VCAM-1 blockade delays disease onset, reduces disease severity and inflammatory cells in an atopic dermatitis model

Abstract

We investigated the functions of critical adhesion molecules ICAM-1 and VCAM-1 in a keratin-14 IL-4-transgenic (Tg) mouse model of atopic dermatitis, the skin lesions of which are characterized by prominent inflammatory cell infiltration, significantly increased mRNAs and proteins of ICAM-1, VCAM-1, E-selectin, P-selectin, L-selectin, and PSGL-1, and significantly increased numbers of dermal vessels expressing these adhesion molecules. We tested the hypotheses that deletion or blockade of these molecules may impede the inflammation by examining the disease progresses in the Tg mice crossed with ICAM-1-knockout mice and Tg mice received anti-VCAM-1-neutralizing antibody. Although the findings of the ICAM-1-knockout Tg mice (Tg/ICAM-1(-/-)) developed skin lesions similar to wide-type ICAM-1 Tg mice (Tg/ICAM-1(+/+)) were surprising, a compensatory mechanism may account for it: the frequency of VCAM-1 ligand, CD49d, on CD3(+) T cells in the lesional skin significantly increased in the Tg/ICAM-1(-/-) mouse, compared with the Tg/ICAM-1(+/+) mice. In contrast, anti-VCAM-1-treated Tg/ICAM-1(-/-) or Tg/ICAM-1(+/+) mice had significantly delayed onset of skin inflammation compared with isotype antibody-treated groups. Moreover, anti-VCAM-1 significantly reduced the skin inflammation severity in Tg/ICAM-1(+/+) mice, accompanied with reduction of mast cell, eosinophil, and CD3(+) T cell infiltration. VCAM-1 is more critical in developing skin inflammation in this model.

Figure 1

Upregulations of adhesion molecules in the skin of IL-4-Tg mice parallel the disease progression. (A) mRNA expressions: Total RNA from skin samples of Non-Tg, Tg-BO, Tg-EL, and Tg-LL mice were extracted and reverse transcripted to cDNA. Relative quantities of ICAM-1, VCAM-1, E-selectin, P-selectin, PSGL-1, and L-selectin mRNA expression were determined by quantitative real time PCR. (B) Protein expressions: Skin protein extracts were obtained from the skins of Non-Tg, Tg-BO, Tg-EL, and Tg-LL mice. After the SES-PAGE and nitrocellulose membrane protein transfer, ICAM-1 was detected by an anti ICAM-1 antibody followed by an Alexa Fluor 680-labeled secondary antibody. The membrane was then scanned by an Odyssey infrared scanner (Li-cor). *p<0.05 compared to non-Tg mice.

Figure 2

The increase of number of adhesion molecules-positive dermal blood vessels and inflammatory cells correlate with the disease progress. (A) Representatives of immunofluorescence photomicrographs of positively stained ICAM-1, VCAM-1, E-selectin, and P-selectin in the skin sections of Non-Tg, Tg-BO, Tg-EL and Tg-LL mice (Original magnification X100). (B) Bar graph presentations of the average number of positively stained vessels (per HPF). (C) Representatives of immunofluorescence photomicrographs of positively stained L-selectin and PSGL-1 in the skin sections of Non-Tg and Tg-LL mice (Original magnification X100). (D) Bar graph presentations of the average number of positively stained inflammatory cells for L-selectin and PSGL-1. * p<0.001 compared to non-Tg mice.

Figure 3

Generation and genotyping of Tg/ICAM-1 knockout mice. (A) Scheme for the generation of Tg/ICAM-1 knockout mice: IL-4-Tg mice were crossed with purchased homozygous ICAM-1−/− mice to produce heterozygous Tg/ICAM-1+/− and non-Tg/ICAM-1+/− mice. The subsequent cross between heterozygous Tg/ICAM-1+/− mice and heterozygous non-Tg/ICAM-1+/− mice produced homozygous Tg/ICAM-1−/− mice (our testing group), non-Tg/ICAM-1−/− mice (our negative control) and the other littermate control mouse groups (B) Genotyping of Tg/ICAM-1 KO mice: Genotyping of ICAM-1 mice (homozygous and heterozygous) was performed according to the instructions from the ICAM-1−/− mice vendor. Photo shows the results of ICAM-1 genotyping, ICAM-−/−, ICMA-1+/−, and ICAM-1+/+ (3 mice for each group) resulted from our crossing. C1: PCR product using control DNA for ICAM-1−/−, C2: PCR product using control for ICAM-1+/+ (wild type), C3: PCR product without DNA template (negative control).

Figure 4

Clinical phenotypes of IL-4-Tg mice with normal or deficient ICAM-1. (A) Representative clinical phenotypes of Tg/ICAM-1−/−, Tg/ICAM-1+/+, Tg/ICAM-1+/− (7 weeks after disease onset), and non-Tg/ICAM-1+/+ mice. (B) Comparison of skin inflammation severity scores of Tg/ICAM-1−/−, Tg/ICAM-1+/−, and Tg/ICAM-1+/+ mouse groups (7 weeks after disease onset. N=15 for each group), p>0.05 (among these three groups).

Figure 5

Upregulation of CD49d and CD24 on T cells in the lesional skin and skin-draining lymph nodes. Single cell suspensions from lesional skin (A) and skin draining lymph nodes (B) of Tg/ICAM-1−/− and Tg/ICAM-1+/+ mice were stained with FITC-CD3/PE-CD24/PE-CD49d for skin cells (A), and FITC-CD4/FITC-CD8/PE-CD49d/PE-CD24 for lymph node (B), then analyzed by flow cytometry. Results were the averages of three experiments. *p<0.01 compared to Tg/ICAM-1+/+ mice in A; # p<0.05 compared to Tg/ICAM-1+/+, or non-Tg/ICAM-1+/+, or non-Tg/ICAM-−/− mice in B.

Figure 6

VCAM-1 neutralizing antibody reduces skin inflammation in Tg ICAM-1+/+ not in Tg ICAM-1−/− mice. (A) Timeline of anti-VCAM-1 treatment. (B) Neutralizing antibody to VCAM-1 delays the onset of skin inflammation and reduces the severity of skin disease (C) in comparison to those mice treated by non-specific isotype control (* p<0.01, #p<0.05 compared to their isotype control antibody treated groups. (D) Representative clinical phenotypes of Tg/ICAM-1−/− and Tg/ICAM-1+/+ mice treated either with anti-VCAM-1 or isotype control. (E) Histopathology of Tg/ICAM-1+/+ mice treated either with anti-VCAM-1 or isotype control (Hematoxylin/eosin stain, Original magnification X100). (F) Mast cells and eosinophils in the dermis after anti-VCAM-1 or isotype control treatment in Tg/ICAM-1+/+ mice. N=5 for each group, *p<0.01 compared to IgG control groups). (G&H) CD3+ T cell infiltrates in Tg/ICAM-1+/+ mice treated either with anti-VCAM-1 or isotype control. * p<0.01 compared to IgG control groups (G, Original magnification X250). (I) mRNA expression of IL-1β, IL-6, IL-23 and CCL24 in the lesional skin of anti-VCAM-1 treated Tg/ICAM-1++ mice. * p<0.01 compared to IgG control groups. (J) Total serum IgE in anti-VCAM-1 treated Tg/ICAM-1++ mice. n=10 in each group, *p<0.05 compared to IgG control groups.