Purinergic Signaling on Leukocytes Infiltrating the LPS-Injured Lung

<div><p>Extracellular nucleotides and nucleosides have been implicated as important signaling molecules in the pathogenesis of acute lung injury (ALI). While adenosine is known to inhibit T cell activation, little information is available as to ATP and NAD degrading enzymes, the expression of ATP and adenosine receptors/transporters in different T cell subsets. ALI was induced by challenging mice with intra-tracheal instillation of 60 µl (3 µg/g) LPS. After 3 d and 7 d blood, lung tissue and bronchoalveolar lavage was collected and immune cells were analyzed using flow cytometry. The transcriptional phenotype of T helper cells, cytotoxic and regulatory T cells sorted by FACS was assessed by measuring the expression profile of 28 genes related to purinergic signaling using TaqMan Array Micro Fluidic Cards. Catabolism of ATP, NAD and cAMP by activated CD4⁺ T cells was evaluated by HPLC. CD73 was found to be highly abundant on lymphoid cells with little abundance on myeloid cells, while the opposite was true for CD39. After ALI, the abundance of CD39 and CD73 significantly increased on all T cell subsets derived from lung tissue and bronchoalveolar space. Expression analysis in T cell subsets of the lung revealed ATP (<i>Cd39</i>, <i>Cd73</i>) and NAD (<i>Cd38</i>, <i>Cd157</i>, <i>Cd296</i>, <i>Pc-1</i>) degrading enzymes. However, only transcription of <i>Cd38</i>, <i>Cd39</i>, <i>Cd73</i>, <i>Ent1</i> and <i>A2a receptor</i> was significantly upregulated after ALI in T helper cells. CD4⁺ T cells from injured lung rapidly metabolized extracellular ATP to AMP and adenosine but not NAD or cAMP. These findings show that lung T cells – the dominant cell fraction in the later phase of ALI – exhibit a unique expression pattern of purinergic signaling molecules. Adenosine is formed by T cells at an enhanced rate from ATP but not from NAD and together with upregulated A2a receptor is likely to modulate the healing process after acute lung injury.</p></div

"Imposition et liberté d’exercice du culte », note sous 8 décisions CEDH (depuis CEDH, 29 sept. 2010, n° 8916/05, Les Témoins de Jéhovah c. France) et une Cass. (Com, 15 janv. 2013, n° 12-11.642, Assoc. L’Arche de Marie) in chronique de jurisprudence fiscale

<p>(A+B) The percentage of cells expressing CD73 was high within the T cell subsets but low within the myeloid cell, B cell and NK cell populations. The percentage of CD73 expressing cells tended to be increased in T helper cells after ALI. (C+D) As assessed by means of the MFI CD73 was highly expressed on the different T cell subsets and showed a comparatively low expression on myeloid cells, B cells and NK cells. After LPS installation particular T helper cells, NKC and M&M showed an increased abundance of CD73. Data are mean ± SD (n = 5 mice per group). Statistical significance was assessed by one-way ANOVA with Dunnett's post hoc test. *P<0.05, **P<0.01, ***P<0.0001. Under unstressed conditions CD73 staining of regulatory T cells (IS) and T helper cells (BAL) was detected in only n = 2, thus statistical significance was not assessed. ALI  =  acute lung injury, AM  =  alveolar macrophages, APC  =  antigen-presenting cells, BAL  =  bronchoalveolar lavage, BC  =  B cells, CTC  =  cytotoxic T cells, Gr  =  granulocytes, IS  =  interstitial lung tissue, MFI  =  mean fluorescence intensity, M&M  =  monocytes and macrophages, n.d.  =  not detected, NKC  =  natural killer cells, SD  =  standard deviation, THC  =  T helper cells, Treg  =  regulatory T cells.</p

Gene expression of adenosine and ATP receptors in the T cell subsets isolated from the lung under basal conditions and 7d after LPS exposure determined by quantitative <i>real-time</i> PCR.

<p>(A) In the unstressed lung, T cells expressed predominantly the <i>A2a receptor</i>. The <i>P2x4</i> and <i>P2y6 receptors</i> were moderately expressed while <i>P2x5/7</i> and <i>P2y2 receptors</i> displayed a very low expression level. Gene expression was normalized to beta-actin and relative expression levels are depicted. (B) LPS administration significantly induced the <i>A2a receptor</i> expression in T helper cells 7 d post instillation. Since expression of some target mRNAs was below the detection limit in at least one of the conditions, fold changes (control vs. 7 d) were not calculated but the relative expression levels were depicted. Data are mean ± SD (n = 4 mice per group). Expression levels under basal conditions were compared to that in the injured lung 7 d post induction of ALI and statistical significance was then assessed by Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.0001. ALI  =  acute lung injury, ATP  =  adenosine triphosphate, AU  =  arbitrary units, LPS  =  lipopolysaccharide, n.d.  =  not detected, SD  =  standard deviation.</p

Gene expression of distinct ectoenzymes, transporters and channels in the T cell subsets isolated from lung tissue under basal conditions and 7<i>real-time</i> PCR.

