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Ifosfamide, a clinically potent chemotherapeutic agent, causes the depletion of intracellular glutathione (GSH) levels in various cell types. GSH is the major intracellular reductant against oxidative stress. 4-Hydroxyifosfamide (4-OH-IF), the activated form of ifosfamide, depletes GSH levels in T cells and natural killer (NK) cells; this is accompanied by a decrease in T-cell and NK-cell function. Here we demonstrate for the first time that human monocyte-derived dendritic cells (DCs) express higher constitutive levels of GSH and are less sensitive to 4-OH-IF-induced GSH depletion than T cells and NK cells. Treatment of DCs with 4-OH-IF significantly reduced their ability to stimulate allogeneic T-cell proliferation and interferon-γ (IFN-γ) production. Ifosfamide also decreased DC interleukin-12p70 (IL-12p70) production after stimulation with lipopolysaccharide (LPS) and IFN-γ. The decrease in allostimulatory capacity and in IFN-γ and IL-12 production correlated with a decrease in intracellular GSH in the DCs. The responses could be restored by reconstituting DC GSH levels with glutathione monoethyl ester (GSH-OEt). 4-OH-IF had no inhibitory effect on the ability of DCs to present exogenously added tyrosinase peptide to tyrosinase-specific cytotoxic T lymphocytes (CTLs). These studies suggest that in cancer patients treated with ifosfamide, protection strategies based on glutathione reconstitution may enhance DC function. (Blood. 2003;102: 3668-3674)

Ifosfamide, a clinically potent chemotherapeutic agent, causes the depletion of intracellular glutathione (GSH) levels in various cell types. GSH is the major intracellular reductant against oxidative stress. 4-Hydroxyifosfamide (4-OH-IF), the activated form of ifosfamide, depletes GSH levels in T cells and natural killer (NK) cells; this is accompanied by a decrease in T-cell and NK-cell function. Here we demonstrate for the first time that human monocyte-derived dendritic cells (DCs) express higher constitutive levels of GSH and are less sensitive to 4-OH-IF-induced GSH depletion than T cells and NK cells. Treatment of DCs with 4-OH-IF significantly reduced their ability to stimulate allogeneic T-cell proliferation and interferon-γ (IFN-γ) production. Ifosfamide also decreased DC interleukin-12p70 (IL-12p70) production after stimulation with lipopolysaccharide (LPS) and IFN-γ. The decrease in allostimulatory capacity and in IFN-γ and IL-12 production correlated with a decrease in intracellular GSH in the DCs. The responses could be restored by reconstituting DC GSH levels with glutathione monoethyl ester (GSH-OEt). 4-OH-IF had no inhibitory effect on the ability of DCs to present exogenously added tyrosinase peptide to tyrosinase-specific cytotoxic T lymphocytes (CTLs). These studies suggest that in cancer patients treated with ifosfamide, protection strategies based on glutathione reconstitution may enhance DC function. (Blood. 2003;102: 3668-3674)

Members of the heat shock protein (hsp70) family are either constitutively expressed (hsc70) or can be induced by hyperthermic stress (hsp70). Recombinant hsp70 (rhsp70) stimulates cytokine production from monocytes and enhances NK cell proliferation and cytotoxicity. Here we demonstrate that rhsp70 binds to immature dendritic cells (DC) derived from monocyte precursors and induces theirmaturation as evidenced by an increase in CD40, CD86 and CD83 expression. Immature DC stimulated to mature with rhsp70 show an enhanced ability to present tyrosinase peptide to specific CTL. MatureDC did not bind rhsp70, suggesting a down‐regulation in the expression of its receptor. When rhsp70 was added to monocyte precursors at the same time as GM‐CSF and IL‐4 it reduced the differentiation of monocytes into DC as shown by a decrease in the level of CD40, CD83, CD86 and HLA‐DR expression and an increase in CD14 expression. The constitutively expressed hsc70 had neither a stimulatoryeffect on the maturation of immature DC nor did it reduce the differentiation of monocytes into DC. These findings demonstrate the specific ability of rhsp70 to induce the maturation of immature DC. Therefore rhsp70 may be useful for its adjuvant like properties in DC based immunotherapy of certain tumors.

The mechanism whereby inflammatory cells gain access to the retina in posterior intraocular inflammatory disease remains unclear. The chemokine RANTES has the potential to influence the migration of memory T cells and monocytes across the blood–retinal barrier during inflammatory eye disease. We have therefore examined the production of RANTES by cultured human retinal pigment epithelial cells (RPE), which form a part of the blood–retinal barrier, in response to cytokines likely to be present in the microenvironment. IL-1β and TNFα stimulated RANTES production by these cells. IFN-γ acted synergistically with TNFα to increase RANTES production. In contrast, IL-4 down-regulated RANTES production stimulated by TNFα. RT–PCR studies showed that RANTES mRNA from RPE followed the same pattern of expression in response to cytokines as did RANTES production indicating that RANTES production was controlled at, or prior to, transcription. RANTES is producedin vitroby RPE in response to the proinflammatory cytokines IL-1β, TNFα, and IFN-γ and is therefore likely to play a role in the development of the inflammatory eye disease endogenous posterior uveitis.

Purified tumour cells and normal mucosa cells from fresh human colorectal cancer resection specimens, and T-cell-enriched autologous peripheral blood lymphocytes, were mixed in short-term (6 day) mixed lymphocytetumour cell (MLTC) microcultures. Lymphocyte stimulation was measured by 3H-thymidine uptake, and a stimulation index (SI=[lymphocytes vs tumour cells (cpm)-tumour cells (cpm)]/[lymphocytes (cpm)])>3 was regarded as significant. Significant lymphocyte reactivity was found in 10/15 patients with colon carcinoma. However, 1 patient with autologous tumour reactivity, also showed significant stimulation against autologous normal mucosa cells, suggesting tumour-associated reactivity. Maximum stimulation occurred most frequently at a lymphocyte:tumour cell ratio of 2:1 and with nylon wool-passaged lymphocytes.