Chemotactic Migration Triggers IL-8
Generation in Neutrophilic Leukocytes
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J Immunol 1999; 162:1077-1083; ;
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References
Rafat A. Siddiqui, Luke P. Akard, J. G. N. Garcia, Yi Cui
and Denis English
Chemotactic Migration Triggers IL-8 Generation in
Neutrophilic Leukocytes1
Rafat A. Siddiqui,*† Luke P. Akard,* J. G. N. Garcia,‡ Yi Cui,* and Denis English2*§
N
eutrophils activated by various metabolic agonists, including opsonized zymosan, PMA, LPS, granulocytemacrophage-CSF, and TNF-a, synthesize and release
several cytokines including TNF, IL-1, IL-6, IL-8, and IL-12 (1–
6). These factors are important regulators of the inflammatory response. In particular, IL-8 is a potent chemoattractant that promotes the mobilization of neutrophils into sites of infection and
inflammation. Recent studies have demonstrated that, in some instances, protein synthesis rather than release of stored material
accounts for the enhanced secretion of IL-8 by activated neutrophils (7). Activated neutrophils potentially modulate IL-8 synthesis at either the level of mRNA transcription or translation. Northern blot analysis revealed increased expression of IL-8 mRNA
upon stimulation of neutrophils with certain cytokines (8, 9), LPS
(9, 10), culture supernatants (11), and selectin ligands (12). Transcriptional regulation of IL-8 has also been shown in several other
systems, including epithelial cells infected with Helicobacter pylori (13) or Pseudomonas aeroginosa (14) and monocytes responding to cytokines (15). In addition to transcription, IL-8 generation during inflammation may also be regulated at the level of
mRNA translation. Kuhns and Gallin found that IL-8 production in
ionophore-stimulated neutrophils was partially suppressed by cycloheximide, a protein synthesis inhibitor (7). Under similar con†
*Experimental Cell Research Program, Methodist Research Institute, and Department of Biology, Indiana University/Purdue University, Indianapolis, IN 46201; and
Departments of ‡Medicine and §Allied Health Sciences, Indiana University School of
Medicine, Indianapolis, IN 46202
Received for publication April 27, 1998. Accepted for publication September
30, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by project 3 of National Institutes of Health Grant 1 PO1
HL 58064-01, by a grant from the Indiana affiliate of the American Heart Association,
and by a grant from the Phi Beta Psi Sorority.
2
Address correspondence and reprint requests to Dr. Denis English, Experimental
Cell Research Program, The Methodist Research Institute, 1701 North Senate, Indianapolis, IN 46201.
Copyright © 1999 by The American Association of Immunologists
ditions, the RNA synthesis inhibitor actinomycin-D had little effect, suggesting that the synthesis of IL-8 was under translational
rather than transcriptional control.
After migration to sites of infection and inflammation, neutrophils putatively release enhanced amounts of IL-8 and other cytokines in response to metabolic stimulation. At these sites, cytokines secreted by chemotactic neutrophils exert potent effects on
many aspects of the inflammatory response (16 –18). However, the
ability of chemotactically responsive cells to release IL-8 upon
further stimulation has not, to our knowledge, been directly tested.
In preliminary experiments, we observed that neutrophils recovered after chemotactic migration not only displayed a remarkably
enhanced ability to release IL-8 upon metabolic stimulation but
also possessed markedly elevated levels of IL-8 in comparison
with nonmotile cells that were exposed directly to chemoattractants, suggesting that IL-8 synthesis may have occurred during
chemotactic migration. The present investigation was undertaken
to explore this possibility. The results indicate that chemotactic
migration induces IL-8 synthesis by activating a cellular process
that results in enhanced translation of mRNA present in resting
cells.
Materials and Methods
Human IL-8, ELISA IL-8 assay kits, and monoclonal anti-IL-8 Abs were
obtained from R&D systems (Minneapolis, MN). Platelet-activating factor
(PAF)3 was obtained from Calbiochem (San Diego, CA). L-[4,5-3H]leucine
(140 Ci/mmol) was from New England Nuclear (Boston, MA). Tissue
solubilizer NSC-II was from Amersham Canada (London, Ontario, Canada). Precast gels and reagents for SDS gel electrophoresis were from BioRad (Hercules, CA). Endothelial cell culture reagents were from Cell Systems (Kirkland, WA). Protein A/G plus beads were from Oncogene
Science (Cambridge, MA). Dulbecco’s minimal essential media (DMEM)
was from Grand Island Biological (Grand Island, MI). Zymosan, FMLP,
recombinant C5a, PMA, casein, DMSO, HBSS, cytochalasin-D, actinomycin-D, and cycloheximide were obtained from Sigma (St. Louis, MO).
Cycloheximide was dissolved in distilled water at a concentration of 1
3
Abbreviations used in this paper: PAF, platelet-activating factor; DMEM, Dulbecco’s minimal essential media; ZAS, zymosan-activated serum.
0022-1767/99/$02.00
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Neutrophils recovered from inflammatory exudates possess increased levels of IL-8, but exposure of neutrophils to chemoattractants results in only a modest stimulation of IL-8 generation. This study was undertaken to explore the hypothesis that IL-8
generation in these cells is dependent upon the process of migration. Neutrophils synthesized up to 30 times as much IL-8 during
migration in response to a gradient of diverse chemoattractants than they did when stimulated directly by the attractants in the
absence of a gradient. This IL-8 response was dependent on migration since it was not observed in cells exposed to concentration
gradients of chemoattractants under conditions that prevented cell movement. While actinomycin-D (1 mg/ml) had little influence
on the generation of IL-8 during chemotaxis, the protein synthesis inhibitor cycloheximide (10 mg/ml) markedly blunted the
accumulation of cell-associated IL-8, suggesting that new protein synthesis from preexisting mRNA was responsible for the effect.
Consistent with this interpretation, migrating cells incorporated over 10 times as much [3H]leucine into IL-8 as did nonmotile
neutrophils exposed to chemoattractants. A substantial portion of the IL-8 generated during chemotaxis was released upon
subsequent metabolic stimulation. Thus, the IL-8 synthesized during chemotaxis is uniquely positioned to exert a regulatory
influence on inflammatory processes governed by neutrophilic leukocytes responding to inflammatory and infectious stimuli. The
Journal of Immunology, 1999, 162: 1077–1083.
1078
CHEMOTACTIC INDUCTION OF IL-8 SYNTHESIS
mg/ml. Actinomycin-D was dissolved in ethanol at a concentration of 100
mg/ml. Cytochalasin-D and PMA were dissolved in DMSO at a concentration of 1 mg/ml. Chemoattractant solutions were prepared as described
below.
Isolation of neutrophils
Neutrophils were isolated from the blood of healthy donors using FicollHypaque density gradient centrifugation as previously described (19). After erythrocyte sedimentation at room temperature, buffy coats were layered on a 3-ml cushion of Ficoll-Hypaque and centrifuged at 800 3 g for
20 min. The pellets were washed once with HBSS, and contaminating
erythrocytes were removed by isotonic ammonium chloride lysis. Neutrophils were washed and suspended at a final concentration of 1.0 3 107/ml
in PBS unless otherwise indicated.
Preparation of chemoattractants
Endothelial cell culture
Bovine pulmonary aortic endothelial cells were cultured as described previously (21). In brief, bovine pulmonary aortic endothelial cells were
grown in DMEM containing 100 mM nonessential amino acids, 2 mM
glutamine, 20% colostrum-free bovine serum, 20 mg/ml penicillin, 20
mg/ml streptomycin, 0.05% mg/ml fungisone, 0.2 ml of sodium heparin/
100 ml, and 2 mg/ml endothelial cell growth supplement under a humidified atmosphere of 95% air and 5% CO2 in gelatin-coated flasks. Between
their 16th and 22nd passage, the cells were trypsinized and dislodged.
Single cell suspensions were then prepared in DMEM. These suspensions
were seeded onto collagen-treated 3-mm pore size polycarbonate filters at
the base of inserts of 24-mm Transwell chambers (Costar, Cambridge,
MA) at an average density of 104 cells/well, as previously described (21).
The inserts were returned to wells containing 0.5 ml of endothelial cell
growth media and cultured until confluent growth was attained (3–5 days),
at which time the inserts were rinsed and used for chemotaxis assays as
described below.
Chemotaxis assay
Chemotaxis was performed using 24-mm Transwell chambers (Costar),
wherein the chemoattractant was separated from cells within replaceable
inserts by either an untreated or endothelialized 3-mm pore size polycarbonate membrane. To expose neutrophils to chemoattractant gradients under conditions that physically limited migration, a 0.4-mm pore size filter,
through which no migration was observed, was used. Preliminary experiments revealed that the smaller pore size did not markedly restrict the
diffusion of macromolecules from lower to upper compartments of chemotaxis chambers, as assessed with blue dextran (Fig. 1). While the pore
size of these filters is not small enough to impede the transfer of even large
molecules, the pore density of the 0.4-mm filters is markedly greater than
that of the 3.0-mm filters, as assessed microscopically. Thus, cells in the
upper compartments of chambers with either 3.0- or 0.4-mm filters were
exposed to a continuously increasing concentration gradient of chemoattractant at approximately the same rate. In experiments designed to examine neutrophil responses during transendothelial migration, an endothelial
monolayer was grown on the filters as described above. Chemoattractants
were deposited in lower compartments in a final volume of 1.5 ml and
prewarmed to 37°C. After warming, 1 3 107 neutrophils in 1.0 ml of HBSS
were aliquoted into the detachable inserts, which were placed over the
chemoattractant solutions. Loaded chambers were incubated for 90 min at
37°C in a humidified atmosphere of 5% CO2 in air. At the end of incubation, the cells that migrated into the bottom chambers were dislodged by
gentle scraping, harvested, and enumerated electronically. In some experiments, cells and media from upper chambers were recovered after incubation and processed for IL-8 analysis. Suspensions of recovered cells were
cooled on melting ice and pelleted by centrifugation at 500 3 g for 5 min.
Supernatants were recovered and saved for assay of IL-8 levels. Washed
FIGURE 1. Kinetics of diffusion of macromolecules from lower to upper compartments of chambers with 3.0- and 0.4-mm pore size filters.
Lower compartments of 12-mm diameter Transwell chambers were loaded
with 0.9 ml of blue dextran (m.w. 5000, 20 mg/ml, in HBSS; Sigma),
inserts were positioned, and upper compartments were filled with 0.2 ml of
HBSS. At various times thereafter, upper compartments were aspirated,
and the blue dextran content within them was measured by adsorption at
595 nm to assess the rate of diffusion of macromolecules from lower to
upper compartments. The results of one experiment that was repeated twice
are shown. Each data point reflects OD values assessed from an individual
chamber. The OD of the blue dextran solution introduced into lower compartments was 7.2.
pellets were lysed with 0.2% Triton X-100 on ice for 10 min and centrifuged, and the supernatants were stored at 270°C for further analysis.
IL-8 assay
Quantitation of IL-8 was performed using a commercially available ELISA
kit (R&D Systems) that employs IL-8 Ab-coated 96-well plastic microtiter
plates. After incubation with recovered extracts or IL-8 standards, the wells
were washed three times and then incubated with a peroxidase-conjugated
anti-IL-8 Ab to ligate IL-8 bound to the fixed Ab. After washing, a peroxidase substrate (tetramethyl benzidine) and hydrogen peroxide were
added, and oxidation of the substrate was monitored spectrophotometrically. This method was capable of detecting IL-8 levels as low as 100
pg/ml (10 pg/106 cells), and linear responses were detected between 1 ng
and 10 mg IL-8/ml. Where indicated, cells were preincubated for 10 min at
37°C with 10 mg/ml cytochalasin-D, 1 mg/ml actinomycin-D, or 10 mg/ml
cycloheximide before chemotactic stimulation.
Measurement of IL-8 synthesis
Neutrophils were labeled with L-[4,5-3H]leucine (100 mCi/ml) for 3 h at
37°C in a humidified 5% CO2 and 95% air in HBSS containing 10% dialyzed FCS. After incubation, cells were washed extensively with warm
(37°C) HBSS and used in chemotaxis chambers as described above. After
migration, cells were recovered and lysed with 0.2% Triton X-100. Lysates
from 106 cells were spiked with 5 mg of nonlabeled IL-8 to facilitate
identification and recovery of labeled IL-8 after immunoprecipitation and
gel electrophoresis. Extracts (1 ml) were incubated with 5–10 mg monoclonal anti-IL-8 overnight at 4°C and subsequently precipitated with 30 ml
of protein A/G Plus beads for 2 h at room temperature. Beads were centrifuged and washed three times with RIPA buffer (20 mm HEPES, 150
mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS,
pH 7.4), and the resulting pellets were boiled with 20 ml of 23 sample
buffer for 5 min. Supernatants obtained after boiling were then separated on
a 5–20% gradient polyacrylamide gel. After staining with silver nitrate,
IL-8 bands were identified, excised, and solubilized in NSC-II. Solubilized
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Zymosan-activated serum (ZAS) was prepared by incubating 15 mg of
zymosan in 1 ml of fresh serum for 1 h at 37°C as described (20). After
incubation, the reaction was heat inactivated at 56°C for 30 min, and the
supernatant was collected after centrifugation at 500 3 g for 15 min at 4°C.
The activated serum was stored at 270°C until use. Casein (5 mg/ml) was
dissolved in HBSS heated at 56°C for 10 min. The solution was then cooled
at 4°C and centrifuged to remove undissolved particles. The solution was
stored at 220°C until used. PAF was dissolved in ethanol at a final concentration of 2 mM and stored at 220°C. Stock solutions of FMLP (10
mM) were prepared in DMSO and stored at 270°C. Stock solutions of
recombinant C5a (10 mM) were prepared in HBSS containing 1% BSA and
stored at 270°C until use.
The Journal of Immunology
1079
Table I. Levels of IL-8 in chemotactic- and chemoattractant-treated neutrophils
IL-8 Response of
Chemoattractant-stimulated cellsb
Chemotactic cellsc
Chemoattractant
Chemotaxisa
(cells 3 106)
Cell associated (pg/106 cells)
Released
Cell associated (pg/106 cells)
Released
None
ZAS
PAF (5 nM)
FMLP (10 nM)
C5a (2.9 3 1028 M)
Casein (0.5 mg/ml)
,0.1
4.55 6 1.1
2.40 6 0.7
4.40 6 1.1
4.50 6 0.9
5.30 6 0.9
51 6 7.2d
648 6 40
143 6 18
120 6 12
160 6 15
560 6 32
NDe
ND
ND
ND
ND
ND
—
6777 6 363
903 6 102
3918 6 214
2302 6 220
5608 6 353
—
1989 6 120
ND
ND
620 6 47
1465 6 217
a
The number of responding cells was determined by electronic particle analysis of an aliquot of lower chamber fluid.
Neutrophils were stimulated by direct addition of chemoattractants in the absence of a chemotactic gradient for 90 min at 37°C.
c
Cells were recovered after chemotactic migration through 3-mm filters. Released activity reflects levels found in lower compartment fluids after removal of cells
by centrifugation and corrected for cell concentration. Cell-associated levels reflect levels recovered in washed cells.
d
Value represents IL-8 levels observed in resting cells (mean and SD of three experiments).
e
ND, not detectable (,10 pg/106 cells).
b
from upper chambers after migration did not possess similarly increased levels of IL-8 (not shown).
Results
Involvement of migration in the IL-8 response
Effect of chemoattractants on IL-8 synthesis
The results of the experiments described above indicate that exposure of neutrophils to gradients of chemoattractants potentiates
IL-8 synthesis to an extent that is not observed in cells exposed to
attractants in the absence of a gradient. While exposure of cells to
chemoattractant gradients may directly induce IL-8 synthesis, the
response may be secondary to processes activated during migration induced by these gradients. Experiments were designed to
explore the actual role of chemotactic migration in induction of the
IL-8 response. In the first approach, we employed a system to
expose cells to a gradient of chemoattractant under conditions that
prevented migration by limiting the pore size of the membrane
within chemotaxis chambers to 0.4 mm (see Fig. 1). As shown in
Table II, cells exposed to chemoattractant gradients produced
higher levels of IL-8 than did cells directly exposed to chemoattractants, whether or not migration was allowed to proceed. However, cells recovered after migration displayed markedly higher
levels of IL-8 than did cells exposed to chemoattractant gradients
under conditions in which migration was prevented.
In a second approach, we examined the influence of the actin
polymerization inhibitor cytochalasin-D (22) on chemoattractantinduced IL-8 synthesis. Preliminary experiments demonstrated
Several chemoattractants including ZAS, PAF, FMLP, C5a, and
casein were characterized for their ability to induce IL-8 synthesis
in neutrophils. With the exception of PAF, each of these chemoattractants induced a potent chemotactic response resulting in the
migration of 44 –53% of upper chamber cells into the lower compartments. The response induced by PAF, which attracted 24% of
upper chamber cells, was lower than that induced by other chemoattractants but still markedly greater than that observed in
chambers without chemoattractant, wherein less than 1% of the
cells deposited in the upper chambers were recovered in lower
compartments (Table I).
When added to neutrophils in the absence of a chemotactic gradient, each of the chemoattractants used induced a moderate increase in cell-associated IL-8; levels of released IL-8 remained
undetectable (Table I). The chemoattractants that induced the
strongest migratory responses, ZAS and casein, induced the highest increase in cell-associated IL-8, increasing constitutive levels
over 10-fold. With other chemoattractants, migratory responses,
and IL-8 synthesis did not appear to correlate. Thus, PAF, FMLP,
and C5a each induced a similar increase in IL-8 levels over that
found in unstimulated cells but varied considerably in chemotactic
potency (Table I). Lower levels of chemoattractants (1:10, 1:100,
and 1:1000 dilutions of levels of ZAS, C5a, and FMLP reported in
Table I) did not result in marked increase in the IL-8 response (not
shown).
Neutrophils recovered after migration to each of the chemoattractants were found to possess far greater levels of IL-8 than levels observed in cells exposed directly to chemoattractants. ZAS
and casein were again the most potent attractants in this respect;
levels of IL-8 in cells responding to these attractants were over 100
times greater than levels found in unstimulated cells and 10- to
30-fold higher than levels found in cells stimulated with chemoattractants in the absence of a gradient. Cells migrating in response
to these chemoattractants released a small portion of this increased
IL-8. Cells that migrated in response to other chemoattractants
(PAF, FMLP, C5a) also possessed levels of IL-8 that were markedly greater than those of cells that were directly stimulated with
these chemoattractants but did not secrete detectable levels of IL-8
into the extracellular medium during chemotaxis. Cells retrieved
Table II. Requirement of migration for the chemoattractant-induced
IL-8 response
IL-8 Response
Treatment Conditionsa
Cell associated
(pg/106 cells)
Released
No gradient
Gradient/permissive conditions
Gradient/migration physically limited
Gradient/migration biochemically limited
436 6 76
4327 6 357
1881 6 97
706 6 95
ND
1328 6 86
529 6 28
494 6 43
a
Cells were exposed either to a single concentration of chemoattractant (10%
ZAS) in the absence of a gradient or to a concentration gradient of chemoattractants
under either permissive conditions or conditions that physically or biochemically
limited migration. Physical limitation of migration was effected by employing a
0.4-mm pore size filter in chemotaxis chambers. Biochemical inhibition was effected
by addition of the actin polymerization inhibitor cytochalasin-D (10 mg/ml). IL-8
analyses were carried out using cells recovered from lower chambers when permissive conditions were used and from upper chamber under restrictive conditions. IL-8
responses in recovered cells were assessed as described in Table I.
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samples were then mixed with 10 ml of scintillation fluid, and the samples
were assayed for radioactivity in a liquid scintillation counter.
1080
CHEMOTACTIC INDUCTION OF IL-8 SYNTHESIS
Table III. Effect of cytochalasin-D on chemoattractant-stimulated IL-8 generationa
Chemoattractant
ZAS
FMLP
C5a
Treatment
Cell associated
Released
Cell associated
Released
Cell associated
Released
Untreated
Cytochalasin-D
281 6 8
324 6 6
140 6 8
130 6 10
62 6 18
174 6 12
50 6 3
74 6 5
255 6 4
213 6 20
21 6 10
55 6 18
a
Untreated or cytochalasin-D (10 mg/ml)-treated neutrophils were stimulated by direct addition of chemoattractants for 10
min at 37°C and assayed for IL-8 generation as described in the text. Values (pg/106 cells) reflect levels above those observed
in unstimulated cells (mean 6 SD of three determinations). Chemoattractant concentrations were the same as those used for the
experiments described in Table I.
Effect of RNA and protein synthesis inhibitors on the
chemotaxis-dependent IL-8 response
The studies described above demonstrate that chemotactic neutrophils possess enhanced quantities of IL-8 as a result of processes
activated during migration. However, the origin of this IL-8 is not
clear. To investigate the origin of chemotaxis-induced IL-8, we
examined IL-8 levels of cells induced to migrate in the presence of
the protein and RNA synthesis inhibitors cycloheximide and actinomycin-D, respectively. Actinomycin-D (1 mg/ml) had little effect on IL-8 increases associated with chemotactic migration (Fig.
2). In contrast, cycloheximide (10 mg/ml) markedly attenuated increases in both cell-associated and released IL-8 during chemotactic migration. These observations are consistent with the proposal that increases in IL-8 levels observed in migrating
neutrophils result from increased protein synthesis directed by preexisting mRNA.
the surrounding medium. Exposure of neutrophils to an endothelial
cell monolayer in the absence of chemoattractants did not result in
enhanced levels of neutrophil IL-8. The amount of IL-8 released
by endothelial cells cultured on inserts and exposed to 10% ZAS
in either the absence or the presence of neutrophils (1.0 3 107 cells
in 1.0 ml) was below the limits of detection of the assay used.
Thus, the elevated IL-8 levels observed in neutrophils migrating
through the endothelial monolayer could not be attributed to uptake of IL-8 released from stimulated endothelial cells.
Release of IL-8 by metabolic stimulation of chemotactic
neutrophils
Finally, we examined the ability of neutrophils to release the IL-8
that was generated during chemotaxis in response to metabolic
stimulation. Cells were recovered from the lower compartment of
chemotaxis chambers after migration induced by 10% ZAS,
washed, and exposed to 100 nM PMA for 30 min at 37°C. Results
were compared with those obtained with neutrophils from the
same preparation that were not subject to chemotactic migration or
chemoattractant exposure. Upon exposure to PMA, chemotactic
cells released 3132 6 156 pg IL-8/106 cells while control cells
IL-8 synthesis in migrating neutrophils
To demonstrate the de novo synthesis of IL-8 during chemotactic
migration, we employed neutrophils preincubated with L-[4,53
H)]leucine as described under Materials and Methods. The results
shown in Fig. 3 demonstrate that chemotactic migration resulted in
over a 10-fold increase in [3H]leucine incorporation into IL-8 as
compared with cells treated with chemoattractant directly. Furthermore, when cells were treated with cycloheximide (10 mg/ml), the
incorporation of L-[4,5-3H)]leucine into IL-8 during migration was
reduced by 40%.
IL-8 synthesis by neutrophils during transendothelial migration
To evaluate chemotaxis-dependent IL-8 synthesis in a physiologically relevant system, we examined cells after migration through
endothelial monolayers. The results demonstrated that migration of
neutrophils through endothelial monolayer results in markedly potentiated increases in cell-associated IL-8. In these experiments,
neutrophils that migrated through endothelial monolayers were
found to possess 6989 6 1250 pg of IL-8/106 cells. During migration, these cells released 2050 6 125 pg of IL-8/106 cells into
FIGURE 2. Effects of cycloheximide and actinomycin-D on the chemoattractant-induced IL-8 response. Neutrophils were incubated with either cycloheximide (10 mg/ml) or actinomycin-D (1 mg/ml) or vehicle
before introduction into upper compartments of chemotaxis chambers containing 10% ZAS in lower compartments. Chemotaxis was allowed to proceed for 90 min, and responsive cells were recovered from lower chambers.
Concentrations of IL-8 were measured by ELISA as described in the legend of Table I. Results are the mean 6 SD of three experiments.
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that, under the conditions used, cytochalasin-D did not inhibit IL-8
generation in neutrophils exposed to a single stimulatory concentration of chemoattractant (Table III). As shown in Table II, cytochalasin-D treatment markedly attenuated IL-8 synthesis induced by exposure of neutrophils to gradients of chemoattractants
within chemotaxis chambers. In the presence of cytochalasin-D,
IL-8 levels induced by chemotactic gradients were similar to those
induced in neutrophils by direct exposure to chemoattractants in
the absence of gradients. These results are consistent with the
possibility that the IL-8 response in neutrophils exposed to chemoattractant gradients depends, in large part, upon actual cellular migration.
The Journal of Immunology
released 31.7 6 1.6 pg IL-8/106 cells. Since the amount of IL-8
released after chemotaxis is much greater than the total amount of
IL-8 observed in cells exposed to chemoattractants directly (see
Table I), the IL-8 generated during chemotaxis accounts for the
increased ability of chemotactic cells to release the cytokine upon
metabolic stimulation.
Discussion
Neutrophil IL-8 generation is a key component of the inflammatory response (16, 17). Released from migrating cells, IL-8 is a
potent autokine that possesses the ability to attract other leukocytes
and potentiate cellular function. The IL-8 that is retained by the
neutrophil presumably exerts its effects later, after the cell has
reached its target. In addition, IL-8 released by damaged neutrophils may exert important effects at the site of inflammatory reactivity, possibly acting in concert with lipids and other protein mediators confined to membrane fragments of disrupted cells. How
the neutrophil is prompted to generate IL-8 along its path to the
inflammatory sites was the focus of the present investigation.
IL-8 synthesis by metabolically stimulated neutrophils has been
well characterized (1–12, 16 –18, 23, 24), and phagocytosing cells
release enhanced levels of the cytokine (25, 26). Since cells recovered from inflammatory exudates possess elevated levels of
IL-8 (7), we examined the IL-8 response of chemotactic neutrophils. Not surprisingly, we observed that neutrophils recovered
from lower compartments after chemotactic migration displayed a
remarkably enhanced capacity to release IL-8 upon metabolic
stimulation. Chemotactic cells released far greater levels of IL-8
than resting cells possessed. In addition, the extent of IL-8 release
by these cells could not be accounted for by direct chemoattractant-stimulated IL-8 synthesis. These considerations led us to explore the possibility that IL-8 was generated in neutrophils during
chemotaxis as a result of cellular processes dependent upon or
activated by chemotactic migration. The experiments presented in
this report demonstrate that this clearly is the case; chemotaxis per
se is an effective and potent stimulus of neutrophil IL-8 generation.
Neutrophils migrating in response to a gradient of chemoattractant
consistently displayed increased levels of cell-associated IL-8.
While some of this IL-8 was released into the surrounding media,
the majority was retained by the cells. A number of different chemoattractants that are known to activate widely divergent signaling
pathways (27) induced a similar IL-8 response, suggesting that the
response depended more on the functional consequence of stimulation than on the manner by which that response was triggered. As
noted above, cells exposed directly to chemoattractants in the absence of a gradient displayed a markedly lower IL-8 response than
that observed in migrating cells. In addition, physical limitation of
migration markedly limited the IL-8 response of cells exposed to
concentration gradients of chemoattractants. Finally, no response
was observed in cells exposed to gradients of chemoattractants in
the presence of the inhibitor of actin polymerization, cytochalasin-D, used at levels that effectively prevented migration. While
cytochalasin-D may have effects on cells that potentially influence
their reactivity independently of the disruption of actin polymerization, it did not inhibit IL-8 generation in cells exposed to a
single concentration of chemoattractant (Table III). Thus, IL-8treated cells retain the machinery necessary to generated IL-8 in
response to chemoattractant stimulation. Therefore, it certainly appears that migration itself rather than a biochemical process activated by either chemoattractants or concentration gradients of chemoattractants (as discussed in Refs. 28 and 29) was necessary for
optimal expression of the response documented in this report. Neutrophils arriving at sites of infection and inflammation may be
expected to possess enhanced levels of IL-8 not as a result of
enhanced metabolic reactivity after migration but rather as a result
of cellular process activated by migration.
Although cellular migration apparently played a major role in
inducing IL-8 synthesis, we cannot entirely discount the possibility
that migration facilitated a response that was triggered by a more
traditional mechanism. Many cells, including neutrophils, are encumbered by an “adaptation” response that continuously resets the
magnitude of responses to certain stimuli, including chemoattractants (see Ref. 28 and references therein). In some noteworthy
instances, the rate at which the cell resets is such that no response
is initiated by small rates of stimulus increase; at higher rates of
stimulus increase, the cell will respond maximally. Our data (Fig.
1) demonstrate that nonmigrating neutrophils in upper compartments of chemotaxis chambers are exposed to a linear increase in
chemoattractant levels. It is possible that such a linear increase is
not sufficient to trigger an IL-8 response. However, when the cells
are migrating through the filter toward the chemoattractant, the rate
of increase of attractant to which they are exposed would increase
appreciably, probably in an exponential manner. Such an exponential rate of increase may be sufficient to trigger the maximal
IL-8 response. If this is the explanation for the results observed, it
should be possible to initiate with chemoattractants a maximal response in static cells by ramping the stimulus concentration exponentially. Experiments are underway to determine whether this interesting hypothesis provides the basis for our dramatic results.
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FIGURE 3. IL-8 synthesis during chemotactic migration. Neutrophils
were labeled with L-[4,5-3H]leucine (100 mCi/ml) for 3 h at 37°C in a
humidified 5% C02 and 95% air incubator in HBSS containing 10% dialyzed FCS. After incubation, cells were washed extensively with warmed
(37°C) HBSS. Washed cells were exposed directly to chemoattractant
(chemoattractant-treated cells) or recovered after chemotaxis induced by
10% ZAS (chemotactic cells) as described in Table I. Where indicated,
chemotaxis was conducted in the presence of cycloheximide (10 mg/ml).
Recovered cells were lysed with 0.2% Triton X-100 and immunoprecipitated with monoclonal anti IL-8 Ab, and the immunoprecipitates were separated on a 5–20% gradient polyacrylamide gel. The bands corresponding
to authentic IL-8 were localized, excised, and assayed for radioactivity.
Results reflect mean and SD of three determinations.
1081
1082
rupted after chemotaxis. In addition, cells with enhanced levels of
IL-8 may possess heightened functional reactivity as a result of
endogenous cytokine or in response to low but continuous levels of
released cytokine. Finally, intracellular IL-8 potentially serves a
paracrine-signaling function when intact but apoptotic neutrophils
are ingested by macrophages.
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While heterogeneity in neutrophil responses to chemoattractants
may have influenced our results, we do not believe neutrophil subpopulations can explain the dramatic increase in IL-8 levels observed in cells that have migrated. Indeed, there are well-documented precedents for heterogeneous responses of neutrophil
subpopulations to chemoattractants and other metabolic stimuli
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in a subpopulation that displays a very brisk IL-8 response to chemoattractants, independent of cell migration. However, it should
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The nature of the processes activated during migration that lead
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Thus, while the protein synthesis inhibitor cycloheximide markedly attenuated the response, the RNA synthesis inhibitor actinomycin-D had little effect. In addition, it is clear that de novo synthesis of IL-8 contributed to the response, since radiolabeled amino
acids were incorporated into the parent molecule during migration.
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CHEMOTACTIC INDUCTION OF IL-8 SYNTHESIS
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