Nucleotides are emerging as an ubiquitous family of extracellular signaling molecules. It has been known for many years that adenosine diphosphate is a potent platelet aggregating factor, but it is now clear that virtually every circulating cell is responsive to nucleotides. Effects as different as proliferation or differentiation, chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species are

Anedoctal evidence accumulated over almost 20 years has shown that many different cell types are killed by sustained exposure to high concentrations of extracellular ATP. The plasma membrane receptors involved have been pharmacologically characterized and cloned during the last 3 years, and named purinergic P2X. P2X receptors share an intriguing structural relatedness with Caenorhabditis elegans degenerins and mammalian amiloridesensitive Na channels (ENaCs). Depending on the ATP dose, length of stimulation and receptor subtype, P2X receptor stimulation may cause necrosis or apoptosis. The intracellular pathways activated are poorly known, but the perturbation in intracellular ion homeostasis clearly plays a major role. ICE proteases (caspases) are also triggered, nonetheless their activation is not requested for ATP-dependent cell death. The physiological meaning of P2X receptor-dependent cytotoxicity is not understood, but an involvement in immune-mediated reactions is postulated.

Human leukocytes can express the P2X 7 purinergic receptor, an ionic channel gated by extracellular ATP, for which the physiological role is only partially understood. Transfection of P2X 7 cDNA into lymphoid cells that lack this receptor sustains their proliferation in serum-free medium. Increased proliferation of serumstarved P2X 7 transfectants is abolished by the P2X 7 receptor blocker oxidized ATP or by the ATP hydrolase apyrase. Both wild type and P2X 7 -transfected lymphoid cells release large amounts of ATP into the culture medium. These data suggest the operation of an ATP-based autocrine/paracrine loop that supports lymphoid cell growth in the absence of serum-derived growth factors.Human and mouse leukocytes express the purinergic P2X 7 receptor (1-4). There is evidence that this plasma membrane receptor/pore participates in various macrophage, microglia, and dendritic cell responses such as plasma membrane permeabilization, cytokine release, multinucleated giant cell formation, and apoptosis (5-10), but its physiological function in lymphocytes is unknown. Several authors have proposed extracellular ATP as a novel mediator of cell proliferation, and evidence for such a role also in lymphoid cells has been presented in the past (11)(12)(13)(14). Although the P2 receptor subtype involved has never been clearly defined, it is generally believed that the growth stimulating effects of ATP are mediated by P2Y receptors. In a previous study we showed that human peripheral T lymphocytes express a purinergic receptor of the P2X 7 subtype, and we made the surprising observation that its blockade severely decreased the proliferation stimulated by anti-CD3 antibodies, phytohemagglutinin, or allogeneic cells (15). Therefore, we put forward the suggestion that P2X 7 receptors could also mediate a proliferation signal. During the last 2 years, cDNAs encoding the rat, mouse, and human P2X 7 receptor have become available, thus allowing investigation of the effect of P2X 7 transfection on cell proliferation (16 -19). In our laboratory we have thoroughly characterized the P2 receptor of several human lymphoid cell lines, among which two, K562 and LG14, lack endogenous P2X 7 receptors as well as functional P2Y receptors (19). In the present study we have compared the proliferation rate and measured ATP release from wild type and P2X 7 -transfected K562 and LG14 cells. Our data show that P2X 7 transfection enhances cell proliferation in the absence of exogenous growth factors and that this effect depends on autocrine/paracrine stimulation by released ATP. EXPERIMENTAL PROCEDURESCells and Solutions-K562 leukemic cells and the LG14 B-lymphoblastoid cell line were grown in Iscove's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 10 units/ml penicillin. [Ca 2ϩ

The expression and importance of the non-classical human leukocyte antigen (HLA) class Ib gene, HLA-G, at the feto-maternal interface have been recognized. The HLA-G molecule is almost monomorphic and expressed in both membrane-bound and soluble isoforms. It has been shown to inhibit NK-mediated cell lysis and influence cytokine expression. Recently, a possible boarder immunoregulatory function of HLA-G also in adult life has been recognized. HLA-G gene polymorphism has been linked to differences in gene expression profile of alternatively spliced HLA-G transcripts and levels of specific HLA-G mRNA isoforms. On this background it is of general interest to further elucidate any associations between HLA-G polymorphism and protein expression. We have HLA-G genotyped 85 individuals attending IVF treatment, and further studied sHLA-G1/HLA-G5 and interleukin-10 (IL-10) in serum samples. In 21% of the serum samples sHLA-G1/HLA-G5 could be detected. There was no correlation between sHLA-G1/HLA-G5 and IL-10 concentrations in serum. Soluble HLA-G1/HLA-G5 was not detected in any samples homozygous for a 14-bp insertion polymorphism in exon 8 of the 3'-untranslated region (3'UTR) of the HLA-G gene ( P=0.03; Fisher's exact test). Polymorphisms in the 5'-upstream regulatory region (5'URR) of the HLA-G gene were also studied. In conclusion, this study indicates that polymorphisms in the 3'UTR and the 5'URR of the HLA-G gene may influence the expression of sHLA-G of possible importance in pathological pregnancies and also in organ transplantation.

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