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Research Communications |
production and accelerated IL-10 expression
Department of Surgery, Research Laboratories,
a Institute of General and Experimental Pathology,
b Department of Medical Computer Sciences, University of Vienna, A-1090 Vienna, Austria
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-)
and interleukin-1ß (IL-1ß) stimulate the progression of septic
shock whereas the anti-inflammatory cytokine IL-10 has
counterregulative potency. The amino acid glycine (GLY) has been shown
to protect against endotoxin shock in the rat by inhibiting TNF-
production. In the current study we investigated the role of GLY on
lipopolysaccharide (LPS) -induced cell surface marker expression,
phagocytosis, and cytokine production on purified monocytes from
healthy donors. GLY did not modulate the expression of HLA-DR and CD64
on monocytes, whereas CD11b/CD18 expression (P<0.05) and
E. coli phagocytosis (P<0.05) decreased
significantly. GLY decreased LPS-induced TNF- production
(P<0.01) and increased IL-10 expression of purified
monocytes. Similarly, in a whole blood assay, GLY reduced TNF-
(P<0.0001) and IL-1ß (P<0.0001) synthesis
and increased IL-10 expression (P<0.05) in a
dose-dependent manner. The inhibitory effects of GLY were neutralized
by strychnine, and the production of IL-10 and TNF- was augmented by
anti-IL-10 antibodies. Furthermore, GLY decreased the amount of IL-1ß
and TNF--specific mRNA. Our data indicate that GLY has a potential
to be used as an additional immunomodulatory tool in the early phase of
sepsis and in different pathophysiological situations related to
hypoxia and reperfusion.—Spittler, A.,Reissner, C. M., Oehler,
R., Gornikiewicz, A., Gruenberger, T., Manhart, N., Brodowicz, T.,
Mittlboeck, M., Boltz-Nitulescu, G., Roth, E. Immunomodulatory effects
of glycine on LPS-treated monocytes: reduced TNF- production and
accelerated IL-10 expression.
Key Words: sepsis • cytokines • antigen expression • tumor necrosis factor • whole blood cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-),2
produced from lipopolysaccharide (LPS)
-activated monocytes/macrophages and T cells, is the key mediator in
the early phase of septic shock 3-5)
. The
anti-inflammatory cytokine interleukin 10 (IL-10) is synthesized rather
late, at a time when TNF- secretion seems to be suppressed
6-8)
. In addition to the well-documented capacity of Th1
cells to inhibit interferon-
(IFN-) production and TNF- by
activated peripheral blood mononuclear cells, IL-10 has a capacity to
inhibit the proliferation of T cells when monocytes are used as antigen
presenting cells 9-12)
. Administration of IL-10 in animal models has been shown to protect against the lethal endotoxemia, and treatment with anti-TNF monoclonal antibodies (mAb's) prevent septic shock (13 , 14 ). However, clinical phase II/III studies with several anti-TNF mAb's and the TNF:Fc fusion protein could not confirm this effect (15 , 16 ). Therefore, new therapeutical strategies must be found to influence the mortality of septic patients.
Recent studies have shown that a supply of the amino acid glutamine in
pharmacological quantities was able to reduce mortality in intensive
care unit patients, possibly via an influence on monocyte phenotype and
function 17-19)
. Enteral administration of glycine (GLY)
decreased the release of TNF- from isolated Kupffer cells almost
completely and significantly reduced mortality in an experimental model
of endotoxemia (20)
. Kupffer cells contain
voltage-dependent Ca2+ channels, and increases in
intracellular Ca2+ concentration are necessary for LPS to
induce synthesis of TNF-. Additional studies from the same group
showed that GLY blunts the elevation of Ca2+ through
actions on chloride channels (21)
. This effect was
reversed by strychnine, leading to the conclusion that Kupffer cells
contain a GLY-gated chloride channel. These studies delivered the
pathophysiological explanation of earlier findings, in which GLY has
been shown to be protective against hypoxia and ischemia in hepatocytes
in a low-flow perfusion model and against various cytotoxic substances
in renal tubules 22-25)
. Moreover, GLY has been shown to
be a useful additive to organ preservation solutions: it improves graft
function, increases survival after rat liver transplantion, and reduces
the nephrotoxicity of cyclosporine by interfering with oxygen radical
metabolism (26)
.
The aim of our study was to investigate the time and dose dependency of GLY on cytokine expression, marker expression, and phagocytosis of isolated human monocytes and cells cultured in a whole blood assay.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whole blood cell culture
At each time point, venous blood from healthy individuals was
collected in heparin containing purple vacutainer (Becton Dickinson,
San Jose, Calif.). A 100 mmol/l GLY stock solution was prepared in
sterile NaCl 0.9% and further diluted to the final concentrations. All
tubes were then preincubated for 6 h in a shaking 37°C water
bath. Probes were also slightly rotated every 30 min. Subsequently,
whole blood was stimulated with 1 µg/ml or 1 ng/ml LPS. GLY receptor
blocking experiments were performed using 1 µmol/l strychnine (Sigma,
St. Louis, Mo). IL-10 blocking experiments were performed with an
anti-IL-10 Ab (R&D Systems, Inc., Minneapolis, Minn.), and 1 µl/ml
neutralizing 5 ng/ml of IL-10. Tubes were then prepared for cytokine
detection or mRNA isolation. Dead cells were determined by propidium
iodide staining and fluorescence-activated cell sorting (FACS; Coulter,
Hialeah, Fla.) analysis.
Immunofluorescence and flow cytometric analysis
Cultured monocytes were harvested, washed, and resuspended in
phenol red-free Hanks' balanced salt solution containing 0.3% bovine
serum albumin and 0.1% NaN3. After preincubation of 5 x 105 cells for 30 min on ice with 20% human AB-serum,
cells were washed and then incubated with 50 µl of mAb solution for
30 min on ice. The following mAb's were used: anti-Fc IgG receptor
type I (FcRI/CD64; clone 32.2) obtained from Medarex Inc. (W.
Lebanon, N.H.) and anti-HLA-DR (clone L243) from Becton Dickinson (San
Jose); anti-CD14 (clone My 4) was from Coulter, and anticomplement
receptor type 3 (CR3, CD11b/CD18) was from Immunotech (Marseille,
France). Whole blood (100 µl) was incubated with mAb for 30 min at
room temperature in the dark. Each probe was double stained with the
anti-CD14 mAb. Red blood cells were lysed and leukocytes were
stabilized and fixed by a Multi-Q-Prep (Coulter, Miami, Fla.). Finally,
cells were washed three times and resuspended in 300 µl medium. At
least 104 CD14+ gated monocytes were analyzed
on a FACS-XL (Coulter). The data were collected with 4 decade
logarithmic amplification and expressed as arbitrary units of mean
channel fluorescence of CD14+ cells. Identical instrument
settings were used.
Detection of intracellular cytokine production
PBMC from 30 ml of heparinized blood from healthy donors were
isolated by Ficoll-Paque density gradient and incubated for 40 h
in 0, 2, and 10 mmol/l GLY, as mentioned above. Intracellular cytokine
production using the CytoStain Kit with GolgiStop (Pharmingen, San
Diego, Calif.) was detected as described earlier (28)
. In
brief, PBMC were stimulated with 1 µg/ml LPS for 6 h in the
presence of monensin to block their intracellular transport processes.
Subsequently cells were harvested, and stained with the fluorescein
isothiocyanate (FITC) -labeled anti-CD14 mAb for 30 min on ice. Cells
were then washed twice, fixed with paraformaldehyde, permeabilized, and
stained with the phycoerythrin (PE) -conjugated isotype-control, the
anti-TNF- (PE), or the anti-IL-10 mAb (PE), respectively, for 30 min
on ice (all mAb were from Pharmingen, Germany). After two additional
washings, 1 x 104 CD14+ cells were
analyzed by flow cytometry with a two-color dot plot.
Determination of phagocytosis
Immunoglobulin-opsonized and FITC-conjugated Echerichia
coli (Phagotest, Orpegen, Heidelberg, Germany) were opsonized with
complement-containing autologous serum. Cells (2x106) and
bacteria (2x108) were incubated for 10 min at 37°C,
washed, quenched, and fixed according to the manufacturer's procedure.
The percentage of CD14+ phagocytic cells and the MCF were
determined by FACS analysis. FITC-labeled latex beads (Fluoresbrite
plain YG, 0.75 µm in diameter) were obtained from Polysciences Inc.
(Warrington, Pa.). Monocytes (2x105) and beads
(8x106) were incubated for 1 h at 37°C,
centrifuged, and washed. The percentage of CD14+ monocytes
ingesting E. coli was analyzed by FACS (19)
.
Detection of TNF-, IL-1ß, and IL-10 by ELISA
Aliquots of supernatants from cultured monocytes or from plasma
were collected before and after LPS stimulation. Supernatants were
frozen immediately at -70°C until analysis. The concentrations of
cytokines were measured by the use of commercially available
enzyme-linked immunoassay (ELISA) kits (Amersham, Southampton, U. K.). Assays were performed in duplicate and analyzed in a plate reader
(Dynatech, Chantilly, Va.).
RNA extraction and Northern blots
PBMC were purified by a Ficoll gradient and total RNA was
extracted with trizol reagent (Gibco BRL, New York, N.Y.) according to
the manufacturers protocol. Total RNA (20 µg) was denatured with 5.5
M glyoxal at 50°C and RNA was separated on a 1.5% agarose gel by
electrophoresis. RNA was blotted overnight onto a nylon membrane in a
buffer containing 20 xSSC. The RNA was fixed on the membrane by
UV-cross-linking and cytokine mRNA was hybridized to radiolabeled cDNA
probes obtained by reverse transcription polymerase chain reaction
(RT-PCR). The primer sets for cytokines were 5'-CAC ACC CTG ACA AGC TGC
CAG GC-3' and 5'-TTC CTA AGC AAC CTT TAT TTC TCG CC-3' (for TNF-);
5'-ACC AAC CTC TTC GAG GCA CAA GG-3' and 5'-TTG CTC ATT TAT AAA TAT TCC
C-3' (IL-1ß); 5'-CTT TCT TTC AAA TGA AGG ATC AGC TGG-3' and 5'-GGA
AAA CAG CTC AAC AGC TAG AAA GCG-3' (for IL-10). For ß-actin, the
primer pair at positions 294-325 and 1131-1100 (Clontech, Palo Alto,
Calif.) was used. RT-PCR products were gel-purified on a low melting
agarose gel. Purified cDNA was labeled with a random priming kit
(Amersham, England, U.K.) and 32P-dCTP. Labeled cDNA was
then purified by nick columns (Pharmacia Biotech, Uppsala, Sweden) and
measured in a ß-counter. RNA was hybridized (5x105
counts/ml) overnight at 65°C in a buffer containing 5 x TEN,
5 x Denhardt
solution, and 0.2% sodium dodecyl sulfate
(SDS). Membranes were washed twice with 2 x TEN and 0.2% SDS at
65°C for 15 min, followed by one wash with 0.2 x TEN and 0.2%
SDS at 65°C for 30 min. Bands obtained by autoradiography were
quantitated with a densitometer.
Statistical methods
Data are described and visualized with means ±SD
for each group and every time point. Comparisons between control and 2
mmol/l GLY with 1 ng LPS were performed using a paired
t-test. For comparisons with more than two groups, a blocked
analysis of variance with random factors were made using the procedure
MIXED of the statistical package SAS (1990, SAS/STAT User's
Guide. Version 6. SAS Institute, Cary, N.C.). In case of
measurements at several time points, the time effect and group–time
interactions were also tested. For multiple comparisons, adjustments
according to Tukey-Kramer were made. Probability values are two-sided
and P<0.05 is considered to be statistically
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and enhances IL-10 secretion by
whole blood cells (P<0.0001, Fig. 1B
) were
significantly reduced with increasing GLY concentrations over the whole
time period. We found no changes in cell viabilities or in leukocyte
populations before and after the incubation procedure (data not shown).
Stimulating whole blood with 1 ng/ml of LPS led to production of
IL-1ß (1.47±0.52 ng/ml) and TNF- (1.05±0.23 ng/ml). This effect
was significant reduced by 2 mmol/l GLY (IL-1ß: 0.92±0.34 ng/ml;
P<0.05; TNF-: 0.74±0.28; P<0.05).
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To further investigate whether GLY influences secretion of anti-inflammatory cytokines in the whole blood assay, supernatants were analyzed for IL-10 by ELISA. GLY (2 mmol/l) in whole blood led to a significant increase of IL-10 (P<0.05) production for up to 6 h after LPS (1 µg/ml) stimulation and reached nearly the same values after 12 h (Fig. 2 ). At 1 ng/ml, LPS significantly enhanced IL-10 production by cells cultured in the presence of 2 mmol/l GLY (from 0.46±0.28 to 0.84±0.24 pg/ml; P<0.05).
|
Inhibitory effects of blocking anti-IL-10 Ab or strychnine
To test the specifity of the GLY-dependent effects, additional
experiments were performed; results are shown in Fig. 3
.As expected, a reduction in IL-1ß and TNF- and enhanced IL-10
production were measured. Anti-IL-10 Ab augmented TNF- and IL-10
production, whereas IL-ß synthesis was slightly diminished. Secretion
of these cytokines was not significantly modulated by strychnine.
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Intracellular TNF- and IL-10 production
The two-color dot plots, depicted in Fig. 4
,show intracellular TNF- and IL-10 production by LPS-stimulated PBMC.
The percentage of TNF- secreting CD14+ cells cultured in
GLY-free medium was 72.1%±5.4%, and was significantly reduced at 2
mmol/l GLY (62.7%±6.1%) to 10 mmol/l GLY (58.4%±5.4%),
P<0.01. In contrast, a significant increase of IL-10 from 0
mmol/l GLY (55.7%±2.7%) to 2 mmol/l GLY (60.1%±2.9%) and to 10
mmol/l GLY (64.2%±2.7%) was measured (P<0.01). Only a
small amount of CD14- cells produced TNF- or IL-10;
thus, the CD14+ cells are the main source of these
cytokines.
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Nothern blot analysis of IL-1ß and TNF-
The results presented in Fig. 5
show that GLY at 2 mmol/l reduced the amount of IL-1ß and
TNF--specific mRNA, an effect reversed by strychnine. In cells
treated with anti-IL-10 Ab, the level of IL-1ß was not changed but a
marked increase in TNF--specific mRNA was found.
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Influence of GLY on TNF- and IL-10 production by purified
monocytes
The influence of various GLY concentrations on TNF- and IL-10
production by monocytes was also kinetically studied. Monocytes were
purified by counterflow centrifugation and precultured in different GLY
concentrations for 40 h after LPS stimulation. The results
depicted in Fig. 6A
show that TNF- production was reduced by GLY in a time- and
dose-dependent manner (P<0.01).
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By varying the GLY concentrations, secretion of IL-10 showed a time-dependent course (Fig. 6B ). High amounts of GLY (10 mmol/l) led to a threefold increase of IL-10 after 6 h, with maximum production after 12 h; at 2 mmol/l, a higher IL-10 level was measured at 12 and 24 h; and in the absence of GLY, maximum IL-10 production was detected after 24 h.
Influence of GLY on monocyte phenotype and phagocytosis
Treatment of monocytes with LPS caused a significant decrease in
HLA-DR and CD11b/CD18 expression, whereas CD64/FcRI was influenced
only slightly (Table 1
). Varying the GLY concentration did not change HLA-DR and CD64
expression of either LPS-treated or untreated monocytes, but a
significantly lower expression of CD11b/CD18 could be detected. In
addition, enhancing the GLY concentration significantly reduced
phagocytosis of opsonized E. coli by LPS-untreated
monocytes, whereas no GLY-dependent effect was seen after LPS
stimulation. Moreover, no effects were detected on latex beads
phagocytosis of LPS-treated or untreated monocytes (Table 2
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-1ß at both the protein and mRNA level and enhanced expression
of the anti-inflammatory cytokine IL-10. A significant reduction in the
constitutive expression of CD11b/CD18 and the phagocytosis of opsonized
E. coli by LPS-untreated monocytes, as measured by flow
cytometry, could be detected. The expression of HLA-DR and CD64 on
monocytes and their capacity to phagocytose latex beads were not
modulated by GLY.
Several therapeutic trials have been performed in septic patients, but
until now these studies have all failed to show clinical efficacy.
Antibodies to endotoxin have been prepared to counteract and block the
primary agents responsible for the inflammation, and therapies against
TNF-, IL-1, and platelet-activating factor have been developed
(14
, 29-34
). Although these mAb's were
effective in animal models, they did not have the potency to reduce
mortality in humans. It can be assumed that a total block of
proinflammatory cytokines is contraindicative in the septic state,
because they are necessary for a sufficient and prolonged immune
response and also have positive effects on protein and energy
metabolism by delivering amino acids and lactate to splanchnic organs.
Enteral administration of GLY could possibly be effective because
supply of GLY to endotoxin-boostered mice reduced but did not abolish
plasma TNF- levels by blunting increases in intracellular
Ca2+ in Kupffer cells (20)
. Chloride influx
causes hyperpolarization of the plasma membrane and is most likely
involved in the mechanism of action of GLY by decreasing open
probability of voltage-dependent Ca2+ channels. LPS
depolarizes the plasma membrane and induces Ca2+ influx in
Kupffer cells. This effect was blunted by GLY and reversed by low-dose
strychnine, an antagonist of a GLY-gated chloride channel. Our study
revealed that GLY modulates the secretion of TNF-, IL-1ß, and
IL-10, effects reversed by low-dose strychnine that therefore support
the existence of GLY-gated chloride channels on human monocytes.
The production of TNF- by purified monocytes was diminished by GLY
in a time- and dose-dependent manner, whereas IL-10 secretion showed a
biphasic course. Monocytes cultured in 10 mmol/l GLY showed an
increased IL-10 production after 6 h, with a maximum at 12 h,
whereas monocytes cultured in 2 mmol/l GLY reached maximum IL-10
secretion after 24 h. The secretion of IL-10 by whole blood cells
was significantly accelerated by GLY. In contrast to IL-1ß and
TNF-, expression of IL-10-specific mRNA was similar in cells
incubated with or without 2 mmol/l GLY supplementation (data not
shown). As seen at the protein level, there are overlapping time- and
dose-dependent effects of GLY on IL-10 expression. Our results suppose
that IL-10 alterations after GLY administration are not regulated at
the level of transcription.
Earlier results have shown that administration of IL-10 influences
markedly the cytokine network by inhibiting proinflammatory cytokine
production of monocytes after activation with IFN-, LPS, or
combinations of IFN- and LPS (35)
. In animal models,
the administration of an anti-CD28 Ab protects mice from the bacterial
toxin-induced septic shock by inducing the expression of the
anti-inflammatory cytokine IL-10. Treatment with an anti-IL-10 Ab has
been shown to increase and sustain the production of TNF- in plasma
and to dramatically increase bacterial toxin-induced death in mice
(36
, 37
). Incubation of whole blood
supplemented with 2 mmol/l GLY and an anti-IL-10 Ab, enhanced the
production of TNF- and IL-10 in our study by abolishing the
autoregulatory role of IL-10. Therefore, we hypothesize that the
reduced mortality, as found after GLY administration in
endotoxin-boostered rats, is possibly caused not only by reduced
TNF- secretion, but also by an increased IL-10 release, which may
counteract the shock situation by diminishing TNF- levels.
Monocytes express cell surface antigens with a wide variety of
functions
Products of the major histocompatibility class II complex, such as
HLA-DR, are essential in presentation of intracellularly processed
antigens to CD4+ T cells. The cell surface markers
CD64/FcRI and CD11b/CD18 (CR3) expressed by monocytes are important
in the phagocytosis of opsonized particles. Several in vitro
studies have demonstrated the influence of TNF- and IL-10 on the
phenotype of monocytes. TNF- is known to strongly up-regulate HLA-DR
(38)
, whereas IL-10 dramatically down-regulates it
(26)
. A decrease in HLA-DR expression on monocytes after
LPS stimulation has been described (33)
and was confirmed
by our study. However, the alterations of cytokine production as found
after GLY supply did not alter constitutive HLA-DR and CD64 expression.
Expression of CD11b/CD18, as well as the capacity of monocytes to
phagocytose opsonized E. coli (see Table 2
), was
significantly reduced by GLY. This contradicts earlier results from our
group showing that the amino acid glutamine given in equimolar amounts
to GLY augmented significantly HLA-DR, CD64, and CD11b/CD18 expression
on peripheral blood monocytes (19)
. Furthermore, these
effects correlated with an increased capacity to present antigen and to
phagocytose.
Until now, regulation of cytokine metabolism has been accomplished mainly by administration of anticytokine antibodies or receptor antagonists. Our study shows that supply of GLY alters especially the production of proinflammatory cytokines under in vitro and ex vivo conditions. In the clinical situation, GLY is administered to critically ill patients by parenteral nutrition as the dipeptide glycine-glutamine. Future studies have to confirm whether the amount of GLY infused to these patients may influence cytokine production and could be used as an additional tool in sepsis therapy and in various pathophysiological situations related to hypoxia and reperfusion.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: CR, complement receptor; ELISA,
enzyme-linked immunoassay; FACS, fluorescence-activated cell sorting;
FcR, Fc receptor for IgG; FCS, fetal calf serum; FITC, fluorescein
isothiocyanate, GLY, glycine; IL, interleukin; IFN, interferon; LPS,
lipopolysaccharide; mAb, monoclonal antibody; PBMC, peripheral blood
mononuclear cells; PE, pychoerythrin; RT-PCR, reverse transcription
polymerase chain reaction; SDS, sodium dodecyl sulfate; TNF, tumor
necrosis factor.
Received for publication May 4, 1998.
Revision received October 30, 1998.
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as a mediator of the lethality of endotoxin and tumor necrosis factor-. J. Immunol. 1992;149:1666-1670.[Abstract]
MAb Sepsis Study GroupAbraham E., Wunderink R., Silverman H., Perl T. M., Nasraway S., Levy H., Bone R., Wenzel R. P., Balk R., Allred, Pennington J. E., Wherry J. C.. Efficiacy and safety of monoclonal antibody to human tumor necrosis factor- in patients with sepsis syndrome. J. Am. Med. Assoc. 1995;273:934-941.[Abstract]
,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cellsrole in cell differentiation, activation and phagocytosis. Immunology 1997;90:286-293.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and 2 mmol/l (
) GLY
or without GLY supplementation (
). All tubes were then preincubated
for 6 h in a shaking 37°C water bath and subsequently stimulated
with 1 µg/ml LPS. After the indicated stimulation times, tubes were
centrifuged and supernatants immediately frozen until analysis by
ELISA. Each value represents the mean from four experiments
±SD. Statistically significant different decrease over
time, with increasing GLY concentrations of IL-1ß
(P<0.0001; panel A) and TNF-
), 2 mmol/l (
), 5 mmol/l (
), or 10 mmol/l
(