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(MIG), IFN-Inducible T Cell 
Chemoattractant (I-TAC), and IFN--Inducible Protein-10 (IP-10) Chemokines by Human Neutrophils1
Departments of
*
General Pathology and
Pediatric Clinic, University of Verona, Verona, Italy;
Molecular Biology and Cell Biology Department, Human Genome Sciences, Inc., Rockville, MD 20850; and
§
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(MIG), IFN-inducible T cell 
chemoattractant (I-TAC), and IFN--inducible protein of 10 kDa
(IP-10) are related members of the CXC chemokine subfamily that bind to
a common receptor, CXCR3, and that are produced by different cell types
in response to IFN-. We have recently reported that human
polymorphonuclear neutrophils (PMN) have the capacity to release IP-10.
Herein, we show that PMN also have the ability to produce MIG and to
express I-TAC mRNA in response to IFN- in combination with either
TNF- or LPS. While IFN-, alone or in association with agonists
such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage
(GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression,
IFN-, in combination with TNF-, LPS, or IL-1ß, resulted in a
considerable induction of IP-10 release by neutrophils. Furthermore,
IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10,
and I-TAC mRNA and the extracellular production of MIG and IP-10 in
neutrophils stimulated with IFN- plus either LPS or TNF-.
Finally, supernatants harvested from stimulated PMN induced migration
and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes;
these activities were significantly reduced by neutralizing
anti-MIG and anti-IP-10 Abs, suggesting that they were mediated
by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and
I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the
ability of neutrophils to produce these chemokines might contribute not
only to the progression and evolution of the inflammatory response, but
also to the regulation of the immune response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein of 10 kDa (IP-10), monokine induced
by IFN- (MIG), and the recently cloned IFN-inducible T cell
chemoattractant (I-TAC) (4) lack the ELR sequence and fail to attract
polymorphonuclear neutrophils (PMN) (1, 2, 3, 4, 5). Interestingly, based on
their structure and function, MIG, IP-10, and I-TAC constitute a group
of three related chemokines that all act upon T lymphocytes (4, 5).
While the receptors for the C-X-C chemokines containing the ELR motif
are expressed on different types of leukocytes, there is thus far only
a single receptor, CXCR3, that is known to bind MIG, I-TAC, and IP-10
(4, 5, 6). Importantly, CXCR3 (and CCR5) seem to be preferentially
expressed on activated T lymphocytes of the Th1 phenotype (7, 8, 9, 10).
Neutrophils are usually the first cells to arrive at a site of
inflammation and are essential for nonspecific host defense via the
release of a variety of proteases, reactive oxygen intermediates, and
arachidonic acid metabolites (11). In addition to their defensive
functions, it has become well established, both in vitro and in vivo,
that neutrophils can synthesize and secrete several cytokines and
chemokines (12). Among the various chemokines, IL-8 (13, 14),
MIP-1ß (15), MIP-2 (16), GRO (17, 18), and cytokine-induced
neutrophil chemoattractant (CINC) (19) have been reported to be
secreted following activation of PMN. More recently, we have
demonstrated that human neutrophils possess the capacity to release
IP-10 (20), but whether they can also express MIG and I-TAC has yet to
be determined.
In this study, we show that, following stimulation with IFN- in
combination with either TNF- or LPS, neutrophils synthesize and
release MIG and express I-TAC mRNA. In addition, we show that IFN-
together with TNF-, LPS, and IL-1ß, may represent another potent
stimulus for the generation of IP-10 by neutrophils and that IL-10, and
to a lesser extent IL-4, can negatively modulate the expression of MIG,
I-TAC, and IP-10 in human neutrophils. Finally, we provide evidence of
the functional significance of neutrophil-derived MIG and IP-10 in
acting on CXCR3-expressing lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified granulocytes (>99.5%) and PBMC were isolated
under endotoxin-free conditions from buffy coats of healthy donors, as
previously described (21). The granulocyte populations contained
usually <4% eosinophils (n = 23), as revealed by
May-Grunwald-Giemsa staining. Eosinophils (purity >95%) were isolated
by immunomagnetic beads, according to the method described by Hansel et
al. (22). Immediately after purification, cells were suspended in RPMI
1640 medium supplemented with 10% low-endotoxin FCS (<0.009 ng/ml;
Seromed, Biochrom, Berlin, Germany) and usually treated with 100 U/ml
IFN- (21) (Hoffmann-La Roche, Basel, Switzerland), 100 U/ml IL-10
(kindly provided by Dr. K. Moore, DNAX and Schering-Plough, Palo Alto,
CA) (23), 1000 U/ml IFN- (Roferon-A; Hoffmann-La Roche), or 10 ng/ml
IL-4 (Peprotech, Rocky Hill, NJ) in different combinations. Cells were
then stimulated, plated at 5 x 106/ml either in
24-well tissue culture plates (Nunc, Roskilde, Denmark) or in
polystyrene flasks (Greiner, Nurtingen, Germany), and cultured at
37°C, 5% CO2 atmosphere. Cell agonists used were the
following: 1 µg/ml LPS (from Escherichia coli, serotype
026:B6, purchased from Sigma, St. Louis, MO), 5 ng/ml TNF-
(Peprotech), heat-killed yeast particles opsonized with IgG (Y-IgG) at
a particle/cell ratio of 2:1, 10 nM fMLP (Sigma), 10 ng/ml GM-CSF
(Genetics Institute, Boston, MA), 1000 U/ml G-CSF (Granulokine;
Hoffmann-La Roche), 20 ng/ml IL-1ß (Hazleton Laboratories,
Vienna, VA), and 50 ng/ml IL-8 (Sandoz, Vienna, Austria). At the
indicated times, cell-free supernatants were harvested and stored at
-20°C, whereas cell pellets were extracted for total RNA. All
reagents used were of the highest available grade and were dissolved in
pyrogen-free water for clinical use (20, 21, 23).
RNA isolation and Northern blot analysis
Total RNA from PMN and PBMC was extracted by the guanidinium isothiocyanate method, usually from 6–7 x 107 PMN and 2–3 x 107 PBMC per condition, and analyzed as already described (21). Filters were hybridized using MIG, I-TAC, IP-10, IL-1 receptor antagonist (IL-1ra), IL-8, glyceraldehyde 3-phosphate dehydrogenase (GAPD), and actin cDNA fragments, 32P-labeled using a Ready-to-go DNA labeling kit (Pharmacia, Uppsala, Sweden).
MIG, IP-10, and IL-8 antigenic determination
MIG protein was measured in the cell-free supernatants by using
a specific double-determinant RIA, developed in our laboratory.
Briefly, flat-bottom 96-well plates (MaxiSorp, No. 439454; Nunc) were
coated with 50 µl/well of protein-G-purified rabbit serum (No. 5092;
24) (40 µg/ml in 0.1 M carbonate buffer, pH 9.5) for
24 h at 4°C, and then extensively washed with PBS, pH 7.5,
0.05% Tween 20 (washing buffer). Fifty microliters per well of either
MIG standards (Peprotech) or cell-derived culture supernatants were
then added, followed by an incubation for 6 h at 20°C. Plates
were rinsed with washing buffer before addition of 50 µl/well of
125I-labeled rabbit anti-human MIG (No. 500-P50;
Peprotech) (0.5 µg/ml in PBS-Tween with 50% FCS) and
incubated overnight at 4°C. After extensive washings of the plates,
60 µl of 1 N NaOH was added into each well, harvested after 30 min,
and read in a gamma counter. This RIA had a detection limit of 30–50
pg/ml and did not cross-react with 100 ng/ml IP-10, 100 ng/ml
IL-8, 1 ng/ml MIP-1, 10 ng/ml GRO, 10 ng/ml IFN-, 5
ng/ml IL-10, 10 ng/ml IL-1ß, 10 ng/ml TNF-, or 10 ng/ml
GM-CSF. IP-10 and IL-8 were measured in the cell-free
supernatants by, respectively, RIA (detection limit 30 pg/ml)
and ELISA (detection limit 20 pg/ml), according to
the procedures previously published (13, 20).
Estimation of apoptotic cells
Flow cytometric analysis of apoptosis was performed by using the method described by Nicoletti et al. (25). In brief, harvested cells were washed twice with PBS and then suspended in 1.5 ml hypotonic fluorochrome solution (propidium iodide 50 µg/ml in 0.1% sodium citrate and 0.1% Triton X-100). The mixture was placed in the dark overnight at 4°C. The fluorescence of each individual nucleus was measured using a FACScan flow cytometer (XL-Coulter, Hialeah, FL).
In vitro chemotaxis assay
To evaluate the chemotactic activities of neutrophil- and
PBMC-derived supernatants, we used, as target cells, either the
previously described 300-19 mouse pre-B cell clones stably transfected
with human CXCR3 cDNA (6) or PHA-stimulated peripheral T lymphocytes
cultured in the presence of IL-2 for 8–14 days. CXCR3 receptor
expression on transfected cells or activated T cells was checked by
FACScan analysis by using 1C6, an anti-hCXCR3 mAb (7), kindly
provided by Dr. Carlo Agostini (University of Padova, Italy). Migration
of CXCR3 transfectants was assessed in a 48-well modified Boyden
chamber (NeuroProbe, Cabin John, MD) using polyvinylpyrrolidone-free
polycarbonate membranes (Nucleo pore) with 5-µm pores, as we
previously described (26). In brief, leukocyte-derived supernatants (28
µl) or 10 nM recombinant MIG or IP-10 (diluted in RPMI medium
containing 1% FBS) were added to the bottom wells of the chemotaxis
chamber. PBMC-derived supernatants from resting or IFN--treated
cells were usually used undiluted, whereas supernatants from resting or
stimulated PMN were used either undiluted or after (approximately) a
100-fold concentration (by the Centricon Plus 20 device; Amicon,
Beverly, MA). The parental cell line and CXCR3 transfectants were
suspended at 5 x 106/ml in RPMI and added to the top
wells of the chamber in a volume of 50 µl. Chambers were then
incubated for 120 min in a 37°C, 5% CO2 atmosphere.
After the incubation period, the filters were removed, washed with PBS
on the upper side, fixed, and stained with Dif-Quik (Baxter, Deerfield,
IL). The number of cells migrating to the lower surface was
microscopically counted in six randomly high powered fields. All assays
were performed in triplicate. Spontaneous migration was determined in
the absence of samples. In selected experiments, leukocyte-derived
supernatants or recombinant chemokines were preincubated at 37°C for
30 min with 30 µg/ml of rabbit anti-human MIG (No. 500-P50,
Peprotech), goat anti-human IP-10 (No. AF-266-NA; R&D Systems,
Abingdon, U.K.), goat anti-human IL-8 (kindly provided by Dr. M.
Ceska, Sandoz, Vienna), mouse anti-human TNF- and IFN-
(kindly provided by Dr. Giorgio Trinchieri, Wistar Institute,
Philadelphia, PA), and isotype-matched Abs, to neutralize their
chemotactic activities.
Chemotaxis was also assessed using a 24-well transwell chamber (6.5-mm diameter, 5-µm pore size, Costar 3421; Corning Costar, Rochester, NY), essentially as described by Gosling et al. (27). In brief, the parental cell line and CXCR3 transfectants or activated T lymphocytes were suspended at 2 x 106/ml in RPMI 1640, containing 10% FCS and 20 mM HEPES, pH 7.2. One hundred microliters of cell suspension were added to the top chamber, whereas 600 µl of leukocyte-derived supernatants or 10 nM recombinant chemokines were added to the bottom well. The chambers were then incubated for 2 h at 37° in an atmosphere containing 5% CO2. Cells that passed through the membrane were harvested from the lower well and counted by using the CyQuant Cell proliferation assay kit (Molecular Probes, Eugene, OR), according to the manufacturer’s instructions.
Adhesion assay
Human T lymphocytes expressing CXCR3 were used in these assays. T cells were first purified from PBMC by E-rosetting with neuraminidase-treated sheep RBC and then incubated (2 x 106/ml) at 37°C for 3 days in complete medium containing 500 U/ml IL-2 (Proleukin; Chiron Diagnostic, Cassina de’ Pecchi, Italy) and 5 µg/ml PHA. Subsequently, T cells were maintained at a density of 3–5 x 106/ml in fresh media containing 250 U/ml IL-2 to optimize the induction of CXCR3 expression and their responsiveness to IP-10 and MIG, as recently reported by Cole et al. (4) and Loetscher et al. (28). At the days indicated in the results, activated T lymphocytes were washed and resuspended at 5 x 106/ml in PBS, 1 mM Ca2+/Mg2+, 10% heat-inactivated FCS (pH 7.2). Twenty microliters of cell suspension (105 cells) were allowed to settle for 10 min at 37°C on 18-well glass slides precoated for 16 h at 4°C with 20 µl of purified human fibronectin (20 µg/ml in PBS; Sigma, F 6277). After the cells were allowed to settle, lymphocyte adhesion was stimulated for 2 min with 5 µl of leukocyte-derived supernatants or with 5 µl of recombinant chemokines (100 nM final concentration), added at the 12 o’clock position of the well. At the appropriate time point, the slide was dipped twice in ice-cold HBSS/10 mM HEPES to remove nonadherent cells, and bound cells were then fixed in ice cold PBS containing 1.5% gluteraldehyde. Adherent cells in 0.2 mm2 were calculated by computer-assisted enumeration. In selected experiments, specific Abs were used to neutralize the biological effects of recombinant chemokines or leukocyte-derived supernatants (see above). Each experiment was performed in quadruplicate for each condition, and the SD was calculated. Results are representative from at least two independent donors.
Statistical analysis
Data are expressed as means ± SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate whether PMN produce MIG, cells were incubated for
up to 42 h with a series of neutrophil agonists, including LPS,
TNF-, IL-1ß, IL-8, fMLP, G-CSF, and GM-CSF, used either as single
stimuli or in combination with IFN-. As shown in Table I
, only IFN- plus either LPS or
TNF- stimulated a detectable extracellular release of MIG.
Surprisingly, up to 50,000 U/ml IFN- did not induce any significant
extracellular production of MIG from PMN (data not shown).
Dose-response experiments further established that, in IFN--treated
neutrophils, 5 ng/ml TNF- represented an optimal concentration for
MIG release, whereas the stimulatory effect of LPS did not
substantially differ over a wide concentration range, i.e., from 0.01
to 10 µg/ml (Fig. 1). Accurate time
course analyses (not shown) revealed that, in IFN- plus TNF-- or
LPS-stimulated PMN, antigenic MIG started to be released only after
18–21 h of stimulation and progressively accumulated into the
supernatants for up to 42 h (Table I). In contrast, more than 80%
or even 100% of the total antigenic IP-10 was already released within
21 h in response to IFN- plus LPS and IFN- plus TNF-,
respectively (Table II) (20). Moreover,
stimulation of neutrophils for 42 h with IFN- plus TNF-
consistently (in 12 independent experiments) led to the release of
higher amounts of MIG than when the cells were stimulated with IFN-
plus LPS, a pattern that was not always observed in the case of IP-10.
Finally, considerable amounts of IP-10 were released by neutrophils
even when they were cultured in the presence of IFN- in association
with IL-1ß, but not with GM-CSF (Table II).
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To exclude a role for contaminating cells present in our
neutrophil preparations, we determined the ability of eosinophils and
PBMC to release MIG and IP-10. For this purpose, in selected
experiments, eosinophils and PBMC were purified (in addition to
neutrophils) and stimulated for 21 h with IFN- plus either LPS
or TNF-. As illustrated in Fig. 2,
eosinophils did not produce MIG, but they did produce IP-10. However,
substantial release of IP-10 by eosinophils occurred only in response
to IFN- plus TNF-. In contrast to neutrophils and eosinophils,
PBMC treated with IFN- released amounts of MIG higher than those
detected in IFN- plus LPS-treated cells (Fig. 3). However, the highest levels of
antigenic MIG detected in PBMC culture supernatants were observed in
IFN- plus TNF--treated cells (Fig. 3).
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To gain further insights into the molecular mechanisms regulating
MIG production by PMN, neutrophils were stimulated with various
combinations of agonists, and total RNA was analyzed by Northern blot
analysis. In the same experiments, we also investigated whether
neutrophils might express I-TAC mRNA. As shown in Fig. 4A, resting neutrophils, as
well IFN--treated PMN, do not express detectable MIG or I-TAC
transcripts. LPS or TNF-, or other stimuli such as Y-IgG, fMLP,
G-CSF, and GM-CSF, used alone or in the the presence of IFN-, also
failed to induce MIG or I-TAC gene expression (data not shown). In
contrast, a considerable accumulation of MIG and I-TAC mRNA was
observed in PMN stimulated with IFN-, plus either TNF- or LPS,
with maximal expression levels being reached by 21 h, especially
in the case of IFN- plus TNF--treated cells (Fig. 4A).
IP-10 mRNA transcripts were also highly expressed under those
stimulatory conditions, in agreement with our previous data (20).
However, kinetics of IP-10 mRNA expression in IFN- plus LPS-treated
neutrophils differed greatly from those of IFN- plus TNF- cells;
as in the former condition, IP-10 transcripts reached a maximum at
3 h and then declined, whereas in the latter maximal levels of
IP-10 mRNA were detected at 21 h (Fig. 4A).
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B shows a representative Northern blot analysis of
MIG, I-TAC, and IP-10 mRNA accumulation in PBMC, stimulated as
neutrophils. It is evident that IFN- alone represents already an
optimal stimulus to induce the mRNA expression for all three chemokines
and that LPS (but not TNF-) down-modulates the up-regulatory effect
of IFN- (Fig. 4B), in accord with extracellular chemokine
detection (Fig. 3). Effects of IL-10 and IL-4
Because IL-10 and IL-4 are negative modulators of chemokine
production by neutrophils (29), we investigated whether they
might also influence the inducible expression of MIG, I-TAC, and IP-10.
IL-10 greatly reduced the mRNA expression for MIG, I-TAC, and IP-10
induced by IFN- plus either LPS or TNF-, with this inhibitory
effect being much more pronounced in PMN stimulated with IFN- plus
LPS (Fig. 5A), as opposed to
IFN- plus TNF- (Fig. 5B). However, IL-10 potentiated
the stimulatory effect of IFN- plus either LPS or TNF- on IL-1ra
transcripts, confirming and extending previous findings (30, 31).
Consistent with the Northern blot data, a potent inhibitory action of
IL-10 was observed at the level of MIG and IP-10 extracellular release,
both at 21 (not shown) and 42 h poststimulation (Fig. 6), especially on IFN- plus
LPS-treated PMN. Relative to IL-10, IL-4 only moderately suppressed
MIG, I-TAC, and IP-10 mRNA accumulation (not shown), as well as MIG and
IP-10 release in neutrophils. In four independent experiments,
IL-4-mediated inhibition of MIG and IP-10 secretion amounted to
34.5 ± 16.2% and to 24 ± 9%, respectively, in PMN
stimulated for 42 h with IFN- plus LPS, and to 19.5 ±
12% and 15.4 ± 10%, respectively, in cells treated with IFN-
plus TNF-.
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also illustrates that IL-10 markedly inhibits MIG and IP-10
release also in PBMC cultured for 21 h. In three independent
experiments, suppression of MIG and IP-10 release by IL-10 in cells
treated with IFN- amounted to 91 ± 7 and 92 ± 3%,
whereas, in those stimulated with IFN- plus LPS and with IFN-
plus TNF-, inhibition mediated by IL-10 was higher than in
neutrophils (Fig. 6). Assessment of neutrophil apoptosis
Although neutrophils undergo constitutive apoptosis when aged in
vitro, there is considerable evidence to suggest that this process is
substantially delayed if cells are cultured in the presence of agonists
such as LPS, IFN-, G-CSF, or GM-CSF (32). In this context, it was
recently reported that IL-10 seems to block the protective effects of
LPS and other cytokines, including IFN- and TNF-, on the survival
of cultured neutrophils (33). As a result, we sought to determine
whether the reduced chemokine expression by stimulated PMN cultured in
the presence of IL-10 might be the consequence of an accelerated
apoptotic process. To verify this possibility, we measured the rates of
neutrophil apoptosis under our experimental conditions. Table III shows that, after a 21 and 42 h
culture period, recovery of live neutrophils was 69 ± 15% and
48.5 ± 13%, respectively; IFN-, LPS, and, more effectively,
IFN- plus either LPS or TNF- were found to exert significant
protective effects on neutrophil apoptosis. Under those conditions,
IL-10 influenced neither the spontaneous nor the cytokine-regulated
apoptosis rate of neutrophils, yet maintaining its suppressive effect
on IL-8 release (not shown) (23).
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on MIG, I-TAC, and IP-10 mRNA expression and
IP-10 production by neutrophils
In the attempt to identify further stimulatory conditions able to
induce expression of MIG, I-TAC, or IP-10 in neutrophils, we examined
the effect of IFN-, used either alone or in association with other
neutrophil agonists. As shown in Fig. 7,
IFN- alone induced only IP-10 mRNA in neutrophils, while in PBMC it
induced both IP-10 and I-TAC mRNA. MIG mRNA was not up-regulated by
IFN- in either cell type, as expected (34). In both cell types,
GM-CSF up-regulated IL-8 mRNA expression (29), while IFN-
down-regulated the constitutive IL-8 transcripts in PBMC, as previously
reported (35). The effect of IFN- on IP-10 mRNA expression in
neutrophils was synergistically potentiated by the simultaneous
addition of LPS and TNF- (not shown). Subsequent assays on IP-10
protein release demonstrated that treatment of neutrophils for 42
h with IFN- in combination with IFN-, LPS, TNF-, or IL-1ß,
but not alone, resulted in a considerable release of antigenic IP-10
into the supernatants (Fig. 8).
Preincubation of neutrophils for 5 h with IFN- or IFN-
before stimulation with LPS, TNF, IL-1ß, IL-8, fMLP, and GM-CSF did
not augment the release of IP-10 or MIG, as compared with the chemokine
release observed without preincubation (not shown).
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To ascertain whether neutrophil-derived MIG and/or IP-10 were
biologically active, we initially tested the ability of
neutrophil-derived supernatants to recruit CXCR3-positive cells in a
chemotaxis assay in vitro. For this purpose, we used the mouse pre-B
cell line, 300-19, stably transfected with CXCR3 cDNA. These
transfectants represent a reliable experimental model to determine the
chemotactic activities of IP-10, MIG, and I-TAC and have been widely
used by many investigators (4, 6, 28). As shown in Fig. 9, CXCR3-transfected cells readily
migrated toward 10 nM MIG or IP-10, while cells from the
nontransfected, parental cell line did not respond. Supernatants
harvested from neutrophils stimulated with IFN- plus TNF- (or, to
a lesser extent, with IFN- plus LPS) and from IFN--treated PBMC
exerted significant chemotactic activities on CXCR3-transfected, but
not on parental, cells (Fig. 9). By contrast, supernatants harvested
from resting leukocytes or from IFN--stimulated PMN were
ineffective. Of note, neutralizing anti-MIG and anti-IP-10 Abs
partially abrogated the chemotactic activities exerted by supernatants
from both stimulated neutrophils and PBMC, indicating that MIG and
IP-10 were, at least in part, responsible for these chemotactic effects
(Fig. 9). No additive effects were found if neutralizing anti-MIG
and anti-IP-10 Abs were used in combination (not shown). It must be
pointed out that, to detect biological effects of the supernatants
harvested from cultured PMN, it was necessary to concentrate them
(approximately 100-fold) before assaying. Indeed, preliminary tests
revealed that crude undiluted supernatants from agonist-stimulated
neutrophils failed to exert chemotactic activities on CXCR3-transfected
cells. Moreover, supernatants from IFN--stimulated PBMC displayed
chemoattraction on either CXCR3-transfected cells or activated T
lymphocytes (again, partially neutralized by anti-MIG and
anti-IP-10 Abs) also in the transwell assay (not shown).
Unfortunately, we could not test neutrophil-derived supernatants in the
transwell system because it was not feasible to obtain sufficient
volumes (more than 4 ml) of concentrated samples that were necessary to
perform experiments with all appropriate controls.
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, both MIG and IP-10 triggered a
rapid adhesion of activated T lymphocytes to fibronectin that was
completely inhibited by the respective neutralizing Abs. Preliminary
experiments confirmed that IP-10- and MIG-induced rapid and firm
adhesion of T cells was a transient phenomenon and was maximal after 2
min, exactly as recently reported by Piali et al. (37). No binding was
induced in the absence of agonists.
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plus TNF--stimulated PMN and,
to a lesser extent, from IFN--stimulated PBMC also induced the rapid
adhesion of activated T lymphocytes to fibronectin (Fig. 10). In
contrast, no detectable binding was triggered by supernatants from
resting cells or from IFN--stimulated neutrophils (not shown).
Importantly, adhesion triggering by supernatants was almost completely
blocked by Abs to either MIG or IP-10 (Fig. 10), whereas
isotype-matched and IFN--, TNF--, and IL-8-neutralizing Abs were
completely ineffective (not shown), suggesting that MIG and IP-10
present in neutrophil-derived supernatants were responsible for these
biological effects. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in a range of cell types, including monocytes, keratinocytes,
endothelial cells, and astrocytes (4, 5).
We have recently shown that human neutrophils produce IP-10 (20). In
this work, we demonstrate for the first time that neutrophils are also
able to release MIG and to express I-TAC mRNA, but only under specific
conditions. Indeed, extracellular release of MIG in substantial amounts
was found to require costimulation of PMN with IFN- along with
either LPS or TNF-. IFN- plus TNF- reproducibly represented
the most effective combination for this response. Similar data were
obtained with endothelial cells (37, 47). Strikingly, IFN- alone,
which represents the classical trigger of MIG production in other cell
types (5), proved to be ineffective toward the extracellular release of
MIG by neutrophils. Other mediators, including GM-CSF, IL-1ß, IL-8,
and fMLP all failed to induce MIG gene expression and extracellular
release by neutrophils, regardless of the presence of IFN- or
IFN- in the culture medium. Opsonized particles (Y-IgG) used in
combination with IFN-, which are known to induce high amounts of
TNF- production (29), had no effect on the induction of MIG release
(our unpublished observations), thus making it unlikely that the
effects observed with IFN- plus LPS were mediated by the endogenous
TNF- (29).
We could only investigate the gene expression of I-TAC, due to the
availability of only the cDNA. Our experiments showed that I-TAC mRNA
is expressed in neutrophils if they are cultured with IFN- in
association with either LPS or TNF-. No other stimulatory conditions
were found effective in inducing I-TAC mRNA expression in neutrophils.
Kinetic studies of I-TAC mRNA expression in neutrophils treated with
IFN- plus LPS or TNF- revealed that, similarly to MIG
transcripts, maximal levels of I-TAC transcripts occurred at 21 h
after stimulation. However, differently from MIG and IP-10 mRNA, I-TAC
gene expression was not induced if neutrophils were stimulated under
serum-free medium conditions and cultured in FBS-precoated flasks (our
unpublished observations).
We have also greatly extended our previous findings on the production
of IP-10 by neutrophils (20). We now provide evidence that IL-1ß and
GM-CSF represent effective costimuli for IP-10 release by
IFN--treated neutrophils and that a significant release of IP-10 by
neutrophils can be obtained even if cells are stimulated with IFN-
in association with LPS, TNF-, or IL-1ß. The latter data support
the ability of type I IFNs to selectively up-regulate IP-10, which was
previously reported to occur in monocytes and other cells (34, 48, 49, 50).
The distinctive patterns of MIG and IP-10 production in response to
IFN- and IFN- are understandable considering that the regulatory
sequences identified in the MIG and IP-10 promoters are quite different
(51, 52). Since also IFN-, in addition to IL-12, IFN-, TGFß,
and hormones, has been reported to regulate Th1 development (53),
production of IP-10 by IFN--stimulated neutrophils may represent one
of the mechanisms that contributes to Th1 responses essential for
clearance of those pathogens such as viruses.
Interestingly, the kinetics of IP-10 mRNA expression in, and release
by, IFN- or IFN- plus LPS- or TNF--treated PMN were faster
than those of MIG mRNA expression and release, or I-TAC mRNA
expression. Furthermore, neutrophils released much higher amounts of
IP-10 than MIG under those conditions. The significance of a sequential
regulated production of IP-10 and MIG (and likely I-TAC) by neutrophils
is only a matter of speculation, but a similar pattern has been found
in other cell types, for instance, endothelial cells and mouse
macrophages (37, 47, 54).
Another novel observation that we report in this study is that also
eosinophils have the capacity to produce IP-10 but not MIG. However,
differently from neutrophils, eosinophils were observed to release
significant levels of IP-10 only in response to IFN- plus TNF-.
The failure of IFN- plus LPS to induce IP-10 release in eosinophils
is consistent with the poor ability of LPS to affect selected
eosinophil functions, likely due to the lack of CD14 expression on
these cells (55). Furthermore, we highlighted striking differences
between PBMC and PMN with respect to their ability to express or
produce MIG, I-TAC, or IP-10. In fact, although in PBMC the most potent
stimulatory combination for both mRNA accumulation and extracellular
production of MIG, I-TAC, and IP-10 consisted of IFN- plus TNF-,
IFN- alone represented a highly effective stimulus. IFN- was so
powerful that its association with LPS led to a diminished mRNA
expression of MIG, I-TAC, and IP-10 mRNA, and MIG and IP-10 production,
relative to PBMC treated with IFN- alone. Taken together, the latter
findings clearly indicate that the mechanisms governing the expression
of MIG, I-TAC, and IP-10 in neutrophils, eosinophils, and PBMC are
specific and subjected to distinct regulatory pathways. Furthermore,
and not of lesser importance, they demonstrate that the results
obtained in neutrophils with respect to MIG, I-TAC, and IP-10
expression cannot be attributed to a contamination of the PMN
populations with eosinophils or mononuclear cells.
Numerous studies have established that the production of
proinflammatory cytokines and chemokines by neutrophils can be markedly
modulated by immunoregulatory polypeptides such as IL-10 (23, 29) or
IL-4 (31, 56). Herein, we show that both IL-10 and IL-4 negatively
control MIG, I-TAC, and IP-10 mRNA expression and release in
neutrophils, IL-10 being much more suppressive than IL-4. Inhibition of
chemokine expression and release by IL-10 was more pronounced on
IFN- plus LPS-treated neutrophils than on IFN- plus
TNF--treated cells, in agreement with the powerful capacity of IL-10
to negatively influence the effects of endotoxin (57). We did not
investigate further the molecular mechanisms whereby IL-10
down-regulates chemokine production in neutrophils, but we clearly
established that IL-10 does not affect the strong protective effects of
IFN- plus either LPS or TNF- on the rates of neutrophil
apoptosis. These findings completely exclude the possibility that the
suppression of MIG, IP-10, and other cytokine release (29) depends on
an induced decrease of neutrophil survival by IL-10. On the other hand,
our observations are in contrast with those by Keel and colleagues
(33), who reported that the inhibitory effect of LPS, TNF-, IFN-,
G-CSF, and GM-CSF on the spontaneous apoptosis of cultured neutrophils
was significantly decreased by coincubation with IL-10. At present, we
have no explanation for the discrepancies between our results and those
of Keel and coworkers. Although the assay performed to quantify
neutrophil apoptosis was the same in both laboratories, it cannot be
excluded that other different experimental conditions used to isolate,
to lyse, and to culture neutrophils might affect their responsiveness
to IL-10.
In addition to the numerous descriptive findings outlined above, we were also able to provide clear evidence for a functional significance of neutrophil-derived IP-10 and MIG. Indeed, we show that supernatants harvested from stimulated PMN, as well as from stimulated PBMC, exert chemotactic activities toward CXCR3-expressing lymphocytes. These effects were partially neutralized by anti-MIG and anti-IP-10 Abs, consistent with the presence of IP-10 and MIG in leukocyte-derived supernatants, and in agreement with their capability to induce migration of CXCR3-positive cells (4, 5, 6, 28). However, since neutralization of MIG and IP-10 did not completely abolish cell migration, other CXCR3 ligands, probably present in leukocyte-derived supernatants, are likely to contribute to the observed chemotactic activities. According to our gene expression data, one obvious candidate may be I-TAC. We also demonstrate that supernatants from stimulated leukocytes trigger rapid integrin-dependent adhesion of PHA/IL-2-activated T lymphocytes to fibronectin. Rapid integrin triggering is a fundamental step in the overall regulation of leukocyte diapedesis, under physiological as well as pathological conditions. Chemokines are likely to be physiological activators of integrin-dependent adhesion under flow and to direct lymphocyte extravasation and specific microenvironmental targeting in lymphoid tissues. In agreement with very recent findings (37), recombinant IP-10 and MIG were able by themselves to trigger maximal lymphocyte adhesion to immobilized integrin ligands. Interestingly, neutralizing Abs toward either MIG or IP-10 reduced by more than 90% lymphocyte adhesion triggered by supernatants from stimulated leukocytes. This could suggest a threshold effect whereby MIG, IP-10, and likely other CXCR3 ligands (I-TAC?) can cooperate to trigger the required signal leading to integrin activation. Whatever is the case, to our knowledge, these are the first demonstrations of relevant physiological processes attributed to neutrophil-derived chemokines. It may therefore be postulated that neutrophil-derived MIG and IP-10 are important in diapedesis and recruitment of immunocompetent T lymphocytes to sites of inflammation and disease.
In conclusion, the ability of PMN to produce MIG, IP-10, and likely
I-TAC might be significant considering the various biological functions
that these three chemokines possess. Generation of MIG, IP-10, and TAC
by PMN may, for instance, contribute to recruit Th1 lymphocytes to
sites of inflammation. Therefore, along with the well-known capacity of
neutrophils to release IL-8, MIP-1ß, MIP-2, GRO, and
cytokine-induced neutrophil chemoattractants (CINCs) (29), our
data reinforce the concept that neutrophils have the potential to
regulate the migration of various leukocytic cellular types into
inflammatory sites. Alternatively, neutrophil-derived MIG, IP-10, or
I-TAC might negatively regulate hemopoiesis (40, 41). Production of
MIG, IP-10, and I-TAC by neutrophils may also represent one of the
mechanisms whereby these cells exert antitumor effects (58), in light
of the well-established angiostatic properties of IP-10 and MIG
(42, 43, 44, 45, 46). Granulocytes can mediate tumor cell killing through direct or
bystander effects and can participate in the cross-talk with CD8 T
cells, which has been demonstrated to be instrumental in the rejection
of specific cytokine-transduced tumors (59, 60). Interestingly, such
crosstalk was sustained by CD8 cell-produced IFN- (59). Although it
is still too early to speculate on an eventual in vivo role of
neutrophil-derived MIG, I-TAC, or IP-10, the initial question that must
be answered is under which circumstances neutrophils uniquely produce
these factors, considering that their in vitro activities are so
overlapping if not identical. In any case, the selective activity of
MIG, I-TAC, and IP-10 on activated T cells and probably NK cells is
consistent with a role in regulating the trafficking and/or function of
effector cells during an immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marco A. Cassatella, Istituto di Patologia Generale, Strada Le Grazie 4, 37134 Verona, Italy. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; G-CSF, granulocyte-CSF; PMN, polymorphonuclear neutrophil; MIG, monokine induced by IFN-; IP-10, IFN--inducible protein-10; I-TAC, IFN-inducible T cell chemoattractant; GRO, growth-related gene product; GM-CSF, granulocyte-macrophage CSF; Y-IgG, yeast particles opsonized with IgG; IL-1ra, IL-1 receptor antagonist.
Received for publication August 4, 1998. Accepted for publication January 29, 1999.
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T. Akahoshi, T. Sasahara, R. Namai, T. Matsui, H. Watabe, H. Kitasato, M. Inoue, and H. Kondo Production of Macrophage Inflammatory Protein 3{alpha} (MIP-3{alpha}) (CCL20) and MIP-3{beta} (CCL19) by Human Peripheral Blood Neutrophils in Response to Microbial Pathogens Infect. Immun., January 1, 2003; 71(1): 524 - 526. [Abstract] [Full Text] [PDF]
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J. Y. Channon, K. A. Miselis, L. A. Minns, C. Dutta, and L. H. Kasper Toxoplasma gondii Induces Granulocyte Colony-Stimulating Factor and Granulocyte-Macrophage Colony-Stimulating Factor Secretion by Human Fibroblasts: Implications for Neutrophil Apoptosis Infect. Immun., November 1, 2002; 70(11): 6048 - 6057. [Abstract] [Full Text] [PDF]
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G. van Zandbergen, N. Hermann, H. Laufs, W. Solbach, and T. Laskay Leishmania Promastigotes Release a Granulocyte Chemotactic Factor and Induce Interleukin-8 Release but Inhibit Gamma Interferon-Inducible Protein 10 Production by Neutrophil Granulocytes Infect. Immun., August 1, 2002; 70(8): 4177 - 4184. [Abstract] [Full Text] [PDF]
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K. Boztug, M. J. Carson, N. Pham-Mitchell, V. C. Asensio, J. DeMartino, and I. L. Campbell Leukocyte Infiltration, But Not Neurodegeneration, in the CNS of Transgenic Mice with Astrocyte Production of the CXC Chemokine Ligand 10 J. Immunol., August 1, 2002; 169(3): 1505 - 1515. [Abstract] [Full Text] [PDF]
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T. M. Tumpey, R. Fenton, S. Molesworth-Kenyon, J. E. Oakes, and R. N. Lausch Role for Macrophage Inflammatory Protein 2 (MIP-2), MIP-1{alpha}, and Interleukin-1{alpha} in the Delayed-Type Hypersensitivity Response to Viral Antigen J. Virol., July 17, 2002; 76(16): 8050 - 8057. [Abstract] [Full Text] [PDF]
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K. Mohan, Z. Ding, J. Hanly, and T. B. Issekutz IFN-{gamma}-Inducible T Cell {alpha} Chemoattractant Is a Potent Stimulator of Normal Human Blood T Lymphocyte Transendothelial Migration: Differential Regulation by IFN-{gamma} and TNF-{alpha} J. Immunol., June 15, 2002; 168(12): 6420 - 6428. [Abstract] [Full Text] [PDF]
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P. Scapini, L. Nesi, M. Morini, E. Tanghetti, M. Belleri, D. Noonan, M. Presta, A. Albini, and M. A. Cassatella Generation of Biologically Active Angiostatin Kringle 1-3 by Activated Human Neutrophils J. Immunol., June 1, 2002; 168(11): 5798 - 5804. [Abstract] [Full Text] [PDF]
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 K. Wakasugi, B. M. Slike, J. Hood, K. L. Ewalt, D. A. Cheresh, and P. Schimmel Induction of Angiogenesis by a Fragment of Human Tyrosyl-tRNA Synthetase J. Biol. Chem., May 31, 2002; 277(23): 20124 - 20126. [Abstract] [Full Text] [PDF]
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M. Saetta, M. Mariani, P. Panina-Bordignon, G. Turato, C. Buonsanti, S. Baraldo, C. M. Bellettato, A. Papi, L. Corbetta, R. Zuin, et al. Increased Expression of the Chemokine Receptor CXCR3 and Its Ligand CXCL10 in Peripheral Airways of Smokers with Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., May 15, 2002; 165(10): 1404 - 1409. [Abstract] [Full Text] [PDF]
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J. K. Lee, E. H. Lee, Y. P. Yun, K. Kim, K. Kwack, D. S. Na, B. S. Kwon, and C.-K. Lee Truncation of NH2-terminal Amino Acid Residues Increases Agonistic Potency of Leukotactin-1 on CC Chemokine Receptors 1 and 3 J. Biol. Chem., April 19, 2002; 277(17): 14757 - 14763. [Abstract] [Full Text] [PDF]
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L. Michalec, B. K. Choudhury, E. Postlethwait, J. S. Wild, R. Alam, M. Lett-Brown, and S. Sur CCL7 and CXCL10 Orchestrate Oxidative Stress-Induced Neutrophilic Lung Inflammation J. Immunol., January 15, 2002; 168(2): 846 - 852. [Abstract] [Full Text] [PDF]
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 J. M. Ruehlmann, R. Xiang, A. G. Niethammer, Y. Ba, U. Pertl, C. S. Dolman, S. D. Gillies, and R. A. Reisfeld MIG (CXCL9) Chemokine Gene Therapy Combines with Antibody-Cytokine Fusion Protein to Suppress Growth and Dissemination of Murine Colon Carcinoma Cancer Res., December 1, 2001; 61(23): 8498 - 8503. [Abstract] [Full Text] [PDF]
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M.-C. Dieu-Nosjean, C. Massacrier, B. Vanbervliet, W.-H. Fridman, and C. Caux IL-10 Induces CCR6 Expression During Langerhans Cell Development While IL-4 and IFN-{gamma} Suppress It J. Immunol., November 15, 2001; 167(10): 5594 - 5602. [Abstract] [Full Text] [PDF]
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D. D'AMBROSIO, M. MARIANI, P. PANINA-BORDIGNON, and F. SINIGAGLIA Chemokines and Their Receptors Guiding T Lymphocyte Recruitment in Lung Inflammation Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1266 - 1275. [Full Text] [PDF]
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M. Miura, K. Morita, H. Kobayashi, T. A. Hamilton, M. D. Burdick, R. M. Strieter, and R. L. Fairchild Monokine Induced by IFN-{gamma} Is a Dominant Factor Directing T Cells into Murine Cardiac Allografts During Acute Rejection J. Immunol., September 15, 2001; 167(6): 3494 - 3504. [Abstract] [Full Text] [PDF]
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R. L. Robson, R. M. McLoughlin, J. Witowski, P. Loetscher, T. S. Wilkinson, S. A. Jones, and N. Topley Differential Regulation of Chemokine Production in Human Peritoneal Mesothelial Cells: IFN-{{gamma}} Controls Neutrophil Migration Across the Mesothelium In Vitro and In Vivo J. Immunol., July 15, 2001; 167(2): 1028 - 1038. [Abstract] [Full Text] [PDF]
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 A. Shimada, J. Morimoto, K. Kodama, R. Suzuki, Y. Oikawa, O. Funae, A. Kasuga, T. Saruta, and S. Narumi Elevated Serum IP-10 Levels Observed in Type 1 Diabetes Diabetes Care, March 1, 2001; 24(3): 510 - 515. [Abstract] [Full Text]
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K. Tateda, T. A. Moore, J. C. Deng, M. W. Newstead, X. Zeng, A. Matsukawa, M. S. Swanson, K. Yamaguchi, and T. J. Standiford Early Recruitment of Neutrophils Determines Subsequent T1/T2 Host Responses in a Murine Model of Legionella pneumophila Pneumonia J. Immunol., March 1, 2001; 166(5): 3355 - 3361. [Abstract] [Full Text] [PDF]
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R. E. Wiley, K. Palmer, B. U. Gajewska, M. R. Stampfli, D. Alvarez, A. J. Coyle, J.-C. Gutierrez-Ramos, and M. Jordana Expression of the Th1 Chemokine IFN-{{gamma}}-Inducible Protein 10 in the Airway Alters Mucosal Allergic Sensitization in Mice J. Immunol., February 15, 2001; 166(4): 2750 - 2759. [Abstract] [Full Text] [PDF]
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E. Di Carlo, G. Forni, P. Lollini, M. P. Colombo, A. Modesti, and P. Musiani The intriguing role of polymorphonuclear neutrophils in antitumor reactions Blood, January 15, 2001; 97(2): 339 - 345. [Full Text] [PDF]
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L. S. Cauley, E. E. Miller, M. Yen, and S. L. Swain Superantigen-Induced CD4 T Cell Tolerance Mediated by Myeloid Cells and IFN-{gamma} J. Immunol., December 1, 2000; 165(11): 6056 - 6066. [Abstract] [Full Text] [PDF]
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M. T. Liu, B. P. Chen, P. Oertel, M. J. Buchmeier, D. Armstrong, T. A. Hamilton, and T. E. Lane Cutting Edge: The T Cell Chemoattractant IFN-Inducible Protein 10 Is Essential in Host Defense Against Viral-Induced Neurologic Disease J. Immunol., September 1, 2000; 165(5): 2327 - 2330. [Abstract] [Full Text] [PDF]
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F. N. Lauw, D. Pajkrt, C. E. Hack, M. Kurimoto, S. J. H. van Deventer, and T. van der Poll Proinflammatory Effects of IL-10 During Human Endotoxemia J. Immunol., September 1, 2000; 165(5): 2783 - 2789. [Abstract] [Full Text] [PDF]
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F. N. Lauw, A. J. H. Simpson, J. M. Prins, S. J. H. van Deventer, W. Chaowagul, N. J. White, and T. van der Poll The CXC Chemokines Gamma Interferon (IFN-gamma )-Inducible Protein 10 and Monokine Induced by IFN-gamma Are Released during Severe Melioidosis Infect. Immun., July 1, 2000; 68(7): 3888 - 3893. [Abstract] [Full Text] [PDF]
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E. Brandt, G. Woerly, A. B. Younes, S. Loiseau, and M. Capron IL-4 production by human polymorphonuclear neutrophils J. Leukoc. Biol., July 1, 2000; 68(1): 125 - 130. [Abstract] [Full Text]
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D. P. Widney, Y.-R. Xia, A. J. Lusis, and J. B. Smith The Murine Chemokine CXCL11 (IFN-Inducible T Cell {alpha} Chemoattractant) Is an IFN-{gamma}- and Lipopolysaccharide- Inducible Glucocorticoid-Attenuated Response Gene Expressed in Lung and Other Tissues During Endotoxemia J. Immunol., June 15, 2000; 164(12): 6322 - 6331. [Abstract] [Full Text] [PDF]
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M. M. Mazanet, K. Neote, and C. C. W. Hughes Expression of IFN-Inducible T Cell {alpha} Chemoattractant by Human Endothelial Cells Is Cyclosporin A-Resistant and Promotes T Cell Adhesion: Implications for Cyclosporin A-Resistant Immune Inflammation J. Immunol., May 15, 2000; 164(10): 5383 - 5388. [Abstract] [Full Text] [PDF]
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M. Centola, G. Wood, D. M. Frucht, J. Galon, M. Aringer, C. Farrell, D. W. Kingma, M. E. Horwitz, E. Mansfield, S. M. Holland, et al. The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators Blood, May 15, 2000; 95(10): 3223 - 3231. [Abstract] [Full Text] [PDF]
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