Influence of CD137L Reverse Signaling on Myelopoiesis in Acute and Chronic Inflammation

Ph.DDOCTOR OF PHILOSOPH

Species Difference of CD137 Ligand Signaling in Human and Murine Monocytes

BACKGROUND: Stimulation of CD137 ligand on human monocytes has been shown to induce DC differentiation, and these CD137L-DCs are more potent than classical DCs, in stimulating T cell responses in vitro. To allow an in vivo evaluation of the potency of CD137L-DCs in murine models we aimed at generating murine CD137L-DCs. METHODOLOGY/PRINCIPAL FINDINGS: When stimulated through CD137 ligand murine monocytes responded just as human monocytes with an increased adherence, morphological changes, proliferation and an increase in viable cell numbers. But CD137 ligand signaling did not induce expression of inflammatory cytokines and costimulatory molecules in murine monocytes and these cells had no T cell stimulatory activity. Murine monocytes did not differentiate to inflammatory DCs upon CD137 ligand signaling. Furthermore, while CD137 ligand signaling induces maturation of human immature classical DCs it failed to do so with murine immature classical DCs. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that both human and murine monocytes become activated by CD137 ligand signaling but only human and not murine monocytes differentiate to inflammatory DCs

Alternative myeloid cell populations and CD137 ligand agonists.

<p>(A) Source of cells: Balb/C CD11b⁺ cells from bone marrow and spleen and total bone marrow cells were treated with immobilized Fc or CD137-Fc protein. (B, C) Source of CD137 protein: CD11b⁺, Ly6G⁻ monocytes were treated with immobilized Fc or human or murine CD137-Fc protein. Cells differentiated to immature and mature classical DCs were included as controls. (B) After 7 days the cells were used as stimulators in a MLR with C57/Bl6 T cells at a ratio of 1∶10. Proliferation was determined by ³H-thymidine incorporation at day 3 of coculture. (C) Expression of CD14 and F4/80 was determined at day 7 of the culture. iDC: immature DC. *p<0.05. Data are representative of three independent experiments.</p

Activation of murine monocytes by recombinant CD137 protein.

<p>Monocytes were cultured under indicated conditions for 7 days. (A) Morphological changes were documented by photography at 20x (upper panel) and 63x (lower panel) magnifications. (B) Numbers of viable cells were determined by flow cytometry using Sphero Accucount Blank Particles. (C) Proliferation was determined by ³H-thymidine incorporation at 3 day of culture. *: p<0.05. Data are representative of three independent experiments.</p

Absence of DC characteristics.

<p>Monocytes were cultured under indicated conditions for 7 days. (A) Expression of CD80, CD86, F4/80 and CD14 were determined by flow cytometry. Numbers in the graphs represent the percentages of positively stained cells (grey histograms) compared to the isotype control (open histograms). (B) Expression of CD11c and MHC-II were determined by flow cytometry. Numbers in the graphs represent percentage of the population in each quadrant. (C) Cytokine secretion. The concentrations of (A) IL-10, (B) IL-12, and (C) soluble CD137 in the supernatants were measured by ELISA. *p<0.05. N.D: Not detectable. (D) Phagocytosis was determined by adding flourescent beads to cells at a ratio of 50∶1. The flourescence was determined by flow cytometry. Numbers in the graphs indicate the percentages of positive cells and mean fluorescence intensities (MFI). Half of the harvested cells were trypsinized to remove non-phagozytosed beads that might have stuck on the surface of the cells. Data are representative of three independent experiments.</p

Comparison of CD137-Fc, TNFRI-Fc and CD134-Fc.

<p>Monocytes were cultured under indicated conditions for 7 days. (A) Morphological changes were documented by photography at 20x magnification. (B) Numbers of viable cells were determined by flow cytometry using Sphero Accucount Blank Particles. (C) The concentrations of IL-10 and MCP-1 in the supernatants were measured by ELISA. N.D: Not detectable. *p<0.05. Data are representative of three independent experiments.</p

CD137-treated monocytes exert no DC activity.

<p>(A) Monocytes from Balb/C mice were cultured for 7 days on plates with immobilized Fc or CD137-Fc protein and where indicated stimulated by LPS for the last 24 h. The cells were cocultured with T cells from C57/Bl6 mice in an allogeneic MLR at a ratio of 1∶10. The rate of proliferation was determined by ³H-thymidine incorporation three days later. iDC: immature DC: monocytes treated for 7 days with GM-CSF + IL-4. DC: iDC stimulated with LPS for 24 h. (B) Maturation of classical iDCs. Immature DCs were treated as indicated for 3 days. Expression of costimulatory molecules (grey histograms) was determined by flow cytometry. Isotype control (open histograms). Numbers in the graphs indicate the percentages of positive cells. *p<0.05. Data are representative of three independent experiments.</p