Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0803379 on June 14, 2004

Published online before print June 14, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0803379v1
76/3/685    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dajotoy, T.
Right arrow Articles by Egesten, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dajotoy, T.
Right arrow Articles by Egesten, A.
(Journal of Leukocyte Biology. 2004;76:685-691.)
© 2004 by Society for Leukocyte Biology

Human eosinophils produce the T cell-attracting chemokines MIG and IP-10 upon stimulation with IFN-{gamma}

Terese Dajotoy*, Pia Andersson*, Anders Bjartell{dagger}, Claes-Göran Löfdahl{ddagger}, Hans Tapper§ and Arne Egesten*,1

* Departments of Medical Microbiology and
{dagger} Urology, Malmö University Hospital,
§ Cell and Molecular Biology, Lund University, and
{ddagger} Respiratory Medicine, University Hospital, Lund, Sweden

1 Correspondence: BMC B 14, Tornavägen 10, SE-221 84 Lund, Sweden. E-mail: Arne.Egesten{at}mikrobiol.mas.lu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils participate in allergic inflammation, where expression of T helper cell type 2 (Th2) cytokines such as interleukin (IL)-4 and IL-5 are seen. However, eosinophils sometimes accumulate during disease with expression of Th1 cytokines [i.e., interferon-{gamma} (IFN-{gamma}), tumor necrosis factor {alpha} (TNF-{alpha}), and IL-1ß]. In this study, we investigated whether eosinophils can respond with expression of the IFN-inducible C–X–C chemokines monokine induced by IFN-{gamma} [MIG; CXC chemokine ligand 9 (CXCL9)], IFN-{gamma}-inducible protein (IP-10/CXCL10), and IFN-inducible T cell {alpha} chemoattractant (I-TAC/CXCL11). These chemokines share the ability to recruit and activate T cells and natural killer cells to sites of inflammation. We found that IFN-{gamma} induced rapid and sustained gene expression of MIG, IP-10, and I-TAC in eosinophils, as detected by quantitative reverse transcriptase-polymerase chain reaction. During incubation, IFN-{gamma}-stimulated eosinophils released MIG and IP-10, as detected by enzyme-linked immunosorbent assay, while I-TAC could not be detected in the medium. TNF-{alpha} but not IL-1ß enhanced the IFN-{gamma}-induced production of MIG and IP-10. Conversely, addition of the Th2 cytokine IL-4 down-regulated IFN-{gamma}-induced synthesis of MIG and IP–10 in eosinophils. Crohn’s disease is characterized by a Th1-polarized inflammation and presence of eosinophils. In lesions from this disease, MIG was detected in eosinophils by immunohistochemistry. Taken together, the results point to immunoregulatory roles for eosinophils during some diseases with Th1-polarized inflammation.

Key Words: CXCL9 • CXCL10 • CXCL11 • CXCR3


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are peptides that regulate leukocyte trafficking, and their importance in inflammatory processes is illustrated by their potent effects on recruitment and activation of specific subsets of leukocytes. One group of chemokines is the C–X–C chemokines, which can be subdivided further into two classes depending on the presence of a glutamate-leucine-arginine (ELR) motif preceding the first two cysteines of the molecules. Interleukin-8 (IL-8), growth-related oncogene (GRO)-{alpha}, and epithelial cell-derived neutrophil-activating peptide (ENA)-78/CXC chemokine ligand 5 (CXCL5) are chemokines that contain the ELR motif, and they exert stimulatory and chemotactic activities toward neutrophils [1 ]. Monokine induced by interferon-{gamma} (IFN-{gamma}; MIG/CXCL9), IFN-{gamma}-inducible protein (IP-10/CXCL10), and IFN-inducible T cell {alpha} chemoattractant (I-TAC/CXCL11), conversely, are ELR-negative C–X–C chemokines. MIG, IP-10, and I-TAC all share the ability to signal through a G protein-coupled receptor, CXC chemokine receptor 3 (CXCR3) [2 ], which is present on subsets of lymphocytes, i.e., T cells and natural killer cells, and activation of the receptor results in recruitment and activation of these cells [1 ].

