HIF-1 expression in healing wounds: HIF-1alpha induction in primary inflammatory cells by TNF-alpha

Jorge E. Albina, Balduino Mastrofrancesco, Joseph A. Vessella, Claudine A. Louis, William L. Henry Jr., and Jonathan S. Reichner

Division of Surgical Research, Department of Surgery, Rhode Island Hospital and Brown Medical School, Providence, Rhode Island 02903


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The expression of the hypoxia-responsive transcription factor hypoxia-inducible factor (HIF)-1 during acute inflammation was investigated in experimental wounds. HIF-1alpha mRNA was maximally expressed in wound cells 6 h after injury. HIF-1alpha 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)-alpha , but not interleukin-1beta , increased the HIF-1alpha protein content of cells isolated 1 and 5 days after injury, and also of glycogen-elicited peritoneal cells, but not HIF-1alpha mRNA. HIF-1alpha did not accumulate in TNF-alpha -treated HeLa, NIH/3T3, NR8383, or RAW 264.7 cells. Nitric oxide from S-nitrosoglutathione did not induce HIF-1alpha accumulation or modulate the response to TNF-alpha . TNF-alpha 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-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha and HIF-1beta , 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-1alpha mRNA in wound cells during the initial 24 h after wounding and of HIF-1alpha 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)-alpha , but not interleukin (IL)-1beta or NO, induces HIF-1alpha 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha (BioSource International, Camarillo, CA) or recombinant human IL-1beta (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-1alpha 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-1alpha . 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-1alpha , 5'-TGCTCATCAGTTGCCACTTCC-3', 5'-CGCTGTGTGTTTTGTTCTTTACCC-3'; TNF-alpha , 5'-GGTTCTCTTCAAGGGACAAGGC-3', 5'-GGGCTCTGAGGAGTAGACGATAAAG-3'; IL-1beta , 5'-TCCATGAGCTTTGTACAAGG-3'; 5'-GGTGCTGATGTACCAGTTGG-3'; and VEGF, 5'-CGACAGAAGGGGAGCAGAAAG-3', 5'-GCAAGTACGTTCGTTTAACTC-3'.

Results are reported as an "inflammatory index." This index represents the ratio between 18S-normalized mRNA in wound cells and that in circulating leukocytes [(wound cell mRNA/18S)/(circulating white blood cell mRNA/18S)].

Wound fluid analyses. TNF-alpha 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-1alpha 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-1alpha antibody (Novus Biologicals, Littleton, CO) was used for HIF-1alpha 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-alpha . 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HIF-1alpha 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-1alpha 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-1alpha mRNA in early wound cells did not appear to result from systemic hypoxia or anesthesia because no increase in HIF-1alpha mRNA was detected in liver or kidney samples from the same animals. Murine wound cells harvested 24 h after wounding also showed substantial HIF-1alpha mRNA.


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Fig. 1.   Evidence that hypoxia-inducible factor (HIF)-1alpha mRNA accumulates in early wound cells. Northern blot analysis shows HIF-1alpha mRNA expression in rat wound cells harvested 1-10 days after injury as well as in liver and kidney sampled from nonwounded animals (0) and animals on day 1 postinjury (1). Rel OD, relative optical density. Wound cells harvested from mice 1 day after injury also contained abundant HIF-1alpha mRNA.

A more detailed temporal analysis of the expression of HIF-1alpha mRNA in wound cells was performed with semiquantitative RT-PCR. As indicated in the preceding paragraph, cells infiltrating the wound within the initial 24-48 h after injury are mostly blood-derived polymorphonuclear leukocytes. HIF-1alpha mRNA found in early wound cells could reflect either the arrival at the wound of circulating cells containing high-level message or the induction of HIF-1alpha mRNA in the cells on their entry into the wound.

To distinguish between these two possibilities, HIF-1alpha message was determined in circulating white blood cells from wounded animals and in wound cells that were normalized in each case for 18S abundance and then expressed as an inflammatory index [(wound cell HIF-1alpha mRNA/18S)/(circulating white blood cell HIF-1alpha mRNA/18S)]. Results of those calculations, shown in Fig. 2, demonstrate that arrival at the inflammatory site results in a marked increase in cellular mRNA for HIF-1alpha . Wound cells harvested 6 h after injury were found to contain 25 times more HIF-1alpha mRNA than circulating leukocytes from wounded animals. The message content of the cells declined thereafter so that by 24 h, message abundance was the same as in cells harvested 3 h after wounding but was still greater than 8 times higher than that in circulating white blood cells.


