title: Cytokine-mediated effects of peripheral blood mononuclear cells from patients with atopic eczema on keratinocytes (HaCaT) in a new coculture system.
abstract: Interactions between keratinocytes and mononuclear cells via cytokines and adhesion molecules are thought to play a crucial part in inflammatory skin diseases. The cytokine-mediated effects of peripheral blood mononuclear cells (PBMC) from patients with atopic eczema (AE) and healthy individuals on keratinocytes (HaCaT) were investigated in vitro. A new coculture model (Transwell system) which consists of a lower and an upper compartment, which are separated by a polycarbonate-treated membrane, was established. 3[H]thymidine incorporation of keratinocytes and lymphocytes, as well as IL-6, IL-8 and IFN-gamma synthesis, were measured. Keratinocyte proliferation was significantly enhanced in the presence of PBMC from patients with AE. In contrast, PBMC from normal donors did not enhance HaCaT cell proliferation when they were cocultured. Lymphocytes from patients with AE showed a significantly enhanced proliferation after coculture with keratinocytes. However, PBMC from normal donors did not proliferate in the presence of HaCaT cells. Keratinocyte supernatants incubated with PBMC from either atopic or normal volunteers induced a suppression of lymphocyte 3[H]thymidine incorporation. In supernatants from cocultures of PBMC from patients with AE and keratinocytes, significantly enhanced amounts of IL-6 and IL-8, compared with normal donor's lymphocytes and HaCaT cells, were measured. No differences in IFN gamma production were observed. When PBMC were cultured without HaCaT cells, supernatants contained equal levels of IL-6, IL-8 and IFN-gamma in normal donors and in patients with AE. Interestingly, HaCaT cells spontaneously secrete measurable amounts of IL-6, IL-8 and IFN-gamma. Blocking experiments with neutralizing antibodies against these interleukins showed a complete inhibition of keratinocyte proliferation when PBMC from normal donors were used whereas the proliferative potency of PBMC supernatants from patients with AE on keratinocytes remained. Our data indicate that (i) PBMC from patients with AE stimulate keratinocyte proliferation via soluble factor(s) that are different from IL-6, IL-8 and IFN-gamma; (ii) probably, HaCaT cells spontaneously produce lymphocyte/monocyte inhibitory soluble factors and IL-6, IL-8 as well as IFN-gamma; and (iii) secretion and/or activity of keratinocyte-derived inhibitory mediators is regulated via cytokines of PBMC infiltrating inflammatory skin.
authors.affiliation: Department of Dermatology and Allergology, University Hospital Hamburg-Eppendorf, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Neuber
authors.forename: K
authors.initials: K
authors.lastname: Steinrücke
authors.forename: L
authors.initials: L
authors.lastname: Kowalzick
authors.forename: I
authors.initials: I
authors.lastname: Köhler
authors.forename: J
authors.initials: J
authors.lastname: Ring
mesh_terms: D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018929:Cell Culture Techniques; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism*; D003876:Dermatitis, Atopic / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D016207:Cytokines; D015850:Interleukin-6; D016209:Interleukin-8; D007371:Interferon-gamma; D013936:Thymidine