Division of Surgical Research, Department of Surgery, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903
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ABSTRACT |
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The expression of the
hypoxia-responsive transcription factor hypoxia-inducible factor
(HIF)-1 during acute inflammation was investigated in experimental
wounds. HIF-1 mRNA was maximally expressed in wound cells 6 h
after injury. HIF-1
protein was detectable in wound cells 1 and 5 days after injury. Cells from 1-day-old wounds were not hypoxic, as
determined by lack of pimonidazole hydrochloride adduct formation.
Tumor necrosis factor (TNF)-
, but not interleukin-1
, increased
the HIF-1
protein content of cells isolated 1 and 5 days after
injury, and also of glycogen-elicited peritoneal cells, but not
HIF-1
mRNA. HIF-1
did not accumulate in TNF-
-treated HeLa,
NIH/3T3, NR8383, or RAW 264.7 cells. Nitric oxide from
S-nitrosoglutathione did not induce HIF-1
accumulation or
modulate the response to TNF-
. TNF-
did not increase oxygen consumption or result in the production of reactive oxygen
intermediates by day 1 wound cells. Vascular endothelial
growth factor mRNA in wound cells peaked 24 h after wounding.
HIF-1 expression in early wounds may contribute to the regulation of
inducible nitric oxide synthase and vascular endothelial growth factor,
two HIF-1-responsive genes intimately related to the process of repair.
inflammation; wound; hypoxia-inducible factor 1; tumor necrosis
factor-
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INTRODUCTION |
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HYPOXIA-INDUCIBLE
FACTOR 1 (HIF-1), a heterodimeric transcription
factor composed of the basic helix-loop-helix-PER-ARNT-SIM (PAS)-domain-containing proteins HIF-1 and HIF-1
, was originally identified as an activator of the hypoxia-responsive element of the
erythropoietin gene enhancer (31, 34). Since then, HIF-1 has been shown to regulate the expression of other hypoxia-responsive genes including the inducible form of nitric oxide (NO) synthase (iNOS), vascular endothelial growth factor (VEGF), glucose
transporter-1, and several glycolytic enzymes (31).
Although the current paradigm supports HIF-1 as a prominent regulator
of the genetic response to hypoxia, it has recently been shown that
certain proinflammatory cytokines are able to activate HIF-1 in
selected cell types in culture (10, 33). These
observations led to the proposal that HIF-1 may be involved in the
regulation of gene expression during inflammation (10). To
test this hypothesis in vivo, the work reported here examined the
expression of HIF-1 in experimental wounds. The results to be shown
demonstrate the prominent presence of HIF-1 mRNA in wound cells
during the initial 24 h after wounding and of HIF-1
protein in those isolated from the wound 1 and 5 days after injury. Interestingly, no evidence for hypoxia was detected in the wound at the
early time point. Additional work demonstrated that tumor necrosis
factor (TNF)-
, but not interleukin (IL)-1
or NO, induces HIF-1
protein expression in primary inflammatory cells.
The temporal correlation between the expression of HIF-1 and that of its target genes iNOS and VEGF supports a role for HIF-1 in the regulation of gene expression in acute inflammation. HIF-1 induction in early inflammation, in turn, appears to be mediated by cytokines rather than by hypoxia.
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METHODS |
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Animals and cells. Male Fischer rats (150-200 g; VAF Plus; Charles River Laboratories, Wilmington, MA) and B6D2F1 mice (Taconic, Germantown, NY) were used in all experiments. Animals were kept in barrier cages and allowed chow and water consumption ad libitum. Brown University/Rhode Island Hospital veterinary personnel monitored animal welfare. Peritoneal inflammatory cells were harvested from rats 12-16 h after the intraperitoneal injection of 10 ml of 1% oyster glycogen (Sigma Chemical, St. Louis, MO). The cells obtained were >95% polymorphonuclear leukocytes. Cell lines used in these experiments were obtained from the American Type Culture Collection (Manassas, VA).
Wound model. The subcutaneously implanted polyvinyl alcohol (PVA) sponge wound model has been described previously (2). The infiltration of the sponge material by inflammatory cells and the subsequent accumulation of collagen in the sponges models precisely for soft tissue wounds (2). Cells were retrieved from the sponges at specified times after wounding with the method described (2) and were used immediately or after overnight culture. Liver and kidney samples were obtained from nonwounded animals and from wounded animals one day after wounding.
Cell culture.
