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Blood, Vol. 108, Issue 10, 3580-3589, November 15, 2006
Apoptotic cell thrombospondin-1 and heparin-binding domain lead to dendritic-cell phagocytic and tolerizing states
Blood Krispin et al.
108: 3580
Supplemental materials for: Krispin et al, Vol 108, Issue 10, 3580-3589
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 21.6 KB)
- Figure S1. Evaluation of apoptotic monocyte-iDC interaction using flow cytometry (PDF, 79.6 KB) -
(A) iDCs interact with apoptotic monocytes. DiI-stained monocytes were offered to iDCs. Ratios of iDCs to apoptotic monocytes are indicated above each histogram. Acquisition of DiI by CD1a+ iDCs following DiI-stained monocyte engulfment or adherence was measured using flow cytometry (bold line). Noninteracting iDCs are shown by the black line. Median fluorescence is indicated in each histogram. Data are representative of 3 experiments. (B) iDC acquisition of DiI from apoptotic monocytes was linearly proportional to the number of interacting apoptotic cells. DiI-stained apoptotic monocytes were offered to iDCs in triplicate at the indicated ratios. The median fluorescence of DiI-stained CD1a+ iDCs was linearly proportional to the number of apoptotic cells offered. R2 = 0.9824.
- Figure S2. N-terminal domain of TSP-1 is secreted from apoptotic monocytes (PDF, 95 KB) -
(A) A 26-kDa protein is found in the supernatant of apoptotic monocytes. Supernatants were collected from 3 × 107 monocytes, which were either undergoing serum withdrawal apoptosis or treated with 20 µM pan-caspase inhibitor zVAD-fmk for the times indicated. Proteins were separated as explained in “Materials and methods” under the subheading “Proteomic-based identification of secreted proteins from cells undergoing apoptosis,” and were loaded on 4% to 20% gradient polyacrylamide gel at 15 µg per lane. Proteins were stained with colloidal Coomassie. A 26-kDa band was identified (thin arrow) as specific to the apoptotic process, as shown by its significant decrease in the presence of pan-caspase inhibitor zVAD-fmk (thick arrow). (B) The 26-kDa band is the heparin-binding domain of TSP-1. Sequences corresponding to these peptides are bolded. The 26-kDa fragment is identical to N-terminal heparin-binding domain of TSP-1.
- Figure S3. Expression of 26-kDa HBD of TSP-1 is serine protease dependent (PDF, 10.8 KB) -
Monocytes (Mono) were exposed to serum withdrawal (SW), anti-Fas (Fas), or staurosporine (Stauro) to induce apoptosis. All 3 apoptosis patterns led to significant expression of TSP-1, which was inhibited in the presence of zVAD-fmk (zVAD). Neutrophil (PMN) serum withdrawal apoptosis led to TSP-1 secretion by apoptotic neutrophils 12 and 24 hours following apoptosis induction. TSP-1 was measured by EIA. Hrs indicates hours.
- Figure S4. TPCK inhibits secretion of TSP-1 and its heparin-binding domain (PDF, 26.5 KB) -
(A) Apoptotic monocytes were exposed to chymotrypsin inhibitor TPCK at 0 to 100 µM as described in “Materials and methods,” under “Induction and detection of apoptosis.” After 10 hours, a dose-dependent decrease in culture media TSP-1 concentration was evident, as measured by EIA. (B) A Western blot analysis (see Figure 3B-C for details) of 3 × 107 apoptotic monocyte culture media shows a dose-dependent decrease in 26-kDa cleavage in the presence of 0 to 100 µM TPCK. At 100 µM, a marked reduction in cellular protein secretion was observed, and cells were absent or necrotic, as verified by counting and Trypan blue staining. Purified TSP-1 was used in the left lane as a positive control.
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