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Originally published online as doi:10.1189/jlb.0806541 on January 30, 2007

Published online before print January 30, 2007
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(Journal of Leukocyte Biology. 2007;81:1236-1244.)
© 2007 by Society for Leukocyte Biology

Ascorbate deficiency results in impaired neutrophil apoptosis and clearance and is associated with up-regulation of hypoxia-inducible factor 1{alpha}

Margret C. M. Vissers1 and Rachel P. Wilkie

Free Radical Research Group, Pathology Department, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand

1 Correspondence: Free Radical Research Group, Pathology Dept., Christchurch School of Medicine and Health Sciences, P.O. Box 4345, Christchurch, New Zealand. E-mail: margret.vissers{at}chmeds.ac.nz


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Some cells, including neutrophils, accumulate high intracellular ascorbate concentrations, which suggests that they have an important function in these cells. In this study we have used L-gulono-{gamma}-lactone oxidase (Gulo)–/– mice, which are unable to synthesize ascorbate, to generate ascorbate-deficient neutrophils and have used these to investigate the effect of ascorbate on neutrophil function. Peritoneal neutrophils from ascorbate-deficient animals had normal morphology and respiratory burst activity but failed to undergo spontaneous apoptosis, determined by morphology and the surface expression of phosphatidylserine. Initially, there was increased cell survival, but death eventually occurred by necrosis within 48 h. Neutrophils persisted in thioglycollate-induced inflammation in Gulo–/– mice with the later appearance of necrotic cells, suggesting that apoptosis was also affected in vivo. Also, ascorbate-deficient neutrophils were not recognized by macrophages in an in vitro assay for phagocytosis, providing further evidence for defective apoptosis and clearance. Neutrophils from Gulo–/– mice had elevated levels of hypoxia-inducible factor (HIF)-1{alpha}, a transcription factor regulated by Fe2+-dependent hydroxylases which require ascorbate for optimal activity. HIF-1{alpha} has been shown previously to inhibit neutrophil apoptosis under hypoxic conditions. Our results suggest that in ascorbate deficiency, up-regulation of HIF-1{alpha} blocks neutrophil apoptosis under normoxic conditions and that this represents a novel and important function for vitamin C in inflammatory cells.

Key Words: vitamin C • neutrophil necrosis • HIF hydroxylases • neutrophil clearance


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vitamin C (ascorbate) is a small, water-soluble compound, which readily acts as a one or two electron-reducing agent for many radicals and oxidants, and this forms the basis of its diversity of action [1 ]. Humans, along with primates, guinea pigs, and fruit bats, cannot synthesize ascorbate as a result of a loss of function in the gene for L-gulono-{gamma}-lactone oxidase (Gulo), the enzyme required for the terminal step of its synthesis, and therefore require daily supplementation from the diet to maintain good health [2 ]. Intracellular levels of ascorbate are tightly controlled and vary between tissues [1 , 3 ]; for example, the adrenal glands and retina have more than 100 times the plasma concentration of ~60 µM ascorbate, whereas skeletal and smooth muscle cells have approximately 10 times the plasma concentration [1 , 4 ]. High concentrations are thought to be indicative of an essential metabolic function. Many functions have been ascribed to ascorbate as the result of in vitro studies, but there is still a great deal of uncertainty about the role it may play in disease prevention and in the pathology of disease [5 , 6 ]. Recently, mice deficient in the ascorbic acid transporter Svct2 were found to die within minutes of birth with respiratory failure and brain hemorrhage, suggesting an essential but uncharacterized function for ascorbate in the perinatal period [7 ].

The ascorbate level is high in mature peripheral blood neutrophils, the immune cells primarily responsible for bacterial killing, reaching millimolar concentrations [4 ]. However, how ascorbate affects neutrophil metabolism and function is unclear, although it has been shown to affect chemotaxis and phagocytosis [8 ] and to influence the reactivity of the microbicidal oxidants H2O2 and hypochlorous acid [9 , 10 ]. An important function of neutrophils is their ability to undergo apoptosis. These cells are released from the bone marrow in a proapoptotic state and are cleared from the circulation by spontaneous apoptosis within 8–10 h [11 , 12 ]. This process is responsible for clearing 1010–1011 cells daily [13 ] and ensures their safe disposal without release of the many cytotoxic and hydrolytic enzymes present in the cytoplasmic granules [14 , 15 ]. Neutrophil apoptosis can be delayed under conditions of active inflammation by certain cytokines and growth factors such as TNF-{alpha} and GM-CSF, whereas actively phagocytosing cells undergo an accelerated program, which results in their being removed from inflammatory sites and the successful resolution of inflammation [13 , 16 , 17 ]. More recently, neutrophil apoptosis was shown to be inhibited markedly under hypoxic conditions [18 , 19 ]. This effect involves hypoxia-inducible factor (HIF)-1, a ubiquitous transcription factor that regulates most aspects of the hypoxic response [19 ].

