HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jahnsen, F. L. Articles by Johansen, F.-E. Search for Related Content PubMed PubMed Citation Articles by Jahnsen, F. L. Articles by Johansen, F.-E.

Glucocorticosteroids Inhibit mRNA Expression for Eotaxin, Eotaxin-2, and Monocyte-Chemotactic Protein-4 in Human Airway Inflammation with Eosinophilia¹

Frode L. Jahnsen^2,*, Rolf Haye, Einar Gran, Per Brandtzaeg^* and Finn-Eirik Johansen^*

^* Laboratory for Immunohistochemistry and Immunopathology (LIIPAT), Institute of Pathology, and Department of Ear, Nose, and Throat, University of Oslo, The National Hospital, Rikshospitalet, Oslo, Norway

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
How eosinophils are preferentially recruited to inflammatory sites remains elusive, but increasing evidence suggests that chemokines that bind to the CCR3 participate in this process. In this study, we investigated the transcript levels and chemotactic activity of CCR3-binding chemokines in nasal polyps, a disorder often showing prominent eosinophilia. We found that mRNA expression for eotaxin, eotaxin-2, and monocyte-chemotactic protein-4 was significantly increased in nasal polyps compared with turbinate mucosa from the same patients, or histologically normal nasal mucosa from control subjects. Interestingly, the novel CCR3-specific chemokine, eotaxin-2, showed the highest transcript levels. Consistent with these mRNA data, polyp tissue fluid exhibited strong chemotactic activity for eosinophils that was significantly inhibited by a blocking Ab against CCR3. When patients were treated systemically with glucocorticosteroids, the mRNA levels in the polyps were reduced to that found in turbinate mucosa for all chemokines. Together, these findings suggested an important role for CCR3-binding chemokines in eosinophil recruitment to nasal polyps. Such chemokines, therefore, most likely contribute significantly in the pathogenesis of eosinophil-related disorders; and the reduced chemokine expression observed after steroid treatment might reflect, at least in part, how steroids inhibit tissue accumulation of eosinophils.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue eosinophilia is a characteristic feature of several chronic inflammatory airway disorders, and increasing evidence suggests that these cells can cause tissue damage by release of their cytotoxic granule proteins and lipid mediators (1). Mechanisms that control leukocyte extravasation to sites of inflammation are only partially understood, but a recently identified large family of chemotactic cytokines, or chemokines, probably controls several steps in the transmigratory process (2, 3, 4, 5, 6). In vitro studies have shown that chemokines presented on the surface of endothelial cells mediate integrin activation in rolling leukocytes within seconds (7, 8). This enables their strong adhesion through activated integrins that bind to endothelial ligands. When the leukocyte firmly adheres at the endothelial surface, concentration gradients of chemokines produced beneath the vessel wall supposedly direct the cell across the vascular wall and guide the subsequent migration within the tissue toward an inflammatory target.

Compared with classical chemoattractants that act on most leukocytes, chemokines show specificity for different leukocyte subsets (9). For example, a clear distinction can be made between chemokines that act on neutrophils (the CXC family) and eosinophils (the C-C family). Normodense eosinophils express high levels only of the C-C chemokine receptor CCR3 (10, 11), which is expressed by eosinophils, basophils (12), and a T cell subset (13). Several chemokines of the CC family, including eotaxin, RANTES, monocyte-chemotactic protein-3 (MCP-3)³ and MCP-4, have been shown to attract and activate eosinophils via high affinity binding to CCR3 (14, 15, 16, 17, 18). However, only eotaxin binds selectively to this receptor, which supposedly explains that eotaxin is relatively selective in eosinophil recruitment (19). More recently, another CCR3-specific chemokine termed eotaxin-2 has been identified (20, 21), which functionally is quite similar to eotaxin (20, 21, 22).

In various eosinophil-associated disorders such as asthma and allergic rhinitis, increased levels of several eosinophil-activating chemokines have been detected in the inflammatory lesions (9, 23), suggesting that these mediators are involved in the pathogenesis. However, the relative roles of various chemokines remain poorly defined, and particularly the expression of eotaxin-2 has not as yet been examined.

Nasal polyps share several histopathological features with chronic allergic reactions such as bronchial asthma, including a prominent and often selective infiltration with eosinophils. Another common feature of eosinophil-related disorders is the remarkable anti-inflammatory effect of glucocorticosteroids, including reduction of airway eosinophilia (24, 25, 26). To investigate preferential chemokine association with eosinophil recruitment, we examined the transcript levels and chemotactic activity of CCR3-binding chemokines in nasal polyp tissue. The putative pathogenic involvement of the same chemokines was furthermore studied by measuring the effect of systemic glucocorticoid treatment on their mRNA levels in this inflammatory disorder.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Subjects

Nasal polyp patients included in this study (Table I) were part of a clinical trial evaluating whether preoperative systemic glucocorticosteroid treatment could facilitate the surgical procedure and reduce postoperative complications. All patients required surgical treatment for extensive nasal polyposis and were randomly selected for steroid treatment. The treatment group (n = 12) received 40 mg prednisone once daily for 7 days before surgery. The untreated group (n = 15) had not used systemic or topical steroids for at least 1 mo before admission. Patients with seasonal allergic rhinitis were subjected to surgery when being out of their allergen season. Blood samples were obtained from the treatment group the day before medication start and from all patients at the day of surgery. Polyp size was subjectively estimated by rigid endoscopy before and after treatment. Histologic evaluation of the material showed characteristic polyp pathology; the lower turbinate specimens obtained in parallel displayed either normal nasal mucosa (n = 11) or slight to moderate inflammation (n = 9). Histologically normal mucosal control samples from the lower turbinate were in addition obtained from seven individuals undergoing nasal surgery for septum deviation, but otherwise being healthy.

Nasal polyps and mucosal biopsy specimens from the lower turbinate were obtained by surgical excision. The specimens were immediately divided in two parts: one was placed on a thin slice of carrot for appropriate orientation and handling, embedded in OCT (Tissue-Tek; Miles Laboratories, Elkhart, IN), and snap frozen in liquid nitrogen, as detailed elsewhere (27); the remainder was immediately frozen in liquid nitrogen and stored at -70°C until subjected to RNA extraction.

In some cases, tissue fluid was isolated from the polyps. For this purpose, the polyp samples were finely minced with scissors and then centrifuged at 1880 x g for 10 min at 4°C. The supernatant fluids were stored at -70°C until tested for chemotactic activity.

Immunofluorescence staining

To determine the tissue density and numbers of eosinophils and neutrophils relative to all leukocytes, we applied a three-color immunostaining technique on acetone-fixed serial cryosections (4 µm), as described elsewhere (28). Briefly, a mixture of mAbs specific for human CD45 and CD38 (the IgG1 clones PD27/26 and 2B11 combined, Dako, Glostrup, Denmark; and the IgG1 clone HB-7, Becton Dickinson, Mountain View, CA, respectively) and CD15 (the IgM clone FP-298A3, Biosys, Compiègne, France) with addition of FITC-labeled BSA (28) was applied for 1 h. The sections were next incubated with a mixture of indocarbocyanine-labeled (red fluorescence) goat anti-mouse IgG (1/1000; Jackson ImmunoResearch, West Grove, PA) and biotinylated goat anti-mouse IgM (1/20; Southern Biotechnology Associates, Birmingham, AL) conjugates for 1.5 h, followed by 7-amino-4-methylcoumarin-3-acetic acid-labeled (blue fluorescence) streptavidin (0.02 g/L; Vector Laboratories, Burlingame, CA) for 30 min. This method identified in the same field the total number of leukocytes as CD45 or CD38 positive (the latter including plasma cells) by their red staining, all neutrophils as CD15 positive by their blue staining, and all eosinophils by their green staining.

Negative controls were obtained by omission of primary mAbs or incubation with irrelevant isotype- and concentration-matched primary mAbs.

The immunostained tissue sections were examined at x400 magnification in a fluorescence microscope (Model E800; Nikon, Tokyo, Japan). All sections were examined blind by the same investigator (F.L.J.). Eosinophils were numerically recorded per basement membrane length unit (1 mm) to a stromal depth of 300 µm by superimposing a grid (200 x 300 µm); all positive cells were counted in at least one section (>10 fields) from each specimen. Also, to determine the proportion of eosinophils and neutrophils relative to all leukocytes in nasal polyps, triple-stained cryosections were evaluated with regard to red, blue, and green cells by including at least 10 random fields; this approach provided a cell number sufficiently large to obtain a stable accumulative mean for the granulocyte-to-leukocyte ratio.

Isolation and quantification of mRNA

Two internal standards for competitive RT-PCR were employed. For this purpose, we constructed a synthetic DNA that contained cassettes with both forward and reverse PCR primer binding sites for chemokines (MCP-1, MCP-3, MCP-4, eotaxin, RANTES, lymphotactin, macrophage-inflammatory protein-1), cytokines (IL-3, IL-13, IL-14, IL-16), adhesion molecules (VCAM-1, P-selectin), and the housekeeping gene GAPDH, using oligonucleotides essentially as described (29). The cassettes of forward and reverse primer binding sites were constructed separately and subcloned into pCR2 (Invitrogen, Leek, The Netherlands). The two cassettes were subsequently combined into pHCQ1 that had been digested with EcoRI and PstI to remove the insert but leave the synthetic poly(A) tail (30). A standard for eotaxin-2 was constructed by subcloning the segment from the forward primer to an internal EcoRI site into pCR2, and a segment from the reverse primer to the internal EcoRI site into pBS(KS⁺) (Stratagene, La Jolla, CA). The two segments were combined into pHCQ1, as described above. Both standard DNAs were sequenced by the dideoxy chain termination method with Sequenase (Amersham, Slough, U.K.).

Total RNA was isolated by the guanidine/CsCl₂ procedure from tissue specimens directly frozen in liquid nitrogen (see earlier) (30, 31). The two standard DNAs were linearized with HindIII and used as template for RNA synthesis according to the Megascript T7 protocol (Ambion, Austin, TX), and combined at equal molar ratios. Eight serial dilutions of 0.5 log intervals from 10⁷ to 3.3 x 10³ copies were used as template for cDNA synthesis together with a constant amount of 500 ng cellular RNA in a 20-µl reaction in 96-well plates. Synthesis of cDNA was primed by oligo(dT) and reverse transcribed with SuperscriptII, according to the manufacturer’s instructions (Life Technologies, Paisley, U.K.).

Primers, conditions, and product sizes for the PCRs are listed in Table II. All matching forward and reverse primers were designed with software from Genetics Computer Group (Madison, WI) and chosen from separate exons. All reactions were performed with Dynazyme (Finnzymes Oy, Espoo, Finland) in 96-well plates containing 1 µl cDNA and 10 pmol primers in 25 µl reaction volume. Each PCR cycle included the following incubation temperatures for 1 min each: 95°C denaturing; annealing temperature as indicated; 72°C extension; and a final extension of 10 min at 72°C. PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide staining (Fig. 1). Relative band intensities were determined by densitometry on Polaroid 665 negatives (Kodak, Rochester, NY). All target mRNA levels were related to the level of GAPDH message in the same RNA sample to normalize for inaccuracies in quantifying the cellular RNA.

View larger version (54K): [in this window] [in a new window]   FIGURE 1. Visualization of competitive quantitative RT-PCR. Varying numbers of standard RNA molecules were reverse transcribed together with a constant amount of cellular RNA (500 ng). The cDNA reaction mixture from the same cellular source was amplified with primers specific for GAPDH (upper panel) or eotaxin (lower panel). PCR products were separated by electrophoresis and visualized with ethidium bromide staining. When the ratio of band intensities equals one, the number of target RNA molecules matches the number of standard RNA molecules. In this example, 1 µg of cellular RNA contained 6 x 106 and 1.5 x 105 molecules of GAPDH and eotaxin mRNA, respectively, as assessed by densitometry.

Peripheral blood was obtained from healthy individuals with normal eosinophil counts. Mononuclear cells were removed by Lymphoprep (Nycomed Pharma, Oslo, Norway) and erythrocytes were lysed by a NH₄Cl solution, as previously described (32). Eosinophils were further purified by negative selection with anti-CD16-coated immunomagnetic microbeads and a magnetic cell separation (MACS) system (Miltenyi Biotec, Bergish Gladbach, Germany), according to the manufacturers’ directions. The purity of eosinophils was always >95%, with viability >98%, as assessed by ethidium bromide (2.5 µg/ml) staining.