<p>(A) Under basal conditions, T cell subsets expressed various ectoenzymes of ATP and NAD degradation cascade as well as nucleotide and nucleoside transporters and channels. Gene expression was normalized to beta-actin and relative expression levels are depicted. (B) In the diseased state, <i>Cd38</i>, <i>Cd39</i> and <i>Cd73</i> were significantly upregulated on T helper cells and tended to be increased in cytotoxic and regulatory T cells. <i>Ent1</i> transcripts were significantly increased in T helper cells. Data are mean ± SD (n = 4 mice per group). Fold changes were calculated by comparing the expression under basal conditions to that in the injured lung 7 d post induction of ALI and statistical significance was then assessed by Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.0001. ALI  =  acute lung injury, Art2.b  =  ADP-ribosyltransferase 2b, ATP  =  adenosine triphosphate, AU  =  arbitrary units, Cnt  =  concentrative nucleoside transporter, Cx  =  connexin, Ent  =  equilibrative nucleoside transporter, LPS  =  lipopolysacch, NAD  =  nicotinamide adenine dinucleotide, Panx-1  =  pannexin-1, SD  =  standard deviation.</p

Ratios ( = fold changes) of CD39 and CD73 expression (percentage and MFI) on immune cell subsets in IS compared to IV (IS/IV) and in BAL compared to IS (BAL/IS) 7 d post induction of ALI.

<p>(A+B) Portion of CD39 and CD73 expressing cells is particularly increased within the T cell subsets from IS compared to that from IV 7 d post induction of ALI. Analysis of the ratio BAL/IS revealed that CD73 expressing cells were significantly augmented within the T cells subsets while changes in CD39 positive cells were small. (C+D) Expression levels of CD39 and CD73 assessed by MFI were significantly upregulated on nearly all immune cell subsets from IS compared to that from IV 7 d post induction of ALI. A significant increased expression of CD73 was found on granulocytes and cytotoxic T cell when analyzing the ratio BAL/IS. Data are mean ± SD (n = 5 mice per group). Ratios were calculated by dividing the fraction of positive cells or MFI of immune cell subsets in IS by that in IV (IS/IV) or in BAL by that in IS (BAL/IS) 7 d after induction of ALI. Statistical significance was assessed by Mann-Whitney U test. *P<0.05, **P<0.01, ***P<0.0001. ALI  =  acute lung injury, AM  =  alveolar macrophages, APC  =  antigen-presenting cells, BAL  =  bronchoalveolar lavage, BC  =  B cells, CTC  =  cytotoxic T cells, Gr  =  granulocytes, IS  =  interstitial lung tissue, IV  =  intravascular space, MFI  =  mean fluorescence intensity, M&M  =  monocytes and macrophages, n.d.  =  not detected, NKC  =  natural killer cells, SD  =  standard deviation, THC  =  T helper cells, Treg  =  regulatory T cells.</p

Dynamic changes of distinct leukocyte subpopulations in lung tissue (IS) and bronchoalveolar lavage (BAL) 3 d and 7 d after intratracheal instillation of LPS.

<p>(A) Representative plots illustrating the gating strategy used to identify different leukocyte subpopulations by flow cytometry. Non-viable immune cells were excluded from the CD45⁺ cells on the basis of a positive DAPI-staining. Viable CD45⁺ cells were then divided into subpopulations using a panel of cell-specific fluorochrome-labeled antibodies. Lymphocytes were gated for CD45R(B220)⁺ cells (B cells) and CD3⁺ cells (T cells). T cells were subdivided into CD4⁺ cells (T helper cells) and CD8⁺ cells (cytotoxic T cells). Myeloid cells were defined as CD11b⁺ cells and further subdivided into CD11c⁺ cells (APCs) and Ly6g⁺ cells (granulocytes) (B) In IS the early phase of inflammation (3 d) was characterized by a transient increase in myeloid immune cell subsets. In the later phase of inflammation (7 d), different T cell subsets were significantly enhanced. (C) Similar to the lung tissue, myeloid immune cell subsets were increased in BAL in the early phase while T cells were enhanced only in the later phase of inflammation. Data are mean ± SD (n = 5 mice per group) Statistical significance was assessed by one-way ANOVA with Dunnett's post hoc test. *P<0.05, **P<0.01, ***P<0.0001. ALI  =  acute lung injury, AM  =  alveolar macrophages, APC  =  antigen-presenting cells, BAL  =  bronchoalveolar lavage, BC  =  B cells, CTC  =  cytotoxic T cells, Gr  =  granulocytes, IS  =  interstitial lung tissue, LPS  =  lipopolysaccharide, M&M  =  monocytes and macrophages, n.d.  =  not detected, NKC  =  natural killer cells, SD  =  standard deviation, THC  =  T helper cells, Treg  =  regulatory T cells.</p

Extracellular degradation of ATP, AMP, NAD, cAMP, ADPR (substrate concentration: 20 µM; 37°C) by activated CD4⁺ T cells isolated from the lung 7 d post induction of ALI.

<p>(A) Rapid ATP degradation to ADP and AMP and slow adenosine formation. (B) AMP only was added as substrate was slowly degraded with concomitant formation of adenosine and inosine. (C) NAD is degraded to ADPR at a moderate rate. (D) CD4⁺ T cells do not degrade ADPR and cAMP. ADO  =  adenosine, ADP  =  adenosine diphosphate, ADPR  =  adenosine diphosphate ribose, ALI  =  acute lung injury, AMP  =  adenosine monophosphate, ATP  =  adenosine triphosphate, cAMP  =  cyclic adenosine monophosphate, INO  =  inosine, NAD  =  nicotinamide adenine dinucleotide.</p