Effects of IL-2 on circulating immune cells. (a) Changes in peripheral blood cell count following administration of one dose of IL-2. (b) Cell counts in PBMC obtained by apheresis. PBMC were further purified by density gradient and stimulated in vitro with IL-2 (6,000 IU/ml). At each time point, adherent and non-adherent cells were harvested and counted with an automated cell counter. Symbols as in (a). (c) Effect of density-gradient separation on peripheral blood-cell populations. Monocytes and lymphocytes are enriched after gradient separation (Post-) compared with before (Pre-), whereas polymorphonuclear cells, eosinophils and basophils are excluded. (d) Expression of marker genes for leukocytes derived from a 6,500-spot cDNA array analysis. Expression profiles are displayed in an in vivo time course. Samples were obtained from blood 1, 2, 3, 4, 6 and 8 h after the first dose of IL-2 (green bar); from a group of PBMC obtained from different patients 3 h after IL-2 administration (gray bar); and from FNAs of melanoma metastases obtained 3 h after one (light blue) or four (dark blue) doses of IL-2. In the clusterograms, colors are presented according to the central method for display using a normalization factor as recommended by Ross et al. . The results represent the ratio of hybridization of fluorescent test samples to reference samples. Ratios are depicted according to the color scale bar underneath indicating the degree of gene upregulation (red) or downregulation (green). (e) Effect of IL-2 and/or lymphocyte/monocyte-enriched PBMC on HUVEC permeability. Confluent monolayers of HUVEC were incubated for 3 h (white bars) or 24 h (black bars) with IL-2 (6,000 IU/ml) and/or PBMC. Permeability was measured as increase of optical density in abluminal chamber medium. PBMC increased HUVEC permeability in all conditions, compared with those in which no PBMC were present (Student t-test, p < 0.01). IL-2 addition to PBMC increased permeability significantly compared to PBMC alone only after 24 h incubation (Student t-test, p = 0.02). (f) Effect of IL-2 and/or lymphocyte/monocyte-enriched PBMC on HUVEC gene expression. HUVEC were exposed for 3 h to IL-2 (column 1), culture medium alone (column 2) or supernatant obtained 3 h (column 3) or 24 h (column 4) after exposure of PBMC to IL-2. Controls: supernatant obtained from PBMC after 3 and 24 h of culture (columns 4 and 6) was used for HUVEC conditioning. Total RNA was extracted from HUVEC immediately after 3 h exposure to supernatants. Six signatures encompassing all genes specifically upregulated by IL-2-stimulated PBMC are shown (Student t-test, p < 0.05, supernatant from IL-2-conditioned PBMC for 24 h compared to PBMC alone).