29,30 The growth characteristics of human cutaneous KS are distinctive, with early lesions showing an infiltrative pattern by bland FXIIIaDD. as well as Ia antigen on exposure to interferon-. Cells so treated exhibit significant ability to present alloantigens in a manner that resembles Kaposis sarcoma, a condition known to involve proliferation of human dermal dendrocytes. Factor XIIIa-positive dermal dendrocytes (FXIIIaDD) are bone marrow-derived spindle cells that are diffusely distributed within the dermis of normal human skin. 1 Discovered in 1986, this distinctive cell type is usually individual from fibroblasts 2 and expresses the transglutaminase, coagulation Factor XIIIa, known to be important in crosslinking of fibrin and fibronectin. 3 The physiological function of FXIIIaDD has not been established definitively, although roles in wound healing and MHC class II-restricted antigen presentation have been proposed. 4-6 Nickoloff and Griffiths 7 have implicated these cells in the histogenesis and pathogenesis of AIDS-related Kaposis sarcoma (KS). KS is an infiltrative angioproliferative spindle cell tumor made up of, in addition to proliferating endothelium, numerous FXIIIaDD that, in this pathological setting, also may express the endothelial antigen, vascular cell adhesion molecule-1 (VCAM-1). 8 Experimental studies of the biology of FXIIIaDD and their potential role in KS have been limited by inability to obtain purified cultures of normal FXIIIaDD and paucity of animal models, CAB39L respectively. However, mouse skin recently has been shown to harbor cells analogous to human FXIIIaDD. 9 In addition, KS-like lesions may develop in murine skin of transgenic animals expressing genes that encode for human immunodeficiency virus (HIV)-associated Tat protein or KS-associated herpesvirus (HHV-8 K1). 10,11 The cell line 12E2 was originally cloned from a murine dermal cell line, DFB1, derived from spindle cells within the superficial dermis that adhered to enzymatically removed epidermal sheets. 12,13 This spatial localization directly beneath the epidermal layer coincides with a known subpopulation of FXIIIaDD, 4 and thus we hypothesized that this 12E2 clone may represent a pure population of murine FXIIIaDD, and as such, have biological relevance to the pathogenesis of KS. Our data indicate that 12E2 cells 1) express FXIIIa, VCAM-1, and inducible class II antigen, 2) produce mRNA for cytokines relevant to human dendritic cells and KS lesions, 3) express a glycoprotein receptor involved in antigen processing and present alloantigen similar to spindle cells in human KS. Materials and Methods Cell Culture The murine dermal cell clone, 12E2, was grown at 37C with 5% CO2 in RPMI 1640 made up of 10% heat-inactivated fetal bovine serum (Mediatech, Herndon, VA), 2 mmol/L L-glutamine (Life Technologies, Grand Island, NY), 0.1 mmol/L non-essential amino acids (Life Technologies), 1 mmol/L sodium pyruvate (Life Technologies), 10 mmol/L HEPES (Life Technologies), 1 g/ml indomethacin (Sigma Chemical, St. Louis, MO), 100 IU penicillin/100 g/ml streptomycin (Mediatech) and 5 10?5 M 2-mercaptoethanol (Sigma) as previously described. 13 Cells were routinely produced in T75 culture flasks and Dinoprost tromethamine subcultured using 0.025% trypsin/0.01% ethylene diaminetetraacetic acid in Hanks balanced salt solution (HBSS) at 37C for 5 to 7 minutes (Mediatech) at approximately 70 to 80% confluency. Murine fibroblast BALB/3T3 (clone A31), keratinocyte PAM212, and endothelial 3B-11 cell lines (ATCC, American Type Culture Collection, Manassas, VA) were grown according to ATCC protocols. In certain experiments, cells were exposed to recombinant murine interferon- (IFN-; PeproTech Inc, Rocky Hill, NJ) at 500 (12E2 and 3T3) or 5000 IU/ml (12E2) for 48 hours. 12E2 Growth Characteristics Doubling time was determined by plating 7.5 104 cells per T75 flask (1000 cells per cm2) in complete media. Cells were collected by trypsinization from triplicate flasks at 24, 48, 72, and 96 hours and viable cell number using trypan blue exclusion was decided with a hemacytometer. Doubling time was calculated from data generated during logarithmic phase of growth. Morphology Cultured cells were evaluated by phase contrast Dinoprost tromethamine microscopy and conventional transmission electron microscopy. injected cells were analyzed Dinoprost tromethamine via hematoxylin and eosin (H&E) staining of paraffin-embedded and/or frozen sections. Antibodies Antibodies for immunohistochemistry included 1:25 dilution of rabbit anti-human factor XIIIa and rabbit control (Biogenex, San Ramon, CA), 1:50 dilutions of rat monoclonal anti-murine CD31, CD34, Mac-1 (CD11b), VCAM-1 (CD106), isotype-matched irrelevant control (BD Pharmingen, San Diego, CA), and NLDC-145 antibody (rat monoclonal anti-DEC-205, Bachem Bioscience, King of Prussia, PA), 1:100 dilutions for rat monoclonal anti-murine I-A (b,d,q haplotypes) and Dinoprost tromethamine panendothelial antibodies (clone MECA-32), hamster monoclonal anti-murine ICAM (CD54), and hamster isotype-matched irrelevant control (BD Pharmingen); 1:50 dilution of rabbit anti-human factor XIIIa (Calbiochem, La Jolla, CA); and 1:25 dilution of mouse monoclonal anti-human CD31 (Dako, Carpinteria, CA). Immunohistochemistry 12E2 cells were analyzed using BD Falcon culture.
