Cutting Edge: HMG-1 as a Mediator of Acute
Lung Inflammation
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J Immunol 2000; 165:2950-2954; ;
doi: 10.4049/jimmunol.165.6.2950
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References
Edward Abraham, John Arcaroli, Aaron Carmody, Haichao
Wang and Kevin J. Tracey
●
Cutting Edge: HMG-1 as a Mediator of
Acute Lung Inflammation1
Edward Abraham,2* John Arcaroli,* Aaron Carmody,*
Haichao Wang,† and Kevin J. Tracey†
A
cute lung injury, called the acute respiratory distress
syndrome in its most severe clinical manifestation, affects ⬃150,000 patients per year in the U.S., with recent
mortality rates being ⬎30% (1–3). At present, there is no effective
treatment. Acute lung injury often develops after the onset of injury
or severe infection (4 – 6). The pathogenesis involves increased production of inflammatory mediators, such as TNF-␣ and IL-1␤, and is
characterized histologically by accumulation of large numbers of neutrophils and the development of interstitial edema (1–3, 6).
*Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado
Health Sciences Center, Denver, CO 80262; and †Laboratory of Biomedical Science,
North Shore University Hospital-New York University School of Medicine, Manhasset, NY 11030
High mobility group protein-1 (HMG-1)3 is a highly conserved
protein with ⬎95% amino acid identity between rodents and humans (7, 8). HMG-1 was initially characterized as a nonhistone
nuclear protein that binds to the narrow minor groove of AT sequence-rich B form DNA. It has been implicated in the regulation
of gene transcription and in stabilizing nucleosome formation (7–
11). HMG-1 also is present in a membrane associated form, termed
amphoterin, that mediates neurite outgrowth (8, 12). Amphoterin
can interact with macrophage cell surface receptors for advanced
glycation end products to enhance expression of tissue-type plasminogen activator (12–14). HMG-1 was recently identified as a
late mediator of endotoxin lethality (15). Circulating levels of
HMG-1 rose after the administration of endotoxin, and injection of
HMG-1 itself was lethal. Abs to HMG-1 attenuated mortality associated with endotoxemia, even when the Abs were administered
2 h after the onset of endotoxemia, when the TNF peak had already
occurred (15). Moreover, in patients with severe infection, increased serum HMG-1 levels correlated with nonsurvival (15).
Because endotoxin and proinflammatory cytokine release is important in the mediation of acute lung injury (1– 6), we reasoned
that HMG-1 might also be involved in the development and progression of this entity. The present experiments show that HMG-1
itself can cause an acute pulmonary inflammatory response, manifested by neutrophil accumulation, interstitial edema, and increased production of proinflammatory cytokines in the lungs. Anti-HMG-1 Abs attenuated endotoxin-induced lung injury, but not
the early release of TNF-␣ and IL-1␤, indicating that HMG-1 is a
late mediator of endotoxin-induced acute lung injury.
Materials and Methods
Mice
Male C3H/HeJ mice, 8 –12 wk of age, were purchased from The Jackson
Laboratory (Bar Harbor, ME), and BALB/c mice, 8 –12 wk of age, were
purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were
kept on a 12-h light/dark cycle with free access to food and water. All
experiments were conducted in accordance with institutional review boardapproved protocols.
Model of endotoxin exposure and anti-HMG-1 treatment
1
This work was supported in part by National Institutes of Health Grants HL50284
and HL62221 (to E.A.).
Methoxyfluorane-anesthetized BALB/c mice received 5 ␮g Escherichia
coli O111:B4 endotoxin (Sigma, St. Louis, MO) intratracheally (i.t.) in 50
␮l PBS. Control mice were given 50 ␮l PBS i.t. without LPS. In experiments using anti-HMG-1 Abs, mice were given 0.2 ml preimmune (control) or postimmune (anti-HMG-1) rabbit antiserum i.p. either 30 min before and 12 h after LPS administration or 2 and 12 h after LPS
administration. Rabbit anti-HMG-1 antiserum was assayed for specificity
2
Address correspondence and reprint requests to Dr. Edward Abraham, Division of
Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Box C272, 4200 East Ninth Avenue, Denver, CO 80262. E-mail address: [email protected]
3
Abbreviations used in this paper: HMG-1, high mobility group protein-1; MIP-2,
macrophage inflammatory protein-2; i.t., intratracheally; MPO, myeloperoxidase.
