Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin

Abstract

Stromal-cell derived factor or SDF-1 is a CXC chemokine constitutively expressed by stromal bone marrow cell cultures that binds to the G-protein-coupled receptor CXCR4. SDF-1/CXCR4 represents a unique, nonpromiscuous ligand/receptor pair that plays an essential role in prenatal myelo- and lymphopoiesis as well as in cardiovascular and neural development. SDF-1 prevents entry of CXCR4-dependent (X4) HIV viruses in T lymphocytes, by binding and internalizing CXCR4. The expression pattern of SDF-1 protein in normal tissues is not known. Here we describe an analysis of SDF-1 mRNA and protein in normal and inflamed skin by in situ hybridization and immunohistochemistry, using a novel anti-SDF-1 monoclonal antibody. We also describe the expression pattern of CXCR4 receptor by immunohistochemistry. Our results show that SDF-1 protein and mRNA are normally expressed by endothelial cells, pericytes, and either resident or explanted CD1a+ dendritic cells. Epithelial cells of sweat glands but not keratinocytes also express SDF-1. In various inflammatory skin diseases, a large number of mononuclear cells and fibroblasts in close contact with CXCR4-positive lymphocytic infiltrates also express SDF-1. CXCR4 was also detected in many different normal cell types, including endothelial and epithelial cells, which points to a role for SDF-1/CXCR4 cell signaling in vascular and epithelial homeostasis. The demonstration of SDF-1 expression in dendritic and endothelial cells provides new insights into the mechanisms of normal and pathological lymphocyte circulation and makes it possible to envisage a role for locally secreted SDF-1 in the selective incapacity of mucosal dendritic cells to support and propagate infection by X4 HIV isolates.

Figure 1.

Expression and detection of SDF-1 gene products in mammalian cells by the K15C MAb. a: TIGEF cells were transfected with pCDNA3, SDF-1α, or SDF-1β. Forty-eight hours after transfection cell supernatants (pcDNA, SDF-1α, or SDF-1β) were collected, and their ability to promote CXCR4 down-regulation from the surface of lymphoblastoid Jurkat T cells was assessed. Supernatants from untransfected TIGEF cell cultures (Untr.) were used as controls. The expression of CXCR4 was analyzed by cytofluorometry, using the CXCR4-antibody 12G5 or an isotype-matched mouse immunoglobulin (Ctrl). b: TIGEF cells were transfected as indicated above and treated or not for the last 4 hours in culture with Brefeldine A (Bref. A). Cells were collected 48 hours after transfection, and cytoplasmic proteins (60 μg) were analyzed by Western blot with the K15C antibody. The ability of the K15C mAb to recognize TIGEF-expressed or synthetic (100, 50, or 25 ng) SDF-1 isoforms was compared.

Figure 2.

Detection by confocal microscopy of the intracellular accumulation of SDF-1β by the K15C mAb. TIGEF cells cotransfected with SDF-1β (or an insertless pcDNA3 plasmid) and GFP (green) expression vectors were incubated with K15C mAb and a secondary antibody conjugated to cye-3 (red). Cultures were treated with Brefeldine A. A and B: Cotransfection of SDF-1β and GFP expression vectors. C and D: Cotransfection of insertless pcDNA3 and a GFP expression vector. A and C: Simultaneous detection of GFP (green) and SDF-1β (red). B and D: Detection of SDF-1β (red).

Figure 3.

SDF-1 immunostaining in normal skin. A: Epidermal scattered DC were immunostained by anti-SDF-1 K15C MAb (arrow). B: A parallel section shows the identical morphology and distribution of SDF-1-positive and CD1a-positive epidermal DC (Langerhans cells). C: Dermis also contains scattered SDF-1 immunostained DC (arrow). EC and pericytes of dermal capillary vessels (arrowhead) were also immunostained by anti-SDF-1 MAb.D: Both EC (arrow) and pericytes (arrowhead) of small capillary vessels were immunostained. E: EC of larger vessels displayed weak immunostaining with anti-SDF-1 MAb. F: Epithelial cells of sweat glands showed strong SDF-1 immunostaining (arrow). Sweat gland immunostaining was not uniform, and there were unstained areas (arrowhead).

Figure 4.

SDF-1 mRNA expression by ISH in normal skin. A: SDF-1 sense riboprobes did not produce any signal above background (arrowheads mark the dermoepidermal union). B: SDF-1 mRNA expression was detected in epidermal Langerhans cells (marked by arrows). C: Both small capillary vessels (arrow) and larger blood vessels (arrowhead) displayed EC labeling. D: A small capillary vessel with higher magnification shows labeling of both EC (arrow) and pericytes (arrowhead). E: Sweat gland epithelial cells showed uneven mRNA SDF-1 labeling, with alternating labeled (arrow) and unlabeled (arrowhead) areas.

Figure 5.

Expression of SDF-1 and CXCR4 in purified CD1a+ DC explanted from skin. Cytofluorometric analysis of CD1a+ cells labeled with the anti-SDF K15C mAb, 12G5 anti-CXCR4 mAb, or isotype-matched immunoglobulin (control). SDF-1 was only detected in permeabilized cells. CXCR4 was present at low levels on the surface of nonpermeabilized cells, and its detection increased in permeabilized cells.

Figure 6.

Detection of SDF-1 mRNA in CD1a+ DC explanted from the skin. Foreskin fibroblasts and PBMC were used, respectively, as sources of positive and negative controls for the expression of SDF-1 gene. cDNAs were generated by reverse transcription (RT) of total RNA and amplified by PCR. Aliquots of each sample were similarly processed for the expression of β-actin, which was used as a cDNA loading control. To rule out DNA contamination and verify the specificity of the PCR-amplified signal, addition of reverse transcriptase was omitted where indicated (RT).

Figure 7.

SDF-1 and CXCR4 immunostaining in inflammatory infiltrates from skin autoimmune diseases. A, C: SDF-1 immunostaining is present in a large number of fibroblasts (arrows) and large mononuclear cells (arrowheads) but not in small lymphocytes of inflammatory infiltrates. B, D: Virtually all lymphocytes and mononuclear cells within inflammatory infiltrates were CXCR4 positive. A, C: Cutaneous lupus. B, D: Dermatomyositis.

Figure 8.

CXCR4 immunostaining in normal skin. A: Normal epidermal keratinocytes were uniformly stained by anti-CXCR4 antibodies. EC and pericytes (arrowhead) and dermal fibroblasts (arrow) were also immunostained. B: Normal sweat gland epithelial cells were strongly and uniformly immunostained. C: Larger vessels also showed strong CXCR4 staining (arrowhead). Cells with dendritic (arrow) and fibroblastic morphology around blood vessels are also immunostained.