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-Inducible Protein-10 and Monokine Induced by IFN-1
*
Laboratory of Medical Allergology, Allergy Unit, National University Hospital, and
Department of Medical Physiology, University of Copenhagen, Copenhagen, Denmark; and
Department of Immunology, Anhui Medical University, Hefei, People’s Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein-10 ( IP-10) and monokine induced by
IFN- (Mig). We report a novel finding that CXCR3 is also expressed
on eosinophils. IP-10 and Mig induce eosinophil chemotaxis via
CXCR3, as documented by the fact that anti-CXCR3 mAb blocks
IP-10- and Mig-induced eosinophil chemotaxis. IP-10- and
Mig-induced eosinophil chemotaxis are up- and down-regulated by IL-2
and IL-10, respectively. Correspondingly, CXCR3 protein and mRNA
expressions in eosinophils are up- and down-regulated by IL-2 and
IL-10, respectively, as detected using flow cytometry,
immunocytochemical assay, and a real-time quantitative RT-PCR
technique. IP-10 and Mig act eosinophils to induce chemotaxis via
the cAMP-dependent protein kinase A signaling pathways. The fact that
IP-10 and Mig induce an increase in intracellular calcium in
eosinophils confirms that CXCR3 exists on eosinophils. Besides
induction to chemotaxis, IP-10 and Mig also activate eosinophils to
eosinophil cationic protein release. These results indicate that
CXCR3- IP-10 and -Mig receptor-ligand pairs as well as the effects
of IL-2 and IL-10 on them may be especially important in the
cytokine/chemokine environment for the pathophysiologic events of
allergic inflammation, including initiation, progression, and
termination in the processes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein-10 ( IP-10) and monokine induced by
IFN- (Mig) with Ki values of 0.14
and 4.9 nM, respectively. IP-10 and Mig are two members of the CXC
chemokine superfamily whose expression is dramatically up-regulated by
IFN-. Both chemokines have been shown to be functional agonists of
CXCR3 (1). The proteins act largely on NK cells and
activated T cells and have been implicated in mediating some of the
effects of IFN- and LPS as well as T cell-dependent anti-tumor
responses. Recently, the CC chemokine 6Ckine and IFN-inducible T cell
chemoattractant have been identified as new ligands for CXCR3
(2, 3). IP-10 and Mig induce rapid and transient
adhesion of human IL-2-stimulated T lymphocytes to immobilized integrin
ligands through their receptor CXCR3, which is selectively expressed on
activated T cells (4). Naive T cells expressed only CXCR4,
whereas the majority of memory/activated T cells expressed CXCR3, and a
small proportion expressed CCR3 and CCR5 (5). CXCR3 was
expressed at high levels on Th0 and Th1 lymphocytes and at low levels
on Th2 lymphocytes. In contrast, CCR3 and CCR4 were found on Th2
lymphocytes (5). Circulating blood T cells, B cells, and
NK cells also express CXCR3 (6). Blood T cells expressing
CXCR3 were mostly CD45RO+ and generally expressed
high levels of ß1 integrins. CXCR3 and CCR5 are
markers for T cells associated with certain inflammatory reactions,
particularly Th1-type reactions such as rheumatoid arthritis. CXCR3 and
CCR5 appear to identify subsets of T cells in blood with a predilection
for homing to these sites (6). Interestingly, Mig was
reported to promote tumor necrosis in vivo (7).
In the present study we have observed that IP-10 and Mig induce
eosinophil chemotaxis via CXCR3, as documented by the fact that
anti-CXCR3 mAb blocks IP-10- and Mig-induced eosinophil
chemotaxis. IP-10- and Mig-induced eosinophil chemotaxis are up-
and down-regulated by IL-2 and IL-10, respectively. Interestingly,
CXCR3 protein and mRNA expressions in eosinophils are up- and
down-regulated by IL-2 and IL-10, indicating that IL-2 and IL-10
control IP-10- and Mig-induced eosinophil chemotaxis via regulation
of CXCR3 expression. It has been demonstrated that IP-10 and Mig
induce eosinophil chemotaxis via the cAMP-dependent protein kinase A
signaling pathway. The fact that IP-10 and Mig induce an increase
in [Ca2+]i in eosinophils
confirms that CXCR3 exists on eosinophils. Besides induction to
chemotaxis, IP-10 and Mig also activate eosinophils to eosinophil
cationic protein (ECP) release. These results indicate that CXCR3-
IP-10 and -Mig receptor-ligand pairs and modulation of CXCR3 expression
by IL-2 and IL-10 may be especially important in the cytokine/chemokine
environment that controls initiation, progression, and termination of
allergic and other eosinophil-dominated forms of inflammation
processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human peripheral eosinophils were purified from healthy,
nonallergic volunteers as described in detail previously (8, 9). Briefly, the method was based on Percoll gradient
centrifugation (density, 1.082 g/ml; Pharmacia, Uppsala, Sweden) to
isolate granulocytes, lysis of RBC with 155 mM ammonium chloride
(NH4Cl), and immunomagnetic depletion of
neutrophils by the magnetic cell separation system (MACS) using
anti-CD16-coated MACS particles (Miltenyi Biotech, Bergisch
Gladbach, Germany). The purity of eosinophil preparations was
invariably 
97%, as judged by eosin staining. Throughout the
purification procedure, the cells were kept at 4°C in a
Ca2+- and Mg2+-free
medium.
Stimulation of cells
For chemotaxis regulation, purified eosinophils were
preincubated with Th1-associated cytokines (IL-2, IFN-, or TNF-;
optimal concentration at 10 ng/ml) (10, 11) and
Th2-associated cytokines (IL-4, IL-5, or IL-10; optimal concentration
at 10 ng/ml) (11) for 24 h at 37°C in 5%
CO2 before chemotaxis assay. For measurement of
CXCR3 mRNA, purified eosinophils were preincubated with IL-2 (10 ng/ml)
or IL-10 (10 ng/ml) for 24 h at 37°C. Then the cells were
subjected to mRNA isolation for real-time quantitative RT-PCR. All
cytokines used were purchased from R&D Systems Europe (Abingdon, U.K.).
For investigation of signaling pathway of IP-10 and Mig to induce
eosinophil chemotaxis, the cells were preincubated for 45 min at room
temperature with pertussis toxin (PT; 1 µg/ml), staurosporine (Sta; 1
µM), tyrphostin 23 (1 µM),
N-(2-(methylamino)ethyl)-5-isoquenolinesulfonamide
dihydrochloride (H-8; 30 µM),
N-(2-(
-bromocinnamylamino)ethyl)-5-isoquenilesulfonamide
(H-89; 30 µM), or bisindolylmaleimide I (BIM I; 1 µM),
respectively, before the cells were subjected to additional
experiments. All signaling pathway inhibitors used were purchased from
Sigma (St. Louis, MO). For measurement of ECP, the purified human
eosinophils were stimulated with chemokines at different
concentrations, as indicated, for 4 h at 37°C in 96-well plates.
The supernatants were collected after stimulation for ECP
measurement.
Chemotaxis assay
The following human recombinant chemokines were studied:
IP-10, Mig, and eotaxin (R&D Systems Europe). The chemotaxis assay was
performed using a 48-well microchamber (Neuro Probe, Bethesda, MD)
technique (12). Briefly, chemokines were diluted in RPMI
1640 with 0.5% pooled human serum and placed in the lower wells (25
µl). Fifty microliters of the cell suspension at 1 x
106 cells/ml was added to the upper well of the
chamber, which was separated from the lower well by a 5-µm pore size,
polycarbonate, polyvinylpyrolidone-free membrane (Nucleopore,
Pleasanton, CA). The cells were freshly isolated eosinophils or
eosinophils incubated with reagents as indicated. The chamber was
incubated for 60 min at 37°C in an atmosphere containing 5%
CO2. The membrane was then carefully removed,
fixed in 70% methanol, and stained for 5 min in 1% Coomassie
brilliant blue. The cells that migrated and adhered to the lower
surface of the membrane were counted using a light microscopy.
Approximately 6% of eosinophils will migrate spontaneously (known as
migrating cells on negative control, MCNC) (10),
corresponding to between 2500 and 4000 cells. It may vary from day to
day, but very little within the same day’s experiments. The results
were expressed as a chemotactic index (C. I.), which is the ratio
between the number of migrating cells in the sample and that in the
medium control (12), and with the SD. For blocking tests
of unstimulated or stimulated eosinophil chemotaxis toward the
chemokines indicated, the cells were preincubated with either
anti-CXCR3 mAb (5 µg/ml; clone 49801.111, R&D Systems, Oxon,
U.K.) or isotype IgG1 (5 µg/ml) for 60 min at room temperature before
chemotaxis assay.
Flow cytometry
As previously described (13), eosinophils either freshly isolated or stimulated with cytokines were first incubated with a mouse anti-human CXCR3 mAb or anti-CCR3 mAb (R&D Systems Europe, clone 49801.111; or Leukocite, Cambridge, CA, clone 7B11) at 5 µg/ml or with 5 µg/ml matched isotype mouse IgG1 (Dako, Glostrup, Denmark) in PBS containing 2% BSA and 0.1% sodium azide for 20 min. The cells were then washed twice in staining buffer and resuspended in FITC-conjugated F(ab)2 donkey anti-mouse mAb (1/250, v/v; Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 min, followed by washing twice in staining buffer. All procedures were conducted at 4°C. The cells were then fixed with 1% paraformaldehyde. The analyses were performed with a flow cytometer (Coulter XL; Coulter, Miami, FL).
