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The Journal of Immunology, 2006, 177: 7740-7749.
Copyright © 2006 by The American Association of Immunologists, Inc.

TLR2/TLR4-Independent Neutrophil Activation and Recruitment upon Endocytosis of Nucleosomes Reveals a New Pathway of Innate Immunity in Systemic Lupus Erythematosus1

Viktoria M. Rönnefarth*, Annika I. M. Erbacher*, Tobias Lamkemeyer{dagger}, Johannes Madlung{dagger}, Alfred Nordheim{dagger}, Hans-Georg Rammensee* and Patrice Decker2,*

* Department of Immunology and {dagger} Proteom Center Tübingen, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The nucleosome is a major autoantigen in systemic lupus erythematosus (SLE); it can be detected as a circulating complex in the serum, and nucleosomes have been suggested to play a key role in disease development. In the present study, we show for the first time that physiological concentrations of purified nucleosomes trigger innate immunity. The nucleosomes are endocytosed and induce the direct activation of human neutrophils (polymorphonuclear leukocytes (PMN)) as revealed by CD11b/CD66b up-regulation, IL-8 secretion, and increased phagocytic activity. IL-8 is a neutrophil chemoattractant detected in high concentrations in the sera of patients, and IL-8 secretion might thus result in enhanced inflammation, as observed in lupus patients, via an amplification loop. Nucleosomes act as free complexes requiring no immune complex formation and independently of the presence of unmethylated CpG DNA motifs. Both normal and lupus neutrophils are sensitive to nucleosome-induced activation, and activation is not due to endotoxin or high-mobility group box 1 contamination. In mice, i.p. injection of purified nucleosomes induces neutrophil activation and recruitment in a TLR2/TLR4-independent manner. Importantly, neutrophils have been suggested to link innate and adaptive immunity. Thus, nucleosomes trigger a previously unknown pathway of innate immunity, which may partially explain why peripheral tolerance is broken in SLE patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Systemic lupus erythematosus (SLE)3 is a systemic autoimmune disease of unknown etiology that affects over 1 million people in the United States. It is described as an inflammatory rheumatic disease and is characterized by the production of a large amount of circulating autoantibodies. Some of these Abs, in the form of immune complexes, cause tissue damage upon deposition. Among the targets recognized by these autoantibodies is the nucleosome, a molecular complex composed of 180-bp DNA and the five histone proteins H1, H2A, H2B, H3, and H4. Significantly, circulating nucleosomes are found in the sera of SLE patients (1), and the level of circulating nucleosomes has been shown to be associated with disease activity (2). The levels of circulating nucleosomes are raised in SLE patients with active CNS and renal involvement. Only circulating nucleosomes are usually found in SLE patients but not free DNA (1), and autoreactive nucleosome-specific B and Th lymphocytes can also be detected in these patients (3, 4). Interestingly, the level of circulating anti-nucleosome autoantibody has been shown to be associated with disease activity (5, 6), and anti-dsDNA Abs represent a disease marker. Increased levels of IgG3 anti-nucleosome Abs are associated with active nephritis (7). Finally, anti-nucleosome autoantibodies are detected at a very early stage of the disease and are potentially nephritogenic in lupus mice (8). Altogether, these results suggest that nucleosomes play a key role in lupus development, but it is still not known why antinucleosome autoimmunity is triggered.

Recently, new functions have been attributed to autoantigen, some of which are directly associated with innate immunity. In addition to their role as targets for autoantibodies, they have been shown to have a positive and active effect on the immune system. Thus, histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, two autoantigens in myositis, have been shown to activate chemokine receptors on T lymphocytes and immature dendritic cells (9). Similarly, a chemoattractive function has been demonstrated for other autoantigens, including lupus autoantigens (10). Likewise, small nuclear ribonucleoprotein particles in the form of immune complexes indirectly stimulate plasmacytoid dendritic cells (11, 12) and B lymphocytes (13). Regarding nucleosomal Ags, chromatin-containing immune complexes activate rheumatoid factor-positive B lymphocytes as well as dendritic cells (see Ref. 14 for review). Free nucleosomes have also been shown to be potentially pathogenic, as reviewed in Ref. 15 . Finally, we have shown that purified nucleosomes directly activate dendritic cells (16). In contrast to the studies described above in which nucleosomes activate dendritic cells in the form of immune complexes, we found that free nucleosomes induce cell activation without the need for immune complex formation, which points to a new mechanism of dendritic cell activation by nucleosomes.

Neutrophils represent the first line of defense against invading pathogens and are the first cells recruited during an inflammatory response. Since SLE is an inflammatory disease, polymorphonuclear leukocytes (PMN) might play an important role in the disease. Accordingly, neutrophils from patients with active disease express higher levels of CD11b/CD18, a process used as a neutrophil activation marker (17). Moreover, sera from lupus patients in active phase contain an activity responsible for neutrophil activation (18). Likewise, serum levels of IL-8, a chemoattractant for neutrophils that is also secreted by neutrophils, have been shown to be elevated in lupus patients and to correlate with disease activity (19). Finally, activated neutrophils have been shown to interact with dendritic cells and have been suggested to link innate and adaptive immunity (20, 21, 22). This led us to the assumption that circulating nucleosomes might be one of the serum factors involved in neutrophil activation in lupus. As a consequence, we examined whether purified nucleosomes, a key autoantigen in SLE, could directly activate PMN in the absence of lupus autoantibodies, which would strengthen the role of this autoantigen and indicate a new activation pathway of innate immunity in SLE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

BALB/c mice were purchased from Charles River Laboratories. TLR2-knockout (KO) mice (described in Ref. 23) on a C3H/HeJ background (C3H/HeJ/TLR2-KO) were obtained from Prof. H. Schild (Institute for Immunology, Johannes Gutenberg University, Mainz, Germany) who originally received them from Prof. H. Wagner (Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany). Experiments were approved by the local animal ethics committee (Ref. IM 2/03).

