Published online before print August 3, 2007
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-dependent mannerDivision of Pediatric Surgery, Children’s Hospital of Pittsburgh, and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
1 Correspondence: Children’s Hospital of Pittsburgh, Department of Surgery, 3705 Fifth Avenue, 4A485 DeSoto, Pittsburgh, PA 15213, USA. E-mail: david.hackam{at}chp.edu
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(HIF-1) in macrophages, which was reversed after p38 inhibition, suggesting a link between p38 activation and HIF-1 expression. It is striking that small interfering RNA knockdown of HIF-1 reversed the effects of hypoxia on phagocytosis, and overexpression of HIF-1 caused a surprising increase in phagocytosis compared with nontransfected controls, demonstrating a specific role for HIF-1 in the regulation of phagocytosis. These data indicate that hypoxia enhances phagocytosis in macrophages in a HIF-1-dependent manner and shed light on an important role for HIF-1 in host defense.
Key Words: critical illness • necrotizing enterocolitis • hypoxic stress • bacterial clearance • p38 • bacterial translocation
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Evidence in support of a possible role for hypoxia in the modulation of phagocytosis includes the finding that hypoxia itself is known to cause the activation of intracellular signaling pathways, which themselves may regulate phagocytosis. For instance, hypoxia has been shown to increase the activation of the p38-MAPK pathway in macrophages [10
], a network of signaling molecules, which has been shown to regulate the early events required for particle internalization [11
]. Exposure of many cell types to hypoxia also leads to an increase in the expression of hypoxia-inducible factors (HIF), a family of transcription factors, which are activated under conditions of low oxygen tension [12
, 13
]. Under normoxic conditions, HIF-1 protein is degraded rapidly to undetectable concentrations [14
, 15
], and under hypoxic conditions, HIF-1 is stabilized and translocates from the cytoplasm to the nucleus, where it dimerizes with HIF-1β, allowing the HIF-1 complex to bind to hypoxia response elements in the regulatory regions of target genes to induce their expression. Although Johnson and co-workers [16
] have shown such genes to influence various aspects of myeloid cell function, including myeloid cell aggregation, motility, and invasiveness, the role of HIF-1, if any, in phagocytosis remains uncertain. It is noteworthy that mouse embryonic fibroblasts deficient in p38-MAPK are unable to activate HIF-1 under conditions of hypoxia [17
], raising the intriguing possibility that if hypoxia can exert effects on phagocytosis, a link between p38 and HIF-1 may play a role.
In the current study, we sought to define whether hypoxia could regulate phagocytosis by macrophages and specifically predicted that phagocytosis would be impaired after hypoxic treatment. It is surprising that we now demonstrate that hypoxia leads to an increase in the phagocytic and microbicidal abilities of macrophages, that activation of p38-MAPK signaling is required for this to occur, and that the expression of HIF-1 plays a necessary and sufficient role for the hypoxia-mediated increase in phagocytosis by macrophages.
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were from Upstate (Lake Placid, NY, USA). SRBCs were from Fitzgerald Industries (Concord, MA, USA). Dynabeads (3 µm) were from Dynal Biotech (Oslo, Norway). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA). Where indicated, cells were pretreated with the MAPK inhibitor SB202190 (10 µM, 1 h, Calbiochem, San Diego, CA, USA). To induce hypoxia in vitro, macrophages were plated in six-well dishes (Sigma-Aldrich) and placed in a hypoxic chamber (flushed in advance with 1% oxygen, 37°C, Coy Laboratory Products, Grass Lake, MI, USA) for durations between 15 min and 2 h as indicated. Cells were maintained at 37°C at all times during internalization.