In experiments requiring cell culture, cells were suspended in RPMI
1640 medium (Life Technologies, Grand Island, NY) supplemented with 1%
fetal bovine serum (HyClone, Logan, UT) and antibiotics and dispensed
into Permanox (Nunc, Naperville, IL) 60-mm culture plates. The plates
were placed in modular incubator chambers (Billups-Rothenberg, Del Mar,
CA) equilibrated with room air or with certified gas containing 1%
O2, 6% CO2, and 93% N2. The
O2 tension in the culture medium was monitored with an
O2 electrode as described (15). When so
indicated, recombinant rat TNF- (BioSource International, Camarillo,
CA) or recombinant human IL-1
(R&D Systems, Minneapolis, MN) was
added to the cultures at 50 ng/ml.
Detection of pimonidazole HCl adducts. Pimonidazole hydrochloride (hydroxyprobe-1; Natural Pharmacia International, Research Triangle Park, NC) was administered intraperitoneally at 70 mg/kg 3 h before animals were killed. Cells harvested from the wounds were fixed with 1% paraformaldehyde and centrifuged onto glass slides (Cytospin; Shandon, Pittsburgh, PA). Endogenous peroxidase activity was quenched by immersion in PBS containing 3% H2O2, and the cells were permeabilized with PBS-0.2% Brij 35. Blocking was performed with 5% normal goat serum in PBS-0.2% Brij 35, and pimonidazole HCl adducts were detected with hydroxyprobe-1 monoclonal antibody (Natural Pharmacia International) antibody diluted 1:50 in PBS-0.2% Brij 35 and incubated overnight at 4°C. Control slides were similarly treated with MOPC 21 (IgG1; Sigma). After a washing step, antibody binding was detected with the Universal Elite Avidin-Biotin system (Vector Laboratories, Burlingame, CA), and peroxidase activity was visualized with Vector VIP (Vector Laboratories).
Northern blot analysis.
The probe to detect rat HIF-1 mRNA (GenBank accession no. Y09507)
was generously provided by Dr. Thomas Kietzmann, Institut fur Biochimie
und Molekulare Zellbiologie, Goettingen, Germany (13), in
a plasmid (pCRII) containing an 800-bp fragment of HIF-1
. The insert
was excised by EcoRI digestion. For 18S ribosomal RNA, a
pUC830 plasmid containing the mouse 18S ribosomal cDNA was obtained
from the American Type Culture Collection. Sph1 and BamH1 digestion of the plasmid yielded a 752-bp cDNA insert
that detects 18S ribosomal RNA in rat and mouse cells. Northern blot analysis was performed exactly as described (16). Probes
were radiolabeled with [32P]dCTP by random priming
(Pharmacia). Total RNA was isolated from 15-20 × 106 cells or from 1 g of kidney or liver with
Ultraspec (Biotecx, Houston, TX), fractionated by 1% agarose-0.66%
formaldehyde gel electrophoresis, and transferred to a nylon membrane
and immobilized by ultraviolet cross-linking. After hybridization and
autoradiography, mRNA levels were quantitated by densitometry with NIH
Image version 1.6.2.
Semiquantitative RT-PCR.
RT-PCR was performed with target mRNA-to-18S ratios with the Quantum
RNA 18S internal standard method, following the manufacturer's instructions (manual version 9907, Ambion, Austin, TX). Primer pairs
were as follows: HIF-1, 5'-TGCTCATCAGTTGCCACTTCC-3',
5'-CGCTGTGTGTTTTGTTCTTTACCC-3'; TNF-
, 5'-GGTTCTCTTCAAGGGACAAGGC-3',
5'-GGGCTCTGAGGAGTAGACGATAAAG-3'; IL-1
,
5'-TCCATGAGCTTTGTACAAGG-3'; 5'-GGTGCTGATGTACCAGTTGG-3'; and VEGF,
5'-CGACAGAAGGGGAGCAGAAAG-3', 5'-GCAAGTACGTTCGTTTAACTC-3'.
Wound fluid analyses.
TNF- and IL-1 bioactivities in wound fluids were determined with an
L-929 cytotoxicity assay and a thymocyte proliferation assay,
respectively, as previously reported (17).
Western blot analyses.
Western blot analysis of HIF-1 was performed as described
(16). Cell lysates were fractionated in a 7.5%
SDS-polyacrylamide gel and transferred to a nitrocellulose membrane.
