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Dendritic cells (DC) express a number of P2X receptors including the P2X₇/P2Z receptor whose activation by extracellular adenosine 5′-triphosphate (ATP) induces the influx of calcium, DC maturation, cytokine release and apoptosis. In B lymphocytes ATP induces the rapid shedding of CD23 and CD62 ligand by activating a membrane metalloprotease. In this study, we examined the expression and early effects of P2X₇ receptor activation on monocyte-derived DC, generated from individuals either wild-type or homozygous for a loss-of-function single nucleotide polymorphism at position 1513 of the P2X7 gene. Labeling with an anti-human P2X₇ receptor mAb demonstrated that DC express the P2X₇ receptor at a lower level than macrophages. Short-term incubations (5 min) of DC with ATP induced an influx of ethidium⁺ (314 Da) into wild-type DC, but not into DC homozygous for the loss-of-function polymorphism. In contrast to results with ethidium⁺, ATP did not induce the influx of the viability dye propidium²⁺ (415 Da) into DC in short-term incubations. Addition of ATP also induced a rapid loss of CD23 from the surface of wild-type DC (t_1/2 < 120 s), and this loss was inhibited by oxidized ATP and KN-62 which are known P2X₇ receptor antagonists. Moreover, ATP-induced shedding of CD23 was slower from DC homozygous for the loss-of-function polymorphism than from wild-type DC. The data show that monocyte-derived DC express the P2X₇ receptor whose activation opens a cation-selective channel, and which leads to rapid and near complete shedding of CD23. Both of these functions of the P2X₇ receptor are impaired on DC from subjects who are homozygous for the loss-of-function 1513 polymorphism.

The permeability of lymphocytes to NH+4was examined by measuring intracellular pH using the fluorescent pH-sensitive dye BCECF. Addition of 20 InM NH4Cl produced a rapid phase of alkalinization. This was followed by a slow return to resting pHidue to NH+4influx. The rate of NH+4was increased many fold by extracellular ATP and the increment showed features consistent with NH+4being a permeant for the P2Zpurinoceptor operated ion channel. Cytosolic pH measurements showed monomethylammonium+and dimethylammonium+were also permeants, but trimethylammonium+(69 Dalton) was excluded by this channel. Since our previous data showed ethidium+(314 Dalton) is a permeant it appears that molecular conformation rather than molecular weight determines entry of cationic solutes through the channel.

It is now recognized that extracellular ATP can open a receptor-operated ion channel in a variety of cell types. In human lymphocytes this P₂Z purinergic channel conducts Na+, K+, Rb+, Li+, and Ca2+ but its permeability to larger cations is not known. Fluorometric measurements were used to show that ATP4− induced the entry of Sr2+ (87 Da) and Ba2+ (137 Da) into human lymphocytes loaded with fura-2. Flow cytometry was used to show that ATP4− induced the entry of ethidium+ (314 Da) but that the larger propidium2+ cation (414 Da) was excluded. ATP4− induced entry of both Ba2+ and ethidium+ showed features previously demonstrated for smaller cation permeants: (i) inhibition by amiloride analogs, (ii) sigmoid dependence of flux on ATP concentrations, and (iii) inhibition by extracellular Na+ ions. Specific inhibitors of L-type voltage-gated Ca2+ channels (nisoldipine and diltiazem) had no effect on ATP4−-induced Ba2+ influx. Suramin and reactive blue 2, which are recognized antagonists of ATP-operated purinergic receptors in other tissues, inhibited ATP-induced uptake of ethidium+ in lymphocytes with K1/2 of 61 and 69 µM, respectively. However, hexamethylene amiloride was a more potent inhibitor of ATP-induced ethidium+ uptake with a K1/2 of 13 µM. These data show that the ATP4− receptor-operated channel of human lymphocytes allows influx of cations as large as Ba2+ or ethidium+ and that this influx is inhibited by suramin and reactive blue 2.Copyright 1993, 1999 Academic Press

Platelet-associated immunoglobulin (IgG) has been measured directly on platelets from normal and thrombocytopenic subjects by a modified radioactive Coombs technique. Platelet-associated IgG was detected using 125l-staphylococcal protein A after which platelets were separated from unbound 125l-protein A by centrifu-gation through oil. Elevated values of platelet-associated IgG were observed in 17 of 19 patients with autoimmune thrombocytopenic purpura. All patients with thrombocytopenia associated with systemic lupus erythematosus or rheumatoid arthritis showed elevated platelet-associated IgG, while normal values were found in diverse thrombocytopenias which were non-immunological in origin. Measurement of anti-platelet antibody levels by an indirect test was less reliable in diagnosing autoimmune thrombocytopenic purpura, since only 7 of the 19 patients gave elevated values. However, the indirect test was reliable in the diagnosis of quinine-dependent immune thrombocytopenia; all 6 patients with suspected quinine purpura gave strongly positive indirect tests when 1mM quinine was added to the patient's plasma. The use of 125I-protein A to detect platelet-associated IgG provides a rapid and simple technique for the reliable diagnosis of immune thrombocytopenia.

The binding of the cardiac glycoside, ouabain, to cells had been used to quantify the number of active cation pumps. In this study, lymphocytes were incubated with 3H-ouabain and the equilibrium binding analyzed for the maximal number of specific binding sites. Lymphocytes from normal peripheral blood bound 44,200 +/- 9920 molecules/cell, compared with 29,200 +/- 8370 molecules/cell for the lymphocytes of chronic lymphocytic leukemia (CLL) subjects. This difference was significant (p less than 0.01) and did not reflect a lower number of sites on B cells than T cells, since B-cell-enriched lymphocytes from normal peripheral blood showed the same ouabain binding characteristics as the standard T-cell-rich preparation. Although monocytes bind threefold more ouabain than lymphocytes, the small monocyte contamination (3.0%) in normal lymphocyte preparations could not account for the difference between normal and CLL. The fewer ouabain binding sites on CLL lymphocytes may reflect both their smaller size (by 10%) and lower mitotic activity compared with lymphocytes from normal peripheral blood.