Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0506349 on September 15, 2006

Published online before print September 15, 2006
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(Journal of Leukocyte Biology. 2007;81:129-136.)
© 2007 by Society for Leukocyte Biology

HMGB1-secreting capacity of multiple cell lineages revealed by a novel HMGB1 ELISPOT assay

Heidi Wähämaa*,1, Therese Vallerskog*, Shixin Qin{dagger}, Carolina Lunderius{ddagger}, Gregory LaRosa{dagger}, Ulf Andersson§ and Helena Erlandsson Harris*

* Rheumatology and
{ddagger} Clinical Immunology and Allergy Units, Department of Medicine, and
§ Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden; and
{dagger} Critical Therapeutics Inc., Lexington, Massachusetts, USA

1Correspondence: Rheumatology Research Unit, Karolinska Institutet CMM L8:04, Karolinska Sjukhuset, Stockholm, Sweden. E-mail: heidi.wahamaa{at}cmm.ki.se

ABSTRACT

High mobility group box protein 1 (HMGB1) exerts different biological functions dependent on its cellular localization. Nuclear HMGB1 maintains chromatin architecture and is required for undisturbed transcription activity, and extracellularly released HMGB1 mediates inflammation and tissue regeneration. A present paucity of readily accessible methods to quantify released HMGB1 represents a problem concerning the exploration of HMGB1 biology. We have now developed a HMGB1-specific ELISPOT assay enabling enumeration of individual HMGB1-releasing cells. The method also allows automated, semiquantitative assessment of released HMGB1 by evaluating areas of single HMGB1 spots. Actively secreted HMGB1 as well as cells passively releasing the protein following necrotic cell death are visualized distinctly using this ELISPOT assay. Kinetics of HMGB1 secretion after different stimuli was studied using cell lines of various lineages. IFN-{gamma} already induced substantial HMGB1 secretion from the monocytic cell line RAW 264.7 within 24 h and even more so after 48 h. LPS only stimulated a modest HMGB1 release within 24 h, but this increased considerably by 48 h. TNF-induced HMGB1 release was unexpectedly low. Mast cells, which share the secretory, lysosomal pathway with macrophages/monocytes, did not secrete HMGB1 in response to any studied mode of activation. Most transformed cells overexpress HMGB1, but the ELISPOT assay revealed that all transformed cell lines will not actively secrete the protein. We believe the ELISPOT method provides a novel tool to study pathways promoting or inhibiting HMGB1 secretion.

Key Words: monocytes • macrophages • mast cells • HCT 116 cells • immunocytochemistry

INTRODUCTION

High mobility group box protein 1 (HMGB1) is a 25-kD protein with a highly conserved sequence among mammals. Intranuclearly, HMGB1 binds double-stranded DNA, facilitating numerous nuclear functions including transcription, replication, and recombination [1 , 2 ].

It is now known that HMGB1 can also be secreted from cells and exert extracellular functions as a proinflammatory cytokine. HMGB1, actively secreted by monocytes and macrophages, acts as a truly potent proinflammatory cytokine, mediating lethal, systemic inflammation [3 , 4 ], is involved in the pathogenesis of arthritis [5 6 7 ] in acute lung injury [8 ], and is indicated as a pathogenic factor in atherosclerosis [9 ] and myositis [10 ]. Stimulation of PBMC with HMGB1 induces the production of proinflammatory cytokines such as TNF, IL-1{alpha} and IL-1ß, IL-6, and MIP-1{alpha} [3 , 4 , 8 ]. Furthermore, HMGB1 also induces the expression of adhesion molecules on endothelia [11 , 12 ] and induces migration of smooth muscle cells and mesangioblast stem cells [13 , 14 ].

Intranuclear HMGB1 can reach the extracellular space via two different routes: active secretion by stimulated macrophages and monocytes, mature myeloid dendritic cells [15 ], and NK cells [16 ] and passive release from necrotic and damaged cells [17 , 18 ]. The active secretion of HMGB1 by monocytes and macrophages occurs in response to inflammatory stimuli such as LPS, TNF, and IL-1ß [4 , 19 ]. In most cells, HMGB1 shuttles continually between the nucleus and the cytoplasm. Activation of macrophages/monocytes leads to the acetylation of specific lysine residues on HMGB1, which then begins to accumulate in the cytoplasm, as its re-entry into the nuclear compartment is blocked [20 ]. Gardella and coworkers [21 ] originally discovered that HMGB1 accumulated in the cytoplasm after activation of monocytes and was redistributed into secretory lysosomes before release into the extracellular space [20 ]. This mode of release via secretory lysosomes is also used by IL-1ß. Although the secretion of IL-1ß is induced earlier in an autocrine manner by ATP [22 , 23 ], the HMGB1 secretion can be triggered by lysophosphatidylcholine [21 ], generated later at the inflammation site. In comparison with cytokines such as TNF and IL-1ß, HMGB1 can thus be denoted a late mediator of inflammation, as its active secretion occurs after more than 16 h after stimulation.

As HMGB1 binds loosely to chromatin in living cells, it readily diffuses into the extracellular space when cells undergo unprogrammed cell death caused by hypoxia, mechanical, or thermal damage or ATP depletion. HMGB1 released from necrotic cells is presumably not acetylated but still evokes inflammatory responses in vivo and in vitro. It is presently unknown whether acetylated and unacetylated HMGB1 mediates identical or only partially overlapping functions. Polyclonal antibody blockade of HMGB1 in vivo reduces leukocyte recruitment in a model of hepatic necrosis, indicating that HMGB1 is an important contributor to the necrotic inflammation [17 ].

An accumulating body of evidence thus supports that HMGB1 is an important mediator of inflammation with functions and properties needing further clarification. Methods to detect and quantitatively measure active HMGB1 secretion from living cells have until now been laborious. Detection of HMGB1 in supernatants by Western blot or ELISA requires serum-free conditions and high HMGB1 concentrations, which can only be obtained in the presence of high cell numbers in the cultures. Dense cell cultures induce cell stress and cell death, which make it difficult to discriminate between active HMGB1 secretion and passive HMGB1 release from necrotic cells. Immunocytochemistry can be applied to visualize the cellular HMGB1 localization of nuclear HMGB1 and cytoplasmic HMGB1 accumulation, but immunocytochemistry is not suitable as a method to measure and quantify HMGB1 secretion.

In this study, we present a HMGB1-specific ELISPOT assay, which allowed us to define promoters of active HMGB1 secretion as well as the magnitude and kinetics thereof. The ELISPOT assay allows quantitative measurement of frequency of secreting cells at a single-cell level directly ex vivo, allowing minimal in vitro handling of cell populations. The bulk of our work has been performed using monocytic RAW 264.7 cells, but the method can be applied to adherent and nonadherent cells including primary cells. Activated cells produce spots that have specific size, intensity, and morphology, characteristics that allows quantification and determination of the kinetics of HMGB1 release. The count of spots per number of cells plated enables discrimination between a high HMGB1 release response from few cells and a low release from a large number of cells. We could define that IFN-{gamma} stimulation was as potent as LPS stimulation in inducing HMGB1 secretion, although demonstrating faster kinetics. Furthermore, we could demonstrate that mast cells, despite their high content of secretory lysosomes, do not release HMGB1 upon stimulation, and the human colon cancer cell line HCT 116 released large amounts of HMGB1 in an unstimulated state.

MATERIALS AND METHODS

Generation of recombinant HMGB1 (rHMGB1)
rHMGB1 was generated as described previously [24 ]. Briefly, GST-tagged rat HMGB1 was expressed in Escherichia coli and purified using a glutathione sepharose column, which was then treated with protease to remove the GST tag and to release purified rHMGB1.

Generation of mAb against HMGB1
Anti-HMGB1 mAb were generated at Critical Therapeutics Inc. (CTI; Lexington, MA) by a conventional method. Briefly, BALB/c mice were immunized with 100 µg rat rHMGB1 mixed with Freund’s adjuvant. Three i.p. injections were given at 2-week intervals. A final boost of 10 µg soluble HMGB1 was given i.v. 4 weeks later. Four days after the boost, fusion was carried out using spleen cells from immunized animals, using standard hybridoma technique. Direct ELISA was used to screen fusion to select antibodies against rat rHMGB1. Hybridoma clones were established by limiting dilution. Clone 2G7 has an isotype of mouse IgG2b. It recognizes rHMGB1 from a number of sources, as well as HMGB1 purified from calf thymus (S. Qin, unpublished data). It cross-reacts with human, mouse, and bovine HMGB1, but it does not bind rHMGB2 (Abnova, Taiwan). Overlapping, 18 amino acid peptides covering the entire HMGB1 were synthesized by BioSource International (Camarillo, CA). Clone 2G7 binds to an epitope within amino acids 53–63 in the A-Box subunit of the HMGB1 molecule, based on ELISA, using synthetic peptides from BioSource International. mAb were purified from spent cell culture supernatant using Protein G.

To further validate the specificity of the 2G7 antibody, murine wild-type and Hmgb1–/– fibroblasts (gift from Marco E. Bianchi, San Rafaelle Scientific Institute, Milan, Italy) were stained for HMGB1 expression according to the immunocytochemistry protocol below using a goat antimouse antibody, 2 µg/ml (Jackson ImmunoResearch Lab, West Grove, PA), as the secondary antibody. Only the wild-type fibroblasts stained positively for nuclear HMGB1.

Cell lines
The following cell culture media described contained 10 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Paisely, Scotland). RAW 264.7 mouse monocytic cell line was cultured in DMEM supplemented with 5% heat-inactivated FCS (PAA Laboratories, Linz, Austria) and 50 µM 2-ME (all reagents from Life Technologies). RAW 264.7 cells were split 2 days prior to the experimental set-up, rendering a cell confluence of 80% at the beginning of the experiment.

HCT 116, human colon cancer cell line, was cultured in McCoy’s 5A medium (Gibco, Paisley, Scotland, UK) supplemented with 10% heat-inactivated FCS. The mouse mast cell line C1MC/C57.1 (C57) was cultured in RPMI (Gibco) supplemented with 10% heat-inactivated FCS. The human mast cell line HMC-1.2 [25 , 26 ] was cultured in IMDM (Gibco) supplemented with 10% FCS, 2 mM L-glutamine (Gibco), and 1.2 mM {alpha}-thioglycerol (Sigma-Aldrich, Steinheim, Germany).

HMGB1 ELISPOT
Multiscreen 96-well HTS Plate Clear (MSIPS4510, Millipore, Stockholm, Sweden) was prewet with 70% ethanol, washed immediately with sterile H2O before being coated with 20 µg/ml mouse mAb 2G7 (noncommercial antibody, available upon request, CTI), and diluted in sterile PBS (Gibco) overnight at 4°C in a moist chamber. The plates were washed three times with sterile PBS followed by blocking for 2 h with cell-specific medium containing 5–10% heat-inactivated FCS. Indicated concentrations of cells were added. Three different cell concentrations of cells were always assayed, and the cell concentration giving a maximal number of spots less than 600 spots/well was used: RAW 264.7, 1500, 2000, and 2500 cells/well; C57 cells, 2500–5000 cells/well; HMC-1.2, 2500–5000 cells/well; and HCT 116, 1250–5000 cells/well, and stimulated for 6–48 h with: 0.1–10 µg/ml LPS L-6529 (Sigma Chemical Co., St. Louis, MO), 100 U mouse rIFN-{gamma} (Sigma Chemical Co.) used for RAW 264.7 and C57 cells, 100 U human rIFN-{gamma} (BioSite, Täby, Sweden) used for HMC 1.2 cells, 50 ng/ml mouse rTNF (Sigma Chemical Co.), 1 ng/ml PMA (Sigma-Aldrich) + 1 µM ionomycin (Calbiochem Novabiochem, San Diego, CA), 50 ng/ml p53 reactivation and induction of massive apoptosis (PRIMA-1; Sigma-Aldrich), or mouse anti-TNP IgE antibody (BD Biosciences PharMingen, San Diego, CA), cross-linked by addition of 100 ng/ml TNP(15)-BSA (Biosearch Technologies, Inc., Novato, CA). Cell viability was assessed at every experimental set-up and determined to be 90–100% using Trypan blue (Merck, Darmstadt, Germany) exclusion. The blocking and stimulation steps were performed in an incubator at 37°C with 5% CO2. Supernatants were collected for lactate dehydrogenase (LDH) and IL-6 analyses at time-points indicated, and the plates were washed seven times with PBS/0.05% Tween 20 (PBS/Tw; Merck, Hohenbrunn, Germany) with an ELISA washer flushing with high speed to remove adherent cells. Polyclonal antigen affinity-purified rabbit anti-HMGB1 antibodies (BD Biosciences PharMingen), 0.5 µg/ml, in PBS/Tw were added and incubated overnight at 4°C in a moist chamber. Plates were washed three times with PBS/Tw and incubated with biotinylated donkey anti-rabbit antibody (Jackson ImmunoResearch Lab), 0.8 µg/ml, in PBS/Tw at room temperature for 3 h in a moist chamber. Plates were washed three times with PBS/Tw, followed by two washes with PBS alone, and further incubated with Streptavidin-alkaline phosphatase (Mabtech AB, Stockholm, Sweden), diluted 1/1000 in PBS for 2 h at room temperature in a moist chamber, followed by washing three times with H2O. Wells were developed for 2–10 min with 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP)/nitroblue tetrazolium chloride chromogen liquid substrate (Sigma-Aldrich), which was filtered through a 0.20-µm filter to avoid precipitates. The reaction was stopped by washing six times with H2O, and the plates were allowed to dry at room temperature overnight, analyzed using an AID EliSpot Reader System (AID, Strassberg, Germany), and evaluated with the following settings: for the low-intensity spots, spot size, 8; spot intensity, minimum 3 and maximum 15; and the high-intensity spots, spot size, 8; spot intensity, minimum >15.