Isolation of murine peritoneal macrophages
Male C3H/HeOUJ mice (Jackson Laboratory, Bar Harbor, ME, USA), at 4–8 weeks of age, were housed in accordance with the Children’s Hospital of Pittsburgh Animal Care and Use Committee guidelines (Protocol 08-05, Pittsburgh, PA, USA). Animals were selected at this age, as they yield a robust number of primary macrophages for subsequent experiments and are able to withstand systemic hypoxia without significant toxicity. Where indicated, animals were maintained in a normoxic environment or were exposed to hypoxic treatment in a hypoxic chamber (5% O2, 95% N2, Billups-Rothenberg, Del Mar, CA, USA) three times/day for 3 min duration over 48 h. The degree of hypoxia, which was induced in mice under these conditions, was measured using a portable arterial blood gas monitor (i-STAT 1 Analyzer, Model MN-300, Abbot Point of Care Inc., East Windsor, NJ, USA).
After the induction of hypoxia, animals were killed, and macrophages were then harvested by peritoneal lavage as described [19 ]. Briefly, after sacrifice, the skin was cleansed with 70% ethanol, and an incision was made in the lower abdomen through the skin, leaving the peritoneum intact. Animals were then injected with 10 ml ice-cold PBS followed by gentle massage to allow for the distribution of the lavage fluid throughout the peritoneal cavity. The PBS lavage fluid was then removed using a 10-ml syringe attached to a 21-guage needle, and cells were washed three times in DMEM + 10% FBS. Macrophages were identified by adherence to glass coverslips (1 h, 37°C) and morphology characteristics under light microscopy, and cell viability was confirmed by exclusion of Trypan blue.
Phagocytosis assays
To accurately quantify the rate and extent of phagocytosis, it was critical to reliably distinguish particles, which were internalized, from those that were merely adherent to the macrophage surface. To do so, three separate approaches were used: an in vitro SRBC assay, an in vitro latex bead (confocal-based) assay, and an in vivo-labeled bacteria assay. The details follow. For SRBC, the following experimental protocol was adapted from that of Grinstein and colleagues [20
]: In brief, SRBCs (Fitzgerald Industries) were opsonized by incubating with IgG (Sigma-Aldrich; 1/10 dilution from stock, 20 min, 37°C) and were then added to RAW264.7 or murine peritoneal macrophages, which had been plated overnight on glass coverslips at 60% confluence (SRBC:macrophage ratio 10:1; 30 min incubation at 37°C). Noninternalized SRBCs were then removed by hypotonic lysis with ice-cold water for 30 s. After washing with ice-cold PBS, cells were fixed in 4% paraformaldehyde for 1 h. Phagocytosis was then assessed using a Nikon Eclipse TS100 microscope under phase-contrast optics, under which only internalized particles appeared as a result of the previous hypotonic lysis step. The number of macrophages, which had internalized at least one SRBC, was then measured. For the latex bead assay, in parallel, macrophages were incubated with Dynabeads under the above conditions in the presence of 10% FCS. After allowing phagocytosis to occur (20 min, 37°C), noninternalized particles were removed by copious washes with PBS. Internalized particles were distinguished from extracellular particles using an Olympus Fluoview 1000 confocal microscope under differential interference contrast (DIC) filters, during which slices were obtained throughout the plane of the cell. A three-dimensional (3D) reconstruction was developed for each field, and planes were obtained from the top of the cell to the coverslip, which reliably identified noninternalized particles. For an in vivo phagocytosis assay and for the determination of the effects of hypoxia on phagocytosis in vivo, C3H/HeOUJ mice were subjected to hypoxia as described above. On the 3rd day after the final hypoxia treatment, mice were injected i.p. with nonpathogenic Escherichia coli (8x108 CFU/mouse, DH5a, Invitrogen Life Technologies, Carlsbad, CA, USA) labeled with Oregon Green (Invitrogen Life Technologies, 1 mg/ml in PBS for 1 h at 37°C), as per the manufacturer’s instructions [21
]. Bacteria themselves were not subjected to hypoxia. After allowing 2 h for internalization of labeled bacteria, peritoneal macrophages were harvested as described above by lavage. Harvested macrophages were plated on glass coverslips, and after allowing 20 min for adherence, cells were washed with copious amounts of ice-cold PBS to remove noninternalized bacteria. Macrophages were then fixed using 4% paraformaldehyde and examined using confocal microscopy (Olympus Fluoview 1000) under DIC and FITC filter sets, which could reliably detect internalized, fluorescent particles.
