Flow cytometric analysis of IL-4, IL-13 and IFN-γ expression in peripheral blood mononuclear cells and detection of circulating IL-13 in patients with atopic dermatitis provide evidence for the involvement of type 2 cytokines in the disease

doi:10.1016/S0923-1811(01)00174-8

Journal of Dermatological Science

Volume 29, Issue 1, 1 May 2002, Pages 19-25

https://doi.org/10.1016/S0923-1811(01)00174-8 Get rights and content

Abstract

Recent studies have shown increased T-helper (Th) 2 cytokine expression and decreased IFN-γ expression in peripheral blood from patients with atopic dermatitis (AD). In the present study, we examined the cytokine expression in peripheral blood mononuclear cells in patients with AD and tested how the cytokine profile correlated with the patients’ age, severity and laboratory findings. Peripheral blood mononuclear cells obtained from 20 patients with AD were stimulated with phorbol 12-myristate 13-acetate and ionomycin, and were examined for the frequencies of CD4+ cells expressing IL-4, IL-13 and IFN-γ at the single cell level using intracellular cytokine staining and flow cytometry. There was a significant positive correlation between IL-4 and IL-13 expression in CD4+ cells. Expression levels of both IL-4 and IL-13 in CD4+ cells were significantly correlated with peripheral blood eosinophilia. These findings suggest that CD4+ cells producing IL-4 and IL-13 play important roles in the pathogenesis of AD. IFN-γ expression did not correlate with either IL-4 or IL-13 expression, or with blood eosinophilia. We also measured serum levels of IL-13 in 144 patients with AD using enzyme-linked immunosorbent assay, and found 16 patients with detectable levels of serum IL-13. IL-13+ patients had significantly higher serum IgE levels than IL-13− patients, suggesting a direct association between serum levels of IL-13 and IgE. It was also shown that the IL-13+ group was significantly younger in age than the IL-13− group, although the implication of this finding is not clear at present. The sum of these findings suggested that the predominance of Th2 cells and the consequent overexpression of IL-4 and IL-13 in peripheral blood may be deeply involved in the pathogenetic process of AD.

Introduction

Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease characterized by typically distributed eczematous skin lesions with lichenification, pruritic excoriations and xerosis [1]. Serum levels of immunoglobulin E (IgE) are frequently elevated in patients with AD [1], [2]. IgE production is strongly influenced by cytokines produced by activated T cells, and among the cytokines, interleukin (IL)-4, IL-13 and interferon-gamma (IFN-γ) have important roles. IL-4 acts on B cells as an isotype switch factor for the induction of IgE synthesis [3]. IL-13 is closely related to IL-4, and the two factors share many biological and immunoregulatory functions including stimulation of IgE synthesis [4]. On the other hand, IFN-γ inhibits IgE production induced by IL-4 [5].

The cytokine pattern in AD has been extensively studied. A number of studies using enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction methods found increased IL-4 and IL-13 production and decreased IFN-γ production in peripheral blood mononuclear cells (PBMC) from patients with AD [6], [7], [8], [9], [10]. Based on these findings, it is currently presumed that the immune response in AD is driven by T-helper (Th) 2 cytokines (IL-4, IL-5, IL-10, IL-13) and that a Th1/Th2 imbalance plays a role in the pathogenesis of AD [1], [11].

Recent advances in flow cytometric analysis enabled us to measure cytokine production at the single cell level [12]. It has the advantage of being able to measure rapidly the cytokine production of thousands of individual cells and to detect simultaneously two or more cytokines in the same cell. Using this method, recent studies have shown increased Th2 cytokine expression and decreased IFN-γ expression in peripheral blood from patients with AD compared with healthy controls [13], [14], [15], [16].

Most of these studies have been mainly focused on the comparison between AD patients and healthy individuals. Therefore, little is known about the relation between the intracellular cytokine profile of AD patients measured at the single cell level and patients’ age, severity, and laboratory findings. In the present study, we examined PBMC from AD patients for the frequencies of CD4+ cells expressing IL-4, IL-13 and IFN-γ using intracellular cytokine staining and flow cytometry, and tested how the cytokine profile correlated with each patient's clinical background. In addition, we measured serum levels of IL-13 by ELISA to determine whether serum IL-13 levels are associated with the disease.

