Effect of culture PO2 on macrophage (RAW 264.7) nitric oxide production

Cynthia M. Otto1 and James E. Baumgardner2,3

Departments of 1 Clinical Studies-Philadelphia and 2 Anesthesia, University of Pennsylvania, Philadelphia 19104; and 3 SpectruMedix, State College, Pennsylvania 16803


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Macrophages are commonly cultured at a PO2 of 149 Torr, but tissue macrophages in vivo live in an environment of much lower oxygen tension. Despite the many potential mechanisms for changes in oxygen tension to influence nitric oxide (NO) synthesis, there have been few reports investigating the effect of PO2 on macrophage NO production. With the use of a culture chamber designed to rigorously control oxygen tension, we investigated the effects of culture PO2 on macrophage NO production, inducible nitric oxide synthase (iNOS) activity, iNOS protein, and tumor necrosis factor production. NO production and iNOS activity were linearly related in the range of 39.4 to 677 Torr, but not in the range of 1.03 to 39.4 Torr. Therefore, results obtained in vitro for the high oxygen tensions commonly used in cell culture were quantitatively and qualitatively different from results obtained in cells cultured at the lower oxygen tensions that more accurately reflect the in vivo environment. The influence of oxygen tension on NO production has implications for cell culture methodology and for the relationship between microcirculatory dysfunction and inflammatory responses in rodent models of sepsis.

inducible nitric oxide synthase; hypoxia; diffusion; inflammation; tumor necrosis factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

NITRIC OXIDE (NO) is widely recognized as an important messenger in numerous physiological and pathophysiological functions. Tissue macrophages are a major source of NO. Bacterial lipopolysaccharide- (LPS) and cytokine- [e.g., interleukin-1, tumor necrosis factor (TNF)] stimulated macrophages express the enzyme inducible nitric oxide synthase (iNOS) (41), which in turn generates NO from molecular oxygen and L-arginine (21, 28). The cytotoxic effects of iNOS-generated NO are thought to contribute to tissue injury in atherosclerosis, arthritis, cancer, and the systemic inflammatory response syndrome (SIRS) (6).

In vitro culture of isolated macrophages or macrophage-related cell lines has been invaluable in the study of inducible NO production. Typical environmental PO2 for in vitro cell culture, however, is much higher than the PO2 that macrophages are exposed to in vivo. For convenience, cell culture PO2 is frequently maintained in the range of room air or 149 Torr. Tissue macrophages in vivo live in an environment of much lower oxygen tension. Estimates for typical nonpulmonary tissue PO2 range from 5 to 71 Torr (5, 15, 17, 22, 38).

Variations in tissue PO2 within the physiological range, and the wider variations that accompany pathology, have many potential opportunities to influence macrophage NO production. For example, the Michaelis-Menten constant (Km) for NO production from oxygen for isolated macrophage iNOS has been reported as 5 Torr (32). A comparable Km for iNOS in vivo would imply that decreases in tissue PO2 within a physiological range could decrease macrophage NO production via simple substrate dependence. In contrast, a hypoxia-responsive element has been reported in the iNOS promoter (25), and hypoxia inducible factor is involved in the induction of iNOS in pulmonary artery endothelial cells (29). The presence of these promoter elements predicts that low oxygen would increase iNOS gene expression, which would increase NO production at low PO2. Additionally, iNOS protein is subject to posttranscriptional regulation by the protease calpain (39). Hypoxia upregulates calpain expression and activity (42), suggesting a mechanism for increased iNOS degradation during hypoxia.

We therefore tested the hypothesis that cell culture PO2 influences NO production, iNOS activity, and iNOS protein in the RAW 264.7 macrophage cell line. With the use of a culture system that was specially designed to rigidly control headspace PO2 (the oxygen tension of the gas phase in the culture chamber) and to minimize cell handling and activation, we investigated the effects of culture PO2 for oxygen tensions ranging from pathological hypoxia to supraphysiological hyperoxia.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell line. All studies were performed with the mouse macrophage cell line RAW 264.7 (American Type Culture Collection, Manassas, VA). Cells were initially cultured under standard conditions (5% CO2 in air in a humidified environment at 37°C) with DMEM (BioWhittaker, Walkersville, MD), 10% heat-inactivated FCS (Life Technologies, Rockville, MD), and 1% antibiotic/antimycotic (penicillin, streptamycin, and fungizone; Life Technologies). When cells reached 90% confluence, they were plated onto 13-mm-diameter cell culture-treated coverslips (Nunc, Naperville, IL), placed in 60-mm2 dishes with DMEM/FCS, and cultured under standard conditions. When the cells on the coverslips were 90% confluent with a viability of >85%, each coverslip was transferred to the bottom of a 15-mm × 45-mm sterile glass vial (Kimble Glass, Vineland, NJ).

