HMGB1 and microparticles as mediators of the immune response to cell death - PubMed (original) (raw)

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HMGB1 and microparticles as mediators of the immune response to cell death

David S Pisetsky et al. Antioxid Redox Signal. 2011.

Abstract

In a wide variety of diseases, cell death represents both an outcome and an important step in pathogenesis. This duality occurs because cell death leads to the extracellular release of molecules and structures that can potently induce the innate immune system. These mediators include the alarmins which are endogenous cellular constituents that exit activated or dying cells to stimulate toll-like receptors (TLRs) as well as non-TLR receptors. Of alarmins, the nonhistone protein HMGB1 is the prototype. Like DNA and RNA, HMGB1 can translocate from cells as they die. The activity of HMGB1 may reflect its interaction with other molecules such as LPS, DNA, and cytokines. In addition to alarmins, dead and dying cells can release subcellular organelles called microparticles that contain cytoplasmic and nuclear constituents, including DNA and RNA. These particles can impact on many cell types to induce inflammation. The release of HMGB1 and microparticles shows important similarities, occurring with cell death as well as stimulation of certain but not all TLRs. Furthermore, nitric oxide can induce the release of both. These observations suggest that the products of dead cells can serve as important mediators to drive immune responses and promote inflammation and autoreactivity.

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Figures

FIG. 1.

FIG. 1.

Western blot analysis of high mobility group box 1 protein (HMGB1) release from apoptotic Jurkat cells. Jurkat cells were induced to undergo apoptosis (A) by treatment for the indicated times with staurosporine (STS), etoposide (Etop), or camptothecin (Camp). Apoptosis was inhibited by a 30-min pretreatment with 100 μ_M_ Z-VAD-fmk. Necrosis (B) was induced by three cycles of freeze-thawing or heating cells at 56°C for 30 min. Supernatants were concentrated and run on SDS-PAGE gels and stained with an anti-HMGB1 antibody. Figure adapted with permission from Bell et al (2).

FIG. 2.

FIG. 2.

The effect of sex on the generation of blood DNA from apoptotic cells. Jurkat cells (108 cells) were treated with etoposide in vitro to induce apoptosis and then administered by the intraperitoneal route to normal male and female BALB/c mice. Blood was obtained at various times subsequently and DNA levels were determined by fluorimetry using the PicoGreen dye. Values are expressed as mean (±SD). Figure adapted with permission from Pisetsky and Jiang (40).

FIG. 3.

FIG. 3.

Comparison of flow cytometric measurement of microparticles by SYTO 13 staining and side scatter. HL-60, Jurkat, and MOLT-4 cells (107 cells) were treated with 1 μ_M_ staurosporine and supernatants harvested 24 h later for analysis. The MPs produced by these cells were detected in cell-free supernatants by fluorescence from SYTO 13 staining (shaded bars) or side scatter (clear bars) in unstained samples. MP counts by SYTO 13 detection or SSC detection were analyzed and were significantly different. *=p<0.0004 in all cases. Figure adapted with permission from Ullal et al (55).

FIG. 4.

FIG. 4.

Detection of microparticles by light scatter (SSC) and SYTO 13 staining. Cell-free supernatants containing MPs from Jurkat cells induced to undergo apoptosis by treatment with 1 μ_M_ staurosporine were analyzed by flow cytometry. The dot-plot compares MPs detected by SSC (black dots) with those detected by SYTO 13 staining (gray dots). Numbers in bold face denote percentage of MPs detected by SSC in each of the quadrants (Q1–Q4), and numbers in regular font indicate percentage of MPs detected by fluorescence. The populations of MPs detected by both SSC and fluorescence appear in quadrant Q2. Figure adapted with permission from Ullal et al (55).

FIG. 5.

FIG. 5.

The phenotype of microparticles released into cultures of apoptotic cells. MPs released from staurosporine-treated Jurkat cells were isolated at the intervals of 0–2 h (dark gray peak), 2–4 h (solid line), 4–6 h (broken line), and 6–8 h (light gray peak) after treatment and assessed for propidium iodide, PI (top panel) and annexin V-binding (bottom panel). Figure adapted with permission from Ullal and Pisetsky (56).

FIG. 6.

FIG. 6.

Production of microparticles by RAW 264.7 cells in response to stimulation by LPS, poly (I:C), or CpG DNA. RAW 264.7 cells were treated with 0.05 (gray), 0.5 (stipple), 5 (black), or 50 (stripe) μg/ml LPS, or 0.25 (gray), 2.5 (stipple), 25 (black), or 250 (stripe) ng/ml P(I:C), or 0.1 (gray), 1.0 (stipple), 10 (black), or 100 (stripe) μ_M_ CpG DNA oligonucleotide, or medium alone (white) for 24 h. MPs were measured with flow cytometry using a side scatter threshold. Figure adapted with permission from Gauley and Pisetsky (19).

FIG. 7.

FIG. 7.

The role of nitric oxide in microparticle production by RAW 264.7 cells. (A) RAW 264.7 cells were stimulated with 50 μg/ml LPS or 0.25 μg/ml poly(I:C) for 24 h following pre-incubation with (black bars) or without (white bars) the inducible nitric oxide synthase (iNOS) inhibitor, 1400W. (B) RAW 264.7 cells were treated with 0 (C), 200, 400, 600, 800, or 1000 μ_M_ of the nitric oxide donor, DPTA (dipropylenetriamine NONOate) for 24 h. Microparticles were measured with side scatter using flow cytometry. *p<0.05 control vs. treatment. Error bars represent SD of the mean. Figure adapted with permission from Gauley and Pisetsky (19).

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