Novel functional multiparameter flow cytometric assay to characterize proliferation in skin

Abstract

Keratins are a group of cytoskeletal proteins that are found in human epidermis and other stratified squamous epithelia. Several different types of keratins 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 differentiation and hyperproliferation are its hallmarks. In order to study the hyperproliferation associated keratinization in both well differentiated and poorly differentiated keratinocytes, and in order to assess the proliferative activity of all K10 and K6 subpopulations, simultaneous assessment of K6, K10, 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 simultaneous measurement of K6, K10, and DNA content, which enables the characterization of the proliferative activity of several cellular subpopulations in epidermis. From 16 patients with psoriasis and from 15 healthy volunteers, punch biopsies were obtained. After preparation of single cell suspensions, cells were stained with the anti-keratin 10 IgG(1)-isotype monoclonal antibody RKSE60, with the anti-keratin 6 IgG(2a)-isotype monoclonal antibody LHK6B, and with the DNA fluorochrome TO-PRO-3 iodide. Isotype specific secondary antibodies conjugated with phycoerythrein (PE) and fluorescein isothiocyanate (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 from both psoriatic patients and healthy volunteers were than measured. Owing to the IgG specificity of RKSE60 and LHK6B, no cross-reactivity was observed between these antibodies. The triple staining with RKSE60, LHK6B, and TO-PRO-3 iodide showed subpopulations of K10 expressing cells, K10/K6 co-expressing cells, and K6 only expressing cells. There was a significant difference in the proportion of K6 expression and K10/K6 co-expression between psoriatic and normal skin. Moreover, the proliferative activity of these subpopulations could be quantified by this protocol. We concluded that a triple staining protocol for the assessment of K6, K10, and DNA content, using the monoclonal antibodies LHK6B, RKSE60, and TO-PRO-3 iodide, supplies reliable and reproducible data for cellular studies on these keratins and for studying the proliferative activity of the subpopulations of these keratins in epidermis. Moreover, the present study showed that with respect to the proportion of K6, significant differences are present between psoriatic and healthy human skin.

DRAQ5‐based DNA content analysis of hematolymphoid cell subpopulations discriminated by surface antigens and light scatter properties

“…Moreover, although DRAQ5 fluorescence is optimally excited at 647 nm, it is sufficiently energized at 488 nm, and is thus suitable for excitation with argon-ion lasers that are found in most clinical instruments. Because no fixation or permeabilization is needed, DRAQ5 is technically advantageous over other dyes that do require these extra steps, such as PI, Topro-3, and 7-AAD (4,7,8,15), which have previously been used in multiparameter DNA and surface antigen analysis. Hoechst 33342 is a DNAbinding fluorochrome that can be used without cell permeabilization since it enters viable cells by active uptake FIG.…”

“…For example, three-color analysis with the DNA binding dye 7-amino-actinomycin D (7-AAD), in conjunction with FITC-and PE-conjugated antibodies, has been performed with the efficient use of a single 488 nm laser (7). Additionally, 3-color (8) and 4-color analysis using TO-PRO-3 iodide has been reported, with minimal emission into the green, orange, and deep red fluorescence detectors (8).…”

Monitoring hyperproliferative disorders in human skin: Flow cytometry of changing cytokeratin expression

“…The K10 ϩ K6 Ϫ cells represent the terminally differentiated keratinocytes, and an increase in this subpopulation of keratinocytes implies a normalization of keratinization in the psoriatic lesion. The K10 Ϫ K6 Ϫ cells correspond to the basal keratinocytes, and proliferative activity is believed to take place in this compartment (7,18). However, assessment of the cells in the S/G 2 /M phase of the cell cycle did not show statistically significant changes in this compartment.…”

“…Earlier work from our group resulted in multiparameter flow cytometric assays, which enabled the assessment of various aspects of epidermal proliferation and differentiation in psoriasis and allowed monitoring of the effect of antipsoriatic therapies (5)(6)(7)(8).…”

“…Van Hooijdonk et al (9) and later Glade et al (5) developed a flow cytometric triple-parameter method by using markers for K10 (RKSE60), vimentin for non-keratinocytes (mesenchymal cells including inflammatory cells; VIM 3B4), and DNA content (TO-PRO-3 iodide). To define further keratinization associated with hyperproliferation, Mommers et al replaced the inflammation marker anti-vimentin with the hyperproliferation marker anti-K6 (7). However, by using this method, additional information supplied by the inflammation marker anti-vimentin was lost.…”