The specificity of the ELISPOT assay was tested by coating the plate with an isotype mouse control IgG2b mAb against glucose oxidase Aspergillus niger (Dako Cytomation, Glostrup, Denmark), by using an irrelevant polyclonal rabbit IgG fraction (Dako Cytomation) at the detection step, or by omitting the detection anti-HMGB1 antibody.

TNF ELISPOT
The TNF ELISPOT was performed according to the manufacturer’s instructions (R & D Systems, Minneapolis, MN). Briefly, multiscreen 96-well HTS Plates were pretreated as in the HMGB1 ELISPOT assay and coated overnight at 4°C with the capture antibody diluted 1/60 in PBS. RAW 264.7 cells at concentration of 1.4 x 104 cells/ml were added and stimulated for 7 h with LPS, IFN-{gamma}, TNF, or PMA + ionomycin, as in the HMGB1 ELISPOT assay. Cells were removed, and the biotinylated TNF detection antibody diluted 1/60 in PBS/Tw was added and incubated overnight at 4°C in a moist chamber. The plates were also developed using the Streptavidin-alkaline phosphatase-paraquat/BCIP system.

Immunocytochemistry
In parallel with the ELISPOT method, we studied intracellular HMGB1 expression using immunocytochemistry. RAW 264.7 cells were cultured for 6–48 h in chamber slides (Nalge Nunc International, LAB-TEK, Naperville, IL; with a concentration of 50,000 cells/ml) and stimulated with LPS, IFN-{gamma}, TNF, or PMA + ionomycin. Slides were washed with PBS and fixed with 4% paraformaldehyde for 10 min and stored in PBS at 4°C until staining. Cells were permeabilized with 20 mM Hepes, pH 7.4, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, and 0.5% (vol/vol) Triton X-100 (Sigma Chemical Co.) for 3 min followed by washing in PBS containing 0.1% saponin. Throughout the staining procedure, the slides were washed with PBS/saponin, and all incubations were performed at room temperature. The slides were blocked with 5% FCS followed by avidin and biotin-blocking (Vector Laboratories, Burlingame, CA) for 30 min and 15 min, respectively. Cells were stained with the polyclonal anti-HMGB1 antibody (2 µg/ml) for 1 h, followed by incubation with biotinylated donkey antirabbit IgG (2 µg/ml) for 30 min. Cells were stained with Avidin-Oregon Green 1:500 (Molecular Probes, Eugene, OR) for 30 min followed by nuclear staining with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), diluted 1/2000 in PBS. Coverslips were mounted with PBS/glycerol, and microscopic analysis was conducted using a Polyvar 2 UV microscope (Reichert-Jung, Vienna, Austria).

IL-6 ELISA
Supernatants from the ELISPOT plates were collected after 24 h of stimulation and stored at –20°C until analysis. An IL-6 ELISA (R & D Systems GmbH, Germany) was performed according to the manufacturer’s instructions. Absorbance was determined at 405 nm using a Molecular Devices Emax precision spectrophotometer (Workingham, UK), and the IL-6 concentrations were determined using a standard curve ranging from 7.8 to 500 pg/ml.

Cytotoxicity detection
At the end of stimulation, the supernatants were collected from the ELISPOT plates, and the cell viability was determined using a colorimetric LDH assay (Roche, Mannheim, Germany) and performed according to the manufacturer’s instructions.

Apoptosis assay
The ability of rTNF to induce apoptosis in RAW 264.7 cells was determined by incubating the cells in 4 ml polypropylene tubes for 90 min in complete DMEM, with or without the addition of 5 ng/ml, 0.5 ng/ml, or 0.05 ng/ml rTNF. Cells were stained with Annexin V-FITC conjugates and propidium iodide (R & D Systems Abingdon, UK), performed according to the instructions from the supplier, and analyzed by flow cytometry (FACSort, Becton Dickinson, Franklin Lakes, NJ).

