Dear Vengadeshprabhu,

Attached please find a paper which covers the answer to your question.

I have copied some important text, see below.

Abstract

We have examined the expression of chemokines and their receptors in the atopic dermatitis–like (AD-like) lesions of NC/Nga mice. Such lesions develop when the mice are kept in conventional conditions, but not when they are kept isolated from specific pathogens. The thymus- and activation-regulated chemokine TARC is unexpectedly highly expressed in the basal epidermis of 14-week-old mice with lesions, whereas it is not expressed in the skin without lesions. Production of TARC by keratinocytes was confirmed by culturing murine keratinocytic cell line cells (PAM212) with TNF-α, IFN-γ, or IL-1β. Expression of another Th2 chemokine, macrophage-derived chemokine (MDC), was observed in the skin from mice kept in both conventional and pathogen-free conditions, but expression of MDC was increased severalfold in the skin with lesions. The cellular origin of MDC was identified to be dermal dendritic cells. Infiltration of the skin by IL-4–producing T cells and mast cells, and the increase of CCR4 mRNA in the skin, coincided with the development of AD lesions. These observations indicate that TARC and MDC actively participate in the pathogenesis of AD-like lesions in NC/Nga mice and that these Th2 chemokines could be novel targets for intervention therapy of AD in humans.

Methods

Mice.

SPF male and female, 8-week-old, NC/Nga mice were purchased from SLC (Tokyo, Japan). These were divided into 2 groups, one group kept in SPF environment and the other under conventional conditions. In each of the 2 groups, the mice were mated and the offspring were used for experiments. All animal experiments complied with the standards set out in the guidelines of the University of Tokyo.

Steroid treatment.

Mice kept in normal conditions with fully developed skin lesions were treated with steroid (0.05% clobetasolepropionate) ointment (Glaxo, Tokyo, Japan), by applying the ointment to the involved region from the ears to the middle of the back. The treatment was repeated once every day for 7 days from the age of 14 weeks. As a negative control, mice of the same age with the same lesions were treated with vehicle (petroleum jelly alba) alone.

Skin biopsies.

The mice were sacrificed by dislocation of the neck, upon which the hairs of the back had been removed using depilator cream (Kanebo, Tokyo, Japan). An area of approximately 1.5 × 1.5 cm2 was excised, and the subcutaneous fat and blood vessels were carefully dissected away.

Histology.

Skin biopsies were fixed with 10% formalin in neutral buffer for at least 16 hours and embedded in paraffin. Deparaffinized sections (3–5 μm thick) were stained with hematoxylin and eosin and analyzed by light microscopy.

Preparation of antibodies against MDC.

A 21 amino acid–long MDC peptide from amino acids 38–58 was synthesized (PerSeptive Biosystems/Vestec Products, Tokyo, Japan) and coupled to keyhole limpet hemocyanin (KLH; Calbiochem-Novabiochem Corp., San Diego, California, USA) using maleimidobenzoyl-N-hydroxysuccinimideester (MB; Pierce Chemical Co., Rockford, Illinois, USA). The amino acid sequence of the fragment was QDYIRHPLPSRLVREPPWTS. The resulting KLH-MB domain–fused protein was isolated using Sephadyl S-200HR column (Pharmacia Biotech AB, Uppsala, Sweden), after which the fused protein was emulsified in CFA and used for immunization of 2 New Zealand white rabbits after 3-, 2-, 2-, and 1-week intervals. The rabbits were bled and sera obtained, and the resultant antibody specificity was tested in an ELISA. No cross-reactivity was found with other murine chemokines, including TARC, JE, and macrophage inflammatory protein-2 (MIP-2).

Immunohistochemistry.

Skin biopsies were embedded in Tissue-Tek OCT compound (Miles Inc., Elkhart, Indiana, USA), snap-frozen in liquid nitrogen, and stored at –80°C until use. The tissue was cut by a cryostat to 8-μm sections and was thereafter incubated with the first antibody. The antibodies used were rabbit anti-mouse MDC polyantibody, a rat anti-mouse CD4 mAb (RM4-5, PharMingen, San Diego, California, USA), a rat anti-mouse CD8 mAb (53-6.7; PharMingen), a rat anti-mouse DEC-205 mAb (NLDC-145; BMA Biomedicals, Geneva, Switzerland), a rat anti-mouse MHC II (ER-TR3; BMA Biomedicals), a rat anti-mouse c-kit mAb (ACK45; PharMingen), a rat anti-mouse IL-4 mAb (BVD6-24G2; PharMingen), a rat anti-mouse LOM 14 mAb (30), and a hamster anti-mouse TARC mAb (31). As negative control, a rat IgG, a hamster IgG, or a rabbit IgG was used. After staining with the first antibodies, the samples were incubated with either a horseradish peroxidase–conjugated (HRP-conjugated) goat anti-hamster IgG (Southern Biotechnology Associates Inc., Birmingham, Alabama, USA), an HRP-conjugated goat anti-rat IgG (BioSource International, Camarmillo, California, USA), or an alkaline phosphatase–conjugated donkey anti-rat IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, Pennsylvania, USA). The reaction was viewed with either an AEC Substrate Kit for peroxidase or a Vector Red Alkaline Phosphatase Substrate Kit I (both from Vector Laboratories, Burlingame, California, USA).

