Interferon (IFN)-gamma is a main mediator of keratinocyte (HaCaT) apoptosis and contributes to autocrine IFN-gamma and tumour necrosis factor-alpha production.
BACKGROUND
Apoptosis of keratinocytes or intestinal epithelial cells is an important pathophysiological mechanism of organ damage during acute graft-versus-host disease.


OBJECTIVE
To analyse in detail the mediators and their mutual interaction leading to keratinocyte apoptosis.


METHODS
Experiments were performed using a keratinocyte cell line (HaCaT) and human skin explant cultures.


RESULTS
Supernatants (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLCs) induced apoptosis in HaCaT cells and also in keratinocytes from skin biopsies. Although both interferon (IFN)-gamma and Fas ligand (FasL) were detected in MLC-SN by enzyme-linked immunosorbent assay, the apoptosis-inducing capacity could be fully abrogated by neutralization of IFN-gamma, but not by neutralization of FasL. Recombinant (r) IFN-gamma induced HaCaT keratinocyte apoptosis in a dose- and time-dependent manner. Induction of HaCaT apoptosis by rFasL alone was induced only at higher doses than present in MLC-SN, but apoptosis was dramatically enhanced in the presence of rIFN-gamma. Further synergistic effects with IFN-gamma in the induction of apoptosis were also observed with agonistic antitumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 2 antibody, soluble TRAIL and TNF-alpha. However, in contrast to FasL and TRAIL, TNF-alpha alone did not induce HaCaT apoptosis. Interleukin-1beta and lipopolysaccharide did not enhance the apoptosis-inducing effect of IFN-gamma. Beside its apoptosis-inducing capacity in HaCaT cells, rIFN-gamma also induced autocrine IFN-gamma production, and combined treatment with IFN-gamma and TNF-alpha induced autocrine TNF-alpha production. Neutralization of autocrine IFN-gamma protected HaCaT cells from apoptosis.


CONCLUSIONS
Taken together, our data suggest a central role for IFN-gamma in HaCaT keratinocyte apoptosis but also show the importance of co-acting mediators such as TNF-alpha, TRAIL and FasL, which potentiate the effect of paracrine and autocrine IFN-gamma and TNF-alpha release.
Konur
A
A
Department of Haematology and Oncology, University of Regensburg, Germany. konur@3-med.klinik.uni-mainz.de
Schulz
U
U
Eissner
G
G
Andreesen
R
R
Holler
E
E
D017209:Apoptosis; D001323:Autoantibodies / Q000494:pharmacology; D019898:Autocrine Communication; D002460:Cell Line; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000494:pharmacology / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology; D019169:Jurkat Cells; D015603:Keratinocytes / Q000276:immunology / Q000473:pathology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D007959:Lymphocyte Culture Test, Mixed; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000276:immunology; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000276:immunology
D001323:Autoantibodies; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007375:Interleukin-1; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; C505639:TNFRSF10B protein, human; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma