CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells

Abstract

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA(+) memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA(+) T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.

Figure 1

(a) Amino acid sequence alignment of mouse (m) and human (h) CTACK. (b) Amino acid sequences of CTACK aligned with other CC chemokines, hTECK, hMIP-3β, h6Ckine, and human regulated on activation normal T expressed and secreted (hRANTES). Dark-shaded boxes indicate identical amino acids. Conserved amino acids are shaded light gray. Arrow denotes the predicted signal peptide cleavage site. Asterisks are above conserved cysteines. (c) Mouse CTACK maps in the proximal region of mouse chromosome 4. CTACK was placed on mouse chromosome 4 by interspecific backcross analysis. The segregation patterns of CTACK and flanking genes in 98 backcross animals that were typed for all loci are shown at the top of the figure. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus) F₁ parent. The shaded boxes represent the presence of a C57BL/6J allele, and white boxes represent the presence of an M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 4 linkage map showing the location of CTACK in relation to linked genes is shown at the bottom of the figure with recombination distances between loci in centimorgans shown to the left and the positions of loci in human chromosomes shown to the right.

Figure 2

Expression of CTACK in skin. (a) Southern blot analysis of CTACK expression in cDNA libraries generated from human samples including: PBMC and PBMC-act, resting or activated (act), with anti-CD3 and phorbol 12-myristate 13-acetate (PMA); T cells (MOT72 and MOT72-act) resting or activated with anti-CD28 and anti-CD3; total splenocytes resting or activated with anti-CD40 and IL-4; pooled B cell lines, pooled natural killer (NK) cell clones, and U937 (premonocytic cell line), all resting or activated with PMA and ionomycin; elutriated monocytes activated with lipopolysaccharide (LPS), IFN-γ, and anti-IL-10; monocyte-derived dendritic cells (mono-derived DCs) resting or activated with either LPS or TNF-α plus IL-1α; CD34⁺ stem cell-derived dendritic cells (CD34⁺-DCs) resting or activated with PMA and ionomycin; MRC5 cells (lung fibroblast sarcoma); CHA cells (kidney epithelial carcinoma); A549 cells (lung epithelial) treated with IL-1β, adult organs and tissues, diseased and normal as indicated above each lane. (b) Northern blot analysis of CTACK expression in mouse tissues and organs, including liver, spleen, thymus, peripheral lymph nodes (PLN), epidermis of ear skin, and ear draining lymph nodes (Auricular LN). Ethidium bromide-stained 28S rRNA is shown as a loading control.

Figure 3

Expression of CTACK. (a) Frozen sections of mouse skin (ears) were immunostained with a panel of anti-CTACK mAbs. A representative mAb (isotype IgG2b) is shown and revealed predominant staining in the epidermis and scattered positive cells in the dermis. (b) A control rat mAb (IgG2b) is shown for comparison. (c) RT-PCR analysis of CTACK expression in primary keratinocytes, primary melanocytes, 7-17 cells (epidermal γδ T cell line), and primary dermal fibroblasts either untreated or treated with TNF-α and IL-1β for the indicated times.

Figure 4

CTACK preferentially attracts CLA⁺ memory T cells. (a) Migration (% of each phenotype added to the upper chamber) of CD4⁺ memory T cell populations in response to CTACK. (b) Migration of CD8⁺ memory T cell populations in response to CTACK. Chemotaxis to the optimal concentration of SDF-1α (5 × 10⁻⁸ M) is shown for comparison. Chemokinesis was measured by using the optimal chemotactic concentration of CTACK on both sides of the filter. Data from a representative experiment are shown. The composite mean and SD of the migration indices of seven independent experiments at the maximally effective concentration of CTACK for CD4⁺ memory cells: CLA⁺ = 10.14 ± 1.13 vs. CLA⁻ = 1.02 ± 0.19 (P < 2 × 10⁻⁵), and for CD8⁺ cells: CLA⁺ = 4.26 ± 1.93 vs. CLA⁻ = 1.07 ± 0.09 (P < 0.005). (c) CTACK does not attract CD4⁺ or CD8⁺ naive T cells, B cells, monocytes, or neutrophils. As positive controls, the following chemokines were used: 6Ckine (5 × 10⁻⁷) for naive T cells, SDF-1α (5 × 10⁻⁸ M) for B cells, monocyte chemoattractant protein-3 (1 × 10⁻⁹ M) for monocytes, and IL-8 (1 × 10⁻⁹ M) for neutrophils.