1 Departments of Immunology, 2 Physiology, and 3 Endocrinology, University of Tübingen, D72076 Tübingen, Germany
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ABSTRACT |
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A
hypertonic environment, as it prevails in renal medulla or in
hyperosmolar states such as hyperglycemia of diabetes mellitus, has
been shown to impair the immune response, thus facilitating the
development of infection. The present experiments were performed to
test whether hypertonicity influences activation of T lymphocytes. To
this end, peripheral blood lymphocytes (PBL) of cytomegalovirus (CMV)-positive donors were stimulated by human leukocyte antigen (HLA)-A2-restricted CMV epitope NLVPMVATV to produce interferon (IFN)- at varying extracellular osmolarity. As a result, increasing extracellular osmolarity during exposure to the CMV antigen indeed decreased IFN-
formation. Addition of NaCl was more effective than
urea. A 50% inhibition was observed at 350 mosM by addition of NaCl.
The combined application of the Ca2+ ionophore ionomycin (1 µg/ml) and the phorbol ester phorbol 12-myristate 13-acetate (PMA; 5 µg/ml) stimulated IFN-
production, an effect again reversed by
hyperosmolarity. Moreover, hyperosmolarity abrogated the stimulating
effect of ionomycin (1 µg/ml) and PMA (5 µg/ml) on the
transcription factors activator protein (AP)-1, nuclear factor of
activated T cells (NFAT), and NF-
B but not Sp1. In conclusion,
osmotic cell shrinkage blunts the stimulatory action of antigen
exposure on IFN-
production, an effect explained at least partially
by suppression of transcription factor activation.
cell volume; activator protein-1; nuclear factor of activated T
cells; nuclear factor-B; CD69
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INTRODUCTION |
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ANTIGEN EXPOSURE is a
strong stimulator of lymphocytes that triggers a variety of events
including formation of cytokines such as interferon (IFN)-
(7). The signaling linking antigen binding to IFN-
expression includes activation of protein kinase C (PKC), increase of
intracellular Ca2+ activity (Ca
B, and
nuclear factor of activated T cells (NFAT) (7, 23, 29, 36,
37). These events are crucial for adequate defense against
pathogens (14, 43).
The defense against pathogens is impaired at high ambient osmolarity
such as in the hyperosmolar renal medulla (5, 8, 12, 15,
26). The susceptibility of the hyperosmolar kidney medulla to
bacterial infection is clearly higher than that of the isosmolar renal
cortex (10). Moreover, the immune response was reported to
be impaired in diabetes mellitus (9), which typically
leads to increase of extracellular osmolarity (9). Information on the cellular mechanisms accounting for defective immune
defense in a hypertonic environment is, however, scarce. It was shown
that neutrophil O production, as an indicator for lymphocyte activity. The results indeed disclose a profound inhibition of IFN-
expression in
osmotically shrunken cells. Further experiments were performed to
elucidate the cellular mechanisms involved.
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EXPERIMENTAL PROCEDURES |
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Cell preparation and culture conditions. Peripheral blood lymphocytes (PBL) were separated from blood by Ficoll gradient. After two washes in PBS, PBL were resuspended in RPMI 1640 supplemented with 10% fetal calf serum (FCS), pH 7.4, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. All experiments were performed at 37°C.
Peptide preparation. The immunodominant peptide NLVPMVATV of pp65 protein (41) from cytomegalovirus (CMV) was chosen for cell stimulation. Reaction against the peptide YLLPAIVHI (13) was considered nonspecific. Peptides were synthesized in automated peptide synthesizer 432A (Applied Biosystems, Weiterstadt, Germany) following the Fmoc/tBu strategy. Synthesis products were analyzed by HPLC (System Gold; Beckman Instruments, Munich, Germany) and MALDI-TOF mass spectrometry (G2025A; Hewlett-Packard, Waldbronn, Germany). Peptides of <80% purity were purified by preparative HPLC. Peptides were dissolved in 100% DMSO in a concentration of 10 mg/ml and further diluted to a final concentration of 1 mg/ml peptide with bidistilled H2O.
As stimulator cells, transporter associated with antigen processing (TAP)-deficient human leukocyte antigen (HLA)-A2-positive T2 cells (35) were incubated with 50 µg/ml peptide for 8 h in FCS-free culture medium. The CMV antigen NLVPMVATV is bound on the HLA-A2 molecule of T2 cells. When this major histocompatibility complex (MHC)-peptide complex is recognized by peptide-specific CD8+ T cells, they are activated and produce IFN-Enzyme-linked immunospot analysis.