Received for publication May 31, 2000. Accepted for publication July 21, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Copyright © 2000 by The American Association of Immunologists
●
0022-1767/00/$02.00
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Acute inflammatory lung injury is often a delayed complication of critical illness and is associated with increased mortality. High mobility group-1 (HMG-1) protein, in addition to its
role as a transcriptional regulatory factor, has recently been
identified as a late mediator of endotoxin lethality. In the
present studies, HMG-1 given intratracheally produced acute
inflammatory injury to the lungs, with neutrophil accumulation, the development of lung edema, and increased pulmonary
production of IL-1␤, TNF-␣, and macrophage-inflammatory
protein-2. In endotoxin-induced acute lung inflammation, administration of anti-HMG-1 Abs either before or after endotoxin exposure decreased the migration of neutrophils to the
lungs as well as lung edema. These protective effects of antiHMG-1 were specific, because pulmonary levels of IL-1␤,
TNF-␣, or macrophage-inflammatory protein-2 were not decreased after therapy with anti-HMG-1. Together, these findings indicate that HMG-1 is a distal mediator of acute inflammatory lung injury. The Journal of Immunology, 2000, 165:
2950 –2954.
The Journal of Immunology
2951
and titer to HMG-1 by ELISA and immunoblotting, as previously described (15). The anti-HMG-1 Abs contained in the antiserum reacted specifically with HMG-1 and did not cross-react with LPS, other bacterial
proteins, TNF-␣, or IL-1␤. In these experiments, lungs were harvested 24 h
after i.t. administration of PBS with or without endotoxin as indicated.
HMG-1 exposure
FIGURE 1. HMG-1 increases lung neutrophil accumulation and edema
in C3H/HeJ mice. MPO concentrations were assayed in lungs from control
mice and from mice given 1, 10, or 100 ␮g HMG-1 i.t. 8 h (A) or 24 h (B)
previously. The relative increase in lung wet-dry ratios as compared with
control unmanipulated mice was determined in mice given 1, 10, or 100 ␮g
HMG-1 i.t. 8 h (A) or 24 h (B) previously. The increase in wet-dry ratio was
calculated by subtracting the mean value for control, unmanipulated animals from the value obtained in each HMG-1 treated mouse. ⴱ, p ⬍ 0.05,
ⴱⴱ, p ⬍ 0.01, and ⴱⴱⴱ, p ⬍ 0.001 vs control.
Methoxyfluorane-anesthetized C3H/HeJ mice received 1, 10, or 100 ␮g
HMG-1 i.t. in 50 ␮l sterile water. Control mice were given 50 ␮l sterile
water i.t. without HMG-1. As a control, 1, 10, or 100 ␮g of rat albumin
(Sigma) in 50 ␮l distilled water were given i.t., and no increases in lung
myeloperoxidase (MPO), wet-dry ratio, or cytokine levels compared with
control, unmanipulated mice were found 1, 8, or 24 h after such treatment.
Recombinant HMG-1 was prepared as previously described (15), and contained ⬍2.5 ng of LPS per ␮g of rHMG-1. Similar doses of HMG-1 have
been given i.p. in previous studies (15) examining the systemic effects of
this molecule. In the present experiments, lungs were harvested 8 or 24 h
after the i.t. injections.
MPO assay
MPO activity was assayed as reported previously (16). Excised lungs from
three to four mice per treatment group were frozen in liquid nitrogen,
weighed, and stored at ⫺86°C. Lungs were homogenized for 30 s in 1.5 ml
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FIGURE 2. HMG-1 increases proinflammatory cytokine concentrations
in the lungs. Lung homogenates were obtained from control, unmanipulated mice and from mice given 1, 10, or 100 ␮g HMG-1 i.t. 24 h previously. ⴱ, p ⬍ 0.05, ⴱⴱ, p ⬍ 0.01, and ⴱⴱⴱ, p ⬍ 0.001 vs control.
2952
20 mM potassium phosphate, pH 7.4, and centrifuged at 4°C for 30 min at
40,000 ⫻ g. The pellet was resuspended in 1.5 ml 50 mM potassium phosphate, pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide,
sonicated for 90 s, incubated at 60°C for 2 h, and centrifuged. The supernatant was assayed for peroxidase activity corrected to lung weight.
Wet-dry lung weight ratios
All mice used for lung wet-dry weight ratios were of identical ages. Lungs
were excised, rinsed briefly in PBS, blotted, and then weighed to obtain the
“wet” weight. Lungs were then dried in an oven at 80°C for 7 days to
obtain the “dry” weight.
Cytokine ELISA
Histochemistry
Control or HMG-1 mice were euthanized by cervical dislocation under
methoxyfluorane anesthesia 24 h after i.t. injections, and then the chest was
opened and the lung vascular bed was flushed by injecting 5 ml cold PBS
through the right ventricle of the heart. The lungs were gently infiltrated
through the trachea with 1% low melting point agarose (SeaKem, FMC
Bioproducts, Rockland, ME) at 42°C. The lungs were removed en bloc and
fixed in 4% paraformaldehyde, 0.23 M sucrose solution overnight. Tissue
was then embedded, and 5-␮m sections were prepared for staining with
Gill’s hematoxylin (Fisher, Springfield, NJ).
