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Originally published online as doi:10.1189/jlb.0303105 on May 22, 2003

Published online before print May 22, 2003
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(Journal of Leukocyte Biology. 2003;74:197-205.)
© 2003 by Society for Leukocyte Biology

Hypoxia reduces CD80 expression on monocytes but enhances their LPS-stimulated TNF-{alpha} secretion

Nitza Lahat*, Michal A. Rahat*, Mouna Ballan*,{dagger}, Lea Weiss-Cerem{dagger}, Miri Engelmayer* and Haim Bitterman{dagger}

* Immunology Research Unit and
{dagger} Ischemia-Shock Research Laboratory, Carmel Medical Center, Rappaport Family Institute for Research in the Medical Sciences, and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel

Correspondence: Michal A. Rahat, D.Sc., Immunology Research Unit, Carmel Medical Center, 7 Michal St., Haifa, 34362, Israel. E-mail: rahat_miki{at}clalit.org.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocytes/macrophages in ischemic tissues are involved in inflammation and suppression of adaptive immunity via secretion of proinflammatory cytokines and reduced ability to trigger T cells, respectively. We subjected human mononuclear cells and mouse macrophages to hypoxia and reoxygenation, the main constituents of ischemia and reperfusion, and added lipopolysaccharide (LPS) to simulate bacterial translocation, which frequently accompanies ischemia. We monitored the secretion of tumor necrosis factor {alpha} (TNF-{alpha}) and the surface expression of human leukocyte antigen-DR and the costimulatory molecules CD80 and CD86 on monocytes/macrophages. Hypoxia selectively reduced the surface expression of CD80 (P<0.01), and synergistically with LPS, it enhanced TNF-{alpha} secretion (P<0.003). Reoxygenation reversed both phenomena. In the mouse macrophage cell line RAW 264.7, hypoxia reduced the surface expression of CD80 and increased its concentrations in the supernatants (P<0.01). Down-regulation of the mRNA coding for the membrane-anchored CD80 was observed, suggesting that hypoxia triggers alternative splicing to generate soluble CD80. Cumulatively, these results suggest that hypoxia simultaneously affects monocytes/macrophages to enhance inflammation and reduce their ability to initiate adaptive-immunity responses associated with ischemic injury.

Key Words: reoxygenation • CD86 • HLA-DR


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ischemia, caused by occlusion of blood vessels or insufficient blood flow to organs, occurs in a wide variety of pathological conditions (e.g., severe hemmorhage, acute infarcts, intestinal injury, low flow states and circulatory shock, trauma, burns, solid tumors). It results in local cellular hypoxia and tissue damage [1 ], involving induction of high levels of inflammatory mediators [2 3 4 ] and activation of leukocytes and their subsequent migration to afflicted targets [5 6 ]. Frequently, local ischemia or generalized processes (e.g., shock, trauma, burns), which decrease splanchnic perfusion and cause secondary nonocclusive intestinal ischemia, are associated with translocation of bacteria or their toxins to the peripheral blood, leading to the development of systemic inflammatory response (SIR), sepsis syndrome, and multiple organ failure [7 8 9 ]. The excessive systemic inflammatory response in these conditions reflects activation of the body’s nonspecific innate immunity, in which tumor necrosis factor {alpha} (TNF-{alpha}) plays a central role [10 ]. In the critically ill patient, it coexists with profound suppression of the adaptive T helper cell type 1 (Th1)-mediated immunity [11 ]. The use of biological response modifiers targeting inflammatory responses has shown only modest clinical benefit, and interest has shifted toward therapies aimed at reversing the accompanying inhibited-specific immunity [12 ]. However, the mechanisms involved in the immune suppression, which characterizes ischemia and reperfusion (I/R), are not yet clear.

Reperfusion of ischemic tissues results in cellular reoxygenation and is critical for cell salvage but may temporarily aggravate the ischemic injury, partly as a result of increased generation of reactive oxygen species [6 , 13 , 14 ]. Hypoxia and reoxygenation (H/R) are major components of I/R and frequently serve as model systems to study the mechanisms at work during I/R.

Monocytes/macrophages play a pivotal role in the pathophysiology of I/R by participating in inflammation [e.g., secretion of regulatory mediators such as TNF-{alpha}, nitric oxide, or interleukin-6 (IL-6); refs. 15 16 17 ] and in initiation of adaptive-immune responses [e.g., presentation of exogenous antigens in the context of major histocompatibility complex (MHC) class II to Th cells]. Expression of MHC class II as well as a second signal provided by costimulatory molecules, mainly the B7 molecules CD80 and CD86, is required for productive antigen presentation [18 ]. In the absence of B7 molecules on antigen-presenting cells, antigen recognition by the T cell receptor may lead to anergy. It has been suggested that B7.1 (CD80)-binding stimulates a Th1 response, resulting in the secretion of cytokines that promote cellular immune responses (e.g., IL-2, interferon-{gamma}), whereas binding of B7.2 (CD86) evokes a Th2 response, which is characterized by anti-inflammatory and prohumoral cytokines (e.g., IL-4, IL-10, transforming growth factor-ß) [19 ]. This paradigm has recently been expanded by evidence describing the involvement of CD80 and CD86 in Th1 and Th2 responses [20 , 21 ]. As monocytes constitutively express CD86 molecules, whereas CD80 molecules are induced on their surface, it has also been suggested that CD86 is involved in initiation, and CD80 may participate in advanced stages of immune response [22 ].

