Interferon-gamma inhibits endocytosis of soluble animated beta-1,3-D-glucan and neutral red in mouse peritoneal macrophages.

We previously demonstrated that soluble animated beta-1,3-D-glucan (AG) is internalized after binding to a specific beta-glucan receptor on macrophages. Internalization, but not binding, of AG is reduced when the macrophages are treated with IFN-gamma. Because our data indicated that AG is taken up by macrophages through beta-glucan receptor-mediated endocytosis, we wanted to characterize further the inhibitory effect of IFN-gamma on endocytosis. We compared the internalization of AG and neutral red (NR). NR is internalized by macrophages through fluid-phase endocytosis. AG and NR showed a similar influx/efflux pattern. The initial rate of accumulation was much larger for AG than for NR, however, probably because of the involvement of the beta-glucan receptor in the uptake of AG. Internalized AG was associated with membranes of the endocytic vesicles and formed characteristic rings on confocal laser scanning microscopy (CLSM) images. Both the influx and efflux of AG and NR was inhibited by treatment of macrophages with IFN-gamma. Phorbol myristate acetate (PMA) added to the cell cultures increased the accumulation of AG and NR and reversed the inhibitory effect of IFN-gamma. The effect of PMA was dependent on functionally intact microfilaments and microtubules. CLSM showed that the accumulated AG was localized mostly in small vesicles (size < 2 microns) in IFN-gamma-treated cells, in large and small vesicles in untreated cells, and mostly in large vesicles (size > 2 microns) in PMA-treated cells. In conclusion, IFN-gamma inhibits both the beta-glucan receptor-mediated endocytosis of AG and the fluid-phase endocytosis of NR, probably by inhibiting the formation of large vesicles.

Morphological modifications of apoptosis in HL-60 cells: effects of homocysteine and cytochalasins on apoptosis initiated by 3-deazaadenosine.

Using electron microscopy, confocal laser scanning microscopy and measurements of intact DNA we have studied the morphology and DNA degradation of human leukaemia HL-60 cells undergoing drug initiated apoptosis. Apoptosis was initiated by 100 microM 3-deazaadenosine (c3Ado), 25 microM c3Ado plus 1 mM homocysteine thiolactone (Hcy) and 100 microM c3Ado plus 5 micrograms/ml cytochalasin B (CB). Two different phenotypes of apoptotic cells (APC), blebbed and non-blebbed, were present in the cultures. Blebbed APC dominated in cultures exposed to c3Ado, whereas most APC in cultures treated with c3Ado plus Hcy and all the APC in cultures treated with c3Ado plus CB displayed a non-blebbed phenotype. A more pronounced reduction of the chromatin/cytoplasm ratio, lower volume fractions of uncondensed chromatin and higher volume fractions of highly condensed chromatin (micronuclei) were found in cultures exposed to c3Ado and c3Ado plus Hcy when compared with cultures exposed to c3Ado plus CB. A partial inhibition of c3Ado apoptosis by CB was confirmed by measurements of intact DNA. The inhibitory effect of CB was not reproducible by CE, indicating that CB exerts its effect by an actin independent mechanism. Both blebbed and non-blebbed APC displayed nuclear fragmentation, segregation of organelles and cytoplasmic vesiculation, suggesting that the differences between the phenotypes were restricted to the cytoplasmic membrane. We were not able to demonstrate the presence of F-actin by fluorescein isothiocyanate-phalloidin staining in blebbed APC nor in non-blebbed APC in cultures treated with c3Ado plus Hcy. Non-blebbed APC in cultures treated with c3Ado plus CB displayed foci of F-actin at the internal part of the cytoplasmic membrane. This suggests that F-actin is preserved by the mechanism by which CB inhibits blebbing, and may indicate that blebbing of the cytoplasmic membrane during apoptosis is associated with F-actin deficiency rather than a result of actin-myosin interactions.

Dynamics of blood components and peritoneal fluid during treatment of murine E. coli sepsis with beta-1,3-D-polyglucose derivatives. II. Interleukin 1, tumour necrosis factor, prostaglandin E2, and leukotriene B4.

The influences of pretreatment with beta-1,3-D-polyglucose derivatives on levels of cytokines and arachidonic acid metabolites in body fluids in experimental peritonitis in mice are reported. Peritonitis was induced by an intraperitoneal injection of 10(8) live Escherichia coli. Pretreated animals survived the infection, untreated animals died about 12 h after inoculation with E. coli. Levels of IL-1 in plasma and peritoneal fluid, measured by cytotoxicity assay of the HT-2 cell line, increased significantly during the first 48 h after intraperitoneal treatment with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble, aminated beta-1,3-D-polyglucose (AG). After subsequent challenge with E. coli, the levels of IL-1 were significantly lower than in untreated animals. There was no increase in levels of TNF after treatment with GDM or AG, measured by cytotoxicity assay of the WEHI clone 13 cell line. After challenge with E. coli, TNF in plasma and peritoneal fluid was significantly lower compared with untreated animals. Both PGE2 and LTB4, measured by radioimmunoassay kits, were increased in peritoneal fluid after treatment with GDM and AG. After challenge with E. coli, PGE2 and LTB4 in peritoneal fluid increased to about half the concentration of infected control animals. Intraperitoneal injection of indomethacin to pretreated animals resulted in increased levels of IL-1 and TNF and decreased levels of PGE2 following challenge with E. coli. The levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seem to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.

Differential expression of major histocompatibility complex class II variants in cells infiltrating the meth A sarcoma.

CD4+ T cells are involved in immune responses against the Meth A sarcoma and infiltrate tumours arising from Meth A cells inoculated intradermally in (BALB/c x C57BL/6)F1 (H-2d/b) mice. In order to investigate whether the prerequisites for tumour antigen presentation to CD4+ T cells are fulfilled within the malignant lesion, expression of class II major histocompatibility complex (MHC) molecules and the costimulatory ligand B7 were examined. The I-Ab and I-Ed allomorphs were abundantly expressed by virtually all B cells and 50% of macrophages infiltrating the tumours. In striking contrast, the I-Ad variant was hardly detectable. This pattern of class II expression appeared to be unique for the tumour microenvironment. Thus the proportion of I-Ad+ spleen B cells and peritoneal macrophages did not significantly differ from the proportion expressing I-Ab and I-Ed, and these extratumoral I-Ad+ cells stained as brightly as did cells from healthy mice. Expression of B7 was weak by tumour-infiltrating cells. The profound capacity of the Meth A sarcoma to confer low local I-Ad and B7 expression might preclude T-cell-dependent anti-tumour immune responses and thus promote survival of malignant cells. Whereas MHC class II genes are generally found to be co-ordinately transcribed, this study demonstrates that the expression of MHC class II allelic variants can be differentially regulated in vivo.