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The Journal of Immunology, 2001, 166: 7128-7135.
Copyright © 2001 by The American Association of Immunologists

An Essential Role of the NF-{kappa}B/Toll-Like Receptor Pathway in Induction of Inflammatory and Tissue-Repair Gene Expression by Necrotic Cells1

Ming Li*, David F. Carpio*, Ye Zheng*, Peter Bruzzo*, Veena Singh*, Fateh Ouaaz*, Ruslan M. Medzhitov and Amer A. Beg2,*

* Department of Biological Sciences, Columbia University, New York, NY 10027; and Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tissue damage induced by infection or injury can result in necrosis, a mode of cell death characterized by induction of an inflammatory response. In contrast, cells dying by apoptosis do not induce inflammation. However, the reasons for underlying differences between these two modes of cell death in inducing inflammation are not known. Here we show that necrotic cells, but not apoptotic cells, activate NF-{kappa}B and induce expression of genes involved in inflammatory and tissue-repair responses, including neutrophil-specific chemokine genes KC and macrophage-inflammatory protein-2, in viable fibroblasts and macrophages. Intriguingly, NF-{kappa}B activation by necrotic cells was dependent on Toll-like receptor 2, a signaling pathway that induces inflammation in response to microbial agents. These results have identified a novel mechanism by which cell necrosis, but not apoptosis, can induce expression of genes involved in inflammation and tissue-repair responses. Furthermore, these results also demonstrate that the NF-{kappa}B/Toll-like receptor 2 pathway can be activated both by exogenous microbial agents and endogenous inflammatory stimuli.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cell death can take place by apoptosis or necrosis (1). Importantly, apoptotic cells retain membrane integrity and can be rapidly phagocytosed by macrophages or by surrounding cells before lysis (2). In contrast, cells dying by necrosis readily lose membrane integrity and release intracellular contents, which may cause an inflammatory response (3). Innate immunity mediated by induction of an inflammatory response provides an important defense mechanism against both invading microbes and tissue damage. These protective effects are mediated by infiltrating neutrophils and macrophages, which phagocytose microbes and tissue debris. Neutrophils are readily detected within areas of tissue necrosis, and their presence has been used as evidence of inflammatory properties of necrotic cells (3). However, as recently discussed (4, 5), it is not known how necrotic cells are able to recruit neutrophils and induce inflammation. Nonetheless, studies of other inflammatory models have revealed an important role for polypeptides called chemokines in neutrophil recruitment (6, 7, 8, 9). Neutrophil-specific chemokines important for recruitment in situ include the human IL-8 and growth-related oncogene molecules and their functional counterparts in mice, (KC) and macrophage-inflammatory protein 2 (MIP-2)3 (10). Thus, one possible mechanism by which necrotic cells may allow neutrophil infiltration is by inducing expression of neutrophil-specific chemokines.

Tissue damage can initiate not only an inflammatory response but also a tissue-repair and remodeling process mediated by induction of metalloproteinases (11) and factors involved in angiogenesis, such as vascular endothelial growth factor (VEGF; Ref. 12). Interestingly, enhanced VEGF expression was detected in areas adjacent to necrotic cells in various disease models (13, 14), although the inducing mechanisms are still largely uncharacterized. Two recent studies have proposed that necrotic cells, but not apoptotic cells, can potently induce dendritic cell (DC) maturation in vitro (15, 16). Sauter et al. (16) have further demonstrated that the immunostimulatory activity was present in necrotic tumor cells but not in primary necrotic cells. Thus, it is not known whether primary necrotic cells have any immunoregulatory effect on DCs.

The NF-{kappa}B family of transcription factors are key regulators of genes involved in immune and inflammatory reactions (17, 18, 19). Studies with mice deficient in the RelA (p65) subunit of NF-{kappa}B also have shown that these proteins are important in the regulation of apoptosis. The absence of RelA results in massive apoptotic death of hepatocytes and embryonic lethality at E15 (20), which appears related to the cytotoxic effect of TNF-{alpha} on RelA-/- cells (21, 22). Among the most potent NF-{kappa}B inducers are microbial products such as bacterial LPS (18). NF-{kappa}B activation by LPS is dependent on members of the mammalian Toll-like receptor (TLR) protein family (homologous to Drosophila Toll; Refs. 23, 24, 25), and downstream signaling molecules which include TNFR-associated factor (TRAF) 6, myeloid differentiation primary response gene and IL-1 receptor-associated kinase (26, 27, 28). Our recent studies have demonstrated a key role for RelA in activation of many inflammatory genes in response to LPS stimulation, including those encoding proinflammatory cytokines and chemokines (29).

