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1.
Figure 9

Figure 9. A proposed model for signaling mechanisms of MDL-1–mediated shock.. From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

Binding of MDL-1 by DV or anti–MDL-1 agonist mAb on CD11b+Gr-1+ immature myeloid cells triggers activation of adaptor proteins DAP12 and DAP10, as well as the downstream kinases Syk, PI3K, and Akt. Akt physically interacts with and phosphorylates eNOS to produce NO. NO in turn triggers TNF-α production, at least in part by increasing TACE activity. Both NO and TNF-α are important mediators for MDL-1–mediated lethal shock.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
2.
Figure 6

Figure 6. NO generated by eNOS is a critical mediator of MDL-1–induced shock.. From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A and B) Mice were administered ConA and pretreated with (A) NO scavenger (Carboxy-PTIO) or (B) NOS inhibitor (L-NAME) and control vehicle (saline) (n = 4) prior to stimulation with anti–MDL-1 mAb, and survival was monitored. **P < 0.01 compared with saline. (C) WT or iNOS–/– mice (n = 10) were treated with ConA, followed by anti–MDL-1 mAb, and survival was monitored. (D) Mice were administered ConA and pretreated with eNOS inhibitor (L-NIO) (n = 5) or vehicle (saline) (n = 4) prior to stimulation with anti–MDL-1 mAb, and survival was monitored. **P < 0.01 compared with saline. (E) WT or eNOS–/– mice (n = 10) were treated with ConA, followed by anti–MDL-1 mAb, and survival was monitored. ***P < 0.001 compared with WT.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
3.
Figure 4

Figure 4. CD11b+Gr-1+ cells are pathogenic in MDL-1–mediated shock. . From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A) CD11b+ cells were isolated from livers of ConA-treated WT or MDL-1–/– mice and injected into naive WT recipient mice via tail vein (n = 10). Anti–MDL-1 mAbs were injected 30 minutes after transfer, and survival was monitored. *P < 0.05 compared with IgG1 or transfer cells from MDL-1–/– mice. (B) Mice were pretreated with anti–Gr-1 mAb (n = 5) or IgG2b (n = 4) prior to administration of ConA, followed by anti–MDL-1 mAb, and survival was monitored. **P < 0.01 compared with IgG2b. (C) Liver sections from B were immunostained with anti–MDL-1 mAb, and positively stained cells were quantitated (n = 5). ***P < 0.001 compared with IgG2b.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
4.
Figure 2

Figure 2. G-CSF is important for MDL-1–mediated shock.. From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A) Serum and liver homogenates were obtained from mice treated with ConA for the indicated times (n = 5), and G-CSF levels were quantitated by Luminex. *P < 0.05, **P < 0.01 compared with 0-hour time point. (B) Mice were pretreated with anti–G-CSF neutralizing antibodies (n = 5) or IgG1 (n = 3) prior to administration of ConA, followed by anti–MDL-1 mAb, and survival was monitored. **P < 0.01 compared with IgG1. (C) Blood and livers were collected from the mice represented in B, and G-CSF levels in the serum and liver homogenates were measured to evaluate the efficiency of G-CSF neutralization. **P < 0.01, ***P < 0.001 compared with IgG1. (D) Liver sections collected from the mice represented in B were immunostained with anti–MDL-1 mAb, and positively stained cells were quantitated (n = 5). ***P < 0.001 compared with IgG1.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
5.
Figure 5

Figure 5. DV-activated MDL-1+ cells produce NO and TNF-α in vitro, ex vivo, and in vivo. . From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A and B) CD11b+ cells harvested from livers of ConA-treated WT and MDL-1–/– mice were plated and stimulated with (A) anti–MDL-1 mAb or (B) DV for 24 hours. NO and TNF-α in the conditioned media were measured. Data are mean ± SEM of triplicates and are representative of 3 independent experiments. *P < 0.05, ***P < 0.001 compared with naive cells from WT or stimulated cells from MDL-1–/– mice. (C and D) Precision-cut liver slices from ConA-treated WT or MDL-1–/– mice were plated and stimulated with (C) anti–MDL-1 mAb or (D) DV for 4 hours. NO and TNF-α in the conditioned media were measured. Data are mean ± SEM of triplicates and are representative of 2 independent experiments. *P < 0.05, ***P < 0.001 compared with naive liver slices from WT mice or stimulated liver slices from MDL-1–/– mice. (E and F) Serum from WT or MDL-1–/– mice treated with ConA, followed by (E) anti–MDL-1 mAb or (F) DV (n = 5), was collected. NO and TNF-α in the serum were measured. *P < 0.05, ***P < 0.001 compared with serum of IgG1-treated or naive mice.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
6.
Figure 8

Figure 8. Blockade of DAP12, DAP10, Syk, PI3K, and Akt confers protection from MDL-1–triggered shock.. From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A and B) WT and (A) DAP12–/– or (B) DAP10–/– mice (n = 5) were treated with ConA, followed by anti–mMDL-1 mAb, and survival was monitored. ***P < 0.001 compared with WT. (CE) Mice were administered with ConA and pretreated with inhibitors for (C) Syk (BAY 61-3606) (n = 4) or (D) PI3K (wortmannin) (n = 5), (E) Akt (MK-2206) (n = 16) or vehicle (DMSO) prior to stimulation with anti–mMDL-1 mAb, and survival was monitored. ***P < 0.001 compared with vehicle control. (F) MDL-1 activates Akt and eNOS to form a signaling complex. Leukocytes isolated from livers of ConA-treated mice were stimulated with anti–MDL-1 mAb or IgG1, and cell lysates were immunoprecipitated with anti-eNOS and immunoblotted with antibodies against phosphorylated eNOS(Ser1177) (p-eNOS), eNOS, phosphorylated Akt(Ser473) (p-Akt), and Akt. Data are representative of 2–3 independent experiments with at least 3 mice per group.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
7.
Figure 1

