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*
Clinical Services Program, Science Applications International Corp. (SAIC) Frederick, Frederick Cancer Research and Development Center, Frederick, MD 21702;
Laboratory of Experimental Immunology, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702;
State University of New York Health Science Center, Brooklyn, NY 11203; and
§
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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200-fold more cell-associated IL-8 than
isolated peripheral blood neutrophils (4). These observations, together
with the demonstration that neutrophils undergoing phagocytosis release
IL-8 (5), suggest that stimulation of IL-8 production by the neutrophil
may be a paracrine mechanism for recruitment of additional neutrophils,
as well as other immune cells such as lymphocytes (6) and basophils
(7), to inflammatory sites.
While the mechanism(s) controlling IL-8 production and release have not
been fully delineated, the observation that the Ca2+
ionophore, A23187, increased neutrophil IL-8 synthesis and release (4)
implicates a role for elevation of the intracellular Ca2+
concentration,
[Ca2+]i,3
in the signaling pathway. To further characterize the role of
[Ca2+]i in IL-8 production by neutrophils,
this report examines the effect of thapsigargin on the production and
release of IL-8 and other cytokines by neutrophils. Thapsigargin is a
naturally occurring sesquiterpene lactone that inhibits a microsomal
Ca2+-ATPase, and thus results in the release of microsomal
Ca2+ stores. In neutrophils (8) and many other cells
(9, 10, 11), thapsigargin not only causes the release of intracellular
Ca2+ stores, but also opens a Ca2+ influx
pathway. These events can lead to cell activation in the absence of
receptor-ligand interactions, e.g., the induction of IL-2 (12) and the
-chain of the IL-2R in lymphocytes (13) and IL-6 production in
murine macrophages (14).
This report demonstrates that the thapsigargin-induced synthesis and release of IL-8 in neutrophils is relatively specific and requires a sustained Ca2+ influx. Studies with inhibitors of the immunophilins that target calcineurin implicate this calmodulin-dependent phosphatase in the signaling cascade leading to thapsigargin-induced IL-8 synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following reagents were purchased from the indicated
sources: recombinant human IL-1ß (R&D Systems, Minneapolis, MN);
recombinant human IL-8 and recombinant human TNF- (PeproTech, Rocky
Hill, NJ); HEPES and HBSS (BioWhittaker, Walkersville, MD); and
1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic
acid pentaacetoxymethyl ester (Indo1-AM) and BAPTA-AM,
1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra(acetoxymethyl) ester (BAPTA-AM) (Molecular Probes,
Eugene, OR). Anti-human cytokine neutralizing Abs were obtained from
the following sources: MAB201 (anti-IL-1ß) and MAB208
(anti-IL-8, R&D Systems); and mAb 1 (anti-TNF-) and 107.3
(purified mouse IgG1 isotype control Ig, PharMingen, San Diego, CA).
All other chemicals used were of reagent grade and were purchased from
Sigma (St. Louis, MO). Stock thapsigargin was dissolved in tissue
culture grade, endotoxin-free DMSO at 1 mM and stored until use at
-80°C.
Isolation of peripheral blood neutrophils
Peripheral blood neutrophils were isolated from blood obtained from normal volunteers (anticoagulant citrate dextrose solution USP formula A, Baxter Healthcare, Deerfield, IL). Whole blood was diluted with HBSS without divalent cations and layered over a discontinuous gradient cushion (Histopaque-1083; Sigma). The sample was spun for 30 min at 500 x g at room temperature. After discarding the supernatant fluid, the neutrophil/erythrocyte pellet was suspended in an equal volume of HBSS. The cell suspension was then diluted with dextran ([1.5%]final, m.w. 200,000–500,000 g/mol; Pharmacia, Uppsala, Sweden) and allowed to sediment at 1 x g for 20 min. The neutrophil-rich supernatant fluid was harvested and spun at 200 x g for 10 min. Contaminating erythrocytes in the pellet were removed by two sequential hypotonic lyses using 0.25x PBS for 30 s followed by an equal volume of 1.75x PBS to restore isotonicity. The neutrophils were then counted on an automated cell counter (model T540, Coulter, Hialeah, FL). The final preparation of neutrophils was >95% pure with 4% eosinophils and <1% monocytes and lymphocytes as assessed by differential staining.