Immunocytochemistry assay
For detection of chemokine receptors on eosinophils, the freshly isolated resting cells were spun down on a glass slide at 800 rpm for 4 min. Then the slide was moved into a fixation dish immediately to avoid drying out. The fixation liquid was a mixture of methanol and acetone (1/1, v/v). After 5-min fixation the preparation was washed twice in PBS for 5 min. Blocking buffer (PBS with 1% BSA and 0.3% Triton X-100) was added for 5 min at 20°C to avoid unspecific binding, followed by primary Ab (a mouse anti-human CXCR3 mAb (R&D Systems Europe, clone 49801.111) or CCR3 mAb (Leukocite, clone 7B11) at concentration of 10 µg/ml. The preparation was incubated overnight at 4°C. The next day the preparation was washed twice in PBS for 5 min each time, followed by addition of secondary Ab and was visualized using the alkaline phosphatase staining system (Dako) according to the manufacturer’s instruction. Finally, the preparation was sealed and stored in the dark until observation under a microscope.
Real-time quantitative RT-PCR assay
All real-time quantitative RT-PCR reactions were performed as described previously (9, 14, 15). Briefly, total RNA from peripheral eosinophils (1 x 106; purity, >99%) was prepared using the Quick Prep Total RNA Extraction Kit (Pharmacia Biotech, Piscataway, NJ), and any potential contaminating chromosomal DNA was digested with DNase I according to the manufacturer’s instructions. For RT, the RNA was reverse transcribed using oligo(dT)12–18 and Superscript II reverse transcriptase (Life Technologies, Grand Island, NY), according to the manufacturer’s instructions. RT was performed for 60 min at 37°C, and any potential contaminating protein was denatured by incubation for 10 min at 95°C. The real-time quantitative PCR was performed in special optical tubes in a 96-well microtiter plate (Perkin-Elmer/Applied Biosystems, Foster City, CA) with an ABI PRISM 7700 Sequence Detector Systems (Perkin-Elmer/Applied Biosystems), according to the manufacturer’s instructions. By using the SYBR Green PCR Core Reagents Kit (Perkin-Elmer/Applied Biosystems, P/N 4304886), fluorescence signals were generated during each PCR cycle via the 5'- to 3'-endonuclease activity of AmpliTaq Gold (14) to provide real-time quantitative PCR information. The CXCR3 genes were generated by connecting the following sequences of the specific primers (purchased from DNA Technology, Aarhus, Denmark): sense, 5'-GGAGCTGCTCAGAGTAAATCAC-3'; and antisense, 5'-GCACGAGTCACTCTCGTTTTC-3'.
All unknown cDNAs were diluted to contain equal amounts of ß-actin
cDNA. The standards, no template controls, and unknown samples were
added in a total volume of 50 µl/reaction. PCR retain conditions were
2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C
and 60 s at 60°C for each amplification. Potential PCR product
contamination was digested by uracil-N-glycosylase, because
dTTP is substituted by dUTP (14). All PCR experiments were
performed with a hot start. In the reaction system,
uracil-N-glycosylase and AmpliTaq Gold (Perkin-Elmer/Applied
Biosystems) were applied according to the manufacturer’s instructions
(14, 15). To analyze data for PCR products two terms were
used to express the results: 
Rn,
the normalized reporter signal minus the baseline signal established in
the first few cycles PCR; and CT (threshold
cycle), the PCR cycle at which an increase in reporter fluorescence
signal above a baseline can first be detected.
Changes in [Ca2+]i in single eosinophils
[Ca2+]i in single eosinophils was measured as described previously (16). Briefly, purified human eosinophils (1 x 105 cells/ml) were loaded with fura-2/AM at 2 µM at 37 C° for 30 min in PIPES buffer. One-milliliter eosinophil suspensions were placed in a specially constructed chamber (Nunc, Roskilde, Denmark) without any coating for cell attachment. The changes in [Ca2+]i were determined using a digital imaging system consisting of a Zeiss Axiovert 135 microscope (New York, NY), a light-sensitive video camera (Genesys, DAGE MTI, Michigan City, IN), and software from Universal Imaging (Media, PA). The stimuli were added at the concentrations indicated. The [Ca2+]i concentrations were recorded in the individual cells before and after stimulation. Ionomycin stimulation (1 nM) was followed to record the maximal increase in [Ca2+]i. The gray scale values from each cell were converted into [Ca2+]i by dividing fluorescence emissions measured following 340 and 380 nm excitation. The Rmax value was 1.1, and the Rmin was 0.1. The Kd for fura-2 was 224 nM, and the proportionality coefficient, sf2/sb2, measured as the fluorescence intensity exciting at 380 nm from the solutions containing low concentrations of free and Ca2+-saturated dye amounting to 2.0, was 2.