In all cases, the rate of phagocytosis was determined by counting the number of cells per high power field (hpf), which had internalized at least one particle as a ratio of the total number of cells per field. Over 100 fields were enumerated to give the "phagocytosis rate" for a typical experiment. To assess the statistical variability, at least three separate experiments were performed, and the mean and SE were calculated for each of the experiments. The precise number of times that each experiment was repeated is stated in the corresponding figure legends.
Determination of macrophage bactericidal activity
To measure bacterial killing, the following gentamicin protection assay was used as described previously [22
]. Briefly, RAW264.7 macrophages were plated in 3 cm dishes (Sigma-Aldrich) and then exposed to nonpathogenic E. coli (8x107 CFU/dish, DH5a, Invitrogen Life Technologies). After 20 min to allow for phagocytosis, cells were washed three times with PBS and then treated with gentamicin (5 µg/ml in DMEM supplemented with 10% FBS) for 20 min to eliminate extracellular bacteria. Cells were then lysed in 100 µl vol lysis buffer, as described above, and the entire cell lysate from each condition was plated on separate, gram-negative-selective, MacConkey agar plates overnight. The extent of microbicidal activity was quantified by counting the number of CFUs in each group cultured from each cell lysate sample.
Determination of Rho activation
RhoA activity was quantified using an ELISA-based detection assay (Cytoskeleton, Denver, CO, USA). Briefly, RAW264.7 macrophages were serum-starved overnight, exposed to hypoxia, and subjected to hypotonic lysis at 4°C. The relative expression of activated RhoA-GTP to inactive RhoA-GDP was determined spectrofluorometrically, based on the increased avidity of RhoA-GTP for the effector protein rhotekin, as described [23
]. Positive controls included samples enriched with GTP-
S to maximize RhoA activation.
Knockdown and overexpression of HIF-1
The expression of HIF-1 was silenced in RAW264.7 macrophages using a small interfering (si)RNA transfection approach with lipofectamine as a carrier. Briefly, RAW264.7 macrophages were plated onto six-well culture dishes (500,000 cells/well, Sigma-Aldrich). After allowing 24 h for adhesion, the medium was changed, and cells were treated with 10 µl 40 µM HIF-1 siRNA and 10 µl Lipofectamine 2000 (Carlsbad, CA, USA) in OptiMem media (Invitrogen Life Technologies). Six hours later, wells were repleted with DMEM containing 10% FBS. After 24 h, cells were retreated with the original concentration of siRNA. Five days after the initial plating, cells were harvested using cell lysis buffer (Cytoskeleton), and total protein concentration was determined using the bicinchoninic acid (Sigma-Aldrich) assay and then subjected to SDS-PAGE for detection of HIF-1 expression using specific antibodies as described above. A nontargeting siRNA engineered against no specific gene (40 µM) was used in all cases as a negative control. To overexpress HIF-1, RAW264.7 macrophages were exposed to GFP-tagged HIF-1 cDNA (the generous gift of Dr. George Simos, University of Thessaly, Larissa, Greece) [24
] using Lipofectamine 2000 as a carrier molecule. The expression of HIF-1 was confirmed by SDS-PAGE. To assess the effects of GFP-HIF-1 on cell proliferation, RAW264.7 macrophages were plated (200,000 cells per 12-well dish) and allowed to adhere overnight. The next day, cells were counted and then transfected with enhanced GFP (Clontech, Mountain View, CA, USA) cDNA alone, or GFP-tagged HIF-1 cDNA, and cell counts were taken over the ensuing 48 h. After transfection, cells were assessed for their ability to undergo phagocytosis as described above. Where indicated, bands were detected with ECL-Super Signal (Pierce, Rockford, IL, USA), and images on radiographic film were quantified using a GS700 BioRad densitometer and QuantityOne analysis software (Hercules, CA, USA).
Determination of the effects of hypoxia on the release of proinflammatory molecules from macrophages
To assess whether exposure of macrophages to hypoxia affected the release of proinflammatory molecules, the supernatants of RAW 264.7 macrophages, which had been plated on 3 cm dishes overnight and exposed to hypoxia as described above in the presence or absence of the MAPK inhibitor SB202190 (10 µM, 1 h), were harvested and assessed by ELISA for IL-1 and TNF- (R&D Systems, Minneapolis MN, USA), according to the manufacturer’s directions. In parallel, nitrite concentrations were assessed by the Greiss reaction as described [25
], and the concentration of reactive oxygen species (ROS) was assayed using the ferricytochrome c method (Sigma-Aldrich) as described [26
, 27
]. The ability of these proinflammatory molecules to modulate phagocytosis was assessed using the SRBC assay described above after treatment of RAW264.7 macrophages under the following conditions: The NO donor deta-NONOate (Sigma-Aldrich, 1 mM, 4 h); TNF- (Sigma-Aldrich, 1 ng/ml, 4 h); IL-1β (Sigma-Aldrich, 1 nM, 4 h); hydrogen peroxide (100 µM, 4 h). As internal controls, cells were exposed to hypoxia as above or were treated with LPS (Sigma-Aldrich, 10 ng/ml, 4 h).
Statistics
Phagocytosis was quantified by dividing the number of cells per hpf, which had internalized at least one particle (SRBC, fluorescent bacteria, or latex bead) by the total number of cells per hpf. In parallel, the number of intracellular particles per macrophage was determined under fluorescent or bright-field optics and averaged over at least 250 cells per experiment. For quantification of phagocytosis, at least 100 fields were examined and enumerated per experimental condition. All experiments were repeated at least four times. Comparisons were made by Student’s t-test and ANOVA where appropriate.
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Figure 1. Hypoxia leads to an increase in phagocytosis and bacterial killing by peritoneal macrophages. (A and B) Peritoneal macrophages were harvested from C3H/HeOUJ mice, which had been exposed to hypoxic or normoxic conditions, and phagocytosis was assessed as the ability to internalize opsonized SRBCs. (A) Representative image depicting five macrophages, two of which had internalized an opsonized SRBC (black arrows show internalized particles; original size bar=10 µm). (B) Quantification of phagocytosis; *, P < 0.05, representative of three separate experiments with over 100 cells per experiment. (C–F) RAW264.7 macrophages were maintained under normoxic conditions or were exposed to hypoxic conditions in a modular hypoxic chamber and allowed to undergo phagocytosis. Representative images showing the internalization of latex particles by normoxic (C) and hypoxic (D) cells are shown. Original size bars = 10 µm. White arrows correspond to internalized particles, as determined by confocal microscopy with 3D reconstruction. (E) Quantification of phagocytosis; *, P < 0.05 representative of three separate experiments with over 100 cells per experiment. (F) Number of intracellular particles per macrophage in normoxic (open bars) and hypoxic (solid bars) cells; *, P < 0.05, representative of three separate experiments. (G) Gentamicin protection assay indicating the microbicidal activity of RAW264.7 macrophages under normoxic (open bars) and hypoxic (solid bars) conditions; greater microbicidal activity correlates with a decreased retrieval of bacteria on agar. Representative of three separate experiments; *, P < 0.05.
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Systemic hypoxia leads to an increase in phagocytosis by peritoneal macrophages in vivo
Given the results of the preceding experiments, we next sought to assess whether systemic hypoxia would increase phagocytosis by mouse peritoneal macrophages in vivo. To do so, mice were exposed to hypoxia and then injected i.p. with Oregon green-labeled E. coli. The extent of phagocytosis by peritoneal macrophages in vivo could then be quantified by confocal microscopy by measuring the number of fluorescent bacteria within harvested macrophages. As shown in Figure 2A
, the exposure of animals to systemic hypoxia led to a significantly higher rate of phagocytosis of labeled bacteria by peritoneal macrophages compared with normoxic controls (see Fig. 2B
and 2C
, for representative images). These findings indicate that despite our initial prediction, hypoxia causes an increase in the phagocytic ability of macrophages in vitro and in vivo.