Membranes were blocked in PBS, 5% nonfat dry milk, and 0.05% Tween
20. An anti-HIF-1
antibody (Novus Biologicals, Littleton, CO) was
used for HIF-1
detection at a dilution of 1:1,000 in blocking
buffer. Blots were washed and incubated with horseradish
peroxidase-conjugated rabbit anti-mouse IgG (Amersham Pharmacia
Biotech, Piscataway, NJ) diluted 1:2,000 in blocking buffer and were
then detected by chemiluminescence with enhanced chemiluminescence
reagent (ECL; Amersham Pharmacia Biotech).
Cellular respiration.
For measurements of O2 consumption, day 1 wound
cells (6 × 106 cells/ml) were dispensed into the
chamber of an Instech 203 (Instech, Plymouth Meeting, PA)
O2 uptake system. Cells were stimulated (or not) with 50 ng/ml of rat TNF-. O2 content of the medium was
continuously monitored and recorded as described (20).
Reactive O2 species detection. The detection of reactive O2 species formation by lucigenin chemiluminescence was performed exactly as described (20).
Data presentation. Experiments were repeated at least three times. In all RT-PCR experiments, RNA was extracted from cells harvested from at least three animals and pooled. Results shown are means ± SD of normalized densitometric measurements from at least three independent experiments. Cytokine bioactivities in wound fluids were determined in samples from at least 6 animals/time point and are shown as means ± SD. For Northern and Western blot analysis and for pimonidazole adduct staining, the results shown are from a representative experiment.
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RESULTS |
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HIF-1 mRNA and protein in inflammatory cells.
The subcutaneous implantation of PVA sponges resulted in the rapid
infiltration of the sponge material by circulating polymorphonuclear leukocytes and monocytes (19). Maximal HIF-1
mRNA was
detected by Northern blot analysis in cells isolated from rat wounds
24 h after injury, a time when >90% of cells present in the
wound were neutrophils (19) (Fig.
1). The high-level expression of HIF-1
mRNA in early wound cells did not appear to result from systemic
hypoxia or anesthesia because no increase in HIF-1
mRNA was detected
in liver or kidney samples from the same animals. Murine wound cells
harvested 24 h after wounding also showed substantial HIF-1
mRNA.
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Hypoxia is not detectable in day 1 wound cells.
Hypoxia is the canonical regulator and activator of HIF-1
(31). Although wounds are known to be relatively hypoxic,
the expression of HIF-1
in wound cells appeared to occur earlier than the reported development of hypoxia in wounds (see
DISCUSSION). Wound cells were harvested from animals
previously injected with the hypoxia marker pimonidazole hydrochloride
(24). This compound forms intracellular protein adducts
under conditions of O2 tension of <10 Torr and has been
used by others (9) to establish the time course of hypoxia
in wounds. As shown in Fig.
4A, cells harvested from 24-h
wounds failed to stain for pimonidazole adducts. In contrast, cells
harvested from 5-day-old wounds demonstrated substantial specific
staining (Fig. 4B), thus confirming the hypoxic status of
the wound at that time.
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Nonhypoxic induction of HIF-1 in primary inflammatory cells.
Recent reports (10, 33) have indicated that TNF-
and
IL-1 are capable of inducing HIF-1
protein accumulation in selected cell types. Figure 5 reports TNF-
and
IL-1 mRNA abundance in wound cells and bioactivities in cell-free
fluids harvested from implanted PVA sponges 6 h to 10 days after
injury. The results shown in the figure illustrate that maximal
expression for both cytokines in this wound model occurs within 12 h of injury.
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Potential gene regulation by HIF-1 in early wounds.
iNOS and VEGF are genes known to be induced by HIF-1 and to be involved
in the regulation of inflammation and wound healing (14).
Previous work from this laboratory (25) demonstrated the
expression of iNOS in the wound model here employed correlates temporally with the enhanced expression of HIF-1 mRNA. Shown in Fig.
8 are results demonstrating that
peak expression of VEGF mRNA in the wound was detected 12 h after wounding, that is, 6 h after maximal HIF-1
mRNA
accumulation and before the establishment of significant hypoxia.
