In order to examine the regulatory effects of major Th1-derived cytokines, such as IL-12, and Th2 cytokines, IL-4 and IL-10, on the formation of neopterin and degradation of tryptophan, two metabolic pathways induced by interferon-gamma (IFN-gamma) in human monocytes/macrophages, we investigated the human monocytic cell line THP-1, primary human macrophages, and peripheral blood mononuclear cells (PBMC). Neopterin formation and tryptophan degradation were induced similarly by IFN-gamma in all three cell types investigated, but the effects of interleukins were different between THP-1, primary macrophages and PBMC. In PBMC, but not in THP-1 cells and primary macrophages, IL-12 was found to be additive to the effects of IFN-gamma to superinduce neopterin formation and tryptophan degradation. IL-4 and IL-10 reduced the effects of IFN-gamma on monocytic cells, and both cytokines were additively antagonistic to IFN-gamma in PBMC and THP-1 cells. Finally, on preincubation, but not on addition of IL-12, the effects of IL-4 and IL-10 on PBMC could be abrogated, whereas no such effect was seen in THP-1 cells. The results show that IL-12 up-regulates neopterin formation and tryptophan degradation by inducing additional IFN-gamma production by Th1 cells, while a direct effect of IL-12 on monocytes/macrophages appears to be absent. Similarly, IL-4 and IL-10 inhibit neopterin production and tryptophan degradation in PBMC by down-regulating Th1-type cytokine production and possibly also via direct deactivation of IFN-gamma effects towards monocytes/macrophages. The results clearly show how Th1 cell-mediated immunity may be up- or down-regulated by endogenous cytokine production.

Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P-glycoprotein (P-gp), we developed a functional flow cytometric assay for the detection of multidrug-resistant (MDR) cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established that enabled demonstration of significant differences in Rh123 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two-colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rh123 was associated with P-gp expression, the leukaemic cells were stained with the monoclonal antibody MRK-16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P-gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two-colour flow cytometry was much easier to perform with Rh123 than with daunorubicin. Our results indicate that flow cytometric measurement of Rh123 accumulation/efflux proves applicable to detect MDR cells in heterogenous clinical samples.

Summary.To better understand the phenomenon of Pglycoprotein (P-170) expression we investigated lymphocyte subpopulations for P-170 function in healthy volunteers. Studies were based on three-colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P-170. Marked Rh123 efflux was detected in CD8 þ T lymphocytes with CD8 þ /CD45RA þ T cells (naive cells) showing significantly higher P-170 activity as compared with CD8 cells (memory cells) demonstrated less P-170 activity than CD8þ /CD45RO ¹ cells (P < 0·04). P-170 function was less prominent in CD4 þ T cells, however, Rh123 efflux was higher in the CD4 þ /CD45RA þ and CD4 þ / CD45RO ¹ subpopulations (P < 0·025) corresponding to the CD8 þ results. Dye efflux differed significantly between activated and non-activated CD8 þ and CD4 þ as well as CD8 þ /CD11b þ and CD8 þ /CD11b ¹ T lymphocytes. Since CD16 þ natural killer cells (NK) expressed the highest level of P-170, the NK cytotoxicity against 51 Cr-labelled K562 target cells was assayed in the presence or absence of P-170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R-verapamil and dexnigaldipine-HCP in a dose-dependent manner.The differential expression of P-170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR-modulators suggest a physiological role of P-170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.

Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.

It has been shown previously that verapamil and other calcium antagonists and calmodulin inhibitors can reverse multidrug resistance. We compared the potency of the dihydropyridine derivatives (4R)-3-[3-(4,4-diphenyl-1-piperadinyl)-propyl]-5-methyl-1,4-dihydr o-2,6- dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (B859-35), a metabolite of B859-35, niguldipine and (R)-nitrendipine to that of (RS)-verapamil in reversing multidrug resistance. The accumulation of the fluorescent dye rhodamine 123, which is transported by the P-glycoprotein, was determined by a flow cytometer. Multidrug-resistant human HeLa KB-8-5 and Walker rat carcinoma cells were incubated in the presence and in absence of the drugs indicated above. We found that 0.1 microM B859-35 increases the accumulation of rhodamine 123 in multidrug-resistant KB-8-5 and Walker cells more effectively than 1 microM (RS)-verapamil. In sensitive KB-3-1 cells addition of the drugs had no significant influence on the accumulation of rhodamine 123. IN KB-8-5 cells, 10 nM Adriamycin caused a reduction of cell growth to 85% compared to untreated controls (= 100%). If 1 microM B859-35, B859-35 metabolite, niguldipine, verapamil or (R)-nitrendipine was added to 10 nM Adriamycin, growth reduction compared with untreated controls increased to 12%, 11%, 23%, 63%, and 82% respectively. The effect of 0.1 microM B859-35 was a reduction in proliferation to 38%, that of 0.1 microM verapamil to 72%. These data illustrate that B859-35, a compound with antitumor activity in several tumors, is at least ten times more potent than racemic verapamil in reversing multidrug resistance.