RESULTS

Specificity controls of the ELISPOT assay
A HMGB1-specific ELISPOT assay was developed to investigate HMGB1 secretion from adherent and nonadherent cells. This was achieved by coating the ELISPOT plate with the HMGB1-specific mAb 2G7, which recognizes the amino acid sequence 53–63 of the HMGB1 molecule. The captured HMGB1 was then detected using peptide affinity-purified polyclonal antibodies recognizing the 166–181 segment of the HMGB1 molecule. The amino acid sequence 166–181 is distinct from that in HMGB2. The specificity of the assay was tested by replacing the capture or the detection anti-HMGB1 antibodies with an isotype control antibody or an irrelevant polyclonal rabbit antibody, respectively, or by omitting the detection anti-HMGB1 antibody. All these controls resulted in the absence of spots for stimulated and unstimulated RAW264.7 cells. As a background, control wells without the addition of cells but with all other reagents added were always included in the experiments. Such control wells generally resulted in two spots/well, and this background number of spots was subtracted from presented data. Supernatants collected from the ELISPOT assays did not reveal increased LDH activity in the stimulated cell cultures versus unstimulated cells, demonstrating that the accumulated HMGB1 spot formation was not caused by increased cell death in the cell cultures (data not included).

Active secretion of HMGB1 by RAW 264.7 cells
To determine the potency and the kinetic pattern caused by known inducers of active HMGB1 secretion, RAW 264.7 cells were cocultured with LPS, LPS + IFN-{gamma}, IFN-{gamma}, TNF, or PMA + ionomycin for different lengths of time (Fig. 1 ). By analyzing the number of low-intensity spots (reflecting the initial phase of HMGB1 secretion) and the number of high-intensity spots (defining well-established, high-output HMGB1 secretion) over time, it was evident that IFN-{gamma} alone was as strong an inducer of HMGB1 secretion as the prototype inducer LPS (high-intensity spots 251±14 vs. 298±23 at 48 h). In addition, IFN-{gamma} stimulation induced a faster release of the HMGB1 than any of the other investigated, stimulating agents (IFN-{gamma} 33±8 vs. LPS 17±2 at 6 h and IFN-{gamma} 252±8 vs. LPS 112±6 low-intensity spots after 24 h of stimulation). IFN-{gamma} alone was as potent as the combined activity of LPS + IFN-{gamma} at 6 h and 24 h, measured as low- and high-intensity spots. LPS-induced HMGB1 release was thus slower than that induced by IFN-{gamma} stimulation (Figs. 1 and 2 ). Stimulation with TNF, a well-described inducer of HMGB1 release, resulted in a markedly lower number of high-intensity spots at 24 h (TNF 5±2 vs. IFN-{gamma} 114±22) and after 48 h (TNF 58±16 vs. IFN-{gamma} 251±14). The number of spots in TNF-stimulated wells differed only slightly from the number of spots formed in wells with unstimulated cells. The bioactivity of the TNF was verified in an apoptosis assay in which TNF induced an increased binding of Annexin V to the cell surface of TNF-treated RAW 264.7 cells. The number of late apoptotic cells, Annexin V, and propidium iodoide-positive cells increased with 14% compared with untreated cells during 90 min of treatment. PMA combined with ionomycin activate many functions expressed by macrophages but did not cause increased numbers of HMGB1-forming spots in the cultures as compared with unstimulated cells (Fig. 1A and 1B) .


Figure 1
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  		Figure 1. ELISPOT detection of HMGB1 secretion from unstimulated and PMA + ionomycin (PI)-, TNF-, LPS-, LPS + IFN-{gamma}-, and IFN-{gamma}-stimulated RAW 264.7 cells. Determination by (A) high-intensity spots at 24 h and 48 h of stimulation and (B) low-intensity spots at 6 h and 24 h of stimulation. A representative experiment of three performed is depicted. Results are expressed as means ± SD of three different well counts.

 

Figure 2
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  		Figure 2. ELISPOT assay visualizing spontaneous or LPS-, IFN-{gamma}-, and TNF-induced HMGB1 secretion from 1500 RAW 264.7 cells per well after (A) 24 h or (B) 48 h of stimulation. A representative experiment out of three performed is presented.

