leukocytes were fixed.
- The data of at least 5000 monocytes were measured using a FACS Calibur flow cytometer (Becton-Dickinson).
- Monocytes were gated on the basis of their scatter properties on a side versus CD14 dot plot.
- The FL2 mean fluorescence intensity (MFI) of the leukocytes was determined.
- The MFI measured in the presence of the proper isotype control mAb (background) was subtracted from the MFI value of the respective sample containing the receptor-specific mAb to correct nonspecific binding.
- The absolute receptor count was determined using mAb-coated standard beads (Qifikit; Dako).
- A set of beads coated with different and known amounts of an anti-human CD5 mAb was incubated for the same time and with the same concentration of the phycoerythrin-labeled goat anti-mouse IgG conjugate as that used for cell surface staining.
- After one washing step, the FL2 fluorescence intensity of 50 000 beads (TLR2) was measured by flow cytometry, under the same fluorescence setting as that used for the cells.
- The MFI of the various standard beads was plotted against the given number of mAb per bead (in a log/log scale) and, by linear regression, converted into a standard curve.
The MFI of the cells was converted into a number of mAb-binding sites per cell using this standard curve 12.
Data were collected, revised, verified, and then edited on a PC Data were then analyzed using the statistical package for social sciences (SPSS), version 15 (IBM, Chicago, Illinois, USA). The data were summarized using descriptive statistics: mean, SD, minimal, and maximum values for quantitative variables, and number and percentage for qualitative values. Statistical differences between groups were tested using the χ2-test for qualitative variables. The nonparametric Mann–Whitney test and the Kruskal–Wallis test were used for quantitative variables that were not normally distributed. Correlations were determined to test for linear relations between variables. P-value greater than 0.05 was considered not to be significant, whereas a P-value of 0.05 or less was considered as significant and P-value of 0.001 or less as highly significant.
The age of onset of AD patients (14 males and 16 females), ranged from 2 months to 16 years (mean±SD 3.00±3.52) as shown in Table 1.
On the basis of the serum total IgE concentrations, 11 patients had intrinsic AD (mean±SD 51.6±24.5 kU/l) and 18 patients had extrinsic AD (mean±SD 349±89.5 kU/l) as shown in Table 2. There was no statistically significant difference in the age of onset or sex in patients with AD, either intrinsic or extrinsic.
The Nottingham Eczema Severity Score
Surface area measurement using tick boxes
Record a tick in each box if more than 2 cm2 (size of a 10 pence coin) is involved with atopic eczema. Calculate the total ticks by adding together the number of recorded ticks for both the front and the back of the surface diagram. The final score was calculated using the tables below:
According to the Nottingham Eczema Severity Score (NESS) 13, patients were divided into three groups: mild, moderate, and severe, ranging from 5 to 13 (the minimum possible value is 3 and the maximum possible value is 15) (mean±SD 9.47±2.39) as shown in Table 3.
Table 4 shows that there was a statistically significant difference between the patients’ age and the severity of the eczema as measured by the surface area score. Also, there was a statistically significant positive correlation between the duration since onset and the surface area score. The relation between the age of onset and the items of the Nottingham eczema score was not statistically significant.
Serum IgE levels ranged from 12.10 to 440.20 IU/l (mean±SD 236.19 IU/l±163.24) in patients and from 22.30 to 52.80 IU/l, (mean±SD 39.05 IU/l±6.69) in controls as shown in Table 5; significantly higher levels were found in patients with AD compared with the controls.
The number of TLR2 on the surface of monocytes showed no statistically significant different values in AD patients compared with those of the controls (Table 6).
The number of TLR2 on the surface of monocytes showed no statistically significant difference in patients with extrinsic AD (6.99±4.98) compared with patients with intrinsic AD (10.29±8.88) (Table 7).
There was no statistically significant correlation between TLR2 and serum IgE with age, age of onset, and duration since onset. A highly statistically significant positive correlation was found between serum IgE levels and the surface area score, but not between TLR2 levels and items of the NESS as shown in Table 8.
There was no statistically significant difference between TLR2 or IgE levels with different grades of NESS as shown in Table 9.
Table 10 shows that the TLR2 levels were statistically higher in patients with a personal history of other allergic diseases compared with those without. The difference in the IgE level was not statistically significant.
There was no statistically significant difference between a family history of AD or other allergic diseases with both IgE and TLR 2 levels (Table 11).
AD is one of the most frequent chronic inflammatory skin diseases, with an increasing prevalence, affecting 10–20% of children and 1–3% of adults in industrial countries 14.
