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Articles in PresS. Am J Physiol Cell Physiol (April 23, 2008). doi:10.1152/ajpcell.00572.2007
HYPOXIA ENHANCES LYSOSOMAL TNF
DEGRADATION IN MOUSE PERITONEAL
MACROPHAGES
Nitza Lahat1*, Michal A. Rahat1*, Amalia Kinarty1, Lea WeissCerem2, Sigalit Pinchevski1, and Haim Bitterman2.
1
Immunology Research Unit and 2Ischemia-shock Research Laboratory, Carmel
Medical Center, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel
Institute of Technology, Haifa, 34362, Israel.
Running Title: Hypoxia inhibits LPS-induced TNF, secretion.
Corresponding Author: Michal A. Rahat, D.Sc., Immunology Research Unit,
Carmel Medical Center, 7 Michal St., Haifa, 34362 Israel. Tel: 972-4-8250404 Fax:
972-4-8250408, e-mail: [email protected]
*
The first and second authors are equally contributing senior authors.
Copyright © 2008 by the American Physiological Society.
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ABSTRACT
Infection, simulated by LPS, is a potent stimulator of tumor necrosis factor ,
(TNF,) production, and hypoxia often synergizes with LPS to induce higher levels of
the secreted cytokine. However, we show that in primary mouse peritoneal
macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7,
J774A.1 and PMJ-2R), hypoxia (O2 <0.3%) reduces the secretion of LPS-induced
TNF, (p<0.01). In RAW 264.7 cells this reduction was not regulated
transcriptionally, as TNF, mRNA levels remained unchanged. Rather, hypoxia and
LPS reduced the intracellular levels of TNF, by 2-fold (p<0.01) by enhancing its
degradation in the lysosomes and inhibiting its secretion via secretory lysosomes, as
shown by confocal microscopy and verified by the use of the lysosome inhibitor
Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the
soluble receptor TNFRII, it increased its binding to the secreted TNF, by 2 fold
(p<0.05). We suggest that these two post-translational regulatory checkpoints co-exist
in hypoxia, and may partially explain the reduced secretion and diminished biological
activity of TNF, in hypoxic peritoneal macrophages.
Key words: Macrophages, Cytokines, Inflammation, Trafficking, Secretory
lysosomes.
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INTRODUCTION
Hypoxia is a central feature of ischemic, inflamed, and infected tissues, and a
principal determinant of the pathophysiology of local and generalized systemic
inflammatory responses (SIR) in these conditions (27). In infection, increased oxygen
consumption by proliferating bacteria and inflammatory/immune cells as well as
hemodynamic alterations are some of the proposed mechanisms that underlie this
phenomenon (27). Among all causes of septic SIR, intraperitoneal infection is a
relatively common clinical entity that frequently occurs in chronic liver diseases, and
complicates abdominal trauma, operations, and bowel perforation. The latter are
usually caused by a mixed bacterial population and have been shown to cause
peritoneal hypoxia (35). Hence, both exposures to hypoxia and bacterial proinflammatory mediators are important components in the pathophysiology of local
and systemic inflammatory responses (28), in clinical sepsis in general and in
infectious peritonitis in particular. . Tumor necrosis factor-, (TNF,) secreted mostly
by macrophages plays a central role in mediating excessive inflammatory responses in
these conditions (13; 33). Since hypoxia is a major constituent of ischemia, and LPS
is a potent transcriptional and translational inducer of TNF, (5; 48) and a key
mediator of SIR, their combined effect is commonly employed to mimic a clinically
relevant in vivo scenario of inflammatory response in ischemia and infection. So far, a
number of studies have shown that hypoxia synergizes with LPS to enhance the
production and release of TNF, from human, rat and mouse monocytes and
macrophages (7; 16; 21; 26; 36; 46). This synergism was mainly attributed to
transcriptional up-regulation involving NF B (7; 24), although other mechanisms
such as reduction in cAMP were also suggested (26). However, hypoxia has also been
demonstrated to inhibit or not to change the amounts of secreted LPS-induced TNF,
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(29; 46). The reduction was explained by enhanced sensitivity of these macrophages
to hypoxia-mediated apoptosis. Furthermore, the effects of circulating or secreted
TNF, may also be regulated by its binding to the soluble forms of the two TNF
receptors, sTNFR-I and sTNFR-II, which can bind and inhibit its activity (4). Indeed,
elevated levels of these receptors have been found in patients with different
pathologies, whose common denominator is hypoxia (8; 12; 42), and hypoxia was
shown to increase their secretion in vitro (36).
In this study we evaluated changes in the secretion and activity of TNF, from
thioglycollate (TG)-elicited primary peritoneal macrophages and the mouse peritoneal
macrophage cell lines RAW 264.7, PMJ-2R and J774.A1 following exposure to LPS
and hypoxia, where LPS represents gram-negative infection or ischemia-associated
bacterial translocation,
and hypoxia is characteristic of an inflammatory
microenvironment or simulates ischemia. In our model of simulated hypoxia/infection
in peritoneal macrophages we show that hypoxia reduced the secretion and biological
activity of TNF, without associated cell death, and suggest that at least two posttranslational mechanisms, enhanced lysosomal degradation of TNF, and increased
extracellular binding to soluble TNF receptor II, are responsible for this phenomenon.
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MATERIALS AND METHODS
Cells: Primary peritoneal macrophages, isolated from both female and male
BALB/c and C57Bl/6 mice, were collected 4 days after an i.p. injection of 3ml of 24
mg/ml solution thioglycollate (TG), by injecting their peritoneal cavity 10ml of
phosphate-buffered saline (PBS), massaging, and drawing back to the syringe the fluid
now containing the macrophages. Washed TG-elicited peritoneal macrophages were
plated at a concentration of 0.8x106 cells/well for 2 hours in Dulbecco's modified
Eagle's medium (DMEM) and 20% fetal calf serum (FCS) to facilitate adherence,
following replacement of the medium with DMEM without FCS (serum starvation) for
1 hour before their exposure to the experimental conditions, in order to avoid possible
masking of signals initiated by the exogenous stimuli in the FCS. Purity of all the
adherent primary macrophages was routinely I84±2.9% as was evaluated by labeling
the cells with FITC-conjugated rat anti-mouse F4/80 (SeroTec, Oxford, UK) and
analyzing its surface expression by flow cytometry. This part of the study was
performed in adherence to the NIH “guide for the care and use of laboratory animals”.
The mouse peritoneal macrophage cell lines RAW 264.7, J774.A1 (derived from
BALB/c mice) and PMJ2R (derived from C57BL/6J mice), were cultured in DMEM
with 10% FCS and antibiotics, and were similarly serum-starved 1 hour before the
beginning of the experiment. All cells were subjected to normoxia or hypoxia for 24
hours, with or without the addition of LPS (1 µg/ml, E. Coli 055:B5, Sigma, St. Louis,
MO). In some experiments cells were incubated with increasing amounts of
bafilomycin A1 (Calbiochem, Darmstadt, Germany) and MG132 (Calbiochem), that
did not cause cell death, as assayed by the XTT kit. In all experiments cell viability
was determined using the XTT kit (Biological industries, Beit-Haemek, Israel).
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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),
where the hypoxic environment (O2 <0.3%, 5% CO2, 95% N2), the temperature (37°C)
and humidity (90%) are kept constant. 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). Mean PO2
values in the supernatants were 146±1.9 mmHg in normoxia and 26±0.9 mmHg in
hypoxia, whereas the mean values of PCO2 (32.5±0.3 mmHg) and the pH values
(7.4±0.07) did not change in normoxia and hypoxia.
ELISA: Commercial TNF,, soluble TNF receptor I and soluble TNF receptor II
ELISA kits (R&D Systems, Minneapolis, MN) were carried out according to the
manufacturer's instructions.
TNF biological activity: TNF secretion from RAW 264.7 cells was determined
by its biological activity as well as by an ELISA assay. To measure the biological
activity, L-929 fibroblast cells at a concentration of 5x103 cells/well were plated in 96well flat bottom plates. When the monolayer became confluent, 4 pg/ml actinomycin
D (Sigma) together with either the mouse recombinant TNF
or the experimental
supernatants were added. After 24h incubation, XTT (30 µl/well) was added and
incubated for 4h 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 doseresponse curves, in which the OD read was proportional to the reciprocal of the
concentration of biologically active TNF- .
Quantitative real-time PCR analyses: Total RNA was extracted from 4x106 RAW
264.7 cells exposed to the experimental conditions using TriReagentTM (Molecular
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Research Center, Cincinnati, OH) according to the manufacturer's instructions. Five
micrograms of total RNA were transcribed to cDNA at 37°C for 1 hour using 200µM
deoxynucleotides (Sigma), 5µM random hexamers (Amersham Pharmacia Biotech,
Piscataway, NJ), 20 U RNAguard (Amersham), and 200 U/µl of MMLV-RT (US
Biochemicals, Cleveland, OH). TNF, mRNA expression was determined by
quantitative real-time PCR on the cDNA samples using the TaqMan assay on
demand™ kit with the ABI-PRISM 7000 (Applied Biosystems, Foster City, CA).
Analysis was carried out in triplicates in a volume of 20µl (2 min at 50ºC, 10 min at
95ºC, and a total of 40 cycles, each of 15 sec at 95ºC and 1 min at 60ºC) for both
TNF, and the endogenous reference gene 18S rRNA, which does not change in
hypoxia, and the comparative CT method was used. In each experiment the normoxic
non-stimulated RNA sample was used as a calibrator to allow comparison of relative
quantity between the samples.
Western blots analyses: RAW 264.7 cells were lysed in RIPA buffer and their
supernatants were collected, concentrated 30-fold by VivaSpin2 (Vivascience,
Lincoln, UK) and equal protein amounts or equal volumes were loaded on a 10%
SDS-PAGE. Proteins were separated and transferred onto cellulose nitrate membranes
(Schleicher & Schuell, Germany). The membranes were incubated for 1 hour in
blocking buffer (20% skimmed milk, 1% BSA, 0.01% Tween-20, 10mM Tris pH 8.0,
150mM NaCl) at room temperature, probed for 1 hour with the diluted (1:250) rabbit
polyclonal anti-TNF, (Genzyme, Cambridge, MA), washed three times in 1XTBST
(10mM Tris pH 8.0, 150mM NaCl, 0.5% Tween-20), and incubated with HRPconjugated goat anti-rabbit IgG (Jackson ImmunoReasearch Laboratories, West
Grove, PA), diluted 1:5000 for additional 1 hour. The enhanced chemiluminescence
(ECL) system (Amersham) was used for detection. The optical density of the bands
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was quantified using Bio-Imaging system (Dinco & Rhenium, Jerusalem, Israel) and
TINA software (Raytest, Straubenhardt Germany).
