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HMG-1 as a Late Mediator of Endotoxin Lethality in Mice

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Science  09 Jul 1999:
Vol. 285, Issue 5425, pp. 248-251
DOI: 10.1126/science.285.5425.248

Abstract

Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia). Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis. Here a potential late mediator of lethality is identified and characterized in a mouse model. High mobility group–1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1. Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure. Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal. Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target.

Mortality rates for systemic bacterial infection have not declined significantly, despite advances in antibiotic therapy and intensive care. Bacteria do not directly cause lethal shock and tissue injury. Rather, bacterial endotoxin (lipopolysaccharide, LPS) stimulates the acute, early release of cytokines such as TNF and IL-1β from macrophages, and it is these host products that mediate damage (1). Macrophages from C3H/HeJ mice do not release TNF and IL-1 when stimulated by LPS; these animals are resistant to LPS lethality (2). Normal, LPS-responsive mice can be protected from lethal endotoxemia by therapeutic agents that selectively inhibit cytokine action or prevent cytokine release (3).

Translating these pathogenic insights into clinical therapy has proved difficult, in part because these “early” mediators (TNF and IL-1) are released within minutes after LPS exposure (4). Thus, even a minimal delay in treatment directed against TNF or IL-1 is ineffective (3, 5). Paradoxically, LPS-responsive mice treated with lethal doses of LPS succumb at latencies of up to 5 days, long after serum TNF and IL-1 have returned to basal levels. Moreover, mice deficient in TNF die within several days of LPS administration (6), suggesting that mediators other than TNF might contribute causally to endotoxin-induced death.

To identify potential “late” mediators of endotoxemia, we stimulated murine macrophage-like RAW 264.7 cells with LPS and analyzed the conditioned culture medium by SDS–polyacrylamide gel electrophoresis (PAGE). LPS stimulation for 18 hours induced the appearance of a 30-kD protein that was not apparent at earlier time points. The NH2-terminal sequence of this late-appearing factor (Gly-Lys-Gly-Asp-Pro-Lys-Lys-Pro-Arg-Gly-Lys-Met-Ser-Ser) was identical to murine HMG-1, a 30-kD member of the high mobility group (HMG) nonhistone chromosomal protein family (7,8). Based on the HMG-1 sequence in GenBank (accession no. M64986), we designed primers and isolated HMG-1 cDNA after polymerase chain reaction (PCR) amplification. Recombinant HMG-1 (rHMG-1) protein was expressed inEscherichia coli, purified to homogeneity, and used to generate polyclonal antibodies (9).

Immunoblot analysis revealed that large amounts of HMG-1 were released from RAW 264.7 cells in a time-dependent manner (Fig. 1A), beginning 6 to 8 hours after stimulation with LPS. Cell viability, as judged by trypan blue exclusion and lactate dehydrogenase release, was unaffected by LPS concentrations that induced the release of HMG-1, indicating that HMG-1 release was not due to cell death. HMG-1 mRNA levels were unaffected by LPS treatment (Fig. 1B), indicating that HMG-1 release is unlikely to be linked to increased transcription of the gene. Stimulation of RAW 264.7 cells for 18 hours with TNF (5 to 100 ng/ml) or IL-1β (5 to 100 ng/ml) also induced HMG-1 release in a cytokine dose-dependent manner. In contrast, stimulation with interferon-γ (IFN-γ) alone did not induce HMG-1 release, even at concentrations up to 100 U/ml; however, IFN-γ increased by three- to fivefold the amount of HMG-1 released by stimulation with either TNF or IL-1 (10, 11). Pulse labeling experiments with 35S-methionine revealed that most of the HMG-1 released during the first 12 hours after TNF and IFN-γ stimulation was derived from a preformed protein pool. Radioactivity was incorporated into newly synthesized HMG-1 from 12 to 36 hours after macrophage stimulation (10, 11).

