Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0704411 on December 15, 2004

Published online before print December 15, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0704411v1
77/3/296    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, N.
Right arrow Articles by Pisetsky, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, N.
Right arrow Articles by Pisetsky, D. S.
(Journal of Leukocyte Biology. 2005;77:296-302.)
© 2005 by Society for Leukocyte Biology

The effect of inflammation on the generation of plasma DNA from dead and dying cells in the peritoneum

Ning Jiang and David S. Pisetsky1

Division of Rheumatology, Duke University Medical Center, and Medical Research Service, Durham VA Hospital, Durham, North Carolina

1 Correspondence: Durham VA Medical Center, Box 151G, 508 Fulton St., Durham, NC 27705. E-mail: dpiset{at}acpub.duke.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To assess the effects of inflammation on the generation of circulating DNA from dead and dying cells, plasma DNA levels were determined in BALB/c mice, administered apoptotic or necrotic Jurkat cells following induction of peritonitis by treatment with thioglycollate (TG), peptone (PT), or sodium periodate (NaIO4). In mice receiving TG or NaIO4, plasma DNA levels following intraperitoneal administration of Jurkat cells were significantly reduced compared with controls, whereas they were not affected in mice receiving PT. To determine the basis of these differences, the cellular composition of peritoneal fluids prior to the administration of the dead cells was analyzed. Among agents tested, TG administration led to the largest increase in cells, both neutrophils and monocytes. As shown by flow cytometry, the exudates contained apoptotic neutrophils and macrophages, with the highest levels in the TG-induced exudates. Analysis of DNA and caspase 3 in the fluids also showed differences. TG exudates showed increases in DNA and capase 3, while NaIO4-induced exudates had an increase only in DNA. Fluid from PT-treated mice did not have increases in DNA or caspase 3. Together, these results indicate that prior inflammation can affect the generation of blood DNA from apoptotic or necrotic cells, although this effect may vary depending on the composition of the exudates with respect to cells as well as DNA.

Key Words: apoptosis • necrosis • neutrophils • peritonitis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA is a nuclear macromolecule that can exist in an intracellular and extracellular form. In its extracellular form, DNA can appear in the blood as well as other biological fluids, with its level rising in a variety of clinical conditions [1 2 3 4 5 6 7 8 ]. These conditions include systemic lupus erythematosus (SLE), malignancy, pregnancy, and trauma among many others. Although often considered inert, extracellular DNA, depending on context and binding to other molecules, can display potent effects on the immune system [9 , 10 ]. In SLE, for example, immune complexes containing DNA can stimulate interferon-{alpha}/ß production by their action on plasmacytoid dendritic cells [11 , 12 ]. Similarly, in mice expressing a rheumatoid factor transgene, such immune complexes can activate B cells by a mechanism involving Toll-like receptor 9 [13 , 14 ]. Understanding the origin of blood DNA is therefore important for elucidating the mechanisms of autoimmune and other immune-mediated diseases.

In view of the conditions in which its levels are elevated, blood DNA has generally been considered the result of cell death, and apoptosis is implicated as the major mechanism for DNA release. Indeed, DNA in the blood shows low molecular weight and size-laddering, the hallmarks of the apoptotic process [3 , 6 , 15 , 16 ]. Furthermore, in vitro, apoptotic cells can release DNA into the extracellular milieu. In contrast, necrotic cells do not release DNA under in vitro conditions [17 ]. The role of apoptosis in DNA release is strengthened by observations that treatment of mice with an anti-Fas antibody, which induces widespread hepatic apoptosis, causes a large increase in blood DNA [18 ].

The settings in which blood DNA levels are elevated are, in general, complex and involve cell activation and cell death, often occurring concomitantly [2 , 7 , 8 , 19 ]. Thus, in tissue injury from trauma or toxin exposure, inflammation may follow the death-inducing insult. Similarly, in tumors, areas of necrosis and apoptosis may coexist adjacent to surrounding inflammation. These processes are dynamic, and the cellular composition of an inflammatory site as well as the activation state of cells present evolve over time. Furthermore, as these processes progress, the elaboration of cytokines and other inflammatory mediators may provoke subsequent waves of cell death and activation [20 21 22 ].

In a previous study, we used a murine model to elucidate the mechanisms of DNA release, testing whether cell death is sufficient to induce a blood DNA response and whether apoptotic and necrotic cells are similar in their behavior [23 ]. For this purpose, normal mice were administered apoptotic or necrotic Jurkat cells, and plasma DNA was determined using the fluorimetric dye PicoGreen. Results of these studies indicated that, in vivo, apoptotic and necrotic cells can produce blood DNA and that the blood DNA arising from these cells shows laddering with both. Furthermore, we showed that in mice, in which macrophages were eliminated by clodronate treatment, a rise in blood DNA did not occur after administration of the dead cells. Together, these results indicate that the generation of blood DNA depends on macrophages and may not simply reflect the occurrence of cell death.