It has been shown that multiple trauma and hypovolemic shock reduce the expression of MHC class II molecules on monocytes/macrophages [23 24 25 26 27 ] associated with immunosuppression. Other studies have shown increased secretion of TNF-{alpha} during different types of tissue ischemia leading to SIR and shock [16 , 17 , 28 ]. Although Th2 responses have been shown to contribute to immunosuppression observed in sepsis [29 , 30 ], their relationships with the expression of B7 molecules are not yet clear. The expression of B7 molecules under stress conditions in vivo has hardly been investigated, with the exception of studies demonstrating elevated CD80 expression 3 days after cold I/R injury [31 ] and reduced CD86 expression on monocytes derived from patients with septic shock [32 ]. The isolated influences of H/R on monocytes function have only scarcely been evaluated, and only few studies revealed hypoxia-induced enhancement of TNF-{alpha} production by human and murine monocytes/macrophages [33 34 35 ]. No data are available on the isolated effects of H/R on the monocyte/macrophage expression of MHC class II and costimulatory molecules.

In this study, we evaluated H/R-induced simultaneous changes in human and mouse monocytes/macrophages in the secretion of TNF-{alpha} as a representative of proinflammatory cytokines and in the expression of molecules required for antigen presentation: MHC class II, CD80, and CD86.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Blood (50 ml) was taken from healthy volunteers in the presence of 0.2% EDTA. Peripheral blood mononuclear cells (PBMC) were separated on Ficoll-Hypaque (Uni-Sep, NovaMed, Israel) and were washed three times with phosphate-buffered saline (PBS). PBMC were counted and plated in 60-mm dishes at a concentration of 1 x 106 cells/plate. The cells were subjected to one of the following conditions: normoxia (N); short-term hypoxia for 2 h (H2); prolonged hypoxia for 24 h (H24); hypoxia (short-term or prolonged) followed by short-term reoxygenation for 2 h (R2); and hypoxia (short-term or prolonged) followed by prolonged reoxygenation for 24 h (R24). All experiments were performed with or without lipopolysaccharide (LPS; 1 µg/ml, Escherichia coli 055:B5, Sigma Chemical Co., St. Louis, MO). After exposure to H/R, cells were harvested for analysis of surface markers, and supernatants were collected for determination of TNF-{alpha} levels. The local and national Helsinki committees approved this part of the study.

Primary resident peritoneal macrophages were isolated from female BALB/c mice that were 7–11 weeks old. Mice were killed, and their peritoneal cavity was injected with 5 ml PBS and massaged. The fluid containing the macrophages was drawn back to the syringe, washed once with PBS, and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal calf serum (FCS). Cells were counted, plated in 96-well plates at a concentration of 0.2 x 106 cells/well, and incubated for 2 h. The medium was then replaced with DMEM with no FCS, and cells were serum-starved for 1 h before beginning the experiment. Peritoneal macrophages were subjected to normoxia or hypoxia for 24 and 48 h, with or without addition of LPS (1 µg/ml, E. coli 055:B5, Sigma Chemical Co.). The culture consists of 90–95% macrophages, as determined by rat anti-mouse F4/80 staining (SeroTec, Oxford, UK) using a Coulter-XL flow cytometer (Coulter Electronics, UK). This part of the study was performed in adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

In addition, the mouse peritoneal macrophage-like cell line RAW 264.7 was cultured in DMEM with 10% FCS and antibiotics and was subjected to the same experimental conditions, with or without LPS. RAW 264.7 cells also were incubated in the presence of the internalization inhibitor cytochalasin B at nontoxic concentrations (0.1–5 µM, Sigma Chemical Co.) or with different protease inhibitors: aprotinin (0.5–10 µM), leupeptin (50–200 µM), pepstatin A (0.1–10 µM), and phenantrolin (10–100 µM, all from Sigma Chemical Co.). In these experiments, cell viability was determined using the 2,3-bis(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT) kit (Biological Industries, Beit-Haemek, Israel).