HIF-1 is a heterodimer of two helix–loop–helix proteins, HIF-1{alpha} and HIF-1ß. The {alpha}-subunit protein levels and activity are controlled by post-translational hydroxylation of the Pro 402 and Pro 564 residues in the oxygen-sensing domain and Asn 803 at the COOH terminal [20 , 21 ]. Proline hydroxylation promotes binding of HIF-1{alpha} to von Hippel-Lindau tumor-suppressor protein with subsequent ubiquitination and degradation by the proteasome, whereas asparagine hydroxylation by a factor inhibiting HIF prevents recruitment of the p300 coactivator, which is required for transcription [20 , 21 ]. When hydroxylation is compromised, there is stabilization and activation of the HIF-1{alpha} protein, with increased expression of many gene products, including key glycolytic enzymes, angiogenic factors such as vascular endothelial growth factor, erythropoietin, and cell survival proteins [20 ]. The hydroxylases responsible for these reactions belong to the 2-oxoglutarate-dependent superfamily, which includes the enzymes involved in collagen hydroxylation, and they are known to require ascorbate for optimal activity [20 , 21 ]. It is likely then, that decreased hydroxylation of HIF-1{alpha} could occur in ascorbate deficiency.

In this study, we have investigated the effect of intracellular ascorbate on neutrophil apoptosis. This work stems from our previous findings that ascorbate can influence apoptosis in endothelial cells under oxidative stress [22 ]. Neutrophils are terminally differentiated cells and cannot be maintained in culture, and to obtain these cells deficient in ascorbate, it was necessary to harvest the cells from a deficient animal donor. For this purpose, we used Gulo–/– mice, which have no ascorbate oxidase activity and require dietary vitamin C [23 ]. We found that apoptosis is inhibited profoundly in neutrophils lacking ascorbate, with increased survival within the first 24 h, followed by death by necrosis. As neutrophil apoptosis is delayed under conditions of hypoxia [18 , 19 ], we monitored HIF-1{alpha} protein and found elevated levels in ascorbate-deficient cells under normoxic conditions. Neutrophils were found to persist in the peritoneal cavity of Gulo–/– mice after thioglycollate challenge, suggesting that clearance was impaired, and this was supported by findings that ascorbate-deficient, aged neutrophils were not recognized or phagocytosed by macrophages in vitro. Our results suggest that ascorbate plays an important role in neutrophil apoptosis and may be vital for the resolution of inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mutant mice
Mutant C57BL/6 mice lacking a functional gene for Gulo were obtained from the Mutant Mouse Resource Center, University of California (Davis, CA, USA), as heterozygous Gulo+/– breeding pairs [23 ]. Animals were handled in accordance with guidelines from the Animal Ethics Committee of the University of Otago (Dunedin, New Zealand). All animals were fed ad libitum with regular chow, and homozygote Gulo–/– mice were given water supplemented with vitamin C (330 mg ascorbate/liter with 10 µM EDTA), which was renewed twice per week. For genotyping by PCR, three primers, P2 (5'-CGCGCCTTAATTAAGGATCC-3'), P3 (5'GTCGGTGACAGAATGTCTTGC-3'), and P4 (5'-GCATCCCAGTGACTAAGGAT-3'), were used, with a 230-bp fragment derived from the targeted locus and/or a 330-bp fragment derived from the endogenous locus, used to distinguish among homozygote, heterozygote, and wild-type mice [23 ].