Figure 1

(A) Release of HMG-1 from cultured macrophages after stimulation with LPS. Murine macrophage-like RAW 264.7 cells (American Type Culture Collection, Rockville, Maryland) were cultured in RPMI 1640 medium, 10% FBS, and 1% glutamine. When 70 to 80% confluence was reached, cells were resuspended in serum-free OPTI-MEM I medium and seeded onto tissue culture plates (5 × 106 cells per well). After 2 hours, RAW 264.7 cells were treated with LPS (E. coli 0111:B4, 100 ng/ml) and proteins in the cell-conditioned medium were fractionated by SDS-PAGE, excised from Coomassie- stained SDS-PAGE gels, and subjected to NH2-terminal sequencing analysis (Commonwealth Biotechnologies, Richmond, Virginia). Polyclonal antisera against purified recombinant HMG-1 were generated in rabbits (Biosynthesis, Lewisville, Texas); immunoblotting showed that antiserum reacted with native HMG-1 released by RAW cells (inset). HMG-1 levels were measured by optical intensity of bands on immunoblots with NIH 1.59 image software, with reference to standard curves generated with purified rHMG-1. Data are shown as the mean ± SE (n = 3). (B) Expression of HMG-1 mRNA in macrophages. Murine macrophage-like RAW 264.7 cells were cultured in RPMI 1640, 10% FBS, and 1% glutamine, and stimulated with LPS (1 μg/ml) for 0, 8, 12, and 16 hours as indicated. Total RNA was isolated with the SV Total RNA Isolation System (Promega) and levels of HMG-1 mRNA were determined by reverse transcriptase (RT)–PCR with the Access RT-PCR System (Promega; β-actin primers, 5′-TCATGAAGTGTGACGTTGACATCCGT-3′ and 5′-CCTAGAAGCATTTGCGGTGCACGATG-3′; and HMG-1 primers, 5′-ATGGGCAAAGGAGATCCTA-3′ and 5′-ATTCATCATCATCATCTTCT-3′). (C) Accumulation of HMG-1 in serum of LPS-treated mice. Male Balb/C mice (20 to 23 g) were treated with LPS [10 mg/kg, intraperitoneally (ip)]. Serum was assayed for HMG-1 by immunoblotting; the detection limit is ∼50 pg. Data are shown as the mean ± SE (n = 3).

We next examined the inducible release of HMG-1 from other cell types. LPS triggered HMG-1 release from human primary peripheral blood mononuclear cells and primary macrophages from LPS-sensitive mice (C3H/HeN), but not from macrophages from LPS-resistant C3H/HeJ mice (11, 12). Human primary T cells, rat adrenal (PC-12) cells, and rat primary kidney cells did not release HMG-1 after stimulation with LPS, TNF, or IL-1β. Like other macrophage products (for example, TNF, IL-1β, and macrophage migration inhibitory factor), HMG-1 lacks a classical secretion signal sequence, so the mechanism of release remains to be determined.

To determine if HMG-1 was released systemically during endotoxemia in mice, we measured serum HMG-1 levels after LPS administration. Serum HMG-1 was readily detectable 8 hours after administration of a median lethal dose (LD50) of LPS and was maintained at peak, plateau levels from 16 to 32 hours after LPS treatment (Fig. 1C). About 20 to 50 μg of HMG-1 was released into the murine circulation within 24 hours after endotoxin administration [assuming a distribution half-life (t1/2) of 3 min and an elimination t1/2 of 20 min]; this is comparable to the quantity of TNF and IL-1 released by LPS treatment. The kinetics of HMG-1 appearance in the blood of LPS-treated mice differs from that of previously described lethal LPS-induced mediators.

Passive immunization of unanesthetized mice with a single dose of antibodies to HMG-1 (anti–HMG-1) 30 min before a lethal dose (LD100) of LPS did not prevent LPS-induced death (Fig. 2A). Based on the kinetics of HMG-1 accumulation in serum (Fig. 1C), and the relatively short biological half-life of antibodies to cytokines (3, 13), we reasoned that complete neutralization of a late-appearing mediator might require repeated dosing. Administration of anti–HMG-1 in two doses (one 30 min before LPS and one 12 hours after LPS) increased the survival rate of the mice to 30%. With three doses of antiserum (−30 min, +12 hours, +36 hours), 70% of the treated mice survived, as compared with 0% survival in controls treated with three matched doses of preimmune serum (P < 0.05). No late death occurred over 2 weeks, indicating that anti–HMG-1 did not merely delay the onset of LPS lethality, but provided lasting protection.