 
HMGB1 translocation in RAW 264.7 cells
To verify that the recorded spots resulted from an active secretion of HMGB1, we performed immunocytochemistry using stimulated and unstimulated RAW 264.7 cells, and cells activated with LPS or LPS + IFN-{gamma} displayed a vesicular pattern of HMGB1 accumulation in the cytoplasm of the cells, most likely corresponding to the formation of secretory lysosomes containing HMGB1 translocated from the nucleus. This pattern was already visible 6 h poststimulation but reached its highest intensity after 24 h of stimulation. IFN-{gamma} or TNF induced a more diffuse cytoplasmic staining of HMGB1 at 24 h, which for the IFN-{gamma}-stimulated cells, converted to a granular pattern after a culture period of 48 h (Fig. 3 ). Thus, our cellular immunostainings implicate that the active secretion recorded by the ELISPOT assay correlated with the formation of secretory lysosomes, a secretion pathway described previously by Bonaldi et al. [20 ] and Gardella et al. [21 ]. However, potent IFN-{gamma}-induced HMGB1 secretion also occurred at time-points when the granular pattern was not yet evident, as judged microscopically. It is interesting that the diffuse cytoplasmic staining detected in cells stimulated with TNF appeared similar to that seen with IFN-{gamma} stimulation, although this treatment did not progress to robust HMGB1 secretion.


Figure 3
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  		Figure 3. Immunocytochemistry visualizing the HMGB1 pattern (in green) in unstimulated and TNF-, LPS + IFN-{gamma}-, and IFN-{gamma}-stimulated RAW 264.7 cells after 24 h of culture; cell nuclei are stained in blue with DAPI. LPS + IFN-{gamma} stimulation induced a granular HMGB1 pattern, whereas IFN-{gamma} stimulation alone resulted in a diffuse, cytoplasmic HMGB1 pattern.

 
Apoptotic cells do not release HMGB1
HMGB1 has previously been demonstrated by other methods to be released from necrotic cells but not from apoptotic cells. To verify these results, we treated RAW 264.7 cells with the apotosis-inducing agent PRIMA-1 or with distilled water to induce osmotic cell damage. Complete cell death could be verified in both groups by trypan blue exclusion analysis. Confirming earlier data, the apoptotic PRIMA-1-treated cells did not produce any spot formation above background numbers, whereas osmotic cell damage resulted in 421 ± 33 low- and high- intensity spots/1000 cells (Fig. 4A ). Thus, by using the ELISPOT assay, we can detect passively and actively released HMGB1. However, the appearance of the spots differed. Actively secreted HMGB1 gave rise to smaller and more defined spots as compared with passively released HMGB1 from necrotic cells, which gave rise to larger, more diffuse spots (Fig. 4B) .


Figure 4
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  		Figure 4. HMGB1 is passively released from necrotic but not from apoptotic cells. (A) HMGB1 release from RAW 264.7 cells or subjected to water-mediated cell lysis or PRIMA-1-mediated apoptosis determined by ELISPOT assay, measuring the low- and high-intensity spots. (B) Photos of ELISPOT wells demonstrating differences in HMGB1 spot morphology after passive release (water-induced) and active release (IFN-{gamma}+LPS-induced) of HMGB1 from 5000 RAW 264.7 cells. Results of high- and low-intensity spots are expressed as means ± SD of triplicates.

 
TNF and IL-6 production in RAW 264.7 cells
Having established by ELISPOT that LPS, LPS + IFN-{gamma}, and IFN-{gamma} were good inducers of HMGB1 secretion, we next investigated whether these stimuli also induced TNF and IL-6 production in RAW 264.7 cells. We figured that these comparative studies might elucidate whether similar or separate intracellular, activation-transducing events were involved in HMGB1 and TNF/IL-6 secretion. TNF release was measured by ELISPOT, and IL-6 production was determined by ELISA. LPS induced TNF and IL-6 production and showed a synergistic effect when combined with IFN-{gamma} regarding IL-6 release. In contrast, IFN-{gamma} alone was a poor inducer of TNF production and did not stimulate any detectable IL-6 (Table 1 ). Thus, the secretion of HMGB1 did not parallel the induction of TNF and IL-6 production.


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  		Table 1. Quantification of HMGB-1, TNF, and IL-6 Secretion from LPS-, LPS + IFN-{gamma}-, and IFN-{gamma}-Stimulated RAW 264.7 Cells Analyzed by ELISPOT or ELISA