Culture system. It has been demonstrated that exposure of cells to one stimuli can lead to either priming (enhanced response) or tolerance (diminished response) to a second stimulus (8). It is possible that methods to elicit and isolate native macrophages result in priming or activation (8, 11). We minimized handling and potential stimulation of the cells by allowing them to grow on coverslips, which were then transferred to the 15-mm × 45-mm vials, eliminating the effects of adherence (11). Four hundred microliters of serum-free DMEM (+ 1% antibiotic/antimycotic) were added to each vial, thereby covering the cells with ~2-3 mm of media. The media was overlaid with 50 µl of sterile cell culture-tested mineral oil (Sigma, St. Louis, MO) to prevent evaporation. The vials were held upright in dry 10-ml beakers (Kimble Glass), which were then placed in a 5- to 10-mm layer of water in the bottom of a sealed 500-ml glass Ehrlemeyer flask fitted with gas inlet and outlet ports. Flasks were submersed in a 37°C waterbath.

Control of headspace PO2. A common obstacle to the study of cultured cells in low-oxygen environments is the difficulty of reliably producing headspace PO2 in the physiological range. Mean PO2 in various tissues has been estimated from 5 to 71 Torr (5, 15, 17, 22, 38). These are estimates for PO2 in the tissue that would typically surround nonpulmonary macrophages. Accounting for macrophage oxygen consumption and the diffusional gradient of oxygen from the surrounding tissue to the cell would result in even lower estimates for typical macrophage oxygen tensions in vivo. Tissue PO2 is further decreased by the microcirculatory dysfunction (13) or diffusion impairment (4) that accompanies many diseases. Therefore, the typical PO2 of macrophages in vivo is substantially lower than common cell culture conditions, including cell culture conditions typically defined as "hypoxic" (1, 2, 18). Achievement of a reduced oxygen environment in cell culture is a substantial challenge because even small convective or diffusive leaks in the walls of the culture chamber will admit relatively large amounts of oxygen. Oxygen also dissolves in most plastics and can thereafter remain a source of oxygen for hours (35). Consequently, purging and sealing culture chambers with anoxic gas mixtures frequently does not result in headspace PO2 low enough to simulate tissue hypoxia, nor does it result in steady-state oxygen tensions. Oxygen tensions reported for cultures purged with anoxic gas mixtures of 95% N2 and 5% CO2 range from 9 Torr (9) to 31.7 Torr (2) after 24 h.

We overcame some of these limitations by use of a culture chamber that we designed to provide minimal convective leaks, with wall materials that resisted oxygen permeation by diffusion. We avoided plastics inside the culture chamber that could absorb oxygen on exposure to room air. After an initial purge flow of 2.4 l/min for 3 min, headspace PO2 was maintained with a constant flow of 8 ml/min, bubbled through the thin layer of water in the bottom of the 500-ml flask that held the sample vials. Maintenance of headspace PO2 within 0.35 Torr of the specified oxygen partial pressure was confirmed by micropore membrane inlet mass spectrometry (3).

Culture environment. Cells were cultured in eight different oxygen tensions: 1.03, 7.91, 24.1, 39.4, 80.0, 141, 356, and 677 Torr. Certified premixed compressed gas cylinders, all with 5% CO2 and balance nitrogen (BOC gases; Murray Hill, NJ), provided the headspace gas for each of the eight culture conditions. For each headspace gas, two of the four vials per flask were stimulated with 100 ng/ml LPS (Escherichia coli, 0111:B4, Sigma) and 100 U/ml recombinant mouse interferon (IFN)-gamma (Life Technologies); the remaining two vials were unstimulated. At least five experiments were performed for each gas tension. We also evaluated the effect of IFN (100 U/ml) in the absence of LPS on NO production and iNOS activity in cells cultured at 7.91 and 24.1 Torr (n = 5).

Sample collection. At the end of the 18-h culture period, the supernatants were removed and frozen at -70°C. The cells were rinsed twice with PBS to remove any residual oil, media, and dead cells. One hundred fifty microliters of lysis solution [5 µg/ml pepstatin A, 5 µg/ml aprotinin, and 100 µl/l phenylmethylsulfonyl fluoride in deionized H2O (ICN Biomedicals, Aurora, OH)] were added to the vials before freezing at -70°C. Cells were subjected to three freeze cycles at -70°C followed by thaw on ice. At each thaw cycle, the cells were vigorously mixed. At the final thaw cycle, samples were removed for iNOS activity, Western blot analysis, and protein assay.

Protein assay. Protein concentration of the cell lysates was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). A protein standard curve was generated using bovine serum albumin. To determine the relationship between cell lysate protein and cell number, RAW 264.7 cells were counted and immediately lysed using the freeze-thaw protocol as described above. A regression analysis demonstrated a strong correlation between cell number and lysate protein concentration (r2 = 0.9996). All samples were run in duplicate. In a separate experiment, the effect of hypoxia on the relationship between total cellular protein and cell number was evaluated. Cells were cultured for 18 h under constant flow for PO2 of 1, 40, and 141 Torr. Cells were cultured in serum-free DMEM with LPS and IFN in 100-mm-diameter plates placed in a modified version of the glass culture chambers. At the termination of the experiment, cells were scraped and counted. Known numbers of cells (1 × 105, 5 × 105, and 1 × 106) were then lysed and protein content measured. Four separate experiments were performed for each gas tension.

Nitrite analysis. Nitrite, a stable byproduct of NO production, was measured in the culture supernatant using the Greiss reagent as previously described (7). Fifty microliters of the culture supernatant were added to 96-well plates that contained 50 µl of freshly mixed Greiss reagent. All samples were run in duplicate. A standard curve with 1.95 to 250 µmol/ml sodium nitrite (Sigma) in DMEM was used. After a 10-min incubation at room temperature, the plates were read on a Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA) at 550 nm. The results were computed using the Softmax program (Molecular Devices). Because of variation in initial cell number and cell proliferation during the culture time, nitrite values were normalized to cell protein concentration.

iNOS activity. The assay of iNOS activity was adapted from Weinberg et al. (40). Briefly, 30 µl of the cell lysis solution were removed and incubated with 20 µl of reaction buffer [final concentration 50 mM HEPES (pH 7.4), 200 µM NADPH (Sigma), 1 mM dithiothreitol (Fisher Scientific, Pittsburgh, PA), 10 µM FAD (ICN), 100 µM tetrahydrobiopterin (BH4, Sigma), 10 µM L-arginine (Sigma), 0.4 µl [14C]arginine labeled in the guanido position at a radioactive concentration of 50 µCi/ml, and specific activity of 396 µCi/µmol (Amersham, Arlington Heights, IL)]. Samples were incubated in duplicate for 30 min at room temperature and then loaded on a 0.4-ml Dowex AG cation exchange resin (50W-X8, Bio-Rad) column and washed twice with 3 ml of deionized water. Measurement of L-citrulline was determined by lack of adherence to the Dowex resin. Specific activity was measured following the addition of 16 ml of Luminex (ICN) scintillation fluid on a Beckman LS6500 scintillation counter. Counts per minute were normalized to cell protein concentration.

iNOS Western blot. In six experiments, each at 1.03, 7.91, 24.1, 39.4, 80.0, and 141 Torr, we collected cell lysates for iNOS protein content. In addition, cell lysates were pooled from several LPS/IFN-stimulated cultures performed under standard conditions (149 Torr O2). The pooled lysates from standard conditions were used to demonstrate that the iNOS protein content was linearly related to the amount of protein loaded and to provide a standard designated as 100%. Equal amounts of protein (8 µg) from each experiment and the pooled standard were loaded on 12% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with PBS that contained 0.03% Tween 20 for a minimum of 90 min and then washed three times with PBS that contained 0.05% Tween 20. Western blot analysis was performed by incubating the membrane with a polyclonal rabbit anti-iNOS antibody (SC651; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1,000 in 5% dried milk in PBS with 0.05% Tween 20 for 30 min. After three washes, the membrane was incubated with a horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (Amersham Pharmacia Biotech, Piscataway, NJ) 1:5,000 in 5% dried milk in PBS with 0.05% Tween 20 for 30 min. After three washes, signal density was visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) and quantified (mean optical density times area, corrected for background) using analysis software (Bio-Rad Multi-Analyst/PC version 1.1). For statistical analysis, values were expressed as the ratio of density of iNOS protein from lysates of LPS/IFN-stimulated cells to the density of 8 µg of a pooled lysate derived from LPS/IFN-stimulated cells cultured under standard conditions.

Measurement of the O2 gradient. In a separate set of experiments, we measured the PO2 gradient in representative culture media. We mounted a 0.3-mm fiberoptic, phosphorescence-quenching oxygen sensor (Foxy AL300 fiberoptic oxygen sensor; Ocean Optics, Dunedin, FL) on a micropositioning stage with 10-µm resolution (Parker-Daedal 4592M). The oxygen probe was calibrated in media equilibrated with either pure nitrogen or 39.4 Torr oxygen. The cell culture system used to determine the oxygen gradient was identical to that described above with the following additions 1) the flask was covered in aluminum foil to eliminate ambient light and 2) before covering, the probe was passed retrograde through the gas exit into the flask with alignment along the sidewall of one of the vials of unstimulated cells.

The media thickness was measured at the center (1.9 mm) and at the edge (4.0 mm) of the vial. To calculate the average media thickness, we assumed that the meniscus was a portion of a sphere and derived an expression for the media thickness as a function of radial distance from the center. This expression was integrated to calculate an area-weighted average thickness of 2.87 mm. The coordinates of the media surface were also used to calculate the area-weighted average PO2 at the cell surface based on the measured average oxygen tension gradient. PO2 at the cell surface was estimated (assuming the same diffusivity, solubility, and convection in the media for all experimental cultures and the cultures used to measure the oxygen gradient) and corrected for measured cell density. Uniform oxygen consumption for PO2 of 24.1-677 was assumed, with a correction for the reported decrease in specific metabolic rate after LPS stimulation (14).

Lactate dehydrogenase. Randomly selected samples of cell lysate and paired supernatant were assayed for lactate dehydrogenase (LDH) activity using a cytotoxicity detection kit (Boehringer Mannheim, Roche Molecular Biochemicals, Indianapolis, IN). At least six samples for stimulated and two samples for unstimulated cells were assayed for every PO2.

pH. The pH of the media was determined by insertion of a pH microelectrode with a 16-gauge needle tip (Orion Research, Beverly, MA) into an average of four randomly selected samples of supernatant (both stimulated and unstimulated) for each experimental oxygen tension. Samples were equilibrated with 5% CO2 at 37°C. The value for pH was recorded when the reading on the pH meter (Orion model 520A, Orion Research) reached steady state.

TNF assay. TNF was quantified in four or five randomly selected culture supernatants from LPS/IFN-stimulated cells for each PO2 using the Quantikine M Mouse TNF-alpha immunoassay kit (R and D Systems, Minneapolis, MN). Supernatants were diluted 1:500 in PBS and assayed according to the manufacturer's instructions. TNF concentrations were normalized to cell protein concentration.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The PO2 dependence of macrophage NO production is shown in Fig. 1. NO production by unstimulated cells was negligible at all oxygen tensions. NO production by stimulated cells decreased as culture oxygen tension decreased throughout the entire range of PO2 (P < 0.01, one-way ANOVA). Variations in the activity of extracted iNOS with culture PO2 are shown in Fig. 2. iNOS activity for unstimulated cells was insignificant for all PO2. For stimulated cells, iNOS activity varied directly with PO2 in the range 39.4 to 677 Torr. The addition of IFN alone to the media did not increase iNOS activity at either 7.91 or 24.1 Torr.


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Fig. 1.   Effect of PO2 on nitric oxide (NO) production. Nitrite production (normalized to cell protein) vs. PO2 for lipopolysaccharide/interferon (LPS/IFN)-stimulated () and unstimulated (open circle ) RAW 264.7 cells. Symbols represent means ± SE of the mean (minimum n = 8, maximum n = 12) for each headspace oxygen tension from 1.03 to 677 Torr. In the stimulated cells, the oxygen tension had a significant effect on NO production. P < 0.01, one-way ANOVA.



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Fig. 2.   Effect of PO2 on inducible nitric oxide synthase (iNOS) activity. iNOS activity (normalized to cell protein) vs. PO2 for LPS/IFN-stimulated and unstimulated RAW 264.7 cells. Symbols represent means ± SE of the mean (minimum n = 7, maximum n = 12) for each headspace oxygen tension from 1.03 to 677 Torr. For stimulated cells, there was a significant effect of oxygen tension on NO production. cpm, Counts per minute. P < 0.01, one-way ANOVA.

To assess the impact of variations in iNOS activity on the PO2 dependence of NO production, NO production as a function of iNOS activity has been plotted in Fig. 3. In the range of PO2 from 39.4 to 677 Torr, NO production and iNOS activity were linearly related with extrapolation through the origin, suggesting that iNOS activity determines NO production at high PO2. To investigate the potential for substrate limitation in the lower oxygen tensions, we normalized NO production to the measured iNOS activity for each experiment. Figure 4 shows the PO2 dependence of NO production when the NO production is scaled according to the measured changes in iNOS activity, with a logistic equation fit to the data by nonlinear regression. NO production was 50% of maximal at a headspace PO2 of 30 Torr.


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Fig. 3.   Relationship between iNOS activity and NO production as culture PO2 varies. iNOS activity in cpm [14C]citrulline per milligram is plotted vs. nitrite in nanomoles per milligrams over 18 h for LPS/IFN-stimulated cells. The upper 5 headspace oxygen tensions (, 39.4-677) and the lower 3 (open circle , 1.03-24.1) represent means ± SE of the mean of iNOS activity and NO production. The line is a forced linear regression through the origin for the top 5 headspace oxygen concentrations.



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Fig. 4.   Scaled NO vs. log10(PO2). Data points are nitrite production divided by the measured iNOS activity for each experiment (with error bars representing SE of the mean) vs. log10(PO2). The solid line is a weighted nonlinear regression (Marquardt-Levenberg) using a logistic equation. The sigmoid relationship between NO and iNOS activity in the range 7.91 to 39.4 Torr is consistent with regulation of NO production by oxygen substrate limitation. The continued production of NO as PO2 approaches 0 suggests iNOS-independent NO production (43) via reduction of nitrite by xanthine oxidase (10) or mitochondria (19).

To determine whether the extracted iNOS activity was representative of iNOS protein, we measured iNOS protein in cell lysates by Western blot analysis. The mean iNOS protein density for stimulated cells decreased with decreasing PO2 (Fig. 5). iNOS protein was significantly lower at 1.03 and 7.91 Torr than at 141 Torr (one-way ANOVA, pairwise comparison by Student-Newman-Keuls test; P < 0.05). There was no detectable iNOS protein in unstimulated cells.


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Fig. 5.   Effect of PO2 on iNOS protein. Representative Western blot of lysates (8 µg loaded/lane) from LPS/IFN-stimulated cells cultured under standard (Std) conditions, 1.03, 7.91, 24.1, 39.4, 80.0, and 141 Torr using anti-iNOS antibody (1:1,000).

To investigate the effects of diffusion limitation in causing the cell PO2 to be different from headspace PO2, we measured PO2 of the media as a function of depth from the media surface (Fig. 6) in representative cell cultures. The average PO2 gradient (n = 4) for unstimulated cells with a headspace PO2 of 39.4 Torr was 6.9 ± 5.5 Torr/mm (mean ± SD). For comparison, if transport had been solely by diffusion with no convection as reported by Metzen et al. (27), the calculated gradient would be 84.0 Torr/mm. Estimated PO2 at the cell surface based on this average PO2 gradient is presented in Table 1. For headspace PO2 of 1.03 and 7.91 Torr, the estimated cell PO2 was negative. These cells must have reduced their metabolic uptake to adapt to their low-PO2 environment (33), and the estimated PO2 in Table 1 for these cultures has been listed as <1 Torr. The oxygen gradient measured in the 7.91 Torr culture (Fig. 6) shows a reduced PO2 gradient and a cell surface PO2 <1 Torr. To examine the influence of different culture PO2 on cell survival, we assayed LDH activity in supernatant and cell lysates. The ratio of supernatant to lysate LDH activity in Fig. 7 provides an index of cell death. There was a significant increase in the supernatant:lysate LDH ratio in stimulated cells cultured at 1.03 and 7.91 Torr compared with 141 Torr (ANOVA on ranks, post hoc pairwise comparison using Dunn's method, P < 0.05). Figure 7 also shows the effect of culture PO2 on cell density. There was a trend for the density of stimulated cells to decrease at 1.03 and 7.91 Torr (Fig. 7).


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Fig. 6.   PO2 as a function of depth in the media. The media surface is at x = 0; the cells are at a depth of ~4.0 mm. Measurements were made at the end of an 18-h culture period in 2 separate cultures of unstimulated RAW cells exposed to 39.4 Torr oxygen (open circle , ) and 1 culture exposed to 7.91 Torr (). The average gradient for 4 sets of measurements was 6.9 ± 5.5 Torr/mm in the 39.4 Torr cultures. Lines are linear regressions forced through the surface PO2 for each experiment.


                              
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Table 1.   Calculated PO2 at the cell for each headspace gas



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Fig. 7.   Lactate dehydrogenase (LDH) ratio and cell density vs. PO2. The mean ratio of supernatant LDH to lysate LDH ± SE of the mean is represented by  for stimulated cells (n = a minimum of 6) and open circle  for unstimulated cells (n = a minimum of 2). The mean cell density ± SE of the mean is represented by black-triangle for stimulated cells and triangle  for unstimulated cells. Results from all experiments (average n = 13) are included. *LDH ratios (for stimulated cells) are different from value at PO2 of 141. P < 0.05, ANOVA on ranks, post hoc pairwise comparison by Dunn's method.

The relationship between total cell protein content and cell number was linear and similar at all three culture PO2 tested, suggesting that normalization to protein content is equivalent to normalization to cell number, despite reported decreases in new protein synthesis in hypoxia (12, 34). These results are consistent with the findings of Simon et al. (34) for macrophages cultured for 24 h in low concentrations (2%) of serum. The pH of the media was not related to PO2.

To determine whether the PO2-dependent decrease in NO production reflected a global decrease in macrophage LPS responsiveness, we measured TNF in the supernatant of LPS/IFN-stimulated cells. There was no relationship between PO2 and LPS/IFN-stimulated TNF production (ANOVA on ranks; P = 0.388; Fig. 8).


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Fig. 8.   Effect of PO2 on tumor necrosis factor (TNF). TNF (normalized to cell protein) in the supernatants from stimulated () cells vs. PO2. Symbols represent mean ± SE of the mean for a minimum of 4 experiments for each headspace oxygen tension from 1.03 to 677 Torr.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The oxygen tension of the cell cultures had a major influence on macrophage production of NO. The data in Fig. 3 clearly show a linear relationship between NO production and iNOS activity in the higher (39.4-677 Torr) PO2 ranges, including those typically used in cell culture. NO production was not closely related to iNOS activity, however, in lower PO2 representative of the physiological and pathophysiological ranges (1.03-39.4 Torr). Therefore, results obtained in vitro for the high oxygen tensions commonly used in cell culture were quantitatively and qualitatively different from results obtained from cells cultured at the lower oxygen tensions that more accurately reflect the in vivo environment. These data emphasize the importance of careful characterization and control of the cell culture oxygen tension if in vitro responses are to be extrapolated to predict function in vivo.

No studies of variation in NO production with culture PO2 in the range of 141 to 677 are available for comparison with the data in Fig. 1. There are, however, a few prior studies of inducible NO production at low oxygen tensions. Melillo et al. (26) showed a 70% decrease in nitrite production in the ANA-1 mouse macrophage cell line cultured at low PO2, similar to our findings of Fig. 1. Albina et al. (1) reported a reduction of citrulline synthesis at low PO2 for native rat macrophages. Reductions in NO production have also been demonstrated in rat mesangial cells (2) and rat cardiac myocytes (16) cultured in low-oxygen environments.

In contrast to the consistent relationship between low PO2 and NO production, the reported effects of PO2 on the iNOS enzyme are conflicting. Albina et al. (1) reported an increase in iNOS protein in wound-derived but not resident macrophages cultured in low PO2. Their observation of iNOS protein in unstimulated wound-derived macrophages, however, suggests that these cells were primed and may have responded differently than naïve macrophages. Melillo et al. (26) reported a parallel increase in cytosolic iNOS activity and iNOS protein in IFN-stimulated macrophages, but not in unstimulated cells, cultured in low O2. Although the signal transduction pathways for IFN- and LPS-mediated iNOS expression are different (30), our studies evaluating IFN alone suggest that the differences between the results reported here and those of Mellilo et al. (26) are not the result of an IFN-specific effect. In contrast to the reports of increased iNOS in low-PO2 cultures, Kacimi et al. (16) demonstrated that low oxygen tensions inhibited iNOS protein in interleukin-1beta -stimulated cardiac myocytes. Our results from Fig. 2 demonstrate that, similar to Kacimi (16) and Melillo (26), hypoxia did not induce iNOS activity in the unstimulated RAW 264.7 cells. In stimulated RAW cells cultured in serum-free media and rigorously controlled headspace PO2, iNOS activity (Fig. 2) and iNOS protein (Fig. 5) decreased with decreasing oxygen, similar to the results reported by Kacimi et al. (16) for myocytes cultured under similar conditions. Conflicting results concerning iNOS production at reduced PO2 may be due to differences in cell type, control of the culture PO2, and the use of serum vs. serum-free media.

In addition to the difficulty in controlling headspace PO2, an additional explanation for the discrepancies between results generated by different culture apparatus is that the relationship between headspace PO2 and cellular PO2 is highly dependent on the amount of convective mixing in the culture media. Macrophage PO2 is determined not only by the PO2 of the headspace but also by the gradient in oxygen tension from the headspace to the cell surface. This PO2 gradient is determined by cell density, specific oxygen consumption, thickness of the media, diffusivity of oxygen in the media, and convective mixing of the media. Reports of cell culture conditions rarely mention any of these factors. Metzen et al. (27) mathematically modeled diffusion of oxygen through media and showed the potential for PO2 at the cell to be substantially lower than headspace PO2 if cell density and oxygen consumption were large and oxygen transport was purely by diffusion with no convection. They also measured very low oxygen tensions in the vicinity of cultured cells, implying a large gradient in the media. In contrast, the gradients we measured (Fig. 6) were substantially smaller than gradients predicted purely from diffusive transport, most likely due to convective effects (thermal convection currents and vibrational convection) that can substantially increase transport beyond pure diffusion (20).

Numerous mechanisms could potentially be responsible for the decrease in macrophage NO production as culture oxygen tension is reduced (Fig. 1). The mechanisms can be broadly divided into two categories: 1) reductions in the total amount of iNOS or in the capacity for a given amount of iNOS to produce NO, as oxygen tension is reduced; and 2) reduced substrate availability for NO production at low oxygen tensions.

Several mechanisms could have reduced the total amount of iNOS or iNOS efficacy as cell culture PO2 decreased. The simplest explanation for the PO2 dependence of NO production (Fig. 1) would be that hypoxia led to decreases in cell density that reduced the total amount of iNOS. Variations of cell density with culture PO2, however, were minimal over the entire range of PO2 (Fig. 7). Additionally, the small changes in cell density due to different culture PO2, as well as changes in density due to variation between experiments, were accounted for by normalizing the nitrite, iNOS activity, and TNF data for measured cell protein.

Another possible mechanism for the influence of culture oxygen tension on macrophage iNOS production is a nonspecific inhibition of the cellular response to LPS as culture PO2 is reduced. The data in Fig. 8, however, show that culture PO2 had no influence on production of TNF, one of the predominant inflammatory cytokines expressed by macrophages in response to LPS stimulation. This makes generalized downregulation of the inflammatory response an unlikely explanation for the influence of PO2 on iNOS.

Another potential mechanism for reduced NO production at low PO2 is a specific iNOS inhibitory effect of mediators released from dying cells. Although not statistically significant, there was a trend toward reduced cell density at 1.03 and 7.91 Torr (Fig. 7). The increased LDH ratio at low PO2 (Fig. 7) suggests that these reductions in cell density were due to a slightly increased rate of cell death in the lowest oxygen environments. The pH of the culture media was not decreased at a PO2 of 1.03 Torr. Therefore, it is unlikely that the increased cell death was a consequence of energy failure and lysis after a period of anaerobic metabolism.

The decrease in iNOS activity with decreasing PO2 in the range of 677 to 39.4 Torr (Fig. 2) and the decrease in iNOS protein in the range of 141 to 7.91 Torr (Fig. 5) could also be mediated by increases in calpain expression in hypoxia (42), contributing to increased iNOS degradation (39).

In addition to these potential mechanisms for alterations in iNOS activity, the decrease in NO production as oxygen tension is reduced (Fig. 1) could also be mediated by effects of oxygen tension on substrate availability for NO production. Because the reaction that produces NO and citrulline from arginine includes molecular oxygen as a substrate (21), the most obvious explanation for reduced substrate availability at low oxygen tensions is that oxygen became the rate-limiting substrate for the production of NO. The data in Fig. 4, where NO production has been scaled according to the amount of measured iNOS activity, is consistent with oxygen as a rate-limiting substrate for culture PO2 in the range 7.91 to 39.4 Torr. The calculated PO2 at 50% reaction velocity (30 Torr for the headspace gas with an estimate of 14 Torr at the cell surface) is close to the reported Km for isolated iNOS of 5 Torr (32). The relationship between the Km of the isolated enzyme in vitro and the Km of iNOS in intact cells, however, is unknown. In particular, cytoplasmic levels of the many known cofactors (i.e., BH4, NADPH, FAD, and heme) (36) for the reaction have not been measured. Additionally, we did not assess many of the other potential mechanisms that would need to be controlled or accounted for to establish that oxygen substrate availability is the sole mechanism in this range of PO2.

Alterations in culture PO2 could also have led to changes in the availability of arginine, the other substrate for NO synthesis (28). Hypoxia is reported to increase arginase but also increases the arginine transporter (24).

There are several limitations of the current study that could influence interpretation of results. Similar to many other studies, we used measurements of nitrite, a product of NO degradation, to estimate production of NO. It is possible that hypoxia affected the relative conversion of NO to nitrate vs. nitrite (2), which would impact our estimates of NO production.

We used a standard technique to measure extracted iNOS activity in an excess of cofactors (26, 32, 40). Although this should provide a reasonable estimate of relative changes in iNOS activity in the cells, the role of hypoxia in regulation of cofactors necessary for iNOS activity is unknown. For example, depletion of BH4 could result in failure of enzyme dimerization and thus decreased iNOS activity in situ (36), which would have been reversed when BH4 was provided in excess.

NO production was estimated as the integral of NO production over the whole 18-h culture period. In contrast, iNOS was assessed at the end of the culture period. Macrophage NO production was reported to reach steady state within 9 h of LPS/IFN stimulation under standard conditions (23), suggesting that most of the 18-h culture period represented steady state. Depending on the kinetics of both NO production and iNOS production, however, it is possible that important features in the relationship between NO production and iNOS activity were missed.

The PO2 dependence of NO production, iNOS activity, and iNOS protein in Figs. 1, 2, and 5 has significant implications for cell culture as well as for the interrelationship of microcirculatory and immune dysfunctions in disease. Macrophage NO production was linearly dependent on iNOS activity when the cells were cultured in a range of PO2 from 39.4 to 677 Torr. Macrophage NO production was not closely related to iNOS activity, however, when the cells were cultured in a more physiological range of PO2. These results indicate the importance of reproducing a realistic oxygen environment in vitro if in vivo functional behavior is to be accurately predicted.

These results also imply a potential link between microcirculatory derangements and inflammatory responses in the pathogenesis of disease. Alterations in both microcirculatory function (37) and inflammatory response (31) are prominent components of the rodent cecal ligation and puncture model of sepsis and SIRS. The data in Fig. 1 demonstrate that macrophage NO production was influenced by oxygen tension over the range of physiological and pathophysiological PO2. These results, therefore, suggest a potential connection between microcirculatory derangements, which alter tissue PO2 within this range, and the inflammatory response, as modulated by macrophage NO production. It is also possible for signaling to occur in the other direction, i.e., it is possible that changes in macrophage NO production could alter microcirculatory control owing to the role of NO in the control of vascular smooth muscle. So little is known about NO transport in tissue in vivo, however, that it is impossible to predict whether the changes in rates of macrophage NO production we observed in cell culture could influence vascular smooth muscle.


    ACKNOWLEDGEMENTS

The authors thank Maureen Callaghan, Julia Fox, and Troy Hallman for technical assistance.


    FOOTNOTES

This work was supported by Grant 97-08283A from The American Heart Association, Southeastern Pennsylvania Affiliate.

Address for reprint requests and other correspondence: C. M. Otto, Dept. of Clinical Studies-Philadelphia, Univ. of Pennsylvania, 3900 Delancey St., Philadelphia, PA 19104-6010 (E-mail: cmotto{at}vet.upenn.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 10 January 2000; accepted in final form 12 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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