E2F1-/- C57BL/6 mice overexpressing a human Bcl-2 transgene in B cells develop a mild autoimmune syndrome.

Bcl-2, besides having an anti-apoptotic function, delays cell cycle progression at G1 to S. Overexpression of Bcl-2 in B cells induces an autoimmune syndrome (AIS) in pro-autoimmune genetic backgrounds. E2F1, a member of the E2F transcription factors, controls cell cycle, but it also induces cell death. E2F1(-/-) mice show an altered negative thymic selection but a conserved peripheral tolerance. As a consequence, these mice do not develop autoimmunity. Our aim was to evaluate whether deregulation of both apoptosis and cell cycle alters the mechanisms of tolerance and induces an AIS. C57BL/6 E2F1(-/-) mice were crossed with C57BL/6 mice overexpressing a human Bcl-2 transgene in B cells to obtain E2F1(-/-) hbcl-2 Tg mice. These mice were followed for up to 15 months of age with bleedings every three months to obtain serum and whole blood. The production of an AIS was assessed by quantitation of serum anti-DNA antibodies, renal light microscopy, and direct immunofluorescence in search of immunoglobulin deposits. E2F1(-/-) hbcl-2 Tg mice developed an AIS characterized by anti-DNA autoantibody production with renal damage observed after the 9th month of age. The lesions consisted mainly on cellular proliferation and mesangial deposits, compatible with a mesangial glomerulonephritis. The composition of deposits was predominantly of IgA, followed by IgM and IgG. Despite the development of renal damage, the AIS observed did not induce an accelerated mortality. The coexistence of an altered B cell apoptosis, together with the lack of E2F1, induces a mild AIS in the non-autoimmune background of C57BL/6 mice.

Activation of phospholipase D-2 by P2X(7) agonists in rat submandibular gland acini.

Exogenous ATP stimulated phospholipase D (PLD), but not sphingomyelinase in rat submandibular gland (SMG) acini. PLD activation was dependent upon extracellular Ca(2+) and did not involve intracellular Ca(2+) mobilization or phosphoinositide-specific phospholipase C activation. ATP-stimulated PLD was attenuated by inhibition or downregulation of protein kinase C (PKC). PLD activation was fully blocked by the cytosolic phospholipase A(2) (PLA(2)) inhibitor ONO-RS-082 and partially attenuated by the selective Ca(2+)-dependent cytosolic PLA(2) inhibitor, arachidonyl trifluoromethylketone (AACOCF(3)), or by bromoenol lactone, an inhibitor of Ca(2+)-independent cytosolic PLA(2). Magnesium, which decreases the concentration of ATP(4-), and nickel, which blocks nonspecific cation channels coupled to purinergic receptors, inhibited PLD activation by ATP. Using reverse transcription-polymerase chain reaction and Northern blotting techniques, we demonstrated that the PLD isoform stimulated by ATP was PLD-2. Among various ATP analogs, only the P2Z/P2X(7) purinergic receptor agonist benzoyl-benzoyl ATP stimulated PLD-2. The response to ATP was inhibited by the nonselective P2X purinergic antagonist suramin and by oxidized ATP, a potent P2Z/P2X(7) receptor antagonist. It is concluded that in rat SMG acinar cells, PLD-2 is upregulated by exogenous ATP through a mechanism involving Ca(2+) influx, cytosolic PLA(2), and PKC. Also, the data suggest an involvement of P2X(7) receptors in PLD-2 stimulation by ATP.