Here we have identified necrotic cells as novel and potent inducers of NF-{kappa}B. NF-{kappa}B activation by necrotic cells resulted in dramatic induction of expression of genes involved in the inflammatory response, such as neutrophil-specific chemokines, and genes involved in the tissue-repair and remodeling process. Interestingly, necrotic cell-induced NF-{kappa}B was found dependent on the TLR2 signaling pathway. In contrast to necrotic cells, apoptotic cells did not induce expression of chemokines because they did not significantly activate NF-{kappa}B. Thus, our results provide the first evidence for an NF-{kappa}B-dependent mechanism responsible for the strikingly different pathophysiological affects of necrotic and apoptotic cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and materials

DCs were prepared from bone marrow suspensions obtained from femurs and tibias of C57BL/6 mice (Taconic Farms, Germantown, NY). Cells were grown in 6-well plates at 3 x 106 cells in 2 ml of RPMI 1640 supplemented with 5% FBS, 2-ME (50 µM), IL-4 (5 ng/ml; R&D Systems, Minneapolis, MN), and GM-CSF (10 ng/ml; R&D Systems). Cells were "fed" IL-4 and GM-CSF every 2 days. On day 5, nonattached cells (DCs) were replated in fresh medium and treated with either LPS (5 µg/ml; Sigma, St. Louis, MO) or necrotic cells (106). Mouse embryonic fibroblasts (MEFs) or fetal liver macrophages were obtained and cultured as described previously (21). TNF-{alpha} and IL-1 were purchased from R&D Systems and used at 10 and 4 ng/ml, respectively. pBIIX-luc is a firefly luciferase report construct driven by a minimal murine c-fos promoter with two Ig{kappa} {kappa}B motifs upstream. pRL-TK is a renilla luciferase control reporter construct driven by the HSV thymidine kinase promoter. The expression vector pLPC was a gift of S. Lowe (Cold Spring Harbor Laboratory, NY). The pI{kappa}B{alpha}(S-A) plasmid was constructed by cloning an I{kappa}B{alpha} mutant (Ser32, Ser36 to Ala) into pLPC. pIKK{beta}DN was constructed by inserting a dominant-negative mutant of I{kappa}B kinase (IKK{beta}; a mouse IKK{beta} mutant consisting of a point mutation of Lys44 to Ala) into pLPC. pTRAF2DN, pTRAF6DN, pMyD88DN, and pTLR2DN were described previously (26, 30). pTLR2, pTLR4, and pTLR6 were constructed by cloning murine TLR2, TLR4, and TLR6 cDNA into the pIRES vector (Clontech Laboratories, Palo Alto, CA). Constructs were confirmed by sequencing.

Generation of necrotic and apoptotic cells

To obtain necrotic cells, MEFs or thymocytes were washed and resuspended at 107 cells/ml of PBS. Dounce-lysis was carried out by subjecting MEFs or thymocytes to 10 strokes with a Teflon homogenizer, after which complete cell lysis was evident by trypan blue inclusion. Freeze-thaw lysis was carried out by subjecting cells to five cycles of freezing on dry ice followed by thawing at 37°C. Such treatment generally resulted in lysis of 90% of the cells as judged by trypan blue inclusion. Apoptotic thymocytes were generated by {gamma}-irradiation. Briefly, thymocytes were subjected to 750 rad of {gamma}-irradiation. The cells then were cultured for 10 h, after which 90% of the cells showed typical apoptotic morphology and were annexin V positive. These cells were collected and resuspended in PBS. Greater than 80% of these cells were typically trypan blue negative. Necrotic or apoptotic cells were added to DC suspensions, MEFs, or macrophages for indicated periods. All experiments were carried out under sterile conditions. All cells were tested to be mycoplasma-free with a mycoplasma detection kit from American Type Culture Collection (Manassas, VA).

ELISA

Supernatants from cultured DCs, MEFs, or macrophages were used to determine the amount of IL-12 or KC. ELISA was carried out following recommendations of the manufacturer (R&D Systems).

EMSA and Western and Northern blots

EMSA was carried out as described previously (20). RelA-specific antiserum was purchased from Rockland (Gilbertsville, PA). Western blotting for I{kappa}B{alpha} was carried out with cytosolic extracts from ~105 MEFs by using a rabbit polyclonal IgG against the carboxyl terminus of I{kappa}B{alpha} (Santa Cruz Biotechnology, Santa Cruz, CA). Northern blotting was carried out as described previously (29) with probes made from cDNA fragments amplified by RT-PCR with gene-specific primers.