Vitamin C-deficient neutrophils
To create ascorbate-deficient animals, Gulo–/– mice were given normal water [23 , 24 ]. When the animal’s weight had dropped by approximately 20%, 1 ml aged, sterile, 4% thioglycollate solution was injected into the peritoneum. After 16–18 h, the mice were killed by cervical dislocation, and the peritoneal cavity was flushed with 5 ml sterile HBSS containing 1% BSA. The cell preparation was layered onto 3 ml Ficoll-Hypaque (density=1.077 g/ml) and centrifuged for 20 min at 1000 g. The neutrophil pellet was washed twice with HBSS and resuspended at 106/ml in RPMI-culture medium. The proportion of neutrophils present was determined by analyzing Giemsa-stained cytospin preparations.

The time-course of thioglycollate-induced inflammation was monitored by harvesting the peritoneal cells at intervals over 48 h. Cell counts were performed immediately, and cytospins were prepared to determine differential counts of neutrophils and macrophages. At least 300 cells were counted per slide.

The ability of the cells to mount an oxidative burst was determined by measuring the production of superoxide (O2–) by cytochrome C reduction in response to challenge with phorbol myristate acetate (PMA) [25 ]. The vitamin C content was measured by solubilizing the cells in 150 µl 0.27 M perchloric acid and analyzing by HPLC using an Aqua C18 µm column with electrochemical detection [26 ].

Neutrophil apoptosis, viability, and necrosis
The status of the neutrophils was determined by their morphology, monitored in cytospin preparations by phase-contrast microscopy and by transmission electron microscopy. Externalization of phosphatidylserine (PS) was measured by double-labeling the cells with Annexin V-FITC as a marker of apoptosis and uptake of propidium iodide (PI) as a marker of necrosis (Nexins Research, BV, The Netherlands) [25 , 27 ]. The cell populations were washed free of RPMI with HBSS and resuspended in Annexin –FITC, according to the manufacturer’s instructions, and the fluorescence of 10,000 cells was analyzed on a bivariate flow cytometer.

Caspase-3 activity
Neutrophils (106) were incubated in RPMI with 10% mouse serum for stated periods, washed twice with HBSS, pelleted, and frozen at –80°C. Pellets were resuspended in 100 µl buffer [100 mM HEPES with 10% (w/v) sucrose, 1% CHAPS, 5 mM dithiothreitol (DTT), 10–4 % Nonidet P-40] with 50 µM Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC), and fluorescence changes were monitored at 370 nm (ex) and 445 nm (em) [28 ]. The amount of AMC liberated was determined from a standard curve [22 ].

Ascorbate supplementation of deficient neutrophils
Myeloid cells, including neutrophils, can transport dehydroascorbate via the glucose transporters [29 ]. We have restored ascorbate to deficient neutrophils as detailed [29 ]: A 2-mM solution of ascorbate was incubated with ascorbate oxidase (1 unit/ml) at pH 5.6 for 2 min to generate dehydroascorbate. The neutrophils were suspended in HBSS without glucose at 2 x 106/ml, and dehydroascorbate was added to give a final concentration of 0.4 mM. The cells were incubated at 37°C for 20 min, then washed by centrifugation in HBSS, and resuspended in full medium. This treatment resulted in restoration of intracellular ascorbate to levels within the normal range as measured by HPLC (data not shown).

In vitro assay for phagocytosis of aged neutrophils by macrophages
Peritoneal neutrophils from ascorbate-deficient Gulo–/– and from control heterozygote Gulo+/– or homozygote +/+ mice were aged for 24 h in RPMI with 10% mouse serum at 37°C. Macrophages were isolated from heterozygote Gulo+/– mice 48 h after thioglycollate infusion, washed, and resuspended in RPMI. These cells were plated in a 24-well plate at 0.5 x 106 cells/well and allowed to adhere for 5 h. Nonadherent cells were washed away, and suspensions of aged neutrophils were added to each well (6x105 per well). After 3.5 h incubation at 37°C, the wells were washed three times with HBSS, and the adherent cells were fixed with 4% paraformaldehyde for 20 min at 37°C. After two further washes, 300 µl peroxidase stain (1 mM o-dianisidine, 5 mM H2O2, 50 mM sodium phosphate buffer, pH 6) was added to each well and incubated for 20 min. The cells were then washed again and visualized by phase-contrast microscopy. Two people independently scored phagocytic macrophages (cells that had internalized peroxidase activity). At least 300 cells were counted each time, and repeat estimates were made on every sample.