Blood donors

Heparin-blood from random, healthy individuals and from lupus patients was used in the present study. Lupus blood and clinical data were provided by Dr. I. Kötter (Department of Internal Medicine II(Haematology, Oncology, Immunology, and Rheumatology), University Hospital, Tübingen, Germany). Lupus patients fulfilled the American College of Rheumatology criteria. Experiments with human cells were approved before the study by the local ethics committee (Ref. 146/2001V), and samples were obtained after receiving the informed consent of the donors.

Nucleosomes, histones, DNA, and high-mobility group box 1 (HMGB1)

Nucleosomes were prepared from calf thymus as previously described (16, 24) under sterile conditions. H1-depleted nucleosomes were obtained after additional incubation in 0.5 M NaCl. Mononucleosomes were obtained after ultracentrifugation on to sucrose gradients. DNA and protein contents were verified on a 1.5% agarose gel and an 18% SDS-PAGE, respectively. We have verified that the amount of nonhistone proteins was reduced in H1-depleted nucleosomes (data not shown). If unspecified in the text, nucleosomes refer to mononucleosomes. Importantly, histones are among the most conserved proteins. For instance, there is no difference in protein sequence between histone H4 from calf, human, and mouse. To analyze cell surface binding and endocytosis, nucleosomes were dialyzed and FITC conjugated as described previously (25, 26). In some cases, PMN were activated with DNA purified from mononucleosome fractions by phenol/chloroform extraction, followed by ethanol precipitation, as well as with purified histones (Roche). In both cases, the material was resuspended in the buffer used for nucleosome purification. DNA and histone contents were verified as described above. Purified HMGB1 of cytokine-quality grade was purchased from HMGBiotech.

The level of endotoxin contamination was tested using a Limulus amebocyte lysate assay (BioWhittaker) and was determined to be low, ~10–50 IU/mg nucleosomes. The same endotoxin concentration (in IU/ml) was measured in purified nucleosomes and in the purification buffer that was used as a negative control.

Two-dimensional gel electrophoresis

Protein (150 µg) of a nucleosome preparation was desalted by ultrafiltration using Nanosep spin columns (molecular weight cutoff 3 kDa; Pall Life Sciences) and rehydration solution (8 M urea, 2 M thiourea, 2% CHAPS, 0.5% pharmalytes 3/10, and 50 mM DTT) as the new buffer. Additionally, 5 µg of purified HMGB1 were diluted into rehydration solution to a final volume of 125 µl. Proteins were focused in 7-cm pH 3–10 immobilized pH gradient strips (Bio-Rad) after active rehydration (15 h, 50 V) until a total of at least 16,000 V-h was reached (maximum 4,000 V; Protean isoelectric focusing cell; Bio-Rad). After focusing, immobilized pH gradient strips were equilibrated (6 M urea, 2% (w/v) SDS, 30% (w/v) glycerol, 50 mM Tris (pH 8.8), containing 1% DTT and 4% iodoacetamide, respectively; 15 min each). The second dimension was performed on 18% separating gels (pH 8.8) using SeeBlue molecular mass markers (Invitrogen Life Technologies). The gels were subjected to silver staining compatible to subsequent analysis by mass spectrometry (MS).

MS and analysis of spectrometric data

To check whether HMGB1 was present in the nucleosome preparations, protein spots occupying positions similar to the reported molecular mass and isoelectric point (pI) of HMGB1 from calf thymus (27) and similar to that of purified HMGB1 were excised from two-dimensional gels loaded with nucleosomes and digested in-gel using trypsin (sequencing grade; Promega). The eluted, trypsin-generated peptides were subsequently analyzed by nanoflow liquid chromatography tandem MS (nano-LC-MS/MS), and proteins were identified by correlating the data from the MS/MS spectra with the NCBI.nr-protein sequence database mammalia (see Ref. 28 for details). All results from two-dimensional experiments and MS experiments and all search results where stored in a Laboratory Information Management System database (Proteinscape 1.3; Bruker Daltonics).

Neutrophil preparation

Neutrophils were prepared from heparinized blood by dextran sedimentation using Polymorphprep (Axis-Shield PoC-Progen), according to the manufacturer’s recommendations and as described previously (29). Contaminating RBC were lysed using a hypotonic lysis buffer (150 mM ammonium chloride, 1 mM potassium bicarbonate, and 0.1 mM EDTA in distilled water (pH 7.3)). Purity of the cell preparation was analyzed by flow cytometry by estimating the cell surface expression of CD66b, a marker for neutrophils. Cells were stained with a CD66b-specific mAb or the corresponding isotype control (BD Pharmingen). At least 85% of the cells were CD66b+.

Cell culture

Purified neutrophils were cultured in RPMI 1640 medium (BioWhittaker) supplemented with 10% heat-inactivated FCS (PAA) at 106 cells/ml in 96-well plates (round-bottom). Cells were incubated in medium alone or medium supplemented with either purified nucleosomes, purification buffer (as a negative control), purified DNA, purified histones, HMGB1, or LPS (Salmonella typhimurium; Sigma-Aldrich). At different time points, cell culture supernatants were harvested and frozen at –80°C until use, and neutrophil activation and survival were estimated. In some cases, PMN were cultured in the presence of the LPS-inhibitor polymyxin B sulfate (25 µg/ml; Fluka).

