HYPOXIA INDUCES THE EXPRESSION AND RELEASE OF INTERLEUKIN 1 RECEPTOR ANTAGONIST IN MITOGEN-ACTIVATED MONONUCLEAR CELLS

doi:10.1006/cyto.2001.0842, available online at http://www.idealibrary.com on

HYPOXIA INDUCES THE EXPRESSION AND RELEASE OF INTERLEUKIN 1 RECEPTOR ANTAGONIST IN MITOGEN-ACTIVATED MONONUCLEAR CELLS Antonella Naldini, Annalisa Pucci, Fabio Carraro Hypoxia modulates the expression of inflammatory mediators in a variety of cell types. Since interleukin (IL-)1 receptor antagonist (Ra) is a cytokine widely associated with an inflammatory state and is expressed by activated mononuclear cells, we investigated whether hypoxia induces IL-1Ra expression in human peripheral blood mononuclear cells (PBMC) activated by phytohaemagglutinin (PHA). RNase protection assay, conducted on PHA-activated PBMC cultured under hypoxic conditions (2% O2) for 16–40 h, revealed that hypoxia enhances IL-1Ra mRNA expression. Further, IL-1Ra release was significantly affected by hypoxia, as determined by ELISA. Concomitantly, hypoxia enhanced, even though at a lesser extent, both IL-1
and IL-1 mRNA expression and release, as determined by RPA and ELISA. However, at 40 h of treatment, hypoxia did not affect cell viability and DNA fragmentation, but caused an inhibition of the proliferation index after PHA stimulation, obtained by MTT assay. These results suggest that activated mononuclear cells tend to respond to hypoxic stress by modulating the expression of IL-1Ra and IL-1-related molecules and their release in the surrounding microenvironment.  2001 Academic Press

Hypoxia is a common modification of the extracellular environment that can affect cellular physiology. However, cells tend to maintain homeostasis by responding to this basic environmental stress in different ways.1,2 They reduce protein synthesis but, at the same time, increase the expression of stress-responsive genes, such as hypoxia-inducible factor (HIF)-1
.3 Hypoxia affects the release of different cytokines by peripheral blood mononuclear cells (PBMC)4 and the expression of interleukin (IL-)6 and IL-1
in endothelial cells.5,6 We previously reported that hypoxia affects the biological activities of tumour necrosis factor (TNF)-
7 and regulates the release of cytokines as well as cell proliferation in PBMC.8 IL-1 is a cytokine well known for its pleiotropic activities on a variety of cell types.9 There are three molecules associated with IL-1: IL-1
, IL-1 and IL-1 receptor antagonist (Ra); IL-1Ra blocks the activities From the Institute of General Physiology, University of Siena, Siena, Italy Correspondence to: Dr Antonella Naldini, Institute of General Physiology, University of Siena, Via Aldo Moro, 53100 Siena, Italy. E-mail: [email protected] Received 22 November 2000; accepted for publication 9 January 2001  2001 Academic Press 1043–4666/01/060334+08 $35.00/0 KEY WORDS: cytokines/hypoxia/IL-1Ra/inflammation/ischemia 334

of IL-1
and IL-1 by binding to IL-1 receptors. IL-1-related molecules are present in several physiological and physio-pathological situations, where hypoxia also usually occurs, and IL-1 is considered a highly inflammatory cytokine. In fact, hypoxia is a condition that resembles inflammation;5 it induces the expression of inflammatory cytokines10 and molecules11 and is usually associated with a poor outcome in several human disorders. In neuronal hypoxia, IL-1 is involved in controlling cell apoptosis by an IL-1-converting enzyme (ICE)-dependent mechanism12 and hypobaric hypoxia has been associated with cerebral oedema, due to changes in vascular permeability.13 It has been recently reported that high altitude increases circulating IL-1Ra and this may be related to the development of high-altitude pulmonary oedema.14 In addition, IL-1 is also involved in hypoxia-induced vascular smooth muscle cell and endothelial cell proliferation and it may contribute to the onset of certain vascular diseases, such as arteriosclerosis and chronic pulmonary hypertension.6,15 For these reasons we decided to determine the effects of hypoxia on the expression of IL-1Ra, as well as of IL-1
and IL-1, in PBMC stimulated with the lectin phytohaemagglutinin (PHA), a well known PBMC activator. The expression of IL-1Ra, IL-1
and IL-1 was evaluated by RNase protection assay. CYTOKINE, Vol. 13, No. 6 (21 March), 2001: pp 334–341

Hypoxia enhances IL-1Ra expression / 335

16 h Medium N H

N

40 h PHA H

Medium N H

N

PHA H

IL-1Ra L32

*

150 Area units

Hypoxia enhanced the expression of IL-1Ra and, even though at a lesser extent, of IL-1
and IL-1 in PHA-activated PBMC, suggesting that IL-1-related molecules might play a role in hypoxic conditions. Enhanced IL-1Ra, IL-1
and IL-1 mRNA expression was associated with an increased release of IL-1Ra, as well as of IL-1
and IL-1, as determined by ELISA. Finally, to rule out the possibility that hypoxia-induced IL-1 molecule expression was the consequence of apoptotic cell death,12 we tested the effect of hypoxia on cell viability and DNA fragmentation. The present results confirm the hypothesis that hypoxic cells respond to extracellular environmental stress by expressing and releasing different molecules involved in defence and rescue mechanisms and that IL-1Ra expression by hypoxic PBMC is particularly indicated for the study of cellular responses during hypoxia.

100

*

50

0

16

40 Time (h)

RESULTS Hypoxia upregulates the expression of IL-1Ra Since high altitude increases circulating IL-1Ra and mononuclear cells are sensitive to the effects of hypoxia, IL-1Ra gene expression was assessed in resting and PHA-activated PBMC. IL-1Ra mRNA levels were not detectable in resting PBMC. However, exposure to hypoxia enhanced the expression of IL-1Ra mRNA in PHA-activated PBMC (Fig. 1). After either 16 or 40 h of hypoxic treatment, PHA-activated PBMC expressed higher amounts of IL-1Ra mRNA. When compared with the corresponding time control-PBMC, IL-1Ra mRNA levels were increased in hypoxic PBMC by 2.3- and four-fold, respectively, after 16 and 40 h of exposure to 2% O2.

Hypoxia enhances IL-1
and IL-1 expression To investigate further the effect of hypoxia on the IL-1 system, IL-1
and IL-1 gene expression was assessed in resting and PHA-activated PBMC. Both IL-1
and IL-1 RNA levels were not detectable in resting PBMC. Hypoxia induced the expression of IL-1
mRNA (Fig. 2) in PHA-stimulated PBMC after 16 h of hypoxic treatment (2.8-fold). However, IL-1
mRNA was not detectable after 40 h of treatment in either normoxic or hypoxic activated PBMC. After 16 h of exposure to hypoxia, IL-1 mRNA (Fig. 3) was elevated in both normoxic and hypoxic PHA-activated PBMC. However, after 40 h of exposure to hypoxia IL-1 mRNA levels increased 2.7-fold compared with the levels in PHA-activated PBMC incubated at 20% O2.

Figure 1.

Effect of hypoxia on IL-1Ra expression.

PBMC were cultured at a concentration of 1106 cells/ml in the presence or absence of PHA (5 g/ml) and exposed to either an aerobic (N, ) or hypoxic environment (H, ) for 16 and 40 h. Cell lysates were analyzed by RNase protection assay as reported in the Materials and Methods section. L32 was used as housekeeping gene. Data presented in the upper panel are the results of one of three similar experiments. Quantification was achieved with the Sigma Gel analysis software; the results, representing the percentage of area of each band calibrated on the L32 housekeeping gene band, are shown in the lower panel. Data presented are the meanSEM of three independent experiments. Asterisks indicate statistically significant (P<0.05) differences between IL-1Ra mRNA expressed in PHA-activated PBMC incubated in hypoxia vs aerobic controls.

Hypoxia enhances IL-1Ra release Since hypoxia increases the expression of IL-1Ra mRNA, we next investigated whether hypoxic PBMC release a higher amount of IL-1Ra in the extracellular compartment. To test this hypothesis, the presence of IL-1Ra was assessed in the supernatant of hypoxic PBMC (Fig. 4). After 40 h of hypoxic treatment, PHA-activated PBMC released higher amounts of IL-1Ra (4.5-fold) with respect to controls. These results indicate that hypoxic PHA-activated PBMC express higher levels of IL-1Ra mRNA and effectively release higher amounts of IL-1Ra in the extracellular compartment.

