A. Santoni

Effects of cadmium on lymphocyte activation.

The effects of cadmium (Cd) on phytohemoagglutinin or phorbol myristate acetate-induced lymphocyte activation were investigated and a dose-dependent inhibition of cell proliferation was found. Kinetic studies revealed that the Cd-sensitive step is an early event of T cell stimulation. Failure of IL2 secretion and reduction of IL2 receptor expression in the Cd-treated cells are also reported. Regardless of which mechanism is responsible for Cd effects, our studies show that the inhibition of lymphocyte activation is associated with reduced [3H]phorbol dibutyrate binding to Ca2+-phospholipid-dependent protein kinase and altered breakdown of phosphatidylinositols. Thus, Cd interferes with two biochemical events which play a critical role in lymphocyte signal transduction and activation.

In vivo cadmium treatment alters natural killer activity and large granular lymphocyte number in the rat.

The effects of oral exposure for nearly 6 months to Cd (200 or 400 ppm) on rat natural killer (NK) activity were investigated. No significant differences in body and thymus weights were observed. Peripheral blood lymphocyte (PBL) number was consistently higher in treated rats throughout the treatment; the number of spleen cells decreased during the first 50 days, and then reached a level higher than in controls. NK activity, evaluated in a 4-h chromium release assay against YAC-1 target cells, was altered in treated rats: lower up to day 30, and then higher. In parallel, a reduction of the large granular lymphocytes (LGL) was found initially in the peripheral blood of Cd-treated rats, followed by a persistent marked increase. These changes were closely correlated with the altered distribution of CD8+ aGM1+ CD5- cells, which mostly consist of NK cells. Fluorescence-activated cell sorter (FACS) analysis revealed a decrease of cell subset with a typical NK phenotype during the first 30 days of treatment and a clear increase thereafter. Post-exposure observations indicated that all these effects disappeared with a return to control values 2 months after cessation of treatment. These findings suggest that in vivo administration of Cd induces both inhibitory and stimulatory effects on rat NK cell number and cytotoxic activity, depending on time of exposure.

Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): Role of PK-C on natural killer (NK) activity

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.

The growth-related gene product β induces sphingomyelin hydrolysis and activation of c-Jun N-terminal kinase in rat cerebellar granule neurones

The growth-related gene product β (GROβ) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GROβ receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GROβ receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GROβ activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GROβ-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.

Natural killer activity and antibody-dependent cellular cytotoxicity in progressive systemic sclerosis

Enhanced natural killer (NK) activity and normal lymphocyte antibody‐dependent cellular cytotoxicity (ADCC) were observed in 16 patients with a diagnosis of progressive systemic sclerosis (PSS). Higher NK activity levels were observed against NK‐sensitive K562 target cells, while the NK‐resistant P815, Daudi and Raji cell lines were not lysed. Cytofluorimetric studies and morphological analysis of peripheral blood lymphocytes (PBL) showed an increased number of CD16 positive cells and large granular lymphocytes (LGL), indicating that the enhancement observed was probably attributable to an increase in the number of circulating NK cells.

Laminin synthesis by NK cells and modulation of its expression by TPA (12-O-tetradecanoylphorbol-13-acetate)

Natural killer (NK) cells have been suggested to play a major role in resistance against metastatic spread of tumors. This study was aimed at understanding whether laminin (LM), a component of the extracellular matrix involved in the mechanism of tumor invasion and cell interaction, is expressed by NK cells. The results indicate that NK cells can synthesize and display on the cell surface LM and that TPA can modulate its expression. Our findings suggest that the presence of LM on NK cells could be relevant in the control of tumor invasion by NK cells.