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Fig. 2.   Temporal analysis of HIF-1alpha mRNA expression in rat wound cells. Circulating white blood cells and wound cells harvested 3 h to 2 days after wounding were analyzed by semiquantitative RT-PCR for HIF-1alpha mRNA. Data are shown as inflammatory index, which represents the ratio between 18S-normalized HIF-1alpha mRNA in wound cells and that in circulating leukocytes [(wound cell HIF-1alpha mRNA/18S)/(circulating white blood cell HIF-1alpha mRNA/18S)].

Figure 3 shows the results of Western blot analysis of wound cells harvested 1 and 5 days after injury with anti-HIF-1alpha antibody. As shown in the figure, the antibody detected a triplet with an approximate molecular mass of 110 kDa, which is slightly less than that of HIF-1alpha in HeLa cells. Cells isolated from 5-day-old wounds contained ~30% less HIF-1alpha protein than those harvested 1 day after injury. Figure 3 also shows that HIF-1alpha content of day 1 wound cells could be further upmodulated in vitro by culture in hypoxic (1% O2) conditions and decreased by culture in room air.


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Fig. 3.   Western blot analysis of HIF-1alpha protein expression in rat wound cells harvested 1 (lane 1) and 5 (lane 2) days after injury. Wound cells harvested 1 day after injury also were cultured for 6 h in normoxic culture medium (lane 3) or in culture medium equilibrated with 1% O2 (lane 4) before cell lysis and Western analysis. Extracts from HeLa cells cultured in 1% O2 for 6 h were analyzed as positive controls (lane 5). No. at right, molecular mass.

Hypoxia is not detectable in day 1 wound cells. Hypoxia is the canonical regulator and activator of HIF-1alpha (31). Although wounds are known to be relatively hypoxic, the expression of HIF-1alpha 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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Fig. 4.   Wound cells harvested 1 day postinjury from pimonidazole HCl-injected rats do not demonstrate pimonidazole adduct staining (A). Cells harvested 5 days after wounding, in contrast, stain prominently for these compounds (B; arrows), thus demonstrating local hypoxia. As previously reported (25), cells found in 1-day-old wounds are >90% polymorphonuclear leukocytes, whereas those retrieved on day 5 are >80% macrophages. Bar, 40 µM.

Nonhypoxic induction of HIF-1alpha in primary inflammatory cells. Recent reports (10, 33) have indicated that TNF-alpha and IL-1 are capable of inducing HIF-1alpha protein accumulation in selected cell types. Figure 5 reports TNF-alpha 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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Fig. 5.   Temporal expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta mRNA and bioactivity in experimental wounds in rats. Cytokine mRNA was determined by semiquantitative RT-PCR in cells harvested 3 h to 10 days after injury. Data are shown as inflammatory index (see text and legend to Fig. 2). Cytokine bioactivity in cell-free wound fluids harvested from the same wounds was determined as described in METHODS.

To determine whether TNF-alpha and/or IL-1 could regulate HIF-1alpha in primary inflammatory cells, cells isolated from the wound 24 h after injury were cultured overnight in room air with 6% CO2 to allow HIF-1alpha protein content to decrease to baseline levels and were then exposed (or not) to hypoxia, TNF-alpha , or IL-1beta for up to 6 h. Hypoxia and TNF-alpha , but not IL-1beta , were effective in increasing the HIF-1alpha content of the cells (Fig. 6). Neither hypoxia nor TNF-alpha modulated the expression of HIF-1alpha mRNA in cultures of up to 6 h (data not shown). In identical experiments, TNF-alpha and hypoxia, but not IL-1beta , induced HIF-1alpha accumulation in wound cells harvested 5 days after injury (>85% macrophages; data not shown). Moreover, TNF-alpha and hypoxia increased the HIF-1alpha protein content of inflammatory neutrophils obtained from the peritoneal cavity of glycogen-injected rats (Fig. 7). In contrast, species-specific recombinant TNF-alpha failed to induce the accumulation of HIF-1alpha in a variety of human, rat, and murine cell lines (HeLa, NR8383, NIH/3T3, and RAW 264.7; data not shown).


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Fig. 6.   TNF-alpha induces HIF-1alpha accumulation in wound cells harvested 1 day after wounding. Western blot analysis shows HIF-1alpha protein expression in cultured day 1 wound cells exposed to hypoxic (equilibrated with 1% O2) culture medium or to normoxic medium containing 50 ng/ml of rat TNF-alpha or IL-1beta for indicated times. nd, Not determined.



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Fig. 7.   Western blot analysis of HIF-1alpha protein expression in peritoneal inflammatory cells cultured in room air (lane 1), 1% O2 (lane 2), room air + TNF-alpha (50 ng/ml, lane 3), or IL-1beta (50 ng/ml, lane 4) for 6 h immediately after harvest from the peritoneal cavity.

