Figure 4.

sFasL secreted by L. major-stimulated CL PBMCs induce apoptosis in Fas-expressing keratinocytes. a and b: Supernatants from L. major-stimulated CL PBMCs induced apoptosis in human keratinocytes. Keratinocytes (HaCaT) were incubated with medium alone (a) or supernatants from stimulated CL PBMCs for 40 hours. Annexin V/propidium iodide stainings were performed to assess apoptosis. Typically apoptosis (reflecting cell turnover in the culture) in control cultures was ∼15 to 20 cells/10 fields. The number of Annexin V+/propidium iodide− cells were evaluated with a ×25 fluorescence lens in a Nikon Optiphot 2 UV microscope equipped with suitable barrier filters. The results shown in b are apoptosis exceeding the turnover apoptosis. Annexin V-positive cells are seen in white. c: Levels of sFasL in supernatants from stimulated CL PBMCs influenced the level of apoptosis induced in keratinocytes. Supernatants from six different CL PBMCs were incubated with HaCaT. All samples were performed in duplicates. Two samples were repeated on two different occasions with similar results. There was a strong correlation between the levels of sFasL in the supernatants used (x axis) and the observed levels of apoptosis in keratinocytes (y axis) (r = 0.7, P < 0.001). sFasL was measured by enzyme-linked immunosorbent assay. d: Adding Fas-blocking antibodies (ZB4, open bars) to stimulated supernatants and HaCaT blocked keratinocyte apoptosis in two of three supernatants (CL12 and CL4) containing high levels of sFasL. In one case, CL3, apoptosis was not blocked. The positive control CH11 induced apoptosis at 1 μg/ml and was used as a positive control. CH11 was effectively blocked in the presence of ZB4 at 1 μg/ml. e: Individual data for the supernatants included in the HaCaT experiments. The expression of Fas or levels of apoptosis in restimulated PBMCs did not correlate with the levels of HaCaT apoptosis induced. CL5 was not included in the HaCaT experiments. The FasL staining in some of the donors were performed with an inefficient batch of FasL antibody and thus inconclusive (i).