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. 2014 Jul 29;143(1):33–41. doi: 10.1111/imm.12286

Absence of the adaptor protein Shb potentiates the T helper type 2 response in a mouse model of atopic dermatitis

Karin Gustafsson 1, Elsa Willebrand 1, Michael Welsh 1,✉
PMCID: PMC4137953  PMID: 24645804

Abstract

Aberrant regulation of T helper (Th) cell maturation is associated with a number of autoimmune conditions, including allergic disorders and rheumatoid arthritis. The Src homology domain protein B (Shb) adaptor protein was recently implicated as a regulator of Th cell differentiation. Shb is an integral component of the T-cell receptor (TCR) signalling complex and in the absence of Shb the TCR is less responsive to stimulation, resulting in the preferential development of Th2 responses under conditions of in vitro stimulation. In the present study, we extend those observations to an in vivo situation using a murine model of atopic dermatitis. Shb knockout mice develop more pronounced symptoms of atopic dermatitis with increased localized oedema, epidermal hyperplasia and IgE production. Dermal infiltration of mast cells, eosinophils, CD4+ Th cells and F4/80+ macrophages was also significantly increased in Shb-deficient mice. This correlated with elevated transcription of the hallmark Th2 cytokines interleukin-4 and interleukin-5. The loss of Shb therefore alters TCR signalling ability, thereby favouring the development of Th2-driven inflammation and exacerbating symptoms of allergy.

Keywords: atopic dermatitis, cytokines, Src homology domain protein B, T helper type 2 responses, T-cell receptor signalling

Introduction

Activity of the immune system is largely regulated through the production of different types of cytokines by T helper (Th) cells. Based on cytokine secretion profiles, several Th cell subsets have been identified.1–3 Th2 cells partake in the host's defence against extracellular parasites such as helminths,2,4–7 and are characterized by production of high levels of interleukin-4 (IL-4), IL-5 and IL-13. A meticulous balance between different populations of Th cells is, however, essential for optimal pathogen clearance as well as for proper immune regulation. Several autoimmune conditions are linked to deregulated cytokine profiles.8–10 Individuals suffering from allergic disorders such as asthma and atopic dermatitis often have an aberrant Th2 response with an elevation in eosinophil and basophil blood counts as well as increased levels of IgE.11–15

A wide variety of factors influence naive Th cells as they differentiate toward mature effectors in the periphery. The cytokine microenvironment in combination with antigen avidity and affinity will contribute to the mature cytokine profile of the Th cell.16–20 T-cell receptor (TCR) signalling also appears to be of great importance since removal of central TCR pathway components has demonstrated that Th cell responses can be altered if the balance in the TCR signalling complex is disrupted.21–24

The association between allergic disease and Th2 driven inflammation is well established both in humans and in murine models. Patients suffering from asthma or atopic dermatitis show linkage to specific polymorphisms in the IL-4 receptor (IL4R) gene locus as well as to downstream effectors of the IL-4 signalling chain.25,26 Additionally, deletion of typical Th2 cytokines results in milder symptoms in murine experimental models of asthma and atopic dermatitis.27–31 Thymic stromal lymphopoietin (TSLP) has recently emerged as a key regulator of Th2 responses and allergic conditions.32–34 TSLP acts on a wide variety of immune cells, including dendritic cells, basophils, mast cells and T cells, inducing a potent Th2 promoting microenvironment.35–38 Epithelial cells secrete TSLP in response to danger signals such as mechanical injury or exposure to microbes and allergens.37–39 TSLP secretion, however, is not only triggered by potentially harmful agents. Epidermal application of vitamin D or vitamin D analogues can elicit robust TSLP expression in skin keratinocytes.40 Mice subjected to daily exposures of a vitamin D analogue develop skin lesions reminiscent of human atopic dermatitis with dermal hyperplasia and significant infiltration of CD4+ T cells, so providing a convenient model system to study allergic inflammation.35,40,41

The Src-homology 2 domain containing adaptor protein Shb is mainly involved in mediating signals from activated tyrosine kinase receptors such as the vascular endothelial growth factor receptor-2, the platelet-derived growth factor receptor as well as the TCR.42–44 Shb's Src-homology 2 domain preferably binds to phosphotyrosine residues in the cytoplasmic tail of receptor tyrosine kinases and subsequently recruits further signalling mediators.45 Shb associates with the ζ-chain in the activated CD3-complex of the TCR and engages LAT, Grb2 and SLP76.44,46 This complex can in turn promote the phosphorylation and activation of several central TCR signalling components including PLC-γ, Vav-1 and nuclear factor of activated T cells.46,47

An Shb knockout mouse was recently generated and a study on that mouse further confirmed the importance of Shb as a modulator of T-cell activation.48 Shb null CD4+ Th cells are hyperproliferative and display a bias towards a Th2 profile under in vitro stimulation. TCR signalling is aberrant in the absence of Shb, with increased basal activation of Vav-1, Cbl and p38 mitogen-activated protein kinase but blunted stimulation-induced phosphorylation of several signalling mediators including PLC-γ.49 Together, these findings prompted the current investigation of Shb's role in in vivo regulation of the immune system under Th2 promoting conditions.

Materials and methods

Animals

The generation of Shb knockout mice has been described previously.48 Since the Shb knockout was embryonically lethal on a C57BL/6 background the animals were initially maintained on a mixed genetic background consisting of 129/SvJ, FVB/N and C57Bl/6. To conduct experiments in a Th2 promoting environment the Shb knockout was bred on to the BALB/c background, known to be Th2 biased. BALB/c females (Scanbur AB, Sollentuna, Sweden) were mated with Shb knockout males and the resulting Shb+/− pups were continuously backcrossed with BALB/c for seven generations. At this point Shb+/− matings were set up to generate Shb−/− and Shb+/+ animals. Subsequent experiments were all conducted on these Shb knockout (−/−) or wild-type (+/+) animals on the BALB/c background. All experiments were approved by the local animal ethics committee at Uppsala University.

Atopic dermatitis induction

The low calcaemic vitamin D agonist MC903 (Sigma-Aldrich, St Louis, MO) was dissolved in ethanol at a concentration of 50 μm; 1 nmol in a volume of 10 μl was applied to the inner and outer surfaces of the ear as previously described.40 Ten microlitres of ethanol was used as vehicle control treatment. The animals were monitored daily for the development of symptoms and ear thickness was recorded every third day using digital callipers. MC903 and vehicle-treated mice were killed on day 9, and ears and auricular draining lymph nodes were isolated.

Histology

Upon isolation, ears were cut in half. One half was fixed in 4% formalin overnight and subsequently paraffin embedded whereas the other half was frozen and embedded in section media (Richard Allan Scientific, Thermo Scientific, Waltham, MA). Paraffin-embedded tissue was cut in 5-μm sections and mounted on polylysine microscope slides (Menzel Gläser, Braunschweig, Germany) and stained with haematoxylin & eosin, alkaline 0·5% Congo red or toluidine blue. Frozen tissue was cut into 8-μm sections and mounted on SuperFrost glass slides (Menzel Gläser) and fixed for 10 min in ice cold methanol before immunofluorescence staining.

Immunofluorescence

Frozen sections were stained with primary antibodies directed towards Th cell marker CD4 or macrophage marker F4/80 (both primary antibodies were from eBioscience, Hatfield, UK) in a concentration of 1 : 300. Secondary anti-rat Alexa Fluor 488 antibodies (Invitrogen, Carlsbad, CA) were used in a 1 : 500 dilution. Sections were mounted with Vectashield HardSet mounting medium (Vector Laboratories, Burlingame, CA) containing 4′,6-diamidino-2-phenylindole (DAPI) for staining of nuclei. A Nikon Eclipse TE2000-U fluorescence inverted microscope (Nikon, Tokyo, Japan), with a Nikon D Eclipse C1 camera was used to take photos and nikon act-1c v1.02.10 (Nikon) and imagej v. 1.45s (NIH, Bethesda, MD) software programs were used for image analysis.

IgE ELISA

Immunoglobulin E was determined on serum samples from five wild-type and five Shb knockout mice either subject to MC903 treatment or not for 9 days using an IgE ELISA according to the manufacturer's recommendation (BioLegend, San Diego, CA).

RNA isolation and real-time reverse transcription-PCR

Auricular draining lymph nodes were fixed and preserved in RNALater (Qiagen, Solna, Sweden) immediately after isolation. RNA was extracted using an RNAeasy mini kit (Qiagen) following the instructions provided by the manufacturer and gene expression was determined by one-step real-time reverse transcription PCR using QuantiTect™ SYBR® Green RT-PCR kit (Qiagen). The PCR conditions were as follows; reverse transcription at 50° for 20 min, inactivation at 95° for 15 min, 50 cycles of denaturation at 94° for 15 seconds, annealing for 25 seconds at the various temperatures indicated in Table 1, and extension at 72° for 15 seconds. Primer sequences are listed in the Supporting information, Table S1. All reactions were run on a LightCycler™ real-time PCR machine (Roche Diagnostics, Basel, Switzerland). Cycle threshold (CT) values were estimated with the lightcycler software v 4.1 (Roche, Basel, Switzerland) and transcript levels were normalized by subtracting the corresponding β-actin values.

Miles assay

To determine the effects of acute histamine permeability a modified Miles assay was performed.50,51 Mice were shaved on the back 2 days before the experiment. On the day of the experiment, the mice were anaesthetized with avertin at a dose of 0·02 ml/g body weight. Then, 1% Evans Blue (Sigma Aldrich, St Louis, MO) dissolved in sterile PBS was injected at 2 μl/g body weight via the tail vein. After 30 min, the mice were intradermally injected with 3 μg histamine in 20 μl on their right side and 20 μl PBS on the left side, serving as a negative control. The animals were killed 30 min later and the affected tissue was collected and weighed. Evans Blue was extracted in 1·5 ml formamide at 70° and the optical density was measured at 630 nm using a FLUOStar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany) to quantify the amount of Evans Blue extracted.

Statistical analysis

All values are presented as mean ± SEM. Comparisons between groups were performed by one-way analysis of variance followed by post hoc analysis with Bonferroni's test. Statistical significance was set to P < 0·05.

Results

Shb knockout mice develop more severe symptoms in a model of atopic dermatitis

Initial attempts to induce atopic dermatitis in Shb knockout mice by ovalbumin exposure to tape-stripped skin failed because the mice rapidly dismantled the bandage holding the ovalbumin-containing patch.52,53 However, atopic dermatitis-like symptoms40 can be induced by exposure to the vitamin D analogue MC903, which via TSLP transcription results in increased levels of Th2 cytokines. MC903 was therefore applied daily to the ears of Shb knockout and wild-type mice. A second group of mice treated with ethanol served as a vehicle treatment control group. All MC903-treated mice developed localized oedema by day 6; however, the ear swelling was significantly pronounced in the Shb-deficient mice, suggestive of an aggravated reaction (Fig. 1a). The treatment continued over 9 days and the increased oedema persisted in the Shb knockout mice at each recorded time-point (Fig. 1a). The vehicle-treated group remained unaffected throughout the course of the experiment (Fig. 1a). Histological examination of the ears on day 9 revealed more epidermal hyperplasia and dermal infiltration of what appeared to be leucocytes in Shb null mice (Fig. 1b). MC903 augmented ear TSLP expression (Fig. 1c) and increased serum IgE in Shb knockout mice (Fig. 1d). To confirm that the cells infiltrating the dermis of the oedematous ears were leucocytes, frozen ear sections were stained for Th cell marker CD4 and macrophage marker F4/80.54,55 Wild-type as well as Shb knockout MC903-treated ears contained increased numbers of CD4+ Th cells and macrophages compared with the vehicle group (Fig. 2a,b), but the degree of increase was higher in the knockout (Fig. 2a,b). Mast cells are important effector cells in allergic inflammation; ears were therefore stained with toluidine blue to enable identification of mast cells.56 Shb-deficient atopic dermatitis mice also displayed increased numbers of mast cells compared with their wild-type counterparts (Fig. 3a). The minimal increase of mast cell numbers observed in the wild-type situation is likely to reflect a milder disease and that mast cell infiltration is a late event in MC903-induced allergic dermatitis. There was also an increased infiltration of eosinophils in MC903-treated Shb knockout ears (Fig. 3b) further reinforcing the view that a more severe inflammatory reaction prevails in the absence of Shb. The loss of Shb therefore appears to affect the immune response under Th2 inflammatory conditions by enhancing the infiltration of various immune system effector cells.

