The ATP-gated P2X7 purinergic receptor (P2X7R) is predominantly expressed in cells of hematopoietic origin including macrophages and T lymphocytes. Roles for the P2X7R have been identified in the regulation of various pro-inflammatory and immune responses. An unusual feature of the P2X7R is its high threshold for activation by extracellular ATP (EC50 ~ 500 μM); this contrasts with much lower activation thresholds for the other six members of the P2X family (EC50 ~ 10 μM). Recent studies have demonstrated that extracellular NAD induces the ATP-independent activation of the P2X7R in murine T lymphocytes via ADP-ribosylation of arginine residues on the P2X7R ecto-domain. This modification is catalyzed by ART2.2, a GPI-anchored ADP¬ribosyltransferase (ART) that is constitutively expressed in murine T cells. Although the NAD-induced, ART2-dependent mechanism is clearly a major pathway for P2X7R activation in mouse T lymphocytes, it is unclear whether this mechanism is operative in macrophages, another class of leukocytes which natively express P2X7R at high levels. The studies described in this dissertation addressed this question in following two folds.
The first series of studies (Chapter 3) demonstrated that bone-marrow derived macrophages (BMDM) from BALB/c mice selectively up-regulate thiol-dependent ART2.1 in response to multiple proinflammatory mediators including agonists for toll-like receptors (TLR) and type-1/2 interferons. LPS-induced ART2.1 expression involved regulation by multiple kinase pathways or possibly IFN-β autocrine signaling. A related group of studies (Chapter 5) showed expression of ART2.1 in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid antigen-prensenting leukocytes.
In contrast with T cells, stimulation of naïve or inflammatory macrophages with NAD alone did not activate the P2X7R. Rather, NAD potentiated ATP-dependent P2X7R activation in inflammatory macrophages as indicated by a left-shift in the ATP dose-response relationship (Chapter 4). These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X7R signaling in murine macrophages and that the cellular context in which P2X7R signaling occurs differs between myeloid versus lymphoid leukocytes.
Collectively, this dissertation demonstrated that the regulation of ATP-dependent P2X7R activation by NAD/ART2.1 provides an additional layer of regulatory control in multiple phases of innate and adaptive immunity.