Measurement of cell activation and cell death in human neutrophils

Activation of human PMN was estimated by flow cytometry by measuring cell surface molecule up-regulation using FITC-conjugated anti-CD66b and PE-conjugated anti-CD11b-specific mAb and the corresponding isotype controls (BD Pharmingen) on a FACSCalibur apparatus with CellQuest software (BD Biosciences). Cells were costained with 7-aminoactinomycin D (7-AAD; BD Pharmingen) to exclude dead cells. Cell activation was also estimated by measuring the phagocytic activity. Briefly, ingestion of PE-labeled polystyrene microspheres (1 µm in diameter, Fluoresbrite Plain Microspheres PCRed; Polysciences) was evaluated as previously described (30) using 2 x 105 freshly purified PMN preincubated in the presence of microspheres (diluted 1/100) for 30 min at 37°C and then incubated with the stimuli for several hours. Cells were washed twice, fixed in 1% formaldehyde in PBS, and analyzed by flow cytometry. Activation of PMN was confirmed by measuring IL-8 secretion as estimated by a sandwich ELISA kit (BD Pharmingen) according to the manufacturer’s recommendations.

Neutrophil cell death was estimated by flow cytometry after staining with FITC-conjugated anti-CD66b mAb, PE-conjugated annexin V (BD Pharmingen), and 7-AAD according to classical procedures. Early apoptotic cells are annexin V positive but 7-AAD negative, whereas living cells are annexin V negative and 7-AAD negative.

In vivo neutrophil recruitment and activation

Mice were i.p. injected with either 100 µg of purified nucleosomes, the same volume of purification buffer or 50 µg of LPS dissolved in purification buffer. After 18 h, mice were sacrificed to prepare peritoneal exudate cells. Briefly, the peritoneal cavity was washed twice with 5 ml of PBS containing 1 mM EDTA. Cells were then harvested, and any possible contaminating RBC were lysed with 5 ml of hypotonic buffer, followed by two washes with PBS. Neutrophil recruitment and activation was estimated by measuring the percentage of living Ly-6GhighCD11bhigh cells among peritoneal cells, i.e., activated mature neutrophils, in each individual mouse. Peritoneal exudate cells were first incubated with an FcR-blocking mAb (BD Pharmingen) and then stained with FITC-conjugated anti-Ly6G and PE-conjugated anti-CD11b-specific mAb or the corresponding isotype controls (BD Pharmingen) in the presence of 7-AAD. Cells were analyzed by flow cytometry, gating out dead cells (7-AAD-positive cells). At least 50,000 events were acquired for each sample.

Cell surface binding and endocytosis

The fate of exogenous nucleosomes was first examined by flow cytometry. PMN (105) were incubated with or without 10 µM FITC-conjugated nucleosomes (FITC-nucleosomes) on ice or at 37°C in 100 µl of 10% FCS-containing PBS for 30 min. Cells were then washed, incubated with propidium iodide, and analyzed.

To confirm the results, cells were analyzed by confocal microscopy. PMN were incubated as described above on ice or at 37°C. After two washes, cells were stained with a PE-conjugated anti-CD11b mAb, fixed in 4% formaldehyde, and analyzed using an inverted LSM510 confocal laser scanning microscope (Carl Zeiss) fitted with a Plan-Neofluar x40/0.75 objective. For double detection of fluorescein and PE fluorescence, the 488-nm line of an argon ion laser and the light of a 543-nm helium/neon laser were used, and fluorescence was detected using a 505- to 530-nm band pass filter for fluorescein and a 560-nm long pass filter for PE.

To exclude the possibility that FITC-nucleosomes behave differently from unconjugated nucleosomes due to structural changes, the above experiments were reproduced, and binding/endocytosis was detected by indirect staining using 35 µg/ml unconjugated nucleosomes. After two washes, cells were permeabilized and stained with a mouse anti-histone mAb (Roche) known to recognize nucleosomes. After another series of washes, cells were incubated with a FITC-conjugated goat anti-mouse secondary Ab, fixed, and analyzed by confocal microscopy as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Nucleosomes directly activate human neutrophils without the need for immune complex formation

Enriched human neutrophils from healthy donors were cultured with low endotoxin containing purified nucleosomes, and cell activation was analyzed. We first estimated CD66b and CD11b up-regulation by flow cytometry, CD66b being a neutrophil-specific marker in humans and CD11b being an activation marker. As shown in Fig. 1A, nucleosomes induced a clear CD66b and CD11b up-regulation by PMN as compared with nonactivated cells. As a positive control, LPS also activated PMN (Fig. 1B, without polymyxin B). Nucleosome-induced CD66b up-regulation was as strong as LPS-induced up-regulation. As far as CD11b up-regulation is concerned, LPS-induced activation was stronger than nucleosome-induced activation. Nevertheless, nucleosomes induced a significant 2-fold increase in CD11b expression (see Fig. 1, A and B, without polymyxin B). CD66b/CD11b up-regulation was induced by nucleosomes in a dose- and time-dependent manner (starting at 10 µg/ml and after 4 h; data not shown), whereas the buffer used for nucleosome purification did not induce cell activation (Fig. 1B). This buffer, and not the medium alone, is the appropriate negative control for nucleosomes. It corresponds to an empty sucrose gradient unloaded with chromatin (see Materials and Methods). For CD11b and CD66b, both the percentage of positive cells, as well as the level of cell surface expression, was increased with nucleosomes. The purification buffer contains a similar level of endotoxin than the nucleosome concentrations used, as determined by Limulus amebocyte lysate assay, but does not activate cells.