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Figure 2. Systemic hypoxia leads to an increase in phagocytosis by peritoneal macrophages in vivo. (A–C) Mice were exposed to hypoxic or normoxic conditions and then injected i.p. with fluorescently labeled E. coli. Peritoneal macrophages were then harvested, plated on glass cover slips, and examined by confocal microscopy under DIC and FITC optics to evaluate the presence of internalized fluorescent bacteria. (A) Quantification of phagocytosis; *, P < 0.05; n = 3 separate experiments with three mice per experiment involving over 100 macrophages per mouse. Representative images showing a merged DIC and fluorescent image from macrophages, which had been obtained from normoxic (B) and hypoxic (C) mice. Original size bars = 10 µm. White arrows indicate internalized bacteria.
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Figure 3. Hypoxia increases phagocytosis in RAW264.7 macrophages in vitro in a p38 MAPK-dependent manner. (A) RhoA activation assay as assessed by ELISA in RAW264.7 macrophages, which had been exposed to hypoxic conditions for the time-points indicated. No significant differences were noted. Representative of three separate experiments. (B) SDS-PAGE of lysates from RAW264.7 macrophages, which had been exposed to the conditions indicated and then immunoblotted with antibodies against phospho-p38 (P-p38) MAPK. Blots were stripped and reprobed using antibodies against total p38 MAPK, and f-actin. SB, Cells pretreated with the p38 inhibitor SB202190 (10 µM, 1 h). Cells were exposed to hypoxia for 1 h as described in Materials and Methods. (C) RAW264.7 macrophages remained under normoxic conditions (normoxia) or were exposed to hypoxia (Hyp; 1 h), hypoxia plus the p38 MAPK inhibitor SB202190 (Hyp+SB; 1 h), or inhibitor alone (SB; 1 h) and were subjected to a SRBC phagocytosis assay as described in Materials and Methods; *, P < 0.05, by ANOVA, representative of at least three separate experiments with over 100 cells counted per experiment.
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-dependent manner is known to increase the activation of a variety of activities by myeloid cells [16
], although a link between HIF-1 and phagocytosis remains largely unexplored. Previous authors have demonstrated a link between the activation of p38-MAPK and the expression of HIF-1 in several cell types [17
, 34
]. Based on these findings and having shown that the inducing effects of hypoxia on phagocytosis require p38-MAPK (Fig. 3)
, we next considered whether a link exists between hypoxia-induced p38 activation and the induction of HIF-1 in regulating phagocytosis by macrophages. As shown in Figure 4A
, exposure of RAW264.7 macrophages to hypoxia resulted in an increase in HIF-1 expression. To define whether HIF-1 was involved in phagocytosis after hypoxia treatment, RAW264.7 macrophages were treated with HIF-1 siRNA, and the extent of phagocytosis after hypoxic exposure was assessed. Transfection of RAW264.7 cells with HIF-1 siRNA resulted in a >80% inhibition of protein expression as compared with RAW264.7 cells treated with control siRNA, which had been engineered against no known targets (both groups were exposed to hypoxia; see Fig. 4A
). As shown in Figure 4B
, inhibition of HIF-1 led to a significant reversal of the enhancing effects of hypoxia on phagocytosis. Moreover, pretreatment of RAW264.7 macrophages with the p38-MAPK inhibitor SB202190 prevented the hypoxia-induced increase in the expression of HIF-1 (Fig. 4C)
, which we had observed previously (see Fig. 4A
). Taken in aggregate, these results suggest that hypoxia increases the expression of HIF-1, in part through p38 activation, leading to an increase in phagocytosis.