The addition of TNF-alpha to cultured day 1 wound cells did not change their respiratory rate as measured by the continuous monitoring of O2 consumption by the cells (data not shown). Thus the induction of HIF-1alpha accumulation in wound cells during their in vitro stimulation with TNF-alpha did not result from O2 depletion of the culture medium.

It has been proposed that HIF-1alpha is induced by reactive O2 intermediate production during hypoxia (4, 5). Arguing against a role for O2 radicals in the induction of HIF-1alpha by TNF-alpha , the addition of 50 or 100 ng/ml of this cytokine did not induce lucigenin-dependent chemiluminescence in cultured day 1 wound cells (data not shown).

Additional experiments investigated the potential regulation of HIF-1alpha expression by NO. These experiments were performed because it was recently reported that NO, as derived from the NO-donor S-nitrosoglutathione (GSNO), increases the HIF-1alpha content of cultured kidney cells (29, 30) and because maximal expression of iNOS and accumulation of NO in wounds occurs within 24 h of wounding (25). Addition of GSNO (1 mM) to day 1 wound cell cultures containing (or not) TNF-alpha (50 ng/ml) did not, however, change the kinetics or magnitude of HIF-1alpha protein accumulation (data not shown).

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-1alpha 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-1alpha mRNA accumulation and before the establishment of significant hypoxia.


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Fig. 8.   Vascular endothelial growth factor (VEGF) mRNA expression in wound cells. Wound cells harvested 3 h to 10 days after injury were analyzed by semiquantitative RT-PCR for VEGF mRNA. Data are expressed as inflammatory index (see text and legend to Fig. 2).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

HIF-1 was originally described by Wang and Semenza (34) as a heterodimeric nuclear factor composed of HIF-1alpha and HIF-1beta (ARNT) (34). HIF-1beta has been found to be constitutively expressed in most cells. HIF-1alpha , 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-1alpha -HIF-1beta 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-1alpha mRNA and protein in wound cells over time. Findings here reported demonstrate the prominent expression of HIF-1alpha mRNA during the initial 24-48 h of acute inflammation in wounds and that of HIF-1alpha protein in cells harvested from the wound 1 day after wounding. The close temporal correlation between HIF-1alpha 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-1alpha 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-1alpha 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-1alpha mRNA by in situ hybridization to basal keratinocytes in cutaneous wounds in mice. The close proximity of the HIF- 1alpha -positive cells to capillaries in their specimens prompted the authors to propose a nonhypoxic mechanism of HIF-1alpha 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-1beta and TNF-alpha could induce moderate HIF-1 DNA binding activity in human HepG2 cells, whereas only IL-1beta was capable of increasing cellular HIF-1alpha protein content. Further work from the same laboratory (3) showed a modest increase in HIF-1alpha protein in whole cell and nuclear extracts of human tubular epithelium kidney cells treated with IL-1beta , 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-1beta , TNF-alpha , or lipopolysaccharide.

The results just shown indicate that maximal TNF-alpha 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-alpha , but not IL-1beta , induces HIF-1alpha protein accumulation in cultured day 1 and day 5 wound cells. The capacity of TNF-alpha to enhance HIF-1alpha accumulation was found not to be restricted to wound cells. Inflammatory peritoneal neutrophils also showed increased HIF-1alpha content after TNF-alpha stimulation (Fig. 7). In interesting contrast, TNF-alpha failed to increase HIF-1alpha in a variety of cell lines, including rat (NR8383) and mouse (RAW 264.7) macrophage cell lines.

The mechanism(s) by which TNF-alpha determined HIF-1alpha accumulation in the cells was investigated. Of concern was the possibility that TNF-alpha 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-alpha to the cultures.

The O2 sensor used by cells to detect decreases in environmental O2 tension and to trigger HIF-1alpha 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-1alpha stabilization. TNF-alpha 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-1alpha accumulation in kidney tubular epithelial cells. Given the great abundance of NO in very early wounds (26), the possibility that NO could induce HIF-1alpha accumulation in cultured wound cells or potentiate the effects of TNF-alpha 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-1alpha 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-1alpha in early wounds by immunostaining were not successful. Staining with anti-HIF-1alpha 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-1alpha 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-alpha exposure with HIF-1alpha accumulation. Moreover, because 49% of neutrophils harvested from the experimental wounds 1 day after injury stain positive for intracellular TNF-alpha (Albina, unpublished observation), it could be proposed that neutrophil TNF-alpha initiates a positive autocrine loop that results in the accumulation of HIF-1alpha in these cells.

In conclusion, then, the present results demonstrate expression of HIF-1alpha 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-alpha , a cytokine prominently expressed in the wound at that time, can induce HIF-1alpha protein accumulation in primary inflammatory cells but does not modify HIF-1alpha 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-1alpha protein expression in wound cells 5 days after injury, a time at which wound hypoxia could contribute to HIF-1alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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