Luciferase assays

Transfections in the mouse macrophage cell line, RAW 264.7, were carried out by using Fugene6 (Roche, Gipf-Oberfrick, Switzerland). 293 Cells were transfected by using calcium phosphate. 24 h after transfection, cells were either left untreated or treated with 106 dounce-lysed MEFs. After another 10 h, cellular extracts were made and tested for luciferase activity.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of KC expression by necrotic but not apoptotic cells

We wished to investigate whether necrotic primary cells have any immunoregulatory effect on DCs. DC activation induced by microbial agents, such as LPS, results in enhanced production of IL-12, a key cytokine that promotes differentiation of Th1 cells. Thus, we first tested whether necrotic primary MEFs also could induce IL-12 production by DCs. To this end, 106 dounce-lysed MEFs were used to stimulate mouse bone marrow-derived DCs. After 12 h, supernatants were collected and the amount of IL-12 produced by DCs determined by ELISA. Only minimal levels of IL-12 were detected in necrotic cell-treated supernatants (Fig. 1GoA), whereas LPS-treated DCs produced high levels of IL-12 (Fig. 1GoA). Necrosis in vivo often is associated with recruitment of neutrophils (3). Therefore, we tested whether necrotic cell treatment of DCs could result in production of KC, a chemokine involved in neutrophil recruitment during inflammation (6, 8, 9). Significantly, necrotic MEF-treated DCs secreted high levels of KC (Fig. 1GoA). Interestingly, unlike IL-12, the amount of KC produced by necrotic MEF-treated DCs was higher than that produced by LPS-treated DCs (Fig. 1GoA), suggesting that necrotic cells may preferentially induce genes involved in innate immunity. To determine whether necrotic cells also could induce KC production in macrophages and fibroblasts, two cell types that play a critical role in regulation of innate immunity, we treated fetal liver-derived macrophages and MEFs with dounce-lysed MEFs for 6 h. Significant amounts of KC also were produced by these cells (Fig. 1GoB), demonstrating that necrotic cells can induce KC production in multiple cell types.



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  		FIGURE 1. Necrotic cells, but not apoptotic cells, induce KC expression. A, Mouse bone-marrow DCs were left untreated (UT), treated with 106 dounce-lysed MEFs (DLF), or 5 µg/ml LPS for 12 h. The amounts of IL-12 and KC in the supernatant (pg/ml) were determined by ELISA. B, Mouse fetal liver macrophages or MEFs were either left untreated (UT) or treated with 107 DLF for 6 h. Supernatants were collected and the amount of KC determined as in A. C, Macrophages or MEFs were left untreated (UT), treated with 107 dounce-lysed thymocytes (DLT), 107 apoptotic thymocytes (AT), or 107 dounce-lysed apoptotic thymocytes (DLAT) for 6 h. The amount of KC in the supernatant was assayed as in A. D, Macrophages or MEFs were stimulated as in C. RNA was extracted and tested for expression of KC, MIP-2, and GAPDH genes by Northern blotting.

 
Apoptotic cell death is generally not accompanied with induction of inflammation or recruitment of neutrophils. To determine the ability of apoptotic cells to trigger KC production, we used apoptotic thymocytes generated by {gamma}-irradiation. These apoptotic cells were annexin V positive and retained membrane integrity for several hours (data not shown), making them suitable for these experiments. In contrast to dounce-lysed thymocytes, no significant increase in KC was detected in either macrophages or MEFs treated with apoptotic thymocytes (Fig. 1GoC). Importantly, KC production took place if apoptotic thymocytes were rendered necrotic by dounce-lysis (Fig. 1GoC), although the levels of KC were lower than those induced by dounce-lysed thymocytes. These results suggest that intracellular contents are critical and sufficient for induction of KC expression. These results also demonstrate that intracellular contents from different primary cell types (fibroblasts or thymocytes) can induce KC production.

To determine whether the increased KC production was a consequence of enhanced mRNA levels, Northern blot analysis was performed with RNA from both macrophages and fibroblasts treated with necrotic thymocytes. In both cell types, a significant increase in KC transcripts were detected (Fig. 1GoD). Consistent with ELISA results, no significant change in KC levels were detected in either macrophages or fibroblasts treated with apoptotic thymocytes (Fig. 1GoD), whereas dounce-lysis of apoptotic thymocytes resulted in enhanced KC transcription (Fig. 1GoD). Consistent with enhanced production of KC by DCs, increased KC transcripts were detected in necrotic MEF-treated DCs (data not shown). Another neutrophil-specific chemokine, MIP-2, also was induced by necrotic thymocytes in either macrophages or fibroblasts (Fig. 1GoD). Increased KC and MIP-2 transcripts also were detected in necrotic MEF-treated fibroblasts and macrophages (see below). These observations suggest that the intracellular contents released from necrotic cells can induce neutrophil-specific chemokine gene expression at the transcriptional level in multiple cell types.