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Figure 4. Hypoxia increases phagocytosis in RAW264.7 macrophages in vitro in a HIF-1-dependent manner. (A and B) RAW264.7 macrophages were subjected to hypoxia (1 h) in the presence or absence of siRNA against HIF-1 (HIF-1 siRNA) or against no known targets (Control siRNA). (A) SDS-PAGE of lysates, which were immunoblotted against HIF-1. Blots were then reprobed with antibodies against actin. (B) Quantification of phagocytosis by a SRBC assay of RAW264.7 macrophages, which were normoxic (normox) or hypoxic (hypox; 1 h) and were pretreated with siRNA as above; *, P < 0.05, compared with normoxia; representative of at least three separate experiments. (C) RAW264.7 macrophages were maintained under normoxic or hypoxic conditions (1 h) in the presence or absence of the p38-MAPK inhibitor SB202190, electrophoresed, and then subjected to SDS-PAGE using antibodies against HIF-1. Blots were reprobed with antibodies against actin. The mean band intensity in the individual groups is shown. ctrl, No inhibitor; *, P < 0.05, versus hypoxia-treated cells, which were exposed to SB202190; representative of three separate experiments.
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in RAW264.7 macrophages increases phagocytosis plays a critical role in the regulation of phagocytosis in RAW264.7 macrophages, a "knock-in" approach was undertaken. To do so, cells were transfected with GFP-HIF-1, as described in Materials and Methods, and the effects on the rate of phagocytosis were assessed. As is shown in Figure 5A
, the overexpression of GFP-HIF-1 in RAW264.7 macrophages was determined by SDS-PAGE. It is remarkable that transfection of RAW264.7 macrophages with GFP-HIF-1 led to a significant increase in the phagocytic ability of the macrophages over nontransfected cells (Fig. 5B
and 5C)
or cells transfected with GFP alone (not shown). Transfection with GFP-HIF-1 did not result in a significant change in the number of macrophages after 48 h compared with nontransfected cells (not shown). Taken in aggregate, these results demonstrate a critical role for hypoxia, acting via HIF-1, in the modulation of phagocytosis by macrophages.
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Figure 5. Overexpression of HIF-1 results in increased phagocytic ability in RAW 264.7 macrophages, which were transfected with GFP-HIF-1 and assessed for the ability to undergo phagocytosis of latex beads. (A) SDS-PAGE of RAW264.7 macrophages, which were nontransfected (control) or were transfected with GFP-HIF-1 cDNA (HIF-1). (B) Representative images of nontransfected (Control; i and ii) and GFP-HIF-1-transfected (GFP-HIF-1; iii and iv) RAW264.7 macrophages, which had been incubated with latex particles and evaluated by confocal microscopy under GFP (i and iii) and DIC (ii and iv) filter sets. Original size bars = 10 µm. White arrows indicate extracellular particles, and red arrows indicate internalized particles. (C) Quantification of phagocytosis; *, P< 0.05, of three separate experiments of over 100 cells per experiment.
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has been shown to induce the expression of a variety of proinflammatory cytokines, including TNF- and IL-1 [35
], and increases in ROS/nitrogen species have been shown to stabilize HIF-1 [36
]. These findings raise the intriguing possibility that HIF-1 could act through these proinflammatory molecules, leading to changes in phagocytosis. To examine this possibility directly, macrophages were exposed to hypoxia, and the release of IL-1 and TNF- and ROS and reactive nitrogen species was measured. Of note, hypoxia did not induce the release of these proinflammatory molecules significantly, and as expected, treatment with SB202190 did not affect the release (data not shown). Although hypoxia clearly increased the expression of HIF-1, treatment of macrophages with IL-1, TNF-, the ROS donor H2O2, or the NO donor detaNONOate did not increase the expression of HIF-1 (Fig. 6A
). The rate of phagocytosis was increased after treatment with hypoxia and LPS (an additional positive control), yet treatment of macrophages with IL-1, TNF-, or ROS/nitrogen species did not affect phagocytosis significantly. Taken in aggregate, these findings do not support the possibility that hypoxia-induced cytokine release can account for the increase in phagocytosis observed under hypoxic conditions yet suggest a more direct role for HIF-1 in activating phagocytosis through other pathways, likely requiring p38 activation.