Normoxic and hypoxic conditions
For normoxic conditions, cells were incubated in a regular incubator (21% O2, 5% CO2, 74% N2). Hypoxic incubation was performed in a sealed anaerobic workstation (Concept 400, Ruskin Technologies, Leeds, UK/Jouan, Saint Herblain, France), where the hypoxic environment (O2<0.3%, 5% CO2, 95% N2) is kept constant and so are the temperature (37°C) and humidity (90%). Samples from the culture medium were taken at the end of the exposure to hypoxia to determine the partial pressures of O2 and CO2, as well as pH, using a blood gas analyzer ABL510 (Radiometer, Denmark). Hypoxic PO2 values were 23 ± 1.4 mmHg, PCO2 values were 37.8 ± 0.28 mmHg, and pH values were 7.28 ± 0.12. Reoxygenation was performed by removing the cells from the hypoxic chamber and immediately transferring them to the normoxic incubator.

Expression of surface molecules on human peripheral blood monocytes
After exposure to the experimental conditions, the cells were harvested by centrifugation, and adherent cells were scraped after incubation with 2.5 mM EDTA in cold PBS for 20 min. Viability of the adherent cells before and after scraping was assessed by trypan blue staining and was not changed. The cells were then incubated with phycoerythrin-conjugated monoclonal anti-CD14 and fluorescein isothiocyanate-conjugated monoclonal anti-CD80, anti-CD86, or anti-human leukocyte antigen (HLA)-DR (PharMingen, San Diego, CA). After washing, the cells were fixed in 0.1% formaldehyde, and cells were analyzed using a Coulter-XL flow cytometer (Coulter Electronics). Dead cells were excluded from the analysis by their forward- and sideway light-scattering properties. Only the double-stained cells were analyzed to ensure that the expression of surface markers was measured on monocytes/macrophages.

Expression of surface molecules on RAW 264.7 cells
Expression of CD80 on RAW 264.7 cells was measured by radioimmunoassay (RIA) to enable the multiple measurements required in these experiments. Cells (4x104) were plated in 96-well plates in DMEM and subjected to the experimental conditions with or without incubation with cytochalasin B or with the various protease inhibitors. Cells were incubated for 40 min at 4°C with the 1:200 diluted rat anti-mouse CD80 (Southern Biotechnology, Birmingham, AL). After washing, cells were incubated for 40 min at 4°C with 200,000 cpm of the 125I-conjugated sheep anti-rat immunoglobulin (Ig; Amersham Pharmacia Biotech, Piscataway, NJ). The cells were then tyrpsinized, and radioactive counts were measured and normalized to the optical density (OD) of viable cells as determined by XTT (Biological Industries, Beit Haemek, Isreal).

Secretion of TNF-{alpha}
TNF-{alpha} secretion from human and mouse monocytes was determined by its biological activity and by an enzyme-linked immunosorbent assay (ELISA). To measure the biological activity, L-929 fibroblast cells at a concentration of 5 x 103 cells/well were plated in 96-well flat-bottom plates. When the monolayer became confluent, 4 pg/ml actinomycin D (Sigma Chemical Co.), together with human or mouse recombinant TNF-{alpha} or with experimental supernatants, was added. After 24 h incubation, 30 µl/well XTT was added and incubated for 4 h at 37°C. The absorbance at 450 nm (with reference at 620 nm) was then read, and measurements for the dilution series were plotted to produce dose-response curves, in which the OD read was proportional to the reciprocal of the concentration of biologically active TNF-{alpha} [36 ]. In addition, the concentration of secreted TNF-{alpha} in the supernatants of human and mouse monocytes was measured using the ELISA DuoSet system (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions, with essentially similar results. In both assays, recombinant TNF-{alpha} served as a positive control, as well as for the standard curves.

