Beta-(1-->3)-D-glucan modulates DNA binding of nuclear factors kappaB, AT and IL-6 leading to an anti-inflammatory shift of the IL-1beta/IL-1 receptor antagonist ratio.

BACKGROUND: Beta-1-->3-D-glucans represent a pathogen-associated molecular pattern and are able to modify biological responses. Employing a comprehensive methodological approach, the aim of our in vitro study was to elucidate novel molecular and cellular mechanisms of human peripheral blood immune cells mediated by a fungal beta-1-->3-D-glucan, i.e. glucan phosphate, in the presence of lipopolysaccharide (LPS) or toxic shock syndrome toxin 1 (TSST-1). RESULTS: Despite an activation of nuclear factor (NF) kappaB, NFinterleukin(IL)-6 and NFAT similar to LPS or TSST-1, we observed no significant production of IL-1beta, IL-6, tumor necrosis factor alpha or interferon gamma induced by glucan phosphate. Glucan phosphate-treated leukocytes induced a substantial amount of IL-8 (peak at 18 h: 5000 pg/ml), likely due to binding of NFkappaB to a consensus site in the IL-8 promoter. An increase in IL-1receptor antagonist (RA) production (peak at 24 h: 12000 pg/ml) by glucan phosphate-treated cells positively correlated with IL-8 levels. Glucan phosphate induced significant binding to a known NFIL-6 site and a new NFAT site within the IL-1RA promoter, which was confirmed by inhibition experiments. When applied in combination with either LPS or TSST-1 at the same time points, we detected that glucan phosphate elevated the LPS- and the TSST-1-induced DNA binding of NFkappaB, NFIL-6 and NFAT, leading to a synergistic increase of IL-1RA. Further, glucan phosphate modulated the TSST-1-induced inflammatory response via reduction of IL-1beta and IL-6. As a consequence, glucan phosphate shifted the TSST-1-induced IL-1beta/IL-1RA ratio towards an anti-inflammatory phenotype. Subsequently, glucan phosphate decreased the TSST-1-induced, IL-1-dependent production of IL-2. CONCLUSION: Thus, beta-1-->3-D-glucans may induce beneficial effects in the presence of pro-inflammatory responses, downstream of receptor binding and signaling by switching a pro- to an anti-inflammatory IL-1RA-mediated reaction. Our results also offer new insights into the complex regulation of the IL-1RA gene, which can be modulated by a beta-1-->3-D-glucan.

Anti-myeloma activity of natural killer lymphocytes.

Natural killer (NK) cells are assumed to contribute to a graft-versus-leukaemia effect. In vitro experiments have shown that many leukaemic cells are NK-cell sensitive. Nevertheless, no data concerning the influence of purified NK cells on malignant myeloma (MM) cells exist. We co-incubated NK cells with three different MM cell lines and fresh bone marrow samples of nine MM patients. The proportion of vital MM cells was determined before and after co-cultivation by a flow-cytometry-based assay. All MM cells tested, with the exception of one cell line (NCI H929), were susceptible to a NK-cell attack even without exogenous interleukin 2 (IL-2). The mean killing of the native MM samples was 23.1 +/- 5.4% and 34.5 +/- 6.5% at 10:1 and 20:1 effector:target ratio respectively, This corresponded to about 2/3 of those values obtained with the highly sensitive line K562. In contrast, CD34-positive haematopoietic stem cells as well as peripheral mononuclear cells were completely resistant under similar experimental conditions (1.3% killing). To elucidate the underlying triggering mechanisms, we measured human leucocyte antigen (HLA)-class I expression of the MM cells. No evidence for HLA loss, which could have explained the NK-cell recognition if it occurred, was demonstrated. These findings may contribute to the understanding of in vivo NK-cell activation and encourage clinical applications of NK cells for MM patients.

Large-scale generation of natural killer lymphocytes for clinical application.

Natural killer (NK) lymphocytes can be used for adoptive immunotherapeutic strategies. Alternatively, they may be employed as adjuvants for stem cell/bone marrow transplantation, either to re-induce remission, or to purge autografts of contaminating malignant cells. We developed a new protocol that enables the generation of NK cells on a clinical scale in a closed system that enables good manufacturing practice (GMP) conformity. Aside from the initial NK cell inoculum, our protocol includes activated feeder cells [irradiated peripheral blood mononuclear cells (PBMC) and no transformed blasts], cytokines [interleukin-2 (IL-2) and IL-15], human serum, and a complex basic media formulation. During the whole expansion period of approximately 14 days, the cells were handled in PTFE (Teflon) bags, whereby fresh medium was added without opening the system. The use of immortalized or virus-transformed feeder cells, as used in many other current research protocols, was completely avoided. A precise controlling of a number of environmental factors was necessary to achieve reproducible results. Increases in NK cell number ranged between 80- and 200-fold. The resulting NK cells were CD56(+), CD3(-), and CD16(+) (75%). They were highly cytotoxic against different malignant target cells and did not produce significant levels of interferon-gamma. Therefore, they belonged to the cytotoxic rather than the immunoregulatory NK subpopulation. No non-specific activation against normal allogenous lymphocytes occurred. This work might permit the realization of future protocols for evaluating the clinical effect of NK lymphocytes in human disease.

A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells.

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.