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Figure 6. The release of proinflammatory molecules by macrophages does not readily account for the effects of hypoxia on phagocytosis. (A) SDS-PAGE of RAW264.7 macrophages, which were untreated (ctrl), exposed to hypoxia, or were treated with the NO donor deta-NONOate (NO; 1 mM), H2O2 (100 µM), IL-1β (1 nM), or TNF- (1 ng/ml) as indicated. Blots were then probed for HIF-1 and then stripped and reprobed for actin. (B) RAW264.7 macrophages were untreated (open bar), exposed to hypoxia (solid bars), or treated with LPS (10 ng/ml), deta-NONOate (1 mM), H2O2 (100 µM), TNF- (1 ng/ml), or IL-1β (1 nM; cross-hatched bars), as described in Materials and Methods. Phagocytosis was then assessed by SRBC assay; *, P < 0.05, of three separate experiments.
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, in part through activation of p38-MAPK. It is important that the overexpression of HIF-1 alone is sufficient to lead to an increase in phagocytosis. Taken together, these findings now demonstrate a critical role for HIF-1 in phagocytosis and illustrate the positive effects of hypoxia on particle internalization.
It is important to note that the experimental protocol, which was used to induce hypoxia in the current studies, was carefully selected to mirror the situations in which hypoxia may be encountered clinically. Specifically, after treatment of mice with hypoxia three times per day, we measured an arterial pO2 of 20–25 mmHg, significantly below that of normoxic mice. It is important that this degree of hypoxia is similar to that observed in critically ill patients [37
] and is consistent with tissue levels of oxygen, which are observed in models of endotoxemia [38
], at sites of necrosis [39
], as well as in murine and human peritonitis [40
, 41
]. Although we now find an important role for hypoxia in the regulation of phagocytosis, we acknowledge the possibility that hypoxia may induce the release of mediators from the lung or other distant organs, which could themselves induce the increase in phagocytosis that is observed. However, the fact that hypoxia induces an increase in phagocytosis in vitro through a mechanism that requires the expression of HIF-1 supports the concept that hypoxia exerts a direct role in the induction of phagocytosis. Moreover, the fact that we were not able to detect an increase in the release of proinflammatory cytokines from macrophages after induction of hypoxia further supports this conclusion, although the possibility remains that other factors may be released during hypoxia, which serve to augment phagocytosis, independent of HIF-1, which we simply did not detect.
A major finding of the current study relates to the specific role of HIF-1 in the regulation of phagocytosis. The finding that inhibition of HIF-1 using siRNA prevents the hypoxia-induced increase in phagocytosis and that overexpression of HIF-1 enhances phagocytosis strongly indicates that the expression of HIF-1 plays a necessary and sufficient role in this process. This important function for HIF-1 likely underscores the broad array of genes, which are induced in response to HIF-1, expected to enhance internalization. Although the current studies do not specifically identify a HIF-1 target responsible for the increase in phagocytosis, several candidate genes may be attractive targets. Previous studies have demonstrated that HIF-1 may be required for RhoA activation [42
43
44
], although the fact that we do not measure changes in RhoA activity in response to hypoxia makes RhoA an unlikely target in macrophages. In contrast, HIF-1 expression is known to lead to the activation of calcium/calmodulin signaling [45
, 46
], as well as the PI-3K/AKT pathway [47
, 48
], each of which has been shown to lead to an increase in phagocytosis. Further studies to identify the downstream gene products, which specifically orchestrate the increase in phagocytosis, may serve to elucidate the previously unrecognized role(s) of HIF-1 in host defense.