Western blotting of HIF-1{alpha} protein
Nuclear fractions of neutrophils from control or Gulo–/– ascorbate-deficient mice were prepared after disruption by nitrogen cavitation [30 ]. The cells (approximately 107) were washed twice in ice-cold PBS and resuspended in 1.5 ml relaxation buffer [10 mM HEPES, pH 7.3, with 30 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 1.25 mM EGTA, 0.5 mM DTT, and Complete inhibitors (Roche, Indianapolis, IN, USA)]. The cell suspension was maintained on ice and equilibrated to 350 pounds per square inch (psi) for 20 min in the nitrogen bomb before release. Vitamin C-deficient, hypoxia-treated, and CoCl2-treated cells appeared more fragile, and a pressure of 250 psi could not be exceeded to obtain a nuclear pellet. The lysed cells were spun at 500 g for 10 min to pellet nuclei. These were then resuspended in gel-loading buffer with 1% SDS, 100 mM DTT, and Complete inhibitors, heated to 95°C for 5 min, and samples were separated by SDS-PAGE. Western blots were developed with polyclonal goat antihuman/mouse HIF-1{alpha} (1 µg/ml, R&D Systems, Minneapolis, MN, USA) and peroxidase-labeled, rabbit antigoat IgG, and bands were visualized using ECL reagent. Positive control samples were generated by incubating control neutrophils for 8 h with 100 µM CoCl2 or under hypoxic conditions (by equilibrating the cell suspension with 95% N2/5%CO2).

Statistics
Significant differences between observations were determined using the Student’s t test for unpaired data points.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Neutrophils from vitamin C-deprived Gulo–/– mice contain no detectable ascorbate
On removal of vitamin C-supplemented water, the health status of the Gulo–/– mouse was apparently unchanged for 3 weeks, with no obvious distress or change in weight, despite the levels of ascorbate reportedly falling to 10–15% of normal during this time [23 , 24 ]. Typically, during the fourth week, the animal’s weight began to drop sharply (Fig. 1 ), and ascorbate was undetectable in serum. When the animal’s weight had dropped by approximately 20%, peritoneal neutrophils were collected 16–18 h after installation of 1 ml sterile thioglycollate solution. The cells were fractionated through Ficoll-Hypaque to yield cell populations containing similar numbers of neutrophils (Table 1 ). The cells from vitamin C-deficient mice contained no detectable ascorbate (Table 1) but were indistinguishable from neutrophils from heterozygous or wild-type littermates by morphology having normal, ring-shaped nuclei (Fig. 1B) . They were also equally able to generate O2– when challenged with the phorbol ester PMA (Table 1) .


Figure 1
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  		Figure 1. Induction of ascorbate deficiency in Gulo–/– mice. (A) A representative profile of Gulo–/– ({circ}) and heterozygote Gulo+/– or +/+ (•) mouse weights. At 4–5 weeks after removal of the vitamin C supplement, Gulo–/– animals lose weight rapidly. When body mass was approximately 80% of initial weight, neutrophils were harvested by peritoneal lavage 18 h after injection of 1 ml sterile 4% (w/v) thioglycollate solution. (B) Morphology of freshly isolated neutrophils from Gulo–/– and Gulo+/– mice. The results shown are representative of at least 15 experiments, all of which showed similar results.

 

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  		Table 1. Characteristics of Semi-purified Peritoneal Neutrophil Populations Isolated after Thioglycollate Challenge

 
Constitutive apoptosis is defective in ascorbate-deficient neutrophils
The morphology of cells kept in culture medium overnight was monitored by light and electron microscopy and by the externalization of PS by flow cytometry after double-labeling with Annexin V-FITC and PI [25 ]. Neutrophils from control mice showed changes in morphology which were consistent with apoptosis: loss of multi-lobed nuclear appearance with condensation of chromatin, shrinking, loss of cytoplasm, and the formation of membrane blebs (Fig. 2A 2B 2C ). In contrast, ascorbate-deficient cells maintained recognizable, ring-shaped nuclei. Cell viability was difficult to determine in cytospin samples, but there was an obvious deterioration in morphology, and many cells became enlarged and lost cell integrity (Fig. 2A) . By phase-contrast and electron microscopy, the cells appeared to be viable or necrotic (Fig. 2B and 2C) . The lack of apoptotic changes in the ascorbate-deficient cells was noticeable at 5 h and was marked by 24 h (Fig. 2D) .


Figure 2
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  		Figure 2. Morphology changes in mouse peritoneal neutrophils after 24 h in vitro incubation. Neutrophils were harvested 18 h after injection with thioglycollate and incubated in full medium for 24 h. (A) Giemsa staining of cytospun cells viewed by differential interference contrast. Control mouse cells showed signs of apoptosis after 24 h with condensed nuclei (black arrows), whereas many of the ascorbate-deficient neutrophils retained a recognizable, multilobed nucleus but with a significant loss of cell integrity (white arrows). Bars = 20 µm. (B) Phase-contrast microscopy. After 24 h, many control cells are shrunken and show membrane blebbing (arrows), whereas vitamin C-deficient cells are swollen and necrotic. Bars = 25 µm. (C) Electron microscopy confirms cell shrinkage and condensation of multilobed nuclei in control cells, whereas in the vitamin C-deficient population, the cells retain a viable morphology or appear necrotic. Bars = 2 µm. (D) The number of viable, apoptotic, and necrotic cells from heterozygote (solid bars) or ascorbate-deficient (shaded bars) mice was estimated in Giemsa-stained samples determined by two investigators in a blinded manner. Means ± SEM for four or five separate animals are shown. Statistical significance for all data was determined using the Student’s t-test for unpaired data. **, P < 0.005; *, P < 0.05.

 
The decrease in apoptotic morphology was matched by decreased externalization of PS (Fig. 3 ). Within 5 h, there was a significant difference between the ascorbate-deficient and control cells, and this became marked after 24–48 h. Whereas normal mouse neutrophils progressed to become mostly apoptotic (Annexin V-positive, PI-negative), significantly more ascorbate-deficient cells remained viable or became necrotic (PI-positive; Fig. 3 ). Activated caspase-3 was detected in normal and ascorbate-deficient neutrophils (Fig. 4 ). However, this did not result in any morphological signs of apoptosis in the ascorbate-deficient cells, and there was no PS exposure in these cells, even at early time-points (Fig. 3) . Therefore, despite activation of caspase-3, which is considered to be central to the apoptotic program [31 , 32 ], vitamin C-deficient neutrophils did not progress to apoptosis.


Figure 3
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  		Figure 3. Apoptosis is inhibited in ascorbate-deficient mouse neutrophils. Peritoneal cells were harvested 18 h after injection with thioglycollate. (A) Neutrophil viability, apoptosis, and necrosis, as detected by flow cytometry after double-labeling with Annexin V-FITC and PI immediately upon isolation and after 24 h. Viable cells do not bind Annexin V or take up PI and are found in the lower left quadrant. Apoptotic cells with surface expression of PS bind Annexin V but exclude PI and are found in the lower right quadrant, whereas necrotic cells are permeable to PI and are found in the upper panels. Small fragments of cells that were PI-positive but did not stain with Annexin V were included in the ungated populations of cells that were analyzed. (B) Combined means ± SEM of four to eight experiments with control heterozygote (solid bars) and ascorbate-deficient (shaded bars) neutrophils, incubated in RPMI. Apoptosis was inhibited significantly in the ascorbate-deficient cells, with a corresponding increase in the number of viable or necrotic cells. **, P < 0.005; *, P < 0.05. Significant differences were detectable at all times assayed and as early as 5 h with more cells in the viable populations at this time.

 

Figure 4
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  		Figure 4. Caspase-3 activation in mouse peritoneal neutrophils, which when isolated 18 h after thioglycollate injection, were incubated in full medium, and activated caspase-3 was detected with the fluorogenic peptide DEVD-AMC. There was an eight- to tenfold increase in activity after 5 h, which returned to baseline by 24 h, with no significant difference between the control (•) and ascorbate-deficient ({circ}) neutrophils. Means ± SEM of five experiments are shown.

 
Neutrophils can accumulate ascorbate by active transport of dehydroascorbate [29 ]. When we restored ascorbate to deficient cells, the inhibition of apoptosis was reversed, as evidenced by the morphological appearance of the cells. Shrunken, apoptotic cells were readily apparent in the ascorbate-supplemented population, and this was accompanied by a significant increase in PS exposure (Fig. 5 ).