Figure 2

(A) Anti–HMG-1 protect against LPS lethality in mice. Polyclonal antibodies against rHMG-1 were generated in rabbits, and antiserum was assayed for specificity and titer by enzyme-linked immunosorbent assay and immunoblotting. Antibodies reacted specifically with HMG-1 and did not cross-react with LPS, other bacterial proteins, TNF, or IL-1β. Immunoblots of lysates of macrophages or E. coli transformed with plasmid containing HMG-1 cDNA revealed only one band of immunoreactivity. Male Balb/C mice (20 to 23 g) were randomly grouped (10 mice per group) and treated with an LD100 of LPS (25 mg/kg). Anti–HMG-1 (Ab) or preimmune serum (0.2 ml per mouse, ip) was administered 30 min before LPS. Additional doses of preimmune (0.4 ml, ip) or anti–HMG-1 (0.4 ml, ip) were administered at 12 and 36 hours after LPS as indicated. (B) Delayed administration of anti–HMG-1 protects against LPS lethality in mice. Male Balb/C mice (20 to 23 g) were randomly grouped (seven mice per group) and treated with an LD100 of LPS. Anti–HMG-1 or preimmune serum (0.4 ml per mouse) was administered at 2, 24, and 36 hours after LPS. (C) Administration of rHMG-1 is lethal to mice. Recombinant HMG-1 was purified and LPS content determined by the Limulus Amoebocyte Lysate Test (Bio-Whittaker, Walkersville, Maryland). Purified rHMG-1 protein contained <2.5 ng of LPS per microgram of rHMG-1. Male Balb/C mice (20 to 23, 10 animals per group) were injected with a nonlethal dose of LPS (3.1 mg/kg, ip). Purified rHMG-1 protein was administered intraperitoneally in the doses indicated at 2, 16, 28, and 40 hours after LPS.

To investigate whether antibody treatment could be delayed until after administration of LPS, we injected anti–HMG-1 beginning 2 hours after LPS (followed by additional doses at 12 and 36 hours after LPS). This delayed treatment conferred significant protection against an LD100 of LPS (Fig. 2B). Preimmune serum–treated controls all developed lethargy, piloerection, and diarrhea before death, whereas anti–HMG-1–treated mice remained well groomed and active, had no diarrhea, and were viable. To clarify that anti–HMG-1 protected mice from LPS lethality, we purified the immunoglobulin G (IgG) fraction from anti–HMG-1 and administered it to mice exposed to an LD100 of LPS (11, 14). The highest dose of anti–HMG-1 IgG tested, 5 mg per mouse, conferred complete protection against an LD100 of LPS, whereas all control mice given comparable doses of rabbit IgG died (Table 1). Treatment with anti–HMG-1 IgG (2 mg per mouse) significantly reduced serum HMG-1 levels, whereas no reduction was observed after treatment with a lower dose of antibodies (0.5 mg per mouse) or with control IgG (5 mg per mouse). Antiserum against a chemically synthesized peptide corresponding to the first 12 amino acids of HMG-1 also significantly attenuated the lethality of endotoxemia in mice (15).

Table 1

Protection against LPS lethality by anti–HMG-1 IgG. Balb/C mice (male, 20 to 23 g, three to six mice per group) were injected intraperitoneally with IgG purified from anti–HMG-1 or control rabbit IgG 30 min before injection of an LD100 of LPS. All mice were then treated with additional doses of anti–HMG-1 IgG or control IgG at 12 and 24 hours after LPS. Serum HMG-1 levels were determined by immunoblots (under denaturing conditions) 14 hours after LPS challenge (n = 3 per group). ND, not determined.

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To determine if HMG-1 was toxic, we administered highly purified rHMG-1 to unanesthetized Balb/C mice (10 to 50 μg per mouse). Within 2 hours, the mice developed signs of endotoxemia, including lethargy, piloerection, and diarrhea. At higher doses (500 μg per mouse), three of five mice died at 18, 30, and 36 hours after rHMG-1 administration. Toxicity and lethality were not observed in control mice treated with a protein fraction purified fromE. coli transformed with a plasmid devoid of HMG-1 cDNA (9), indicating that the toxicity we observed was specific to HMG-1. To exclude the possibility that endotoxin contamination of HMG-1 preparations mediated lethality, we injected rHMG-1 into LPS-resistant mice. rHMG-1 (500 μg per mouse) was lethal within 16 hours both to C3H/HeJ (n = 4) and C3H/HeN (n = 3) mice, indicating that HMG-1 itself is toxic even in the absence of LPS signal transduction. When sublethal doses of rHMG-1 were injected into Balb/C mice together with sublethal doses of LPS, the combined challenge was lethal to 90% of the mice, as compared with 0% lethality in mice exposed to LPS or HMG-1 alone (Fig. 2C). Thus, HMG-1 itself mediates lethality in both LPS-sensitive and LPS-resistant mice.

Animal models of human sepsis, including the murine endotoxemia model used here, have inherent limitations (16). As an initial step in determining whether HMG-1 participates in the pathogenesis of human sepsis, we studied 8 normal subjects and 25 critically ill septic patients with bacteremia and sepsis-induced organ dysfunction. HMG-1 was not detectable in the serum of normal subjects, but significant levels were observed in critically ill patients with sepsis (Fig. 3), and these levels were higher in patients who succumbed as compared to patients with nonlethal infection.

Figure 3

Increased serum HMG-1 levels in human sepsis. Serum was obtained from 8 healthy subjects and 25 septic patients infected with Gram-positive [Bacillus fragilis(one patient), Enterococcus faecalis (one patient),Streptococcus pneumoniae (four patients), Listeria monocytogenes (one patient), or Staphylococcus aureus(two patients)], Gram-negative [E. coli (seven patients),Klebsiella pneumoniae (one patient), Acinetobacter calcoaceticus (one patient), Pseudomonas aeroginosa(one patient), Fusobacterium nucleatum (one patient),Citrobacter freundii (one patient)], or unidentified pathogens (five patients). Serum was fractionated by SDS-PAGE, and HMG-1 levels were determined by immunoblotting analysis with reference to standard curves of purified rHMG-1 diluted in normal human serum; the detection limit is ∼50 pg. *P < 0.05 versus normal. **P < 0.05 versus survivors.

HMG-1 is a highly conserved protein with >95% amino acid identity between rodent and human (17–20). It has previously been characterized as a nuclear protein that binds to cruciform DNA (21), and as a membrane-associated protein termed “amphoterin” that mediates neurite outgrowth (19,20). Extracellular HMG-1 interacts directly with plasminogen and tissue type plasminogen activator (tPA), which enhances plasmin generation at the cell surface; this system plays a role in extracellular proteolysis during cell invasion and tissue injury (19). In addition, HMG-1 has been suggested to bind to the receptor for advanced glycation end products (RAGE) (22).

As with other inflammatory mediators such as TNF and IL-1, there may be protective advantages of extracellular HMG-1 when released in nontoxic amounts. Macrophages release HMG-1 when exposed to the early, acute cytokines, indicating that HMG-1 is also positioned as a mediator of other inflammatory conditions associated with increased levels of TNF and IL-1 (for example, rheumatoid arthritis and inflammatory bowel disease). Indeed, in most inflammatory scenarios, LPS is probably not the primary stimulus for HMG-1 release; it seems more likely that TNF and IL-1 function as upstream regulators of HMG-1 release. The delayed kinetics of HMG-1 release suggest that serum HMG-1 levels may be a convenient marker of disease severity. Moreover, the observations that HMG-1 itself is toxic, and that anti–HMG-1 prevents LPS lethality, point to HMG-1 as a potential target for therapeutic intervention.

  • * To whom correspondence should be addressed. E-mail: hwang{at}picower.edu

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