Metalloprotease inhibitors GM6001 and Ro 31–9790 inhibit PMA/Iono- and ATP-induced shedding of CD62L on MRL^+/+ T lymphocytes.

<p>Spleen cells from MRL<i>^+/+</i> and MRL/<i>lpr</i> mice (n = 5 mice/group) were preincubated with or without 10, 25, 50, 100 µM GM6001 or 3, 30, 100 µM Ro 31–9790 for 15 min at 37°C, and then either left unstimulated (□) or stimulated with 500 µM ATP (▴) or 5 ng/ml PMA plus 0.5 µg/ml Ionomycin (○) for 45 min at 37°C. Cells were subsequently stained with anti-CD90 and anti-CD62L mAb. Cell surface expression of CD62L was assessed in the gated CD90⁺ T cells by flow cytometry.</p

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220⁺ Double-Negative T Lymphocytes of Autoimmune MRL/<em>lpr</em> Mice

<div><p>Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including <em>Fas Ligand</em> (FasL) and <em>P2X7 receptor</em> (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/<em>lpr</em> mice (<em>lpr</em> mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220⁺ CD4^–CD8^– (DN) T lymphocytes. The precise ontogeny of B220⁺ DN T cells is unknown. B220⁺ DN T lymphocytes could be derived from effector CD4⁺ and CD8⁺ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/<em>lpr</em> mice carry a <em>P2X7R</em> allele, which confers a high sensitivity to ATP. However, during aging, the MRL/<em>lpr</em> T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220⁺ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220⁺ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220⁺ T cells observed in normal MRL^+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220⁺ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.</p> </div

P2X7R antagonist KN-62 inhibits ATP-induced CD62L shedding and pore formation in MRL^+/+ T lymphocytes.

<p>Spleen cells from 3 to 4-mo-old MRL<i>^+/+</i> mice (n = 4 mice/group) were preincubated for 15 min at 37°C with or without 0.5, 1 and 5 µM KN-62 and then stimulated with 500 µM ATP for 30 min. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L or YO-PRO-1 to assess by flow cytometry: (A) CD62L shedding or (B) pore formation in the gated CD90⁺ T-cell population stimulated (▪) or not (□) with ATP. Asterisks denote statistically significant differences between ATP-stimulated and unstimulated groups: *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

Real-time kinetic analysis of calcium influx in B220^– and B220⁺ T lymphocytes stimulated with ATP.

<p>Spleen cells from 4-mo-old MRL<i>^+/+</i> (<i>red</i>) MRL/<i>lpr</i> (<i>blue</i>) and B6<i>^P2X7−/−</i> (<i>black</i>) mice (n = 3 mice/group) were loaded with calcium-reactive fluorescence probe, Oregon Green 488 BAPTA-1. After loading, cells were stained with anti-CD90, anti-CD19 and anti-B220 mAbs. Baseline MFI of the calcium probe was recorded in the gated B220^–CD90⁺ (○) and B220⁺CD90⁺ (▵) T cells during 50 s by flow cytometry, and was followed by the addition of 500 µM ATP (at time t0, arrow). Changes over time in MFI values of the calcium probe were monitored in the T-cell subsets by flow cytometry. MFI values were normalized by subtracting the baseline MFI.</p

Expression of P2X7R and CD39 in T-cell subsets.

<p>(A) The levels of CD39 expression on B220^– (either CD4⁺ or CD8⁺) (□) and B220⁺ DN (▪) T-cell subsets as well as on CD19⁺ B cells (▪) from MRL/<i>lpr</i> mice (n = 3) were analyzed by flow cytometry. Results shown on histogram are normalized MFI (nMFI) calculated as MFI of positive cells × percentage of positive cells. Asterisks denote statistically significant differences between B220⁺ DN T cells or B220^–CD8⁺ T cells and B220^–CD4⁺ T cells: *<i>p</i>≤0.05; **<i>p</i>≤0.01. (B) P2X7R mRNA expression was analyzed in FACS-purified B220^– (□) and B220⁺ (▪) CD19^–CD90⁺ T-cell subsets from individual B6, MRL^+/+ and MRL/<i>lpr</i> mice by real-time RT-PCR. Specific P2X7R cDNA was quantified by standard curves based on known amounts of PCR-amplified P2X7R cDNA. Data are expressed as amount (pg) of P2X7R RNA per cell, and histograms represent the mean ± SE of three independent experiments (n = 8 mice/group). (C) P2X7R protein levels in whole LN cells from 3 to 4-mo-old MRL<i>^+/+</i>, MRL/<i>lpr</i> and B6<i>^P2X7−/−</i> mice, and FACS-sorted B220^– and B220⁺ CD19^–CD90⁺ T-cell subsets from MRL/<i>lpr</i> mice were analyzed by western blotting using affinity-purified rabbit anti-P2X7R polyclonal antibodies (1∶1000; Alomone Laboratories). A non-specific band (*) is frequently observed with this polyclonal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052161#pone.0052161-Auger1" target="_blank">[22]</a>. The blot was stripped and reprobed with anti-actin mAb. (D and E) Flow cytometric analysis of P2X7R expression levels on B220^– and B220⁺ T-cell subpopulations. Spleen cells from MRL<i>^+/+</i>, MRL/<i>lpr</i> and B6<i>^P2X7R−/−</i> mice (n = 3 mice/group) were stained with anti-CD90 mAb, anti-B220 mAb, rabbit polyclonal anti-P2X7R antiserum (1∶100) generated by genetic immunization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052161#pone.0052161-Gonnord1" target="_blank">[34]</a> and with either anti-CD4 mAb, anti-CD8 mAb or anti-CD4 plus anti-CD8 mAbs. (D) Overlay histograms showing the expression levels of P2X7R on gated CD4 T cells or CD8 T cells from MRL<i>^+/+</i> mice (open histogram) and B6<i>^P2X7R−/−</i> mice (shaded histogram). (E) Overlay histograms comparing the expression levels of P2X7R on B220^– (–) and B220⁺ (–) T cells, either CD4⁺ (<i>left</i>) or CD8⁺ (<i>middle</i>), from MRL/<i>lpr</i> mice. <i>Right</i>, overlay histogram comparing the expression levels of P2X7R on B220⁺ DN T cells (–) and B220^– CD4⁺ T cells (–) from MRL/<i>lpr</i> mice. The results shown are representative of at least four independent experiments.</p

P2X7R activity in T lymphocytes from MRL/lpr mice at early and late stages of the disease, and in B220⁺ and B220^– T lymphocytes from MRL^+/+ and MRL/lpr mice.

<p>(A and B) Spleen cells from 4-mo-old MRL<i>^+/+</i> and 2- and 4-mo-old MRL/<i>lpr</i> mice (n = 3 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) or 50 µM NAD (▪) for 45 min at 37°C. Cells were then double-stained with anti-CD90 mAb and either Annexin V or YO-PRO-1 to assess PS exposure and pore formation, respectively, in the gated CD90⁺ T-cell population by flow cytometry. (C–E) Spleen cells from 3 to 5-mo-old MRL^+/+ and MRL<i>/lpr</i> (n = 5 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) or 50 µM NAD (▪) for 45 min at 37°C. Cells were then triple-stained with anti-CD90, anti-B220 and either anti-CD62L mAb, YO-PRO-1 or 7-AAD to assess CD62L shedding (C), pore formation (D) or cell death (E) in B220^–CD90⁺ and B220⁺CD90⁺ T-cell subsets. Asterisks denote statistically significant differences between ATP- or NAD-stimulated and unstimulated groups: **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

Dose–response experiments for ATP-induced CD62L shedding on T lymphocytes.

<p>Spleen cells from 3 to 4-mo-old MRL<i>^+/+</i> (<i>solid line</i>), MRL/<i>lpr</i> (<i>dashed line</i>) and P2X7-deficient B6<i>^P2X7R−/−</i> (<i>dotted line</i>) mice (n = 3 mice/group) were treated for 45 min at 37°C with doses of ATP ranging from 100 to 5000 µM. Spleen cells were then triple-stained with anti-CD90, anti-CD19 and anti-CD62L mAb to assess by flow cytometry: (A) the percentage of CD62L-expressing CD19^–CD90⁺ T cells and (B) MFI of CD62L on CD19^–CD90⁺ T cells. Results are expressed as the mean percentage of initial expression ± SE.</p

ATP-induced shedding of CD62L in CD4⁺, CD8⁺ and DN T lymphocytes expressing the transmembrane tyrosine phosphatase B220.

<p>Spleen cells from 3 to 5-mo-old MRL<i>^+/+</i>, MRL<i>/lpr</i>, B6 and B6/<i>lpr</i> mice (n = 5 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) for 45 min at 37°C. Spleen cells were then stained with anti-CD90, anti-B220, anti-CD62L and anti-CD4 or anti-CD8 mAb to assess CD62L expression on B220<i>^–</i> and B220⁺ CD90⁺ T cells (either CD4⁺ or CD8⁺) from MRL<i>^+/+</i>, MRL<i>/lpr</i>, B6 and B6/<i>lpr</i> mice as well as on B220⁺ DN CD90⁺ T cells from MRL<i>/lpr</i> and B6/<i>lpr</i> mice. Asterisks denote statistically significant differences between ATP-stimulated and unstimulated groups: **<i>p</i>≤0.01; ***<i>p</i>≤0.001.</p

P2X7R activity in T lymphocytes from normal MRL^+/+ and autoimmune MRL/lpr mice.

<p>Spleen cells from 3 to 4-mo-old MRL<i>^+/+</i> (□) and MRL/<i>lpr</i> (▪) mice (n = 5 mice/group) were incubated with or without 500 µM ATP for 45 min (A–C) or 4 h (D) at 37°C. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L mAb, YO-PRO-1, Annexin V or 7-AAD to assess in T cells by flow cytometry: (A) CD62L shedding; (B) pore formation; (C) cell surface PS exposure; (D) cell death. Numbers reported in the dot plots indicate the percentages of CD62L⁺CD90⁺ or YO-PRO-1⁺CD90⁺ cells in the gated CD90⁺ T-cell population. Histograms correspond to the mean percentages ± SE (n = 5 mice/group) of double-stained cells in the gated CD90⁺ T-cell population. Asterisks denote statistically significant differences (***<i>p</i>≤0.001) between ATP-stimulated and unstimulated groups.</p

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<p>A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5′-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RB^low Tconvs, but not CD45RB^high Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RB^low Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RB^highCD44^low to effector/memory CD45RB^lowCD44^high stage. Maximum levels of upregulation are reached on recently activated CD69⁺ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69⁺ CD45RB^highCD44^low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RB^highCD44^low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RB^low Tconvs, CD45RB^lowFoxp3⁺ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.</p