Novel functional multiparameter flow cytometric assay to characterize proliferation in skin

Keratins are a group of cytoskeletal proteins that are found in human epide rmis and other stratified squamous epithelia. Several different types of ke ratins have been described. Keratin 10 (K10) is a keratin that is expressed in well differentiated, suprabasal keratinocytes, and keratin 6 (K6) is a keratin which is associated with hyperproliferation. Psoriasis is a chronic inflammatory skin disease, and besides inflammation, disturbed differentia tion and hyperproliferation are its hallmarks. In order to study the hyperp roliferation associated keratinization in both well differentiated and poor ly differentiated keratinocytes, and in order to assess the proliferative a ctivity of all K10 and K6 subpopulations, simultaneous assessment of K6, K1 0,:and DNA content is required. So far, a triple staining protocol had not been available. In the present study, we established a novel protocol for s imultaneous measurement of K6, K10, and DNA content, which enables the char acterization of the proliferative activity of several cellular subpopulatio ns in epidermis. From 16 patients with psoriasis and from 15 healthy volunt eers,:punch biopsies were obtained. After preparation of single cell suspen sions, cells were stained with the anti-keratin 10 IgG(1)-isotype monoclona l antibody RKSE60, with the anti-keratin 6 IgG(2a)-isotype monoclonal antib ody LHK6B, and with the DNA fluorochrome TO-PRO-3 iodide. Isotype specific secondary antibodies conjugated with phycoerythrein (PE) and fluorescein is othiocyanate (FITC) were used as the second step in the staining procedure. Controls were measured omitting the primary antibodies, and gates were set :in order to differentiate between the K10 and K6 subpopulations. Samples f rom both psoriatic patients and healthy volunteers were than measured. Owin g to the IgG specificity of RKSE60 and LHK6B, no cross-reactivity was obser ved between these antibodies. The triple staining with RKSE60, LHK6B, and T O-PRO-3 iodide showed subpopulations of K10 expressing cells, K10/K6 cc-exp ressing cells, and K6 only expressing cells. There was a significant differ ence in the proportion of K6 expression and K10/K6 cc-expression between ps oriatic and normal skin. Moreover, the proliferative activity of these subp opulations could he quantified by this protocol. We concluded that a triple staining protocol for the assessment of K6, K10, and DNA content, using th e monoclonal antibodies LHK6B, RKSE60, and TO-PRO-3 iodide, supplies reliab le and reproducible data for cellular studies on these keratins and for stu dying the proliferative activity of the subpopulations of these keratins in epidermis. Moreover, the present study showed that with respect to the pro portion of K6, significant differences are present between psoriatic and he althy human skin. (C) 2000 Wiley-Liss, Inc.