Abstract

Abstract 1499

Poster Board I-522

Dendritic cells (DCs) are an attractive target for therapeutic manipulation of the immune system due to their potent antigen presentation capacity and ability to induce effective immune response. In steady-state conditions different DC subsets including myeloid DCs (CD11c+CD11b+B220neg) and plasmacytoid DCs (CD11c+CD11bnegB220+) are generated in bone-marrow (BM) from hematopoietic stem cells through a series of differentiation steps. We recently demonstrated that prostaglandin (PGE2), the predominant metabolite of arachidonic acid metabolism by cyclooxygenase (COX) enzymes, enhances the homing, survival and proliferation of hematopoietic stem cells (Hoggatt et. al., Blood 2009). In this study, we examined the requirement of prostaglandins in development of DCs from hematopoietic progenitor cells. In vivo treatment of mice for 4 days with the non-steroidal anti-inflammatory drug (NSAID) indomethacin (2.5 mg/kg/bid), a dual COX1/COX2 inhibitor, produced a 59.5±17.8 % (p<0.02) reduction in total bone-marrow DC number compared to control mice treated with vehicle alone. Interestingly, indomethacin selectively decreased marrow CD11c+CD11b+B220neg myeloid DCs without affecting CD11c+CD11bnegB220+ plasmacytoid DCs. To determine whether fewer DCs in the bone marrow of indomethacin treated mice was due to the impairment of DC differentiation from their hematopoietic progenitor cells, we stimulated differentiation of DCs from lineage depleted (CD5,CD45R, CD11b, Anti-Gr-1, 7-4 and Ter-119) bone marrow cells using Flt-3 ligand for 9 days in vitro and treated cultures with indomethacin (1 microM), SC-560 (10 microM), a selective COX-1 inhibitor or NS-398 (10 microM), a selective COX-2 inhibitor. Indomethacin produced a 1.98±0.38 fold, (p<0.02) reduction and the COX-2 inhibitor NS-398 produced a 1.52±0.04 fold, (p<0.05) reduction in CD11c+CD11b+B220neg myeloid DC generation compared to control, while the COX-1 specific inhibitor SC-560 was without effect. As expected, Flt-3-ligand induced plasmacytoid DC (CD11c+CD11bnegB220+) differentiation was not affected by selective COX inhibitors. Indomethacin also impaired generation of CD11a+CD14neg Langerhans DC from human umbilical cord blood CD34+ cells. Measurement of PGE2 production in culture supernatants from DC-producing cultures demonstrated detectable PGE2 after 6 days of culture and DC generation from BM progenitors in these cultures was impaired when PGE2 synthesis was blocked on day 6 by indomethacin administration. Indomethacin treatment during the first 5 days of Flt3-ligand stimulated DC differentiation cultures did not decrease DC production. To identify mechanisms responsible for this impairment in Flt-3 ligand-induced DC generation from hematopoietic progenitor cells, we analyzed the effect of indomethacin on DC-committed precursor cell proliferation and survival. Survival of DC-committed precursors defined as CD11clow CD11bbrightMHCIIlow was reduced 35±2.5% (p<0.05) in indomethacin treated cultures compared to control. However, indomethacin did not affect DC precursor proliferation as measured by BrdU incorporation assay. To elucidate the signaling mechanisms by which indomethacin impaired the survival of DC precursors, we added selective receptor agonists to each of known PGE receptors, EP1-4, during Flt3-ligand induced DC differentiation. The EP1/EP3 agonist 17-phenyl trinor PGE2 rescued the DC precursors from indomethacin mediated death, whereas Butaprost, a specific EP2 agonist and L902688, a selective EP4 agonist, failed to rescue DC precursor death. DCs developed in the presence of prostaglandin inhibitors did not show any defect in LPS-induced activation and expressed CD40, CD80, CD86 and MHCII levels similar to control as measured by flow cytometry. In addition, DC developed in the absence of endogenous PGE2 production successfully induced T-cell activation as measured by mix lymphocyte reaction assay (MLR). In conclusion, COX-2 mediated prostaglandin production by DC-committed hematopoietic precursors confers resistance to cell death via signaling through EP1/EP3 receptors and promotes dendritic cell development.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.