Figure 1
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  		FIGURE 1. Human neutrophils up-regulate cell surface molecules upon activation with nucleosomes. Freshly isolated PMN were cultured with different stimuli and analyzed by flow cytometry after 16 h using specific mAb. A, Representative up-regulation observed after activation by nucleosomes. Left panel, CD66b expression; right panel, CD11b expression. Bold line, Nucleosome-activated neutrophils stained with specific mAb; thin line, nonactivated neutrophils stained with specific mAb; dashed line, isotype control. B, Comparative CD66b/CD11b cell surface expression in the absence or presence of the LPS inhibitor polymyxin B. CD11b expression was analyzed on CD66b-positive cells. MFI, mean fluorescence intensity; buffer, purification buffer used during nucleosome preparation; Nuc, 25 µg/ml purified nucleosomes; LPS, 5 ng/ml lipopolysaccharides; PB, polymyxin B. Shown is 1 of 10 representative experiments. SDs are indicated.

 
Neutrophil activation was then estimated by measuring the phagocytic activity as determined by flow cytometry using PE-conjugated microspheres. The increase of phagocytic activity is commonly used as an activation marker of neutrophils. As shown in Fig. 2A, a basal phagocytic activity is detected with PMN incubated in medium alone, whereas nucleosomes strongly induced phagocytosis by PMN as compared with untreated cells. Again, nucleosome-induced phagocytosis was strong and dose dependent (data not shown) and comparable to LPS-induced phagocytosis (Fig. 2B, without polymyxin B).


Figure 2
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  		FIGURE 2. Nucleosome-activated neutrophils have an increased phagocytic activity. Freshly isolated human PMN were incubated with different stimuli for 16 h and analyzed by flow cytometry using PE-conjugated microspheres. A, Representative phagocytic activity observed upon activation with nucleosomes. Bold line, Nucleosome-activated PMN with microspheres; thin line, nonactivated PMN with microspheres; dashed line, PMN without microspheres. B, Comparative phagocytic activity in the absence or presence of the LPS inhibitor polymyxin B. See Fig. 1 for abbreviations and conditions, except Nuc, 30 µg/ml purified nucleosomes. Shown is one of six representative experiments. SDs are indicated.

 
Nucleosome-induced neutrophil activation was then confirmed by ELISA by measuring IL-8 secretion, another activation marker. Although neutrophils secreted IL-8 spontaneously, nucleosomes and LPS induced a strong cytokine secretion as compared with cells incubated in medium only, usually reaching a high concentration of 1–5 ng/ml (Fig. 3A, without polymyxin B). Neutrophil activation was detected with as low as 1–5 µg/ml purified nucleosomes (Fig. 3B).


Figure 3
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  		FIGURE 3. Nucleosome-activated human neutrophils secrete IL-8. A, Freshly isolated PMN were cultured with different stimuli for 16 h in the presence or absence of polymyxin B. Cell culture supernatants were then harvested and analyzed for the presence of IL-8 by ELISA. See Fig. 1 for abbreviations and conditions. IL-8 concentrations are expressed in nanograms per milliliter. B, Low nucleosome concentrations also induce IL-8 secretion by neutrophils. Cells were incubated as in A in the presence of only 1 µg/ml (Nuc 1) or 5 µg/ml (Nuc 5) nucleosomes. Shown is 1 of 10 representative experiments. SDs are indicated.

 
Importantly, all these experiments were performed with purified nucleosomes in the absence of anti-nucleosome Abs, indicating that free nucleosomes, and not nucleosome-containing immune complexes, activate neutrophils.

Nucleosome-induced neutrophil activation is not due to endotoxin contamination

Neutrophil activation was then estimated in the presence or absence of polymyxin B, an antibiotic which inhibits LPS activity. As shown in Figs. 1B, 2B, and 3 (with polymyxin B), nucleosome-induced activation (estimated by either CD66b/CD11b up-regulation, phagocytic activity, or IL-8 secretion) was not inhibited by polymyxin B. On the other hand, LPS-induced neutrophil activation was almost completely inhibited, revealing that nucleosome-induced activation to be endotoxin independent. Likewise, cell death inhibition by nucleosomes was not affected by polymyxin B (see below). Finally, nucleosome-induced neutrophil activation was observed in TLR2/TLR4-deficient mice (see below).

An intact core nucleosome structure is necessary for neutrophil activation

To examine whether nucleosome integrity is essential in neutrophil activation, cells were incubated with nucleosomes, purified histones, and DNA purified from the nucleosomes, and the activating properties of each stimulus alone or in combination were compared. PMN were cultured in the presence of polymyxin B because the commercial histone preparation was not endotoxin free. As shown in Fig. 4A, a clear increase in IL-8 secretion was observed with nucleosomes in the presence of polymyxin B as compared with cells cultured in the purification buffer, but not with the individual nucleosomal components, namely histones or DNA, or when histones and DNA were added together. This result shows first, that the nucleosome structure is necessary for neutrophil activation, and second, that nucleosome-induced neutrophil activation occurs independently of the presence of unmethylated CpG DNA motifs. Moreover, it demonstrates that not all proteins are able to activate PMN.


Figure 4
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  		FIGURE 4. The core nucleosome structure is necessary for neutrophil activation. A, Fresh human PMN were activated with nucleosomes or purified nucleosomal components in the presence of polymyxin B (PB). Sixteen hours later, cell culture supernatants were harvested and IL-8 concentrations (expressed in nanograms per milliliter) were determined by ELISA. Buffer, purification buffer; Nuc, nucleosomes; DNA, DNA purified from nucleosomes; histones, commercial purified histones. All stimuli were used at 25 µg/ml. B, Nucleosomes, but not HMGB1, induce neutrophil activation. Neutrophils were cultured as in A in the presence or absence of polymyxin B, and IL-8 secretion was measured. Cells were incubated with nucleosomes, H1-depleted nucleosomes (Nuc-H1; containing a lower amount of nonhistone proteins), or HMGB1, and cell activation was compared in the same experiment. Activation-induced IL-8 secretion was calculated for each stimulus by subtracting the background (cells in medium) and is expressed as percentage of the positive control (LPS without polymyxin B). Numbers refer to the concentration of the stimuli (µg/ml nucleosomes or ng/ml HMGB1). LPS was used at 5 ng/ml. C, Nucleosome preparations were analyzed for HMGB1 contamination by two-dimensional gel electrophoresis. An example of a two-dimensional gel of a nucleosome preparation (left panel) and the spot pattern of purified HMGB1 (right panel) are shown. The position of HMGB1 on the gel as defined by its molecular mass and pI was marked (dashed box; pH 5.6–6.8, molecular mass 30–36 kDa). In the corresponding region on the gel of the nucleosome preparation, only two spots were present (encircled; pI = 5.8 and 6.1, respectively). These spots were identified by electrospray ionization-MS/MS as members of the H2A histone family, proving that nucleosome-induced activation is not due to HMGB1 contamination. The prominent spots at pH 10 were revealed to contain histone proteins. Note the different separation of proteins on the two gels during SDS-PAGE as can be seen by the migration distances of marker proteins. Shown is one of three to five representative experiments. SDs are indicated.

 
HMGB1 is a nonhistone chromatin protein. Although purified mononucleosomes normally only contain trace amounts of HMGB1 (31), some HMGB1 may be copurified with nucleosomes. Since HMGB1 has been shown to activate neutrophils (32), we have designed a series of experiments to exclude that the neutrophil activation we detected was due to HMGB1 contamination. Neutrophils were incubated with nucleosomes, H1-depleted nucleosomes (with a lower nonhistone proteins content), or purified HMGB1 of cytokine-quality grade, and cell activation was estimated by IL-8 secretion. We compared the neutrophil activation induced by each stimulus, and the results were normalized to the positive control (LPS in the absence of polymyxin B). Whereas no activation was detected in the presence of the purification buffer, a clear dose-dependent IL-8 secretion was observed with nucleosomes or H1-depleted nucleosomes, even in the presence of polymyxin B (Fig. 4B). Both types of nucleosomes activate neutrophils in the same range of concentration. In contrast, no IL-8 secretion was observed in the presence of HMGB1, even at higher concentrations known to be biologically active. As a control, LPS induced a strong IL-8 secretion, which was inhibited by polymyxin B. These results show that the core nucleosome structure is enough to induce neutrophil activation and that activation is not detected upon treatment with HMGB1.

We then analyzed our nucleosome preparations for the presence of contaminating HMGB1 by two-dimensional gel electrophoresis. In comparison to the reported molecular mass and pI of HMGB1 from calf thymus (27) and to the two-dimensional gel of purified HMGB1, nucleosome preparations contained only two spots occupying positions similar to that of HMGB1 (Fig. 4C). These two spots were picked from two different gels, digested with trypsin, and analyzed by nanoflow liquid chromatography tandem MS. Automated mascot software was used for searching NCBI.nr protein databases on all available mammalian proteins. The proteins were identified as H2A histone family, member Y isoform 2 of Bos taurus (gi number 87578157; molecular weight search scores: 186.6–320.1, sequence coverage: 8.1–15.1%, 4 and 7 peptides, respectively). These results indicate that no HMGB1 was present in the nucleosome preparations. Altogether, our results strongly suggest that nucleosome-induced activation is not due to HMGB1 contamination.

Lupus neutrophils are also activated by nucleosomes

We then compared the sensitivity of normal and lupus neutrophils against purified nucleosomes to exclude any intrinsic difference between both donor groups. As expected, PMN from SLE patients were also activated by nucleosomes and LPS, as estimated by IL-8 secretion (Table I). The activation observed with lupus PMN was similar to that observed with normal cells. The aim was not to test large cohorts and to determine whether IL-8 concentrations were statistically different between lupus and normal donors, but rather to prove that lupus neutrophils are not defective in nucleosome-induced activation. Thus, lupus neutrophils secreted higher amounts of IL-8 upon activation with nucleosomes as compared with cells incubated with the purification buffer, although in the case of patient SLE 5, the concentration was low (Table I). Lupus neutrophils responded to nucleosomes regardless of the patient’s characteristics: irrespective of gender or age, with a disease activity (SLE disease activity index) of 0 or 6, anti-dsDNA-positive or -negative, and under therapy or not. Thus, these results support the existence of such an activation mechanism in vivo in patients.


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  		Table I. Both normal and lupus human neutrophils are activated by nucleosomesa

 
Nucleosome-induced activation delays neutrophil apoptosis

Delayed neutrophil cell death is often a consequence of neutrophil activation. Since neutrophil activators, such as LPS, have been shown to prolong neutrophil survival, the impact of nucleosomes on neutrophil cell death was investigated. Interestingly, nucleosomes significantly increased cell survival in human PMN as compared with untreated cells (Fig. 5A), although to a lesser extent than LPS. This increase was actually due to delayed apoptosis, as estimated by a reduction in the percentage of early apoptotic PMN (annexin V-positive but 7-AAD-negative cells) upon overnight incubation (Fig. 5B). Importantly, the nucleosome-induced partial inhibition of cell death was polymyxin B independent as opposed to LPS-induced death inhibition, confirming that our purified nucleosomes were not endotoxin contaminated. Increased cell death was not observed when PMN were incubated with nucleosomes. It should be noted that we have used here the decrease of neutrophil apoptosis as an activation marker. We do not claim that nucleosomes diminish neutrophil apoptosis but that nucleosome-induced neutrophil activation only transiently delays apoptosis.


Figure 5
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  		FIGURE 5. Nucleosome-induced neutrophil activation results in decreased cell death. Human PMN were cultured with different stimuli for 20 h in the presence or absence of polymyxin B and analyzed by flow cytometry. A, Cell survival was estimated by measuring the percentage of living cells (annexin V-negative, 7-AAD-negative cells). B, The level of early apoptosis was estimated by determining the percentage of annexin V-positive, 7-AAD-negative cells. Results are expressed as percentages of cells among CD66b-positive cells. See Fig. 1 for abbreviations and conditions, except Nuc, 35 µg/ml purified nucleosomes. Shown is one of six representative experiments. SDs are indicated.

 
Nucleosomes are endocytosed by human neutrophils

To determine the mechanism involved in nucleosome-induced neutrophil activation, we first analyzed the ability of FITC-nucleosomes to bind to the cell surface of human PMN by flow cytometry. When PMN were incubated at 4°C to inhibit endocytosis, no clear signal was observed with FITC-nucleosomes as compared with cells incubated in FCS-containing PBS alone (Fig. 6A). On the other hand, a strong signal was detected at 37°C in the presence of FITC-nucleosomes (Fig. 6B), suggesting that nucleosomes bind only slightly to PMN and are endocytosed instead. It should be noted that using the same protocol, we have already observed that FITC-nucleosomes bind to the cell surface of B lymphocytes at 4°C (26).


Figure 6
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  		FIGURE 6. Nucleosomes are endocytosed by human neutrophils. A and B, Fresh PMN were incubated with or without FITC-nucleosomes for 30 min on ice (A) or at 37°C (B), washed, and analyzed by flow cytometry, gating out dead cells (propidium iodide-positive cells). Bold line, PMN with FITC-nucleosomes; thin line, PMN alone. C and D, Human PMN were incubated with FITC-nucleosomes for 30 min on ice (C) or at 37°C (D), washed, stained with a PE-conjugated anti-CD11b mAb (to stain the cell surface), fixed, and analyzed by confocal laser scanning microscopy. Representative sections are displayed. The size is indicated. Green, FITC-nucleosomes; red, anti-CD11b mAb. E and F, PMN were incubated with unconjugated nucleosomes for 30 min on ice (E) or at 37°C (F), washed, permeabilized, and stained with a mouse nucleosome-recognizing mAb. After a series of washes, cells were incubated with a FITC-conjugated goat anti-mouse secondary Ab (green), fixed, and analyzed by confocal microscopy as described above.

 
To confirm these results, cell surface binding and endocytosis were further investigated by confocal microscopy. A PE-conjugated anti-CD11b Ab was used to stain the cell surface of PMN. Whereas a clear and homogeneous cell surface staining was observed at 4°C with the CD11b-specific Ab, only a weak and nonhomogeneous signal was detected at the cell surface with FITC- nucleosomes (Fig. 6C). On the contrary and in agreement with the above flow cytometry results, FITC-nucleosomes were strongly detected inside neutrophils at 37°C (Fig. 6D), indicating that nucleosomes are endocytosed by human neutrophils.

Finally, to exclude that FITC-nucleosomes behave differently from free nucleosomes due to structural changes upon conjugation, the above experiments were reproduced with PMN incubated with unconjugated nucleosomes. The cell surface binding and endocytosis were investigated by confocal microscopy by indirect staining after permeabilization using a mouse nucleosome-recognizing mAb and a FITC-conjugated goat anti-mouse secondary Ab. In agreement with the above results, a weak and nonhomogeneous signal was observed at the cell surface at 4°C, whereas a strong signal was detected inside the cells at 37°C (Fig. 6, E and F).

Nucleosomes activate and recruit neutrophils in vivo

To support our findings, we evaluated nucleosome-induced neutrophil activation in vivo. To do so, mice were i.p. injected with purified nucleosomes. After 18 h, peritoneal exudate cells were harvested and analyzed by flow cytometry. The percentage of live Ly-6GhighCD11bhigh cells among peritoneal cells, i.e., activated mature neutrophils, was determined for each mouse.

We first injected normal BALB/c mice with different stimuli. As shown in Fig. 7A, nucleosomes, as well as LPS, induced an increase in the percentage of peritoneal activated neutrophils as compared with purification buffer-injected mice. Although not all mice responded to nucleosome or LPS injection, most had more neutrophils in the peritoneum than control mice. None of the buffer-injected mice had a high percentage of neutrophils. This result demonstrates that nucleosomes activate and recruit mature neutrophils in vivo. It also supports the hypothesis that nucleosomes may activate neutrophils in vivo in lupus patients.