Eosinophils have a typical content of cytotoxic granule proteins that are subject to regulated release at sites of inflammation [3 ]. In addition, they may have immunoregulatory roles through expression of several cytokines [4 ]. Presence of eosinophils is typically seen during allergic inflammation in diseases such as asthma and parasitic infestation. Allergic inflammation is characterized by expression of T helper cell type 2 (Th2) cytokines by immunoregulatory cells, for example, IL-4 and IL-5 [5 ]. However, abundant presence of eosinophils is also seen during some states of disease characterized by expression of Th1 cytokines. Crohn’s disease is an example of such an inflammation, where the cytokines IFN-{gamma} and tumor necrosis factor {alpha} (TNF-{alpha}) are key inflammatory mediators [6 7 8 ].

Recently, we demonstrated that eosinophils can express GRO-{alpha} and ENA-78 [9 , 10 ]. In the present study, we show that following stimulation with IFN-{gamma}, eosinophils synthesize and release MIG and IP-10 and express I-TAC mRNA. TNF-{alpha} but not IL-1ß enhanced the expression. In addition, we show that IL-4 can down-regulate the synthesis of MIG and IP-10 in eosinophils. Finally, we provide support for in vivo production of MIG by eosinophils in lesions of Crohn’s disease. These findings point out roles for eosinophils during disease that are chararacterized by expression of Th1 cytokines and presence of eosinophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Special reagents
Recombinant human IFN-{gamma}, TNF-{alpha}, IL-1ß, IL-4, IL-5, mouse monoclonal antibody (mAb) against human MIG (clone # 49106), irrelevant isotype-matched mouse immunoglobulin G (IgG), and enzyme-linked immunosorbent assays (ELISAs) for the detection of MIG, IP-10, and I-TAC were all purchased from R&D Systems (Oxon, UK). The human MIG Quantikine colorimetric sandwich ELISA had a sensitivity of 3.84 pg/mL and range, 31.2–2000 pg/mL; human IP-10 quantikine colorimetric sandwich ELISA, sensitivity, 1.7 pg/mL, and range, 7.8–500 pg/mL; and human I-TAC Quantikine colorimetric sandwich ELISA, sensitivity, 14.9 pg/mL, and range, 62.5–4000 pg/mL. The samples were measured undiluted or diluted in incubation medium in duplicates.

Cell isolation
Citrated blood was obtained from healthy volunteers after informed consent, and eosinophils were isolated essentially as described [11 ]. In short, after isolation of granulocytes on Ficoll-Paque (Pharmacia, Uppsala, Sweden), immunomagnetic beads coated with antibodies to CD16 (Miltenyi, Gladbach, Germany) were used to retrieve the neutrophils in a magnetic column, allowing the isolation of highly purified eosinophils. The purity of eosinophils was more than 98%; contaminating cells were neutrophils and mononuclear cells, as judged by routine May-Grünwald-Giemsa staining. In selected experiments, peripheral blood mononuclear cells (PBMC) were removed from the upper layer after centrifugation of whole blood over Ficoll-Paque. PBMC consisted of 27% ± 6% (mean±SD) monocytes and 73% ± 7% lymphocytes, as judged by flow cytometry using side-scatter and forward-scatter characteristics and labeling of the cells by a combination of CD45 and CD14 mAb (Dakopatts, Glostrup, Denmark).

Measurement of released MIG, IP-10, and I-TAC by ELISA
During prolonged incubation, eosinophils (2x106/mL) were suspended in RPMI 1640 (Gibco Life Technologies, Merelbeke, Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS) and gentamycin (10 µg/mL) and IFN-{gamma} (100 U/mL) in 24- or 96-well plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere containing 5% CO2. In selected experiments, IL-1ß (0.001, 0.01, 0.1, 1, and 10 ng/mL), IL-4 (0.0001, 0.001, 0.01, 0.1, and 1 nM), IL-5 (0.0001, 0.001, 0.01, 0.1, and 1 nM), TNF-{alpha} (0.001, 0.01, 0.1, 1, and 10 ng/mL), IFN-{gamma} (1, 10, 100, and 1000 U/mL), or medium alone was added to the cells, and coincubation was performed for 24 h, whereafter, the plates were centrifuged for 10 min at 500 g, and the supernatants were collected. Immediately after isolation, the cell viability was >99%, >98% after 16 h, >96% after 24 h, and >90% after 48 h of incubation, as judged by trypan blue exclusion. Cells were lysed 5 x 106 cells/mL in 1% Triton X-100 (Sigma Chemical Co., St. Louis, MO) in phosphate-buffered saline for 20 min on ice. The supernatants and cell lysates were stored at –70°C until analyzed by ELISA.

