Hyperthermia increases intercellular adhesion molecule-1 expression and lymphocyte adhesion to endothelial cells

We observed that the synergistic combination of immunotherapy and whole-body hyperthermia is active against large well-vascularized tumors but not microscopic tumors, and we therefore hypothesized that hyperthermia may act on lymphocyte-endothelial cell interactions. We undertook these studies to evaluate the effect of hyperthermia on lymphocyte-endothelial cell adhesion.

Cultured human umbilical vein endothelial cells (HUVEC) and normal peripheral blood lymphocytes were used. HUVEC were cultured to confluence. Treatment groups included control, hyperthermia alone (41 degrees C for 2 hours), interferon-gamma (IFN-gamma) alone, or hyperthermia + interferon-gamma. 51Cr-labeled peripheral blood lymphocytes were allowed to adhere to treated HUVEC, and nonadhering cells were washed away. Adherent cells were lysed and counted in a gamma-counter, calculating an adhesion index compared to controls. The experiment was then conducted with the addition of anti-intercellular adhesion molecule (ICAM) antibody. Cell surface ICAM expression was determined with double monoclonal antibody staining and fluorescence-activated cell sorter analysis, and soluble ICAM secretion was determined with enzyme-linked immunosorbent assay in each group.

In a representative experiment, interferon-gamma increased adhesion by a factor of 1.81 (p < 0.05) compared with control and hyperthermia by 1.38 (p < 0.05) and combined treatment by a factor of 2.43 (p < 0.05). Anti-ICAM antibody abrogated the increased adhesion caused by hyperthermia but did not abrogate the effect of interferon-gamma. Although only 26% of control cells expressed ICAM-1 on the cell surface, interferon-gamma increased expression to 53% (p < 0.05), hyperthermia increased expression to 38% (p < 0.05), and combined treatment increased expression to 61% (p < 0.05). Soluble ICAM-1 was not increased 12 hours after treatment, but by 24 hours significant (p < 0.05) differences (control 0.262 ng/ml, IFN alone 1.50, hyperthermia alone 1.57, and combined 2.71) were noted.

These results suggest that hyperthermia has a significant effect on lymphocyte adhesion to endothelial cells, at least in part mediated by ICAM-1. Cell surface ICAM-1 is increased at 12 hours, and soluble ICAM-1 is increased at 24 hours. These data suggest that hyperthermia may function by increasing lymphocyte adhesion, providing another locus of action to improve clinical results with immunotherapy.