Study of apoptosis in human lymphocytes by toxic substances implicated in toxic oil syndrome.

Toxic Oil Syndrome is a multisystemic disease that occurred in epidemic proportions in Spain in 1981 caused by the ingestion of rapeseed oil denatured with aniline. Several data implicate T cells in the pathogenesis of the disease. We evaluated the mechanisms of cytotoxicity in human lymphocytes of TOS-related products: aniline, 3-(N-phenylamino)-1,2-propanediol and its mono- and di-oleyl esters and eosinophilia myalgia-related product such as 3-(phenylamino)-L-alanine, which is chemically similar to 3-(N-phenylamino)-1,2-propanediol, and has been found in manufactured L-tryptophan. Our results show that only di-oleyl ester of 3-(N-phenylamino)-1,2-propanediol induces apoptosis in human lymphocytes, in a concentration and time-dependent way, confirmed by morphology, expression of phosphatidylserine in membrane and analysis of DNA degradation.

Immunological basis of toxic oil syndrome (TOS).

The toxic oil syndrome (TOS), a multisystemic disease, that occurred in Spain in 1981, was caused by the ingestion of rapeseed oil denatured with 2% aniline. Due to the clinical course of the disease, immunopathological mechanisms have been suspected but a direct connection was never demonstrated. To analyse this possibility, we determined several immunological parameters in the sera of patients with TOS and without the disease, using a case-control design: total immunoglobulins, IgG and IgE antibodies against different toxic agents (oleylanilide, aniline, linoleyl-anilide, and 3-phenylaminopropane-1-2-diol), autoantibodies, cytokines (IL-4, IL-6, TNF, GM-CSF) and soluble receptors (sCD23 and sIL-2R). We detected high levels of sIL-2R in TOS patients compared to controls (P < 0.0001). A higher levels of sCD23 and IgE were also found. In addition, the response to oleyl-anilide of peripheral blood lymphocytes from TOS patients was studied and a significant proliferative response in 30% of TOS patients versus 5% controls was observed. Our data support the implication of the immune system in the acute phase of TOS, with a possible activation of T-cells and release of cytokines, that could explain some of the clinical findings in this phase of the disease.

Modulation of the Fc gamma RII and Fc gamma RIII induced by GM-CSF, IFN-gamma and IL-4 on murine eosinophils.

Murine eosinophils express the low-affinity Fc gamma receptors recognized by the monoclonal antibody (mAb) 2.4G2, which are involved in the activation of these cells. We have studied the membrane and RNA expression of the low-affinity Fc gamma receptors on murine eosinophils stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), and interleukin-4 (IL-4). Flow cytometric analysis (FACS) of eosinophils incubated with GM-CSF and IFN-gamma showed an up-regulation on the expression of these receptors with a maximal effect at 24 hr. IL-4 failed to induce any positive or negative effect in these cells. To assess the pattern of expression of the low-affinity Fc gamma receptors on eosinophils, Northern blot analyses were carried out using specific cDNA probes encoding for the Fc gamma RII and Fc gamma RIII. Murine eosinophils constitutively express transcripts for Fc gamma RII and RIII. Incubation with GM-CSF from 3 to 12 hr produced a potent induction of the two transcripts studied (Fc gamma RII and RIII). IFN-gamma did not induce any important up-regulation of the two transcripts from 3 to 12 hr of incubation. By contrast, IL-4 produced a marked inhibition of both transcripts at 24 hr of incubation. Expression of the gamma-chain transcript which is associated with Fc gamma RIII has been detected on freshly isolated eosinophils. This study demonstrates a different regulation pattern of these receptors depending on the cytokine or growth factor used to stimulate murine eosinophils.

Phosphoinositide breakdown is associated with Fc-gamma RII-mediated activation of 5'-lipoxygenase in murine eosinophils.

In this report we present data on the ability of murine eosinophils to generate inositol phosphate derivatives, and their relationship with the activation of 5'-lipoxygenase by a Fc-gamma R-dependent mechanism. The addition of anti-IgG F(ab')2 to mouse eosinophils, previously sensitized with IgG, induces inositol phosphate generation after 2 min and after 10 min of stimulation. Maximal generation of inositol tris and inositol tetrakis phosphate has been detected after 15 min of stimulation, and the optimal concentration of anti-IgG F(ab')2 was found to be 25 micrograms. Inositol tris phosphate formation is also observed at 5 min after the addition of the calcium ionophore A23187 (5 microM). We also report that neomycin, an inhibitor of phosphoinositide-phospholipase C, inhibits Fc-gamma R-mediated phosphoinositide breakdown in a dose-dependent manner (88% inhibition at 150 microM of neomycin). The possible involvement of phosphoinositide breakdown in the activation of 5'-lipoxygenase has been investigated. Using streptolysin-O permeabilized cells and different doses of neomycin that inhibit phosphoinositide breakdown, we have demonstrated a parallel decrease in LTC4 released by these cells, using either A23187 (86% inhibition at 200 microM of neomycin) or anti-IgG F(ab')2 (82.4% inhibition at 100 microM of neomycin). [Ca2+]i elevation has been observed by loading the cells with the fluorescent calcium indicator Fura-2 penta-acetoxy methyl ester and after stimulating with the anti-Fc-gamma RII mAb (2.4G2). It is likely that the activation of murine eosinophils by a Fc-gamma R mechanism stimulates phosphoinositide breakdown as a primary step that leads to the activation of murine 5'-lipoxygenase, producing the formation of leukotriene C4.