Statistical analysis
To limit variability and provide appropriate controls, for each experimental
condition, groups of animals were prepared and studied at the same time.
For each experimental condition, mice in all groups had the same birth date
and had been housed together. Separate groups of mice (n ⫽ 3 to 9 per
group) were used for MPO assays and cytokine analysis. Data are presented as mean ⫾ SEM for each experimental group. One way ANOVA
and the Tukey-Kramer multiple comparisons test was used for comparisons
between data groups. p ⬍ 0.05 was considered significant.
Results and Discussion
In previous experiments (15), high doses of HMG-1 (i.e., 500 ␮g)
were lethal to mice. Lower doses (10 to 50 ␮g/mouse) produced
signs of endotoxemia, including lethargy, piloerection, and diarrhea, but the organ-specific effects of HMG-1 were not specifically
examined. To determine whether HMG-1 might induce acute lung
injury, we administered HMG-1 i.t. to C3H/HeJ mice and then
determined lung MPO and wet-dry ratios 8 or 24 h later. Lung
neutrophil accumulation was increased in a dose-dependent manner 8 h after HMG-1 administration, starting with doses as low as
1 ␮g/mouse (Fig. 1A). Further increases in lung MPO were present
24 h after HMG-1 exposure (Fig. 1B). Lung edema, as measured
by wet-dry ratio, was significantly increased 8 and 24 h after
HMG-1 administration, compared with controls (Fig. 1). These
effects could not be attributed to the trace amount of endotoxin
coadministered with the HMG-1, because C3H/HeJ mice do not
respond to low doses of endotoxin.
Tissue levels of IL-1␤, TNF-␣, and MIP-2 were significantly
elevated in the lungs after administration of doses of HMG-1 as
low as 1 ␮g/mouse (Fig. 2). Histological examination of tissue
sections prepared from the lungs of animals treated with 100 ␮g
HMG-1 24 h previously revealed evidence of an acute diffuse inflammatory response, with accumulation of neutrophils in the interstitial and intraalveolar areas, interstitial edema, and protein exudation into the alveolar space (Fig. 3). These pathological
changes are typically observed in response to acute lung injury
mediated by endotoxin, TNF-␣, and other proinflammatory stimuli
(6, 17, 22–24). Because the doses of HMG-1 applied here fall
within the pathologically relevant range seen in sepsis patients or
endotoxemic mice (15), these findings provide direct evidence that
HMG-1 can participate in the mediation of acute lung injury.
A widely used model of acute lung injury is intratracheal administration of endotoxin (17). This model is characterized by a
pulmonary inflammatory response with neutrophil infiltration and
early increases in proinflammatory cytokines, including TNF-␣,
IL-1␤, and MIP-2. Treatment of mice with anti-HMG-1 Abs either
before or after endotoxin administration significantly decreased
endotoxin-induced neutrophil accumulation into the lungs (Figs.
4A and B). Further, passive immunization of mice with antiHMG-1 Abs before or after endotoxin exposure significantly attenuated the severity of lung edema produced by intratracheal instillation of endotoxin (Fig. 4). It was theoretically possible that
the protective effects of anti-HMG-1 were due to decreasing the
expression of TNF-␣, IL-1␤, and MIP-2 in the lung. However,
anti-HMG-1 treatment had no effect on endotoxin-induced increases in IL-1␤, TNF-␣, or MIP-2 protein concentrations (Fig. 5).
Thus, the protection by anti-HMG-1 in endotoxin-induced acute
lung injury is specific. These results demonstrate that HMG-1 is a
mediator of acute inflammatory lung injury.
HMG-1 has recently been identified as a late mediator of endotoxin lethality, because its systemic release during endotoxemia is
FIGURE 3. Effects of HMG-1 on lung histology. Sections from the lungs of a representative control, unmanipulated mouse (A) and from a mouse given
100 ␮g HMG-1 i.t. 24 h previously (B) are shown. The pulmonary histology in HMG-1-treated mice shows accumulations of interstitial and intraalveolar
neutrophils, interstitial edema, as well as leakage of fibrin and RBC into the alveolar space, consistent with acute inflammatory injury.