The double immunostaining was performed as described by Matsuno et al. (32). Acetone-fixed 6-μm frozen sections were incubated with the first rat or rabbit antibodies for 1 hour. After sequential incubation with alkaline phosphatase–conjugated anti-rat or rabbit IgG for 45 minutes, labeled cells were colored red or blue with Vector Red or Vector Blue (Vector Laboratories). Samples were then incubated with the second rat IgG, and bound mAb’s were detected with an HRP-labeled anti-rat IgG and colored brown or red with 3,3′-diaminobenzidine (DAB; Wako Chemicals, Dallas, Texas, USA) substrate solution or Vector Nova Red. Slides were counterstained with Mayer’s hematoxylin.

Expression of chemokines, cytokines, and chemokine receptors in the skin biopsies.

Total RNA was isolated from the skin biopsies using RNAzol (Biotecx Laboratories, Houston, Texas, USA) according to the manufacturer’s instruction, and reverse-transcribed into DNA using M-MLV RT 200 U/20 μL reaction volume (Sawaday, Tokyo, Japan), RNase inhibitor 40 U/20 μL reaction volume (Sawaday), random primers (Promega Corp., Madison, Wisconsin, USA) 100 μg/20 μL reaction volume, and 3 μg RNA, according to the manufacturers’ instructions. One microliter of the resulting cDNA was amplified by PCR, using AmpliTaq in the buffer with MgCl2 (Perkin-Elmer Corp., Norwalk, Connecticut, USA; Roche Molecular Systems, Branchburg, New Jersey, USA). The sense primer for TARC was 5′-CAGGAAGTTGGTGAGCTGGTATA-3′, and the antisense primer was 5′-TTGTGTTCGCCTGTAGTGCATA-3′. The sense primer for MDC was 5′-TCTGATGCAGGTCCCTATGGT-3′, and the antisense primer was 5′-TTATGGAGTAGCTTCTTCAC-3′. The sense primer for IL-4 was 5′-CAGCTAGTTGTCATCCTGCTCTTC-3′, and the antisense primer was 5′-GCCGATGATCTCTCTCAAGTGA-3′. The sense primer for IFN-γ was 5′-CTCAAGTGGCATAGATGT-3′, and the antisense primer was 5′-GAGATAATCTGGCTCTGCAGGATT-3′. The sense primer for eotaxin was 5′-AGAGCTCACAGCGCTTCTATT-3′, and the antisense primer was 5′-GGTGCATCTGTTGTTGGTGATT-3′. The sense primer for CCR3 was 5′-TTGCAGGACTGGCAGCATT-3′, and the antisense primer was 5′-CCATAACGAGGAGAGGAAGAGCTA-3′. The sense primer for CCR4 was 5′-TCTACAGCGGCATCTTCTTCAT-3′, and the antisense primer was 5′-CAGTACGTGTGGTTGTGCTCTG-3′. The sense primer for CCR5 was 5′-CATCGATTATGGTATGTCAGCACC-3′, and the antisense primer was 5′-CAGAATGGTAGTGTGAGCAGGAA-3′. The sense primer for CXCR3 was 5′-ATCAGCGCTTCAATGCCAC-3′, and the antisense primer was 5′-TGGCTTTCTCGACCACAGTT-3′. The sense primer for GAPDH was 5′-AGTATGACTCCACTCACGGCAA-3′, and the antisense primer was 5′-TCTCGCTCCTGGAAGATGGT-3′. The reaction products were viewed on a 3% polyacrylamide gel with ethidium-bromide.

Quantitative PCR.

To quantitate the mRNA in the skin of the aforementioned cytokines, chemokines, and chemokine receptors, quantitative PCR using the ABI 7700 sequencer detection system (Perkin-Elmer Applied Biosystems, Foster City, California, USA) was performed. The reaction mixture was prepared according to the manufacturer’s instructions to yield final concentrations of 1 × PCR buffer A; 200 μM dATP, dGTP, and dCTP each; 400 μM dUTP; 4 mM MgCl2; 1.25 U AmpliTaq Gold DNA polymerase; and 200 μM of each primer. To the separate reactions were also added the following target hybridization probes (100 μM) for specific detection. For MDC, the probe 5′-CCAATGTGGAAGACAGTATCTGCTGCCA-3′ was added. For CCR5, the probe 5′-TACCTGCTCAACCTGGCCATCTCTGA-3′ was added. For GAPDH, the probe 5′-AACGGCACAGTCAAGGCCGAGAAT-3′ was added. The probe was labeled with a reporter fluorescent dye, FAM (6-carboxyflourescein), at the 5′-end. The thermal cycling conditions were set to 50°C for 2 minutes and 95°C for 10 minutes, followed by 50 cycles of amplification at 95°C for 15 seconds and 55°C for 1.5 minutes for denaturing and annealing/extension, respectively. To compare mRNA expression, the results were compared as relative values using GAPDH as an internal reference and the samples from mice kept in SPF conditions as overall reference. The relative value was calculated after the following formula: relative expression = 2[–(ΣVs/Ns – ΣGAPDHs/Ns)-(ΣVr/Nr – ΣGAPDHr/Nr)], where Vs denotes the ABI Prism signal from a given sample, Ns is the number of samples, GAPDHs is the ABI Prism signal for GAPDH in the same sample, Vr is the ABI Prism signal for the same sample but in the overall reference sample, andGAPDHr is the ABI Prism GAPDH signal in the reference sample. The number of samples was n = 3 in each group.

Hoping this will be helpful,

Rafik