For enzyme-linked immunospot (ELIspot) assay nitrocellulose 96-well
plates (MAHA 45; Millipore, Bedford, MA) were coated with 50 µl/well
anti-human IFN- antibody (20 µg/ml; Biosource, Camarillo, CA)
diluted in coating buffer (in mM: 35 sodium bicarbonate, 15 sodium
carbonate, and 3 sodium azide). After incubation for 3 h at 37°C
the unbound antibody was removed by four washing steps with PBS.
Remaining protein binding sites of the nitrocellulose plates were
blocked with culture medium for 1 h at 37°C. T2 cells were
incubated with the antigen NLVPMVATV and the control peptide YLLPAIVHI
described in Peptide preparation. The peptide-loaded T2 cells (70,000/well) were subsequently cocultured with 50,000 PBL/well in duplicate in 96-well plates under isotonic and hypertonic conditions. In a second series of experiments 20,000 PBL were cocultured in duplicate in 96-well plates under isotonic and hypertonic conditions with 1 µg/ml Ca2+ ionophore and 5 µg/ml
phorbol 12-myristate 13-acetate (PMA). After 6-h incubation, the medium
was replaced and cells were cultured in coated nitrocellulose plates
for 6 h under different conditions. Cells were removed by washing
seven times with PBS containing 0.05% Tween 20 (PBS-T). Fifty
microliters of biotinylated anti-human IFN-
antibody (Biosource),
diluted to 2 µg/ml in PBS containing 0.5% bovine serum albumin (BSA)
and 0.02% sodium azide, was used for detection of bound IFN-
. After
3 h, unbound antibodies were removed by six washes with PBS-T and
50 µl of avidin peroxidase complex (ABC Vectastain-Elite kit; Vector
Laboratories, Burlingame, CA) was added. Two hours after addition of
avidin peroxidase the plate was washed three times with PBS-T and three
times with PBS. In the last washing step the complete plate was
submerged in PBS. Subsequently, the reaction was developed with
3-amino-9-ethylcarbazole (Sigma, St. Louis, MO). The color reaction was
stopped after 5 min by rinsing with water.
Intracellular IFN- staining and FACS analysis.
For intracellular IFN-
staining the Cytofix/Cytoperm kit
(PharMingen, San Diego, CA) was used. After Ficoll gradient separation 106 PBL/ml were stimulated in 4.5-ml polysterol tubes
(Greiner, Frickenhausen, Germany) with 1 µg/ml Ca2+
ionophore (ionomycin calcium salt; Sigma Aldrich, Taufkirchen, Germany)
and 5 µg/ml PMA (Sigma Aldrich) at 37°C. To stop IFN-
secretion,
monensin (0.7 µl/ml Golgi Stop, Cytofix/Cytoperm kit; PharMingen), an
inhibitor of intracellular protein transport (31), was
added. After 6-h incubation cells were washed with PBS and stained with
anti-human CD3 antibody (Coulter Immunotech, Hamburg, Germany) for 30 min at 4°C. After unbound antibodies were removed, cells were fixed
and permeabilized with 300 µl of Cytofix/Cytoperm solution
(PharMingen) for 20 min at 4°C. Cells were washed two times with 1 ml
of Perm/Wash solution (Cytofix/Cytoperm kit) and stained in 100 µl of
Perm/Wash solution with PE-labeled mouse anti-human IFN-
antibody
(PharMingen) diluted to 1.5 µg/ml. After 30 min of incubation the
antibodies were removed by two washes and cells were resuspended in PBS.
Electrophoretic mobility shift assay.
Klenow enzyme and poly(dI-dC) were from Boehringer (Mannheim, Germany);
[-32P]dATP was from Hartmann (Braunschweig, Germany);
and antibodies were from Santa Cruz Technologies (Santa Cruz, CA):
c-Jun (Santa Cruz 1694X), JunB (Santa Cruz 73X), JunD (Santa Cruz 74X),
and c-Fos (Santa Cruz 253X).
Semiquantitative RT-PCR.
To evaluate serum- and glucocorticoid-sensitive kinase (sgk)1 mRNA
expression levels, the cells were stimulated at 500 mosM for 2 h.
Total RNA was isolated with the RNeasy RNA isolation kit (Qiagen,
Germany, Hilders) following the manufacturer's instructions. One
microgram of RNA was reverse-transcribed into cDNA with the Advantage
RT-for-PCR Kit (Clontech, Palo Alto, CA). The oligonucleotides used for
amplification of sgk1 and -actin are the following: sgk1, sense
(5'GATGGGTCTGAACGACTTTA3'), antisense (5'GATTTGCTGAGAAGGACTTG3');
-actin, sense (5'TAAGGAGAAGCTGTGCTACG3'), antisense (5'
CCAGACAGCACTGTGTTG3'). Thirteen picomoles of sgk1 primers and five
picomoles of
-actin primers were added to 50 µl of reaction
containing 50 mM KCl, 10 mM Tris · HCl pH 8.4, 15 mM MgCl2, 2 mg/ml BSA, and dNTP mix (each 0.2 mM). PCR
conditions were optimized so that measurements were done in the linear
range of DNA amplification. The mixture was denaturated at 95°C for 2 min, followed by 34 cycles, each consisting of denaturation at 95°C
for 30 s, annealing at 56°C for 30 s, and extension at
72°C for 30 s. After a final extension at 72°C for 5 min,
samples were kept at 4°C. PCR products were then resolved by
electrophoresis in 2.5% agarose (Boehringer Ingelheim) and recorded by
digital camera.
Statistics. All experiments were performed at least in triplicate. Arithmetic means ± SE of independent experiments were calculated, and statistical analysis was made by paired or unpaired t-test, where appropriate.
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RESULTS |
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Osmotic cell shrinkage inhibits antigen-triggered IFN-
production in PBL.
As reported earlier (30), stimulation of PBL leads to
IFN-
production within 4-8 h. Therefore, the effect of
hyperosmolarity on antigen stimulation and IFN-
secretion was tested
after a 6-h exposure of PBL to antigen at different osmolarities. PBL of a HLA-A2-positive CMV-seropositive donor were stimulated with the
CMV peptide NLVPMVATV of pp65 protein. After the 6-h stimulation the
PBL were transferred to an antibody-coated ELIspot plate to determine
IFN-
secretion for another 6 h. PBL reacted with 64.0 ± 3.7 (n = 4) spots per 50,000 PBL against T2 cells
loaded with NLVPMVATV, whereas there was a response of 12.5 ± 8.7 (n = 4) spots per 50,000 PBL against T2 cells loaded
with YLLPAIVHI (Fig. 1). PBL without T2
cells and medium alone reacted with less than five spots per well (data
not shown). Stimulation at 350 mosM for 6 h decreased the spot
frequency to 34.6 ± 4.8 (n = 4) spots per 50,000 PBL, whereas a 6-h stimulation at 500 mosM led to a response of
1.5 ± 0.9 (n = 4) spots against CMV
peptide-loaded T2 cells (Fig. 1). Thus cells stimulated in hypertonic
buffer secreted significantly less IFN-
than cells stimulated in
isotonic buffer, even though secretion was determined in isotonic
buffer irrespective of the osmolarity during stimulation. PBL
stimulated with peptide under isotonic conditions showed no decrease in
specific IFN-
spots, even when IFN-
secretion was determined
under hypertonic conditions (Fig. 1). When PBL were stimulated in
isotonic buffer, no significant difference of IFN-
secretion was
observed between cells subsequently exposed to hypertonic buffer and
those remaining in isotonic buffer (Fig. 1). Thus hyperosmolarity
interfered with stimulation but not with secretion. The blunting effect
of hyperosmolarity on stimulation of PBL was not the result of
increased NaCl concentration. When PBL were stimulated with CMV peptide
at 500 mosM by adding 200 mM raffinose, IFN-
production was also
blunted (Fig. 1B).
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Combined stimulation of PKC and increase of
Ca secretion,
an effect similarly reversed by osmotic cell shrinkage.
The inhibitory effect of hyperosmolarity could have been due to
impaired binding of antigen or to inhibition of Ca2+ entry
into the cells during activation. To circumvent these mechanisms cells
were stimulated with Ca2+ ionophore (1 µg/ml) and PMA (5 µg/ml). As shown in Fig. 2, PMA and
ionomycin added together stimulated IFN-
expression with 139.8 ± 29.8 spots per 20,000 PBL. PBL alone showed 3.5 ± 2.6 (n = 4) spots per 20,000 PBL. Stimulation at 400 mosM
yielded 95.6 ± 6.9 (n = 4) spots per 20,000 cells, whereas during stimulation at 500 mosM IFN-
production
remained completely suppressed. After stimulation at isotonic
conditions and subsequent exposure to hypertonicity, IFN-
secretion
approached 171.6 ± 7.3 (n = 4) spots per 20,000 PBL at 400 mosM and 154 ± 24.9 (n = 4) spots per
20,000 PBL at 500 mosM, both values not significantly different from
those at isotonic conditions. The inhibition of IFN-
secretion after
stimulation with PMA-ionophore in hypertonic extracellular fluid
indicates that osmotic cell shrinkage must exert its inhibitory effect
by inhibition of PKC or a signaling element downstream of PKC or
Ca2+.
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High osmolarity also decreases intracellular IFN-.
Thirteen percent of all CD3+ cells (T cells) and 18% of
all CD3
cells expressed IFN-
after stimulation with
Ca2+ ionophore (1 µg/ml) and PMA (5 µg/ml) for 90 min.
When cells were stimulated at 500 mosM, IFN-
production was
completely abolished (Fig. 3). After a
3-h stimulation, 22% of CD3+ cells and 32% of
CD3
cells produced IFN-
, which again was completely
abolished at 500 mosM. After a 6-h exposure to Ca2+
ionophore (1 µg/ml) and PMA (5 µg/ml) in isotonic conditions, 94%
of all T cells and 90% of CD3
cells produced IFN-
. At
an osmolarity of 500 mosM only 12% of all T cells and 10% of
CD3
cells produced IFN-
. As shown in Fig.
4, IFN-
production could be inhibited
not only by addition of NaCl but also by addition of urea, which,
however, was significantly less effective than isosmolar NaCl.
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Inhibition of IFN- expression is paralleled by inhibition of
CD69.
CD69 is a C-type lectin used as a marker for lymphocyte activation.
After a 4-h stimulation with Ca2+ ionophore (1 µg/ml) and
PMA (5 µg/ml) at 300 mosM 79.5 ± 1.8% (n = 4)
of all PBL expressed CD69, whereas 8.0 ± 1.1% (n = 4) of PBL expressed CD69 without stimulation. After stimulation with PMA and ionomycin at 500 mosM, only 31.3 ± 2.2%
(n = 4) of all PBL expressed CD69 (Fig.
5). Similar to IFN-
production, CD69 production is blunted by addition of urea as well, even though urea is
again significantly less effective than isosmolar NaCl (Fig.
6).
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Influence of osmolarity on apoptotic cell death.
In theory, the increase of osmolarity could have inhibited IFN-
production by induction of lymphocyte apoptosis
(34). However, as shown in Fig.
7, an increase of extracellular
osmolarity up to 500 mosM does not alter Syto16 staining, indicating
that DNA fragmentation did not occur up to 500 mosM. However, as
reported previously (34), an increase of extracellular
osmolarity to 700 mosM induces apoptotic cell death of some 70% of
the cells within 6 h (Fig. 7).
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Hyperosmolarity prevents activation of transcription factors AP-1,
NFAT, and NF-B.
Because activation of AP-1, NFAT, and NF-
B proteins is implicated in
the upregulation of IFN-
gene expression in T cells (7), we studied the activity of these transcription
factors in PMA-ionomycin-stimulated cells in normo- or hyperosmolar
conditions with electrophoretic mobility shift assay (EMSA).
PMA-ionomycin treatment for 2 h at 300 mosM led to strong
increases in DNA binding activities to AP-1, NFAT, and NF-
B binding
sites (Fig. 8, A-C, Fig.
9, A and B). The
activation of AP-1 (Fig. 8, A-C) and of NF-
B (Fig.
9A) was completely and the activation of NFAT (Fig. 9B) was partially inhibited by exposure to 500 mosM. Binding
activities to a Sp1 binding site were not appreciably affected by
PMA-ionomycin or hyperosmolarity (Fig. 9C). The binding to
the AP-1 binding site was investigated in more detail. By supershift
analysis, c-Fos and JunB proteins were identified as major compounds of this complex whereas antibodies against c-Jun and JunD led only to weak
supershifted bands, indicating only a small amount of these proteins in
the DNA-binding complex (Fig. 8D). Whereas in isotonic
extracellular fluid PMA-ionomycin stimulated AP-1 binding within 1 h and even more so after 2 h, no stimulation was observed in
hyperosmolar extracellular fluid (Fig. 8B). Densitometric
quantification (n = 3) revealed a sevenfold induction
of AP-1 binding activity after PMA-ionomycin treatment (Fig.
8D). Furthermore, the specificity of the bands indicated as
AP-1, NFAT, and NF-
B were confirmed by competition studies with
unlabeled oligonucleotides (Fig. 8D, Fig. 9, A
and B). To explore whether the transcription of a gene known
to be upregulated by osmotic cell shrinkage is impaired, transcripts
for sgk1 (17) were determined. As shown in Fig. 9D, sgk1 transcript levels increased after osmotic cell
shrinkage (Fig. 9D).
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DISCUSSION |
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The present study reveals an inhibitory action of osmotic cell
shrinkage on IFN- production. As the production of CD69 is similarly
impaired, an increase of extracellular osmolarity apparently has a
broader effect on lymphocyte activation. The effect of hypertonic NaCl
is mimicked by hypertonic urea even though the effect of urea is
significantly smaller than the effect of NaCl at similar osmolarities.
Urea may shrink cells even after rapid entry and dissipation of the
gradient across the cell membrane, and it has been suggested that urea
alters the set point of cell volume regulation by interference with
intracellular protein stability (for review see Ref. 16).
Nevertheless, the possibility remains that increase of osmolarity or
NaCl or urea concentrations rather than cell volume as such interfere
with lymphocyte activation.
In theory, the inhibitory effect of hyperosmolarity could have been secondary to apoptotic cell death (3, 4, 19, 25, 28, 33, 34). However, in agreement with previous reports (38), an increase of osmolarity to 500 mosM did not induce apoptosis but only an increase to 700 mosM led to the expected triggering of apoptotic cell death.
The impaired formation of IFN- could further be due to
inhibition of protein synthesis. Osmotic shrinkage of hepatocytes has
indeed been shown to inhibit protein synthesis (16).
However, increase of osmolarity after exposure to the antigen does not significantly alter IFN-
production, indicating that once the cell
is triggered the machinery toward IFN-
production remains largely
insensitive to alterations of osmolarity. Moreover, osmotic cell
shrinkage stimulates the expression of TNF-
(20). Thus the influence of ambient osmolarity on lymphocyte cytokine expression is not uniform.
The activation of lymphocytes on antigen binding involves PKC
(23, 29) and increase of Ca
production by combined stimulation of PKC by phorbol esters and
increase of Ca
Among the downstream events of lymphocyte activation by antigen or
Ca2+ are the activation of transcription factors such as
AP-1 (7), NF-B (36), and NFAT (22,
36). Therefore, the sensitivity of transcription factor
activation has been tested. Indeed, activation of AP-1 and NF-
B is
completely abolished and activation of NFAT blunted by exposure of the
cells to 500 mosM, at least contributing to the inhibition of
lymphocyte activation and subsequent IFN-
and CD69 production. The
constancy of SP1 binding demonstrates that the inhibition of the
transcription factors AP-1, NFAT, and NF-
B is not a general
phenomenon uniformly affecting all transcription factors. Moreover,
hyperosmolarity increases the transcript levels of the serum- and
glucocorticoid-sensitive kinase sgk1, which was shown previously to be
upregulated by cell shrinkage (2, 39, 40). Osmotic cell
shrinkage is known to trigger the expression of a variety of further
proteins (6, 16). Interestingly, IFN-
downregulates
Na+/H+ exchanger (NHE)2 and NHE3
(32). Thus the downregulation of IFN-
expression during
osmotic cell shrinkage may facilitate enhanced
Na+/H+ exchanger activity, a major
volume-regulating mechanism in shrunken cells.
The impaired activation of AP-1, NFAT, and NF-B leading to blunted
formation of IFN-
and CD69 should severely impede the immune
response against pathogens in kidney medulla. As significant effects
are observed even at moderately increased osmolarity, blunted IFN-
and CD69 production may contribute to the impaired immune response in
hyperosmolar states such as hyperglycemia of diabetes mellitus. Along
those lines, evidence for lymphocyte cell shrinkage and subsequently
impaired immune response in diabetes mellitus was presented previously
(9, 18). Moreover, the cell volume-sensitive (2, 39,
40) kinase sgk1 (17) is upregulated in diabetes
mellitus (18).
In conclusion, osmotic cell shrinkage inhibits the activation of the
transcription factors AP-1, NFAT, and NF-B, leading to a decrease of
antigen-induced IFN-
production. These effects may lead to an
impaired immune response in renal medulla on the one hand and in
hyperosmolar states such as diabetic hyperglycemia on the other.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the technical assistance of E. Faber and the meticulous preparation of the manuscript by Tanja Loch.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft (La 315/4-3 and La 315/6-1) and the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Center for Interdisciplinary Clinical Research; 01 KS 9602).
Address for reprint requests and other correspondence: F. Lang, Physiologisches Institut der Universität Tübingen, Gmelinstr. 5, D72076 Tübingen, Germany (E-mail: florian.lang{at}uni-tuebingen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00259.2002
Received 3 June 2002; accepted in final form 26 August 2002.
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