NF-{kappa}B activation by necrotic cells but not apoptotic cells

Our recent studies have demonstrated a critical role for NF-{kappa}B in regulation of KC and MIP-2 (29). Thus, one intriguing possibility was that induction of KC expression by necrotic cells was mediated by activation of NF-{kappa}B in viable cells, after interaction with necrotic cells. To test this possibility, we determined whether NF-{kappa}B could be activated in viable cells exposed to necrotic cells. NF-{kappa}B normally is sequestered in the cytoplasm bound to the inhibitory I{kappa}B proteins (18). Degradation of I{kappa}B proteins by NF-{kappa}B activators allows free NF-{kappa}B to translocate to the nucleus and activate target gene expression (18). EMSA analysis showed that significant nuclear translocation of NF-{kappa}B took place in MEFs exposed to dounce-lysed necrotic thymocytes (Fig. 2GoA). In striking contrast, a similar number of apoptotic thymocytes (Fig. 2GoA) were significantly less capable of activating NF-{kappa}B in MEFs. However, lysis of apoptotic thymocytes resulted in NF-{kappa}B activation to the same extent as with necrotic cells (Fig. 2GoA). These results indicate that cell lysis is required for activation of NF-{kappa}B and demonstrate a key difference between apoptotic and necrotic cells in their ability to activate NF-{kappa}B. Necrotic MEFs also could activate NF-{kappa}B in a dose-dependent manner (Fig. 2GoB). Nuclear NF-{kappa}B also was detected if necrotic MEFs were generated by freeze-thaw lysis, although the activation was less strong than that induced by dounse-lysed cells, suggesting the NF-{kappa}B-inducing agents present in necrotic cells may in fact be quite labile (data not shown). These results indicate that NF-{kappa}B activation is not dependent on the cell type used or the manner in which necrotic cells are generated.



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  		FIGURE 2. Necrotic cells are potent activators of NF-{kappa}B. A, MEFs were left untreated (UT), treated with 107 DLT, 107 AT, or 107 DLAT. Two hours later, nuclear extracts were made and tested for the presence of {kappa}B site binding activity by EMSA. B, DLF (105, 106, or 107) were used to treat MEFs and tested for their ability to activate {kappa}B binding activity. C, RelA+/+ or RelA-/- MEFs were left untreated (UT) or treated with 107 DLF. RelA-specific antiserum was used to determine the presence of RelA (lanes 1–5). A total of 107 dounce-lysed RelA-/- MEFs (DLF(RelA-/-)) were also used to treat RelA+/+ MEFs. Activation of {kappa}B site binding activity and the presence of RelA subunit were tested as above (lanes 6–8). The induced {kappa}B binding activity is indicated by an arrowhead. D, MEFs were left untreated (UT) or treated with 107 DLF for 30 min, 2 h, or 6 h (lanes 1–4). For the 2-h time point, NF-{kappa}B activation was also determined in the presence of cyclohexamide (Cyclo; lanes 5–8).

 
TNF-{alpha} treatment of MEFs results in activation of NF-{kappa}B heterodimers consisting of p50 and RelA (p65) (20). The NF-{kappa}B complex activated by necrotic cells was specifically supershifted by antisera generated against RelA (Fig. 2GoC, lane 3). Furthermore, necrotic cell-treated RelA-/- MEFs showed significantly reduced induction of {kappa}B site binding activity (Fig. 2GoC, compare lanes 2 and 5), as observed previously after TNF-{alpha} treatment (20). When necrotic RelA-/- MEFs were added to RelA+/+ MEFs, a significant increase in {kappa}B binding activity was detected (Fig. 2GoC, lane 7). The induced {kappa}B activity was supershifted by RelA-specific antisera (Fig. 2GoC, lane 8), demonstrating that NF-{kappa}B was activated in viable RelA+/+ MEFs, and not necrotic RelA-/- cells. NF-{kappa}B was activated within 30 min after addition of necrotic MEFs and persisted for at least 6 h in nuclear fractions (Fig. 2GoD, lanes 1–4). Significant NF-{kappa}B activation by necrotic cells also took place in the presence of cyclohexamide (Fig. 2GoD, lanes 5–8), suggesting that activation was not dependent on protein synthesis.

Inflammatory and tissue-repair gene induction in MEFs and macrophages by necrotic cells is dependent on RelA

To determine whether NF-{kappa}B activation by necrotic cells may be an important mechanism for induction of inflammatory genes such as KC and MIP-2, we first investigated expression of I{kappa}B{alpha}, a key NF-{kappa}B target gene. Both RelA+/- and RelA-/- MEFs were treated with necrotic MEFs for different periods after which RNA was isolated to determine levels of I{kappa}B{alpha} mRNA. I{kappa}B{alpha} was strongly induced in RelA+/- but not in RelA-/- MEFs after exposure to necrotic cells (Fig. 3GoA). We then tested whether KC and MIP-2 induction also was dependent on RelA. RelA+/- MEFs showed dramatic induction of both KC and MIP-2 mRNA after a 6-h treatment with necrotic cells (Fig. 3GoA). In contrast, induction of these transcripts was significantly reduced in RelA-/- MEFs (Fig. 3GoA), indicating a key role for RelA in chemokine gene induction by necrotic cell contents. Similar results also were obtained when freeze-thaw MEFs were used (data not shown). These results demonstrate that necrotic cells can induce chemokine gene expression through activation of RelA-containing NF-{kappa}B complexes.