The current findings shed light into the roles of HIF-1 in regulating immune cell activity and build on previous reports in this field. For instance, Peyssonnaux et al. [35
] have demonstrated that activation of HIF-1 under hypoxia enhances bactericidal activity, and HIF-1 pathways are responsive to bacterial stimulation even under normoxia. Cramer et al. [16
] have shown that HIF-1 regulates myeloid cell aggregation, invasion and motility, and internalization of bacteria through effects related in part to intracellular levels of ATP. Although, these prior reports did not specifically examine the effects of hypoxia or HIF-1 on phagocytosis, such studies have clearly elucidated a role for HIF-1 in the effectiveness of the downstream events, which occur after phagocytosis, leading to enhanced intracellular killing. It should also be emphasized that although HIF-1 may be expected to be degraded rapidly after the brief exposure to hypoxia and although HIF-1 may be induced by nonhypoxic stimuli, the current data indicate that the initial induction of HIF-1 is sufficient to activate a signaling cascade, which leads to an enhancement of phagocytosis in vitro and in vivo.
It should be noted that although the finding that hypoxia increases phagocytosis was confirmed in vitro and in vivo, the effect may only apply to Fc-mediated phagocytosis, as opposed to other phagocytic means of particle internalization. This may be particularly relevant in the context of systemic sepsis, during which the expression of phagocytosis receptors on the macrophage membrane may be altered and may have effects on the extent of particle internalization. For instance, Clatworthy and Smith [49
] and Wijngaarden et al. [50
] have demonstrated that FcRIIb function may be influenced by the extent of systemic inflammation present, leading to alterations in macrophage activity and phagocytosis. Similarly, Huber-Lang et al. [51
] and Sundaram et al. [52
] have demonstrated that the complement receptor may be altered during sepsis with changes in the extent of phagocytosis. These findings demonstrate that although the current study highlighted the role of Fc-mediated phagocytosis, alternative pathways may be involved with a significant role in the events leading to particle internalization.
The enhancing effect of hypoxia on phagocytosis is of particular interest to conditions in which low oxygen tension may be experienced and in which the effects on phagocytosis may have a significant impact on the clinical outcome. For instance, in patients with systemic inflammatory diseases in which suprainfection may be a common correlate, the anticipated increase in phagocytosis and microbial killing may lead not only to enhanced host defense but also to the exaggerated release of proinflammatory products, which may be liberated after phagocytosis occurs [53
, 54
] Such inflammatory molecules, including cytokines and reactive oxygen intermediates, may be expected to worsen the extent of inflammation locally and systemically, thereby contributing to increased disease severity despite more effective bacterial clearance. We now propose that although phagocytosis is increased with a corresponding increase in microbicidal cellular activity, these studies may point to a general state of cellular activation, in which not only phagocytosis is increased, a potential beneficial effect, but also other potentially injurious effects of macrophage activation are enhanced. The specific roles of HIF-1, if any, in the general processes, which lead to macrophage activation, remain to be determined.
In summary, contrary to our initial hypothesis, phagocytosis by macrophages is not reduced by hypoxia. In fact, we now report that hypoxia increases phagocytosis in macrophages through the activation of HIF-1, in part through a p38-dependent manner. These findings shed light on a surprising finding of the effects of hypoxia on leukocyte function and raise the possibility that HIF-1 itself may have an enhanced role in host defense during conditions of systemic inflammation during which oxygen delivery may be impaired.
Received March 30, 2007; revised June 25, 2007; accepted June 29, 2007.
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  B. Acosta-Iborra, A. Elorza, I. M. Olazabal, N. B. Martin-Cofreces, S. Martin-Puig, M. Miro, M. J. Calzada, J. Aragones, F. Sanchez-Madrid, and M. O. Landazuri Macrophage Oxygen Sensing Modulates Antigen Presentation and Phagocytic Functions Involving IFN-{gamma} Production through the HIF-1{alpha} Transcription Factor J. Immunol., March 1, 2009; 182(5): 3155 - 3164. [Abstract] [Full Text] [PDF]
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