Potential role of chemerin in recruitment of plasmacytoid dendritic cells to diseased skin

Abstract

Interferon alpha-producing plasmacytoid dendritic cells (pDC) are crucial contributors to pro-inflammatory or tolerogenic immune responses and are important in autoimmune diseases such as psoriasis. pDC accumulate in the lesional skin of psoriasis patients, but are rarely found in the affected skin of patients with atopic dermatitis (AD). While homeostatic chemokine CXCL12 and inducible pro-inflammatory CXCR3 chemokine ligands may regulate pDC influx to psoriatic skin, the mechanism responsible for selective pDC recruitment in psoriasis vs. AD remains unknown. Circulating pDC from normal donors express a limited number of chemoattractant receptors, including CXCR3 and CMKLR1 (chemokine-like receptor 1). In this work, we demonstrate that circulating pDC from normal donors as well as psoriasis and AD patients express similar levels of CXCR3 and responded similarly in functional migration assays to CXCL10. We next found that blood pDC from normal, AD, and psoriasis patients express functional CMKLR1. In contrast to normal skin, however, lesional skin from psoriasis patients contains the active form of the CMKLR1 ligand chemerin. Furthermore, in affected skin from psoriatic patients the level of active chemerin was generally higher than in AD skin. Taken together, these results indicate that local generation of active chemerin may contribute to pDC recruitment to psoriatic skin.

Fig. 1. Circulating pDC from normal, psoriasis and AD patients express similar surface levels of CXCR3 and CMKLR1

PBMC were isolated from the blood of indicated donors and stained for pDC using anti-CD123 and anti-BDCA2 mAbs. The cells were then analyzed by flow cytometry. (a) Data are shown as percentage of pDC among PBMC. Lines indicate the mean value for each data set. Statistically significant differences between normal and psoriasis or AD pDC levels are indicated by asterisks (*, p<0.05; **, p<0.01; Student's t test). (b) Gated pDC were plotted for CXCR3 (left column, black histograms) or CMKLR1 (right column, black histograms). Thin open histograms represent profiles of negative control mAbs. Fluorescence intensity from one representative experiment out of five independent experiments is shown.

Fig. 2. pDC derived from the blood of normal donors as well as psoriasis and AD patients express functional CXCR3 and CMKLR1

PBMC (a) or enriched pDC (b) were tested in transwell chemotaxis, and the migrated cells were stained for CD123 and BDCA2. Migration was assessed to the following chemoattractants, as indicated: CXCL12 (10nM), CXCL10 (115nM) and chemerin/SspB (50pM). Migration to chemotaxis medium served as a negative control (Medium). A live cell gate based on forward and side light scatter was set, and then pDCs were identified based on CD123 and BDCA2 staining. (a) pDC migration is displayed as the mean of eight independent experiments ±S. D. Statistically significant differences in the migration to the negative control (chemotaxis medium) vs. various chemoattractants in pairwise comparisons within patient groups was determined by Student's t test (p<0.05) and indicated by “*”. (b) Migration of pDC (aprox. 90% pure) isolated from eight normal donors is displayed as the mean ± S. D. Statistically significant differences in the migration to CXCL12 vs. CXCL12+chemerin; CXCL12+CXCL10 vs. CXCL12+CXCL10+chemerin; or chemerin vs. chemerin+CXCL10 are indicated by asterisks (*, p<0.05; **, p<0.01; Student's t test).

Fig. 3. Chemerin protein is present in diseased skin

Heparin sepharose enriched plasma fractions (a) and skin-derived protein extracts (b) from the indicated donors were resolved by SDS-PAGE and immunoblotted for chemerin. The position of a molecular weight marker (29.2 kDa) is indicated. Arrows indicate full-length and truncated forms of recombinant chemerin (20 ng), used as a control.

Fig. 4. Chemotactically active chemerin is present in lesional skin of psoriasis patients

Skin-derived extracts containing 0.35 mg of protein were tested in a chemotaxis assay with CMKLR1/L1.2 cells (a) and either CMKLR1/L1.2 or L1.2 parental cells (b). Lines indicate the mean value for each data set. The difference between chemotactic responses to protein extracts derived from normal individuals vs. psoriasis patients in panel (a) was statistically significant as determined by Mann-Whitney test (p<0.05). The difference between chemotactic responses of CMKLR1/L1.2 vs. L1.2 cells to skin extracts derived from psoriasis patients (panel b) was statistically significant as determined by Student's t test (p<0.01).