Figure 5
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  		Figure 5. Supplementation with ascorbate restores apoptosis in deficient neutrophils. Ascorbate levels were restored in neutrophils harvested from deficient mice 18 h after thioglycollate injection, and the cells were incubated in full medium for a further 48 h. (A) The morphology of ascorbate (Asc)-deficient neutrophils after 48 h showed many necrotic cells (white arrows). (B) In contrast, many shrunken, apoptotic cells were apparent in the supplemented cell population (black arrows). (C) Ascorbate supplementation resulted in a significant increase in the number of apoptotic cells as measured by Annexin V binding. The percent of apoptotic cells reflects the Annexin V-positive/PI-negative population as described in Figure 3 . The results shown are the means ± SEM of three estimates. *, P < 0.05; **, P < 0.005.

 
HIF-1{alpha} is up-regulated in ascorbate-deficient neutrophils
The effects of ascorbate deficiency on neutrophil apoptosis and survival closely resemble the effects of hypoxia, which inhibits neutrophil apoptosis markedly [18 , 19 ]. Western blots of vitamin C-deficient mouse neutrophils showed that HIF-1{alpha} protein could be detected in vitamin C-deficient neutrophils under normoxic conditions (Fig. 6A and 6B ). The levels did not reach those inducible by CoCl2 or hypoxia but differed significantly from ascorbate-containing control cells, in which HIF-1{alpha} was undetectable (Fig. 6A and 6B) .


Figure 6
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  		Figure 6. Ascorbate-deficient neutrophils have elevated levels of HIF-1{alpha}. (A) Peritoneal neutrophils isolated 18 h after thioglycollate injection were disrupted by nitrogen cavitation and nuclear pellets obtained. HIF-1{alpha} protein was detected by Western blotting using goat antihuman/mouse HIF-1{alpha}. Positive control samples were normal mouse neutrophils incubated for 8 h under hypoxic conditions (Hyp) or for 8 h with 100 µM CoCl2. HIF-1{alpha} migrates as a broad band with an apparent mass of 120 kD, and the position of the 118-kD marker is shown (arrow). The results of four independent blots are shown. Def1, Def 2, Def3, Individual samples from Gulo–/– mice; C, C1, C2, C3, samples from individual control littermates. (B) Combined results of Western blot detection of HIF-1{alpha}. Each lane shows the mean ± SEM of four to seven estimates.

 
Ascorbate-deficient neutrophils persist at an inflammatory site
To determine whether ascorbate deficiency affects neutrophil apoptosis and clearance in vivo, the progression of thioglycollate-induced peritoneal inflammation was monitored. In control animals, the infiltration of inflammatory cells was as described previously [33 ], with an early influx of mostly neutrophils, followed by a decrease in neutrophil numbers and an increase in monocyte/macrophages. By 48 h after thioglycollate injection, neutrophil numbers had returned to baseline, and mostly, mononuclear cells were present (Fig. 7 ). In contrast, neutrophils persisted in the peritoneal exudates from ascorbate-deficient mice, and significantly higher numbers of polymorphonuclear cells were seen after 24 h (Fig. 7) . After 48 h, many neutrophils with recognizable ring-shaped nuclei were still present (Fig. 7B) , and many showed morphological changes closely resembling the necrotic cells that had been incubated in vitro (Fig. 2A) . These results were also supported by findings that the 48-h peritoneal exudates from the ascorbate-deficient animals contained an increased number of trypan blue-positive cells (27.2±5.0% for ascorbate-deficient and 6.25±1.25% for control mice).


