Combined use of laser capture microdissection and cDNA microarray analysis identifies locally expressed disease-related genes in focal regions of psoriasis vulgaris skin lesions

Abstract

Psoriasis vulgaris is a complex disease characterized by alterations in growth and differentiation of epidermal keratinocytes, as well as a marked increase in leukocyte populations. Lesions are known to contain alterations in messenger RNAs encoding more than 1,000 products, but only a very small number of these transcripts has been localized to specific cell types or skin regions. In this study, we used laser capture microdissection (LCM) and gene array analysis to study the gene expression of cells in lesional epidermis (EPI) and dermis, compared with the corresponding non-lesional regions. Using this approach, we detected >1,800 differentially expressed gene products in the EPI or dermis of psoriasis lesions. These results established sets of genes that are differentially expressed between epidermal and dermal compartments, as well as between non-lesional and lesional psoriasis skin. One of our findings involved the local production of CCL19, a lymphoid-organizing chemokine, and its receptor CCR7 in psoriatic dermal aggregates, along with the presence of gene products LAMP3/DC-LAMP and CD83, which typify mature dendritic cells (DCs). Gene expression patterns obtained with LCM and microarray analysis along with T-cell and DC detection by immune staining suggest a possible mechanism for lymphoid organization via CCL19/CCR7 in diseased skin.

Figure 1. Immunohistochemical staining patterns were correlated with cDNA microarray data

(a) CCL27 was expressed in the basal layer of non-lesional epidermis. (b) CRIP1 was expressed in the granular layer of non-lesional epidermis. (c) STAT-1 stained in the cytosol of cells in non-lesional epidermis, whereas it stained in the nuclei of cells in lesional epidermis and dermis. (d-f) CCR7 and CCL19 were positive in dermal aggregates in lesional skin, whereas CCL21 was detected in neither non-lesional nor lesional skin. (g-h) CXCL13 (3 out of 5 samples) and CXCR5 (6 out of 8 samples) were detected in lesional dermis. (i) CD20+ cells (3 out of 5 samples) were also found in lesional dermis. Arrows indicate the positive layers and cells. Bar=100μm.

Figure 2. Characterization of lymphoid tissue like structures in lesional skin

(a) Both CCR7 and CCL19 were localized in dermal aggregates of the lesional skin. (b) LAMP3/DC-LAMP was co-expressed with CCR7. (c) CD3 was also co-expressed with CCR7. (d-f) CCL19 expression was detected on CD11c+ (d) and LAMP3/DC-LAMP+ DCs (e) as well as CD3+ T cells (f). We also stained CXCR5 in combination with CXCL13 and CD20. (g-h) Both CXCL13 and CXCR5 stained in dermal aggregates of lesional skin (g) and CXCR5 co-stained with CD20 (h). White arrows indicate the double positive cells. White lines represent epidermal dermal junctions. Bar=100μm.

Figure 3. Unique detection of DEGs by LCM samples compared to Bulk skin sample

(a-b) Scatterplots of Log2FCH of Bulk vs. EPI and RD. Black lines: identity lines; gray lines: ±2-FCH; red lines: robust linear regression estimates. (c) Percentage of probe-sets with larger absolute FCH in LCM samples than in Bulk. Pink bars: all probe-sets; red bars: probe-sets considering |FCH|>2.0 (d-e) Venn-Diagrams of Bulk, EPI and RD psoriasis transcriptome. (f-g) Proportion of unique EPI-, RD-, and Bulk-related probe-sets that appear in 1, 2 or 3 of the published Affymetrix studies (considering HU133A2.0 probe-sets) or in the NGS-transcriptome. The width of each bar depicts the number in each category. (h) RT-PCR fold changes of the top eight unconfirmed down-regulated genes using bulk tissue samples (n=9).