In view of the close relationship of inflammation and cell death and the role of macrophages in the clearance of dead and dying cells, we have therefore used an in vivo system to investigate whether prior inflammation can influence the generation of blood DNA from dead and dying cells. For this purpose, we induced peritoneal inflammation in normal mice prior to the administration of apoptotic or necrotic cells and then assessed plasma DNA by fluorimetry. In results presented herein, we show that, in mice with peritonitis induced by thioglycollate (TG) or sodium periodate (NaIO4), a plasma DNA response did not occur following administration of Jurkat cells treated with etoposide to induce apoptosis or ethanol to induce necrosis. In contrast, mice treated with peptone (PT) had blood DNA responses similar to those of untreated mice. Together, these results indicate that inflammation can affect the generation of DNA from dead and dying cells, although the nature of this effect may vary depending on the inflammatory stimulus and local cellular events.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of mice and cells
Female BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were used for experiments at ages 6–12 weeks. To study effect of inflammation on DNA release, mice were treated with 2 ml 4% TG (Sigma Chemical Co., St. Louis, MO) [24 ], 1 ml 10% PT (Becton Dickinson, Sparks, MD) [24 ], or 1 ml 5 mM NaIO4 (Sigma Chemical Co.) [25 , 26 ] at days 1 and 3 and then administered apoptotic or necrotic cells by the intraperitoneal (IP) route. For these experiments, cells were administered at day 4 after TG and NaIO4 treatment and day 3 after PT treatment.

For the induction of apoptosis, Jurkat cells growing in RPMI 1640 with 10% fetal bovine serum were treated with etoposide (Sigma Chemical Co.) at 30 ug/ml for 24 h. For the induction of necrosis, cells were treated with 70% ethanol for 10 min. After treatment, cells were washed twice with phosphate-buffered saline (PBS; Gibco-BRL, Grand Island, NY) and suspended in PBS. The cells were administered IP at 108 cells/mouse. Following these treatments, mice were bled at regular times thereafter as indicated in the figures. The blood samples were collected into tubes with 3–5 µl 0.5 M EDTA, followed immediately by centrifugation. The plasma was removed and stored at –20°C until use. The peritoneal lavage samples were collected in 1 ml cold PBS. The cells were pelleted and stained for flow cytometry analysis. The peritoneal fluids were saved and stored at –20°C until use.

Quantitation of DNA in plasma and peritoneum
Plasma DNA was assessed by a fluorimetric assay as described previously [23 ]. Briefly, plasma in various dilutions were mixed at a 1:1 ratio with the dye PicoGreen (Molecular Probes, Eugene, OR), diluted 1:200 in 10 mM Tris, 1 mM EDTA, pH 8 (TE buffer) in a black, 96-well microtiter plate (Costar, Corning Inc., Corning, NY). The DNA concentration was determined from fluorescence measurements using a TECAN GENios microplate fluorescence reader (Salzburg, Austria) with an excitation wavelength at 485 nm and an emission wavelength at 535 nm. Data were collected as relative fluorescence units. The concentration of DNA in plasma was calculated according to a standard curve using double-stranded calf thymus DNA (Sigma Chemical Co.). Peritoneal fluids were diluted 1:10 in TE buffer, and DNA levels were measured as described above.

Flow cytometry analysis
To assess the effects of various agents on immune cell populations, flow cytometry was performed on preparations of peritoneal washout cells. Briefly, mice treated with TG, PT, or NaIO4 were killed by cervical dislocation, and peritoneal cells were harvested at various time-points by lavage with 1 ml cold PBS. The cells were pelleted at 300 g for 5 min and resuspended in hypotonic lysis buffer to remove red blood cells, followed by centrifugation and two washes with PBS/0.5% bovine serum albumin. The cell concentrations were adjusted to 1 x 107/ml in PBS, and 1 x 106 cells were used for staining. Cells were treated with anti-mouse CD16/CD32 antibody (BD PharMingen, San Diego, CA) and then stained with phycoerythrin (PE)-anti-mouse F4/80 (Serotec, Raleigh, NC) or PE-anti-mouse Ly-6G or Ly-6C (Gr1; BD PharMingen), along with Annexin V-fluorescein isothiocyanate (BD PharMingen). Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, Mansfield, MA). Data analysis was done using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Enzyme assay
Caspase 3 was assayed using a caspase 3 kit (Molecular Probes, E13184). Briefly, plasma samples were diluted 1:100 in PBS, and peritoneal fluids were used neat. Samples were incubated with caspase 3 substrate at room temperature for 30 min. Fluorescence units (FU) were determined using a TECAN GENios microplate fluorescence reader as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To assess the effects of inflammation on the generation of plasma DNA from dead and dying cells, the release of DNA from apoptotic or necrotic Jurkat cells was assessed in mice receiving TG, NaIO4, or PT to induce peritoneal exudates. Although these agents have all been used to peritonitis, they differ in the nature of the inflammatory stimulus, the cellular composition of the resulting exudates, and the time-course of cellular accumulation [24 25 26 ]. In these experiments, 108 Jurkat cells, which were treated with etoposide to induce apoptosis or ethanol to induce necrosis, were administered by the IP route to treated mice.