Figure 7
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  		FIGURE 7. Nucleosomes recruit and activate mouse neutrophils in vivo in a TLR2/TLR4-independent manner. Normal BALB/c (A) or TLR2/TLR4-deficient (B) mice were i.p. injected with purified nucleosomes, the purification buffer or LPS. Eighteen hours later, mice were sacrificed to prepare peritoneal exudate cells. Neutrophil recruitment and activation was estimated by measuring the percentage of living Ly-6GhighCD11bhigh cells among peritoneal cells, i.e., activated mature neutrophils. Cells were first incubated with a FcR-blocking mAb and then stained with FITC-conjugated anti-Ly6G and PE-conjugated anti-CD11b-specific mAb in the presence of 7-AAD. Cells were analyzed by flow cytometry, gating out dead cells. Each bar represents an individual mouse. Buffer, purification buffer used in nucleosome preparation; PEC, peritoneal exudate cells. A, Shown is one representative experiment; B, the results are from two independent experiments. In BALB/c mice, injection with nucleosomes or LPS, but not with buffer, induces neutrophil recruitment and activation in most cases. On the contrary, in TLR2/TLR4-deficient mice, only injection with nucleosomes, and not with LPS or the buffer, induces activation in part of the mice.

 
Having shown that nucleosomes activate neutrophils in mice, we verified that nucleosome-induced activation is not endotoxin dependent and that it occurs also in TLR2/TLR4-deficient mice. For this purpose, we injected C3H/HeJ/TLR2-KO mice i.p. with purified nucleosomes or LPS. As expected, these mice did not respond to LPS injection as compared with buffer-injected mice (Fig. 7B). Significantly, a number of the nucleosome-injected mice had a clearly increased percentage of activated neutrophils in the peritoneum, indicating that nucleosomes also induce neutrophil recruitment and activation in this mouse strain. We did not expect to observe recruitment and activation in every nucleosome-injected mouse, but we did want to verify that these mice can also respond to nucleosomes. Thus, only nucleosome-injected mice had a higher percentage of activated neutrophils in the peritoneum. The results obtained with TLR2/TLR4-deficient mice after injection with nucleosomes or the purification buffer are similar to those observed with BALB/c mice. This suggests that nucleosomes induce neutrophil activation in vivo in a TLR2/TLR4-independent manner and confirms that nucleosome-induced neutrophil activation is endotoxin independent.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we show for the first time that nucleosomes directly induce activation and recruitment of neutrophils in vitro and in vivo. Also, nucleosomes act as free complexes without the need for immune complex formation. These results strengthen the view that autoantigens are not only passive targets for autoantibodies but might also play an active role in the pathogenesis of some autoimmune diseases by inducing innate immunity. Nucleosome-induced neutrophil activation is manifested by CD11b/CD66b up-regulation, IL-8 secretion, increased phagocytic activity, and delayed apoptosis. Only nucleosomes and H1-depleted nucleosomes were able to activate neutrophils; histones or DNA or even a mixture of both failed to do so, suggesting that the core nucleosome structure is necessary for signaling. Moreover, both normal and lupus neutrophils were sensitive to nucleosome-induced activation, supporting such a mechanism in vivo in lupus patients. It is noteworthy that neutrophil activation by nucleosomes was also observed in the presence of polymyxin B and took place in a TLR2/TLR4-independent manner, proving that nucleosome-induced activation was not due to the presence of endotoxins. Importantly, neutrophil activation by purified nucleosomes was not caused by HMGB1 contamination. Since nucleosomes induced the activation of human lupus neutrophils in vitro and in mice in vivo, it suggests that such a mechanism might occur in vivo in SLE patients and thus strengthen the key role of nucleosomes in lupus development.

Recently, several new functions have been attributed to autoantigens, such as activation of plasmacytoid dendritic cells or chemoattraction of immune cells. In accordance with these findings, we demonstrated previously that purified nucleosomes induce dendritic cell activation in vitro and in vivo via a MyD88-independent pathway (16). In contrast to most studies dealing with the effect of autoantigen on diverse cell types, we could show that free nucleosomes, and not nucleosome-containing immune complexes, were involved in neutrophil activation. Most importantly, neutrophil activation was observed using physiological concentrations of purified nucleosomes (as explained in Ref. 26) since activation could be detected with 1–5 µg/ml nucleosomes. This phenomenon might also be induced following a local and transient increase of the nucleosome concentration without any systemic dissemination. Moreover, we found that the whole nucleosomal complex, and not only the DNA moiety, is involved in neutrophil activation. Indeed, although DNA of mammalian origin has been shown to activate some immune cells, it is generally accepted that serum DNA is not found as a free molecule in lupus patients but in the form of circulating nucleosomes (1). Moreover, only extracellular mammalian DNA in the form of immune complexes can activate plasmacytoid dendritic cells (33). Likewise, free (self) mammalian DNA can only activate immune cells when DNA is artificially introduced into cells by transfection (34, 35) or in lipidic vesicles enhancing endosomal translocation (36) or when endogenous DNA from apoptotic cells escapes lysosomal degradation (37). Recently, a novel cytosolic innate immune response to DNA, which can also recognize calf thymus DNA but only after transfection, has been described (38). According to these studies, activation occurs in a TLR9-dependent or -independent manner or even in a fully TLR-independent manner. Likewise, natural phosphodiester oligonucleotides have been shown to activate dendritic cells via TLR9 upon enforced endosomal translocation but independently of CpG motifs (39). In agreement with an absence of cell activation by extracellular mammalian DNA, we found that free DNA purified from our nucleosome preparations does not activate neutrophils, indicating that the nucleosome structure is crucial for neutrophil activation and that unmethylated CpG motifs are not responsible for this cell activation. We already observed that free DNA purified from our nucleosome preparations does not activate dendritic cells (16). Our results thus clearly show that nucleosome-induced activation is not directly due to the DNA moiety of the nucleosome and suggest that a different signaling pathway is used since free nucleosomes do activate neutrophils. It is currently being investigated whether the natural endosomal translocation of nucleosomes is more efficient than that of free DNA and would allow cell activation via TLR9 by the DNA moiety. Self-DNA is indeed able to stimulate TLR9 when it is accessible, as observed with cells expressing a chimeric TLR9 receptor designed to localize at the cell surface (40).

In line with our results is the recent finding that bacterial DNA, but not mammalian DNA, induces human neutrophil activation in a CpG-independent manner and thus via an expected TLR9-independent pathway (41). This indicates that the DNA moiety of the nucleosome is not involved in neutrophil activation and that only the whole nucleosome complex is capable of activating. Likewise, it has been confirmed that calf thymus DNA does not activate human neutrophils (42). We have purified nucleosomes from calf thymus. Moreover, DNA in the form of immune complexes with anti-DNA Abs is phagocytosed by human neutrophils but not free DNA (43). This supports the supposition that free DNA-containing Ag are not recognized by neutrophils. Altogether, these results reveal that we have identified a new mechanism of neutrophil activation by free nucleosomes.

IL-8, a chemokine produced by diverse cell types, including neutrophils, is a neutrophil chemoattractant detected at higher concentrations in the sera of lupus patients where serum IL-8 levels correlate with disease activity (19). Particularly, IL-8 seems to be produced in the kidney in lupus patients, and urinary IL-8 levels are elevated in lupus nephritis, a major complication in SLE, especially during the active phase of the disease (44). One should recall that SLE is an inflammatory disease and that PMN are the first cells recruited to inflammation sites. Nucleosome-induced IL-8 secretion might thus result in increased neutrophil recruitment and activation and subsequently in inflammation via an amplification loop, as observed in patients. In agreement with our results, nucleosomes have been found in the synovial fluid of rheumatoid arthritis patients where high nucleosome concentrations (mean concentration = 14 µg/ml) correlate with high neutrophil concentrations (45), suggesting that the presence of nucleosomes characterizes inflammation.

Interestingly, we have found that nucleosomes bind to the cell surface of human PMN only weakly and are rather endocytosed, suggesting that the signaling cascade for cell activation takes place inside PMN. The intracellular localization of nucleosomes upon endocytosis is currently under investigation in our group and is evaluated in different immune cell types. This will show us whether an intracellular nucleosome-specific receptor exists and will provide information on the mechanism involved in cell activation by nucleosomes. Presently, we learned that, in contrast to other autoantigens, nucleosomes can directly activate PMN in the absence of nucleosome-specific autoantibody.

In conclusion, all these results support the key role of nucleosomes in SLE and suggest that they might be involved in lupus pathogenesis. In accordance with our results, we assume that upon endocytosis by neutrophils, circulating nucleosomes induce IL-8 secretion, leading to increased neutrophil recruitment as well as dendritic cell activation. Activated neutrophils have indeed been shown to induce activation of dendritic cells. The latter, which also endocytose nucleosomes and are thereby activated (16), thus efficiently process and present these autoantigen to autoreactive Th lymphocytes. In agreement with our hypothesis, T cell recruitment and activation has been shown to be facilitated by neutrophil activation. Experiments are being conducted in our laboratory to address the role of dendritic cells upon activation with nucleosomes and to investigate the interplay between neutrophils and dendritic cells upon nucleosome-induced activation. Although circulating nucleosomes may represent only one of the factors triggering SLE in predisposed individuals, this mechanism of innate immunity partly explains why peripheral tolerance is broken in SLE and sustains continuous T cell activation. Interestingly, i.p. injections of nucleosomes in the presence of adjuvant into young lupus prone mice have been shown previously to augment the production of autoantibodies and to markedly accelerate lupus (46).


   Acknowledgments
 
We thank Dr. Markus Radsak (Mainz, Germany) for advice in human neutrophil preparation, Dr. Ina Kötter (Tübingen, Germany) for providing blood samples of lupus patients; Prof. Hansjörg Schild (Institute for Immunology, Johannes Gutenberg University, Mainz, Germany) and Prof. Hermann Wagner (Institute of Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany) for the gift of TLR2/TLR4-deficient mice; Dr. Roland Brock (Tübingen, Germany) for advice in confocal microscopy; Prof. Karolin Luger and Jayanth Chodaparambil (Fort Collins, CO) for providing reconstituted nucleosomes; and Lynne Yakes for critical reading of the manuscript. The technical assistance of Annika Rudzio, Silke Wahl, and Beate Pömmerl is gratefully acknowledged.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a Deutsche Forschungsgemeinschaft Grant DE 879/1-1, as well as Forschungsprogramm der Tübinger Medizinischen Fakultät fortüne Grant 1451-0-0), and Fritz Thyssen Stiftung Grant Az. 10.03.2.123 (to P.D.). The Proteom Centrum Tübingen is supported by the Ministerium für Wissenschaft und Kunst, Landesregierung Baden-Württemberg. Back

2 Address correspondence and reprint requests to Dr. Patrice Decker, Department of Immunology, Institute for Cell Biology, Auf der Morgenstelle 15, D-72076 Tübingen, Germany. E-mail address: patrice.decker{at}uni-tuebingen.de Back

3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; 7-AAD, 7-aminoactinomycin D; FITC-nucleosomes, FITC-conjugated nucleosomes; HMGB1, high-mobility group box 1; KO, knockout; MS, mass spectrometry; pI, isoelectric point; PMN, polymorphonuclear leukocyte. Back

Received for publication May 4, 2006. Accepted for publication September 19, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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