IL-8 production by peripheral blood neutrophils
Isolated peripheral blood neutrophils (2 x 106) were suspended in HBSS with 10 mM HEPES (pH 7.35) and incubated in 2 ml polypropylene screw cap tubes (Sarstedt, Newton, NC) for up to 8 h at 37°C in the presence or absence of thapsigargin. In all inhibitor studies, with the exception of the cell permeant inhibitor BAPTA-AM, inhibitors were added 10 min before the addition of thapsigargin and were present throughout the experiment. In studies with BAPTA-AM, neutrophils (1 x 107/ml HBSS without divalent cations) were incubated with the indicated concentration of BAPTA-AM for 45 min at 37°C, washed twice, and resuspended at the indicated cell concentration in HBSS with HEPES. Neutrophils loaded with as high as 75 µM BAPTA-AM remained viable, evidenced by their ability to a maintain a Ca2+ gradient.
After incubation with thapsigargin for the indicated times, the cell suspensions were spun at 4°C and the supernatant fluid harvested for analysis. Cell pellets were solubilized in 0.2% Triton X-100 using 20, 1-s pulses (minimum setting) with a microtip sonicator (Sonifier II, Branson Ultrasonics, Danbury, CT).
Assays
All of the following cytokines were measured using commercial
enzyme-linked immunosorbent assays (R&D Systems); manufacturer’s
limits of detection are included in parentheses: IL-8 (4.7 pg/ml), IL-6
(0.7 pg/ml), IL-1ß (1.0 pg/ml), IL-1R antagonist (IL-1Ra; 6.5 pg/ml),
TNF- (4.4 pg/ml), RANTES (2.5 pg/ml), growth-related protein
(GRO-; 5.0 pg/ml), and macrophage inflammatory protein-1
(MIP-1; 7.0 pg/ml). Lactate dehydrogenase activity was determined by
monitoring the reduction of NADH at 340 nm using a molar extinction
coefficient of 6220.
Analysis of mRNA
Total cellular RNA was isolated from neutrophil preparations by a single step phenol/chloroform extraction procedure using TRIzol (Life Technologies, Gaithersburg, MD). The OD260/OD280 of the extracted RNA was >1.6; total RNA yields were 20 to 30 µg/5 x 107 neutrophils. RNA concentrations were also determined using the RiboGreen Quantitation Kit with a ribosomal RNA standard (Molecular Probes). In general, the concentrations obtained spectrophotometrically were 2-fold higher than those obtained using the commercial kit. Total cellular RNA (10 µg per sample) was size-fractionated on formaldehyde-denaturing 0.8% agarose gel and transferred to Magnagraph (Micron Separations, Westborough, MA). After UV crosslinking, blots were hybridized in NyloHybe (Fast Pair, Silver Spring, MD) to 32P-labeled cDNA probes prepared by random priming utilizing a commercially available kit (Stratagene, La Jolla, CA). Blots were hybridized for 24 to 36 h at 42°C and then washed for 10 min at room temperature in 2x SSC/0.1% SDS followed by a 10-min wash at 65°C in 0.2x SSC/0.1% SDS. After hybridization with one probe, the blots were stripped by placing in boiling 0.02x SSC/0.01% SDS for 20 min. Blots were then placed in hybridization buffer for 1 to 2 h and the next probe added. All cDNA probes had a specific activity of 2 to 8 x 108 cpm/µg and all hybridizations were performed with 1 x 106 cpm/ml. Blots were exposed to Kodak (Rochester, NY) X-OMAT x-ray film for 2 to 18 h at -70°C. Human IL-8 cDNA was obtained from Dr. Joost Oppenheim (National Cancer Institute/Frederick Cancer Research and Development Center, Frederick, MD) and chicken ß-actin cDNA was obtained from Dr. Donald Cleveland (Johns Hopkins University, Baltimore, MD). Northern blots were quantitated using a imaging densitometer (model GS 670, Bio-Rad, Hercules, CA). Relative levels of IL-8 mRNA were adjusted for unequal loading using ß-actin mRNA expression.
RiboQuant multiprobe RNase protection assays (hCK-5, hCK-2, and hCK-3; PharMingen) were performed as described by the manufacturer. One microgram of neutrophil RNA was added to the reaction mix. A plot of the mobility of the probes vs the nucleotide length was used to predict the migration of the protected probe fragments.
Determination of thapsigargin-induced changes in [Ca2+]i
Neutrophils (1 x 107/ml of HBSS/HEPES) were
incubated with the cell permeant dye, Indo1-AM, in the dark at 37°C
for 45 min. The neutrophils were then pelleted by centrifugation at
200 x g, resuspended in HBSS/HEPES, and the procedure
repeated to remove the residual extracellular Indo1-AM. The cells were
resuspended at 2.5 x 106/ml HBSS/HEPES. Changes in
[Ca2+]i were monitored on a DeltaScan
spectrophotometer (Photon Technology, South Brunswick, NJ) using a
thermostatically controlled cuvette holder. Data were collected as the
ratio (R) of the 
emissions [402
nm/486 nm] using an excitation =
358 nm. [Ca2+]i was determined as described
previously (15). Rmax and
Rmin were empirically determined by addition of
ionomycin (1 µM) and EGTA (12.5 mM), respectively.
The equilibrium composition of free Ca2+ in an EGTA-containing buffer solution was derived using the software EQCAL (BIOSOFT, Cambridge, U.K.) and equilibrium constants cited in the software manual.
Statistics
The significance of difference between test and control groups was analyzed using either Student’s t test or analysis of variance (ANOVA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Maintenance of unstimulated isolated peripheral blood neutrophils
for 8 h in HBSS/HEPES resulted in the accumulation of a total of
0.6 ng of IL-8/106 neutrophils (n = 2),
with only a small fraction of (<10%) found in the extracellular
fluid. In contrast, incubation with thapsigargin (50–100 nM) for
8 h induced the accumulation of 26.4 ng of IL-8/106
neutrophils (n = 2), equally partitioned between the
cellular and extracellular compartments. The induction of IL-8 was
detectable by 15 min and continued to rise throughout the 8-h
measurement period (Fig. 1
). The rate of
total IL-8 production was greatest 1 to 2 h after addition of
thapsigargin. In the first hour, most of the IL-8 (>90%) was found in
detergent extracts of the cells. At 4 h, cellular IL-8 reached a
plateau, while levels of IL-8 in the extracellular fluid continued to
increase. The viability of thapsigargin-treated neutrophils at 4 h
was within 90% of the viability of untreated neutrophils when assessed
by both trypan blue exclusion and lactate dehydrogenase release. In
addition, despite incubation of Indo1-loaded neutrophils with 100 nM of
thapsigargin for 4 h, addition of 1.0 µM ionomycin caused a
further increase in [Ca2+]i (data not shown),
indicating that the cells still maintained a Ca2+ gradient
across the plasma membrane.
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Treatment of neutrophils with the protein synthesis inhibitor, cycloheximide (added at 10 µg/ml at t = -10 min before the addition of thapsigargin and present throughout the course of the experiment), inhibited thapsigargin-induced IL-8 production (measured at t = 4 h; 15.21 ± 2.92 ng IL-8/106 neutrophils in the absence of cycloheximide vs 1.18 ± 0.56 ng IL-8/106 neutrophils in the presence of cycloheximide, p < 0.05), indicating that protein synthesis de novo was responsible for the marked increase in IL-8 production.
Northern blot analysis of total cellular RNA isolated from neutrophils
stimulated with 100 nM thapsigargin showed an increase in mRNA for IL-8
that was detectable at the first time point (5 min), reached a maximum
at 1 h, and remained elevated at 4 h (Fig. 2), suggesting that IL-8 production
resulted from a rapid and sustained expression of IL-8 mRNA levels. As
shown in Figure 2, incubation of neutrophils in buffer alone (resting)
for 1 h caused a slight increase in IL-8 mRNA relative to
ß-actin mRNA compared with the effect observed with thapsigargin.
Densitometric analysis of the Northern blots indicated that neutrophils
treated with thapsigargin exhibited a 7- to 19-fold increase
(n = 3) in the expression of IL-8 mRNA relative to
freshly isolated control neutrophils. Neutrophils incubated in buffer
alone exhibited a 3- to 4-fold increase in IL-8 mRNA.
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Thapsigargin induced a dose-dependent (ED50 =
19.4 ± 4.6 nM) increase in IL-8 production (Fig. 3). Concentrations of thapsigargin as low
as 1 to 10 nM caused significant increases in the levels of cellular
IL-8 with very little extracellular release of IL-8. Higher levels of
thapsigargin (20–100 nM) were associated with increases in both the
cellular and extracellular IL-8. Peak IL-8 production measured at
4 h (18.5 ng/106 neutrophils) was observed with 50 to
100 nM thapsigargin.
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To determine whether the stimulatory effect of thapsigargin was
specific for IL-8, or if other inflammatory cytokines also were
up-regulated by this Ca2+-ATPase inhibitor, cells were
incubated with 50 nM of thapsigargin for 4 h, and both the
cellular and extracellular levels of several different inflammatory
cytokines were measured. The results shown in Table I indicate that the levels of another
C-X-C chemokine, GRO-, and the C-C chemokines, RANTES and MIP-1,
or other cytokines/antagonists such as IL-6 and IL-1Ra were unchanged
by incubation with thapsigargin. However, while IL-8 was the
predominant cytokine induced by thapsigargin, the levels of TNF- and
possibly IL-1ß also increased in response to thapsigargin. These
increases were to a much smaller degree (in terms of total mass) than
the increases measured in the level of IL-8. Because mononuclear cells
can produce large quantities of IL-1ß and TNF- (16), the increased
levels of these cytokines could be accounted for by the contamination
of the neutrophil preparation by as little as 1% mononuclear cells
(the estimate of contamination of our preparations).
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, it was possible
that thapsigargin-induction of IL-8 production was a consequence of
secondary stimulation by either cytokine. Several experimental
protocols were performed to examine this possibility. The first set of
studies demonstrated that incubation of neutrophils with exogenous
IL-1ß or TNF- (at concentrations 1–2 logs higher than those
produced after stimulation of neutrophils with thapsigargin) failed to
induce the IL-8 levels observed with thapsigargin (Table II). The second set of studies
demonstrated that incubation of neutrophils with IL-1ß or TNF- did
not synergize with the effect of a suboptimum dose (20 nM) of
thapsigargin on IL-8 production (Table II). Lastly, the addition of
neutralizing Abs against both IL-1ß or TNF- (at concentrations
which blocked the priming of neutrophils for enhanced FMLP-induced
O2- generation by IL-1ß and TNF-) had no effect
on thapsigargin-induced IL-8 production.
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and MIP-1ß, as well as IL-8. There
was no detectable expression in resting cells, nor increased expression
in thapsigargin-treated neutrophils, of mRNA for the chemokines,
lymphotactin, RANTES, IFN-
-inducible protein 10 (IP-10),
monocyte chemotactic protein-1 (MCP-1), and I309 (Fig. 5). In a single experiment assessing the
expression of mRNA for other cytokines, there was detectable expression
of mRNA for IL-1, IL-1ß, IL-1Ra, TNF-, and TNF-ß in resting
neutrophils. Neutrophils treated with thapsigargin exhibited increased
expression of mRNA for both IL-1 and IL-ß. There was no detectable
expression in resting neutrophils, nor increased expression in
thapsigargin-treated neutrophils, of mRNA for IL-12p35, IL-12p40, IL-6,
IL-10, IFN-, IFN-ß, lymphotoxin-ß, or TGF-ß1–3 (data not
shown).
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Addition of a low dose of thapsigargin (5–10 nM) to Indo1-loaded
neutrophils, a dose that caused a significant albeit submaximal
increase in cellular IL-8 and little IL-8 secretion, caused a small
(50–100 nM) elevation in [Ca2+]i (Fig. 6, inset). In contrast, as
shown in Figure 6, a higher concentration of thapsigargin, 20 to 100
nM, produced a gradual, biphasic rise in the
[Ca2+]i that peaked within 2 to 3 min,
dropped slightly, and then rose again to a maximum level
([Ca2+]i = 1.0 µM) by 10 min that persisted
for at least 4 h (data not shown). As shown by the
Rmax, ionomycin induced an even greater
elevation in the [Ca2+]i, indicating that
neutrophils were still capable of maintaining their Ca2+
gradient across the plasma membrane. Dose-response studies showed that
thapsigargin-induced changes in [Ca2+]i
exhibited an ED50 similar to thapsigargin-induced IL-8
production (Fig. 6 inset compared with Fig. 3
).
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Addition of 2.5 mM EGTA (a dose that negates the Ca2+
gradient across the plasma membrane) before the addition of
thapsigargin (50 nM) had no effect on the thapsigargin-induced
elevation in [Ca2+]i observed within the
first 2 min but blocked a sustained rise in
[Ca2+]i, suggesting that early changes in
[Ca2+]i resulted from the release of
intracellular Ca2+ stores (Fig. 7). Addition of EGTA at 
2 min after
thapsigargin caused a rapid drop in the
[Ca2+]i to levels observed in the presence of
EGTA added at t = 0, suggesting that later increases in
[Ca2+]i involved the influx of
Ca2+ across the plasma membrane.
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To determine whether an influx of extracellular Ca2+
induced by thapsigargin was involved in the stimulation of IL-8
production, experiments were performed in which neutrophils were
exposed to EGTA (2.5 mM) before or at various times after the addition
of thapsigargin (Table III). The addition
of EGTA (2.5 mM) before the addition of thapsigargin caused >95%
inhibition of the thapsigargin-induced increase in IL-8 production.
EGTA also caused a similar inhibition of the thapsigargin-stimulated
induction of mRNA for IL-8 (Fig. 8).
Addition of EGTA up to 2 min after the addition of thapsigargin (and
concurrent with the EGTA-insensitive elevation of
[Ca2+]i]) continued to block 95% of the
thapsigargin-induced IL-8 production (Table III). Addition of EGTA at
later times (t 5 min, and subsequent to
activation of the Ca2+ influx) extended the duration of the
Ca2+ influx and had progressively less of an inhibitory
effect, demonstrating that a sustained influx of extracellular
Ca2+ was required for maximum thapsigargin-induced IL-8
production. In addition to blocking the synthesis of IL-8, the addition
of EGTA also inhibited the secretion of IL-8 into the extracellular
fluid. When EGTA was added 120 min after the addition of
thapsigargin, a time at which EGTA no longer significantly altered the
synthesis of IL-8, the secretion of IL-8 was still significantly
inhibited (35 ± 7% of the total IL-8 in neutrophils treated with
thapsigargin alone vs 25 ± 7% of the total IL-8 in neutrophils
treated with thapsigargin plus EGTA, p < 0.01).
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). However, BAPTA-AM loading did not
significantly alter the percentage of synthesized IL-8 that was
secreted at any of the concentrations tested (data not shown).
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).
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Cyclosporin A and ascomycin are immunosuppressive drugs that bind
to specific immunophilins and interfere with the activation of the
Ca2+-, calmodulin-dependent serine/threonine phosphatase,
calcineurin, or protein phosphatase 2B (19). Preincubation of
neutrophils for 10 min with ascomycin and cyclosporin A, at
concentrations achievable therapeutically, caused significant
inhibition (IC50 values of 2.8 ± 0.1 and 54.2 ±
8.4 nM, respectively) of thapsigargin-induced IL-8 production (Fig. 10) and IL-8 mRNA synthesis (Fig. 8).
Densitometric analyses of the Northern blots indicated that cyclosporin
A and ascomycin reduced the levels of thapsigargin-induced IL-8 mRNA to
50% the level observed in the absence of the inhibitors. Rapamycin,
a member of the same family of immunosuppressants that complexes with
an immunophilin but does not interfere with the activation of
calcineurin (20), did not inhibit thapsigargin-induced IL-8 gene
expression. None of the inhibitors caused any significant alteration in
the percentage of synthesized IL-8 that was secreted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thapsigargin stimulation of IL-8 levels was evident 1 h after exposure, continued for the entire 8-h measurement period, and was blocked by cycloheximide. Northern blot analysis of total RNA isolated from peripheral blood neutrophils demonstrated that the levels of IL-8 mRNA were low in both freshly isolated neutrophils and control neutrophils incubated at 37°C for 1 h, whereas exposure to thapsigargin caused a rapid induction (within 5 min) of IL-8 mRNA that persisted for up to 4 h. Thus, thapsigargin treatment of neutrophils induced both the transcription and the subsequent translation and secretion of IL-8.
These findings are consistent with other studies demonstrating
thapsigargin induces the production of cytokines in both macrophages
and lymphocytes (12, 14). To determine whether thapsigargin selectively
induced production and release of IL-8 from neutrophils, measurement of
cellular and extracellular levels of other inflammatory cytokines were
performed. Thapsigargin increases in IL-8 were relatively specific,
with no detectable increases in the levels of GRO-, RANTES,
MIP-1, IL-6, or IL-1Ra. Small increases in the levels of IL-1ß and
TNF- were observed, but these were at much lower levels (pg of
IL-1ß or TNF-/106 cells vs ng of IL-8/106
cells) and could occur from the 1% contaminating mononuclear cells
(16). Based on studies using Abs that neutralized the activity of
IL-1ß and TNF-, we excluded the possibility that induction of IL-8
by thapsigargin was due to the stimulatory effect of these cytokines.
Although the effect of thapsigargin on the production of cytokines was
relatively specific for IL-8, notable increases were observed in the
concentrations of both TNF- and IL-1ß. However, at the level of
mRNA, RNase protection assays revealed marked increases in the mRNA
levels of MIP-1, MIP-1ß, IL-1, IL-1ß, and TNF-. These data
can be explained by two possibilities. It is possible that these other
cytokines are synthesized but at levels too low for detection.
Alternatively, it is possible that the regulatory mechanisms for
synthesis of these other cytokines differ at the translational level.
Numerous studies have demonstrated that thapsigargin induces depletion
of Ca2+ stores, elevating
[Ca2+]i. In many cell types, including
neutrophils, thapsigargin-induced depletion of Ca2+ stores
activates a Ca2+ influx pathway (9, 21, 22, 23). Because
thapsigargin can activate cells in the absence of receptor ligand
interactions by modulating Ca2+ homeostasis, it has been a
very useful tool for dissecting signal transduction pathways. Our data
confirm other studies in neutrophils showing that thapsigargin induces
an initial EGTA-insensitive [Ca2+]i rise
followed by a sustained [Ca2+]i elevation
that is blocked by EGTA (23). Addition of EGTA at various times after
exposure to thapsigargin rapidly restored
[Ca2+]i to baseline levels, indicating that
the influx of Ca2+ determined the level of
[Ca2+]i at times > 2 min. While
thapsigargin has been shown to induce a Na+ influx, as well
as a Ca2+ influx, in neutrophils (24), the observations
that either BAPTA-AM-loading of neutrophils or the simultaneous
addition of EGTA and thapsigargin inhibited IL-8 production and mRNA
synthesis indicates that the influx of Ca2+, not
Na+ or another cation, triggers IL-8 production. The data
obtained using EGTA also indicate that the release of Ca2+
from thapsigargin-sensitive intracellular stores only minimally
triggers IL-8 production (Table IV). A sustained Ca2+
influx is necessary for maximal release as shown by the experiments in
which addition of EGTA as late as 60 min after thapsigargin
caused some inhibition of the IL-8 response. Similarly, the secretion
of synthesized IL-8 was inhibited by the addition of EGTA, suggesting
that the influx of Ca2+ was necessary for not only IL-8
synthesis but its secretion as well. These findings are similar to
those in lymphocytes where a sustained elevation of
[Ca2+]i is required for the
thapsigargin-induced expression of the early immune response genes such
as IL-2, IL-3, and IL-4 (25).
In lymphocytes, thapsigargin-induced proliferation and differentiation are blocked by the immunosuppressive drug, cyclosporin A, which inhibits calcineurin, a Ca2+-activated serine/threonine phosphatase necessary for the nuclear transport of nuclear factor of activated T cells (NF-AT) (25). Increases in [Ca2+]i activate calcineurin, leading to dephosphorylation of the cytosolic form of NF-AT and its migration to the nucleus where it is involved in the regulation of cytokine gene expression. In lymphocytes, this process can occur within minutes, resulting in the rapid up-regulation of gene expression.
The data indicate that thapsigargin-induced IL-8 production in neutrophils results in the activation of a biochemical pathway that is similar to that activated in thapsigargin-treated lymphocytes. Like lymphocytes, thapsigargin-induced up-regulation of the IL-8 gene in neutrophils has a rapid (within 5 min) time course, requires a prolonged increase in [Ca2+]i levels, and is inhibited by ascomycin and cyclosporin A. These results suggest that calcineurin and NF-AT are involved in thapsigargin-induced IL-8 production in neutrophils. It is possible that inhibition of IL-8 synthesis is an integral part of the immunosuppressive action of cyclosporin A and ascomycin in vivo.
In conclusion, the production of IL-8 by peripheral blood neutrophils provides a paracrine mechanism to recruit more neutrophils into an acutely inflamed site. This mechanism can be triggered by the thapsigargin-induced release of intracellular Ca2+ stores and subsequent Ca2+ influx.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John I. Gallin, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, Bldg 10, Room 11N107, Bethesda, MD 20892-1504.
3 Abbreviations used in this paper: [Ca2+]i, intracellular Ca2+ concentration; Indo1-AM, 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester; BAPTA-AM, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; IL-1Ra, IL-1 receptor antagonist; MIP-1, macrophage inflammatory protein-1; NF-AT, nuclear factor of activated T cells; GRO-, growth-related protein .
Received for publication December 2, 1997. Accepted for publication June 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. Monteseirin, P. Chacon, A. Vega, R. El Bekay, M. Alvarez, G. Alba, M. Conde, J. Jimenez, J. A. Asturias, A. Martinez, et al. Human neutrophils synthesize IL-8 in an IgE-mediated activation J. Leukoc. Biol., September 1, 2004; 76(3): 692 - 700. [Abstract] [Full Text] [PDF]
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M. H. Tarlowe, K. B. Kannan, K. Itagaki, J. M. Adams, D. H. Livingston, and C. J. Hauser Inflammatory Chemoreceptor Cross-Talk Suppresses Leukotriene B4 Receptor 1-Mediated Neutrophil Calcium Mobilization and Chemotaxis After Trauma J. Immunol., August 15, 2003; 171(4): 2066 - 2073. [Abstract] [Full Text] [PDF]
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R. He, H. Sang, and R. D. Ye Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA4R Blood, February 15, 2003; 101(4): 1572 - 1581. [Abstract] [Full Text] [PDF]
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Y. Suzuki, C. Gomez-Guerrero, I. Shirato, O. Lopez-Franco, P. Hernandez-Vargas, G. Sanjuan, M. Ruiz-Ortega, T. Sugaya, K. Okumura, Y. Tomino, et al. Susceptibility to T Cell-Mediated Injury in Immune Complex Disease Is Linked to Local Activation of Renin-Angiotensin System: The Role of NF-AT Pathway J. Immunol., October 15, 2002; 169(8): 4136 - 4146. [Abstract] [Full Text] [PDF]
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K. Itagaki, K. B. Kannan, D. H. Livingston, E. A. Deitch, Z. Fekete, and C. J. Hauser Store-Operated Calcium Entry in Human Neutrophils Reflects Multiple Contributions from Independently Regulated Pathways J. Immunol., April 15, 2002; 168(8): 4063 - 4069. [Abstract] [Full Text] [PDF]
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L. Del Rio, S. Bennouna, J. Salinas, and E. Y. Denkers CXCR2 Deficiency Confers Impaired Neutrophil Recruitment and Increased Susceptibility During Toxoplasma gondii Infection J. Immunol., December 1, 2001; 167(11): 6503 - 6509. [Abstract] [Full Text] [PDF]
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D. B. Kuhns, E. L. Nelson, W. G. Alvord, and J. I. Gallin Fibrinogen Induces IL-8 Synthesis in Human Neutrophils Stimulated with Formyl-Methionyl-Leucyl-Phenylalanine or Leukotriene B4 J. Immunol., September 1, 2001; 167(5): 2869 - 2878. [Abstract] [Full Text] [PDF]
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X. Zhang, J. M. Wang, W. H. Gong, N. Mukaida, and H. A. Young Differential Regulation of Chemokine Gene Expression by 15-Deoxy-{{Delta}}12,1412,14 Prostaglandin J2 J. Immunol., June 15, 2001; 166(12): 7104 - 7111. [Abstract] [Full Text] [PDF]
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J. P. Zoeteweij, A. V. Moses, A. S. Rinderknecht, D. A. Davis, W. W. Overwijk, R. Yarchoan, J. M. Orenstein, and A. Blauvelt Targeted inhibition of calcineurin signaling blocks calcium-dependent reactivation of Kaposi sarcoma-associated herpesvirus Blood, April 15, 2001; 97(8): 2374 - 2380. [Abstract] [Full Text] [PDF]
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M. Geiszt, A. Kapus, and E. Ligeti Chronic granulomatous disease: more than the lack of superoxide? J. Leukoc. Biol., February 1, 2001; 69(2): 191 - 196. [Abstract] [Full Text]
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S.-Y. Choi, H. Ha, and K.-T. Kim Capsaicin Inhibits Platelet-Activating Factor-Induced Cytosolic Ca2+ Rise and Superoxide Production J. Immunol., October 1, 2000; 165(7): 3992 - 3998. [Abstract] [Full Text] [PDF]
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K. K. Jefferson, M. F. Smith Jr., and D. A. Bobak Roles of Intracellular Calcium and NF-{kappa}B in the Clostridium difficile Toxin A-Induced Up-Regulation and Secretion of IL-8 from Human Monocytes J. Immunol., November 15, 1999; 163(10): 5183 - 5191. [Abstract] [Full Text] [PDF]
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L. Liu, P. Ridefelt, L. Hakansson, and P. Venge Regulation of Human Eosinophil Migration Across Lung Epithelial Monolayers by Distinct Calcium Signaling Mechanisms in the Two Cell Types J. Immunol., November 15, 1999; 163(10): 5649 - 5655. [Abstract] [Full Text] [PDF]
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G. K. Koski, G. N. Schwartz, D. E. Weng, B. J. Czerniecki, C. Carter, R. E. Gress, and P. A. Cohen Calcium Mobilization in Human Myeloid Cells Results in Acquisition of Individual Dendritic Cell-Like Characteristics Through Discrete Signaling Pathways J. Immunol., July 1, 1999; 163(1): 82 - 92. [Abstract] [Full Text] [PDF]
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 J. A. Lekstrom-Himes, S. E. Dorman, P. Kopar, S. M. Holland, and J. I. Gallin Neutrophil-specific Granule Deficiency Results from a Novel Mutation with Loss of Function of the Transcription Factor CCAAT/Enhancer Binding Protein {varepsilon} J. Exp. Med., June 7, 1999; 189(11): 1847 - 1852. [Abstract] [Full Text] [PDF]
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), and the total IL-8 content of both compartments (
) from
thapsigargin-treated cells. Resting cells (
) were incubated for
8 h in the absence of thapsigargin. The data from resting cells
represent the total IL-8 content. The data represent the mean ±
SD of three experiments. The levels of significance (ANOVA) represent
comparison to the levels of IL-8 observed at t = 0.
*, p < 0.05; **, p <
0.001
