Figure 4 Staining of human CD19+ B cells (y-axis) with FITC-labelled ML I (x-axis) and FITC-labelled ML III (x-axis). Results are representative for 8 independent experiments.
compared to their CD8+ CD62Lhi counterparts, CD4+ T helper cells and CD19+ B cells is unclear. One explanation could be that the binding sites for ML III may differ in CD8+ CD62Lh "naive" cells and CD8+ CD62L'° "memory" cells. In fact, especially the CD8+ cells bind a higher number of ML I than CD4+ T cells and CD19+ B cells, while the binding capacity of these subsets for ML III were similar (Büssing et al., 1999h). However, CD8+ cells were as sensitive to ML I-mediated cell death as compared to CD4+ T cells, but more sensitive to ML III (Büssing et al., 1998). Also, analysis of ML-binding to the CD62L subsets within the CD4+ T cells and CD8+ cells did not explain the previously observed differences in the selectivity of ML III killing. Yet, there is at present no convincing rationale for these conflicting results. One may suggest that differences in the ML-cell surface affinity and/or intracellular uptake of the toxic proteins, and their subsequent degradation could be determinating factors. This has to be addressed in further investigations. Surprisingly, B cells did not bind adequately ML III but were able to bind ML I (Figure 4). One may suggest a lack of adequate "receptors" with galNAc domains on these cells. Anyway, this may explain the lower sensitivity of B cells to ML IIImediated cytotoxicity. In fact, in leukemic B cells a strong Apo2.7 expression was induced only by ML I but not by ML III (Büssing et al., 1999h). In the light of this finding, the possible impact of ML I- and ML III-rich VA-E in the treatment of B cell neoplasia has to be investigated carefully (Büssing et al., 1999d).
Apart from the onset of apoptotic cell death by the ML, Fas ligand (FasL, CD95L) and TNF-R1 (CD120a) expression increased after incubation with ML I and ML III in the surviving lymphocytes, i.e. CD4+ T cells, CD8+ cells, and CD19+ B cells, while the Fas expression decreased (Büssing et al., 1999e). The FasL belongs to the TNF family and induces apoptosis through its cell surface receptor, Fas (Apo-1, CD95). FasL plays a pivotal role in lymphocyte cytotoxicity but is also involved in the downregulation of immune reactions (reviewed by Nagata and Golstein, 1995). The molecule is expressed in immune privileged body sides such as the retina and testis, and most abundantly in T cells (Suda et al., 1995). Once FasL is expressed on activated T cells, they may kill Fas+ target cells. Malfunction of the Fas system may cause lymphoproliferative disorders and accelerates autoimmune diseases; its exacerbation may cause tissue destruction (Nagata and Golstein, 1995). The observed effect of a FasL-induction by ML may reflect "activation" of surviving cells which did not result in a proliferation response as measured by the expression of interleukin-2 receptors (CD25) and transferrin receptors (CD71), or nuclear Ki-67 antigens (Büssing et al., 1999e).
However, not only tumour-specific cytotoxic T cells may kill Fas-sensitive tumour cells, also FasL+ tumour cells may counteract this attack by the elimination of Fas+ cytotoxic T cells via FasL/Fas-mediated apoptosis. On the other hand, the expression of the Fas molecule slightly decreased on the surface of ML-treated surviving lymphocytes, and thus, these cells might be less sensitive against a counterattack of FasL+ tumour cells. Further, ML I and ML III did not induce FasL expression in cultured Molt-4 cells, T-CLL and B-CLL cells (Büssing et al., 1999e); however, other tumour cell lines have to be tested.
Apart from an induction of pro-inflammatory cytokines such as IL-1, IL-6, TNF-a (Hajto et al., 1990; Männel et al., 1991; Ribereau-Gayon et al., 1996; Joller et al., 1996), and gene expression of IL-10, IFN-y, and GM-CSF (Hostanska et al., 1996) by monocytes/macrophages and fibroblast/keratinocytes stimulated with ML I, recent findings indicate a ML I-induced clonogenic growth of CD34+ haematopoietic progenitor cells when co-cultured with several growth factors (Vehmeyer et al., 1998). These properties may explain the observed release of juvenile granulocytes and monocytes from the bone marrow in response to an intravenous application of VA-E (Hajto, 1986; Büssing et al., 1996f). But are these juvenile cells functionally competent?
The phagocytic activity of human leukocytes reportedly increased in response to the B chain of ML I, however, ML I and A chain had no effect even though they inhibited spontaneous migration of macrophages (Metzner et al., 1985). ML I at 50 and 100 ^g/ml induced superoxide anion in human neutrophils and enhanced menadione-dependent release of H2O2 in rat thymocytes treated with>1 ^g/ml (Timoshenko and Gabius, 1993, 1995). These observations may have no clinical relevance because the concentrations were highly toxic to the cells. Using a physiological stimulus such as Escherichia coli, as opposed to viscotoxins, ML I and ML III did not enhance the oxidative burst of human neutrophils but slightly impaired cell function (Stein et al., 1999a, b; Stein and Schietzel, this book).
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