Section snippets

Patients and sample collection

Heparinized peripheral blood for flow cytometric analysis was drawn from 20 patients with AD (10 males, 10 females; age: 0–38 years; average: 13.5±12.3 years) after informed consent was obtained. They consisted of eight patients at the age of 3 years and under, three patients between 10 and 15 years, and nine patients at the age of 16 years and over. For measurements of serum IL-13 levels, sera were collected from 144 AD patients (78 males, 66 females; age: 0–43 years; average: 13.5±12.2

Correlation between the SCORAD index, serum IgE levels and blood eosinophils

Clinical features of the 20 patients with AD from whom blood samples for flow cytometric analysis were drawn were recorded, and correlations between the SCORAD index, serum IgE levels and blood eosinophils were examined. The SCORAD index ranged from 3.5 to 66.5, with the mean±S.D. of 37.6±19.0. The logarithm of serum IgE level (log₁₀ IgE) was 2.71±1.18 and the percentage of eosinophils in white blood cells (% eosinophils) was 13.0±8.5%. Significant correlation was noted between the SCORAD and log

Discussion

In AD patients, serum levels of IgE are elevated with eosinophilia, which was suggested to reflect the altered expression of cytokines, that is, increased IL-4 and IL-13, and decreased IFN-γ expression [2]. Using flow cytometry, we analyzed the frequencies of CD4+ cells expressing IL-4, IL-13 and IFN-γ in PBMC from AD patients, and have shown a close relation between the percentage of IL-4+ cells and IL-13+ cells in CD4+ cells in individuals with AD. This may suggest the presence of a T cell