Hypoxia enhances IL-1
and IL-1 release Since hypoxia upregulates the expression of IL-1
and IL-1 mRNA, we next investigated whether hypoxic treatment affects IL-1
and IL-1 release. Exposure to hypoxia significantly enhanced the release of both IL-1
and IL-1 in PHA-activated PBMC. After 40 h of hypoxic treatment, PHA-activated

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CYTOKINE, Vol. 13, No. 6 (21 March, 2001: 334–341)

16 h PHA

Medium N H

16 h

40 h

N

H

Medium N H

N

Medium N H

PHA H

IL-1α

IL-1β

L32

L32

120

40 h PHA

N

H

Medium N H

N

PHA H

250 *

Area units

Area units

100 80

40

150 * 100 50

0

0

40

16 Time (h)

Figure 2. Effect of hypoxia on IL-1
expression. PBMC were cultured at a concentration of 1106 cells/ml in the presence or absence of PHA (5 g/ml) and exposed to either an aerobic (N, ) or hypoxic environment (H, ) for 16 and 40 h. Cell lysates were analyzed by RNase protection assay as reported in the Materials and Methods section. L32 was used as housekeeping gene. Data presented in the upper panel are the results of one of three similar experiments. Quantification was achieved with Sigma Gel analysis software; the results, representing the percentage of area of each band calibrated on the L32 housekeeping gene band, are shown in the lower panel. Data presented are the meanSEM of three independent experiments. Asterisks indicate statistically significant (P<0.05) differences between IL-1
mRNA expressed in PHA-activated PBMC incubated in hypoxia vs aerobic controls.

PBMC released higher amounts of both IL-1
(Fig. 5) and IL-1 (Fig. 6). In fact, when compared with the control PBMC, IL-1
and IL-1 levels were increased in hypoxic PBMC by 47% and 50%, respectively. Thus, hypoxic PHA-activated PBMC expressed higher levels of both IL-1
and IL-1 mRNAs and effectively released higher amounts of both IL-1
and IL-1 in the extracellular compartment.

Hypoxia affects cell cycle progression but does not induce apoptosis To investigate whether the enhancement of IL-1related molecule expression in hypoxic PHA-activated PBMC is related to metabolic or proliferative derangements in hypoxic PBMC, we determined the concentrations of glucose and lactate, cell viability and cell proliferation, as well as apoptosis, after 16 and 40 h of exposure to 2% O2. As expected, in hypoxic conditions glucose concentration decreased while lactate increased (data not shown). However, these differences were never significant, indicating that critical metabolic

16

40 Time (h)

Figure 3.

Effect of hypoxia on IL- expression.

PBMC were cultured at a concentration of 1106 cells/ml in the presence or absence of PHA (5 g/ml) and exposed to either an aerobic (N, ) or hypoxic environment (H, ) for 16 and 40 h. Cell lysates were analyzed by RNase protection assay as reported in the Materials and Methods section. L32 was used as housekeeping gene. Data presented in the upper panel are the results of one of three similar experiments. Quantification was achieved with Sigma Gel analysis software; the results, representing the percentage of area of each band calibrated on the L32 housekeeping gene band, are shown in the lower panel. Data presented are the meanSEM of three independent experiments. Asterisks indicate statistically significant (P<0.05) differences between IL-1 mRNA expressed in PHA-activated PBMC incubated in hypoxia vs aerobic controls.

derangements were avoided. In fact, such derangements, e.g. glucose deprivation, may have a direct effect on PBMC cytokine production. However, the amount of glucose present in culture was never below the physiological level of 100 mg/dl. As expected, resting and PHA-activated PBMC were still viable when cultured under hypoxic conditions, even after 40 h of treatment (Table 1). However, hypoxic PBMC were less sensitive to mitogenic stimulation induced by PHA. This is supported by the results from cell proliferation experiments that revealed that the number of PHA-activated PBMC was significantly lower after 40 h of hypoxia, when compared with the aerobic controls. As shown in Figure 7 the proliferation index (PI) of hypoxic and aerobic PBMC was 1.270.07 and 1.500.11, respectively (P<0.05). However, hypoxic PHA-activated PBMC did not yet undergo apoptosis. Results of the DNA fragmentation assay (Fig. 8) indicated that, at least after 40 h of hypoxic treatment, DNA samples did not show an apoptic profile. This rules out the possibility that the effects of hypoxia

Hypoxia enhances IL-1Ra expression / 337

1000 14

*

* 800

10

IL-1α (pg/ml)

IL-1Ra (ng/ml)

12

8 6

600

400

4

200 2 0

0 16

40

40

16 Time (h)

Time (h) Figure 4. Effect of hypoxia on IL-1Ra production by PHAstimulated PBMC.

Figure 5. PBMC.

PBMC from six subjects were treated with PHA (5 g/ml) and exposed to either a hypoxic (filled bars) or aerobic environment (open bars). After 16 and 40 h of culture, cell-free supernatants were obtained and IL-1Ra present in the supernatants was determined by ELISA. Raw data for each subject and the meanSEM are presented. An asterisk indicates statistically significant (P<0.05) differences between IL-1Ra released by PBMC incubated in hypoxia vs aerobic controls.

PBMC from five subjects were treated with PHA (5 g/ml) and exposed to either a hypoxic (filled bars) or aerobic environment (open bars). After 16 and 40 h of culture, cell-free supernatants were obtained and the IL-1
present in the supernatants was determined by ELISA. Raw data for each subject and the meanSEM are presented. Asterisks indicate statistically significant (P<0.05) differences between the amounts of IL-1
released by PBMC incubated in hypoxia vs aerobic controls.

on mRNA expression of IL-1Ra and IL-1-related molecules were due to metabolic or proliferative derangements.

IL-1Ra, IL-1
and IL-1 mRNA levels did not increase in the absence of PHA, indicating that a co-stimulus is required to enhance IL-1-related molecule production in hypoxic mononuclear cells. This is in agreement with previous studies showing that hypoxia induces IL-1 release in mononuclear cells stimulated by LPS4 or in re-oxygenated human mononuclear cells.19 Meanwhile, the fact that, in hypoxia, resting PBMC did not express higher levels of IL-1related molecule mRNA suggested that IL-1 expression was a restrained event. Indeed, uncontrolled release of IL-1 by mononuclear cells may induce several pathological processes; it can certainly be deleterious to the organism and should be prevented. Interestingly, the enhancement of IL-1Ra, IL-1
and IL-1 mRNA expression after exposure to hypoxia followed different patterns. IL-1Ra mRNA expression was increased in hypoxic PHA-activated PBMC, when compared with corresponding controls, after either 16 or 40 h of exposure. IL-1
mRNA expression was enhanced only after 16 h of exposure to hypoxia; IL-1 mRNA was highly expressed in both normoxic and hypoxic PBMC after 16 h of exposure, but the difference between hypoxic and normoxic PBMC was significant only at 40 h of exposure. Furthermore, IL-1Ra release increased dramatically at 40 h of hypoxic treatment, while IL-1
and IL-1

DISCUSSION Hypoxia has long been associated with a generalized reduction of protein synthesis at the transcriptional and translational level.16 Although hypoxic cells tend to decrease energy use since ATP synthesis is reduced at low oxygen tension, the expression of certain proteins is enhanced during hypoxia.3 These proteins include growth factors, such as vascular endothelial growth factor (VEGF),17 glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH),18 inflammatory mediators6 and cytokines.8 Hypoxia enhanced mRNA expression of IL-1related molecules, such as IL-1Ra, IL-1
and IL-1 in human PHA-activated PBMC. Although inflammatory cytokine release has been described in several cell types during hypoxic treatment,6,5 this is the first demonstration of IL-1Ra mRNA expression in concomitance with the expression of IL-1
and IL-1 mRNAs in hypoxic mononuclear cells activated by PHA. IL-1Ra, IL-1
and IL-1 expression increased significantly after exposure to 2% O2. However

Effect of hypoxia on IL-1
production by PHA-stimulated

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CYTOKINE, Vol. 13, No. 6 (21 March, 2001: 334–341)

1.75

2500 *

1.40 * Proliferation index (P.I.)

IL-1β (pg/ml)

2000

1500

1000

0.70

0.35

500

0

1.05

0.00 16

16

40

Figure 6. PBMC.

Effect of hypoxia on IL- release by PHA-stimulated

PBMC from five subjects were treated with PHA (5 g/ml) and exposed to either a hypoxic (filled bars) or aerobic environment (open bars). After 16 and 40 h of culture, cell-free supernatants were obtained and the IL-1 present in the supernatants was determined by ELISA. Raw data for each subject and the meanSEM are presented. Asterisks indicate statistically significant (P<0.05) differences between the amounts of IL-1 released by PBMC incubated in hypoxia vs aerobic controls.

TABLE 1. Culture conditions Medium PHA

40 Time (h)

Time (h)

Effect of hypoxia on PBMC viabilitya

Figure 7.

Effect of hypoxia on PBMC proliferation index (PI).

PBMC were cultured at a concentration of 2105 cells/well with medium or PHA (5 g/ml) and exposed to either a hypoxic ( ) or aerobic ( ) environment for 16 and 40 h. The results are presented as PI (proliferation index, calculated as described in the Materials and Methods section). Data presented are the meanSEM of six independent experiments. An asterisk indicates statistically significant (P<0.05) differences between the PI of PBMC obtained in hypoxia vs aerobic controls.

Medium N

H

PHA N

H bp

Normoxia 0.1820.010 0.2690.012

Hypoxia 0.1860.014 0.2340.013

a PBMC were cultured at a concentration of 2105 cells/well with medium or PHA (5 g/ml) and exposed to either a hypoxic or aerobic environment under the conditions described in Materials and Methods. After 40 h of treatment, cell viability was assessed by the MTT method and expressed as O.D.570/630. Data presented are the meanSEM of six independent experiments. Cell viability of PBMC cultured for 16 h under aerobic conditions was: 0.1710.013.

Figure 8.

release increased at a lower extent after 40 h of exposure to hypoxia. This supports the hypothesis that IL-1Ra is released along with IL-1
and IL-1 in order to promote an anti-inflammatory state by blockade of IL-1 activities.20 The present results suggest that, since hypoxia resembles an inflammatory state, IL-1Ra may help counteract the inflammatory properties of IL-1 released during hypoxic conditions. Since IL-1Ra expression can be considered protective against IL-1
and IL-1-mediated injury, the time course of hypoxia-induced IL-1Ra upregulation (16–40 h) leads to cellular protection during acute ischaemia–reperfusion injury or acute inflammatory conditions. Hypoxia induced a series of responses

←

23,130

← ← ←

9,416 6,557 4,361

← ←

2,322 2,027

Effect of hypoxia on DNA fragmentation assay.

PBMC were cultured at a concentration of 1106 cells/ml with medium or PHA (5 g/ml) and exposed to either a hypoxic (H) or aerobic (N) environment for 40 h. DNA was extracted, isolated and analyzed on a 1.2% agarose gel. Lane 1 and 3: resting and PHAactivated PBMC in normoxia. Lane 2 and 4: resting and PHAactivated PBMC in hypoxia. Lane 5:  DNA/Hind III Fragments as size markers. Data presented are the results of one of four similar experiments.

associated with an inflammatory state,6 such as IL-1
and IL-1 expression and their release into the extracellular compartment. In the same context, hypoxia enhanced the expression and release of IL-1Ra, wellknown for its anti-inflammatory activities, supporting the hypothesis of a rescue mechanism in hypoxic cells.2

Hypoxia enhances IL-1Ra expression / 339

Hypoxia usually induces apoptosis in a variety of cell types.3,21 However, although hypoxia inhibited the susceptibility of PBMC to PHA, in this report we demonstrated that at 40 h of hypoxic treatment PBMC did not show any DNA fragmentation. Nevertheless, form our results we cannot state that hypoxia will not induce DNA fragmentation or apoptosis in PBMC after prolonged treatment. Indeed, we chose this incubation time to keep the cells viable and maintain the pH and levels of glucose and lactate in the media within a physiological range.22 In fact, in our experimental conditions, PBMC were still viable and did not show any DNA fragmentation. From the overall data, it is clear that hypoxia enhanced the expression of IL-1Ra, along with IL-1
and IL-1 mRNA. The presence of IL-1
and IL-1 in the surrounding environment of hypoxic activated mononuclear cells is certainly important in many contexts. These molecules may induce the proliferation of different cell types involved in angiogenesis and wound healing.15,23 At the same time, the presence of IL-1Ra in the environment surrounding hypoxic PHA-activated PBMC in inflamed tissues may prevent some undesirable effects induced by IL-1. In summary, hypoxia induces IL-1-related molecule expression and release in mitogen-activated mononuclear cells. Since IL-1 is a highly inflammatory mediator, the present data may be helpful in understanding the events that induce the expression of IL-1 and have important therapeutic implications in disease states in which IL-1-related molecules contribute to cell injury. The modulated expression of IL-1Ra, as well as of IL-1
and IL-1, by PHA-activated PBMC maintained in a continuous hypoxic environment also provides an interesting model for further testing of the rescue and defence mechanisms adopted by cells to survive hypoxia.

initiated. Hypoxic conditions were established by culturing PBMC in two different incubators as previously described.8 In the control experiments we used an incubator (KW Apparecchi Scientifici, Siena, Italy) set to 5% CO2, 20% O2 (atmospheric oxygen ]140 mmHg) and 37C in a humidified environment. The experiments under hypoxic conditions were performed using a water-jacketed incubator (Forma Scientific, Marietta, OH, USA) that provides a customized and stable humidified environment through electronic control of CO2 (5%), O2 and temperature (37C). In our experiment the O2 tension was set and maintained constantly at 2% (]14 mmHg) by automatically injecting N2 in the chamber to bring the O2 level to the set point. PBMC were cultured for 16 or 40 h in the presence or absence of 5 g/ml of PHA (Biochrom KG, Berlin, Germany) under hypoxic or normoxic conditions.

Cell viability and proliferation experiments To assess the effect of hypoxia on PBMC viability, experiments were performed using a colorimetric method,25 based on the tetrazolium salt 3-(4,5dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT). Briefly, 2105 cells/ well were incubated in 200 l of culture medium and exposed to either hypoxia (2% O2) or normoxia (20% O2) in the presence or absence of PHA. After 16 or 40 h of culture, 100 l of medium were harvested to be assayed later either for cytokine of lactate dehydrogenase (LDH) release and 10 l of a solution of MTT (5 mg/ml) were added to each well and incubated at 37C. After 4 h 100 l of acid propan-2-ol (0.04M HCI in propan-2-ol) were added to each well and after all the formazan crystals were dissolved the plate was read on a microelisa reader (Titertek Multiskan MCC, Labsystems, Helsinki, Finland). The optical density values (O.D.) were obtained using a test wavelength of 570 nm and a reference wavelength of 630 nm. The proliferation index (PI) was calculated as follows: PI=PHA-treated lymphocyte activity (O.D.)/ Untreated lymphocyte activity (O.D.)

DNA fragmentation assay MATERIALS AND METHODS Cell culture Human PBMC were isolated from heparinized venous blood of healthy volunteers by a gradient of Lymphoprep (Nycomed Pharma, Roskilde, Denmark) as previously described.24 The gradients were spun at 400g and the PBMC at the interface were removed, washed twice and resuspended in RPMI-1640, Dutch modification, containing 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin and 100 g/ml streptomycin (Biochrom KG, Germany), at a density of 106 viable cells/ml. Cell viability was measured by the ability to exclude 4.16 mM trypan blue. The PBMC preparations contained only lymphocytes (90%) and monocytes (10%), as determined by flow cytometric analysis. PBMC were plated at 106 cells/ml either in 96-well, flat-bottomed tissue culture plates or 25 cm2 flasks (Costar, Cambridge, MA, USA) and experiments were immediately

Since apoptosis is associated with DNA fragmentation, DNA from hypoxic and normal PBMC was analyzed by agarose gel electrophoresis.26 Briefly, at the appropriate time, cells were pelletted, washed and DNA was extracted and purified by a commercially available kit (Easy DNA Kit, Invitrogen, San Diego, CA, USA). Electrophoresis was performed in 1.2% agarose gels in TAE (0.04M Tris acetate, 0.001M EDTA) buffer, containing ethidium bromide (0.5 g/ml) for 2 h at 50 V. Agarose gels were then photographed on a transilluminator (Fotodyne, Hartland, WI, USA) with a Polaroid camera (FCR-10).

Preparation of cell extracts for RNase protection assay Cell extracts were prepared as follows. Freshly isolated PBMC, at a concentration of 106 cells/ml, were incubated in 25-cm2 tissue culture flasks and exposed to either hypoxia (2% O2) or normoxia (20% O2) in the presence or absence of

340 / Naldini et al.

PHA, as described above. After 16 or 40 h of culture, cells were harvested by centrifugation and lysed in an appropriate buffer (Direct Protect, Lysate Ribonuclease Protection Assay Kit, AMBION, Austin, TX, USA) at a concentration of 107 cells/ml of lysis solution. The extracts were centrifuged for 5 min at 10 000g at 4C and the supernatants were carefully removed, placed in a clean tube and stored at
20C until used.

RNase protection assay IL-1Ra, IL-1
and IL-1 mRNAs were evaluated by RNase protection assay.27 Since hypoxia affects GAPDH expression,18 we used the human ribosomal protein L32 as housekeeping gene. The RNA probes were synthesized according to the suggested procedure (MAXIscript AMBION), using T7 phage polymerase and a biotin RNAlabelling mixture (Boehringer, Mannheim, Germany). DNA templates were purchased from Pharmingen (San Diego, CA, USA) as multiprobe template set (hCK-2, Riboquant RNase protection assay System). The cell lysates obtained as described above and appropriate RNA probes were hybridized, according to the suggested procedure (Direct Protect, Lysate Ribonuclease Protection Assay Kit, AMBION). Briefly, 45 l of cell extract and 5 l of the labelled probe were mixed and incubated at 37C overnight. The hybridized probe and lysate were digested by RNase A/T1 and then treated with proteinase K. The products of RNase protection assay were then separated for analysis on a denaturing polyacrylamide gel (5% acrylamide/8M urea) by running the gel at 200 V for 1 h. Gels were then transferred to a positively-charged nylon membrane, using a semi-dry transfer unit (Hoefer Pharmacia Biotech, San Francisco, CA, USA). The nucleic acids were then immobilized by UV cross-linking. Non-isotopic detection was performed with the BrightStar BioDetect kit (AMBION), following the manufacturer’s instructions. The identity and quantity of each mRNA species in the original sample was then determined from the signal intensities given by the appropriately-sized protected probe fragment bands (protected nucleotide sizes: IL-1Ra: 202 nt; IL-1: 227 nt; IL-1
: 255 nt). Antisense L32 (housekeeping gene) probe transcripts were synthesized and used in a series of parallel reactions, to normalize the amounts of RNA present in the lysates (protected nucleotide size for L32: 112 nt). Gel electrophoretic autoradiographs were then quantitated by Sigma Gel analysis software (Jandel Scientific, Chicago, IL, USA).

IL-1-related protein measurements IL-1Ra, IL-1
and IL-1 concentrations were assessed from PBMC culture supernatants by commercially available ELISA kits (human IL-1Ra, Bachem AG, Bubendorf, Switzerland; human IL-1
and IL-1, Euroclone, Devon, UK). Briefly, cell-free supernatants were obtained at the appropriate time. After centrifugation, aliquots from supernatants were frozen at
20C pending assay. None of the assays showed cross-reactivity with other cytokines. Minimum detectable doses were as follows: IL-1Ra: 0.2 ng/ml; IL-1
: 3 pg/ml; IL-1: 2 pg/ml.

CYTOKINE, Vol. 13, No. 6 (21 March, 2001: 334–341)

Statistical analysis Values presented are the meansSE of the results obtained from six independent experiments, unless specifically described. Each experiment was performed in PBMC from six different donors. Statistical evaluation of the data was by Student’s two-tailed paired t-test. A P value of less than 0.05 was considered significant.

Acknowledgements We thank Prof. V. Bocci at the Institute of General Physiology for his helpful suggestions and encouragement during this work and Mr S. Focardi for the excellent technical assistance. We are also indebted to Dr M. Zazzi at the Department of Molecular Biology for providing the Sigma Gel analysis software and Dr G. Fanetti at the Blood Donor Center in Siena.

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