Western blot analysis
Supernatants were collected at different time points, concentrated 20-fold by using Vivaspin-2 (Sartorius AG, Göttingen, Germany), and equal volumes were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic separation, the proteins were transferred and fixed onto a cellulose nitrate membranes (Schleicher and Schuell, Dassel, Germany) in transfer buffer (25 mM Tris, 180 mM glycine, 20% methanol, pH 8.3). The membranes were incubated for 1 h in blocking buffer (20% skimmed milk, 1% bovine serum albumin, 0.01% Tween-20, 10 mM Tris, pH 8.0, 150 mM NaCl) at room temperature and then probed for 1 h at room temperature with the diluted (1:1000) rabbit polyclonal anti-CD80 (H-208, Santa Cruz Biotechnology, Santa Cruz, CA). After washing three times in 1x 10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoReasearch Laboratories, West Grove, PA), diluted 1:5000 in blocking buffer for an additional 1 h at room temperature, and then washed again. The enhanced chemiluminescence system (Amersham Pharmacia Biotech) was used for detection.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
Total RNA was extracted from RAW 264.7 cells at different time periods using TriReagentTM (Molecular Research Center, Cincinnati, OH), according to the manufacturer’s instructions. Total RNA (1 µg) was transcribed to cDNA at 37°C for 1 h using 200 µM deoxynucleotides (Sigma Chemical Co.), 5 µM random hexamers (Amersham Pharmacia Biotech), 20 U RNAguard (Amersham Pharmacia Biotech), and 20 U/µg Moloney murine leukaemia virus-RT (U.S. Biochemicals, Cleveland, OH). The amplification reactions consisted of 0.5 µM each primer, 100 µM deoxynucleotides, 0.5 U Taq polymerase (AmpliTaq Gold, Roche Molecular Systems, Branchburg, NJ), and 5–100 ng reverse-transcribed RNA in a final volume of 15 µl. CD80 mRNA was amplified in two separate reactions: one of the first exon (33 cycles with annealing temperature of 55°C for 30 s) and the second of the transmembranal region (33 cycles with annealing temperature of 60°C for 30 s). The linear range of amplification was determined for each transcript, and amplification was performed only within that linear range. To allow for relative comparison between the samples, the results were normalized for 18S rRNA. CD80 mRNA primers were for the first exon, sense: 5'-CCAAAGCATCTGAAGCTATGGC, antisense: 5'-TTTCCCAGCAATGACAGACAGC; CD80 mRNA primers for the transmembranal region, sense: 5'-ATGCTCACGTGTCAGAGGA, antisense: 5'-GACGGTCTGTTCAGCTAATG; and 18S rRNA, sense: 5'-CCTCGATGCTCTTAGCTGAGTG, antisense: 5'-GATCGTCTTCGAACCTCCGAC.

Statistical analyses
All values are presented as means ± SE. The data for the PBMC were analyzed using nonparametric repeated measures (ANOVA). To analyze the difference within each group, the nonparametric two-tailed Wilcoxon signed ranks test was used, and to analyze the difference between the groups, the nonparametric two-tailed Mann-Whitney test was used. The data for RAW 264.7 cells were analyzed using repeated measures (ANOVA). The Tukey-Kramer multiple comparison test was used to evaluate significance between experimental groups. The data for the CD80 mRNA were analyzed using a one-tail unpaired t-test. P values exceeding 0.05 were not considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of surface HLA-DR, CD86, and CD80 molecules on human monocytes (Figs. 1 and 2 )
After separation on Ficoll-Hypaque gradient, fresh human monocytes showed high basal expression of HLA-DR (76.1±4.9% positives) and CD86 (68.3±8.1% positives) but did not express CD80 (0.45±0.4% positives). After plating the cells and subjecting them to normoxia, hypoxia, and hypoxia followed by reoxygenation, with or without the addition of LPS, the expression of HLA-DR (Fig. 1A) or CD86 (Fig. 1B) as well as their fluorescent intensity (data not shown) did not change significantly. Following 2 h of culture in normoxia without LPS, 14 ± 2.2% of the monocytes expressed CD80 (Fig. 2) , and addition of LPS increased it further after 2 h (19.4±4.6% positives) and 24 h (23.5±4.6% positives). Activation of monocytes during adhesion to the plastic plates, with or without the presence of LPS, probably led to the surface expression of CD80. Short-term hypoxia or short-term hypoxia followed by short-term reoxygenation markedly reduced the expression of CD80 (2.1±0.5, 1.7±0.5% positives, respectively; P<0.01), and addition of LPS did not change this effect (Fig. 2B) . Normoxic levels of surface CD80 expression were restored only after prolonged reoxygenation (P<0.01), and addition of LPS enhanced this effect. Prolonged hypoxia or prolonged hypoxia followed by short-term reoxygenation also reduced CD80 expression (4.4±2.2 and 2.9±1.1% positives, respectively; P<0.01). Prolonged reoxygenation after prolonged hypoxia restored CD80 expression to normoxic levels (16.7±4.8% positives; P<0.01), and addition of LPS enhanced this effect (27.3±3.8% positives).



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  		Figure 1. H/R did not affect HLA-DR and CD86 expression. PBMC were isolated on Ficoll-Hypaque gradient and were subjected to normoxia, hypoxia, or hypoxia followed by reoxygenation, with or without LPS (n=9). Short-term or long-term H/R, with or without the addition of LPS, did not have any significant effect on the expression of HLA-DR (A) or CD86 (B) on monocytes (recognized by double-staining with anti-CD14). N, Normoxia for 2 h or 24 h; H, hypoxia for 2 or 24 h; R, hypoxia for 2 or 24 h followed by reoxygenation for 2 or 24 h.

 


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  		Figure 2. Hypoxia decreased CD80 expression on monocytes, and reoxygenation restored it. (A) A representative analysis of flow cytometry of PBMC subjected to 2 h normoxia (N2), 2 h hypoxia (H2), and 2 h hypoxia followed by 24 h reoxygenation (H2R24). (B) PBMC were subjected to normoxia, hypoxia, or hypoxia followed by reoxygenation, with and without LPS (n=8). Short and prolonged hypoxia reduced the expression of CD80, and only long-term reoxygenation restored it to normoxic values. Addition of LPS did not significantly change the suppressive effect of hypoxia or the restoring effect of reoxygenation. *, P < 0.01.