As shown previously, plasma DNA rises following administration of either type of dead cell with DNA derived predominantly, but not exclusively, from the Jurkat cells [23 ]. Figures 1 2 3 show the results of these experiments; for NaIO4-treated mice, only apoptotic cells were studied. As these data indicate, consistent with previous observations for control mice, the magnitude and time-course of appearance of plasma DNA were similar with apoptotic and necrotic cells, returning to baseline by 24 h after administration. Among the three groups of mice treated to induce peritonitis, however, the appearance of plasma DNA differed. For mice treated with TG or NaIO4, the plasma DNA levels were reduced markedly compared with controls, whereas for PT-treated mice, the levels of DNA were comparable with controls. These results suggest that local inflammation can affect the generation of plasma DNA from dead and dying cells, although the effect will vary depending on how the peritonitis is induced.



View larger version (9K):
[in this window]
[in a new window]
  		Figure 1. Effect of TG administration on plasma DNA levels from apoptotic or necrotic cells. 108 Jurkat cells, treated with etoposide to induce apoptosis (A) or ethanol to induce necrosis (B), were injected IP into BALB/c mice (control or pretreated with 2 ml 4% TG 4 days before). Levels of DNA were determined as described in Materials and Methods. Results are presented as means (±SD) of six mice (A) and three mice (B).

 


View larger version (10K):
[in this window]
[in a new window]
  		Figure 2. Effect of PT administration on plasma DNA levels from apoptotic and necrotic cells. 108 Jurkat cells, treated with etoposide to induce apoptosis (A) or ethanol to induce necrosis (B), were injected IP into BALB/c mice (control or pretreated with 1 ml 10% PT 3 days before). Levels of DNA in the plasma were determined. Results are presented as means (±SD) of six mice.

 


View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Effect of NaIO4 administration on plasma DNA levels from apoptotic cells. 108 Jurkat cells, treated with etoposide to induce apoptosis, were injected IP into BALB/c mice (control or pretreated with 1 ml 5 mM NaIO4 at day 1 and day 3). Levels of DNA in the plasma were determined. Results are presented as means (±SD) of three to six mice.

 
In these experiments, the dead cells were administered into an inflammatory site, where cell populations are undergoing changes in composition and properties. For example, although TG administration leads to an increase in the number of macrophages, the initial response consists of neutrophils, a population prone to die by apoptosis [27 28 29 ]. Therefore, the cellular composition of peritoneal exudates was characterized in the time-period prior to the administration of the apoptotic and necrotic cells to determine any features that could account for the differences in the results obtained on plasma DNA levels.

Table 1 presents the results of experiments showing the total number of peritoneal exudate cells, the number of neutrophils (Gr1+), and the number of macrophages (F4/80+). As these data indicate, the highest number of peritoneal cells observed occurred in the TG-treated mice at early time-points (6 and 16 h) as well as at later time-points (72 h). At 6 and 16 h, the predominant cells in all groups were neutrophils, and at 72 h, the predominant cells were macrophages. In the TG-treated mice, F4/80+ cells were especially abundant, far exceeding the numbers in the other two treatment groups.


View this table:
[in this window]
[in a new window]
  		Table 1. Cellular Composition of Peritoneal Fluids*

 
The composition of these exudates was further analyzed to identify features that could be correlated with the levels of plasma DNA observed following administration of the apoptotic or necrotic Jurkat cells. This analysis focused on the occurrence of apoptosis among cells accumulating in the exudates, especially among neutrophils, as these are short-lived cells that undergo apoptosis as inflammation proceeds. As also shown in Table 1 , peritonitis provoked by all three stimuli led to an increase in the number of apoptotic neutrophils, as assessed by Gr1+ cells, which were annexin+. These numbers were highest in exudates induced by TG. In particular, the exudates from TG had the highest number of apoptotic F4/80 cells at time-points from 6 h to 72 h, and exudates induced by PT or NaIO4 did not differ in the number of annexin+, F4/80+ cells compared with baseline.