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Atopic dermatitis (AD) is a common yet complex skin disease, posing a therapeutic challenge with increasingly recognized different phenotypes among variable patient populations. Because therapeutic response may vary on the basis of heterogeneous clinical and molecular phenotypes, a shift toward precision medicine approaches may improve AD management. Herein, we will consider biomarkers as potential instruments in the toolbox of precision medicine in AD and will review the process of biomarker development and validation, the opinion of AD experts on the use of biomarkers, types of biomarkers, encompassing biomarkers that may improve AD diagnosis, biomarkers reflecting disease severity, and those potentially predicting AD development, concomitant atopic diseases, or therapeutic response, and current practice of biomarkers in AD. We found that chemokine C-C motif ligand 17/thymus and activation-regulated chemokine, a chemoattractant of T_H2 cells, has currently the greatest evidence for robust correlation with AD clinical severity, at both baseline and during therapy, by using the recommendations, assessment, development, and evaluation approach. Although the potential of biomarkers in AD is yet to be fully elucidated, due to the complexity of the disease, a comprehensive approach taking into account both clinical and reliable, AD-specific biomarker evaluations would further facilitate AD research and improve patient management.
Evolution of pathologic T-cell subsets in patients with atopic dermatitis from infancy to adulthood
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The circulating immune phenotype was defined in adults and young children with early atopic dermatitis (AD), but chronologic changes in the blood of infants and children with AD through adolescence have not been explored.
We sought to compare immune activation and cytokine polarization in the blood of 0- to 5-year-old (n = 39), 6- to 11-year-old (n = 26), 12- to 17-year-old (n = 21) and 18-year-old or older (n = 43) patients with AD versus age-matched control subjects.
Flow cytometry was used to measure IFN-γ, IL-9, IL-13, IL-17, and IL-22 cytokine levels in CD4⁺/CD8⁺ T cells, with inducible costimulator molecule and HLA-DR defining midterm and long-term T-cell activation, respectively, within skin-homing/cutaneous lymphocyte antigen (CLA)⁺ versus systemic/CLA⁻ T cells. Unsupervised clustering differentiated patients based on their blood biomarker frequencies.
Although CLA⁺ T_H1 frequencies were significantly lower in infants with AD versus all older patients (P < .01), frequencies of CLA⁺ T_H2 T cells were similarly expanded across all AD age groups compared with control subjects (P < .05). After infancy, CLA⁻ T_H2 frequencies were increased in patients with AD in all age groups, suggesting systemic immune activation with disease chronicity. IL-22 frequencies serially increased from normal levels in infants to highly significant levels in adolescents and adults compared with levels in respective control subjects (P < .01). Unsupervised clustering aligned the AD profiles along an age-related spectrum from infancy to adulthood (eg, inducible costimulator molecule and IL-22).
The adult AD phenotype is achieved only in adulthood. Unique cytokine signatures characterizing individual pediatric endotypes might require age-specific therapies. Future longitudinal studies, comparing the profile of patients with cleared versus persistent pediatric AD, might define age-specific changes that predict AD clearance.
The blood proteomic signature of early-onset pediatric atopic dermatitis shows systemic inflammation and is distinct from adult long-standing disease
2019, Journal of the American Academy of Dermatology
Despite increasing evidence that adults with long-standing atopic dermatitis (AD) have systemic inflammation, little is known about systemic inflammation in recent-onset early pediatric AD.
To analyze blood inflammatory proteins of early pediatric AD.
Using high-throughput proteomics (proximity extension assay), we assessed 257 inflammatory and cardiovascular risk proteins in the blood of 30 children with moderate to severe AD younger than 5 years of age (within 6 months of onset) compared with age-matched pediatric control individuals and adult patients with AD.
In pediatric AD blood, T helper (Th) type 2 (CCL13, CCL22) and Th17 (peptidase inhibitor-3/elafin) markers were increased, together with markers of tissue remodeling (matrix metalloproteinases 3/9/10, urokinase receptor), endothelial activation (E-selectin), T-cell activation (IL2RA), neutrophil activation (myeloperoxidase), lipid metabolism (FABP4), and growth factors (FGF21, transforming growth factor-α). Total numbers of dysregulated proteins were smaller in pediatric AD (n = 22) than in adult AD (n = 61). Clinical severity scores were positively correlated with receptors for interleukins 33 and 36 and inversely correlated with some Th1 markers (interferon gamma, CXCL11).
Different baseline expression levels in healthy pediatric vs adult samples.
Within months of pediatric AD onset, systemic immune activation is present, with Th2/Th17 skewing but otherwise different proteomic patterns from adult AD. Future correlation of proteomic patterns with disease course, comorbidity development, and drug response may yield predictive biomarkers.
Early immunologic changes during the onset of atopic dermatitis
2019, Annals of Allergy, Asthma and Immunology
Atopic dermatitis (AD), which is commonly called eczema, is the most common chronic inflammatory skin disease. The pipeline of new targeted treatments is currently expanding, a development that is largely based on our increasing understanding of disease mechanisms. Mechanistic insights have long been based on long-standing adult AD. Recently, studies also investigated early pediatric AD at disease onset, and revealed several differences in barrier and immune properties when compared with long-standing adult AD. This review focuses on immunological changes very early in life that predispose to the development of AD, and summarizes characteristics of the molecular AD phenotype in this age group.
Review of published literature.
Studies investigating human AD at disease onset in newborns, toddlers, and young children, in comparison with adults with long-standing disease.
Already in cord blood, increased Th2 and decreased Th1 levels were found to increase the risk of AD development. Both pediatric and adult AD share Th2/Th22 activation and defects in lipid barrier deposition and tight junction formation, but Th1 activation and epidermal differentiation complex defects are largely absent in pediatric AD.
Immune changes predisposing to AD development are present very early in life. During the first months of disease, AD shows various differences in immune and barrier properties from long-standing adult AD, which might necessitate tailored treatment approaches depending on the age of the patient.
Blood endotyping distinguishes the profile of vitiligo from that of other inflammatory and autoimmune skin diseases
2019, Journal of Allergy and Clinical Immunology
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Vitiligo consistently showed the greatest IFN-γ frequencies across CD4+ cells (CLA+: patients with vitiligo = 26% vs control subjects = 19%, P = .002; CLA−: patients with vitiligo = 33% vs control subjects = 26%, P = .02; Fig 2, A and B) and even greater frequencies across CD8+ cells (CLA+: patients with vitiligo = 41% vs control subjects = 33%, P = .05; CLA−: patients with vitiligo = 61% vs control subjects = 45%, P = .03; Fig 2, C and D) subsets. TH2 (but not TC2) cell frequencies were greater in patients with vitiligo than in control subjects (CLA+: patients with vitiligo = 9.2% vs control subjects = 4.5%, P = .02; CLA−: patients with vitiligo = 1.6% vs control subjects = 0.7%, P = .01; Fig 2, E and H) to levels corresponding to (P = .37; Fig 2, E) or even greater than (Fig 2, F) those observed in patients with the well-established TH2-skewed AD.33,46 TH9 and even more pronounced TC9 cell frequencies were greater in patients with vitiligo exclusively among the skin-homing compartment (CD4+: patients with vitiligo = 4.6% vs control subjects = 2.2%, P = .001; Fig 2, I; CD8+: patients with vitiligo = 7.9% vs control subjects = 3.8%, P = .01; Fig 2, K) but not in systemic/CLA− populations (Fig 2, J and L).
Peripheral blood skin-homing/cutaneous lymphocyte antigen (CLA)⁺ T cells emerge as biomarkers of cutaneous immune activation in patients with inflammatory skin diseases (atopic dermatitis [AD] and alopecia areata [AA]). However, blood phenotyping across these subsets is not yet available in patients with vitiligo.
We sought to measure cytokine production by circulating skin-homing (CLA⁺) versus systemic (CLA⁻) “polar” CD4⁺/CD8⁺ ratio and activated T-cell subsets in patients with vitiligo compared with patients with AA, AD, or psoriasis and control subjects.
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Patients with Vitiligo showed the highest CLA⁺/CLA⁻ T_H1/type 1 cytotoxic T-cell polarization, with parallel T_H2/T_H9/T_H17/T_H22 level increases to levels often greater than those seen in patients with AA, AD, or psoriasis (P < .05). Total regulatory T-cell counts were lower in patients with vitiligo than in control subjects and patients with AD or psoriasis (P < .001). Vitiligo severity correlated with levels of multiple cytokines (P < .1), whereas duration was linked with IFN-γ and IL-17 levels (P < .04). Patients and control subjects grouped into separate clusters based on blood biomarkers.
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2019, Annals of Allergy, Asthma and Immunology
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Such biomarkers would be of great value for considering new therapies, as well as for predicting course, responses, and future comorbidities. Although few disease markers have been defined in the pediatric population,5–25 broad blood profiling in early-onset AD is lacking. We analyzed freshly drawn peripheral leukocytes, using a broad microarray-based approach (and complementary RT-PCR), to compare blood of early-onset AD patients with matched healthy controls.
Atopic dermatitis (AD) predominantly affects young children, but our understanding of AD pathogenesis is based on skin and blood samples from long-standing adult AD. Genomic biopsy profiling from early pediatric AD showed significant Th2 and Th17/Th22-skewing, without the characteristic adult Th1 up-regulation. Because obtaining pediatric biopsies is difficult, blood gene expression profiling may provide a surrogate for the pediatric skin signature.
To define the blood profile and associated biomarkers of early moderate-to-severe pediatric AD.
We compared microarrays and reverse transcription polymerase chain reaction (RT-PCR) of blood cells from 28 AD children (<5 years and within 6 months of disease onset) to healthy control blood cells. Differentially expressed genes (DEGs) in blood (fold change [FCH] > 1.2 and false discovery rate [FDR] < 0.05) were then compared with skin DEGs.
Eosinophil and Th2 markers (IL5RA, IL1RL1/ST2, HRH4, CCR3, SIGLEC8, PRSS33, CLC from gene arrays; IL13/IL4/CCL22 from RT-PCR) were up-regulated in early pediatric AD blood, whereas IFNG/Th1 was decreased. Th1 markers were negatively correlated with clinical severity (EASI, pruritus, transepidermal water loss [TEWL]), whereas Th2/Th17-induced interleukin (IL)-19 was positively correlated with SCORAD. Although a few RT-PCR–defined immune markers (IL-13/CCL22) were increased in blood, as previously also reported for skin, minimal overlap based on gene array DEGs was seen.
The whole blood signature of early moderate-to-severe pediatric AD blood cells show predominantly a Th2/eosinophil profile; however, markers largely differ from the skin profile. Given their complementarity, pooling of biomarkers from blood and skin may improve profiling and predictions, providing insight regarding disease course, allergic comorbidity development, and response to systemic medications.

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Flow cytometric analysis of IL-4, IL-13 and IFN-γ expression in peripheral blood mononuclear cells and detection of circulating IL-13 in patients with atopic dermatitis provide evidence for the involvement of type 2 cytokines in the disease

Abstract

Introduction

Section snippets

Patients and sample collection

Correlation between the SCORAD index, serum IgE levels and blood eosinophils

Discussion

Lancet

J. Allergy Clin. Immunol.

J. Invest. Dermatol.

Immunol. Today

J. Immunol. Methods

J. Allergy Clin. Immunol.

J. Invest. Dermatol.

Blood

Cellular and immunologic mechanisms in atopic dermatitis

J. Am. Acad. Dermatol.