Figure 7
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  		Figure 7. Time-course of thioglycollate-induced inflammation. The influx of inflammatory cells into the peritoneum of ascorbate-deficient Gulo–/– ({circ}) and control Gulo+/– or +/+ (•) mice. (A) The total number of cells in the peritoneal exudates increased in response to thioglycollate challenge and then declined after approximately 48 h. At most time-points, there was little or no difference between the ascorbate-deficient and control animals, but at 24 h and 48 h, the number of cells obtained from ascorbate-deficient mice was elevated significantly. (B) These differences reflect the differences in neutrophils, which peaked early in control mice (•) and had returned to baseline levels by 48 h. In contrast, neutrophils in ascorbate-deficient animals ({circ}) remained elevated significantly, even at 48 h (*, P<0.001). A peak in cell numbers at 24 h and elevated cell counts in the deficient animals reflect an influx of macrophages without clearance of neutrophils. Lymphocytes were rarely seen, and the total numbers reflect the numbers of neutrophils and macrophages. Each point represents the combined mean ± SEM of three to 10 separate animals. (C) Composition and morphology of peritoneal exudate cells from control mice and ascorbate-deficient animals 8 h, 18 h, and 48 h after administration of thioglycollate. Elevated numbers of neutrophils were seen in the ascorbate-deficient mice at 18 h and 48 h. At the later time-point, neutrophils were recognizable by their ring-shaped nuclei (arrows), but there was significant loss of morphology, and the cells appeared diffuse and pale. Original bars = 20 µm.

 
Macrophage-mediated phagocytosis of aged neutrophils in vitro
Neutrophil uptake was monitored by detection of myeloperoxidase activity within cultured mouse macrophages, which had been incubated with aged neutrophils. Normal mouse neutrophils were readily taken up (Fig. 8 ), but in contrast, there was no phagocytosis of ascorbate-deficient cells, which failed to adhere to the plated macrophages and were washed away before staining. When aged, ascorbate-deficient neutrophils were incubated, the number of phagocytic macrophages was the same as if no neutrophils had been added to the cultures (Fig. 8C) .


Figure 8
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  		Figure 8. Phagocytosis of aged neutrophils by cultured mouse macrophages. When neutrophils, which had been stored for 24 h, were incubated with mouse peritoneal macrophages, normal cells (A) were phagocytosed rapidly and could be detected with a brown peroxidase stain (arrows). In contrast, ascorbate-deficient cells (B) were not taken up, and these samples could not be distinguished from those to which no neutrophils had been added (B and C), in which the extent of phagocytosis reflects the uptake of neutrophils already present with the macrophages. Results are the means ± SEM of four experiments each performed in triplicate. *, P < 0.0001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neutrophil apoptosis is a vital process that ensures the efficient and safe resolution of inflammation and prevents the release of cytotoxic and hydrolytic neutrophil granule enzymes, which can cause tissue damage [11 , 34 ]. In this study, we have shown that apoptosis is inhibited in ascorbate-deficient neutrophils. We found that the ascorbate-deficient cells failed to undergo morphological changes associated with apoptosis, despite apparently normal activation of caspase-3, and that initially, there was increased survival, but eventually the cells became necrotic. Ascorbate supplementation of the deficient cells was able to reverse these changes. Decreased surface changes reflected by PS exposure resulted in the ascorbate-deficient cells not being recognized and taken up by macrophages. That this affects events in vivo was suggested by the delayed clearance of neutrophils from the inflamed peritoneal cavity. These are novel findings that indicate an important role for ascorbate in neutrophils and may explain why it is maintained at such high concentrations in these cells.

The ascorbate-deficient cells behaved similarly to neutrophils under hypoxic conditions, which also fail to undergo constitutive apoptosis as determined by morphological changes [18 , 19 ]. This inhibition of neutrophil apoptosis can be mimicked with CoCl2 or the hydroxylase inhibitor dimethyloxaloylglycine [19 ]. Using HIF-1{alpha}-deficient myeloid cells, Walmsley et al. [19 ] have provided convincing evidence that inhibition of neutrophil apoptosis under hypoxic conditions is dependent on this transcription factor [19 , 35 ]. We detected increased levels of HIF-1{alpha} in ascorbate-deficient cells and suggest that a similar mechanism occurs in these cells. Ascorbate is known to be required for the hydroxylation of HIF-1 [20 ], and it seems likely that basal levels of HIF-1{alpha} are increased as a result of decreased activity of the proline and asparagine hydroxylases. HIF-1 is a major regulator of myeloid inflammatory cells and can affect the production of granule proteases, NO, and cytokines [36 ]. In neutrophils, however, HIF-1 is clearly antiapoptotic [19 ]. This may be linked to the induction of key glycolytic enzymes, which maintain cellular ATP levels, or to the continued expression and activation of NF-{kappa}B, which has a recognized anti-apoptotic function [19 ]. Our experiments suggest that ascorbate deficiency mimics the hypoxic response and prevents neutrophil apoptosis under normoxic conditions.

The level of HIF-1{alpha} protein in ascorbate-deficient cells was less than was achieved with 8–10 h of hypoxia or by treatment with CoCl2. It is likely that this is a result of the effect of ascorbate on the activity of the prolyl and asparaginyl hydroxylases, which do not have an absolute dependence on ascorbate but require it for optimal activity [3 , 20 ]. Increased basal levels of HIF-1{alpha} protein have been reported in ascorbate-deficient, cultured tumor cells [37 ], human neutrophils treated with the hydroxylase inhibitor dimethyloxaloylglycine [19 ], and ascorbate-deficient, primary cells [38 ]. In all these cases, this increase in basal levels of HIF-1{alpha} resulted in increased, HIF-1{alpha}-associated gene expression [37 , 38 ]. We suggest that the basal levels of HIF-1{alpha} seen in the ascorbate-deficient animals reflect the suboptimal activity of the hydroxylases under these conditions and that inhibition of apoptosis in ascorbate-deficient mouse neutrophils occurs as a result of up-regulation of HIF-1{alpha} in a manner analogous to the conditions of hypoxia.

There are some differences between hypoxia and ascorbate deficiency. In our experiments, although there was an increase in cell survival initially, necrotic cell death was obvious by 24 h, and this has not been reported for neutrophils incubated under hypoxic conditions [18 , 19 ]. That ascorbate-supplemented cells reverted to an apoptotic phenotype provides support for the hypothesis that inhibition of apoptosis was a result of inefficient hydroxylation of HIF-1. We also observed apparently normal levels of caspase-3 activation in ascorbate-deficient neutrophils, and this is generally considered to be a marker for apoptosis. Despite this, the cells did not progress to express surface PS or to undergo morphological changes typical of apoptosis (nuclear condensation, membrane blebbing). It is notable that ascorbate-deficient neutrophils were not recognized or phagocytosed by macrophages, which may explain their delayed clearance from the mouse peritoneal cavity. Whether these cells become necrotic in vivo remains to be determined, but the neutrophils present 48 h after thioglycollate infusion appeared similar to those that had been incubated in vitro, with recognizable, ring-shaped nuclei but significant loss of morphology.

Neutrophil necrosis would be devastating for the tissues if it occurred in vivo. In particular, the release of highly active proteases such as elastase, cathepsin G, and collagenase could degrade many tissues [16 , 17 ]. Whether this occurs in the ascorbate-deficient mice is currently under investigation, but it is interesting that extensive tissue injury is one of the major symptoms of ascorbate deficiency. Scurvy has a rapid onset and devastating consequences and has been described eloquently by James Lind in the 1700s [39 ]. "Bleeding and putrid gums... ulcers coated with blood and gore...patients have abdominal pain, breathe with pain and labor, and may die suddenly. It is not easy to imagine a more dismal and diversified scene of misery" [39 ]. The cause of these symptoms is likely to be multifactorial, but they are thought to have in common the cofactor activity of ascorbate in the reactions of various metal, ion-containing enzymes [1 ], particularly, the proline and lysine hydroxylases involved in collagen and extracellular matrix formation [20 ]. The Cu-containing dopamine ß-hydroxylase, which generates noradrenalin, also requires ascorbate [3 ], and loss of this activity is likely to contribute to the malaise and listlessness associated with scurvy. However, that the disease is a result of decreased collagen hydroxylation has been questioned and remains unproven [3 , 40 ]. As the injury occurs at sites of constant neutrophil activity such as the gums, lungs, and intestines, neutrophil necrosis in these areas may offer a more satisfactory explanation for the sudden and extreme devastation of scurvy. If this is the case, then the essential nature of neutrophil apoptosis and the need to control HIF-1{alpha} may indicate a vital role for ascorbate and could provide a mechanism for the positive effects of vitamin C in infection and inflammation.


   ACKNOWLEDGEMENTS
 
We thank Mark Hampton and Gabi Dachs for comments about the manuscript, Mary Morrison and Prachee Gokhalé for technical assistance, Stephanie Neal for the electron microscopy, and Sarah Walmsley for advice about HIF-1{alpha} detection in neutrophils.

Received August 30, 2006; revised December 4, 2006; accepted December 21, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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