These findings suggest that prior to the administration of the apoptotic and necrotic Jurkat cells, the peritoneal cavity had been the site of cell death, and the extent and cell population involved depend on the inducing agent. To further assess this possibility, levels of caspase 3 and DNA were determined in the peritoneal fluid. Caspase 3 and DNA are released from apoptotic cells and provide a marker of apoptosis in biological fluids [17 , 30 , 31 ]. Figure 4 presents results of these determinations for peritoneal fluid. As these data indicate, each of the stimulating agents led to a distinct pattern of DNA and caspase 3 expression. Whereas exudates induced by TG showed DNA and caspase 3, exudates from mice treated with NaIO4 showed only DNA. In contrast, exudates from mice treated with PT showed neither DNA nor caspase 3.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Levels of DNA and caspase 3 in peritoneal fluids. Peritoneal fluids were obtained from mice treated with 2 ml 4% TG, 1 ml 10% PT, or 1 ml 5 mM NaIO4 IP. Peritoneal fluids were assayed for DNA and caspase 3 as described in Materials and Methods. Results are presented as means (±SD) of three to six mice in each group.

 
These data thus provide an opportunity to assess a correlation between events in the peritoneum and the generation of plasma DNA from apoptotic or necrotic Jurkat cells. Thus, plasma DNA from apoptotic or necrotic cells did not occur in mice receiving TG or NaIO4, the two conditions in which exudates contained DNA. These results suggest that events that lead to the release of DNA into the peritoneal fluid affect the subsequent generation of plasma DNA response from dead and dying cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results presented herein provide new insights into the mechanisms for the generation of circulating DNA from dead and dying cells and indicate that prior inflammation can impact this process significantly. Thus, we have shown in a murine model that the presence of peritoneal inflammation can influence the generation of plasma DNA from apoptotic or necrotic cells administered by the IP route. This effect was not uniform, however, and depended on the inducing agent as well as local events in the peritoneum. These events were reflected in the cellular composition of exudates and the occurrence of local apoptosis, as indicated by the presence of DNA and caspase 3 in the peritoneal fluid. Taken together with previous experiments, these results indicate that the release of DNA into the blood is not the simple consequence of the presence of a large number of dead cells but rather results from a complex interplay with other cell types.

Studies assessing the generation of blood DNA in animals have been of two general types. In the first, an animal is treated with an agent that can induce apoptosis or necrosis in vivo. Agents tested have included lipopolysaccharide (LPS), anti-Fas, and dexamethasone (DEX). It is interesting, that although LPS and anti-Fas cause a blood DNA response, DEX does not [18 ]. These findings suggest that the release of DNA may vary depending on the death stimulus and cell type induced to die. In this regard, systems of this type are potentially confounded by the effects of the inducing agent on the activity of phagocytic cells and clearance of dead and dying cells. For example, LPS may promote the activity of macrophages, and DEX may affect the specific interaction of phagocytes with apoptotic cells [32 , 33 ]. Modeling DNA release by inducing cell death in living animals is also limited by uncertainty in the number and location of the dying cells.

The second general model for modeling the release of DNA into the blood involves the administration of cell treated in vitro to die. In contrast to the administration of an inducer of apoptosis or necrosis, the administration of dead and dying cells provides a more defined system in which the number of dead cells is known more precisely, and the response of the recipient animal can be manipulated. In previous studies exploring this approach, we showed that administration of 108 apoptotic or necrotic Jurkat cells to normal mice leads to the appearance of DNA in the blood in a time- and dose-dependent manner [23 ]. This DNA was observed with two different cell lines made apoptotic or necrotic by a variety of means, suggesting a general property of dead and dying cells. As Jurkat cells are of human origin and were derived from a male, the contribution of this cell type to circulating DNA can be established unequivocally by the demonstration by PCR of Y-chromosomal sequences [23 ].

As shown by many experiments in vivo and in vitro, macrophages play a key role in the clearance of apoptotic and necrotic cells and can engulf these cells with high efficiency [23 , 34 35 36 ]. To explore the role of macrophages in the generation of blood DNA, in previous experiments, we assessed the response to the administration of dead and dying cells of mice in which macrophage function was eliminated by clodronate liposomes. Clodronate is a bisphosphonate, which can induce macrophage apoptosis after uptake and render a mouse deficient in this cell population [37 , 38 ]. Using Jurkat cells as a model, we showed that administration of apoptotic or necrotic cells to a mouse without macrophages fails to produce a blood DNA response. Similar results were obtained in mice treated with silica, which can also eliminate macrophage function [23 ].