The monocytic cell line THP-1 (American Type Culture Collection, Manassas, VA) was grown in RPMI 1640 supplemented with 10% heat-inactivated FCS and gentamycin (10 µg/mL) in 24-well plates at 37°C in a humidified atmosphere containing 5% CO2. Cells (106 cells/mL) were stimulated with IFN-{gamma} (100 U/mL) for 16 h followed by addition of lipopolysaccharide (LPS; 100 ng/mL) for 4 h.

Detection of MIG, IP-10, and I-TAC expression by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR (Q-PCR)
Total cellular RNA was isolated using a kit based on a modified, single-step procedure by acid guandinium thiocyanate-phenol-chloroform extraction (Total RNA IsolationTM, BD Biosciences, Stockholm, Sweden).

The primer sequences for MIG were 5'-TTA AAC AAT TTG CCC CAA GC-3' (sense) and 5'-CTG TTG TGA GTG GGA TGT GG-3' (antisense) and for IP-10 were 5'-AGA GGA ACC TCC AGT CTC AGC-3' (sense) and 5'-CCT CTG TGT GGT CCA TCC TT-3' (antisense). The primer sequences for I-TAC were 5'-GCT ATA GCC TTG GCT GTG ATA T-3' (sense) and 5'-CAG GGC CTA TGC AAA GAC A-3' (antisense). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. The GAPDH primers were 5'-ACC ACC ATG GAG AAG GCT GG-3' (sense) and 5'-CAC AGT GTA GCC CAG GAT GC-3' (antisense).

The RT-PCR reactions were performed using premixed, predispensed reaction tubes to which 100 ng RNA and primer pairs were added (Ready-To-Go RT-PCR BeadsTM, Pharmacia, Uppsala, Sweden). First-strand cDNA synthesis was performed at 39°C for 45 min, followed by denaturation at 95°C for 5 min. The conditions for the PCR were as follows: denaturation at 94°C for 60 s, annealing at 53°C for 90 s, and elongation at 72°C for 120 s for 40 cycles. Samples (10 µl) of the PCR-reaction mixtures were loaded on a 2% agarose gel and stained with ethidium bromide. The PCR products were detected by UV light.

To improve the quantification of mRNA, Q-PCR was performed. First, cDNA was synthesized from RNA, isolated as described above, with TaqManTM RT reagent (Applied Biosystems, Foster City, CA) using 50 ng total RNA as template and random hexamers as primers in a total reaction volume of 100 µL. Q-PCR was run with TaqMan universal mastermix (Applied Biosystems) in an ABI PRISM 7700 sequence detection system in a total reaction volume of 25 µL. Amplification of MIG, IP-10, and I-TAC was performed using premade kits (Assay-on-DemandTM, Applied Biosystems). As endogenous controls, GAPDH mRNA and 18S rRNA were amplified (Applied Biosystems). Because of higher stability, 18S rRNA was chosen as endogenous control throughout the experiments. All Q-PCR experiments were performed following the general guidelines from Applied Biosystems.

Immunohistological detection of MIG during Crohn’s disease
Archived diagnostic biopsies obtained at presentation of disease in patients suffering from Crohn’s disease were used to detect MIG-containing cells and eosinophils. In short, sections, 3 µm in thickness, were cut, and after deparaffinization and rehydration, they were incubated with a mAb against MIG (10 µg/mL) and a polyclonal rabbit antibody against the eosinophil granule protein eosinophil cationic protein (ECP; a gift from Dr. Inge Olsson, Department of Hematology, Lund University, Lund, Sweden). Bound antibodies were detected and visualized by a secondary goat anti-mouse antibody [Alexa Fluor 594 F(ab')2 fragment of goat anti-mouse IgG (H+L)] and a goat anti-rabbit antibody [Alexa Fluor 488 F(ab')2 fragment of goat anti-rabbit IgG (H+L)], respectively (Molecular Probes, Eugene, OR). The secondary antibodies were used 2 µg/mL. Omission of the primary antibodies was used to exclude unspecific binding of the secondary antibodies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IFN-{gamma} induces gene expression of MIG, IP-10, and I-TAC in eosinophils
RNA was extracted from eosinophils immediately after isolation and after 3 and 18 h of incubation, respectively, in the absence or presence of IFN-{gamma}. Thereafter, the RNA preparations were subjected to RT-PCR. As deduced from these experiments, IFN-{gamma} induced a rapid transcription of MIG, IP-10, and I-TAC in eosinophils, and the mRNA expression sustained during prolonged incubation in vitro (Fig. 1 ). To improve the quantification of mRNA, Q-PCR was performed and confirmed the impression from RT-PCR that MIG expression increased comparing the 3-h time-point with 18 h. In contrast, IP-10 and I-TAC expression was higher after 3 h than after 18 h.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 1. Gene expression of MIG, IP-10, and I-TAC in eosinophils during prolonged incubation. Eosinophils (2x106/mL) were incubated in medium with or without IFN-{gamma} (100 U/mL) for a total time of 18 h. At different time-points (0, 3, and 18 h), RNA was isolated from the cells and thereafter, subjected to Q-PCR and RT-PCR (lower insert). 18S rRNA was used as housekeeping in Q-PCR experiments and GAPDH in RT-PCR to ensure equal loading. The RT-PCR data shown are from one representative out of three separate donors and experiments. The Q-PCR data represent the mean from two separate donors and experiments.