Immunoprecipitation: RAW 264.7 cells were cultured and exposed to normoxia or
hypoxia, with or without the addition of LPS for 24 hours. Cells were harvested in
RIPA buffer and protein concentrations of the cellular extracts were measured by
Bradford reagent. To avoid non-specific binding, 200µg of protein were incubated
with 2µg of normal mouse serum and precipitated using protein-A agarose beads. The
remaining proteins were incubated with 2µg of hamster polyclonal anti-mouse TNFRI
or TNFR-II (R&D systems) and rotated overnight at 4°C with protein-A agarose. After
centrifugation the pellet was washed 4 times in PBS, resuspended in loading buffer,
boiled for 15 min and loaded onto a 10% SDS-PAGE. After electrophoretic
separation, TNF, was detected by western blot analysis as described before.
Flow cytometry analysis: RAW 264.7 cells were cultured in normoxia or hypoxia
for 24 hours, with or without the addition of LPS, and then were labeled with goat
anti-mouse TNF, (R&D systems) and FITC-conjugated donkey anti-goat (R&D
systems). After washing, the cells were fixed in 0.1% formaldehyde and were analyzed
using a Coulter-XL flow cytometer (Coulter Electronics, UK). Dead cells were
excluded from the analysis by their forward and sideway light-scattering properties.
Immunofluorescence: RAW 264.7 cells were incubated in the experimental
conditions on cover slips, fixed with 3.7% formaldehyde for 10 min at room
temperature and permeabilized in 0.1% Triton X-100. To avoid non-specific binding
the cells were incubated with 4% donkey normal serum for 30 min at room
temperature following 3 washes with PBS. The primary antibodies goat anti-mouse
TNF, (R&D systems) or rabbit polyclonal anti-Rab8, anti-Rab5 and anti-Rab7, antiRab11, anti-Rab4 and anti-LAMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA)
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were incubated for 1 hour at room temperature following three washes with PBS.
Secondary antibodies (Rhodamine Red-X or Cy3-conjugated donkey anti-goat IgG or
Cy2-conjugated donkey anti-rabbit IgG, Jackson ImmunoResearch Laboratories) were
incubated in the dark for 1 hour and washed three times with PBS. The cover slips
were mounted on a slide with fluoromount G. Immunofluorescent images were
acquired by confocal microscopy, using the Bio-Rad MRC 1000 confocal system, and
the images were analyzed using the ImagePro Plus 4.5 software (Media Cybernetics,
Silver Spring, Maryland).
Statistical analyses: All values are presented as mean±SE. The data were analyzed
using repeated measures analysis of variance (ANOVA). The Student Newman-Keuls
multiple comparisons test was used to evaluate significance between experimental
groups, and p values exceeding 0.05 were not considered significant.
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RESULTS
Hypoxia reduces LPS-induced secretion of TNF': In primary peritoneal
macrophages, incubation in normoxia or hypoxia for 24 or 48 hours was not sufficient
to induce TNF, secretion, and stimulation with LPS was required. In normoxia, LPS
induced TNF, secretion, but although hypoxia did not induce cell death, as evaluated
by the XTT viability test (data not shown), it reduced this secretion by 2.5-folds
(p<0.01) after 24 hours (figure 1A). A similar effect persisted after 48 hours of
incubation (11265±3012 pg/ml in normoxia and 5664±981 pg/ml in hypoxia, p<0.01).
Of note, analysis of these results showed a 2-fold difference in the magnitude of
TNF, secretion between male- and female-derived TG-elicited
peritoneal
macrophages incubated in normoxia and LPS (13253±3867 in males vs. 26877±8822
pg/ml in females, p<0.01), however, hypoxia similarly reduced the levels of the
cytokine by 2 and 2.8 folds, respectively (p<0.05 and p<0.001). Similarly, incubation
of the mouse peritoneal macrophage cell line RAW 264.7 in normoxia and hypoxia,
with or without LPS did not cause cell death after 24h (figure 1B) or 48h (data not
shown). In the absence of LPS, TNF, secretion was hardly detected by ELISA (figure
1C), whereas addition of LPS for 24 and 48 hours induced it (92,970±18,024 and
87,450±30,050 pg/ml, respectively, p<0.001). In contrast, incubation in hypoxia
resulted in a significant 3-fold reduction in TNF, secretion after 24 hours (p<0.001)
and 48 hours (p<0.05) of incubation. This inhibitory effect of hypoxia on RAW 264.7
cells was validated by a biological activity assay (3.9-fold, p<0.001, figure 1D), and
by western blot analysis (2.7-fold, p<0.05, figure 1E). The difference in TNF,
concentrations measured in the ELISA and biological assays may reflect the essential
difference between the techniques. ELISA also revealed that two other peritoneal
macrophage cell lines, J774.A1 (figure 1F) and PMJ2R, also exhibited a similar 2.8-
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fold and 1.5-fold reduction in LPS-induced secretion of TNF, in hypoxia (2,815±772
pg/ml and 1,005±664 pg/ml, p<0.05 for J774A.1 and 10810±721 pg/ml in normoxia
vs. 7238±333 pg/ml in hypoxia, in PMJ2R, p<0.01). Of note, hypoxia, even with LPS,
did not cause any cell death in these two cells lines (0.96±0.05 and 1.02±0.02 fold
from control, for J774A.1 and PMJ2R, respectively). Although concentrations of
TNF, were different in each cell line, probably due to differences in strains and/or
gender, the three peritoneal macrophage cell lines and the primary peritoneal
macrophages exhibited similar reduction in TNF, secretion in hypoxia. We focused
on the RAW 264.7 cell line to further study the mechanistic explanations of this
phenomenon.
Hypoxia does not change the steady state levels of TNF' mRNA in RAW 264.7
cells: To evaluate the effects of hypoxia on transcription of TNF,, we used real-time
PCR to measure the relative accumulation of TNF, mRNA following stimulation
with LPS, in both normoxia and hypoxia. Each sample was normalized to the
endogenous reference 18S ribosomal RNA, which does not change in hypoxia, as do
other housekeeping genes such as T-actin or GAPDH (49). While hypoxia alone
increased TNF, mRNA by 1.7±0.2 folds, this tendency did not reach statistical
significance relative to the normoxic non-stimulated cells (figure 2A). LPS
significantly up-regulated the accumulation of TNF, mRNA (by 4.9±0.7 fold,
p<0.001) in macrophages in normoxia, and these levels did not change in hypoxia.
Moreover, the increase in TNF, mRNA levels following LPS stimulation cannot
explain the thousands fold increment in TNF secretion. These observations do not
support transcriptional regulation as an important mechanism controlling the hypoxiainduced suppression of TNF, secretion, and directed our attention to post-
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transcriptional mechanisms involved in the regulation of TNF, production and
secretion.
Hypoxia does not cause intracellular accumulation of TNF' or disrupt its
trafficking: In non-stimulated cells, intracellular concentrations of TNF, protein
measured by western blot and ELISA were very low (figure 2B, 2C), but addition of
LPS in normoxia increased it by 13- and 11-folds, respectively (p<0.001). However,
in hypoxia the intracellular concentrations of TNF, were significantly reduced by 2.5fold in western blot analysis (p<0.05) and by 2-fold in ELISA (p<0.01) compared to
the stimulated normoxic values, corresponding to the hypoxia-induced inhibition of
TNF, secretion and suggesting that TNF, protein is not accumulating in hypoxic
cells. Furthermore, since TNF, is cleaved off the membrane by TNF, converting
enzyme (TACE) we also checked its expression by FACS. Non-stimulated cells in
normoxia expressed low levels of TACE, and LPS was required to enhance its
expression by 3 folds (mean fluorescence of 0.67±±0.11 and 2.22±0.4, respectively,
p<0.05). Hypoxia alone or hypoxia with LPS did not significantly change TACE
expression (2.7±0.6 and 1.92±0.8, respectively).
Using confocal microscopy, we further evaluated TNF, trafficking in the cells and
its co-localization with several Rab proteins that characterize transport vesicles which
could be implicated in TNF, trafficking: Rab8 which is a marker of secretory vesicles
and vesicles mediating polarized delivery of membrane proteins, Rab5 which is a
marker of early endosomes, Rab4 and Rab11 which are markers of recycling
endosomes that are associated with turnover of membranal proteins, Rab7 which is a
marker of late endosomes linking transport between them to the lysosomes and back,
LAMP-1 which is a marker of lysosomes, and Rab27a which is a marker of secretory
lysosomes that mediate secretion of some proteins in cells of the immune system.
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Isotype-matched control sera yielded no immunostaining or only a very weak
staining. TNF, was not induced in non-stimulated cells, whereas the staining of all
Rab proteins was constitutive and was not affected by hypoxia (data not shown).
Analysis of the images demonstrated that in hypoxia the mean area of the cells was
significantly decreased by 1.6-fold (206±13 µm2 compared to 337±31 µm2 in
normoxia, p<0.01). To correct for changes in the fluorescent signal which may occur
due to the decreased area, we used the integrated optical density (IOD), which takes
into account the area of the cells. The IOD values for each fluorescently-labeled
protein was calculated separately and determined in three repetitions of the
experiment, in at least 5 different fields. Staining for Rab7, and for Rab8 did not
change in hypoxia and was not co-localized with TNF, in either normoxia or hypoxia
(data not shown), suggesting that secretory vesicles and late endosomes do not carry
TNF,. Intracellular TNF, was decreased by 33% in hypoxia compared to normoxia
(p<0.001), confirming our previous results (figure 3, panels B and H, figure 4, panels
B and E, H and K, M and Q), whereas staining for Rab5 (figure 3, panels A and G),
Rab4 (figure 4 panels G and J), Rab11 (data not shown), Rab27a (figure 4, panels A
and D) and LAMP-1 (figure 4, panels N and P) did not significantly change in
hypoxia. In both normoxia and hypoxia TNF, was found co-localized to Rab5 (figure
3, panels C and I), Rab4 (figure 4, panels I and L), and Rab27a (figure 4, panels O and
R), but in hypoxia this co-localization was reduced. In contrast, co-localization of
TNF, with LAMP-1 was increased by 84% in hypoxia (p<0.05).
Hypoxia increases TNF' degradation in the lysosome, involving Rab5
expressing endosomes: Hypoxia-induced degradation of TNF, was investigated by
using the proteosome inhibitor MG132 and the lysosome inhibitor bafilomycin A1.