Although these findings point to a central role of macrophages in the generation of circulating DNA, they do not define the role of these cells in the DNA release reaction. Most studies on macrophage clearance of dead cells have focused on the uptake of these cells and have not addressed the disposition of their contents. Thus, it is possible that the macrophages take up dead cells, degrade their contents, and expel the remnants into the extracellular milieu. In the absence of macrophages, the dead cells may gradually autolyze or disintegrate in a manner that fails to cause a measurable elevation of circulating DNA. In this scenario, the released DNA is an end-product of the clearance process, with levels rising as the burden of material to be eliminated increases.

An alternative possibility posits that DNA release occurs when the phagocytic capacity of the macrophage is exceeded. In this situation, a phagocytic macrophage filled with dead cells and their breakdown products is induced to undergo apoptosis, releasing its own DNA and that of the engulfed DNA cells. Circumstantial evidence for this possibility is derived from data showing that the blood of mice receiving dead and dying cells contains murine DNA sequences as well as that of the administered human cell. Furthermore, the DNA of murine and human origin shows size-laddering, implying a role for nucleases activated during apoptosis [23 ]. In this scenario, DNA release thus reflects macrophage dysfunction and death rather than heightened physiological function.

The current results provide data relevant to distinguish between these mechanisms. Thus, findings presented herein indicate that inflammation can alter the subsequent clearance of apoptotic and necrotic cells, although the nature of the effect may vary depending on the inflammatory stimulus [39 , 40 ]. Thus, we have tested the effects of three different agents that have been commonly used to induce peritonitis. TG and PT represent complex mixtures comprised of many components [24 , 27 28 29 , 41 ]. With TG, advanced glycation products, which develop as the mixture "ages" may be the key inducing agent. The proinflammatory components in PT have not been well defined. In contrast, NaIO4 is a well-defined chemical that oxidizes surface molecules, including terminal sugars, to produce free aldehydes [25 , 26 , 42 ]. These modifications induce macrophage activation and T cell mitogenesis, and these functional changes are associated with subsequent recruitment of cells into the peritoneum [25 , 26 , 42 ].

Because of properties of these irritants, the nature of the exudates following their administration may differ. Indeed, a variety of studies have compared macrophages arising in these settings with respect to their activation state, functional properties, and production of various cellular proteins. These studies have identified important differences in the features of the resulting exudates [27 28 29 ]. Although the mechanisms of cellular recruitment and activation in the peritoneum are not well understood, our findings suggest that patterns of peritonitis and likely other forms of local inflammation may differ significantly in their impact on the clearance and metabolism of dead and dying cells.

Among possible events that occur in inflammatory sites that may affect the subsequent clearance of dead and dying cells is the occurrence of apoptosis as the inflammatory reaction proceeds. Thus, each of the treatments produced an increased number of annexin+, Gr1+ cells, although these numbers were greatest with TG treatment, which also led to the highest levels of annexin+, F4/80+ cells. Furthermore, with TG-induced peritonitis, the occurrence of apoptosis could be inferred from the presence of DNA and caspase 3 in the peritoneal fluid. In mice with this treatment, a DNA response was not observed following administration of Jurkat cells. Similarly, in mice treated with NaIO4, administration of dead and dying cells did not lead to a blood DNA response. Although peritoneal fluid after NaIO4 administration did not contain caspase 3, it did contain DNA. In contrast, PT-treated mice showed an intact blood DNA response and lacked peritoneal fluid DNA as well as caspase 3.

These findings thus suggest that the effect of inflammation on the magnitude of the blood DNA response resulting from administration of dead and dying cells relates to the presence of DNA in the peritoneal fluid. At present, the mechanisms leading to this DNA and its effect on the functional properties of macrophages are speculative. It is possible, however, that DNA itself or other debris from apoptotic cells may affect the capacity of macrophages to clear dead cells that subsequently arise locally or in the case of these experiments, the administered Jurkat cells. In this regard, the exudates of mice treated with TG and NaIO4, despite their content of DNA, differed in the presence of caspase 3. Although apoptosis can account for simultaneous release of DNA and caspase 3, the mechanisms leading to DNA in the absence of caspase by NaIO4 are unknown. Possibilities include DNA release during cell activation, cellular leakiness secondary to membrane oxidation, or inhibition of caspase 3 by this agent. These possibilities are under investigation.

Studies from a number of investigators have suggested that, in contrast to previous notions, apoptotic cells may have proinflammatory effects and modulate the function of macrophages [43 44 45 46 ]. Among these functions, clearance of apoptotic cells may be modulated, an issue to be considered in experiments. For example, studies about the role of macrophages in the clearance of dead and dying cells have involved mice that have been treated previously with TG to increase macrophage number and facilitate the analysis [47 ]. It is possible that such treatment may have affected important elements of this process, and the effects we have observed on DNA released also affected interactions such as binding and uptake of dead and dying cells.

As shown in studies in human and murine systems, DNA in the blood may have important immune effects that influence the pathogenesis of SLE [48 49 50 ]. These effects include the formation of immune complexes that promote cytokine production and can deposit in the kidney to induce glomerulonephritis [51 , 52 ]. Other nuclear molecules released from dead and dying cells may also have pathogenic effects. For example, the high-mobility group protein has powerful, proinflammatory properties and can function as a cytokine once it has been released from necrotic cells [53 54 55 56 ]. Studies are therefore in progress to determine changes in macrophages that occur during inflammation, including the contact with apoptotic and necrotic cells and their impact on subsequent events in disease pathogenesis.


   ACKNOWLEDGEMENTS
 
This work was supported by a VA Merit Review grant, an Alliance for Lupus research grant, Lupus Research Institute grant, and National Institutes of Health Grant AI44808.

Received July 19, 2004; revised November 18, 2004; accepted November 27, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Steinman, C. R. (1984) Circulating DNA in systemic lupus erythematosus. Isolation and characterization J. Clin. Invest. 73,832-841
  2. Stroun, M., Anker, P., Maurice, P., Lyautey, J., Lederrey, C., Beljanski, M. (1989) Neoplastic characteristics of the DNA found in the plasma of cancer patients Oncology 46,318-322[Medline]
  3. Rumore, P. M., Steinman, C. R. (1990) Endogenous circulating DNA in systemic lupus erythematosus. Occurrence as multimeric complexes bound to histone J. Clin. Invest. 86,69-74
  4. Lo, Y. M., Leung, T. N., Tein, M. S., Sargent, I. L., Zhang, J., Lau, T. K., Haines, C. J., Redman, C. W. (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia Clin. Chem. 45,184-188[Abstract/Free Full Text]
  5. Anker, P., Lyautey, J., Lederrey, C., Stroun, M. (2001) Circulating nucleic acids in plasma or serum Clin. Chim. Acta 313,143-146[CrossRef][Medline]
  6. Jahr, S., Hentze, H., Englisch, S., Hardt, D., Fackelmayer, F. O., Hesch, R. D., Knippers, R. (2001) DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells Cancer Res. 61,1659-1665[Abstract/Free Full Text]
  7. Lui, Y. N., Lo, Y. M. (2002) Circulating DNA in plasma and serum: biology, preanalytical issues and diagnostic applications Clin. Chem. Lab. Med. 40,962-968[CrossRef][Medline]
  8. Lam, N. Y., Rainer, T. H., Chan, L. Y., Joynt, G. M., Lo, Y. M. (2003) Time course of early and late changes in plasma DNA in trauma patients Clin. Chem. 49,1286-1291[Abstract/Free Full Text]
  9. Pisetsky, D. S. (1996) Immune activation by bacterial DNA: a new genetic code Immunity 5,303-310[CrossRef][Medline]
  10. Krieg, A. M. (2002) CpG motifs in bacterial DNA and their immune effects Annu. Rev. Immunol. 20,709-760[CrossRef][Medline]
  11. Ronnblom, L., Alm, G. V. (2002) The natural interferon-{alpha} producing cells in systemic lupus erythematosus Hum. Immunol. 63,1181-1193[CrossRef][Medline]
  12. Lovgren, T., Eloranta, M. L., Bave, U., Alm, G. V., Ronnblom, L. (2004) Induction of interferon-{alpha} production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG Arthritis Rheum. 50,1861-1872[CrossRef][Medline]
  13. Leadbetter, E. A., Rifkin, I. R., Hohlbaum, A. M., Beaudette, B. C., Shlomchik, M. J., Marshak-Rothstein, A. (2002) Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors Nature 416,603-607[CrossRef][Medline]
  14. Viglianti, A., Lau, C. M., Hanley, T. M., Miko, B. A., Shlomchik, M. J., Marshak-Rothstein, A. (2003) Activation of autoreactive B cells by CpG dsDNA Immunity 19,837-847[CrossRef][Medline]
  15. Stroun, M., Maurice, P., Vasioukhin, V., Lyautey, J., Lederrey, C., Lefort, F., Rossier, A., Chen, X. Q., Anker, P. (2000) The origin and mechanism of circulating DNA Ann. N. Y. Acad. Sci. 906,161-168[Free Full Text]
  16. Stroun, M., Lyautey, J., Lederrey, C., Olson-Sand, A., Anker, P. (2001) About the possible origin and mechanism of circulating DNA apoptosis and active DNA release Clin. Chim. Acta 313,139-142[CrossRef][Medline]