 
Eosinophils produce MIG and IP-10
After 24 h of incubation, MIG and IP-10 were readily detectable in the medium by ELISA. I-TAC was not detected in supernatants nor in cell lysates. The monocytic cell line THP-1 served as a positive control for I-TAC production. After 16 h of incubation in the presence of IFN-{gamma} followed by 4 h of stimulation with LPS, I-TAC could be detected in the medium at ~4 ng/mL.

Several cytokines were used in combination with IFN-{gamma} to investigate possible enhancing effects on the chemokine expression. TNF-{alpha} showed an enhancing effect on the production of MIG and IP-10. This was not the case for IL-1ß, a cytokine that promotes IFN-{gamma}-induced expression of the chemokines in other cells [12 ]. However, even after stimulation with different cytokines, I-TAC could not be detected in the medium nor in cellular lysates. To exclude that PBMC, contaminating the eosinophil preparations, were the predominating source of MIG and IP-10, eosinophils were incubated with different concentrations of PBMC (i.e., <2%, 5%, and 10%, respectively). This did not significantly affect the chemokine concentrations of the supernatants, thus demonstrating that eosinophils indeed synthesize MIG and IP-10 (Table 1 ).


View this table:
[in this window]
[in a new window]
  		Table 1. Synthesis of MIG and IP-10 in Preparations of Eosinophils Containing Spiked Concentrations of PBMCa

 
In Figure 2 , a and b, increasing concentrations of MIG and IP-10 in medium with time are seen. The amount of released proteins was measured using ELISA after 16, 24, and 48 h of incubation in the presence of IFN-{gamma}.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 2. Extracellular release of MIG and IP-10 by IFN-{gamma}-stimulated eosinophils. (A) Incubation of eosinophils (2x106/mL) in medium in the presence of IFN-{gamma} (100 U/mL) resulted in an increased extracellular release of MIG and IP-10. Aliquots were taken at different time-points (0, 16, 24, and 48 h), and the amount of MIG and IP-10 was measured in the cell-free supernatant using ELISA. The data are from four different donors and experiments. Values are means ± SEM. (b) IFN-{gamma} showed a dose-dependent, stimulating effect on the production of MIG and IP-10 from eosinophils. The cells were incubated for 24 h in the presence of IFN-{gamma} at different concentrations. The data are from four different donors and experiments. Data shown represent mean ± SEM.

 
A dose-response relationship with respect to MIG and IP-10 productions was seen after stimulation of eosinophils with increasing concentrations of IFN-{gamma} for 24 h (Fig. 2c and 2d) . The difference in magnitude was attributed to variations between donors.

TNF-{alpha} but not IL-1ß enhances IFN-{gamma}-induced production of MIG and IP–10
Addition of TNF-{alpha} to the IFN-{gamma}-containing medium caused a dose- and time-dependent increase in the production of MIG and IP-10 compared with cells incubated in the presence of IFN-{gamma} alone (Fig. 3 ). The ratio between the amounts of released chemokines in the presence of IFN-{gamma} and TNF-{alpha} or IFN-{gamma} alone remained constant during the time-course investigated (16, 24, and 48 h, respectively). IL-1ß (in a concentration range of 0.001–10 ng/mL) showed no effect on the IFN-{gamma}-induced production of MIG and IP-10 in eosinophils at any of these time-points.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. TNF-{alpha} stimulated the production of MIG and IP-10 from eosinophils during prolonged incubation. Addition of TNF-{alpha} (10 ng/mL) during incubation of eosinophils (2x106/mL) in IFN-{gamma}-containing medium increased the production of MIG and IP-10. Data are shown as percent (mean±SEM) compared with control (no TNF-{alpha}) at different time-points (16, 24, and 48 h, respectively). The data are from four different donors and experiments.

 
Down-regulation of MIG and IP-10 production by the Th2 cytokine IL-4 but not IL-5
IL-4, a key cytokine in the polarization of inflammatory responses toward a Th2 profile during allergic inflammation, decreased the IFN-{gamma}-induced production of MIG and IP-10. Eosinophils were incubated in medium alone, in the presence of IFN-{gamma}, in the presence of IFN-{gamma} and IL-4, or in the presence of IFN-{gamma} and IL-5 for 24 h. Thereafter, MIG and IP-10 were detected in medium and cell lysates using ELISA. IL-4 showed a strong, dose-dependent inhibitory effect on MIG and IP-10 synthesis (Fig. 4a and 4b ). However, no effect from IL-5 was seen. IL-4 did not affect cell viability nor was secretion inhibited by IL-4 as detected by the MIG and IP-10 content in cell lysates (not shown).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. Th2 cytokine IL-4 has a dose-dependent, inhibitory effect on the release of MIG and IP-10. Eosinophils (2x106/mL) were incubated for 24 h in medium containing IFN-{gamma} (100 U/mL), and IL-4 and IL-5, respectively, were added at different concentrations. After 24 h, the amount of extracellular MIG (a) and IP-10 (b) was measured in the cell-free supernatant using ELISA. IL-4 inhibited the release, and IL-5 had no effect. Values are shown as percent (mean±SEM) of control at different concentrations of cytokine. The data are from four different donors and experiments. In control, no IL-4 or IL-5 is added.

 
In vivo presence of MIG in eosinophils during Crohn’s disease
Patients with Crohn’s disease have an increased number of tissue eosinophils. To investigate whether eosinophils during disease show presence of MIG, colon tissue obtained from patients suffering from Crohn’s disease was subjected to immunohistochemistry. A mouse mAb against MIG and a polyclonal rabbit antibody against the eosinophil granule protein ECP were used. Many of the MIG-containing cells had a phenotype resembling eosinophils, and ECP detected by immunofluorescence on the same sections showed a similar pattern of cellular labeling (Fig. 5a and 5c ). Superposition of the labelings for MIG and ECP showed a high degree of cellular colocalization, thus providing evidence for the presence of MIG in eosinophils present in lesions of Crohn’s disease (Fig. 5e) .



View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. Presence of MIG in Crohn’s disease. (a) Detection of bound mAb against MIG by secondary Alexa-conjugated goat anti-mouse antibody on colon tissue section from a patient suffering from Crohn’s disease. Red fluorescence indicates bound antibodies against MIG (arrows). Original magnification, x40. (b) As a control, tissue sections were incubated with secondary antibody alone, resulting in loss of labeling. Original magnification, x40. (c) Detection of the eosinophil granule protein ECP on the same tissue section as in a. Bound primary polyclonal antibody is detected and visualized by secondary Alexa-conjugated goat anti-rabbit antibody, resulting in green fluorescence. Original magnification, x40. (d) As a control, tissue sections were incubated with secondary antibody alone. Absence of primary antibody resulted in loss of labeling. Original magnification, x40. (e) Overlay from a and c, resulting in orange color, thus demonstrating presence of MIG in eosinophils (arrows). Original magnification, x40.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates that eosinophils, after stimulation with IFN-{gamma}, a prototypic Th1 cytokine, can synthesize and release MIG and IP-10 and express the I-TAC gene. Addition of TNF-{alpha} enhanced the IFN-{gamma}-induced production of MIG and IP-10, and IL-4, a typical Th2 cytokine, inhibited the production of MIG and IP-10.

Eosinophils are seen at sites of inflammation and are known to release toxic granule proteins upon specific stimuli, for example, in the airways during allergic asthma and during host defense against invading parasites [13 ]. However, their role in diseases may not be restricted to degranulation. Eosinophils are potent producers of various inflammatory mediators, such as proinflammatory lipid-derived substances, cytokines, and chemokines [14 ]. Among chemokines, eosinophils can express and release at least three different ELR-positive C–X–C chemokines, IL-8, GRO-{alpha}, and ENA-78 [9 , 10 ]. During inflammation, these chemokines can recruit and activate neutrophils via activation of CXCR2 [1 ]. In the presence of IFN-{gamma}, eosinophil expression of ELR-positive chemokines is down-regulated [9 ]. In the present study, it is shown that IFN-{gamma} induces expression of ELR-negative C–X–C chemokines in eosinophils. MIG, IP-10, and I-TAC attract Th1 T cells via activation of the receptor CXCR3 [15 ].

It has been shown by several investigators that eosinophils do respond to IFN-{gamma}, that they possess the IFN receptor, and that IFN-{gamma} induces phosphorylation of the transcription factor, signal transducer and activator of transcription (STAT)1 in eosinophils [16 , 17 ].

Eosinophils themselves express CXCR3, suggesting that they can be activated and recruited by MIG, IP-10, and I-TAC during Th1-polarized inflammation [18 ]. During allergic inflammation, the chemokine receptor (CCR)3 is important for recruitment of eosinophils and basophils [14 ]. Recently, it was shown that MIG, IP-10, and I-TAC are antagonists for CCR3, thus down-regulating the allergic, inflammatory response [19 ]. Taken together, IFN-{gamma} seems to have a key role in the switch from production of ELR-positive chemokines in favor of ELR-negative chemokine expression in eosinophils.In vitro, MIG and IP-10 show chemotactic activity against CXCR3-bearing cells in the nanomolar range [20 ]. Therefore, the amount of chemokine synthesis reported here is likely to be of biological significance in vivo.

In the present study, eosinophil production of I-TAC could not be detected in the medium, neither in the presence of IFN-{gamma} nor in combination with TNF-{alpha}. One possible explanation is low translation into peptide despite high gene expression.

Further, another Th1 cytokine, TNF-{alpha}, enhanced IFN-{gamma}-induced MIG and IP-10 production in eosinophils. The synergistic action of TNF-{alpha} and IFN-{gamma} may be explained by two different mechanisms regulating the transcriptional activity of the genes. First, the nuclear transcription factor-{kappa}B (NF-{kappa}B), activated by TNF-{alpha}, may synergize with the IFN-induced transcription factor STAT1. Second, NF-{kappa}B can increase the binding of the transcription factor IFN regulatory factor (IRF) to the promoter region, further increasing the transcriptional activity [21 ]. In diseases such as Crohn’s disease, where IFN-{gamma} and TNF-{alpha} are expressed, this synergy is likely to be of pathophysiological importance.

Conversely, IL-4, a prototypic Th2 cytokine, down-regulated synthesis of MIG and IP-10 from eosinophils. A possible explanation for the effect from IL-4 may be a down-regulation of IFN-{gamma}-inducible transcription by activation of STAT6, which in turn inhibits IRF-1 expression. [22 ]. However, we were not able to make firm conclusions with regard to the IL-4 effect on gene expression using Q-PCR. The eosinophil-activating cytokine IL-5, similarly to IFN-{gamma}, activates STAT1, thus explaining the lack of effect from IL-5 on IFN-induced gene expression [23 ]. This switch may be important during some states of disease. During the early stages of Crohn’s disease, there is a Th2-polarized inflammation, and in chronical intestinal lesions, a Th1-type cytokine profile predominates, including high levels of IFN-{gamma} [24 ]. In addition, TNF-{alpha} is a key cytokine in the pathogenesis of Crohn’s disease [25 ]. Further, this disease is associated with an increased number of eosinophils in the inflamed tissue [26 ]. Another important group of diseases, with presence of eosinophils and a mixed Th1/Th2 inflammatory response, are parasitic infestation, for example, Schistsomiasis [27 ]. The importance of a balance between IFN-{gamma} and IL-4 has been shown in an animal model using Schistosoma-infected mice [28 ]. The granulomas formed during Schistosomiasis are a complex mix of cellular phenotypes, where eosinophils are a major constituent, making up ~50% of the population [29 ]. Therefore, eosinophils may have important roles, orchestrating the inflammatory response during parasitic infestation. A fine-tuning of IFN-induced CXC chemokines by IL-4 can be important to counteract excessive inflammation and development of fibrosis.

In this study, we show that eosinophils can express and produce MIG and IP-10 in vitro upon stimulation. In addition, eosinophils in inflamed tissue of Crohn’s disease contain MIG, suggesting proinflammatory roles for eosinophils during Th1 inflammation. The findings suggest that despite being a typical component of Th2-mediated inflammation, eosinophils have the capability to respond to IFN-{gamma} and participate in a Th1-polarized, inflammatory response.


   ACKNOWLEDGEMENTS
 
This study was supported by grants from the T. C. Bergh Foundation, the Grönberg Foundation, the Bengt Ihre Foundation, the Greta and Johan Kock Foundations, and the Swedish Asthma and Allergy Association’s Research Foundation.

Received August 13, 2003; revised April 28, 2004; accepted May 4, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Baggiolini, M. (2001) Chemokines in pathology and medicine J. Intern. Med. 250,91-104[CrossRef][Medline]
  2. Murdoch, C., Finn, A. (2000) Chemokine receptors and their role in inflammation and infectious diseases Blood 95,3032-3043[Abstract/Free Full Text]
  3. Egesten, A., Calafat, J., Janssen, H., Knol, E. F., Malm, J., Persson, T. (2001) Granules of human eosinophilic leucocytes and their mobilization Clin. Exp. Allergy 31,1173-1188[CrossRef][Medline]
  4. Lacy, P., Moqbel, R. (2000) Eosinophil cytokines Chem. Immunol. 76,134-155[Medline]
  5. Lucey, D. R., Clerici, M., Shearer, G. M. (1996) Type 1 and type 2 cytokine dysregulation in human infectious, neoplastic, and inflammatory diseases Clin. Microbiol. Rev. 9,532-562[Abstract]
  6. Jeziorska, M., Haboubi, N., Schofield, P., Woolley, D. E. (2001) Distribution and activation of eosinophils in inflammatory bowel disease using an improved immunohistochemical technique J. Pathol. 194,484-492[Medline]
  7. Hommes, D. W., van Deventer, S. J. (2000) Anti- and proinflammatory cytokines in the pathogenesis of tissue damage in Crohn’s disease Curr. Opin. Clin. Nutr. Metab. Care 3,191-195[CrossRef][Medline]
  8. Camoglio, L., Te Velde, A. A., Tigges, A. J., Das, P. K., Van Deventer, S. J. (1998) Altered expression of interferon-{gamma} and interleukin-4 in inflammatory bowel disease Inflamm. Bowel Dis. 4,285-290[Medline]
  9. Persson-Dajotoy, T., Andersson, P., Bjartell, A., Calafat, J., Egesten, A. (2003) Expression and production of the CXC chemokine growth-related oncogene-{alpha} by human eosinophils J. Immunol. 170,5309-5316[Abstract/Free Full Text]
  10. Persson, T., Monsef, N., Andersson, P., Bjartell, A., Malm, J., Calafat, J., Egesten, A. (2003) Expression of the neutrophil-activating CXC chemokine ENA-78/CXCL5 by human eosinophils Clin. Exp. Allergy 33,531-537[CrossRef][Medline]
  11. Hansel, T. T., De Vries, I. J., Iff, T., Rihs, S., Wandzilak, M., Betz, S., Blaser, K., Walker, C. (1991) An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils J. Immunol. Methods 145,105-110[CrossRef][Medline]
  12. Gasperini, S., Marchi, M., Calzetti, F., Laudanna, C., Vicentini, L., Olsen, H., Murphy, M., Liao, F., Farber, J., Cassatella, M. A. (1999) Gene expression and production of the monokine induced by IFN-{gamma} (MIG), IFN-inducible T cell {alpha} chemoattractant (I-TAC), and IFN-{gamma}-inducible protein-10 (IP-10) chemokines by human neutrophils J. Immunol. 162,4928-4937[Abstract/Free Full Text]
  13. Weller, P. F. (1991) The immunobiology of eosinophils N. Engl. J. Med. 324,1110-1118[Medline]
  14. Rothenberg, M. E. (1998) Eosinophilia N. Engl. J. Med. 338,1592-1600[Free Full Text]
  15. Luster, A. D. (1998) Chemokines–chemotactic cytokines that mediate inflammation N. Engl. J. Med. 338,436-445[Free Full Text]
  16. Ochiai, K., Ishihara, C., Tomioka, H. (1999) Signal transduction through interferon-{gamma} receptor on human eosinophils Int. Arch. Allergy Immunol. 118,443-446[Medline]
  17. Ishihara, C., Ochiai, K., Kagami, M., Takashahi, H., Matsuyama, G., Yoshida, S., Tomioka, H., Koya, N. (1997) Human peripheral eosinophils express functional interferon-{gamma} receptors (IFN-{gamma}R) Clin. Exp. Immunol. 110,524-529[CrossRef][Medline]
