UVB-irradiated T-cells undergoing apoptosis lose L-selectin by metalloprotese-mediated shedding.
OBJECTIVE
L-selectin (CD62L) is a prerequisite for leucocyte adhesion to endothelial cells of blood vessels and consequently for transmigration. Its expression on the cell surface therefore regulates the ability of lymphocytes to enter lymph nodes, to re-enter blood vessels or to invade tissues at sites of inflammation. The aim of this study was to determine the expression of CD62L on apoptotic lymphocytes after UVB irradiation.


METHODS
Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of normal healthy volunteers. Cells were stimulated with phorbol myristate acetate (PMA) and ionomycin for activation. Apoptosis in peripheral T-cells and Jurkat cells was induced by irradiation with UVB (120 mJ/cm2). In addition, T-cells or Jurkat cells were cultured for the indicated time with anti-Fas antibody CH11. The CH11-induced apoptosis was inhibited by the pan-caspase inhibitor zVAD-fmk. For detection of apoptosis, cells were analysed by cytofluorometry for morphological changes typical for apoptosis. The reliability of the apoptotic cell gate was confirmed by staining with FITC-labelled annexin-V in the presence ofpropidium iodide (PI). For FACS analysis of CD62L expression on the cell-surface immunofluorescence was performed using FITC-conjugated anti-CD62L and PE-conjugated anti-CD3 antibodies. Soluble CD62L (sCD62L) in the cell supernatants was measured by standard ELISA technique. Assays were performed in the presence and absence of metalloprotease inhibitor KB8301.


RESULTS
PBMC from healthy volunteers undergoing apoptosis following UVB irradiation selectively shed CD62L, whereas the expression of the lineage-specific marker CD3 showed only minor changes. Shedding was blocked by the hydroxamic acid-based metalloprotease inhibitor KB8301. When Jurkat cells were treated with the caspase inhibitor zVAD-fmk, anti-CD95 antibodies did not induce apoptosis, and the expression of CD62L remained unaltered.


CONCLUSIONS
UVB or ionizing radiation induce apoptosis in lymphocytes. The loss of CD62L is associated with apoptosis and will influence lymphocyte trafficking and, by excluding them from CD62L-mediated adhesion and tissue invasion, might contribute to the regulation of inflammation.
Kern
P M
PM
Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nurember, Germany. pete.kern@med3.med.uni-erlangen.de
Keilholz
L
L
Forster
C
C
Stach
C
C
Beyer
T D
TD
Gaipl
U S
US
Kalden
J R
JR
Hallmann
R
R
Herrmann
M
M
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000528:radiation effects*; D017252:CD3 Complex / Q000378:metabolism; D002448:Cell Adhesion; D002469:Cell Separation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D019041:L-Selectin / Q000096:biosynthesis / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000473:pathology / Q000528:radiation effects; D008666:Metalloendopeptidases / Q000378:metabolism*; D010641:Phenotype; D013601:T-Lymphocytes / Q000473:pathology / Q000528:radiation effects*; D013997:Time Factors; D014466:Ultraviolet Rays; D019014:fas Receptor / Q000276:immunology
D000590:Amino Acid Chloromethyl Ketones; D017304:Annexin A5; D017252:CD3 Complex; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D019041:L-Selectin; D008666:Metalloendopeptidases