CD40 was originally described as a functionally significant B cell surface molecule. However, CD40 is also expressed on monocytes, dendritic cells, epithelial cells, and basophils. We now report that synovial membrane (SM) or dermal fibroblasts also express cell surface CD40 in vitro. Fibroblast CD40 expression declines with increasing time in culture and recombinant interferon-gamma (rINF-gamma) induces fibroblast CD40 up-regulation. This effect of rINF-gamma is augmented by recombinant interleukin-1 alpha or recombinant tumor necrosis factor-alpha. CD40 expression on fibroblasts is functionally significant because CD40L-CD40 interactions induce SM fibroblast CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1) up-regulation. Moreover, ligation of CD40 augments IL-6 production by SM fibroblasts and induces fibroblasts to proliferate. In addition, rINF-gamma enhances the effect of CD40L-CD40 interactions on fibroblast proliferation. Taken together, these studies show that fibroblasts can express CD40, cytokines can regulate fibroblast CD40 expression, and CD40 ligation induces fibroblast activation and proliferation.

We have recently demonstrated that cytolysis of human melanoma cells by immune effectors (both NK and T cells) is associated with release of the nuclear chromatin protein, high mobility group box I (HMGB1). Extracellular HMGB1 mediates a number of important functions including endothelial cell activation, stromagenesis, recruitment and activation of innate immune cells, and also dendritic cell maturation that, in the setting of cancer, lead to a chronic inflammatory response. This reparative inflammatory response promotes tumor cell survival, expansion, and metastases. Release of HMGB1 after chemotherapy-induced cytotoxicity has not been well characterized. We measured the release of HMGB1 after chemotherapy or immune cytolysis and demonstrated that this did not correlate with conventional markers of apoptosis and necrosis in several human colorectal, pancreatic, and melanoma tumor cell lines. Rather, we observed that tumor cells incubated with the platinating agent oxaliplatin, retained HMGB1 within the nucleus for significantly longer periods than other agents used at comparable cytotoxic concentrations or even with potent cytolytic cells. Thus, release of HMGB1 from dying tumor cells treated with chemotherapy or cells with lymphokine activated killer cell activity is not dependent solely on the mode of cell death. Sequestration of the damage associated molecular pattern molecule, HMGB1, may play a role in the clinical efficacy of platinating agents and suggests this as a superior agent for coupling with immunotherapeutic strategies, possibly enhancing their effectiveness.

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