  18. Jinquan, T., Jing, C., Jacobi, H. H., Reimert, C. M., Millner, A., Quan, S., Hansen, J. B., Dissing, S., Malling, H. J., Skov, P. S., Poulsen, L. K. (2000) CXCR3 expression and activation of eosinophils: role of IFN-{gamma}-inducible protein-10 and monokine induced by IFN-{gamma} J. Immunol. 165,1548-1556[Abstract/Free Full Text]
  19. Loetscher, P., Clark-Lewis, I. (2001) Agonistic and antagonistic activities of chemokines J. Leukoc. Biol. 69,881-884[Abstract/Free Full Text]
  20. Proost, P., Schutyser, E., Menten, P., Struyf, S., Wuyts, A., Opdenakker, G., Detheux, M., Parmentier, M., Durinx, C., Lambeir, A. M., Neyts, J., Liekens, S., Maudgal, P. C., Billiau, A., Van Damme, J. (2001) Amino-terminal truncation of CXCR3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties Blood 98,3554-3561[Abstract/Free Full Text]
  21. Paludan, S. R. (1998) Interleukin-4 and interferon-{gamma}: the quintessence of a mutual antagonistic relationship Scand. J. Immunol. 48,459-468[CrossRef][Medline]
  22. Coccia, E. M., Stellacci, E., Marziali, G., Weiss, G., Battistini, A. (2000) IFN-{gamma} and IL-4 differently regulate inducible NO synthase gene expression through IRF-1 modulation Int. Immunol. 12,977-985[Abstract/Free Full Text]
  23. van der Bruggen, T., Caldenhoven, E., Kanters, D., Coffer, P., Raaijmakers, J. A., Lammers, J. W., Koenderman, L. (1995) Interleukin-5 signaling in human eosinophils involves JAK2 tyrosine kinase and Stat1 {alpha} Blood 85,1442-1448[Abstract/Free Full Text]
  24. Desreumaux, P., Brandt, E., Gambiez, L., Emilie, D., Geboes, K., Klein, O., Ectors, N., Cortot, A., Capron, M., Colombel, J. F. (1997) Distinct cytokine patterns in early and chronic ileal lesions of Crohn’s disease Gastroenterology 113,118-126[CrossRef][Medline]
  25. McCormack, G., Moriarty, D., O’Donoghue, D. P., McCormick, P. A., Sheahan, K., Baird, A. W. (2001) Tissue cytokine and chemokine expression in inflammatory bowel disease Inflamm. Res. 50,491-495[CrossRef][Medline]
  26. Egesten, A., Andersson, P., Persson, T. (2002) Eosinophils in gastrointestinal inflammation: from innocent bystanders to offenders Scand. J. Gastroenterol. 37,1117-1125[Medline]
  27. Hoffmann, K. F., Cheever, A. W., Wynn, T. A. (2000) IL-10 and the dangers of immune polarization: excessive type 1 and type 2 cytokine responses induce distinct forms of lethal immunopathology in murine schistosomiasis J. Immunol. 164,6406-6416[Abstract/Free Full Text]
  28. La Flamme, A. C., Patton, E. A., Pearce, E. J. (2001) Role of {gamma} interferon in the pathogenesis of severe schistosomiasis in interleukin-4-deficient mice Infect. Immun. 69,7445-7452[Abstract/Free Full Text]
  29. Metwali, A., de Andres, B., Blum, A., Elliott, D., Li, J., Qadir, K., Sandor, M., Weinstock, J. (2002) Th2-type granuloma development in acute murine schistosomiasis is only partly dependent on CD4+ T cells as the source of IL-4 Eur. J. Immunol. 32,1242-1252[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
M. Lampinen, M. Backman, O. Winqvist, F. Rorsman, A. Ronnblom, P. Sangfelt, and M. Carlson
Different regulation of eosinophil activity in Crohn's disease compared with ulcerative colitis
J. Leukoc. Biol., December 1, 2008; 84(6): 1392 - 1399.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Y. Liu, M. E. Bates, N. N. Jarjour, W. W. Busse, P. J. Bertics, and E. A. B. Kelly
Generation of Th1 and Th2 Chemokines by Human Eosinophils: Evidence for a Critical Role of TNF-{alpha}
J. Immunol., October 1, 2007; 179(7): 4840 - 4848.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0803379v1
76/3/685    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dajotoy, T.
Right arrow Articles by Egesten, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dajotoy, T.
Right arrow Articles by Egesten, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS