Ahmad I. Rasheed - Wafaa I. Rasheed* - Maha A-W. Al-Shaarawi **

Egyptian Dermatology Online Journal 2 (2):5, December, 2006.

mailto:airasheed1@yahoo.com

* Department of Dermatology & Venereology - Ain-Shams University
* Department of Medical Biochemistry - National Research Centre
**Department of Pathology - Ain-Shams University

Accepted for publication in: June, 2006.

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Abstract

Background:

 The atopic reaction is mediated through 3 main types of cells; T-cells, APCs and keratinocytes. The role of these cells is orchestrated by complex interplay of adhesion molecules, cytokines and other inflammatory mediators.

Aim of the study:

was to semiquantitatively assess expression of some keratinocyte-derived chemokines in the epidermis of atopic skin (acute to subacute lesions) and to explore any differences in the degree and pattern of epidermal expression between intrinsic and extrinsic subsets of AD.

Material and methods :

 Twenty seven cases of atopic dermatitis (16 of the extrinsic and 11 of the intrinsic type) plus 14 control subjects were evaluated immunohistochemically for the epidermal expression pattern and intensity of some chemokines namely TSLP, GM-CSF, TARC, CTACK and RANTES and for LC density in the epidermis.

Results:

Enhanced expression of TSLP in the lesional epidermis could be seen in ADi group and significantly more in ADe. For GM-CSF, expression was significantly enhanced in the lesional epidermis of ADe only but not in ADi. Lesional skin of both ADi and ADe showed high TARC expression levels but significantly more in ADe than ADi. Expression of both CTACK and RANTES were faint in atopic epidermis and did not differ from that of normal skin. Decrease in the LC count was seen in the lesional skin of both groups but was significant only in the ADe group. The non-lesional atopic epidermis did not show significantly different expression patterns from normal epidermis except for TARC which was significantly enhanced in ADe but not in ADi.

Conclusion:

 The enhanced expression of TSLP, GM-CSF and TARC by atopic epidermis may play a role in the evolution of atopic lesions in general and in the characteristic T_{h 2}  polarization of the inflammatory reaction in particular. The difference in the expression pattern between both subgroups my play a role in determining the exact lineage of the atopic patient (i.e. intrinsic versus extrinsic). TSLP and GM-CSF are likely to augment LC population number and activity in the skin thus increasing the tendency of the skin to react to external allergens which is characteristic of ADe. On the other hand, higher levels of TSLP and TARC in ADe compared to ADi may be responsible for stronger T_{h 2} polarization of the inflammatory reaction. This may explain some reported differences between ADe and ADi at the tissue or serum levels.

Introduction and Aim of the Work:

Atopic dermatitis is a chronically relapsing eczematous skin disease resulting from complex interactions between genetic and environmental factors.[1] A large number of immunological and non-immunological abnormalities have been reported in AD patients whether in the skin or serum.[2,3]

While the primary defect seems to be genetically determined, many triggering factors have been reported including stress, external irritants, scratching and microbial agents. [4]

In spite of controversies as regards the exact pathophysiology of eczematous lesion (and even the exact type of immune reaction), three main types of cells have been confirmed to play the major role in the evolution of characteristic pathology of AD namely T-lymphocytes, antigen presenting cells (APCs) and keratinocytes.[5]

The role played by these effector cells is orchestrated by a growing list of cytokines (or chemokines), adhesion molecules and other inflammatory mediators that control trafficking and action of the inflammatory cells and subsequently determine the nature, extent and duration of the inflammatory reaction.[6]

In the last few years, more light has been shed on the role of keratinocytes in AD where a lot of constitutive and induced abnormalities have been demonstrated at the immunological (both innate and adaptive) and non- immunological levels (e.g. as regards barrier function and microbial colonization). Immunologically, the expression by epidermal cells of a large number of cytokines has been shown to be dysregulated in atopic skin in general or in lesional areas only. The actions of many of these cytokines could be of significance for the atopic reaction (e.g. for expression of adhesion molecules, recruitment, maturation, survival &/or activation of inflammatory cells especially APCs, determination of the polarity of the reaction and perpetuation of the inflammation).[5]

At the same time, the distinction between two subtypes of AD has been recently suggested which are the extrinsic type (ADe) and intrinsic type (ADi). The former type is mainly characterized by elevated serum IgE levels and polyvalent IgE sensitization against inhalant and/or food allergens in skin test or serum while in the intrinsic type, there is no specific IgE sensitization and total serum IgE is not elevated (i.e. <100 kU/L). Other distinctive features of intrinsic AD include later age of onset, mild female predominance, only mild to moderate eosinophilia, lower serum levels of IL-4 and of CD23+ B cells, lower density of tissue eosinophils, lower tissue expression of IL-1 ß , IL-5 and IL-13 as well as Fc RI/Fc RII expression ratio on the CD1a+ epidermal DCs of less than 0.5 in lesional skin. ADi is estimated to represent only 16-45% of all AD cases.[7,8] Although additional differences between these two types are continuously reported whether at the tissue or serum level,[8,9,10] many other aspects of the immune reaction have not yet been explored in a comparative way between both subtypes.

The lack of external allergy and of elevated serum IgE levels in the intrinsic group may point to some difference(s) in the evolution pathway between these two subtypes. The aim of the study was to explore other possible differences between ADi and ADe. The study has focused on the immunological role of keratinocytes and in-particular the expression of some keratinocyte-derived chemokines that could be relevant to the atopic reaction. 


 

Material and Methods
 

Twenty seven patients with typical atopic dermatitis (AD) and who attended the Dermatology Outpatient Clinic in Bou-Shahri Medical Centre – Kuwait, were randomly selected to participate in the study. Fourteen age-matched healthy individuals with no personal or family history of atopy were included as control . Informed consent was obtained from all patients (or their parents) and from control individuals.

The diagnosis of atopic dermatitis was based on the criteria described by Hanifin and Rajka.[11]

All the patients had not been treated with any systemic or topical drugs for at least 1 month before the beginning of the study.

To investigate whether the patients were ADi or ADe and apart from suggestive clinical data, blood samples from all subjects were tested for total serum IgE titre and eosinophil count.

Total serum IgE was measured using an ultrasensitive enzyme-linked immunoassay, IM x total IgE assay (Abbott Laboratories®, Abbott Park, IL, USA), based on microparticle enzyme immunoassay. The results were expressed in IU/mL –1 (1 IU/·mL = 2.4 µg of IgE/L). The minimum sensitivity of the assay was 0.048 IU/mL.

In addition, all AD patients were also checked in-vitro for ingestant- and inhalant-specific serum IgE levels using the multiple antigen simultaneous testing chemiluminescent assay (MAST-CLA) system (MAST Immunosystems, Mountain View, Calif., USA).

MAST-CLA In vitro testing : A three step technique was applied as described by Brown et al. (1985)[12] , in the form of overnight incubation of the serum with multiple allergens fixed on strands of cellulose, 4 hour incubation with enzyme labeled antibody and 30-minute chemiluminescent reaction, which produces a visible image (immunograph) on high-speed Polaroid instant film. The densities of the bands produced on the film are quantified with a microprocessor-controlled infrared transmittance densitometer. Scoring was performed according to the serum level of specific IgE as shown in table (1). A CLA class greater than or equal to 2+, corresponding to an IgE concentration equal to or more than 71 IU/mL (or KU/L), was considered significant.



 

Histopathologic and immunohistochemical evaluation:

From each patient a 5 mm-punch biopsy was obtained from both lesional and non-lesional skin, preserved immediately in 10% formalin and subsequently embedded in paraffin blocks. Four micron sections were prepared for subsequent hematoxylin & eosin (H&E) as well as for immunohistochemical staining. One biopsy of normal skin (at matching sites) has been also taken from each control subject.

Histopathologic examination of H&E stained sections was carried out under light microscopy to confirm the diagnosis and to record histopathologic features. The density of the dermal mononuclear infiltrate was given a score of 0 to 6 and the average score of four sections per biopsy was calculated.

(N.B. Only lesions that were judged clinically and confirmed histopathologically to be in the acute or subacute stage were subjected to immunohistochemical evaluation.).

