1. Annexin A2 tetramer activates human and murine macrophages through TLR4
by Jennifer F. A. Swisher, Nicholas Burton, Silvia M. Bacot, Stefanie N. Vogel, and Gerald M. Feldman
Blood
Volume 115(3):
January 21, 2010
©2010 by American Society of Hematology

2. Blocking antibody for A2tR fails to reduce A2t-dependent cytokine induction in human macrophages.
Blocking antibody for A2tR fails to reduce A2t-dependent cytokine induction in human macrophages. (A) Seven-day-old macrophages were pretreated with either anti-A2tR–blocking antibody or rabbit polyclonal isotype control (10 μg/mL for 20 minutes at room temperature), followed by treatment with 2nM A2t for 1 hour at 37°C. RNA was isolated and reverse transcribed, and levels of the indicated species were analyzed by real-time polymerase chain reaction. (B) Monocytes were plated and aliquots were stained for A2tR expression at indicated times throughout macrophage differentiation. Histograms shown are representative of 4 donors. Black (filled) histograms = rabbit IgG isotype control, gray (open) = anti-A2tR–blocking antibody (both used at 1 μg/mL, then stained with 1:200 Alexa 488 donkey anti–rabbit). (C) Monocytes/macrophages were treated with 5nM A2t for 1 hour at 37°C at the indicated times (days after plating) during differentiation. mRNA levels were analyzed as in (B). Macrophages from the same donors were stained with goat anti–human CD64 antibody, followed by Alexa 488 donkey anti–goat secondary (values for isotype controls subtracted) at the same times to monitor macrophage differentiation. Results (A,C) are the mean ± SEM of 3 experiments with separate donors.
Jennifer F. A. Swisher et al. Blood 2010;115:
©2010 by American Society of Hematology

3. A2t attenuates macrophage TNF-α production in response to LPS
A2t attenuates macrophage TNF-α production in response to LPS. Macrophages were treated with 10 ng/mL LPS for 24 hours, with or without a 6-hour pretreatment with 2nM A2t.
A2t attenuates macrophage TNF-α production in response to LPS. Macrophages were treated with 10 ng/mL LPS for 24 hours, with or without a 6-hour pretreatment with 2nM A2t. Controls were treated with 2nM A2t alone for 30 hours. Supernatants were collected at 24 hours after LPS treatment, and TNF-α was measured by luminex.
Jennifer F. A. Swisher et al. Blood 2010;115:
©2010 by American Society of Hematology

4. Macrophage activation in response to A2t involves both MyD88 and TRIF
Macrophage activation in response to A2t involves both MyD88 and TRIF. (A) WT or MyD88−/− 7-day-old murine BMDM were treated with 5nM A2t for 24 hours, and levels of the indicated cytokines (in pg/mL) were measured in the supernatants by luminex.
Macrophage activation in response to A2t involves both MyD88 and TRIF. (A) WT or MyD88−/− 7-day-old murine BMDM were treated with 5nM A2t for 24 hours, and levels of the indicated cytokines (in pg/mL) were measured in the supernatants by luminex. Macrophages were pooled from 4 males per genotype in each of 2 separate experiments run in duplicate. Results expressed as the mean ± SEM. (B) Nuclear extracts of 7-day-old human MDM treated with either LPS (2 ng/mL) or A2t (5nM) were tested for p65 or IRF3 activity by binding of plated DNA oligos bearing the specific NF-κB or IRF3 consensus site, followed by colorimetric detection of p65 or IRF3 by horseradish peroxidase–linked antibody.
Jennifer F. A. Swisher et al. Blood 2010;115:
©2010 by American Society of Hematology

5. Reduction of TLR4 on the macrophage surface inhibits A2t-driven cytokine production.
Reduction of TLR4 on the macrophage surface inhibits A2t-driven cytokine production. (A) Seven-day macrophages from 3 donors were transfected with small interfering RNAs: 2 separate sequences specific for TLR4 were used, either alone or in combination (“TLR4 a” and “TLR4 b”), as well as negative control siRNA, and an siRNA for an unrelated gene (glyceraldehyde-3-phosphate dehydrogenase). Three days after transfection, cells were stained with fluorescein isothiocyanate–labeled mouse anti–human TLR4 antibody, and surface expression of TLR4 was analyzed by FACS. Results represent the mean ± SEM(after subtraction of fluorescence from the isotype control) of 3 experiments with separate donors. (B) Macrophages (transfected as in panel A) were treated 2 days posttransfectionwith A2t (5nM), LPS (10 ng/mL), or Pam3CSK4 (100 ng/mL) for 24 hours, and concentration of IL-6 was measured in the supernatants by luminex and expressed as the percentage of the value of IL-6 induced by LPS for that donor. Results are the mean ± SEM; *P < .05.
Jennifer F. A. Swisher et al. Blood 2010;115:
©2010 by American Society of Hematology

6. TLR4-blocking antibodies inhibit macrophage production of TNF-α in response to A2t and LPS, but not Pam3CSK4, a TLR2 ligand.
TLR4-blocking antibodies inhibit macrophage production of TNF-α in response to A2t and LPS, but not Pam3CSK4, a TLR2 ligand. Intracellular TNF-α staining of 7-day-old MDM after treatment with indicated stimuli for 6 hours in the presence of brefeldin A (A), or cytokine levels measured by luminex from 24 hours of culture with the same treatments in the absence of brefeldin A (B). Where indicated, cells were pretreated (15 minutes) with 10 μg/mL designated blocking antibody before stimulus addition. (C) Intracellular TNF-α staining of naive (left) or differentiated (right; 7 days with 15nM PMA) THP-1 cells after 6-hour incubation with indicated stimuli. (D) Seven-day PMA-differentiated THP-1 cells were treated with the indicated stimuli and blocking antibodies and stained for intracellular TNF-α, as in panel A. (E) WT or TLR-deficient (TLR4−/−) 7-day-old murine BMDM were treated with 5nM A2t, 10 ng/mL LPS, or 100 ng/mL Pam3CSK4 for 24 hours, and levels of the indicated cytokines (in pg/mL) were measured in the supernatants by luminex. Macrophages were pooled from 5 females per genotype; results are the mean ± SEM of 2 experiments performed in duplicate.
Jennifer F. A. Swisher et al. Blood 2010;115:
©2010 by American Society of Hematology

7. TLR4 is necessary, but not sufficient to confer A2t responsiveness.
TLR4 is necessary, but not sufficient to confer A2t responsiveness. (A-B) Intracellular TNF-α staining of 3-day-old human monocytes (A) or THP-1 cells differentiated with 15nM PMA for 3 days (B) after 6-hour treatment with indicated stimuli. (C) HEK293 cells stably transfected with a NF-κB–driven GFP reporter plasmid were transiently transfected with plasmids containing no insert (mock), or encoding TLR4 or TLR4 along with MD-2, then treated with the indicated stimuli (A2t = 100nM, LPS = 100 ng/mL, PMA = 5mM), and GFP fluorescence was measured by FACS 24 hours after treatment. Data are expressed as percentage of GFP induction compared with the PMA-induced fluorescence for the corresponding transfection and experiment. Results are the mean ± SEM of 3 experiments.
Jennifer F. A. Swisher et al. Blood 2010;115:
©2010 by American Society of Hematology