| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Internal Medicine, Justus-Liebig University, Giessen, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1
rose proportionally with
increasing cell density. The inhibition of IL-8 secretion was
reproduced by conditioned media of PMN at high cell density, but was
not affected by blocking 2 integrin-dependent adhesion.
When analyzing the supernatant of LPS-challenged neutrophils, large
amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R
antagonist (IL-1RA), rising constantly with the cell density, were
detected. Interestingly, combined blocking of the bioactivities of
these mediators completely restored neutrophil IL-8 secretion at high
cell densities, with the anti-IL-1RA Ab being the more potent
agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to
10 x 106 PMN/ml reproduced the suppression of IL-8
generation. We conclude that neutrophil IL-8 synthesis is
autoregulated, being suppressed under conditions of high cell density.
IL-1RA and sTNFRs, accumulating under these circumstances, seem to be
centrally involved in this regulatory mechanism by interfering with the
IL-1- and TNF--dependent IL-8 generation. This feedback mechanism
may control further neutrophil recruitment and activation in a
neutrophil-rich environment, thereby preventing tissue
destruction. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-1,
M-CSF, and G-CSF, as well as IL-1R antagonist (IL-1RA)
(5, 6, 7). While TNF-, IL-1, and growth factors
represent classic proinflammatory cytokines, IL-1RA inhibits the
proinflammatory effects of IL-1 and IL-1 by competitive binding
to IL-1Rs (8, 9). Moreover, neutrophils have the capacity
to release soluble receptors for TNF- (sTNFRs), which block TNF
activity by competing for it with cell surface TNFRs
(10, 11, 12). As neutrophils predominate over other cell types
in many variants of acute and chronic inflammatory conditions,
PMN-derived cytokines may be centrally involved in the regulation of
inflammatory and immune processes in vivo (13).
Although much attention has been paid to the onset of neutrophil
recruitment and activation, little is known about mechanisms limiting
neutrophil activity. Such mechanisms are, however, of major importance
for the control of inflammation in vivo, as excessive neutrophil
activation may cause severe tissue destruction (14, 15).
Pneumococcal pneumonia is an example of initial massive neutrophil
accumulation and subsequent complete resolution of pulmonary injury
with maintenance of lung structure (16, 17). The
neutrophil microenvironment may be a critical factor for the control of
neutrophil kinetics and activity. In the present study, we investigated
whether the neutrophil density per se regulates inflammatory cell
functions, focusing on neutrophil cytokine release. In essence, an
increase in cell density (from 10 x 106 to
60 x 106 PMN/ml) was found to
dramatically down-regulate the release of the CXC chemokine IL-8 upon
stimulation with LPS. Suppression of neutrophil chemokine generation
was not attributable to 2 integrin-dependent
adhesion, but was mediated by soluble factor(s) arising in the
supernatant of neutrophils cultured at high cell density. Biochemical
analysis and function-blocking Abs identified IL-1RA and sTNFRs as
suppressor agents. The observed down-regulation of neutrophil IL-8
generation at high cell density may represent a negative feedback
mechanism helping to control neutrophil inflammatory activity by
preventing further neutrophil recruitment and activation in a
neutrophil-rich microenvironment.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden), FCS
from Greiner (Frickenhausen, Germany), and all other media and
supplements were from Life Technologies (Eggenstein, Germany), unless
otherwise indicated. LPS (Escherichia coli, 0111:B4),
indomethacin, 1-antitrypsin
(1AT), superoxide dismutase (SOD), and
adenosine deaminase (ADA) were purchased from Sigma (Deisenhofen,
Germany), while MK-886 and CV-6209 were from Calbiochem (La Jolla, CA).
All Abs used for cytokine ELISAs as well as recombinant cytokines were
purchased from R&D Systems (Wiesbaden, Germany): mAbs against TNF-
(mAb 610), IL-1 (mAb 601), IL-8 (mAb 208), and IL-10 (mAb 217);
biotinylated Abs against TNF- (BAF 210), IL-1 (BAF 201), IL-8
(BAF 208), and IL-10 (BAF 417); as well as recombinant human TNF-
(210-TA), IL-1 (201-LB-005), IL-8 (208-TA-010), and IL-10
(217-IL-005). For the detection of IL-1RA, sTNFRs, IL-4, IL-13, and
IFN-
, commercial ELISA kits were used (R&D Systems). Neutralizing
Abs targeting IL-1RA (AF-280-NA) and sTNFRs (mAb 225 for sTNFRI(p55)
and mAb 226 for sTNFRII(p75)) were also from R&D Systems, as well as
recombinant IL-1RA (280-RA-010) and sTNFRs (636-R1-025; 226-B2-025).
Peroxidase-conjugated streptavidin (HRP) and ABTS were purchased from
Zymed Laboratories (San Francisco, CA), while the anti-CD18 Ab
MHM23 was from Dako (Hamburg, Germany). The Dynabeads mRNA direct kit
was from Dynal (Oslo, Norway), and the SYBR Green PCR Core Reagents
were from Applied Biosystems (Weiterstadt, Germany). Cell culture
plasticware was purchased from Falcon (Mannheim, Germany).
Isolation of human neutrophils
Neutrophils were isolated from venous blood of healthy donors by centrifugation over a Ficoll-Paque gradient, as previously described (18). In brief, EDTA-anticoagulated blood was layered over Ficoll-Paque and centrifuged at 400 x g for 35 min. After removal of mononuclear cells, erythrocytes were sedimented in 1% polyvinyl alcohol. Residual erythrocytes were removed by hypotonic lysis, and cells were washed twice in Ca2+/Mg2+-free PBS and finally resuspended in RPMI containing 1% FCS at 10 x 106 to 60 x 106 PMN/ml. Cell purity was >97%, as quantified by flow cytometry, and cell viability was >96%, as assessed by trypan blue dye exclusion.
Cell culture and stimulation
In the standard protocol, neutrophils were resuspended in RPMI
supplemented with 1% FCS, plated in 24-well tissue culture plates at
10 x 106 to 60 x
106/ml (500 µl/well), and incubated at 37°C
in a 5% CO2-humidified atmosphere. Neutrophils
were cultured with media alone (control) or stimulated with 10 ng/ml
LPS. In experiments designed to investigate the effects of IL-1RA or
sTNFRs, neutralizing Abs were coapplied with LPS and used at 20
µg/ml. All other agents were also coapplied with LPS: ADA was used at
400 U/ml; the platelet-activating factor (PAF) receptor antagonist
CV-6209, the cyclooxygenase-inhibitor indomethacin, and the
5-lipoxygenase inhibitor MK-886 were used at 10 µM; whereas the
anti-CD18 Ab (MHM23) was used at 50 µg/ml.
1AT was applied at 100 µg/ml, and the
O2- scavenger SOD
was used at 100 U/ml. After 4, 8, and 16 h of incubation, cells
and cell supernatants were separated by centrifugation at 13,000
x g, and cell supernatants were harvested and stored at
-80°C until further processing. To assess the levels of
intracellularily stored cytokines, cell pellets were resuspended in 500
µl RPMI. Cells underwent two freezethaw cycles using
liquid nitrogen, debris was removed by centrifugation at 13,000 x
g, and lysates were stored at -80°C.
Cytokine ELISA
Release of TNF-, IL-1, IL-8, and IL-10 was determined by
self-developed direct sandwich ELISAs. In brief, immunoassay plates
were coated with mouse anti-human TNF-, IL-1, IL-8, or IL-10
mAbs at a concentration of 4 µg/ml. After a blocking period with 1%
BSA in PBS, samples were added. Recombinant human TNF-, IL-1,
IL-8, and IL-10 were used for standard titration curves. To sandwich
the trapped Ag, biotinylated Abs were applied at the following
concentrations: 400 ng/ml anti-TNF-, 200 ng/ml anti-IL-1,
40 ng/ml anti-IL-8, or 200 ng/ml IL-10. Next, plates were incubated
with HRP-conjugated streptavidin, followed by addition of substrate
(H2O2 and ABTS). Absorbance
was measured at 450 nm in a microplate reader using SLT Lab Instruments
software (Creilsheim, Germany) to analyze the generated data. IL-8 and
IL-10 ELISA were sensitive to 15 pg/ml, IL-1 and TNF- ELISA to 7
pg/ml. IL-1RA, sTNFRs, IL-4, IL-13, and IFN- were determined in a
commercial ELISA system, sensitive to 15 pg/ml each. In separate
recovery experiments, the effect of cell debris on the determination of
IL-8 was evaluated. Therefore, rIL-8 was dissolved in the supernatants
of unstimulated neutrophils cultured at the various cell densities, and
cytokine ELISAs were performed exactly as described. Equal values
(deviation <5%) were obtained for the determination of IL-8 either if
dissolved in the supernatant of 10 x 106
PMN/ml or in that of 60 x 106 PMN/ml.
mRNA extraction
Aliquots of 0.5 x 106 cells derived from neutrophils cultured at the various cell densities were transferred into 1.5-ml reaction tubes. After centrifugation at 300 x g, the supernatant was removed and the pellet was lysed in 300 µl lysis buffer of the Dynabeads mRNA direct kit. Based on magnetic separation, mRNA is caught by attachment to oligo(dT) fragments that are coupled to supermagnetic glass particles. Per sample, 150 µg beads were applied. Isolated mRNA was finally dissolved in 20 µl diethyl pyrocarbonate-treated H2O.
cDNA synthesis and real-time PCR
For cDNA synthesis, reagents and incubation steps were applied
as described (19). Ten microliters each of the isolated
mRNA were applied for reverse transcription. The reactions were set up
with the SYBR Green PCR Core Reagents, according to the manufacturer’s
protocol. Using the oligonucleotide primer pairs given in Table I
, 1 µl of each primer (10 µM) and 2
µl cDNA were added to a final volume of 50 µl. Cycling conditions
were adapted to 95°C for 6 min, followed by 40 cycles of 95°C,
20 s; 58°C, 30 s; and 73°C, 30 s. PCR products were
routinely identified by 2.5% agarose gel electrophoresis.
|
Relative mRNA quantitation was performed by the Sequence Detection System 7700 (Applied Biosystems) and real time PCR. (T0/R0) = K x (1 + E)(CT,R - CT,T) in which T0 is the initial number of target gene mRNA copies, R0 is the initial number of standard gene mRNA copies, E is the efficiency of amplification, CT,T is the threshold cycle of target gene, CT,R is the threshold cycle of standard gene, and K is the constant.
On the basis of the given equation, we used comparative
quantitation (
CT), normalizing IL-8 to an
unregulated internal standard gene (19). Therefore,
mRNA transcribed from the gene encoding hypoxanthine-guanine
phosphoribosyltransferase (HPRT) was used. Afterward, values of
relative mRNA expression were normalized to the value of 10 x
106 PMN/ml after 1 h of LPS challenge. In
preliminary experiments, we could show that amplification efficiency of
HPRT and IL-8 primer sets was approximately equal and amounted to
0.95 ± 0.02 (= 95 ± 2%). K is assumed to be
equal within a definite primer system and thus does not influence the
comparison of calculated relative ratios. Due to the nonselective dsDNA
binding of the SYBR Green, gel electrophoresis was performed to confirm
the exclusive amplification of the expected PCR product.
Statistics
For statistical comparison, one-way ANOVA was performed, followed by Tukey‘s honestly significant difference test when appropriate. A level of p < 0.05 was considered to be significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Whereas unstimulated neutrophils released only small amounts of
IL-8 into the cell supernatant (data not given in detail), stimulation
of 10 x 106 PMN with 10 ng/ml LPS resulted
in progressive liberation of this CXC chemokine into the cell
supernatant (Fig. 1A).
Significant accumulation of IL-8 was already noted after 4 h of
endotoxin stimulation, and rose steadily up to 16 h of
stimulation. Interestingly, with increasing cell density, the levels of
IL-8 in the supernatant volume did not increase, but were instead
dramatically reduced (Fig. 1A). As compared with 10 x
106 PMN/ml, IL-8 liberation was reduced to
35% in 60 x 106 PMN/ml after 8 and
16 h of stimulation (1746 ± 316 vs 677.5 ± 180 pg/ml
after 8 h, and 4350 +/- 1001 vs 1796 ± 448 pg/ml after
16 h, n = 6). When calculations were performed per
cell number, this corresponds to a suppression of IL-8 release to
<10% per single cell under conditions of high cell density.
Importantly, any debris in the cell supernatants of neutrophils at high
cell density did not affect the determination of IL-8 in the currently
used ELISA system, as assessed in separate recovery experiments.
|
B), indicating that down-regulation of
neutrophil IL-8 release at high cell density was attributable to
reduced de novo synthesis of this chemokine. mRNA quantification by
real-time RT-PCR, as performed after 1 and 4 h of LPS stimulation,
revealed a comparable decrease in IL-8 mRNA as normalized to HPRT-mRNA
in 60 x 106 PMN/ml (Fig. 2).
|
and IL-1 are continuously liberated upon increase in
neutrophil density
To assess whether the down-regulation of IL-8 synthesis was
paralleled by the suppression of other neutrophil-derived cytokines,
supernatants of LPS-stimulated PMN at various cell densities were
analyzed for TNF- and IL-1. Contrary to IL-8, the liberation of
these cytokines increased proportionally with increasing PMN density
(Fig. 3). After 16 h of incubation,
LPS-induced release of IL-1 from 10 x
106 PMN/ml was 165 ± 32.5 pg/ml, while
838 ± 201 pg/ml IL-1 was liberated from 60 x
106 PMN/ml (Fig. 3A). When calculating
IL-1 synthesis per single cell, this corresponds to 165 ± 32.5
pg/10 x 106 PMN at the lowest cell density
and 138 ± 19.45 pg/10 x 106 PMN at
the highest cell density, indicating that the liberation of this
cytokine was not undergoing any substantial down-regulation by cell
density. Similarly, LPS-induced liberation of TNF-, quantified after
16 h of stimulation, was 75.6 ± 14.3 pg/ml from 10 x
106 PMN/ml, and increased to 410.3 ± 60.6
pg/ml in 60 x 106 PMN/ml (Fig. 3B), corresponding to a TNF- liberation of 75.6 ±
14.3 pg/10 x 106 PMN cultured at the lowest
cell density, and 68.4 ± 10.1 pg/10 x
106 PMN kept in the highest cell density.
|
Next, we investigated whether the selective down-regulation of
neutrophil IL-8 generation was caused by direct cell-to-cell
interactions, or was due to a soluble mediator arising in the cell
supernatant of neutrophils cultured at high cell density. Inhibition of
2 integrin-dependent adhesion with the
function-blocking Ab MHM23, targeting the -chain of LFA-1, did not
restore neutrophil IL-8 generation at high cell density (Table II), indicating that integrin-mediated
cell-to-cell interactions were not underlying neutrophil
down-regulation. Therefore, a transfer of the putative down-regulatory
mediator derived from neutrophils at high cell density was undertaken
to neutrophils at constant density. For this purpose, constant
concentrations of neutrophils (10 x 106
PMN/ml) were stimulated for 8 h with LPS in conditioned medium
derived from neutrophils (originating from the same donor) cultured at
the varying cell densities (10 x
106–60 x 106
PMN/ml). Conditioned media were obtained after an incubation period
with 10 ng/ml LPS for 8 h. As depicted in Fig. 4, inhibition of IL-8 secretion was
reproduced with conditioned media from neutrophils cultured at high
cell density: when stimulated in conditioned medium derived from
neutrophils at 60 x 106/ml, IL-8 release of
10 x 106 PMN was reduced to 20% of that
upon use of conditioned medium from PMN at the lowest cell density
(431 ± 33 pg IL-8/10 x 106 PMN
cultured in conditioned medium from 60 x
106 PMN/ml vs 1808 ± 220 pg IL-8/10 x
106 PMN cultured in conditioned medium from
10 x 106 PMN/ml).
|
|
To identify the soluble factor(s) involved in the down-regulation
of neutrophil IL-8 generation at high cell density, cell supernatants
were analyzed for potentially inhibitory cytokines or cytokine
antagonists. While the antiinflammatory cytokines IL-4, IL-10, and
IL-13 were not detected (data not given), excessive quantities of
IL-1RA (>30 ng/ml for 60 x 106 PMN/ml) and
both types of soluble TNFRs (>10 ng/ml for 60 x
106 PMN/ml), rising dramatically upon increase in
cell density, were recovered from LPS-treated neutrophils (Fig. 5).
|
and IL-1 (20), we
speculated that down-regulation of neutrophil IL-8 generation was due
to blockage of this autocrine loop by IL-1RA and sTNFRs. Indeed, when
blocking the bioactivity of these mediators, LPS-induced synthesis of
IL-8 was restored, rising proportionally with increasing cell density:
as depicted in Fig. 6, combined
application of neutralizing Abs (20 µg/ml) targeting IL-1RA and both
types of soluble TNFRs was the most efficient approach, provoking a
20-fold increase in IL-8 liberation from neutrophils cultured at the
highest cell density (12,024 ± 1,028 pg/ml in the presence of
neutralizing Abs vs 572 ± 36.4 pg/ml in the absence of
neutralizing Abs). Sole neutralization of IL-1RA induced a 15-fold
increase in IL-8 liberation from 60 x 106
PMN/ml, while sole inhibition of sTNFR bioactivity was less efficient,
but still resulted in the liberation of 4,323 ± 663 pg/ml IL-8
from 60 x 106 PMN vs 572 ± 36.4 pg/ml
IL-8, thus corresponding to a 7.5-fold increase. Application of
neutralizing Abs per se in the absence of LPS challenge did not provoke
neutrophil IL-8 release (data not given).
|
To further prove the inhibitory effect of IL-1RA and sTNFRs on
LPS-induced neutrophil IL-8 release, these mediators were added to
neutrophils at constant concentrations (10 x
106 PMN/ml) exposed to LPS challenge, with the
amounts of exogenous IL-1RA (40 ng/ml) and sTNFRs (20 ng/ml)
corresponding to the levels of these mediators secreted from
neutrophils at high cell density. As depicted for IL-8 release after
16 h of LPS challenge, combined application of IL-1RA and both
types of soluble TNFRs resulted in a most impressive inhibition of
LPS-induced IL-8 generation, decreasing from 3585.3 ± 522.6 pg/ml
IL-8 in the absence of cytokine antagonists to 1003.3 ± 206.6
pg/ml in their presence (Fig. 7). As
anticipated from the studies with function-blocking Abs, sole
application of IL-1RA exerted more efficient inhibition of IL-8 release
than sole sTNFR challenge.
|
In separate experiments, we addressed further neutrophil-derived
mediators concerning a putative role in the down-regulation of IL-8
generation upon increase in cell density. Although the cyclooxygenase
product PGE2 was continuously liberated from
neutrophils upon increase in cell density (data not given in detail),
application of the cyclooxygenase inhibitor indomethacin (10 µM) did
not restore chemokine synthesis at high cell density. Moreover,
interference with neutrophil 5-LO metabolism by using the specific
inhibitor MK-886 (10 µM), or inhibition of PAF activity by the
receptor antagonist CV-6209 (10 µM) was ineffective. Furthermore,
neither scavenging oxygen radicals with SOD (100 µM), nor
antagonizing neutrophil proteases with 1AT
(100 µg/ml) restored neutrophil IL-8 synthesis, and a potential
inhibitory role for endogenous adenosine was excluded by addition of
appropriate concentrations of ADA (400 U/ml). Also upon stimulation of
neutrophils with higher (1 µg/ml) or lower (0.1 ng/ml) LPS
concentrations, a down-regulation of IL-8 generation was observed. All
data are given in Table II.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-1 secretion were
not inhibited by the cell density. Supernatant transfer experiments
suggested that the down-regulation of IL-8 secretion was attributable
to secreted factor(s), and these were identified as IL-1RA and sTNFRs
by immunodetection, by experiments with specific blocking, and by
studies employing exogenous IL-1RA and sTNFRs.
Enhancing neutrophil density, achieved by increasing the total number
of neutrophils in a given volume, exerted a profound influence on the
LPS-induced liberation of the CXC chemokine IL-8, which decreased to
<10% per single neutrophil at the highest (60 x
106 PMN/ml) vs the lowest (10 x
106 PMN/ml) cell density. As intracellular levels
of IL-8 were equally diminished, the down-regulation of IL-8 was
obviously not due to reduced liberation, but was caused by reduced de
novo synthesis of the chemokine. Well in line with this reasoning, a
corresponding decrease in IL-8 message was obtained for the highest
neutrophil density (60 x 106 PMN/ml) when
analyzed by quantitative RT-PCR. As in 20 x
106 PMN/ml, a significant decrease in IL-8 mRNA
could not be detected; a posttranscriptional mechanism may additionally
be involved in the regulation of IL-8 synthesis at that cell density.
Importantly, any debris possibly present in the cell supernatant of
more densely cultured neutrophils did not affect the determination of
IL-8 in the currently used ELISA system, as assessed in separate
recovery experiments. Moreover, the reduced IL-8 generation was
unlikely to be an unspecific toxic effect of the high neutrophil
density, as release of lactate dehydrogenase was consistently below 5%
of the total cellular content, and as other neutrophil metabolic
properties demanding cellular integrity, such as TNF- and IL-1
formation, were unaffected by PMN density.
When attempting to elucidate the mechanism(s) involved in the
down-regulation of chemokine generation upon increase in cell density,
supernatant transfer experiments clearly indicated that a soluble agent
arising from the densely cultured neutrophils was responsible for the
suppression of IL-8 generation. Neutrophil IL-8 generation was
previously shown to be regulated by a wide variety of soluble agonists.
Among the cytokines, IL-4, IL-10, IL-13, and IFN-
(23, 24, 25) have been implicated in inhibition of LPS-induced
IL-8 formation. However, these cytokines were not detected in the cell
supernatants of LPS-treated neutrophils at any cell density; therefore,
an inhibitory role for these agonists could be excluded for the present
experimental system. Interestingly, IL-10 was found in the lysates of
neutrophils, but was not liberated, and addition of neutralizing
anti-IL-10 Abs did not restore neutrophil chemokine generation
(data not given). Besides antiinflammatory cytokines, prostanoids have
been shown to down-regulate neutrophil IL-8 generation
(26). Although PGE2 was continuously
secreted from neutrophils at increasing cell densities, the
cyclooxygenase inhibitor indomethacin had no effect on IL-8 generation.
Moreover, interference with other neutrophil-derived mediators, namely
5-LO-metabolites, PAF, proteases, and reactive oxygen species, was
ineffective. Endogenous adenosine, an autacoid known to accumulate in
neutrophil suspensions (27), has recently been implicated
in the regulation of a variety of neutrophil functions, including
cytokine generation (28), and was thus considered as a
further candidate contributing to the presently observed
down-regulation of neutrophil IL-8 generation. However, the lack of
effect of ADA, added at high concentrations to neutrophil suspensions,
seriously questions a role of endogenous adenosine in the present
setup.
Compelling evidence was, however, provided that the inhibition of IL-8
synthesis was centrally linked to IL-1RA and soluble TNFRs,
secreted progressively upon neutrophil culturing in high cell
densities. First, excessive quantities of IL-1RA (>30
ng/ml) and sTNFRs (>10 ng/ml), rising in parallel with the increase in
cell density, were recovered from the supernatants of LPS-treated PMN.
Second, blocking the bioactivity of these mediators by
application of neutralizing Abs completely restored chemokine
generation under conditions of high neutrophil density, thus resulting
in a proportional increase in IL-8 with increasing cell density. These
studies with function-blocking Abs suggested that the different
cytokine antagonists synergized in inhibiting IL-8 release, with IL-1RA
being a more efficacious agent than the soluble TNFRs, as
antagonizing sole IL-1RA activity provoked a 15-fold increase in
IL-8 secretion from 60 x 106 PMN, vs a
7.5-fold increase in IL-8 observed after neutralizing sTNFRs. And
third, exogenous addition of recombinant IL-1RA and sTNFRs, in
concentrations comparable with those detected in the supernatants of
LPS-treated neutrophils at high cell density, reproduced the
suppression of LPS-induced IL-8 liberation, thus confirming the key
role previously suggested for TNF- and IL-1 as autocrine
facilitators of LPS-induced formation of IL-8 (20, 29).
Thus, at high neutrophil cell density, this autocrine loop is
apparently blocked, as although IL-1 and TNF- are present, their
cell surface receptor occupancy is competitively inhibited by IL-1RA
and sTNFRs.
IL-1RA was produced in an 80-fold excess over IL-1, and sTNFRs
were in 40-fold excess over TNF- in populations with high
neutrophil density. Such an excess over the respective agonists is well
known to be necessary to block the physiological actions of IL-1 and
TNF- (8, 9), probably due to decreased affinity of
IL-1RA to IL-1Rs as compared with the agonist IL-1, and due to
preferred engagement of cell surface TNFRs by circulating TNF-.
Hence, in 10 x 106 PMN/ml,
function-blocking Abs targeting IL-1RA and sTNFRs did not amplify
LPS-induced neutrophil IL-8 generation (Fig. 6), indicating that this
regulatory loop was only operative when a certain excess of the
cytokine antagonists over the respective agonists was given, just as
occurring in high neutrophil cell density. In vivo, such excessive
amounts of neutrophil-derived cytokine antagonists should not only
down-regulate inflammatory neutrophil functions, but exert an overall
antiinflammatory effect on various cellular immune effector cells. In
this respect, release of sTNFR p75 by neutrophils has recently been
postulated to be implicated in the regulation of TNF bioactivity in
vivo (30), and exogenous application of IL-1RA and
sTNFRs has been shown to reduce the mortality of endotoxin shock in
numerous animal models (31, 32, 33).
Neutrophils represent the first line of defense against infections or
nonself agents, and a wide variety of systemic mediators was shown to
regulate neutrophil activity. In the present study, we found that the
neutrophil density per se represents a critically variable-limiting
excessive neutrophil activation. IL-1RA and soluble TNFRs, accumulating
massively under conditions of high neutrophil density, are centrally
involved in this regulatory mechanism by interfering with the IL-1-
and TNF--dependent amplification of IL-8 generation. As a result,
IL-8 synthesis and release are dramatically reduced at high neutrophil
density. This negative feedback mechanism is to be assumed to result in
the inhibition of further neutrophil recruitment and activation in a
neutrophil-rich microenvironment, thereby contributing to the control
of inflammatory functions in vivo.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: PMN, polymorphonuclear leukocyte; 1AT, 1-antitrypsin; ADA, adenosine deaminase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IL-1RA, IL-1R antagonist; s, soluble; SOD, superoxide dismutase.
Received for publication November 27, 2000. Accepted for publication March 5, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. J. Exp. Med. 176:593. by the soluble type I and type II TNF receptors. Cytokine 7:26.[Medline]
in mediating the production of IL-8 triggered by lipopolysaccharide. J. Exp. Med. 178:2207. interferon. Cell. Immunol. 157:448.[Medline]
of endotoxin-stimulated human polymorphonuclear leukocytes. J. Lab. Clin. Med. 126:275.[Medline]
This article has been cited by other articles:
|
|
 |
|
  E. Alfaro-Moreno, T. S. Nawrot, B. M. Vanaudenaerde, M. F. Hoylaerts, J. A. Vanoirbeek, B. Nemery, and P. H. M. Hoet Co-cultures of multiple cell types mimic pulmonary cell communication in response to urban PM10 Eur. Respir. J., November 1, 2008; 32(5): 1184 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Kukulski, F. Ben Yebdri, J. Lefebvre, M. Warny, P. A. Tessier, and J. Sevigny Extracellular nucleotides mediate LPS-induced neutrophil migration in vitro and in vivo J. Leukoc. Biol., May 1, 2007; 81(5): 1269 - 1275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Thorley, P. A. Ford, M. A. Giembycz, P. Goldstraw, A. Young, and T. D. Tetley Differential Regulation of Cytokine Release and Leukocyte Migration by Lipopolysaccharide-Stimulated Primary Human Lung Alveolar Type II Epithelial Cells and Macrophages J. Immunol., January 1, 2007; 178(1): 463 - 473. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. G.-J. Ooi, G. Galatowicz, V. L. Calder, and S. L. Lightman Cytokines and Chemokines in Uveitis - Is there a Correlation with Clinical Phenotype? Clin. Med. Res., December 1, 2006; 4(4): 294 - 309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Gordon, F. Li, X. Zhang, W. Wang, X. Zhao, and A. Nayyar The combined CXCR1/CXCR2 antagonist CXCL8(3-74)K11R/G31P blocks neutrophil infiltration, pyrexia, and pulmonary vascular pathology in endotoxemic animals J. Leukoc. Biol., December 1, 2005; 78(6): 1265 - 1272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rupp, H. Kothe, A. Mueller, M. Maass, and K. Dalhoff Imbalanced secretion of IL-1{beta} and IL-1RA in Chlamydia pneumoniae-infected mononuclear cells from COPD patients Eur. Respir. J., August 1, 2003; 22(2): 274 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Garcia-Ramallo, T. Marques, N. Prats, J. Beleta, S. L. Kunkel, and N. Godessart Resident Cell Chemokine Expression Serves as the Major Mechanism for Leukocyte Recruitment During Local Inflammation J. Immunol., December 1, 2002; 169(11): 6467 - 6473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Endlich, D. Armstrong, J. Brodsky, M. Novotny, and T. A. Hamilton Distinct Temporal Patterns of Macrophage-Inflammatory Protein-2 and KC Chemokine Gene Expression in Surgical Injury J. Immunol., April 1, 2002; 168(7): 3586 - 3594. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