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After the lung vascular bed had been flushed by injecting 5 ml chilled (4°C)
PBS into the right ventricle, the lungs were homogenized for 30 s in lysis
buffer containing 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.6%
ipegal, 5 mM PMSF, 1 ␮g/ml leupeptin, 1 ␮g/ml aprotinin, 10 ␮g/ml
soybean trypsin inhibitor, and 1 ␮g/ml pepstatin. The homogenates were
centrifuged at 10,000 rpm at 4°C for 10 min, and the supernatants were
collected. Protein content of the supernatants was determined using the
bicinchoninic acid protein assay kit from Pierce Chemical Co. (Pittsburgh,
PA). Immunoreactive IL-1␤, TNF-␣, and macrophage-inflammatory protein-2 (MIP-2) were quantitated using commercially available ELISA kits
(R&D Systems, Minneapolis, MN). With these assays, the threshold of
sensitivity for IL-1␤ and MIP-2 is 3 pg/ml, and that for TNF-␣ is 10 pg/ml.
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The Journal of Immunology
2953
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FIGURE 5. Anti-HMG Abs do not affect endotoxin-induced increases
in lung proinflammatory cytokine levels. Lung homogenates were obtained
24 h after the administration of 5 ␮g LPS i.t. in mice pretreated i.p. with
either 0.2 ml preimmune rabbit serum (LPS) or anti-HMG-1 serum 30 min
before and 12 h after i.t. LPS. Endotoxin administration resulted in significant increases in pulmonary concentrations of MIP-2, IL-1␤, and TNF-␣
compared with control, unmanipulated mice. There were no statistically
significant differences in cytokine levels between endotoxin-treated mice
given preimmune or anti-HMG serum.
FIGURE 4. Anti-HMG-1 Abs decrease neutrophil accumulation and
lung edema in BALB/c mice treated with intratracheal endotoxin. Lung
MPO levels were assayed 24 h after the administration of 5 ␮g LPS i.t. in
mice treated i.p. with either 0.2 ml preimmune rabbit serum (LPS) or antiHMG-1 serum 30 min before and 12 h after i.t. LPS (A) or 2 and 12 h after
i.t. LPS (B). MPO levels from control, unmanipulated mice are also shown.
The relative increase in lung wet-dry ratios as compared with control unmanipulated mice was determined 24 h after the administration of 5 ␮g
LPS i.t. in mice treated i.p. with either 0.2 ml preimmune rabbit serum
(LPS) or anti-HMG-1 serum 30 min before and 12 h after i.t. LPS (A) or
2 and 12 h after i.t. LPS (B). ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.01 vs control.
delayed as compared with the rapid increase of the early proinflammatory cytokines, such as IL-1␤ and TNF-␣ (15). The present
experiments show that the delayed release of HMG-1 can participate in the downstream development of lung injury. This role of
HMG-1 in the pathogenesis of acute lung injury appears to be
distinct from any effects on earlier acting proinflammatory cytokines. In particular, despite the ameliorative effects of anti-HMG-1
2954
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Abs on the development of lung injury and neutrophil accumulation, endotoxin-induced increases in pulmonary concentrations of
IL-1␤, TNF-␣, and MIP-2 were not affected by inhibiting HMG-1.
Previous studies (18) demonstrated that proinflammatory cytokines, including IL-1␤ and TNF-␣, induce production of HMG-1.
The present findings are consistent with HMG-1 being a distal
inflammatory mediator, with delayed release after cellular exposure to endotoxin or, more likely, with release induced primarily
by proinflammatory cytokines, such as IL-1␤ and TNF-␣, the expression of which is rapidly increased by endotoxin.
Although anti-HMG-1 treatment significantly decreased lung
edema and neutrophil accumulation, such therapy did not completely ameliorate the development of lung injury as compared
with controls. These results indicate that whereas HMG-1 participates in endotoxin-induced lung injury, other mediators also are
involved. It is likely that IL-1␤, TNF-␣, and MIP-2 participate in
the early development of acute lung injury (3, 6, 16, 19 –21); the
levels of these mediators were increased in the lungs of endotoxintreated mice even when anti-HMG-1 was administered. Blockade
of each of these cytokines ameliorates endotoxin-induced lung
damage (17, 22). Instillation of IL-1␤, TNF-␣, or MIP-2 into the
lungs leads to neutrophil accumulation, interstitial edema, and histological changes consistent with inflammatory injury (23, 24).
These proinflammatory cytokines are present in bronchoalveolar
lavage specimens from animals or patients with acute lung injury
(17, 19 –25). The delayed kinetics of HMG-1 release, associated
with its contributory role in acute lung injury, point to HMG-1 as
a potential target for therapeutic intervention. However, because
inhibition of HMG-1 does not completely prevent inflammatory
injury to the lungs, it is likely that effective strategies for acute
lung injury should focus on inhibiting the pathological effects of
both early (e.g., TNF-␣ and IL-1␤) and late (e.g., HMG-1)
mediators.
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