Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins

Abstract

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (Met-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (0;) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced 0; formation induced by submaximally and maximally stimulatory concentrations of Met-Leu-Phe up to fivefold. On the other hand, Met-Leu-Phe primed neutrophils to generate 0; upon exposure to nucleotides. At a concentration of 100 pM, purine nucleotides enhanced 0; formation in the effectiveness order adenosine 5'-0-[3-thio]triphosphate (ATP[yS]nucleotides were similarly effective potentiators of 0; formation as the corresponding adenine nucleotides. GDP[PS] and UDP[PS] synergistically enhanced the stimulatory effects of ATP[yS], GTP[yS] and UTP[yS]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated Met-Leu-Phe-induced release of P-glucuronidase with similar nucleotide specificities as for 0; formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and Met-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation Human neutrophils play a major role in host defense reactions against bacterial infections and in the pathogenesis of various diseases such as rheumatoid arthritis, glomeruloncphritis, dermatoses, myocardial infarction and asthma [l]. The intercellular signal molecules, PAF, LTB4 and the cheinotactic peptide, Met-Leu-Phe, stimulate NADPHoxidase-catalyzed 0; formation [2 -51. Combinations of Met-Leu-Phe plus PAF or LTB4 synergistically induce 0 : formation [2]. In addition to 0; formation, these agents induce degranulation and aggregation [2 -51. Receptors for Met-Leu-Phe, PAF and LTB4 interact with G proteins lead-

Nucleotide-, Chemotactic Peptide- and Phorbol Ester-Induced Exocytosis in HL-60 Leukemic Cells

“…With respect to O2" formation in Bt2cAMP-differentiated HL-60 cells, both nucleotides are similarly potent (22). Differences concerning the potency and/or effectiveness of purine and pyrimidine nucleotides to activate various cell functions were also observed in human neutrophils (6). We did not analyze in detail the structure/activity relationship of purine and pyrimidine nucleotides with respect to Ca 2+ mobilization, but this may be an interesting task for future studies.…”

“…All cell culture media were obtained from Biochrom (Berlin, FRG). Sources of other materials have been described elsewhere (6,7,22,25,26).…”

“…Fura-2/AM was added at a concentration of 4 JiM and cells were incubated for 10 min at 37 0 C Thereafter, cells were diluted with the above buffer to a concentration of 5 XlO 6 cells/ml and were incubated for a period of 50 min at 37 0 C Subsequently, cells were diluted with the above buffer to a final concentration of 0.5 X IO 6 cells/ml and were centrifuged at 250 x g for 10 min at 20 °C. Cells were suspended at 5 x IO 6 cells/ml in the above buffer and were kept at 20 0 C until measurement of cytosolic Ca 2+ . HL-60 cells (2.5 x IO 6 cells) were suspended in 2 ml of the above buffer using acryl fluorescence cuvettes (Sarstedt, Numbrecht, FRG).…”

“…Aggregation was measured by turbidometry (6,33). Bt2cAMP-differentiated HL-60 cells (IxiO 7 cells) were suspended in 900 ul of the buffer used for the determination of Pglucuronidase release.…”

Human neutrophils do not express purinergic P2X7 receptors

“…Labasi et al [26] show that neutrophils from wild-type or P2X 7 R-deficient mice did not change shape and lack L-selectin shedding in response to ATP, suggesting the absence of surface expression of P2X 7 R. Other groups have found that differentiated HL-60 cells show expression of P2X 7 R, but their function (measured as an increase in intracellular Ca 2+ after stimulation with ATP) is not changed during development [1]. Finally, it has been reported that ATP by itself is unable to generate ROS in human neutrophils and differentiated HL-60 cells [39,43] and that P2X 7 R are stored in intracellular organelles susceptible for recruitment to plasma membrane after neutrophil activation [19]. In our hands, however, activation of human neutrophils (judged by cell migration and changes in cell morphology) with fMLP did not result in ATP-activated currents.…”