Intracellular or secreted amounts of TNF, were not affected by inhibition of the
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proteosome degradation activity (figure 5B, 5D), and remained significantly lower in
hypoxia (p<0.05 from normoxia), regardless of the increasing concentrations of
MG132. In contrast, bafilomycin A1 that did not affect the intracellular levels of
TNF, in normoxic macrophages significantly increased it in hypoxic macrophages in
a dose-dependent manner (figure 5A), so that in 5 and 10nM of bafilomycin A1 the
amounts of intracellular TNF, were equal to those in normoxia (p<0.05 and p<0.01
relative to hypoxic cells without bafilomycin A1). The amounts of secreted TNF, did
not change upon addition of bafilomycin A1, further supporting the retention of TNF,
in the cells (figure 5C). In addition to these results, which were obtained using ELISA
on cellular extracts, the effects of bafilomycin A1 were also examined by confocal
microscopy (figure 3, panels D-F and J-L). In normoxia, addition of the lysosome
inhibitor (10nM) did not alter TNF, staining in LPS-stimulated cells (figure 3, panels
B and E) or its co-localization with Rab5 (figure 3, panels C and F), but in hypoxia it
increased the accumulation of intracellular TNF, by 35% (p<0.01) compared to
hypoxia alone (figure 3, panels H and K), restoring it to normoxic values.
Hypoxia decreases membranal TNF' expression: In normoxia or hypoxia, nonstimulated RAW 264.7 cells exhibited only negligible amounts of membraneassociated TNF, as evaluated by flow cytometry (mean fluorescence <0.009, Figure
6B), while incubation with LPS in normoxia significantly increased it (mean
fluorescence 0.27±0.03, p<0.001). Hypoxia significantly decreased LPS-induced
membranal attachment of TNF, by 1.5-fold (figure 6A and 6B, mean fluorescence
0.18±0.02, p<0.01 relative to LPS-stimulated normoxic cells), excluding the
possibility that augmented membranal association of TNF, leads to the reduced
amounts of TNF, in the supernatants.
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Hypoxia does not promote degradation of secreted TNF', but increases its
binding to the soluble receptor type II: We used western blot analysis in an attempt
to detect an increase in fragmented bands of TNF, in the hypoxic cultures. However,
although hypoxia reduced the secreted level of TNF, by 2.7 fold (p<0.05), as
indicated by the autoradiograms (figure 1E), in accordance with the lower biological
function and immunological reactivity (figure 1D, 1C), no additional degraded bands
of the cytokine were observed.
We measured the effects of hypoxia on the concentrations of the two soluble
TNF, receptors (sTNFR-I, sTNFR-II). Figure 6C shows that in normoxia sTNFR-I
was constitutively secreted from RAW 264.7 cells (110±15 pg/ml), but was
significantly reduced in hypoxia (54±9 pg/ml, p<0.01). Addition of LPS elevated the
concentration of sTNFR-I in normoxia (230±18 pg/ml), however its levels in hypoxia
remained significantly reduced (135±11 pg/ml, p<0.01). Negligible amounts of
sTNFR-II were observed in normoxia and hypoxia in non-stimulated cells (figure
6D), and addition of LPS similarly induced them (to about 400±75 pg/ml) in both
normoxia and hypoxia. We then performed immunoprecipitation (IP) using the
supernatants to evaluate the actual binding of secreted TNF, to each of the soluble
receptors. As shown in figure 6E, binding of TNF, to sTNFR-I in normoxia or
hypoxia, with or without the addition of LPS, was hardly detected. In contrast,
following LPS addition, TNF, was bound to sTNFR-II both in normoxia and hypoxia,
and hypoxia significantly increased this binding by 2-fold (p<0.05).
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DISCUSSION
It has previously been shown that hypoxia alone triggers only small amounts of
TNF,, and it synergizes with LPS to induce high secretion from both primary
macrophages (10; 16; 21) and monocyte/macrophage cell lines (7; 36). This
synergism has previously been attributed to the activation of NF B and reduction of
cAMP levels, leading to enhanced transcription of the TNF, gene (7; 24; 26).
Interestingly, we found suppressive effect of hypoxia on LPS-induced secretion of
TNF, from mouse peritoneal macrophages and three peritoneal cell lines (RAW
264.7, J77A.1 and PMJ2R cells). This inhibitory effect of hypoxia on LPS-induced
secretion of TNF, was described before in RAW 264.7 cells and interpreted as
resulting from a pro-apoptotic influence of hypoxia (46). However, we did not detect
increased cell death in hypoxic RAW 264.7, J774A.1, and PMJ2R cells or in the
primary peritoneal macrophages. Therefore, we suggest that the reduction of TNF,
secretion was a consequence of adaptation to the hypoxic environment and did not
result from decreased cell number. The contrast between the reported synergism of
LPS and hypoxia and our findings, may be attributed to the specific
microenvironmental influences of the peritoneum, as macrophages are known for their
heterogeneous functions and responses dictated by their local microenvironment in
each tissue (23). Some studies report a synergism between LPS and hypoxia despite a
common peritoneal origin of the macrophages. For example, Albina et al showed an
increase in TNF, secretion from resident peritoneal cells exposed to 2% O2 and
IFNV+LPS (2). Likewise, the J774A.1 cell line was shown to secrete more TNF, upon
induction with LPS and 1.5% O2 (7). The differences between these studies and our
results may be explained either by the differences between resident and TG-elicited
peritoneal macrophages, or by the different hypoxic environment, as we exposed our
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cells to severe hypoxia (<0.3% O2). In addition, our findings are in agreement with
others, who found reduction in TNF, secretion in hypoxia (46). Thus, the effects of
hypoxia on macrophage TNF, production and secretion remain controversial.