 
Secretion of TNF-{alpha} from human PBMC (Fig. 3 )
Human mononuclear cells exposed to normoxia for 2 h, with or without LPS, secreted relatively low amounts of TNF-{alpha} (914±321, 374±108 pg/ml, respectively). When incubated for 24 h with LPS, TNF-{alpha} levels in the supernatants increased (3742±2085 pg/ml). Short-term hypoxia followed by short- or long-term reoxygenation, with or without addition of LPS, did not enhance secretion of TNF-{alpha} induced by LPS alone. Likewise, prolonged hypoxia without LPS or when followed by reoxygenation did not enahnce TNF-{alpha} secretion. In contrast, LPS added at the start of prolonged hypoxia markedly and significantly increased TNF-{alpha} levels (19,124±5015 pg/ml; P<0.003 compared with the level secreted from mononuclear cells incubated in normoxia for 24 h with LPS). This elevation was maintained after short-term reoxygenation (18,625±4805 pg/ml; P<0.002 compared with normoxic mononuclear cells incubated for 24 h with LPS). Prolonged reoxygenation reduced TNF-{alpha} levels, although they were still higher than those observed in normoxia with LPS (8110±1901 pg/ml; P<0.03). ELISA also measured TNF-{alpha} secretion by human PBMC, and essentially similar results were obtained (data not shown).



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  		Figure 3. Hypoxia and LPS increased the secretion of TNF-{alpha} in human PBMC, which were subjected to normoxia, hypoxia, or hypoxia followed by reoxygenation, with or without LPS (n=9), and the biological activity of TNF-{alpha} in the supernatants was determined. Secretion of TNF-{alpha} was minimal during normoxia, short-term hypoxia, and short-term hypoxia followed by reoxygenation. However, prolonged hypoxia resulted in a nonsignificant increase in TNF-{alpha} secretion, whereas prolonged hypoxia or prolonged hypoxia followed by short-term reoxygenation synergistically enhanced the LPS-induced secretion of TNF-{alpha}. Prolonged reoxygenation reversed the effects of hypoxia on the LPS-induced TNF-{alpha} secretion. *, P < 0.003.

 
Secretion of TNF-{alpha} from mouse macrophages (Fig. 4 )
Resident peritoneal mouse macrophages subjected to normoxia or hypoxia for 24 or 48 h secreted negligible amounts of TNF-{alpha}. When incubated with LPS in normoxia, TNF-{alpha} secretion was enhanced, and its levels amounted to 35 ± 11.8 pg/ml after 24 h (P<0.01) and to 65 ± 21.8 pg/ml after 48 h (P<0.05). A combination of hypoxia and LPS further increased TNF-{alpha} secretion after 24 h (52±10.2 pg/ml) and after 48 h (135±34 pg/ml; P<0.05 from normoxia). Similar results were measured using TNF-{alpha} biological activity (data not shown). The significant difference between the amounts of TNF-{alpha} secreted by primary human mononuclear cells and primary mouse macrophages may be explained by species differences, effect of other cell types present in the human culture, or by different activation states. Although different in magnitude, the same synergistic effect was observed in human and mouse monocyte/macrophages



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  		Figure 4. Hypoxia and LPS increased the secretion of TNF-{alpha} from mouse macrophages. Resident peritoneal mouse macrophages were subjected to normoxia or hypoxia for 24 or 48 h, with or without addition of LPS (n=8). ELISA measured secretion of TNF-{alpha}, which was enhanced in the presence of LPS during normoxia, and a combination of hypoxia and LPS further increased it. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with normoxia; §, P < 0.05, from normoxia with LPS.

 
Expression of CD80 on mouse macrophages (Fig. 5 )
CD80 was constitutively expressed on the mouse peritoneal macrophage-like RAW 264.7 cells (1506±211 cpm/XTT), and short-term hypoxia of only 2 h, with or without the addition of LPS, did not change this expression (data not shown). However, prolonged hypoxia (24 h) reduced CD80 expression by fourfold (355±125 cpm/XTT; P<0.001). Expression of surface CD80 increased only after prolonged reoxygenation, yet it did not reach normoxic values (857±96 cpm/XTT). Thus, the surface expression of CD80 on RAW 264.7 cells mimics, although with slower kinetics, that of human monocytes. Addition of LPS had no significant effect on CD80 expression in RAW 264.7 cells in all experimental conditions.



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  		Figure 5. Hypoxia reduced the surface expression of CD80 on mouse macrophages. Monocyte-like RAW 264.7 cells were incubated in normoxia or hypoxia, with or without LPS, and RIA (n=6) measured the expression of surface CD80. Prolonged hypoxia reduced CD80 expression by fourfold, and only prolonged reoxygenation restored it. *, P < 0.001.

 
The effect of hypoxia on CD80 expression does not result from increased internalization of the surface molecule (Fig. 6 )
To find whether the effects of hypoxia on the surface expression of CD80 in RAW 264.7 cells resulted from increased internalization, cells were incubated with the internalization inhibitor cytochalasin B, which interferes with cytoskelatal interactions and inhibits intracellular traffic. Different concentrations of cytochalasin B, which had previously been determined by XTT viability assay to be nontoxic to the cells, were used. Surface expression of CD80 was measured after exposure to 24 h of normoxia and hypoxia (Fig. 6) . In normoxia, the surface expression of CD80 was gradually enhanced in a dose-dependent manner and reached a significant increase (1.3±0.19-fold from control; P<0.05) at 5 µM cytochalasin B. This result may reflect the decrease in the normal turnover of the molecule. In contrast, cytochalasin B did not increase the surface expression of CD80 in hypoxia, suggesting that the molecule was not internalized during hypoxia.



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  		Figure 6. Cytochalasin B increased CD80 surface expression in normoxia but not in hypoxia. RAW 264.7 cells were incubated in normoxia or hypoxia, with increasing concentrations of cytochalasin B, and RIA (n=5) measured the surface expression of CD80. Cytochalasin B increased surface expression of CD80 by up to 30% in normoxia, in a dose-dependent manner. However, it had no effect on surface CD80 expression in hypoxia. *, P < 0.05.

 
Increased concentrations of soluble CD80 (sCD80) molecule in the supernatants of mouse macrophages following hypoxia (Fig. 7 )
The possibility of increased shedding or secretion of CD80 during hypoxia was studied by measuring its accumulation in the supernatants. RAW 264.7 cells were incubated for different time periods in normoxia and hypoxia, and the supernatants were collected, concentrated 20-fold, and subjected to electrophoretic separation on a 10% SDS-PAGE. Western blot analysis (Fig. 7) revealed two specific CD80 bands, which differed in their molecular weights. Although the two bands were clearly observed and monitored in the normoxic samples, it was technically impossible to separately assess them quantitatively in the hypoxic samples, suggesting the existence of different sizes of the molecule. In normoxia, no change was detected in the accumulation of the two bands, and they remained relatively constant throughout all time periods. In contrast, the accumulation of total sCD80 in the supernatants of cells subjected to hypoxia increased gradually, reaching values fourfold higher than in normoxia after 18 h (P<0.05 compared with normoxia at the beginning of incubation). These findings suggest that hypoxia but not normoxia caused increased shedding of surface CD80 or increased secretion of sCD80 to the medium.



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  		Figure 7. sCD80 accumulated in supernatants of mouse macrophages subjected to hypoxia but not to normoxia. (A) RAW 264.7 cells were incubated in normoxia or hypoxia for different periods of time, and supernatants were collected, concentrated 20-fold as described in Materials and Methods, and separated on 10% SDS-PAGE. (B) Densitometric analysis of the Western blots (n=3). In normoxia, the levels of sCD80 were unchanged, but hypoxia increased them by 4.4 ± 1-fold after 18 h. *, P < 0.05.

 
Effects of protease inhibitors on surface CD80 expression in hypoxia (Table 1 )
To further explore the mechanism leading to the hypoxia-induced accumulation of sCD80 in the supernatants of RAW 264.7 cells, cells were incubated with different protease inhibitors in normoxia or hypoxia, and the surface expression of CD80 was then measured. Each protease inhibitor was assayed using three concentrations that had previously been verified by a XTT viability assay to be nontoxic to the cells (data not shown). Aprotinin (an inhibitor of trypsin, chymotrypsin, kallikrein, and plasmin), leupeptin (inhibitor of serine and thiol proteases), and pepstatin A (inhibitor of aspartic proteases such as pepsin, renin, and cathepsin D) had no effect on CD80 surface expression in normoxia and in hypoxia (Table 1) . Phenantrolin, which inhibits metalloproteases, showed a nonsignificant decrease in surface CD80 in normoxia but had no effect in hypoxia. Collectively, none of these protease inhibitors were found to affect the surface expression of CD80 in normoxia or hypoxia.