Figure 1.

Figure 1

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Development of MC903-induced dermatitis. (a) Oedema development was monitored by measurement of ear thickness every third day with a digital calliper. (b) Haematoxylin & eosin-stained sections of ears from untreated and MC903-treated mice. Arrows point to the junction between the epidermis and the dermis. Images were obtained with a 20× objective. Data are presented as mean ± SEM and based on six MC904-treated and three vehicle-treated mice of each genotype. (c) TSLP gene expression in response to MC903 treatment. Values are relative gene expression after normalization for actin in three untreated and five MC903-treated mice each group. n.d. = not detectable. Significant statistical comparisons were against untreated groups. (d) Serum IgE after MC903 treatment for 9 days or no treatment for five mice each group. *P < 0·05 and ***P < 0·001 as determined by one-way analysis of variance followed by Bonferroni's test.

Figure 2.

Figure 2

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Analysis of T helper (Th) cell and macrophage recruitment. Frozen sections of MC903-treated and vehicle-treated ears were stained for CD4 (a) and F4/80 (b) to evaluate dermal infiltration of Th cells and macrophages, respectively. The proportions of respective cell type were determined by relating the number of marker-positive cells to the total number of DAPI-stained nuclei in the dermis. The results are presented as mean ± SEM and based on six MC904-treated and three vehicle-treated mice of each genotype. All photos were taken at 20× magnification. *P < 0·05 as determined by one-way analysis o f variance followed by Bonferroni's test.

Figure 3.

Figure 3

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Evaluation of mast cell and eosinophil infiltration. (a) Mast cell numbers in ear dermis were determined in toluidine blue-stained sections of vehicle or MC903-treated ears. Arrows point to the junction between the epidermis and the dermis and the arrowheads point to mast cells. Original magnification was 20×. Values are presented as mean ± SEM with six animals in each group from three independent experiments. (b) Eosinophil infiltration after MC903 treatment for 9 days. Ear sections were stained with Congo red and positive cells were counted per section. Arrowheads point to examples of eosinophils. Values are based on five mice for each genotype after MC903 treatment and three mice each for vehicle control. *P < 0·05 and **P < 0·01, as determined by one-way analysis of variance followed by Bonferroni's test. Both MC903-treated groups were significantly elevated compared with vehicle controls.

Increased production of certain Th2 cytokines in Shb knockout mice with atopic dermatitis

Type 2 cytokines IL-4, IL-5 and IL-13 are mediators of many of the symptoms seen in allergic inflammation. To determine cytokine expression, auricular draining lymph nodes were isolated and RNA was extracted. Both IL-4 and IL-5 were expressed at significantly increased levels in the Shb knockout MC903-treated animals (Fig. 4a,b), whereas there was no major difference in the transcription of IL-13 between wild-type and knockout samples (Fig. 4c). Although Th2 cytokines are essential for the establishment of atopic dermatitis, type 1 cytokines like IL-6 and interferon-γ also play an important role in the development of the chronic phase of disease.57 The transcript levels of these factors was examined, revealing similar expression levels in wild-type and Shb knockout atopic dermatitis mice (Fig. 4d,e), further supporting the notion that the absence of Shb results in a Th2 skewed response. Deficits in the regulatory T (Treg) cell population have been reported to contribute to the development of excessive Th2 responses.58 To this end, IL-10 expression was determined. No differences were, however, observed between wild-type and Shb knockout samples with regards to IL-10 levels (Fig. 4f). This suggests that the Th2 bias is the result of a Th cell inherent defect. In conclusion, Shb deficiency appears to confer a more aggressive development of atopic dermatitis with increased leucocyte infiltration, possibly caused by the elevated expression levels of IL-4 and IL-5.

Figure 4.

Figure 4

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Analysis of cytokine expression patterns in auricular draining lymph nodes. The transcript levels of typical T helper type 2 (Th2) cytokines interleukin-4 (IL-4) (a), IL-5 (b), IL-13 (c), interferon-γ (IFN-γ) (d), IL-6 (e) and IL-10 (f) were evaluated by semi-quantitative real-time RT-PCR. All values were normalized to β-actin and subsequently related to the vehicle-treated wild-type. Means are presented as 2−ΔCt ± SEM to demonstrate fold change in mRNA content. The results are representative of three independent experiments with six mice in each treatment group. ***P < 0·001 and *P < 0·05 as determined by one-way analysis of variance followed by Bonferroni's test.

Histamine-induced vascular permeability is not affected in the absence of Shb

The increased infiltration of macrophages and Th cells displayed by the Shb knockout in the atopic dermatitis model might not be dependent on the local cytokine milieu but could merely be a consequence of increased blood vessel permeability. Several previous reports have established that the Shb knockout exhibits alterations in the microvasculature and in the permeability responses to stimulation with vascular endothelial growth factor A.59–61 In Th2-driven inflammation the principal mediator of vessel permeability is histamine and a modified Miles assay with histamine as the permeability-inducing agent was therefore performed.50 As suggested by the amount of extravasated Evans Blue, wild-type and knockout mice responded with increased vascular permeability to histamine stimulation (Fig. 5). However, there was no difference in the magnitude of the histamine response between the two groups (Fig. 5). The enhanced leucocyte recruitment observed in Shb knockout mice suffering from atopic dermatitis is therefore probably not a consequence of increased histamine-induced vascular permeability, but more likely a reflection of the increased production of Th2 cytokines.

Figure 5.

Figure 5

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Assessment of histamine-induced vascular permeability. Evans Blue was delivered by a tail vein injection into wild-type and Shb knockout mice followed by an intradermal challenge with histamine in the right flank and sterile PBS in the left flank. Vascular permeability was quantified by estimation of micrograms of Evans Blue per gram skin tissue. The values are representative of three independent experiments and based on four mice per genotype. Data are presented as mean ± SEM. **P < 0·01 and *P < 0·05 estimated by one-way analysis of variance followed by Bonferroni's test.

Discussion

T helper cell differentiation has previously been demonstrated to be influenced by Shb, as naive Shb-deficient Th cells preferentially adopt a Th2 fate upon in vitro stimulation. In the present study we expand those findings by evaluating the effect of Shb-dependent signalling on Th cells in an in vivo context and consequently reinforce the previously published in vitro data because Shb knockout mice were found to develop more severe symptoms in a murine model of atopic dermatitis, a disease characterized by an abnormal Th2 response.

The signals that trigger Th cell development have been extensively studied, resulting in a complex model involving factors influencing the fate of the naive Th cell. Signalling via the antigen-recognizing TCR synergizes with the signalling cues from the local cytokine environment.16,18,19 Shb null mice suffering from atopic dermatitis displayed significant changes in their cytokine expression pattern at the site of inflammation with increased transcript levels of IL-4 and IL-5 in the auricular draining lymph nodes. Both IL-4 and IL-5 facilitate the infiltration of leucocytes by inducing the secretion of leucocyte-attracting chemokines and by enhancing the leucocyte responses to chemoattractants.62–66 Interleukin-4 can also directly influence leucocyte recruitment by increasing the expression of vascular cell adhesion molecule 1 on endothelial cells.67 In line with this, Shb knockout MC903-treated mice exhibited an exaggerated local recruitment of CD4+ Th cells, macrophages and mast cells.

Notably, IL-13 was found to be expressed at similar levels in Shb knockout and wild-type mice after atopic dermatitis induction. Up-regulation of IL-4 and IL-5 often occurs concomitantly with increased expression of IL-13. Th2 cells are considered to be the major source of IL-13; however, other immune cells, including macrophages, mast cells, eosinophils and basophils, are capable of producing IL-13.68–71 Since the presently employed Shb knockout is global, it cannot be excluded that defects in other immune cells contribute to the observed Shb-deficient atopic dermatitis phenotype.

Although other cell types may influence the immune responses elicited in the absence of Shb expression, defective Th2 regulation is likely to be a major factor in the currently observed aggravated atopic dermatitis symptoms. Our previous report demonstrating a Th2 skewing in Shb knockout T cells was based on in vitro experiments where purified CD62LHi CD44Lo CD4+ naive Th cells were stimulated with CD28 and CD3 antibodies.72,73 These results are suggestive of a Th cell intrinsic defect in cytokine regulation. Previous results have also confirmed that Shb partakes in the signalling complex that assembles after TCR activation and that TCR signalling is disrupted in the absence of Shb.44,46,47,49 TCR signalling strength has been implicated as a key determinant of Th cell differentiation.74 Low concentrations of antigen that lead to weak TCR activation result in a preferential production of IL-4.75 Similarly, altered peptide ligands with reduced TCR affinity also elicit robust Th2 responses.16 The suboptimal TCR activation appears to alter the regulation of transcription factors that are essential for Th2 cell differentiation such as GATA3, AP-1 and nuclear factor of activated T cells.76–78 Moreover, point mutations in the central TCR signalling mediators ZAP70 and CARMA1, which do not abolish signalling but weaken it, alter the balance in favour of Th2 responses.58,79,80 In our previously published report, TCR cross-linking of Shb-deficient Th cells revealed decreased stimulation-induced tyrosine phosphorylation, suggestive of defective TCR signal transduction. The reduced TCR responsiveness displayed in the absence of Shb is likely to result in a Th cell inherent predisposition to promote the transcription of typical Th2 cytokines, so providing a mechanistic explanation for the more severe atopic dermatitis phenotype seen in the Shb knockout.

In conclusion, the loss of Shb expression disrupts TCR signalling, resulting in aberrant Th cell differentiation and development of exacerbated atopic dermatitis symptoms. The knowledge of how TCR signalling contributes to Th cell differentiation is still limited and mostly based on in vitro experiments. Further evaluation of Shb activity in human allergic subjects could therefore provide useful insights into the qualitative and quantitative differences in TCR signal transduction that underlies the development of allergic disorders.

Acknowledgments

KG contributed to study conception and design, data collection, data analysis and interpretation, and manuscript writing. EW collected data. MW contributed to conception and design, data analysis and interpretation, manuscript writing and financial support. The study was supported by the Swedish Cancer Foundation (120831), Swedish Research Council (54x-10822), Swedish Diabetes fund (DIA 2012) and Family Ernfors fund.

Glossary

Ig

immunoglobulin

IL

interleukin

IL4R

interleukin-4 receptor

TCR

T cell receptor

TSLP

thymic stromal lymphopoietin

Shb

Src homology domain protein B

VEGF-A

vascular endothelial growth factor-A

VEGFR-2

vascular endothelial growth factor receptor-2

Disclosures

The authors declare no financial or commercial conflict of interest.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1

List of primers and Tm used for the real-time RT-PCR.

imm0143-0033-sd1.docx (46.9KB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1

List of primers and Tm used for the real-time RT-PCR.

imm0143-0033-sd1.docx (46.9KB, docx)

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