Whereas studies on the release of nuclear molecules (i.e., HMGB1 and DNA) with immunological activity have analyzed these molecules separately, a reductionist approach can easily ignore the realities of cell structure and the nature of the cytoplasmic and nuclear milieus. Rather than a solution in which molecules float freely, these compartments are organized matrices with multiple protein and other macromolecular components that organize and restrict the environment. During death, these compartments can rearrange, allowing trafficking and intracellular migration of these components which can facilitate release from cells to allow either new biological activity or facilitate clearance.
Of mechanisms for cellular reorganization, the formation of blebs is among the most dramatic, providing a hallmark for cells undergoing stress or death. This process, which is a frequent concomitant of apoptosis, leads to translocation of nuclear and cytoplasmic constituents into bubble-like extrusions on the cell surface (10
). The function of blebbing is unknown but it could help sequester degraded or toxic cell products for safe export. Alternatively, like the release of alarmins, blebbing could represent a stage in the cellular demise process that creates immunostimulatory structures that can signal danger and activate innate immunity.
As currently defined, microparticles are small membrane-bound vesicles that, once released from cells, display pro-inflammatory and pro-thrombotic activities. These structures are generally 0.1–1.0 microns in diameter and contain an ensemble of cytoplasmic and nuclear constituents surrounded by a cell membrane. Since apoptosis is a common setting for particle generation, microparticles may correspond to blebs that have detached from cells, although other mechanisms may lead to particle release. Indeed, while blebs can entirely cover the surface of a cell, each cell releases only a few particles as death proceeds. It is possible therefore that, while blebbing and particle release both accompany apoptosis, they may be distinct processes. Particle release is also a feature of cell activation although, in this setting, the formation of blebs has not been extensively investigated (3
Flow cytometry is the most common analytic approach for assessing the presence of particles in biological fluids. Since microparticles are small structures enclosed by a membrane, their assay can entail measurement either on the basis of size or display of cell surface markers. The assay of particles by flow cytometry can be problematic, however, since most cytometers cannot accurately detect structures in the size range of microparticles using ordinary parameters of light scattering. The smaller particles can simply not be detected since their size profile overlaps with noise. Detection by fluorescent agents to surface markers is also commonly used for particle measurement, although this approach is limited by the very small surface area of particles and uncertainties in the expression of various proteins on the surface. For example, while the presence of phosphatidylserine on the particle surface provides the basis for detection by annexin V binding, not all particles bind annexin V.
In the context of our work on nuclear alarmins, we were interested in the relationship between the release of particles and other nuclear molecules such as DNA, and the extent to which microparticles are a transport vehicle for alarmins. We therefore assessed the content of nuclear molecules in particles generated in vitro by apoptotic cells and determined parameters of particle release. For this purpose, we used some of the same systems we used to investigate HMGB1 and DNA release to determine whether microparticle release has similar mechanisms of extracellular generation.
This work is ongoing, although we can already draw certain important conclusions about the relationship between alarmin and particle release. Thus, we have shown that MPs are produced during in vitro
apoptosis and are an important source of extracellular DNA and RNA (41
). In these experiments, we demonstrated the presence of nucleic acid by immunochemical, biochemical, and flow cytometric assays. Particles contain DNA that shows laddering indicative of apoptotic cleavage; ribosomal RNA, including a degradation product that likely arises during apoptosis; mRNA species that can be amplified by PCR; and microRNA. Because free RNA molecules are very sensitive to nucleases, their presence in microparticles may provide a sanctuary in the extracellular space to allow transfer of genetic information or provide a source of nucleic acid to stimulate TLR and non-TLR sensors.
The content of nucleic acids provides the basis of a new flow cytometric assay which detects particles by the binding of SYTO13, a cell permeable dye that interacts with nucleic acids. Using this assay, we showed that flow cytometry with SYTO13 can detect approximately three times as many particles as does light scattering using preparations of particles from Jurkat cells undergoing apoptosis in vitro
). This increased sensitivity results from the detection of particles too small for accurate measurement by light scattering (). As shown by the effects of DNase and RNase, SYTO13 binds to both DNA and RNA in particles, consistent with the content of both nucleic acids.
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 1μM staurosporine and supernatants harvested 24h later for analysis. (more ...)
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 1μM staurosporine were analyzed by flow cytometry. The dot-plot (more ...)
The phenotypic properties of particles generated in vitro
indicate that these structures are dynamic and that they may undergo membrane changes following release from cells treated with agents to induce apoptosis. With particles harvested from the media of Jurkat cells treated with staurosporine, a protein kinase C inhibitor, we showed differences in the phenotype of particles released within 2 hours of treatment and those obtained in cultures treated for 18–24 hours. Whereas the particles from the longer duration cultures were positive for annexin V and propidium iodide (PI), particles from the shorter duration cultures had much lower levels of binding to both agents. Importantly, the early release particles could mature with subsequent culture so that they bound both annexin V and propidium iodide (). The annexin V and PI negative or low particles were also present in untreated cultures, but their levels were very low (56
These observations may be important in understanding the immune activities of MPs since surface membrane phosphatidylserine (PS) may be a signal for both clearance and immune activity. Indeed, the presence of PS on apoptotic cells may represent one of the molecules leading to the anti-inflammatory activity of this form of cell death; PS is also an “eat me” signal that may restrain immune activity from dying cells by promoting phagocytosis and therefore preventing any transition to necrosis and the potential release of alarmins. If this paradigm also operates with particles, the early release particle may have greater immune activity because of the absence of surface PS. As the particle persists in the extracellular space, its immune properties could diminish as surface PS expression rises. Together, these findings suggest that MPs are packets of potentially immunostimulatory molecules with surface marker expression that influence the overall activity of the structure.
As in the case of HMGB1 where both cell activation and cell death can lead to release, MPs can originate from activated cells as well as those that are dying. To understand particle release with activation, we used the RAW264.7 system to elucidate the effects of TLR stimulation on particle release (19
). Using flow cytometry for MP detection, we showed that stimulation with either LPS or poly (I:C) leads to particle release where CpG DNA under the same conditions failed to elicit a comparable response (). Furthermore, we showed that the release of MPs from RAW264.7 cells resulted from the action of nitric oxide since 1400W, an iNOS inhibitor, blocked particle release elicited by LPS or poly (I:C) while an NO donor caused particle release (). In this system, unlike the results with HMGB1 release, we did not observe an effect of IFN on particle release.
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 (more ...)
Since particle release can occur with apoptosis and activation, we followed the format for the experiments on HMGB1 to assess whether caspase inhibition could affect particle release from cells treated with LPS or poly (I:C), both agents which can induce apoptosis under conditions of stimulation. The results of these studies resembled those with the HMGB1 system since caspase inhibition increased particle release, an effect that appears due most likely to a shift or perturbation of the death pathway that leads to necrosis and more extensive release of particles as well as other intracellular components, including alarmins.
While particles are much smaller than cells, they are nevertheless sufficiently large to incorporate both nuclear and cytoplasmic molecules, including DNA and RNA. Since particles can interact with cells to transfer both membrane and internal components to recipient cells, they are vehicles for immunostimulatory nucleic acids that can activate both TLR and non-TLR receptors as well as other signaling pathways. Our studies show notable similarities between HMGB1, DNA, and particle release, although we have not yet measured all of these analytes in the same in vitro
and in vivo
systems. It is of interest therefore that HMGB1 released by enterocyte-like CaCo-2 cells stimulated with cytomix (a mixture of cytokines) can exist in both soluble as well as particulate forms (38
). In this case, the particles display features of exosomes that are a particle type that originates from the multivesicular body; exosomes are smaller than microparticles and bear different proteins. While the localization of HMGB1 on particles in different systems requires future study, we would suggest that both the alarmins and microparticles are part of the same overall defense system that uses internal components to stimulate immune responses.
When distressed or dying, all cells can generate alarmins, discharging these products into the blood to activate the innate immune system both locally and systemically. For alarmins, the active moiety may be a complex, while for MPs the active moiety may be an organelle. Whether these systems are truly separate is a matter of straightforward molecular and cell biological experiments to define the disposition and location of these nuclear components in the extracellular milieu; importantly, such experiments will determine the extent to which molecules that are nominally soluble are in fact particulates. Future studies will define further the manner in which cells are destroyed during death and how the immune system recycles debris, as a very flexible and effective signaling system.