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  		Table 1. Effects of Protease Inhibitors on Surface CD80 Expression on Mouse Macrophages

 
Effect of hypoxia on the accumulation of CD80 mRNA (Fig. 8 )
To establish whether increased amounts of sCD80 in hypoxia resulted from alternative splicing of the transcript, two different areas of CD80 mRNA were amplified. Amplification of the area coding for the transmembranal region of the CD80 molecule yielded a fragment representing the membrane-anchored molecule, whereas amplification of the first extracellular exon yielded a fragment representing the membrane-anchored molecule and the secreted, alternatively spliced CD80. Amounts of CD80 mRNA amplified by the first exon or the transmembranal region primers in normoxia were very similar, although a small, nonsignificant increase of 20% was observed in the transmembranal region. Amplification of exon 1 revealed no change in the accumulation of CD80 mRNA in normoxia and hypoxia throughout the experiment. In contrast, the accumulated transcripts amplified by the transmembranal region primers revealed a 56% decrease in the accumulation of CD80 mRNA after 24 h of hypoxia (P<0.05), which was also significant compared with the amplification of the first exon in normoxia (P<0.05). These results suggest that hypoxia increases the transcription of the alternatively spliced sCD80.



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  		Figure 8. The membrane-anchored spliced form of CD80 was reduced in hypoxia. RAW 264.7 cells were incubated in normoxia or hypoxia for different periods of time. Total RNA was extracted from the cells and transcribed, and semi-quantitative RT-PCR using primers for the transmembranal region or for the extracellular exon 1 amplified CD80 mRNA. (A) A representative gel depicting the reduction of CD80 mRNA at 24 h of hypoxia, only in the transmembranal region. M, Molecular weight marker; -RT, amplification without addition of RT. (B) A densitometric analysis of the gels at 24 h (n=5). The amplified bands were normalized to the 18S rRNA that is not changed in hypoxia. *, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results obtained in this study demonstrate that hypoxia selectively reduced the surface expression of the CD80 costimulatory molecules on human monocytes but did not affect expression of HLA-DR or the other costimulatory molecule CD86. Prolonged reoxygenation restored CD80 to normoxic levels. Addition of LPS had only a marginal effect on the expression of any of these molecules. In contrast to its inhibitory effects on CD80 expression, prolonged hypoxia synergistically enhanced the LPS-induced secretion of TNF-{alpha} from human PBMC, and prolonged reoxygenation was required to significantly attenuate this effect. Similar results demonstrating the reduction of CD80 expression in hypoxia and the synergistic increase in secretion of TNF-{alpha} were obtained using the mouse macrophage cell line RAW 264.7 and the mouse primary peritoneal macrophages, respectively.

LPS is a known potent transcriptional and translational enhancer of TNF-{alpha} [37 , 38 ]. Accordingly, incubation of human PBMC or mouse macrophages with LPS in normoxia increased the secreted cytokine after 24–48 h of culture. Similarly, in rat alveolar macrophages, hypoxia was shown to synergize with LPS to enhance activation of nuclear factor-{kappa}B, resulting in increased production and release of TNF-{alpha} [39 ]. However, detailed evaluation of the signal-transduction pathways that are evoked by hypoxia and lead to the increased production of the cytokine is still unclear. The synergistic "two-hit" attack provided by hypoxia combined with LPS resembles the in vivo situation, where hypoxia simulates ischemia, and LPS represents ischemia-related translocation of bacteria and/or their toxins [7 ], resulting in simultaneous confrontation with both insults. Cumulatively, our results may indicate a crucial role for LPS in effector functions of monocytes/macrophages, as manifested by TNF-{alpha} secretion, and a less-significant role in antigen presentation, as represented by its marginal effects on surface expression of HLA-DR, CD86, and CD80 molecules.

Local ischemia and more so its systemic complications often cause immunosuppression, where the Th1/Th2 balance is shifted toward a Th2 response, and the Th1 cellular-mediated response is impaired [40 ]. Our findings, using the hypoxia-simulation model, suggest one possible explanation for this phenomenon, as onset of a Th1 response has been considered to involve CD80, and CD86 has been shown to be needed for the initiation of a Th2 response [19 ]. We showed a selective reduction of surface CD80 expression by hypoxia, which may point to a temporary impairment of a Th1 response and therefore a shift toward a Th2 response. Recent studies have demonstrated that CD80 molecules may also have a partial role in the generation of Th2-mediated immune responses, depending on the antigen examined [20 , 21 ]. Thus, it is possible that hypoxia, through down-regulation of monocyte/macrophage CD80, may also affect other facets of the immune response.

Although CD86 molecules are constitutively expressed on monocyte surfaces, CD80 expression requires cell activation. Adhesion of monocytes to tissue-culture plates, as performed in this study, has been demonstrated to provide the activation signals needed [41 ] and to induce functional CD80 expression [42 ]. In our experiments, freshly isolated human monocytes were devoid of detectable surface CD80 molecules, which were rapidly induced during adhesion. Such activation may represent part of the inflammatory adherence of monocytes to endothelium and their recruitment into target ischemic tissue [43 , 44 ]. Although the expression of HLA-DR, CD86, and CD80 molecules was measured only on CD14-positive cells and although monocytes are the main secretors of TNF-{alpha}, direct or indirect effects on the expression of these molecules could arise by nonmonocytic cells in the PBMC culture. Therefore, we have used mouse primary-isolated macrophages to verify the synergistic effects of hypoxia and LPS on TNF-{alpha} secretion. As a result of the large number of cells required, we have used the well-established peritoneal macrophage-like cell line RAW 264.7 to corroborate the hypoxia-mediated effects on CD80 expression and to further delineate the mechanisms involved. Unlike human primary monocytes, this cell line constitutively expressed CD80, and hypoxia reduced it, although with slower kinetics. However, the basically similar phenomenon was observed in human and mouse monocytes/macrophages.