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DISCUSSION |
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HIF-1 was originally described by Wang and Semenza
(34) as a heterodimeric nuclear factor composed of
HIF-1 and HIF-1
(ARNT) (34). HIF-1
has been found
to be constitutively expressed in most cells. HIF-1
, in turn, has
been shown to stabilize and accumulate in cells during hypoxia, mainly
through inhibition of its degradation by the ubiquitin-proteasome
system (28). The HIF-1
-HIF-1
dimer translocates to
the nucleus where it binds to a specific recognition motif, the hypoxia
response element, in a variety of hypoxia-responsive genes
(31).
Interest in the expression of HIF-1 during the early inflammatory phase
of wound healing stemmed from the characterization of the temporal
expression of iNOS in wound cells in this laboratory (26).
Those experiments demonstrated that iNOS is prominently expressed in
wounds only during the first 48-72 h after injury. In this
connection, additional results from this laboratory (1) first demonstrated iNOS to be a hypoxia-responsive gene. Others subsequently identified a hypoxia response element in the iNOS promoter
(18) and demonstrated a requirement for such an
HIF-1-binding domain for enhanced iNOS promoter activity in hypoxic
endothelial cells (23). Based, then, on the hypothesis
that HIF-1 could participate in the regulation of iNOS expression in
healing wounds, experiments determined the expression of HIF-1 mRNA
and protein in wound cells over time. Findings here reported
demonstrate the prominent expression of HIF-1
mRNA during the
initial 24-48 h of acute inflammation in wounds and that of
HIF-1
protein in cells harvested from the wound 1 day after
wounding. The close temporal correlation between HIF-1
and iNOS
expression in early wounds supports the contention that the early
expression of HIF-1 in wounds may promote the expression of iNOS in
wound cells.
The results shown in Figs. 1 and 2 demonstrate that HIF-1 mRNA is
acquired by inflammatory cells after their arrival at the wound.
Because wounds have been shown to be sites of relative hypoxia, it
appeared reasonable to propose that it is local wound hypoxia that
induces HIF-1
expression by the inflammatory cells upon migration
into the wound space (11, 22, 27, 32). This, however, does
not appear to be so for cells harvested 1 day after wounding. In this
connection, Jiang et al. (12) demonstrated that
half-maximal activation of HIF-1 in cultured cells requires an
O2 tension of ~10-14 mmHg and that maximal
activation occurs at O2 tensions <3 mm Hg. Time-course
studies of O2 tension in early wounds reported by
Niinikoski and Hunt (21) showed it to reach a minimum of
15-20 mmHg by days 4-5 after injury. Most recently, Haroon et al. (9) concluded that substantial
hypoxia does not develop in cutaneous wounds until 48 h after
injury and that maximal hypoxia (
10mm Hg) occurs ~4 days after
wounding. The results in Fig. 4, obtained with immunodetection of
pimonidazole adducts in wound cells as a measure of in vivo
O2 availability, confirmed those findings by failing to
detect significant adduct formation in day 1 wound cells. It
appears, then, that the O2 tensions present in very early
wounds should not suffice to induce HIF-1 in inflammatory cells. In
this connection, Elson et al. (6) recently reported the
localization of HIF-1
mRNA by in situ hybridization to basal
keratinocytes in cutaneous wounds in mice. The close proximity of the
HIF- 1
-positive cells to capillaries in their specimens prompted
the authors to propose a nonhypoxic mechanism of HIF-1
induction in
the cells.
Although most information regarding HIF-1 regulation concerns its
activation by hypoxia, Hellwig-Bürgel et al. (10)
recently demonstrated that IL-1 and TNF-
could induce moderate
HIF-1 DNA binding activity in human HepG2 cells, whereas only IL-1
was capable of increasing cellular HIF-1
protein content. Further work from the same laboratory (3) showed a modest increase in HIF-1
protein in whole cell and nuclear extracts of human tubular
epithelium kidney cells treated with IL-1
, as well as increased
HIF-1 DNA binding in nuclear preparations from those cells. More
recently, Thorton et al. (33) reported increased HIF-1
mRNA in human synovial fibroblasts treated in vitro with IL-1
,
TNF-
, or lipopolysaccharide.
The results just shown indicate that maximal TNF- and IL-1
bioactivities in the wound occur within 12 h of injury. The early expression of these cytokines in the wound agrees with the findings of
others (7, 8). Taken together with observations summarized in the preceding paragraph, then, it appeared appropriate to test whether these proinflammatory cytokines could induce HIF-1 in primary
inflammatory cells. The results shown in Fig. 6 demonstrate that
TNF-
, but not IL-1
, induces HIF-1
protein accumulation in
cultured day 1 and day 5 wound cells. The
capacity of TNF-
to enhance HIF-1
accumulation was found not to
be restricted to wound cells. Inflammatory peritoneal neutrophils also
showed increased HIF-1
content after TNF-
stimulation (Fig. 7).