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  		FIGURE 3. The RelA subunit of NF-{kappa}B is involved in induction of proinflammatory and tissue-repair gene expression by necrotic cells. A, RelA+/- or RelA-/- MEFs were left untreated (UT), or treated with DLF or TNF-{alpha} for the time periods indicated. RNA was extracted and tested for expression of I{kappa}B{alpha}, KC, MIP-2, MMP3, VEGF, and GAPDH genes by Northern blotting. B, RelA+/+ or RelA-/- macrophages were either left untreated (UT) or treated with DLF for 6 h. Northern blot analysis was used to determine expression of I{kappa}B{alpha}, KC, MIP-2, and GAPDH.

 
Tissue repair and remodeling after tissue damage are essential features of the wound-healing process. Transcripts encoding stromelysin-1/MMP3 and VEGF, two proteins which play an important role in tissue repair (12, 31), also were induced by necrotic cells in RelA+/- MEFs, whereas induction was reduced in RelA-/- MEFs (Fig. 3GoA). Thus, necrotic cells also can induce expression of genes involved in the tissue-repair process. These results also reveal a previously uncharacterized function for RelA in regulation of genes involved in the wound-healing response. Importantly, induction of KC, MIP-2, and VEGF by necrotic cells was significantly higher than induction by TNF-{alpha}, whereas I{kappa}B{alpha} and MMP3 were similarly induced by both stimuli. These results suggest that necrotic cells may preferentially induce high expression of a subset of NF-{kappa}B target genes.

We then tested whether RelA also plays an essential role in regulating inflammatory genes in macrophages. To this end, fetal liver-derived RelA+/+ and RelA-/- macrophages were treated with necrotic cells for 6 h. Although RelA-/- embryos do not survive past E15 because of TNF-{alpha}-mediated liver degeneration (20, 21, 22), we have readily obtained fetal liver macrophages from E13-E14 RelA-/- embryos (21). Northern blot analysis of RNA from these macrophages demonstrated that induction of I{kappa}B{alpha}, KC, and MIP-2 transcripts in RelA+/+ macrophages was significantly more than the induction in RelA-/- macrophages (Fig. 3GoB). ELISA analysis of supernatants showed that synthesis of both chemokines also was significantly more in RelA+/- MEFs and RelA+/+ macrophages than in RelA-/- MEFs and macrophages (data not shown). Thus, NF-{kappa}B activation by necrotic cell contents results in dramatic induction of chemokine production by fibroblasts and macrophages, two cell types that play a key role in chemokine production.

IL-1/TLR signaling molecules are required for NF-{kappa}B activation by necrotic cells

Because NF-{kappa}B activation by many agents, including the proinflammatory cytokines TNF-{alpha} and IL-1, is dependent on degradation of I{kappa}B proteins (18, 32), we tested whether levels of the I{kappa}B{alpha} protein were affected by necrotic cells. A reduction in cytosolic levels of I{kappa}B{alpha} was noticed, which was further decreased in the presence of cyclohexamide (Fig. 4GoA). I{kappa}B degradation requires initial phosphorylation of two key serine residues by the recently identified IKK proteins (19). NF-{kappa}B activation can thus be inhibited by expression of a phosphorylation-deficient I{kappa}B{alpha} protein. As expected, treatment of the RAW macrophage cell-line with necrotic cells significantly increased {kappa}B luciferase reporter activity (Fig. 4GoB; RAW macrophages also activated NF-{kappa}B and induced target gene expression after necrotic cell stimulation; data not shown). The increase in luciferase activity was dramatically suppressed by expression of a phosphorylation-deficient I{kappa}B{alpha} protein (Fig. 4GoB), suggesting that I{kappa}B phosphorylation may be essential for NF-{kappa}B activation by necrotic cells. We then tested the effect of expression of a dominant-negative mutant of IKK{beta} (DN-IKK{beta}), which appears most important for phosphorylation of I{kappa}B proteins (33, 34), on luciferase activity induced by necrotic cells. Expression of DN-IKK{beta} significantly reduced reporter activity (Fig. 4GoB), suggesting a requirement for IKK{beta} in activation of NF-{kappa}B by necrotic cells. These results demonstrate that NF-{kappa}B activation by necrotic cells appears to be by the "classical" activation pathway, which does not require new protein synthesis and depends on phosphorylation and degradation of I{kappa}B proteins.