The driving force for diffusion of oxygen into the tissue is determined by its
partial pressure gradient between the capillary and tissue cells, and not by oxygen
content. Therefore, the hypoxic oxygen tensions in our in vitro environment (26±0.9
mmHg) are relevant to the environment of the injured tissue, and represent a true
hypoxic environment, as normal reported peritoneal oxygen tensions measured were
45-58 mmHg (6; 35). Furthermore, key functions required for clearance of infection
and repair of tissue (e.g. leukocyte killing activity, necrotic tissue proteolysis,
fibroblast proliferation, collagen accumulation and crosslinking and epitheliazation)
are markedly impaired at tissue oxygen tensions below 30 mmHg, and almost
completely stop at significantly lower values (19; 30). Macrophage adapt well to this
level of hypoxia by elevating the expression of specific proteins and growth factors
(e.g. glycolytic enzymes, TGF,, FGF, VEGF and HIF-1) and reducing the expression
of others (25). We have previously shown that in macrophages hypoxia and a proinflammatory stimulus reduced the surface expression of CD80 (21), the secretion of
MMP-9 (34), and the production of NO despite the elevated expression of iNOS (9).
Thus, the effects of hypoxia and LPS on peritoneal macrophages may not be specific
for TNF,, but reflect a general mechanism designed to reduce or restrain their proinflammatory response, although the molecular mechanisms utilized to achieve this
reduction, may be different for each mediator molecule. This inhibitory effect of
hypoxia on LPS-induced secretion of TNF, may be relevant in physiological
conditions where, for example, septic patients were reported to exhibit an antiinflammatory response (compensatory anti-inflammatory response syndrome-CARS)
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rather than a pro-inflammatory response (SIRS) (20). Hence, there is no one response
to stimulations such as hypoxia and LPS, but rather a spectrum of responses that may
depend on the type of bacterium, the specific tissue involved, the degree of hypoxia,
the origin of the macrophages and the underlying condition of the patient
To further delineate mechanisms responsible for the observed hypoxia-induced
reduction of TNF, secretion, we used the RAW 264.7 cell line, which provided us
with a large number of homogeneous cell population needed for these experiments.
LPS strongly enhances TNF, transcription, but since there was no change in the
steady-state amounts of TNF, mRNA between normoxia and hypoxia, transcriptional
regulation as a mechanism explaining the hypoxia-induced reduction in TNF, levels
was ruled out. We then investigated the possibility that hypoxia post-translationally
interferes with the intracellular secretory mechanisms of the already synthesized
TNF,, implying that TNF, would accumulate in higher amounts inside hypoxic cells
or on their membranes compared to normoxic cells. However, we found reduced
intracellular amounts of all TNF, forms (e.g. the mature 17 kDa form and the 26 kDa
membranal form, or additional dimers or trimers) inside RAW 264.7 cells or on their
membranes following incubation in hypoxia and LPS. Of note, TNF, is known to be
stored only in mast cells (32), whereas in other cells all pro-TNF, is transported to the
membrane and cleaved by TACE. Thus, the minimal levels of TACE found in nonstimulated cells may account for the expression of the 26 kDa membranal form of
TNF, found in these cells (figure 2B). The hypoxia-induced reduction of intracellular
TNF,, together with the lack of change in TNF, mRNA, suggests enhanced
degradation of the protein. To examine the effect of hypoxia on TNF, degradation we
used inhibitors specific for the two major protein degrading organelles, the
proteosome and the lysosome. The proteosome inhibitor MG132 did not affect the
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amounts of intracellular TNF, or secreted TNF, in both normoxia and hypoxia, thus
ruling out ubiquitin-mediated degradation of TNF, following LPS stimulation.
Addition of the lysosome inhibitor bafilomycin A1 did not change the amounts of
secreted TNF,, but dose-dependently inhibited the degradation of intracellular TNF,
only during hypoxia, suggesting that intracellular TNF, is directed from the early
endosomes to the lysosomes for enhanced degradation during hypoxia, and remains in
these compartments when the lysosome and regular trafficking of TNF, is inhibited
by bafilomycin A1. Bafilomycin A1 was shown to increase cytokine production in
normoxic peritoneal macrophages that were not induced by LPS (17), however, only
minute amounts of TNF, were detected, suggesting that this effect was probably
related to the inhibition of TNF, recycling. Although we did not directly check the
effects of hypoxia on the translation of TNF,, the dose-dependent inhibition of TNF,
by bafilomycin A1, that at high concentrations reached normoxic values, renders this
regulatory checkpoint unlikely.