Immunohistochemical Technique

i. Four-micron thick tissue sections cut from the representative paraffin-embedded tissue blocks, overlaid on APES (Sigma, St. Louis, USA) coated slides, were deparaffinized (2 changes of Xylene X 5 minutes each, 1 change of acetone X 1 min) followed by rehydration in decreasing ethanol concentrations (95% ethanol X 3 mins., 70% ethanol X 3 mins., distilled water X 1 min).

ii. For staining with all the antibodies, the tissue sections were then subjected to antigen unmasking by heating the sections immersed in 10 mmol. citrate buffer pH 6.0 (2.1 gm of anhydrous citric acid crystals dissolved in 1L of distilled water and pH adjusted to 6.0, if necessary) inside a 600 watt microwave oven in full power for 35 minutes, allowed to cool to room temperature and then washed briefly with 0.05 M Tris-Hcl buffer pH 7.4.

ii. Endogenous peroxidase activity was then quenched by immersing the sections in methanolic H 2 O 2 (1 part 3% H 2 O 2 plus 4 parts absolute methanol) for 30 minutes.  After brief rinsing, the sections were placed in 0.05 M Tris-Hcl buffer pH 7.4 for 10 minutes.

iii. Excess buffer was tapped off followed by a careful wipe around the specimen. Sections were then overlaid with adequate amount of primary antibody diluted optimally using 0.05 M Tris-Hcl buffer pH 7.4 containing 1% bovine serum albumin (Sigma, St. Louis, USA) followed by incubation at 4 0 C overnight.

iv. The slides were then washed with three changes (5 mins each) of 0.05 M Tris-Hcl buffer pH 7.4 followed by incubation for 30 minutes at room temperature after application of biotinylated secondary (link) antibody in phosphate buffered saline containing carrier protein and 15 mmol. sodium azide (LSAB Plus Kit, DAKO, Denmark).

v. After three washings (5 mins each) in Tris-Hcl buffer, peroxidase conjugated streptavidin was applied to cover the specimens and incubated at room temperature for 30 minutes.

vi. Slides were rinsed with 3 changes of Tris-Hcl buffer for 5 mins each. Sections were then covered with substrate chromogen solution prepared freshly by dissolving 1 mg of 3,3 ׳ – diaminobenzidine tetrahydrochloride (Sigma, St. Louis, USA) in 1 ml of 0.05 M Tris-Hcl buffer pH 7.4 containing 1 μl of hydrogen peroxide. The slides were incubated at room temperature for 5 to 10 minutes under microscopic control till the optimal development of brown colored peroxidase reaction product.

vii. After rinsing in distilled water, the sections were lightly counterstained with Harris’ hematoxylin, followed by mounting with cover slips with DPX as mounting medium.

viii. Precaution was taken so that drying of tissue sections strictly did not occur at any time during the entire procedure of immunostaining. All incubations were done inside humid chambers.

ix. Controls: During each batch of staining, positive and negative controls appropriate for the particular antibody were incorporated.

Table (2) shows the details of antibodies utilized in the study.
 

1ry Ab				2ry Ab
1ry Ab	Manufacturer	source	Working dilution	Manufacturer	source	Working dilution
Anti-TSLP	R&D**	sheep	2-15 μg/ml	Abcam*	Rabbit	0.7361111
Anti-TARC	R&D	goat	5-15 μg/ml	Abcam	Rabbit	0.7361111
Anti-CTACK	R&D	goat	15 μg/ml	Abcam	Rabbit	0.7361111
Anti-Rantes	R&D	goat	0.5-5 μg/ml	Abcam	Rabbit	0.7361111
Anti-GM-CSF	Abcam	mouse	5 μg/ml	Abcam	Rabbit	0.7361111
Anti-Langerin#	R&D	goat	2-15 μg/ml	Abcam	Rabbit	0.7361111

*ABCAM -  Cambridge – UK
**R&D Systems – Minneapolis – MN - USA
# As a marker of Langerhans cells. 13

Scoring method:

The scoring method for chemokines was modified from that described by Sinicrope et al. [14] Four sections per specimen have been examined for each marker evaluated in the study using a light microscope equipped with SIS Image Analysis Computer System. The mean percentage of positive tumor cells was determined in at least five fields per section (at 400-fold magnification) and assigned one of the following 8 categories: 0 = <5%; 1 = 5–12.5%; 2 = 12.5–25%; 3 = 25–37.5%;  4 = 37.5-50%, 5 = 50-67.5%, 6 = 67.5-75%, 7 = 75-87.5%, 8 = >87.5%. (N.B. the score refers to the percent of positively stained cells). The intensity of immunostaining was scored as follows: 1+, weak; 2+, moderate; and 3+, intense. Because the lesions showed heterogeneous staining, the dominant pattern was used for scoring. The scores indicating percentage of positive cells and staining intensity were multiplied to produce a weighted score for each field. The average weighted score for the 20 fields examined for each specimen (5 fields/section X 4 sections) is then calculated. Cases with weighted scores <1 were defined as negative, and cases were otherwise defined as positive.

For Langerin reactivity, only the cell with a nucleus and clear immunoreactivity was judged to be a positive cell. Positive cell count only was recorded.

Statistical analysis.

Comparative analysis between two groups was done through unpaired T-test using graphpad software downloaded from the website: http://www.graphpad.com/quickcalcs/ttest1.cfm [15]

Correlation between different parameters was done through graphpad software downloaded from the website: http://calculators.stat.ucla.edu/correlation.php [16]



 

Results
 

The study included 27 patients suffering from atopic dermatitis (acute to subacute stage) in addition to 14 healthy subjects of matched age and sex as control.

According to the results of laboratory and skin tests, the patients have been divided into 2 subcategories; extrinsic group (16 patients) and intrinsic group (11 patients)

Clinical data of the material of the study and of the biopsied lesions are shown in table (3)
 

Results of laboratory tests are shown in tables (4&5).
 

All biopsied lesions were judged clinically and confirmed histopathologically to be in the acute to subacute stage. The density of dermal mononuclear infiltrate were not significantly different between the two subsets of patients. (table 6).
 

Immunohistochemical examination revealed significantly increased immunoreactivity for TSLP in lesional epidermis of atopic patients compared to control (p<0.001) The expression was also significantly higher in ADe lesional epidermis compared to ADi lesional epidermis (p<0.05) Non-lesional atopic epidermis  showed only occasional staining for TSLP which was, however, not significantly different in either subgroup from normal epidermis (p>0.05).

The staining was frequently seen throughout the lesional epidermis, or only confined to the lower or upper parts of epidermis.

For TARC, Significantly increased immunoreactivity could be seen in atopic epidermis compared to normal epidermis which only occasionally showed very faint focal staining (p<0.001) Non-lesional atopic epidermis showed also significantly enhanced staining for TARC compared to normal skin (p<0.001) but less than that of lesional skin. When evaluated separately, ADe lesional epidermis showed higher reactivity than that of ADi skin (p<0.001) and both were significantly higher than normal skin (p<0.001). Non-lesional epidermis showed also staining for TARC in ADe group that was significantly higher than ADi (p<0.001) and only staining in the ADe group was significantly different from control epidermis (p<0.001).

For GM-CSF, significantly increased expression could be seen in lesional epidermis of ADe only compared to control (p<0.001). Reactivity was significantly higher in ADe than ADi. (p<0.001) Non-lesional epidermis showed only faint staining that was not significantly different in either subgroup from control.

Immunoreactivity for RANTES and CTACK in both AD subgroups was not prominent and only occasional and focal. Overall score for each of them was not significantly different from that of control whether at the level of lesional or non-lesional epidermis (p>0.05).

The average langerin-positive cell density in the epidermis was lower in AD patients than in control epidermis but when the two subgroups were evaluated separately the difference was significant only in lesional ADe epidermis (p<0.05).

Strongly positive correlation could be only seen between TSLP and TARC immunoreactivity of lesional skin (π =0.836). No other significant correlations could be found between any two parameters evaluated in the study.

Results of immunohistochemical examination are shown in tables (7 to 11) and figs (1 to 3) .