The role of caspase activation and mitochondrial depolarisation in cultured human apoptotic eosinophils.

Caspases are key intracellular molecules in the control of apoptosis, but little is known concerning their relative contribution to the cascade of events leading to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation dependent apoptosis induction in the cultured eosinophils (CE). CE cultured alone for 48 hours exhibited constitutive apoptosis (12% ± 1.2). Significant (P < 0.05) enhancement of eosinophil apoptosis was observed following monoclonal antibody (Mab) treatment with CD45 (40% ± 0.7), CD95 (36% ± 1.6), or CD69 (34% ± 0.2). Caspase activity was analysed using the novel CaspaTagTM technique and flow cytometry. CE ligated with CD45 (Bra55), CD95 (Fas) and CD69 Mab resulted in caspase-3 and -9 activation after 16 hours post-ligation. This trend in caspase-3 and -9 activation continued to increase significantly through to the 20 and 24 hours time points when compared to isotype control. Activated up-stream caspase-8 was detected 16 and 20 hours after treatment with CD45, CD95 and CD69 Mab followed by a trend toward basal levels at 24 hours. Ligation of CD95 was followed by mitochondrial permeabilization, as demonstrated by marked increase in mitochondrial transmembrane potential ([Formula: see text]) at all time points. However, ligation with CD45 and CD69 failed to induce a change in [Formula: see text] at 16 hours post-treatment compared to isotype control even though there was an alteration in mitochondrial downstream-caspase activity following ligation with these Mab(s) at this time point. At 20 and 24 hours post-ligation, CD45 or CD69 induce significantly altered levels of [Formula: see text]. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti inflammatory therapy for asthma.

The haemopoietic stem cell: between apoptosis and self renewal.

Self renewal and apoptosis of haemopoietic stem cells (HSC) represent major factors that determine the size of the haemopoietic cell mass. Changes in self renewal above or below the steady state value of 0.5 will result in either bone marrow expansion or aplasia, respectively. Despite the growing body of research that describes the potential role of HSC, there is still very little information on the mechanisms that govern HSC self renewal and apoptosis. Considerable insight into the role of HSC in many diseases has been gained in recent years. In light of their crucial importance, this article reviews recent developments in the understanding of the molecular, biological, and physiological characteristics of haemopoietic stem cells.

Modulation of dendritic cell activation chemokines and cellular injury.

Dendritic cells (DC) are professional antigen presenting cells expressing MHC class II, derived from a common marrow precursor. They are motile, diffused and have a spidery shape with many long cytoplasmic processes. The aim of this project was to test the hypothesis that cellular injury induces the activation and functional maturation of DC. To test the effects of injury on DC activation, immature DCs were used as substrate for DC activation assays. They were obtained from their precursor in peripheral blood mononuclear cells (PBMNCs) by culturing them GM-CSF and IL-4. Expression of surface B7 was measured by immunofluorescence and flowcytometry. beta- chemokines were used as potential injury mediators, including: RANTES, MIP-1alpha, MIP-1beta, MCP-1, -2, -3 and -4, as well as other inflammatory cytokines such as TNF-alpha and IL-1. They were screened on immature DCs to examine whether or not they modulate B7-1 and B7-2. A model of cellular injury was established to investigate whether the injured parenchymal cells deliver signals to initiate DC activation or upregulation of B7-1/B7-2 by release of soluble mediators. H2O2 was used as an injury mediator to injure renal tubular epithelial cells (RTECs). RANTES, MIP-1alpha and MIP-1beta upregulated B7-1. MCP-1, -2, -3 and -4 downregulated the expression of HLA-DR greatly. Furthermore, MCP-1, -2, -3 and -4 upregulated B7.2, while and -4 and MCP2 upregulated B7.1. We observed that immature DCs could not be readily stimulated with chemokines and pro-inflammatory cytokines IL-1 and TNF-alpha unless GM-CSF and IL4 were used continuously. The supernatant of injured renal epithelial cells had an effect on DC activation. These findings may explain the role of DCs as a link between the innate and the adaptive immune response, as well as being an active participant in determining the outcome of an antigen encounter.