TLRs are a family of type I transmembrane protein receptors present on various cell types that function in mammals to recognize conserved molecular patterns to microbial pathogens termed pathogen-associated molecular patterns 15. Host cells are able to produce endogenous TLR ligands under stress conditions 16. TLRs have been implicated in both inflammatory responses and the innate host defense against pathogens. So far, the human TLR family consists of 10 members, and is set to expand 17.
The present study has shown that the eczematous lesions appeared before the age of 2 years in 56.7% of cases without a statistically significant difference in terms of sex (47% for females vs. 53% for males). These results are in agreement with those authors 18–20 who reported that there was no statistically significant difference between males and females or among age groups within each sex. This is in contrast to the results of Mar and Marks 21, who reported a higher prevalence in adult females.
There was a statistically significant positive correlation between the patients’ age and the severity of the eczema as measured by the surface area score in this study. Also, there was a statistically significant positive correlation between the age of onset and the surface area score. This is in contrast to the results of Yazganoglu and Ozkaya 22, who reported that there was no significant statistical correlation between the severity of AD and the age of onset of AD before and after the age of 2 years. They also found no correlation between the severity of AD and the sex of the patients.
In this study, no statistically significant difference was found in the expressions of TLR2 on peripheral blood monocytes of patients with intrinsic AD or extrinsic AD compared with those of the controls, although there was high bacterial colonization in AD patients. Therefore, we suspect an impaired innate immune defense against these bacteria leading to greater colonization and greater susceptibility to bacterial colonization. Niebuhr et al.23 showed that macrophages from patients with AD expressed significantly less TLR2 and they also reported that macrophages had a reduced capacity to produce proinflammatory cytokines such as interleukin (IL)-6, IL-8, and IL-1β after stimulation with TLR2 ligands. These data might contribute to the enhanced susceptibility to skin infections with Staphylococcus aureus in AD. This is not in agreement with the results of Sümegi et al.24, who detected significantly elevated expressions of TLR2 on peripheral blood monocytes of patients with intrinsic AD. They added that different bacterial products of Gram-negative and Gram-positive bacteria are able to induce statistically significant upregulation of TLR2 and TLR4 on peripheral blood leukocytes. Therefore, they concluded that the frequent local bacterial infections associated with altered barrier functions of the skin might result in a leakage of bacterial molecules (superantigens, lipopolysaccharides, peptidoglycan) that stimulate and upregulate the TLR expression of leukocytes.
The present study showed that there was a highly statistically significant positive correlation between a personal history of other allergic diseases and TLR2 levels. However, there was no statistically significant difference between the family history of atopic diseases and TLR2 levels. To our knowledge, no previous studies have compared TLR 2 levels and a personal or a family history of allergic diseases. Our study may highlight the relation of TLR2 to patients with an atopic personality, who have multiple allergic diseases such as AD, allergic rhinitis, and bronchial asthma rather than AD alone. Zheng et al.25 have reported that AD is also part of a process called the atopic march, a progression from AD to allergic rhinitis and asthma. This has been supported by multiple cross-sectional and longitudinal studies and experimental data.
Our results showed that serum total IgE levels were elevated in 73.9% of cases, with statistically significant differences between patients with AD and controls. These results are almost in agreement with the reports of many studies 26–30. Also, a highly statistically significant positive correlation was found between serum IgE levels and the surface area score, which is in agreement with Wu et al.31, who reported that serum levels of total IgE correlated with the SCORAD index in pediatric patients with AD; they added that serum levels of total IgE can serve as serum markers for monitoring disease activity in childhood AD.
The present study has shown that there was no statistically significant difference between the grades of NESS (mild, moderate, or severe) and IgE or TLR2 levels. This is in agreement with the reports of Weber et al.32 and in contrast to the reports of many authors 33–36.
Our findings showed that the expressions of TLR2 on peripheral blood monocytes showed no statistically significant difference between patients and controls despite high bacterial colonization in AD patients; therefore, we suspect an impaired innate immune defense against these bacteria leading to greater colonization and greater susceptibility to bacterial infection. However, TLR2 expression is related to atopic personality who has a personal history and suffers multiple atopic manifestations of AD, allergic rhinitis, asthma and others. Serum IgE was elevated in AD patients rather than in controls, with a statistically significant positive difference between serum IgE and the surface area score for AD.
We recommend further studies on a larger scale of patients to clarify the role of TLR2 in the pathogenesis of AD so that TLRs can be targeted with new therapeutic options for altering the imbalance in innate and acquired immunity.
The authors thank to all staff members and colleagues in Al Zahraa University Hospital and in the National Research Center for their generous help.
Conflicts of interest
There are no conflicts of interest.
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