  17. Choi, J. J., Reich, C. F., III, Pisetsky, D. S. (2004) Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro Scand. J. Immunol. 60,159-166[CrossRef][Medline]
  18. Jiang, N., Reich, C. F., III, Monestier, M., Pisetsky, D. S. (2003) The expression of plasma nucleosomes in mice undergoing in vivo apoptosis Clin. Immunol. 106,139-147[CrossRef][Medline]
  19. Nicotera, P., Leist, M., Ferrando-May, E. (1999) Apoptosis and necrosis: different execution of the same death Biochem. Soc. Symp. 66,69-73[Medline]
  20. Duffield, J. S. (2003) The inflammatory macrophage: a story of Jekyll and Hyde Clin. Sci. (Lond.) 104,27-38[Medline]
  21. Gordon, S. (2003) Alternative activation of macrophages Nat. Rev. Immunol. 3,23-35[CrossRef][Medline]
  22. Fadok, V. A., Bratton, D. L., Guthrie, L., Henson, P. M. (2001) Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases J. Immunol. 166,6847-6854[Abstract/Free Full Text]
  23. Jiang, N., Reich, C. F., III, Pisetsky, D. S. (2003) Role of macrophages in the generation of circulating blood nucleosomes from dead and dying cells Blood 102,2243-2250[Abstract/Free Full Text]
  24. Tannenbaum, C. S., Nurmi-McKernan, L., Largen, M. T. (1987) Differential protein synthesis by murine peritoneal macrophages elicited by various stimuli J. Leukoc. Biol. 41,527-538[Abstract]
  25. Weinberg, J. B. (1981) In vivo modulation of macrophage tumoricidal activity: enhanced tumor cell killing by peritoneal macrophages from mice given injections of sodium periodate J. Natl. Cancer Inst. 66,529-533
  26. Weinberg, J. B. (1983) Macrophage polykaryon formation in vitro by peritoneal cells from mice given injections of sodium periodate Am. J. Pathol. 110,182-192[Abstract]
  27. Beelen, R. H., Walker, W. S. (1983) Dynamics of cytochemically distinct subpopulations of macrophages in elicited rat peritoneal exudates Cell. Immunol. 82,246-257[CrossRef][Medline]
  28. Melnicoff, M. J., Horan, P. K., Morahan, P. S. (1989) Kinetics of changes in peritoneal cell populations following acute inflammation Cell. Immunol. 118,178-191[CrossRef][Medline]
  29. Oghiso, Y., Yamada, Y., Shibata, Y. (1992) Exudation of proliferative macrophages in local inflammation in the peritoneum J. Leukoc. Biol. 52,421-424[Abstract]
  30. Thornberry, N. A., Lazebnik, Y. (1998) Caspases: enemies within Science 281,1312-1316[Abstract/Free Full Text]
  31. Hentze, H., Schwoebel, F., Lund, S., Keel, M., Ertel, W., Wendel, A., Jaattela, M., Leist, M., Kehl, M. (2001) In vivo and in vitro evidence for extracellular caspase activity released from apoptotic cells Biochem. Biophys. Res. Commun. 283,1111-1117[CrossRef][Medline]
  32. Hartmann, G., Krieg, A. M. (1999) CpG DNA and LPS induce distinct patterns of activation in human monocytes Gene Ther. 6,893-903[CrossRef][Medline]
  33. Liu, Y., Cousin, J. M., Hughes, J., Van Damme, J., Seckl, J. R., Haslett, C., Dransfield, I., Savill, J., Rossi, A. G. (1999) Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes J. Immunol. 162,3639-3646[Abstract/Free Full Text]
  34. Henson, P. M., Bratton, D. L., Fadok, V. A. (2001) Apoptotic cell removal Curr. Biol. 11,R795-R805[CrossRef][Medline]
  35. Geske, F. J., Monks, J., Lehman, L., Fadok, V. A. (2002) The role of the macrophage in apoptosis: hunter, gatherer, and regulator Int. J. Hematol. 76,16-26[Medline]
  36. Mosser, D. M. (2003) The many faces of macrophage activation J. Leukoc. Biol. 73,209-212[Free Full Text]
  37. Van Rooijen, N., Sanders, A. (1994) Liposome-mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications J. Immunol. Methods 174,83-93[CrossRef][Medline]
  38. van Rooijen, N., van Kesteren-Hendrikx, E. (2002) Clodronate liposomes: perspectives in research and therapeutics J. Liposome Res. 12,81-94[CrossRef][Medline]
  39. Cook, A. D., Braine, E. L., Hamilton, J. A. (2003) The phenotype of inflammatory macrophages is stimulus dependent: implications for the nature of the inflammatory response J. Immunol. 171,4816-4823[Abstract/Free Full Text]