Transportation of TNF, in specialized secretory and endosomal vesicles directs it
towards secretion, membranal expression and degradation. It is now accepted that
TNF, is synthesized as a 26 kDa protein that is first accumulated in the Golgi
complex (37), where early proteolytic processing into its 17 kDa mature form may
occur (39). Then it is transported by the secretory machinery to the cell surface, where
it is degraded by TNF, converting enzyme (TACE) and secreted as the mature
protein. As part of its turnover, membranal TNF, can be endocytosed and directed to
the recycling endosome, where it is either degraded in the lysosome or directed back
to the membrane (38; 43). However, the detailed pathway of TNF, trafficking in
LPS-stimulated macrophages is yet far from clear. The possibility that hypoxia affects
trafficking of proteins destined for secretion has recently been introduced, with
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emphasis on secretory vesicles (34) and on vesicles mediating recycling of membranal
proteins (45). To explore this, we used the Rab proteins, belonging to the family of
Rab small GTPases that are involved in the regulation of intracellular vesicle
transportation (40; 47). The Rab proteins chosen served as markers for secretory
vesicles that direct vesicle transport from the Golgi complex to the cell surface
(Rab8), late endosomes (Rab7) and early endosomes (Rab5), recycling endosomes
(Rab4 and Rab11), lysosomes (LAMP-1) and secretory lysosomes (Rab27a). In LPSstimulated macrophages in normoxia, TNF, was co-localized with Rab5, Rab4 and
Rab11, indicating a role for early and recycling endosomes, but not for late
endosomes or secretory vesicles, in TNF, trafficking. Furthermore, we were able to
show co-localization between TNF, and Rab27a or TNF, and LAMP-1, suggesting a
role for the lysosomes and secretory lysosomes in TNF, secretion. In hypoxia, colocalization of TNF, with these Rab proteins was significantly reduced, probably due
to the reduced amounts of the intracellular cytokine, as staining for the Rab proteins
did not change. However, co-localization with LAMP-1 was increased. The lysosome
inhibitor bafilomycin A1 did not change the expression of TNF, or its co-localization
with Rab5 in normoxia, but in hypoxia it restored intracellular levels of TNF, and its
co-localization with Rab5. These observations, combined with the ability of
bafilomycin A1 to dose-dependently prevent hypoxia-induced TNF, lysosomal
degradation, suggest that in normoxia TNF, is transported from the early endosome
via the lysosome to the secretory lysosomes, and to the membrane, where it is either
secreted or recycled to the recycling endosomes (figure 7). Our data suggest that
hypoxia shifts the direction of TNF, from the membrane towards the lysosome,
where its degradation is increased. As increased co-localization of TNF, and Rab7
did not occur in these conditions, we speculate that TNF, is directly transported via
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Rab5-carrying endosomes to the lysosomes, thus by-passing the need for late
endosomes, as has been observed before (1). Furthermore, to the best of our
knowledge, this is the first time that secretion of TNF, is localized to secretory
lysosomes, which are specialized vesicles that are specifically used by immune cells
(14; 18)
In addition to intracellular and membranal events, extracellular mechanisms could
also lead to reduced TNF, levels in hypoxic supernatants. Enzymes such as
cathepsins, elastase and stress-induced proteases were shown to degrade TNF, in
supernatants and produce inactive fragments of the cytokine (3; 22; 31). However, we
ruled out the possibility that hypoxia enhanced extracellular degradation of TNF,,
since we detected only the mature form of TNF, (17 kDa) and no additional
degradation products in hypoxic supernatants. An additional explanation for the
reduced amounts of TNF, and its biological activity in hypoxic supernatants could be
its enhanced binding to the soluble TNF receptors I and II (sTNFRI, sTNFRII). These
receptors can bind and neutralize secreted or membranal TNF, (44), as well as induce
reverse signaling in monocytes expressing membranal TNF,, that causes LPS
resistance and down-regulation of TNF, secretion (11). Hypoxia and trauma were
both shown to enhance the release of the soluble TNF receptors, mainly sTNFR-II (8;
15; 36; 41), although LPS-induced TNF, levels were not always reduced. Here we
show that despite the failure of hypoxia to increase the secreted amounts of the two
soluble receptors, the binding of TNF, to sTNFR-II, but not to sTNFR-I, was
enhanced, suggesting that this receptor binds and sequesters the secreted cytokine.
Since the expression and secretion of sTNFR-II remain unaffected by hypoxia, we
speculate that hypoxia causes a change in one or more post-translational
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modifications, in either the receptor or in TNF, itself, which may change the binding
affinity of the ligand to its receptor.
GRANTS:
This work was supported in part by the Rappaport Family Institute for Research in the
Medical Sciences and by research grants 5343 from the Chief Scientist Office of the
Israeli Ministry of Health.
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LEGEND TO FIGURES
Figure 1: Hypoxia inhibits the LPS-induced TNF secretion from macrophages.
Primary TG-elicited peritoneal macrophages (0.8x106 cells), RAW 264.7 cells or
J774A.1 cells (1x106 cells) were plated in a serum free medium, and incubated in
normoxia or hypoxia for 24 hours, with or without the addition of LPS (1 µg/ml).
Supernatants were collected and TNF, concentrations were determined. (A) TNF,
concentrations determined by ELISA from TG-elicited macrophages (n=12). (B)
Viability of RAW 264.7 cells determined by the XTT assay. (C) TNF, concentrations
in RAW 264.7 cells determined ELISA (n=9). (D). TNF, concentrations in RAW
264.7 cells determined by biological activity assay (n=10). (E) A representative gel
and densitometric analysis of TNF, levels determined by western blot analysis (n=5).
PC, positive control of recombinant mouse TNF,. (F) TNF, concentrations in
J774A.1 cells determined by ELISA (n=9). **, p<0.01, and ***, p<0.001 compared to
non-stimulated cells in normoxia. $, p<0.05, $$, p<0.01 and $$$, p<0.001 compared
to LPS-induced cells in normoxia. LPS was necessary to produce detectable amounts
of TNF, in all macrophage cells, but hypoxia inhibited the LPS-induced secretion of
TNF,
Figure 2: Hypoxia does not change TNF mRNA but decreases intracellular
amounts of TNF protein. 3x106 RAW 264.7 cells were plated and incubated as
before. (A) Total RNA was extracted, reversed transcribed and TNF, mRNA was
amplified by real-time PCR and normalized to the endogenous reference 18S rRNA
(n=6). RQ, relative quantification. (B) A representative western blot of cellular
extracts, indicating the 17 kDa, 26 kDa and 33 kDa forms of TNF,, and a
densitometric analysis of the 17 kDa mature TNF, (n=5). PC, positive control of
recombinant mouse TNF,. (C) Determination of intracellular TNF, by ELISA
normalized to the total amount of proteins. **, p<0.01 and ***, p<0.001 compared to
non-stimulated cells in normoxia. $, p<0.05 and $$, p<0.01 compared to LPS-induced
cells in normoxia. LPS induced the transcription of TNF, mRNA, but hypoxia did not
change the steady-state TNF, mRNA, whereas hypoxia reduced the accumulation of
intracellular LPS-induced TNF, protein.
Figure 3: Effects of hypoxia on the co-localization of TNF with Rab5. 106 RAW
264.7 cells were plated and incubated as before. Cells were immunostained with antiTNF, (red, panels B, E, H, K) and anti-Rab5 (green, panels A, D, G, J). The merged
TNF, and Rab images from A+B, D+E, G+H, J+K are shown (panels C, F, I, L), and
areas of co-localization are in orange. Magnification was x1600. These experiments
were repeated three times, and images of at least 5 different fields were taken for
analysis. TNF, was found co-localized to Rab5 in normoxia, indicating its presence in
early endosomes, whereas hypoxia inhibited this co-localization, probably as a result
of decreased levels of intracellular TNF,. Addition of bafilomycin A1 (10nM)
restored intracellular TNF, to normoxic values.
Figure 4: Effects of hypoxia on the co-localization of TNF with Rab4 and
Rab27a proteins. 106 RAW 264.7 cells were plated and incubated as before. Cells
were immunostained with anti-TNF, (red, panels B, E, H, K), and anti-Rab27a
Page 28 of 35
28
(green, panels A, D) or anti-Rab4 (green, panels G,J). The merged TNF, and Rab
images from A+B, D+E, G+H, J+K are shown (panels C, F, I, L), and areas of colocalization are in orange. Magnification was x1600. These experiments were
repeated three times, and images of at least 5 different fields were taken for analysis.
TNF, was found co-localized to Rab27a and Rab4 in normoxia, indicating its
presence in secretory lysosomes and in recycling endosomes, and hypoxia inhibited
this co-localization, probably as a result of decreased levels of intracellular TNF,.
Figure 5: The lysosome inhibitor bafilomycin A1 normalizes the hypoxiareduced intracellular accumulation of TNF . 1-3x106 RAW 264.7 cells were
plated and incubated as before, with the addition of increasing amounts of the
lysosomal inhibitor Bafilomycin A1 (n=7) (A and C) or the proteosome inhibitor
MG132 (n=5) (B and D). TNF, concentrations were determined by ELISA in cellular
extracts (A and B) or in the supernatants (C and D). *, p<0.05 and **, p<0.01
compared to LPS-stimulated cells in hypoxia. The proteosome inhibitor MG132 had
no effect, whereas the lysosome inhibitor bafilomycin A1 gradually increased the
accumulation of intracellular, but not secreted TNF, in hypoxia.
Figure 6: Hypoxia reduces the membranal expression of TNF . 3x106 RAW
264.7 cells were plated and incubated as before. (A) Cells were labeled with antiTNF, and FITC-conjugated donkey anti-goat IgG and subjected to flow cytometry
analysis to show membranal presence of TNF,. Light grey line, isotype control; Black
line, TNF, in normoxia; Light grey line, TNF, in hypoxia (B) The mean fluorescence
of membranal TNF, expression (n=4) was increased in the presence of LPS, and
hypoxia reduced the LPS-induced surface binding of TNF,.. Supernatants were
collected for the determination of (C) soluble TNF receptor I (sTNFR-I) and (D)
soluble TNF receptor II (sTNFR-II) by ELISA (n=8). (E) A representative gel and
densitometric analysis of immunoprecipitation of the soluble receptors secreted to the
supernatants followed by immunoblotting with anti-TNF, (n=5). PC, positive control
of recombinant mouse TNF,. Concentrations of sTNFR-I were reduced in hypoxia
whereas those of sTNFR-II remained unchanged. TNF, was found bound only to
sTNFR-II, and its binding was increased in hypoxia. *, p<0.05, **, p<0.01, and ***,
p<0.001 compared to non-stimulated cells in normoxia; $, p<0.05, $$, p<0.01 and
$$$, p<0.001 compared to LPS-induced cells in normoxia.
Figure 7: A suggested model for TNF trafficking. In normoxia, TNF, is
transported from the Golgi to the early endosomes (EE), and from there it reaches the
membrane via the lysosome (L) and secretory lysosomes (SL). Membranal TNF, is
cleaved by TACE and secreted, or endocytosed into recycling endosomes (RE). The
normoxic route is indicated in thin arrows. Hypoxia enhances degradation of TNF, in
the lysosome (indicated by the large thick arrow), causing less TNF, to be transported
to the secretory lysosomes and to the membrane. The Rab proteins that are used as
specific markers for each compartment are indicated.
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