 
Release of HMGB1 from nonmonocytic cells such as mast cells and HCT 116 cells
As it is well established that mast cells are a source for the release of several multifunctional, proinflammatory factors using the secretory lysosomal pathway, we investigated whether mast cells could also be induced to actively mediate HMGB1 secretion. Two mast cell lines (the murine C57 and human HMC-1.2) were stimulated with LPS, LPS + IFN-{gamma}, IFN-{gamma}, or IgE + antigen for 48 h and analyzed for HMGB1 secretion in the ELISPOT assay. Only occasional HMGB1 spots were demonstrated (one–two spots/1000 cells), and the number of spots did not increase upon stimulation compared with unstimulated cells (Fig. 5 ). The low incidence of spots, irrespective of mode of stimulation, indicated that the studied mast cell lines did not posses the ability to secrete HMGB1. The intracellular HMGB1 staining pattern of C57 cells revealed nuclear and diffuse cytoplasmic staining in stimulated and unstimulated cells, and HMC-1.2 cells displayed a strict nuclear HMGB1 pattern with and without stimulation, as evaluated by immunocytochemistry (data not included). Many transformed tumor cells are known to express high amounts of HMGB1. We studied whether a cancer cell line of nonmyeloid origin, the human colon cancer cell line HCT 116, had the ability to actively secrete HMGB1 apart from intracellular overexpression. We observed marked, spontaneous HMGB1 release from HCT 116 cells in the ELISPOT assay with 430 ± 25 spots/1000 cells after a culture period of 48 h (Fig. 5) . Cell survival was excellent in the cultures, as judged by trypan blue staining and LDH assessment in the supernatants (data not included). Furthermore, extracellular HMGB1 release in the supernatant from HCT 116 cells was also measured by ELISA using the same antibody pair as used in the ELISPOT assay. Although the cell concentration was increased 40 times for the ELISA studies as compared with the cell concentration used in the ELISPOT assay, we could not detect any HMGB1 in the supernatants (data not included). The immunocytochemical analysis of HCT 116 cells revealed a strict nuclear HMGB1 pattern without any obvious granulae formation (data not included). Thus, these results suggest that tumor cells can spontaneously release large amounts of HMGB1 without using the secretory lysosomal pathway.


Figure 5
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  		Figure 5. Not all transformed cell lines are able to actively secrete HMGB1, and HMGB1 secretion was determined after 48 h of culture of the human colon cancer cell line HCT 116, the murine monocytic cell line RAW 264.7, the murine mast cell line C57, and the human mast cell line HMC-1.2, unstimulated or stimulated as indicated. A representative experiment out of two performed is depicted. Results are expressed as means ± SD of triplicates.

 
DISCUSSION

In this report, we introduce a sensitive, HMGB1-specific ELISPOT assay, suitable for studies of HMGB1 secretion by adherent and nonadherent cells. We verified that LPS and IFN-{gamma}, well-known inducers of HMGB1 secretion, as judged by extracellular HMGB1 protein assessment [4 , 27 ], stimulated HMGB1 secretion from the monocytic RAW 264.7 cell line. The present study adds novel information concerning kinetic secretion patterns observed in response to different stimuli. IFN-{gamma} stimulation alone was surprisingly the most effective inducer of HMGB1 secretion, displaying a faster response during the initial 24 h of stimulation than following LPS activation alone or in combination with IFN-{gamma}. It is notable that the immunocytochemical analysis of cells cultured for 24 h with IFN-{gamma} alone demonstrated increased, general cytoplasmic HMGB1 staining without any observable granular pattern. In contrast, cells stimulated with LPS or LPS combined with IFN-{gamma} expressed a granular cytoplasmic HMGB1 pattern 24 h poststimulation, most likely reflecting a secretory, lysosomal, intracellular transport route. However, RAW 264.7 cells that had been cocultured with IFN-{gamma} for 48 h also displayed a distinct, cytoplasmic granular staining. Whether the strong, IFN-{gamma}-induced HMGB1 secretion that initially occurred in the cultures without coincident, demonstrable, intracellular granulae reflects an alternative secretory pathway for HMGB1 is presently an unresolved issue. In contrast, Rendon-Mitchell and co-workers [27 ] reported that IFN-{gamma} stimulation of RAW 264.7 cells resulted in a granular, cytoplasmic HMGB1 pattern within 24 h of stimulation. Future studies are needed to clarify this discrepancy. Regardless of this, the ELISPOT assay enabled us to identify important aspects of HMGB1 biology, which would have been overlooked by only using the intracellular staining approach.

Although IFN-{gamma} was the most efficient stimulus for inducing HMGB1 secretion by RAW 264.7 cells, it was a most modest inducer of TNF release and did not induce any IL-6 secretion at all (Table 1) . These results indicate that IFN-{gamma} activates a separate pathway for HMGB1 secretion than that leading to TNF and IL-6 secretion. Previous studies have demonstrated this specificity to be mediated via TNF- and Janus kinase 2-dependent mechanisms [27 ]. IFN-{gamma} is produced in the innate immune system by activated NK cells and by activated T lymphocytes in adaptive immune responses and may thus contribute to HMGB1 release at different stages of inflammation. It has been reported that TNF on its own acts as a potent inducer of HMGB1 release from macrophages [4 , 19 ]. However, in our experiments, TNF only mediated a weak induction of HMGB1 secretion compared with that induced by LPS or IFN-{gamma}. Bioactivity of the TNF was verified by its apoptosis-inducing capacity, as measured by increased Annexin V binding to the cell surface of TNF-treated RAW 264.7 cells. Regarding the ability of TNF to induce apoptosis and the inability of inducing HMGB1 secretion, we thus confirmed previously reported data demonstrating that apoptotic cells do not release nuclear HMGB1 in contrast to necrotic and damaged cells [17 ]. Additional evidence for the validity of this concept was derived from control experiments in which cells were forced to extensive apoptotic cell death through PRIMA-1 treatment or to unprogrammed death using extensive osmotic cell damage by water exposure. No HMGB1 release was detected in the apoptotic, PRIMA-1-treated cell cultures, and the passively released HMGB1 from water-lysed cells was evident as large HMGB1 spots (Fig. 4) . These results also demonstrate that active and passive HMGB1 release is measurable using the ELISPOT method. TNF has been well documented to be of vital importance in the pathogenesis of sepsis [4 ], a condition in which high release of HMGB1 has been reported. Our data indicate that TNF does not cause such HMGB1 release directly but could rather be involved in HMGB1 release via its induction of other inflammatory mediators such as IL-1ß and IFN-{gamma}. In addition, during sepsis, endotoxins are commonly present. Endotoxins have previously and in our study been reported to be potent inducers of HMGB1 release.

In addition, we examined if the HMGB1 ELISPOT assay was useful for cell types other than monocytes. We chose two different cell types with characteristics known to be important for active HMGB1 secretion: mast cells, which use the secretory lysosomal pathway when secreting proinflammatory cytokines, and a colon cancer cell line, as transformed cells are known to overproduce HMGB1 [28 ]. Neither the murine C57 mast cell line nor the human HMC-1.2 mast cell lines secreted HMGB1 spontaneously or after 24 h or 48 h of LPS, LPS + IFN-{gamma}, IFN-{gamma} or IgE stimulation. It is interesting that the human colon cancer cell line HCT 116 secreted HMGB1 without application of any exogenous stimulus. It has recently been reported that WiDr human colon cancer cells constitutively release high amounts of HMGB1 [29 ] and that transformed human enterocyte-like Caco-2 cells spontaneously release small amounts of HMGB1 with enhanced secretion after cytokine stimulation [30 ]. It is notable that analysis of HCT 116 cells by immunocytochemical staining revealed that the cellular HMGB1 pattern was strictly nuclear. Liu et al. [30 ] recently demonstrated that HMGB1 secreted by Caco-2 cells was detected in exosomes and in exosome-depleted supernatants, suggesting that HMGB1 was released by these cells via several transport routes. Further study is required to elucidate different mechanisms for HMGB1 secretion and potential differences in the bioactivity between HMGB1 secreted by different cell types. Taken together, we have shown that not all transformed cell lines possess the ability to actively secrete HMGB1. Furthermore, we have also demonstrated that HMGB1 secretion does not always proceed by HMGB1 cytoplasmic granulae formation detected by immunocytochemistry, suggesting additional pathways for HMGB1 secretion than the secretory lysosomal route.

Detection and quantification of HMGB1 secretion are important features for assessing the role of HMGB1 in inflammation and for ascertaining its role in infection, immune-mediated diseases, tumor propagation, and tissue regeneration. The ELISPOT assay is a sensitive method to study and verify which cell types are able to secrete HMGB1 and to assess stimuli promoting and agents attenuating active and passive release of HMGB1 (Therese stberg, Karen Palmblad, Heidi Wähämaa, Maria Shoshan, Michael Lotze, Helena Erlandsson-Harris and Ulf Andersson, manuscript in preparation).

ACKNOWLEDGEMENTS

This study was supported by grants from the Swedish Science Council, Swedish Rheumatism Association, and King Gustaf V’s 80-Year Foundation. The authors thank Lotta Aveberger for excellent technical assistance, Associate Professor Maria C. Shoshan for interesting discussions and for the HCT 116 cell line, Professor Klas Wiman for the apoptosis-inducing compound PRIMA-1, and Associate Professor Robert A. Harris for the linguistic advice.

Received May 24, 2006; revised June 27, 2006; accepted July 26, 2006.

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