We first looked into two possible post-translational mechanisms: that hypoxia caused the internalization of CD80 into the cells or that hypoxia increased shedding of surface CD80. In normoxia, incubation of RAW 264.7 cells with the internalization inhibitor cytochalasin B moderately increased the surface expression of CD80, suggesting interruption of a normal turnover of the molecule. In hypoxia, however, the inhibitor did not increase surface CD80 expression, negating the possibility of its hypoxia-induced internalization. The possibility of enhanced CD80 shedding was then examined by comparing the amount of sCD80 in supernatants derived from cell cultures subjected to normoxia and hypoxia. Indeed, a fourfold elevation of sCD80 expression was observed during hypoxia, which was comparable with the fourfold reduction in the surface CD80 expression, whereas no change was detected in normoxic supernatants. Although protease inhibitors with a wide range of specificities did not block the hypoxia-induced reduction of surface CD80 expression, the possibility of increased shedding by a yet-unknown protease cannot be ruled out. A third possible explanation could involve alternative splicing of the CD80 molecule, resulting in a soluble molecule. Naturally occurring, soluble forms of CD80 [45 ] and sCD80 resulting from alternative spliced transcripts lacking exons coding for the cytoplasmic and transmembrane domains [46 ] support this premise. These data as well as our inability to conclusively demostrate post-translational mechanisms responsible for the hypoxia-induced elevation of sCD80 prompted us to investigate the effect of hypoxia on the accumulation of an alternatively spliced CD80 transcript, which lacks the transmembrane sequence. We have shown that the accumulation of the transcript containing the transmembranal domain was reduced in hypoxia but not in normoxia. In contrast, transcripts that contain the first exon (i.e., the alternatively spliced, soluble form and the membrane-anchored form) remained relatively unchanged in normoxia and hypoxia. However, the mechanisms put into motion by hypoxia, which initiates alternative splicing of CD80, are still unknown and warrant further investigation.

The physiological significance of the alternatively spliced sCD80 is not yet clear. In a recent study, an alternatively spliced, soluble form of the porcine CD80 molecule was cloned and characterized and was shown to interact with its CD28 and cytotoxic T-lymphocyte antigen 4 receptors on T cells and to block their activation [46 ]. This is in line with other soluble adhesion molecules [47 ] and cytokine receptors [48 ], which down-regulate inflammation and support our suggestion that this could be one of the mechanisms by which hypoxia leads to immune suppression.

It has repeatedly been shown that ischemia, sepsis, and trauma reduce the expression of MHC class II molecules on monocytes/macrophages [23 24 25 26 27 , 31 ]. This reduction has been associated with defective antigen presentation and immunosuppression. Our results demonstrate that only one costimulation molecule (CD80) was strongly affected by hypoxia, and others (HLA-DR and CD86) were not changed. In addition, we [16 ] and others [7 ] have previously shown rapid (2–4 h) enhancement of TNF-{alpha} secretion following I/R. However, in the present study, prolonged (24 h) hypoxia and LPS were required for high amounts of TNF-{alpha} production. Collectively, these findings suggest that hypoxia does not fully mimic ischemic processes, in which additional factors such as acidosis, hypercapnia, and the lack of nutrients other than oxygen may play a role in the macrophage inflammatory and immune responses to tissue injury. Additionally, hypoxia-induced, enhanced secretion of TNF-{alpha} and inhibited CD80 surface expression were reversed by prolonged reoxygenation, suggesting that in addition to cell salvage, reoxygenation, which simulates reperfusion, serves to restrain the injurious response of macrophage to hypoxia.

In conclusion, we have shown that hypoxia simultaneously influences two molecules, which regulate aspects of monocytes functions: A molecule (CD80) that is necessary for antigen presentation is suppressed, and a molecule (TNF-{alpha}) that is important in inflammatory/cytotoxic functions is enhanced. Finally, the results suggest, for the first time, that hypoxia may regulate CD80 expression by evoking mechnisms leading to alternative splicing of its mRNA.


   ACKNOWLEDGEMENTS
 
This study was supported in part by funds contributed by the Rappaport Institute of Medical Research, the Technion V.P.R. Fund, and the Hedson Fund for Medical Research. N. L. and M. A. R. are equally contributing, senior authors.

Received March 13, 2003; accepted March 20, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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