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  		FIGURE 4. Activation of NF-{kappa}B by necrotic cells requires I{kappa}B phosphorylation and degradation. A, MEFs were left untreated (UT) or treated with 107 DLF in the absence or presence of cyclohexamide (Cyclo) for 2 h. Cytosolic extracts were made and the level of I{kappa}B{alpha} determined by Western blotting. B, RAW macrophages were transfected in duplicate with 0.1 µg of pBIIX-luc, 0.1 µg of pRL-TK, and 1 µg of pLPC, pI{kappa}B{alpha} (S-A), pIKK{beta}DN, pTRAF2DN, pTRAF6DN, or pMyD88DN plasmids. Twenty-four hours later, transfected cells were either left untreated (UT) or treated with 106 dounce-lysed MEFs (DLF) for 10 h. The relative firefly luciferase activities of pBIIX-luc normalized to renilla luciferase activities are shown.

 
Two major receptor-mediated NF-{kappa}B activation pathways important for inducing inflammatory gene expression have been identified: the TNF pathway, which can be inhibited by a dominant-negative mutant of the adapter protein TRAF2 (35) and the IL-1/TLR pathway, which can be inhibited by dominant-negative mutants of MyD88, IRAK, and TRAF6 proteins (26, 27, 28). Expression of a dominant-negative TRAF2 did not significantly inhibit necrotic cell-mediated NF-{kappa}B activation in RAW cells (Fig. 4GoB), suggesting activation may not be dependent on signaling components used by the TNF pathway. In contrast, expression of MyD88 or TRAF6 dominant-negative proteins significantly inhibited NF-{kappa}B activation by necrotic cells (Fig. 4GoB). These results suggest that NF-{kappa}B activation by necrotic cells could be mediated by IL-1/TLR signaling components.

TLR2 is sufficient for NF-{kappa}B activation and IL-8 expression by necrotic cells

To further study the involvement of the IL-1/TLR signaling pathway in necrotic cell-mediated activation of NF-{kappa}B, we used human 293 cells, which were found unable to activate NF-{kappa}B after necrotic cell stimulation (Fig. 5GoA). However, IL-1 treatment of 293 cells resulted in strong NF-{kappa}B activation (Fig. 5GoA), suggesting that IL-1/TLR signaling components were present in 293 cells. Thus, a possible reason for the inability of 293 cells to activate NF-{kappa}B by necrotic cells may be that these cells lack expression of requisite TLRs (27). We tested this by transiently expressing TLR2, TLR4, or TLR6 along with a {kappa}B reporter construct in 293 cells. As expected from EMSA results, virtually no activation of the {kappa}B reporter occurred when 293 cells were transfected with a control GFP construct and treated with necrotic MEFs (Fig. 5GoB). However, expression of TLR2, but not TLR4 or TLR6, resulted in dramatic activation of NF-{kappa}B activity in 293 cells (Fig. 5GoB). TLR2 also was able to mediate necrotic thymocyte-induced NF-{kappa}B activation in 293 cells (data not shown). These observations demonstrate that the intracellular contents from different cell types can activate NF-{kappa}B in a TLR2-dependent manner. A recent study has revealed that TLR2 can functionally cooperate with TLR6 to mediate responses to certain TLR2 activators (36). To test whether TLR2-mediated NF-{kappa}B activation of necrotic cells could also be potentiated by TLR6, we cotransfected 293 cells with constructs encoding TLR2, TLR6, or TLR2 and TLR6. Significant increase of NF-{kappa}B activity was detected when both TLR2 and TLR6 were expressed (Fig. 5GoC), suggesting that TLR6 can facilitate TLR2 responses to necrotic cells.



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  		FIGURE 5. TLR mediate activation of NF-{kappa}B and induction of chemokine expression by necrotic cells. A, 293 Cells were either left untreated (UT), or treated with 107 dounce-lysed MEF cells (DLF) or 4 ng/ml IL-1. Two hours later, nuclear extracts were made and tested for the presence of {kappa}B site binding activity by EMSA (lanes 1–3). MEFs also were treated with DLF for 2 h and tested for {kappa}B site binding activity (lanes 4–5). The induced {kappa}B binding activity is indicated by an arrowhead. B, 293 Cells were transfected in duplicate with 0.1 µg of pBIIX-luc, 0.1 µg of pRL-TK, and 1 µg of pGFP, pTLR2, pTLR4, or pTLR6 plasmids. Twenty-four hours later, transfected cells were either left untreated (UT) or treated with 106 DLF for 10 h. The relative firefly luciferase activities are shown. C, 293 Cells were transfected in duplicate with 0.1 µg of pBIIX-luc, 0.1 µg of pRL-TK, and 1 µg of pGFP, pTLR2, pTLR6, or pTLR2 and pTLR6 (pTLR2, 6) plasmids. Cells were then stimulated and assayed as in B. D, 293 Cells were either left untransfected or transfected with 2 µg of pGFP, 2 µg of pTLR2, or 2 µg of pTLR2 and 4 µg of pI{kappa}B{alpha} (S-A) plasmids. Twenty-four hours later, cells were left untreated (UT) or treated with 107 DLF for 6 h. Nuclear extracts were made and tested for the presence of {kappa}B site binding activity by EMSA. The induced {kappa}B binding activity is indicated by an arrow head. E, 293 Cells were treated as in D. RNA was extracted and tested for expression of IL-8 and {beta}-actin genes by Northern blot analysis. F, RAW macrophages were transfected in duplicate with 0.1 µg of pBIIX-luc, 0.1 µg of pRL-TK, and 1 µg of pLPC or pTLR2DN plasmids. Twenty-four hours later, transfected cells were either left untreated (UT) or treated with 106 dounce-lysed MEFs (DLF) for 10 h. The relative firefly luciferase activities of pBIIX-luc normalized to renilla luciferase activities are shown.