In interesting contrast, TNF-
failed to increase HIF-1
in a
variety of cell lines, including rat (NR8383) and mouse (RAW 264.7)
macrophage cell lines.
The mechanism(s) by which TNF- determined HIF-1
accumulation in
the cells was investigated. Of concern was the possibility that TNF-
could increase O2 consumption by cultured cells and result
in local hypoxia in the cultures despite the use of
O2-permeable Permanox cultureware. This possibility was
negated by experiments demonstrating that the rate of O2
consumption by the cells was not modified by the addition of TNF-
to
the cultures.
The O2 sensor used by cells to detect decreases in
environmental O2 tension and to trigger HIF-1
stabilization has not been identified. Most recently, Chandel et al.
(4, 5) proposed that the production of reactive
O2 species by mitochondria during hypoxia is both required
and sufficient to initiate HIF-1
stabilization. TNF-
did not, at
the concentrations tested in the current experiments, result in
reactive O2 intermediate production by cultured wound cells
as measured by the sensitive lucigenin chemiluminescence method.
Recent reports (29, 30) indicated that NO is capable of
inducing HIF-1 accumulation in kidney tubular epithelial cells. Given the great abundance of NO in very early wounds (26),
the possibility that NO could induce HIF-1
accumulation in cultured wound cells or potentiate the effects of TNF-
was investigated. As
mentioned above, neither case proved to be true for primary inflammatory cells.
In contrast to findings with early wound cells, cells harvested 5 days after wounding contained readily detectable pimonidazole adducts,
indicating that the O2 tension in the wound at that time was <10 mmHg. The finding of HIF-1 protein, albeit at a reduced level, in cells harvested 5 days after wounding, therefore likely was a
result of the hypoxic status of the wound at the time.
Attempts to identify the specific cell types expressing HIF-1
in early wounds by immunostaining were not successful. Staining with
anti-HIF-1
antibodies purchased from Novus Biologicals (NB100-105 and NB100-123), BD Transduction Laboratories (clone 54; San Diego, CA), Neomarkers (clones OZ12 and OZ15; Fremont, CA;), Santa Cruz Biotechnology (N18 and C-19; Santa Cruz, CA), or with those generously provided by other investigators gave inconsistent results. It is
likely, however, that it is neutrophils that express HIF-1
in early
inflammation. This is so because these cells constitute the vast
majority of cells in the early inflammatory infiltrate in the
experimental wound here used and because other inflammatory neutrophils, like those elicited to the peritoneal cavity by oyster glycogen (Fig. 7), respond to TNF-
exposure with HIF-1
accumulation. Moreover, because 49% of neutrophils harvested from the
experimental wounds 1 day after injury stain positive for intracellular
TNF-
(Albina, unpublished observation), it could be proposed that
neutrophil TNF-
initiates a positive autocrine loop that results in
the accumulation of HIF-1
in these cells.
In conclusion, then, the present results demonstrate expression of
HIF-1 mRNA and protein in primary inflammatory cells harvested from
very early wounds at a time that precedes the establishment of
significant hypoxia in the wound. They also demonstrate that TNF-
, a
cytokine prominently expressed in the wound at that time, can induce
HIF-1
protein accumulation in primary inflammatory cells but does
not modify HIF-1
mRNA. In this regard, the mechanism for the
enhanced expression of HIF-1 mRNA in the early wound remains unexplained. In addition, findings show persistent HIF-1
protein expression in wound cells 5 days after injury, a time at which wound
hypoxia could contribute to HIF-1
stabilization. In terms of a
functional role for HIF-1 in early inflammation, the results shown here
and others reported previously suggest that this nuclear factor is
involved in the regulation of VEGF and iNOS expression at the
inflammatory site.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of General Medical Sciences Grant GM-42859 (to J. E. Albina), the Anita Allard Memorial Fund, and allocations to the Department of Surgery by Rhode Island Hospital.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. E. Albina, Dept. of Surgery, Rhode Island Hospital, 593 Eddy St., Providence, RI 02903 (E-mail: Jorge_Albina{at}Brown.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 May 2001; accepted in final form 15 August 2001.
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