Published online before print June 14, 2004
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* Departments of Medical Microbiology and
Urology, Malmö University Hospital,
Cell and Molecular Biology, Lund University, and
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
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(IFN-), tumor necrosis factor 
(TNF-), 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- [MIG; CXC chemokine ligand 9 (CXCL9)], IFN--inducible protein (IP-10/CXCL10), and IFN-inducible T cell 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- 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--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- but not IL-1ß enhanced the IFN--induced production of MIG and IP-10. Conversely, addition of the Th2 cytokine IL-4 down-regulated IFN--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
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, 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- (IFN-; MIG/CXCL9), IFN--inducible protein (IP-10/CXCL10), and IFN-inducible T cell 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- and tumor necrosis factor (TNF-) are key inflammatory mediators [6
7
8
].
Recently, we demonstrated that eosinophils can express GRO- and ENA-78 [9
, 10
]. In the present study, we show that following stimulation with IFN-, eosinophils synthesize and release MIG and IP-10 and express I-TAC mRNA. TNF- 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.
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, TNF-, 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- (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- (0.001, 0.01, 0.1, 1, and 10 ng/mL), IFN- (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- (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.
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induces gene expression of MIG, IP-10, and I-TAC in eosinophils. Thereafter, the RNA preparations were subjected to RT-PCR. As deduced from these experiments, IFN- 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.
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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- (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.
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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- to investigate possible enhancing effects on the chemokine expression. TNF- 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--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
).
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Table 1. Synthesis of MIG and IP-10 in Preparations of Eosinophils Containing Spiked Concentrations of PBMCa
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.
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Figure 2. Extracellular release of MIG and IP-10 by IFN--stimulated eosinophils. (A) Incubation of eosinophils (2x106/mL) in medium in the presence of IFN- (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- 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- at different concentrations. The data are from four different donors and experiments. Data shown represent mean ± SEM.
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for 24 h (Fig. 2c
and 2d)
. The difference in magnitude was attributed to variations between donors.
TNF- but not IL-1ß enhances IFN--induced production of MIG and IP–10
Addition of TNF- to the IFN--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- alone (Fig. 3
). The ratio between the amounts of released chemokines in the presence of IFN- and TNF- or IFN- 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--induced production of MIG and IP-10 in eosinophils at any of these time-points.
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Figure 3. TNF- stimulated the production of MIG and IP-10 from eosinophils during prolonged incubation. Addition of TNF- (10 ng/mL) during incubation of eosinophils (2x106/mL) in IFN--containing medium increased the production of MIG and IP-10. Data are shown as percent (mean±SEM) compared with control (no TNF-) at different time-points (16, 24, and 48 h, respectively). The data are from four different donors and experiments.
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-induced production of MIG and IP-10. Eosinophils were incubated in medium alone, in the presence of IFN-, in the presence of IFN- and IL-4, or in the presence of IFN- 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).
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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- (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.
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a prototypic Th1 cytokine, can synthesize and release MIG and IP-10 and express the I-TAC gene. Addition of TNF- enhanced the IFN--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-, and ENA-78 [9
, 10
]. During inflammation, these chemokines can recruit and activate neutrophils via activation of CXCR2 [1
]. In the presence of IFN-, eosinophil expression of ELR-positive chemokines is down-regulated [9
]. In the present study, it is shown that IFN- 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-, that they possess the IFN receptor, and that IFN- 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- 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- nor in combination with TNF-. One possible explanation is low translation into peptide despite high gene expression.
Further, another Th1 cytokine, TNF-, enhanced IFN--induced MIG and IP-10 production in eosinophils. The synergistic action of TNF- and IFN- may be explained by two different mechanisms regulating the transcriptional activity of the genes. First, the nuclear transcription factor-
B (NF-B), activated by TNF-, may synergize with the IFN-induced transcription factor STAT1. Second, NF-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- and TNF- 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--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-, 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- [24
]. In addition, TNF- 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- 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- and participate in a Th1-polarized, inflammatory response.
Received August 13, 2003; revised April 28, 2004; accepted May 4, 2004.
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and interleukin-4 in inflammatory bowel disease Inflamm. Bowel Dis. 4,285-290[Medline] by human eosinophils J. Immunol. 170,5309-5316 (MIG), IFN-inducible T cell chemoattractant (I-TAC), and IFN--inducible protein-10 (IP-10) chemokines by human neutrophils J. Immunol. 162,4928-4937 receptor on human eosinophils Int. Arch. Allergy Immunol. 118,443-446[Medline] receptors (IFN-R) Clin. Exp. Immunol. 110,524-529[CrossRef][Medline]-inducible protein-10 and monokine induced by IFN- J. Immunol. 165,1548-1556: the quintessence of a mutual antagonistic relationship Scand. J. Immunol. 48,459-468[CrossRef][Medline] and IL-4 differently regulate inducible NO synthase gene expression through IRF-1 modulation Int. Immunol. 12,977-985 Blood 85,1442-1448 interferon in the pathogenesis of severe schistosomiasis in interleukin-4-deficient mice Infect. Immun. 69,7445-7452This article has been cited by other articles:
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