 
We then determined whether TLR2 expression was sufficient for NF-{kappa}B activation and gene induction. Nuclear extracts of TLR2-transfected and necrotic cell-stimulated 293 cells showed significant NF-{kappa}B activation, which was abolished by coexpression of an I{kappa}B{alpha} (S to A) mutant (Fig. 5GoD). Significantly, necrotic cell treatment of TLR2-transfected 293 cells also induced expression of IL-8 (a human neutrophil-specific chemokine), which also was inhibited by coexpression of I{kappa}B{alpha} (S to A; Fig. 5GoE). These results demonstrate that TLR2 expression is sufficient for NF-{kappa}B activation and expression of the IL-8 chemokine by necrotic cells. Importantly, overexpression of a dominant-negative mutant of TLR2 significantly reduced {kappa}B reporter gene activity by necrotic MEFs in RAW cells (Fig. 5GoF), suggesting that TLR2 may play an essential role in mediating necrotic cell-induced NF-{kappa}B activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
NF-{kappa}B is a master regulator of genes involved in immune and inflammatory responses. Induction of these genes typically occurs in response to stimulation of cells with conserved microbial patterns such as LPS or with proinflammatory cytokines. Here we have shown that intracellular contents released from necrotic cells also function as novel and potent inducers of NF-{kappa}B. We believe these results may have significant implications for our understanding of how cell death affects inflammatory and tissue-repair responses.

Foremost, we believe that necrotic cells may provide an endogenously generated "sentinel" signal that leads to production of inflammatory mediators and allows efficient removal of damaged tissue. Importantly, we have shown that exposure of viable cells to necrotic cell contents is sufficient for inducing expression of chemokines specific for neutrophils. Although the ability of neutrophils to infiltrate necrotic tissue is well known, it has not been clear whether necrosis itself causes neutrophil entry (5, 37). This is especially true where tissue damage is associated with microbial infections, because microbial products are themselves strong inducers of neutrophil recruitment (e.g., LPS). Thus, our results provide the first evidence for a direct role of necrotic cells in regulating expression of genes important for inducing inflammation. Importantly, we also have found that necrotic cells are strong stimulators of KC but weak stimulators of IL-12 production in DCs, suggesting that necrotic cells may preferentially induce genes important for innate immunity.

In addition to an inflammatory response, tissue damage can initiate a tissue-repair and remodeling process. Here, we have provided evidence that necrotic cells also may directly regulate the tissue-repair response. Metalloproteinases can be rapidly induced after injury (11) and play an essential role in tissue repair and remodeling (31). Such induction has been proposed to be mediated by proinflammatory cytokines and growth factors (38). Our results indicate that necrotic cells also may function as inducers of metalloproteinase expression and thus directly initiate a wound-healing response after tissue damage. VEGF is an important mediator of angiogenesis, another key component of the tissue-repair pathway (12). Interestingly, VEGF expression could be detected in areas adjacent to necrotic cells in various disease models (13, 14) and in situ analysis of tumor specimens also has demonstrated the localization of VEGF-producing cells proximal to necrotic foci (39). Our results indicate that necrotic cells may directly induce expression of VEGF and thus also participate in stimulating angiogenesis. Similar to chemokine genes, induction of both MMP3 and VEGF expression was dependent, at least in part, on the RelA subunit of NF-{kappa}B. These observations also indicate that NF-{kappa}B proteins may play an important role in regulating expression of tissue-repair genes.

In addition, our results provide a possible explanation for well known differences between apoptotic and necrotic cell death in induction of inflammation. We have shown that KC and MIP-2 expression is induced by necrotic but not by apoptotic cells. The inability of apoptotic cells to induce chemokine expression appears linked to relatively weak NF-{kappa}B activation by apoptotic cells, which is typically at least 10-fold less than by an equivalent number of necrotic cells (Fig. 2GoA and data not shown). However, lysis of apoptotic cells results in similar activation of NF-{kappa}B as seen with necrotic cells, demonstrating a key role for intracellular contents in NF-{kappa}B activation. Apoptotic cells are rarely detected in situ as a consequence of rapid clearance by phagocytic cells such as macrophages or by surrounding cells (2, 37). Our results indicate that rapid removal of apoptotic cells before lysis may be important in preventing release of intracellular contents that can activate NF-{kappa}B and induce expression of inflammatory genes. In addition, macrophages that have phagocytosed apoptotic cells can produce antiinflammatory cytokines such as TGF{beta} and IL-10 (40, 41, 42). Whether these cytokines also can modulate NF-{kappa}B-mediated induction of inflammatory genes will be an interesting area for investigation.

Necrotic cell activation of NF-{kappa}B is dependent on TLRs

TLRs are evolutionarily conserved receptors that regulate innate and adaptive immune responses (43). This is mediated, in large part, by activation of NF-{kappa}B and subsequent induction of expression of inflammatory genes by this transcription factor. We show here that activation of NF-{kappa}B by necrotic cells also requires signaling molecules of the IL-1/TLR pathway. Recent studies have shown that TLR2 can mediate signaling to multiple components of Gram-positive bacteria, mycobacteria, and yeast (30, 44, 45, 46, 47, 48), whereas TLR4 appears specific for LPS (25, 45). Our results demonstrate that TLR2 also is sufficient to mediate responses to necrotic cells. We believe these results are significant because they show that the mechanism for inducing inflammatory gene expression by exogenous agents, such as microbes, and endogenous signals, such as necrotic cells, is remarkably similar. Interestingly, coexpression of TLR6 and TLR2 could potentiate necrotic cell-induced NF-{kappa}B activation, although TLR6 alone had no significant effect (Fig. 5GoC). Thus, similar to microbial TLR2 activators (36), necrotic cells might also engage a combinatorial repertoire of TLRs to induce an inflammatory response. However, expression of a dominant-negative mutant of TLR6 did not significantly inhibit NF-{kappa}B activation by necrotic cells in RAW macrophages (data not shown). Because other TLRs, such as TLR1, can also augment TLR2 signaling (36), it is possible that TLRs besides TLR6 are involved in necrotic cell-induced NF-{kappa}B activation.

Nature of NF-{kappa}B inducing agents present in necrotic cells?

Two recent studies have demonstrated that recombinant heat shock proteins (HSPs) 60 and 70 can activate NF-{kappa}B (49, 50), raising the possibility that these endogenous proteins may be responsible for induction of chemokine gene expression by necrotic cells. A recent study has further demonstrated that TLR4 can mediate responses to HSP60 (51). However, our results show that TLR4 is not sufficient to mediate NF-{kappa}B activation by necrotic cells. In addition, chemokine induction by necrotic cells was unaffected in macrophages derived from C3H/HeJ mice, in which TLR4 is nonfunctional, whereas HSP60- and HSP70-induced chemokine gene expression was compromised (unpublished observations). These results suggest that HSP60 and HSP70 may not be the agents responsible for the induction of chemokine expression by necrotic cells.

To identify the nature of inducing agents present in necrotic cells, we have carried out fractionation studies and have found that both mitochondrial and nuclear fractions can use TLR2 to activate NF-{kappa}B. Unfortunately, we were unable to further characterize these fractions because of a virtually complete loss of NF-{kappa}B-inducing activity when subjected to standard fractionation and purification procedures. It is possible that the labile nature of these activities represents a requirement for maintenance of higher order structures and/or an unusual susceptibility to inactivation following fractionation. Thus, further characterization may first require identification of appropriate conditions that preserve the structural integrity of NF-{kappa}B-inducing agents present in necrotic cells. Characterization of these activities will eventually be required to determine whether NF-{kappa}B activation is mediated by unique molecule-specific interactions or interactions with classes of molecules representing specific structures (or patterns), as seen during recognition of microbial components.

Concluding remarks

We have demonstrated here, for the first time, that necrotic cells can directly enhance expression of genes important for inducing an inflammatory and tissue-repair response, through activation of the NF-{kappa}B and TLR2 pathway. We believe that these results will help us better understand different pathophysiological affects induced by tissue damage. Because cell death forms an integral part of virtually any disease, these findings also may have significant implications for understanding disease mechanisms.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant R01 CA074892 (to A.A.B.). Back

2 Address correspondence and reprint requests to Dr. Amer A. Beg, 1110 Fairchild Center, Department of Biological Sciences, 1212 Amsterdam Avenue, Columbia University, New York, NY 10027. E-mail address: aab41{at}columbia.edu Back

3 Abbreviations used in this paper: MIP-2, macrophage-inflammatory protein 2; VEGF, vascular endothelial growth factor; DC, dendritic cell; TLR, Toll-like receptor; TRAF, TNFR-associated factor; MEF, mouse embryonic fibroblasts; IKK, I{kappa}B kinase; HSP, heat shock protein. Back

Received for publication December 19, 2000. Accepted for publication April 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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