Figure 1. Recruitment and activation of MDL-1+ cells led to lethal shock in mice. . From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A) C57BL/6 mice were injected with inflammation probe after ConA treatment and monitored for inflammation (left panel). After in vivo imaging, mice were sacrificed and livers were excised for ex vivo imaging (right panel). p, photons; sr, steradians. (B) Mice were treated with ConA for the indicated times (n = 5), and liver sections were stained with anti–MDL-1 mAb. The inset shows high magnification of MDL-1+ cells. Scale bars: 10 μm. (C) Quantitation of MDL-1+ cells in B. *P < 0.05, ***P < 0.001 compared with 0 hours. (D and E) MDL-1–/– and WT mice were treated with ConA, followed by (D) DV or (E) anti–MDL-1 mAb (n = 5), and survival was monitored. *P < 0.05, **P < 0.01 compared with WT. (F) Mice were treated with ConA, followed by the indicated doses of anti–MDL-1 mAb (n = 3), and survival was monitored. (G) Mice were treated with ConA, followed by IgG1 (n = 20), anti–MDL-1 agonist (n = 23), or antagonist (n = 5) mAb, and survival was monitored. ***P < 0.001 compared with IgG1.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
8.
Figure 3

Figure 3. MDL-1+ cells are CD11b+Ly6G+Ly6C+, with ring-shaped nuclei. . From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A) Leukocytes isolated from livers of ConA-treated mice were purified using CD45 microbeads and analyzed by flow cytometry for expression of CD11b, Gr-1, Ly6G, Ly6C, and MDL-1 gated on CD45+ cells. Background signal was established in the same population by staining with the matched isotype controls. Numbers indicate the percentages of Ly6G+Ly6Clo (red ellipsoid), Ly6GLy6Chi (green ellipsoid), and Ly6GLy6Clo (blue ellipsoid) cell populations. The individual population was analyzed for MDL-1 expression (dotted line indicates isotype control). (B) Quantitation of MDL-1+Ly6G+Ly6Clo cells isolated from livers of naive and ConA-treated mice by flow cytometry. *P < 0.05, **P < 0.01 compared with naive cells. (C) Morphologic analysis of Diff-Quik–stained Cytospin preparations of Gr-1+ cells isolated from livers of naive and ConA-treated mice. Note the increased number of cells with ring-shaped nuclei (indicated by arrowheads) with ConA treatment. Scale bar: 10 μm. (D) Representative micrographs of H&E-stained liver sections from naive and ConA-treated mice. ConA treatment increased the number of cells with ring-shaped nuclei as indicated by arrowheads. Scale bar: 10 μm. (E) Representative micrographs of immunostained Cytospin preparations of Gr-1+ cells isolated from livers of naive and ConA-treated mice. Cells were stained with antibodies against CD11b (green) and MDL-1 (red). Nuclei were counterstained with DAPI (blue). Scale bar: 10 μm.

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.
9.
Figure 7

Figure 7. TNF-α is an important mediator of MDL-1–induced shock and is modulated by eNOS via regulating TACE activity.. From: Activation of MDL-1 (CLEC5A) on immature myeloid cells triggers lethal shock in mice.

(A) Mice were pretreated with TNF-α neutralizing mAb or IgG1 (n = 5) prior to administration of ConA, followed by anti–MDL-1 mAb, and survival was monitored. *P < 0.05 compared with IgG1. (B) WT or Tnfa–/– mice (n = 7) were treated with ConA, followed by anti–MDL-1 mAb, and survival was monitored. ***P < 0.001 compared with WT. (C and D) Blood and livers were collected from WT or eNOS–/– mice (n = 10) treated with ConA, followed by anti–MDL-1 mAb, as in Figure 6E, and (C) serum TNF-α levels and (D) hepatic TACE activity were measured. *P < 0.05 compared with WT. (E) Mice were treated with ConA and TAPI-1 (n = 5), followed by anti–MDL-1 mAb, and survival was monitored. *P < 0.05 compared with DMSO. (F) Blood collected from the mice represented in E was measured for serum TNF-α. *P < 0.05 compared with DMSO. (G) Leukocytes isolated from livers of ConA-treated mice were analyzed by flow cytometry for expression of TNF-α, eNOS, and MDL-1 gated on CD11b+ cells. Background signal was established in the same population by staining with the matched isotype controls. Numbers indicate the percentages of TNF-α+eNOShi (red box), TNF-α+eNOSlo (green box), TNF-α+eNOS (blue box), TNF-αeNOS+ (purple box), and TNF-αeNOS (black box) cell populations. The individual population was analyzed for MDL-1 expression (dotted line indicates isotype control).

Ricky Cheung, et al. J Clin Invest. 2011 November 1;121(11):4446-4461.

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