Molecular and cellular evidence for T-cell stimulation by allogeneic retinal pigment epithelium cells in vitro.
OBJECTIVE
The rejection of retinal pigment epithelium (RPE) allografts is a major barrier to long-term success after retinal transplantation. The aim of this study was to investigate the action of RPE cells on allogeneic T cells in coculture with or without macrophages. For the detection of T-cell activation the interleukins IL-1beta and IL-2, typical for this process, were investigated.


METHODS
Human RPE cells (6 x 10(5) cells/flask) were used as stimulator cells. To investigate the influence of MHC class II molecules the RPE cells were pre-incubated with different concentrations of interferon-gamma (IFN-gamma; 0, 50, 100, or 250 U/ml) for 4 days. This was followed by coculture with either 6 x 10(6) T cells or, in a second trial, the T cells plus 6 x 10(5) macrophages. The mRNAs of the cytokines under study were detected using a reverse-transcriptase polymerase chain reaction and were quantified by colorimetry after 6 h. The cytokine protein content in the supernatants was measured after 20 h using specific enzyme-linked immunoabsorbent assays.


RESULTS
Cytokine-specific mRNAs and proteins were found in all samples. After coculture the level of IL-1beta mRNA was higher and that of cytokine-specific protein was significantly increased. Furthermore, the addition of macrophages led to increased cytokine secretion but a general influence of the pre-activation with interferon could not be found. Similar results were detected for IL-2; at the highest dose, IFN-gamma preactivation and, in combination with macrophages, a significant increase in the protein level could be found.


CONCLUSIONS
These results show that RPE cells are able to activate allogeneic T cells in vitro. Professional antigen-presenting cells may promote this process, as may pre-treatment with IFN-gamma. The circumstances modelled here are involved in the rejection process after RPE transplantation in humans and help to explain this immune response.
Enzmann
V
V
Department of Ophthalmology, University of Leipzig, Germany.
Hollborn
M
M
Wiedemann
P
P
Kohen
L
L
D000328:Adult; D000938:Antigen-Presenting Cells / Q000502:physiology; D018920:Coculture Techniques; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000502:physiology*; D008264:Macrophages / Q000502:physiology; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000502:physiology*
D016207:Cytokines; D012333:RNA, Messenger; D011994:Recombinant Proteins; D007371:Interferon-gamma