ECP release assay
The eosinophil cationic protein release assay was performed as described previously (17). Briefly, about 10,000 eosinophils were incubated in heat-stable Ag-coated microtiter plates for 4 h at 37°C, followed by harvesting of supernatants. ECP in supernatants and cell extracts were quantified by a solid phase sandwich ELISA method with a biotin-avidin amplification system in microtiter plates (Nunc). Each well in the microtiter plates was coated with 100 µl of rabbit anti-ECP polyclonal Ab (1.5 µg/ml) overnight at 4°C. Before use, the plates were washed three times. The ECP standards were calibrated using an extinction coefficient, E1%1cm, of purified ECP at 280 nm = 15.45, i.e., a 1% solution of the protein has absorbance of 15.45 when using a light path of 1 cm. The samples were incubated overnight, followed by addition of biotin-conjugated rabbit anti-ECP polyclonal Ab for 1.5 h at 37°C, and then were exposed to avidin-peroxidase at room temperature for 30 min, followed by enzyme reaction for 20 min. The absorbance was measured at 492 nm with a 620 nm reference. The percentage of release was the ratio between free ECP in the supernatant and that in the total cell extract.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IP-10 and Mig induce eosinophil chemotaxis via CXCR3
We examined the abilities of IP-10 and Mig to induce
eosinophil chemotaxis. IP-10 and Mig have induced significant
eosinophil chemotactic migration. The results in Fig. 1
A show that IP-10 and Mig
induced chemotactic migration in freshly isolated eosinophils, yielding
typical bell-shaped dose-dependent chemotaxis response curves. The
optimal chemotactic concentrations of IP-10 and Mig were both 100
ng/ml (C. I., 61 4.1 ± 0.37 and 4.2 ± 0.52,
respectively; MCNC, 3104 ± 766). Eotaxin was used as positive
control and induced a similar eosinophil chemotactic migration (C.
I., 61 2.4 ± 0.26). To confirm that the observed eosinophil
chemotaxis was indeed induced by IP-10 and Mig via CXCR3, we used
anti-CXCR3 mAb to block the eosinophil chemotactic activity of
IP-10 and Mig. The anti-CXCR3 mAb could completely block the
chemotaxis of eosinophils toward IP-10 and Mig (Fig. 1B;
C. I., 61 1.1 ± 0.12 and 0.9 ± 0.13, respectively;
MCNC, 3745 ± 359; both at 100 ng/ml), whereas it did not
interfere the chemotaxis of eosinophils toward eotaxin (C. I., 61
3.4 ± 0.42). The anti-CCR3 mAb completely blocked eosinophil
chemotaxis toward eotaxin (data not shown). The isotype Ab had no
blocking effect (Fig. 1C; C. I., 61 3.1 ± 0.38,
3.8 ± 0.53, and 3.9 ± 0.47, respectively; MCNC, 3, 824
± 479), The results of checkerboard analysis (18)
demonstrate that migratory movements of eosinophils toward IP-10
and Mig are chemotactic, but not chemokinetic (data not shown).
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IP-10- and Mig-induced eosinophil chemotaxis are regulated by
IL-2 and IL-10
We also examined the abilities of Th1- and Th2-associated
cytokines to regulate IP-10- and Mig-induced eosinophil chemotaxis.
The results in Table I show that
Th1-associated cytokine IL-2 significantly up-regulated IP-10- and
Mig-induced eosinophil chemotaxis (C. I., 61 6.20 ± 0.45 and
6.22 ± 0.39, respectively; MCNC, 3397 ± 547), whereas other
Th1-associated cytokines IFN- and TNF- showed no such effect.
Moreover, Th2-associated cytokine IL-10 significantly down-regulated
chemotactic migration of eosinophils induced by IP-10 and Mig
(C. I., 61 1.14 ± 0.09 and 1.18 ± 0.15, respectively;
MCNC, 2873 ± 792), whereas other Th2-associated cytokines, IL-4
and IL-5, showed no such effect. None of the tested Th1- and
Th2-associated cytokines had modulated eosinophil chemotaxis toward
eotaxin (100 ng/ml). The regulatory effects of IL-2 and IL-10 on
chemotactic migration of eosinophils induced by IP-10 and Mig could
be blocked by anti-IL-2R mAb (a-IL-2R) and anti-IL-10R mAb,
respectively (data not shown).
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The results from flow cytometric analyses in Fig. 2 document that there were about 48.3%
CXCR3+ cell fractions in freshly isolated
eosinophils (Fig. 2B). After 24-h incubation with
cytokine-free medium, there was no significant change in the
CXCR3+ cell fraction (41.6%; Fig. 2C). Interestingly, IL-2, a Th1-associated cytokine
significantly up-regulated the expression of CXCR3 on eosinophils by up
to 98.8% (Fig. 2D). IL-10, a Th2-associated cytokine,
showed a robust ability to down-regulate the expression of CXCR3 on
human peripheral eosinophils. After 24-h incubation with IL-10 (10
ng/ml), CXCR3+ eosinophil were reduced to 5.4%
(Fig. 2E). CCR3 were constantly expressed on eosinophils
despite the stimulation with IL-2 and IL-10. There were 99.6%
CCR3-positive cells in freshly isolated eosinophils (Fig. 2G), 99.5% in eosinophils cultured in cytokine-free medium
(Fig. 2H), and 98.2% in IL-2-stimulated eosinophils (Fig. 2I) and IL-10-stimulated eosinophils (Fig. 2J).
Fig. 2, A and F, shows isotype controls for CXCR3
and CCR3 mAbs, respectively. Interestingly, we found that the freshly
isolated and stimulated eosinophils are either positive or negative,
with no gradations in the level of CXCR3 expression. The explanation
may be that the positive cells are abundantly expressed CXCR3,
whereas the negative cells are absolutely not expressed CXCR3, or
that CXCR3 on these cells are absolute at a detectable level on flow
cytometer. IL-2 and IL-10 play roles to switch on or switch off CXCR3
expression on the cells. We conducted similar experiments on T
lymphocytes, but did not find up- or down-regulation of CXCR3
expression on these cells (data not shown). The regulatory effects of
IL-2 and IL-10 on CXCR3 expression on eosinophils can be blocked by
anti-IL-2R mAb (a-IL-2 R) and anti-IL-10R mAb, respectively
(data not shown). IL-2R was demonstrated to be expressed on human
eosinophils (19). Although there is no direct evidence of
the existence of IL-10R on human eosinophils, IL-10 was shown to
directly function on human eosinophils (20, 21). Our
results demonstrate that two cytokines, via their receptors on
eosinophils, act to regulate the expression of CXCR3 on the cells and
to further modulate the biological function of IP-10 and Mig on the
eosinophils. To confirm that expression of CXCR3 is indeed on the
eosinophils and to completely rule out the possibility that the
observed effects may be due to contaminating lymphocytes, we therefore
conducted immunocytochemical assay on purified eosinophils to
demonstrate the existence of CXCR3 and CCR3 (as a positive control) on
eosinophils. Because there is autofluorescence in eosinophils, we have
chosen an alkaline phosphatase staining system for visualization in the
immunocytochemical assay to be absolutely sure of the observed positive
results. The results from the immunocytochemical assay document that
CXCR3 is expressed in the human peripheral eosinophils (Fig. 3B), as well as that CCR3 is
expressed (Fig. 3C). Fig. 3A showd the mouse
isotype Ab-negative control. We also conducted similar experiments on T
lymphocytes, and we found CXCR3 expression on these cells
(22), but not CCR3 expression (data not shown). It should
be stressed that we used purified eosinophils with a purity invariably
97% as judged by eosin staining. Morphologically, the CXCR3-positive
cells were identified as eosinophils in the immunocytochemical
assay.
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The results in Fig. 4A
show that mRNA of CXCR3 was detected in human peripheral resting
eosinophils. Compared with the amplification of standard DNA template
(2.0 x 104 copies) with a housekeeping gene
(ß-actin), there were 
2.0 x 103 copies
for CXCR3 in the tested samples of resting eosinophils. There were
1.5 x 104 copies for CXCR3 in the tested
samples of IL-2-stimulated eosinophil in 24 h. There were
6.9 x 102 copies for CXCR3 in the tested
samples of IL-10-stimulated eosinophils in 24 h. The results in
Fig. 4B show that a linear relationship between the
threshold cycle, CT, and the log starting
quantity of standard DNA template or target cDNA (CXCR3) was detected.
In all experiments the correlation coefficient was 0.93. Because
eosinophils can be very fragile cells, we tested the viability of
eosinophils after stimulation with IL-2 or IL-10 (all at 10 ng/ml). The
viability of the cells was invariably 95%, as determined by trypan
blue exclusion test. This experiment was able to exclude the
possibility that the cells studied after 24-h stimulation with IL-2 or
IL-10 are merely a subpopulation of the input cells.
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IP-10- and Mig-induced
eosinophil chemotaxis
To explore which signaling pathways are involved in IP-10- and
Mig-induced eosinophil chemotaxis, we examined whether the interference
with different signaling pathways can affect eosinophil chemotaxis
toward IP-10 and Mig. Before the chemotaxis assay, we pretreated
eosinophils for 30 min at 37°C with tyrphostin 23 (1 µM),
a selective inhibitor of PTK (23); Sta (1 µM), a
selective inhibitor of protein kinase (24); H-89 (30
µM), a selective inhibitor of cAMP-dependent protein kinase
(25); PT (1 µg/ml), a specific inhibitor of certain G
proteins (26); H-8 (30 µM), a selective inhibitor of
cAMP- and cGMP-dependent protein kinase A (27); or BIM I
(1 µM), a selective inhibitor of protein kinase C (28),
respectively. Table II shows that
IP-10- and Mig-induced eosinophil chemotaxis can completely and
selectively be blocked by staurosporine and H-89 at applied
concentrations, whereas eotaxin-induced eosinophil chemotaxis (which is
via its receptor CCR3) can only be blocked by Sta, not by H-89.
Eotaxin-induced eosinophil chemotaxis (which is via its receptor CCR3)
can completely and selectively be blocked by tyrphostin 23, but not by
Sta and H-89. The doses of PT or BIM I (see above and Materials
and Methods) employed did not changed the pattern of IP-10-,
Mig-, and eotaxin-induced eosinophil chemotaxis. Sta and H-89 were
selective inhibitors of protein kinase or selective inhibitors of
cAMP-dependent protein kinase A, respectively, and tyrphostin 23 was a
selective inhibitor of PTK, respectively. Thus, these results strongly
indicate that IP-10 and Mig induce eosinophil chemotaxis via the
cAMP-dependent protein kinase A signaling pathways, and that eotaxin
(which is via its receptor CCR3) induces eosinophil chemotaxis via a
PTK signaling pathway. We prolonged the incubation time of the cells
with PT up to 2 h. The pretreated cells still showed the same
ability to migrate toward IP-10, Mig, and eotaxin, which confirms
that neither IP-10, Mig nor eotaxin induces eosinophil chemotaxis
via certain G proteins signaling pathways (data not shown). In view of
the limited specificity of applied signaling pathway inhibitors, more
complementary experiments are needed to confirm the observed effects of
these inhibitors. It must be mentioned that H-8, a selective inhibitor
of cAMP- and cGMP-dependent protein kinase A, has failed to inhibit
IP-10- and Mig-induced eosinophil chemotaxis at the concentration used
in our experiments. This controversy requires further
clarification.
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IP-10 and Mig induce an increase in
[Ca2+]i in eosinophils
There is an immediate increase in
[Ca2+]i after CXCR3
ligands IP-10 (final concentration, 50 ng/ml) and Mig (final
concentration, 50 ng/ml) stimulation (Fig. 5, A and B).
Eotaxi, which is known to activate eosinophils, has been used as a
positive control (Fig. 5C). A maximal increase in
[Ca2+]i has been
demonstrated by the addition of ionomycin (0.1 nM) after stimulation
with IP-10 and eotaxin. Thus, the results indicate that the ligands
IP-10 and Mig bind to their common receptor CXCR3 on eosinophils to
induce [Ca2+]i changes in
eosinophils. In cross-desensitization in the calcium flux between
IP-10 and Mig, IP-10 can significantly desensitize Mig in terms of
calcium flux in eosinophils (Fig. 5D) and vice versa (data
not shown). These data further support the idea that IP-10 and Mig
act through the same receptor CXCR3.
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IP-10 and Mig induce ECP release
We also examined ECP release from human peripheral eosinophils.
The results in Fig. 6 document that about
2.0% ECP are spontaneously released during the 4-h culture with
cytokine-free medium (760 ng/ml in total in each cell aliquot in our
system). After 4-h incubation with IP-10, there was significant ECP
release in a dose-dependent manner. At 1 x
103 ng/ml, it induced 41.7% ECP release. After
4-h incubation with Mig, it induced significant ECP release in
dose-dependent manner. At 1 x 103 ng/ml, it
induced 38.1% ECP release. After 4-h incubation with eotaxin, there
was significant ECP release in dose-dependent manner. At 1 x
103 ng/ml, it induced 39.1% ECP release. There
were no significant differences among the three chemokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IP-10, Mig, 6Ckine, and
IFN-inducible T cell chemoattractant (2, 3, 30). Due
to the importance of CXCR3 and its ligands in immunity, a number of
interesting and intensive studies have appeared recently. It has been
suggested that CXCR3 and CCR5 are preferentially expressed in human Th1
lymphocytes, whereas Th2 lymphocytes preferentially express CCR4 and
CCR3 (31). CXCR3 has been documented to be expressed on
lymphocytic cells in virtually every perivascular inflammatory
infiltrate in active multiple sclerosis lesions, and cerebrospinal
fluid T cells are significantly enriched for cells expressing CXCR3 or
CCR5 (32). IP-10 even plays a role in the
epidermotropism of cutaneous T cell lymphoma (33).
Currently, it is believed that unlike most other CXC chemokines,
IP-10 and Mig have no activity on either neutrophils or monocytes, but
appear to target stimulated lymphocytes specifically (34).
The gene for CXCR3 is localized on human chromosome Xq13, which is in
clear contrast to all other chemokine receptor genes, suggesting a
unique function(s) for this receptor and its ligands that may lie
beyond their established role in T cell-dependent immunity
(29). To date, there is no evidence in the literature that
IP-10 and/or Mig are necessary for recruitment or activation of
eosinophils in vivo. There is more convincing evidence for IL-5 as well
as eotaxin in many allergic diseases with predominant eosinophilia. Our
results have shown that IP-10 and Mig induce eosinophil chemotaxis
via CXCR3 expression, and that IP-10- and Mig-induced eosinophil
chemotaxis are up- and down-regulated by IL-2 and IL-10, respectively.
Moreover, we have found that CXCR3 mRNA expression in eosinophils is
up- and down-regulated by IL-2 and IL-10, indicating that IL-2 and
IL-10 control IP-10- and Mig-induced eosinophil chemotaxis via
regulation of CXCR3 expression. Besides chemotaxis, we have found other
biological functions of IP-10 and Mig on eosinophils, including ECP
release. To our knowledge, this is the first report that CXCR3 is
expressed on human peripheral eosinophils and that its expression is
regulated by T lymphocyte-associated cytokines. Thus, our results raise
a conceptual issue of the potential biological or pathophysiological
relevance of the presence of CXCR3 in human eosinophils.
ECP, with storage and secreted forms (35), has a variety
of biological activities, interacting with other immune cells and
plasma proteins such as coagulation factors and proteins of the
complement system. It is a major basic granule protein involved in
contact-dependent Ab-mediated cytotoxicity (36). ECP has
been used extensively as a marker for activation and secretion in
eosinophils (37). A number of studies have documented that
local and systemic ECP measurement is a useful way to monitor
eosinophil number and its activation in subjects with allergic
disorders (38, 39). In the present study we have
demonstrated that IP-10 and Mig activate eosinophils to release
ECP. To our knowledge, this is the first direct evidence of ECP release
from eosinophils induced by CXC chemokines. Our data demonstrate the
ECP-releasing capacity of both CXC chemokines, e.g., IP-10 and Mig,
and CC chemokine, e.g., eotaxin. A rather complex picture is now
beginning to take shape of how eosinophils selectively enter allergic
inflammation sites and cause contact-dependent Ab-mediated cytotoxicity
by means of ECP release under association of chemokines and
cytokines.
IL-10, an immunosuppressive and anti-inflammatory cytokine produced
by monocytes and T lymphocytes, regulates both inflammatory/immune
responses by not only modulating the activities of T lymphocyte, B
lymphocyte, and mononuclear phagocyte function, but also by modulating
polymorphonuclear cell-associated chemokine expression
(40). It has been reported that the newly described CC
chemokine HCC-4 is uniquely up-regulated by IL-10 (41),
whereas another novel CC chemokine, designated alternative macrophage
activation-associated CC-chemokine-1, is specifically induced by IL-10
(42). On the other hand, IL-10 inhibits the expression of
IL-8, macrophage inflammatory protein-1, macrophage inflammatory
protein-1ß, and KC in monocytes and macrophages
(43, 44, 45). IL-10 preincubation resulted in the inhibition
of gene expression for several IFN-induced genes, such as IP-10 and
ICAM-1. The reduction in gene expression resulted from the ability of
IL-10 to suppress IFN-induced assembly of STAT factors to specific
promoter motifs on IFN-- and IFN--inducible genes. IL-10 can
directly inhibit STAT-dependent early response gene expression induced
by both IFN- and IFN- in monocytes by suppressing the tyrosine
phosphorylation of STAT1 (46). IL-10 might act indirectly
to suppress IP-10 expression by inhibiting LPS-induced class I IFN
production (47). IL-10 selectively up-regulates the
expression of CCR1, CCR2, and CCR5 in human monocytes by prolonging
their mRNA half-lives, increasing the number of cell surface receptors,
and producing a better chemotactic responsiveness to relevant ligands
(48). CXCR3 is expressed preferentially in Th1 cells and
in lymphoid organs of the
IL-10-/- mouse, which
develops chronic colitis (49). The participation of
eosinophils in inflammation has often been linked to inflammatory
responses with a so-called Th2 profile, such as various forms of
allergic or parasitic diseases. Our findings fit well with the evolving
hypotheses that link chemokine ligand and receptor pairs to Th1, Th2,
or other types of inflammation and suggest that the eosinophil-mediated
inflammation may not exclusively belong to Th2-type patterns. If
stimulated by the Th1-type cytokine IL-2, CXCR3 is up-regulated, and
the cell becomes responsive to IFN--induced chemokines. On the other
hand, if exposed to IL-10, this pathway is inhibited both by reduction
of IFN- induced cytokines and by the expression of their receptor,
CXCR3. Interestingly, the chemotactic activity of eotaxin, which is
believed to be mediated via CCR3, could not be up- or down-regulated by
addition of a number of cytokines (Table I
), suggesting that the
eotaxin-CCR3 ligand-receptor pair is a constitutive chemotactic pathway
for the eosinophils. Thus, IL-10 seems to suppress two different steps
in the inflammatory response mediated by CXC chemokines acting via
CXCR3. This is in concordance with our demonstration that IL-10 can
dramatically down-regulate CXCR3 expression on eosinophils as well as
eosinophil chemotaxis toward IP-10 and Mig.
Our data have pointed out an interesting phenomenon that CXCR3 and its
ligands IP-10 and Mig may play an important role in terms of
eosinophil activation and trafficking during the allergic inflammation.
However, there is no direct evidence of the significance of CXCR3 and
its ligands IP-10 and Mig in vivo and/or pathophysiologically due
to limited observation. A recent study showed that
depletion/neutralization of eotaxin and/or IL-5 in mice is sufficient
to abolish eosinophilia in lung tissue and bronchoalveolar lavage fluid
(50). These results emphasized the importance of the
CCR3-eotaxin receptor-ligand pair in eosinophil recruitment. They also
raise an argument against an important role in vivo for the
CXCR3-mediated recruitment of eosinophils. Therefore, verifying and
clarifying the significance of CXCR3 and its ligands IP-10 and Mig
in vivo in humans in terms of the mechanism of allergic inflammation
will be interesting to examine in further studies.
In summary, we have documented that CXCR3 is expressed on eosinophils
and is up- and down-regulated by IL-2 and IL-10, and that IP-10 and
Mig, via CXCR3, activate eosinophils to chemotaxis, ECP release, and
NF-AT complex nuclear translocation. The present study provides useful
insights into a novel mechanism of the actions of IP-10 and Mig,
which may be especially important in the cytokine/chemokine environment
for the pathophysiologic events of allergic inflammation, including
initiation, progression, and termination of the processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tan Jinquan or Dr. Lars K. Poulsen, Laboratory of Medical Allergology, National University Hospital, DK-2200 Copenhagen N., Denmark.
3 Abbreviations used in this paper: threshold cycle; CXCR, CXC chemokine receptor; CT, threshold cycle; BIM I, bisindolylmaleimide I; C. I., chemotactic index; ECP, eosinophil cationic protein; [Ca2+]i, intracellular calcium; H-8, N-(2-(methylamino)ethyl)-5-isoquenolinesulfonamide dihydrochloride; H-89, N-(2-(-bromocinnamylamino)ethyl)-5-isoquenilesulfonamide; IP-10, IFN--inducible protein-10; a-IL-2R, anti-IL-2R; MCNC, migrating cells on negative control; Mig, monokine induced by IFN-; MPC, magnetic particle concentrator; PKA, protein kinase A; PKC, protein kinase C; PTK, protein tyrosine kinase; PT, pertussis toxin; Sta, staurosporine.
Received for publication October 5, 1999. Accepted for publication May 17, 2000.
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), Mig (
), or
eotaxin (
). The illustrated data are from a single representative
experiment of four performed. All results were determined as described
in Materials and Methods and are expressed as the
chemotactic index (C.I.), based on triplicate determinations of
chemotaxis with each concentration of chemoattractant. The applied
chemokine concentrations (C.C.) are indicated as nanograms per
milliliter. *, Statistically significant difference (all
p < 0.001) in freshly isolated eosinophil
chemotaxis toward 