  40. Wiltrout, R. H., Brunda, M. J., Gorelik, E., Peterson, E. S., Dunn, J. J., Leonhardt, J., Varesio, L., Reynolds, C. W., Holden, H. T. (1983) Distribution of peritoneal macrophage populations after intravenous injection in mice: differential effects of eliciting and activating agents J. Reticuloendothel. Soc. 34,253-269[Medline]
  41. Li, Y. M., Baviello, G., Vlassara, H., Mitsuhashi, T. (1997) Glycation products in aged thioglycollate medium enhance the elicitation of peritoneal macrophages J. Immunol. Methods 201,183-188[CrossRef][Medline]
  42. Duque, F. T., III, Herscowitz, H. B. (1989) Sodium periodate treatment modulates the accessory and regulatory functions of alveolar macrophages in T-cell responses Reg. Immunol. 2,129-135[Medline]
  43. Voll, R. E., Herrmann, M., Roth, E. A., Stach, C., Kalden, J. R., Girkontaite, I. (1997) Immunosuppressive effects of apoptotic cells Nature 390,350-351[CrossRef][Medline]
  44. Fadok, V. A., Bratton, D. L., Konowal, A., Freed, P. W., Westcott, J. Y., Henson, P. M. (1998) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-ß, PGE2, and PAF J. Clin. Invest. 101,890-898[Medline]
  45. Reddy, S. M., Hsiao, K. H., Abernethy, V. E., Fan, H., Longacre, A., Lieberthal, W., Rauch, J., Koh, J. S., Levine, J. S. (2002) Phagocytosis of apoptotic cells by macrophages induces novel signaling events leading to cytokine-independent survival and inhibition of proliferation: activation of Akt and inhibition of extracellular signal-regulated kinases 1 and 2 J. Immunol. 169,702-713[Abstract/Free Full Text]
  46. Maderna, P., Godson, C. (2003) Phagocytosis of apoptotic cells and the resolution of inflammation Biochim. Biophys. Acta 1639,141-151[Medline]
  47. Taylor, P. R., Carugati, A., Fadok, V. A., Cook, H. T., Andrews, M., Carroll, M. C., Savill, J. S., Henson, P. M., Botto, M., Walport, M. J. (2000) A hierarchical role for classical pathway complement proteins in the clearance of apoptotic cells in vivo J. Exp. Med. 192,359-366[Abstract/Free Full Text]
  48. Fournie, G. J. (1988) Circulating DNA and lupus nephritis Kidney Int. 33,487-497[Medline]
  49. Licht, R., van Bruggen, M. C. J., Oppers-Walgreen, B., Rijki, T. P. M., Berden, J. H. M. (2001) Plasma levels of nucleosomes and nucleosome-autoantibody complexes in murine lupus: effects of disease progression and lipopolysaccharide administration Arthritis Rheum. 44,1320-1330[CrossRef][Medline]
  50. Rosen, A., Casciola-Rosen, L. (2001) Clearing the way to mechanisms of autoimmunity Nat. Med. 7,664-665[CrossRef][Medline]
  51. Vinuesa, C. G., Goodnow, C. C. (2002) Immunology: DNA drives autoimmunity Nature 416,595-598[CrossRef][Medline]
  52. Kramers, C., Hylkema, M. N., van Bruggen, M. C., van de Lagemaat, R., Dijkman, H. B., Assmann, K. J., Smeenk, R. J., Berden, J. H. (1994) Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo J. Clin. Invest. 94,568-577
  53. Maher, J. F., Nathans, D. (1996) Multivalent DNA-binding properties of the HMG-1 proteins Proc. Natl. Acad. Sci. USA 93,6716-6720[Abstract/Free Full Text]
  54. Wang, H., Bloom, O., Zhang, M., Vishnubhakat, J. M., Ombrellino, M., Che, J., Frazier, A., Yang, H., Ivanova, S., Borovikova, L., Manogue, K. R., Faist, E., Abraham, E., Andersson, J., Andersson, U., Molina, P. E., Abumrad, N. N., Sama, A., Tracey, K. J. (1999) HMG-1 as a late mediator of endotoxin lethality in mice Science 285,248-251[Abstract/Free Full Text]
  55. Scaffidi, P., Misteli, T., Bianchi, M. E. (2002) Release of chromatin protein HMGB1 by necrotic cells triggers inflammation Nature 418,191-195[CrossRef][Medline]
  56. Andersson, U., Erlandsson-Harris, H., Yang, H., Tracey, K. J. (2002) HMGB1 as a DNA-binding cytokine J. Leukoc. Biol. 72,1084-1091[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc Am Thorac SocHome page
D. S. Pisetsky
The Role of Nuclear Macromolecules in Innate Immunity
Proceedings of the ATS, July 1, 2007; 4(3): 258 - 262.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0704411v1
77/3/296    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jiang, N.
Right arrow Articles by Pisetsky, D. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jiang, N.
Right arrow Articles by Pisetsky, D. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS