Figure 1.
Characterisation of peripheral blood leukocytes from healthy volunteers- I.
Representative dot plots for flow cytometric gating are shown for healthy male volunteers (n = 17). Blood was drawn from the forearm not bearing skin blisters. Erythrocytes were lysed and the remaining leukocytes incubated with antibodies and processed by flow cytometry. Gating strategies firstly identified CD3+ T cells, CD19+ B cells and CD56+ CD16+/− NK cells. The remaining lymphocyte-deplete population was gated into HLA-DR+ and HLA-DR− cells. Arrows indicate gating strategy.
Figure 2.
Characterisation of peripheral blood leukocytes from healthy volunteers- II.
HLA-DR+ and HLA-DR− cells identified in Figure 1 were further analysed. HLA-DR+ cells were characterised into CD14hi/CD16− , CD14hi/CD16+, CD14lo/CD16+ monocytes and CD14−/CD16− dendritic cells. HLA-DR− cells comprised of typical PMNs (CD16hi, CD66+) and a CD16lo population. On extended characterisation, HLA-DR−/CD16lo cells were identified as Siglec-8+ eosinophils and the HLA-DR+/CD14−/CD16− dendritic cells comprised of CD141+ and CD11c+ dendritic cell subpopulations. Arrows indicate gating strategy.
Figure 3.
Characterisation of inflammatory cell infiltrates into skin blisters at 24(onset) – I.
Representative dot plots for flow cytometric gating are shown for healthy male volunteers (n = 17). Blister contents were collected from one blister 24 h after application of cantharidin in 3% sodium citrate, with cells separated from oedema by centrifugation. Leukocytes were enumerated by haemocytometer and oedema volume recorded. Leukocytes were incubated with antibodies and processed by flow cytometry. Gating strategies firstly identified CD3+ T cells, CD19+ B cells and CD56+CD16+/− NK cells. The remaining lymphocyte-deplete population was gated into HLA-DR+ and HLA-DR− cells. Arrows indicate gating strategy.
Figure 4.
Characterisation of inflammatory cell infiltrates into skin blisters at 24(onset) – II.
HLA-DR+ and HLA-DR− cells identified in Figure 3 were further analysed. HLA-DR+ cells were characterised into CD14+/CD16lo monocytes/macrophages and HLA-DR+/CD14−/CD16− dendritic cells. HLA-DR− cells comprised of typical PMNs (CD16hi, CD66+) and a CD16lo population. On extended characterisation, HLA-DR−/CD16lo were identified as a mixture of Siglec-8+ eosinophils and Annexin V/7AAD+ apoptotic/dead PMNs. The HLA-DR+/CD14−/CD16− dendritic cells comprised of CD141+ and CD11c+ dendritic cell subpopulations. Arrows indicate gating strategy.
Figure 5.
Characterisation of inflammatory cell infiltrates into skin blisters at 72(resolution) – I.
Representative dot plots for flow cytometric gating are shown for healthy male volunteers (n = 17). Blister contents were collected from the remaining blister 72 h after application of cantharidin in 3% sodium citrate, with cells separated from oedema by centrifugation. Leukocytes were enumerated by haemocytometer and oedema volume recorded. Leukocytes were incubated with antibodies and processed by flow cytometry. Gating strategies firstly identified CD3+ T cells, CD19+ B cells and CD56+CD16+/− NK cells. The remaining lymphocyte-deplete population was gated into HLA-DR+ and HLA-DR− cells. Arrows indicate gating strategy.
Figure 6.
Characterisation of inflammatory cell infiltrates into skin blisters at 72(resolution) – II.
HLA-DR+ and HLA-DR− cells identified in Figure 5 were further analysed. HLA-DR+ cells were characterised into CD14+/CD16hi monocytes/macrophages and CD14− CD16− dendritic cells. HLA-DR− cells comprised of typical PMNs (CD16hi, CD66b+) and CD16lo population. On extended characterisation, HLA-DR−/CD16lo were identified as Siglec-8+ eosinophils and the HLA-DR+/CD14−/CD16− dendritic cells comprised of CD141+ and CD11c+ dendritic cell subpopulations. Arrows indicate gating strategy.
Figure 7.
Total leukocyte and oedema profile in resolving skin blisters in humans.
Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer while oedema volume recorded. Temporal differences from 24 to 72 h for total cells, leukocyte concentration and oedema volume are shown (n = 17).
Figure 8.
Leukocyte subtype profiles in resolving skin blisters in humans.
Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer. Leukocytes were incubated with fluorescent antibodies, processed for flow cytometry, and gated according to Figures 2 and 3. Temporal changes in cell populations from 24 h to 72 h are shown (n = 17).
Figure 9.
Cytokine profiles in resolving skin blisters in humans.
Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer while oedema volume recorded and processed by assay for cytokine concentration (A). Correlations were made between cytokine concentration and PMN infiltration at 24 h and 72 h (B) (n = 17).
Figure 10.
Lipid mediators in resolving skin blisters in humans.
Blister content was collected in 3% sodium citrate 24 h and 72 h after application of cantharidin with cells separated from oedema by centrifugation. Red blood cells were lysed and the remaining leukocytes counted by haemocytometer while oedema volume recorded and analysed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry for eicosanoid levels (n = 17).
Figure 11.
Correlations in cell profiles from onset to resolution.
A wide range of PMN numbers infiltrate skin blisters early in the response (onset), and an immune response induces appropriate resolution. Correlation between change in PMN numbers (72 h minus 24 h) and total PMN numbers at onset are expressed as either total cells or as percentages.