Neutrophil apoptosis in autoimmune Fas-defective MRL lpr/lpr mice.

The apoptosis-defective lpr (fas) mutation in MRL mice causes the early onset of a lupus-like autoimmune disease with concomitant inflammation. In order to analyse the consequences of the impaired Fas-dependent apoptosis on inflammation, the susceptibility to apoptosis of polymorphonuclear leukocytes (PMN), obtained from MRL lpr/lpr mice, has been studied. Peritoneal PMN from lpr/lpr and control (+/+) mice were recruited with a mild inflammatory stimulus. The number of cells collected from the peritoneal cavity of young lpr/lpr mice was comparable to that obtained from age-matched control mice, indicating that PMN homeostasis is maintained regardless of the loss-of-function Fas mutation. Recruited neutrophils were exposed in culture to apoptosis-inducing stimuli. Treatment with agonist anti-Fas antibody increased apoptosis of +/+ PMN, but did not affect lpr/lpr PMN which do not express Fas on their surface. However, lpr/lpr PMN could undergo both spontaneous and stimulus-induced apoptosis in a fashion comparable to or higher than that of control +/+ mice. Analysis of mRNA expression revealed that lpr/lpr PMN have reduced expression of IL-18, whereas IL-1beta, IFNgamma, caspase 1 and caspase 3 are expressed at levels comparable to those of +/+ cells. However, caspase-3-like activity was higher in PMN from lpr/lpr mice than in +/+ cells, and correlated with enhanced apoptosis. It could be concluded that in young, uncompromised lpr/lpr mice, PMN homeostasis is still fully regulated through the involvement of Fas-independent, compensatory, apoptotic mechanisms. This could include an increased participation of caspase 3 in the apoptotic pathway, consequent to enhanced activation of the enzyme and to the decreased production of IL-18, which acts as a competitive caspase 3 substrate.

Balance between autocrine interleukin-1beta and caspases defines life versus death of polymorphonuclear cells.

The role of endogenous IL-1beta in regulating spontaneous and Fas-triggered apoptosis of human PMN has been studied in relation to the activity of the IL-1beta-generating enzyme ICE (caspase-1), an enzyme also involved in the mechanism of cell death. Upon in vitro culture, PMN undergo spontaneous apoptosis and express increasing levels of IL-1beta, caspase-1- and caspase-3-like enzymes. Endogenous IL-1beta protects PMN from apoptosis, since inhibition of either IL-1beta or caspase-1 activity can accelerate PMN apoptotic death. Thus, in spontaneous PMN apoptosis caspase-1 essentially plays an anti-apoptotic role by inducing maturation of protective IL-1beta, whereas other molecules are responsible of driving apoptosis. Upon Fas triggering, PMN apoptosis is greatly accelerated, in correlation with increased caspase activity, whereas IL-1beta production is not augmented. Inhibition of IL-1beta activity can increase Fas-induced apoptosis, whereas caspase-1 inhibitors are without significant effect. It is hypothesized that in Fas-induced PMN apoptosis caspase-1 has a double role: it can protect from apoptosis through generation of protective IL-1beta, as in spontaneous apoptosis, and it can also exert pro-apoptotic activity which counterbalances the protective effect and allows accelerated apoptosis.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.

Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.