Chemotaxis assay

Experiments were performed with a 96-well microchemotaxis chamber (ChemoTx; Neuroprobe, Cabin John, MD), as recommended by the manufacturer. Purified eosinophils were washed twice in HBSS without Ca²⁺ and Mg²⁺, and then labeled with Calcein-AM (10 µM, 15 min, 37°C). The labeled cells were washed twice and resuspended in HBSS with 2% FCS (assay medium). Tissue supernatants, recombinant human eotaxin (Peprotech, London, U.K.), or recombinant human RANTES (R&D Systems, Abington, U.K.) diluted in assay medium were loaded into the wells (29 µl). The polycarbonate filter (5-µm pore size) was then installed, and 25 µl of labeled eosinophils (3 x 10⁶ cells/ml) was loaded on top of the filter and incubated at 37°C for 60 min. Thereafter, the cells at the top of the filter were gently removed by flushing with media and wiping with a cell harvester. Cells that had migrated into the filter were assessed by recording the fluorescence signal in a Titertek Fluoroscan II fluorescence multiwell reader (Labsystems, Helsinki, Finland).

For blocking experiments, mAb 7B11 (10 µg/ml, IgG1; courtesy of Dr. Charles Mackay, LeukoSite, Cambridge, MA) directed against CCR3 (33) was added to the labeled cells before incubation.

Statistics

Wilcoxon matched pairs sign rank sum test was performed to compare mRNA levels and numbers of eosinophils at various mucosal tissue sites within patient groups. The Mann-Whitney two-tailed test was performed to compare untreated with steroid-treated patients and control subjects. Paired Student’s two-tailed t test was used to compare chemotactic activity of tissue supernatants with or without addition of blocking mAbs. Spearman’s rank correlation coefficient was used to assess the relationship between the percentage of tissue eosinophils and the chemokine mRNA levels.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue eosinophils in untreated patients

The fraction of leukocytes identified as eosinophils was significantly increased in nasal polyps compared with that in their lower turbinate (internal control) or normal control mucosa (Fig. 2). On average, 43% (range 0–80%) of all leukocytes were eosinophils in these samples, whereas a median of only 2% (0–10%) neutrophils was detected. The median number of eosinophils per mm basement membrane was 103 (range 0–375) in polyp tissue compared with 5 (0–281) and 0 (0–1) in lower turbinate (internal control) or normal control mucosa, respectively.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. The fraction of eosinophils relative to all leukocytes in nasal polyps and lower turbinate mucosa from polyp patients untreated or after systemic steroid treatment and normal turbinate control specimens. Medians indicated by horizontal lines. Statistical analyses performed by Wilcoxon matched pairs sign rank sum test and the Mann-Whitney two-tailed test for paired and unpaired comparisons, respectively.

Quantitative RT-PCR showed that mRNA for eotaxin, eotaxin-2, and MCP-4 were significantly increased in nasal polyps compared with lower turbinate mucosa from the same individuals and normal control mucosa (Fig. 3). All three chemokines displayed similar mRNA levels in turbinate mucosa of the patients (1% of GAPDH levels), but in polyps the eotaxin-2 level was considerably higher (40% of GAPDH) than that of eotaxin (5% of GAPDH) and MCP-4 (6.5% of GAPDH). Furthermore, mRNA levels for eotaxin-2 and MCP-4 were significantly elevated in turbinate mucosa of the untreated patients compared with control mucosa from healthy individuals. RANTES displayed approximately the same transcript level as eotaxin and MCP-4 in the polyps, but the same magnitude of mRNA expression was also found in turbinate mucosa from both patients and controls (Fig. 3). For MCP-3, the mRNA levels were low (and in some samples undetectable) in all specimens tested (not shown). The cumulative or individual chemokine mRNA levels did not correlate significantly with the percentage of eosinophils in the polyps. However, virtually no eosinophils were found in the only polyp sample that displayed low mRNA levels for all chemokines tested (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Quantitative determination of mRNA for eotaxin (A), eotaxin-2 (B), MCP-4 (C), and RANTES (D) in nasal polyps and lower turbinate specimens from the same patients (internal controls), untreated or after systemic steroids treatment, compared with normal turbinate control specimens. All target mRNA concentrations are given as ratios of GAPDH transcript levels in the same RNA sample. Medians indicated by horizontal lines. Statistical analyses performed by Wilcoxon matched pairs sign rank sum test and the Mann-Whitney two-tailed test for paired and unpaired comparisons, respectively (ns, nonsignificant).

By endoscopic inspection of both nasal cavities, an average of >50% (range 25–75%) reduction of polyp size was observed after glucocorticosteroid treatment. Steroids also significantly reduced the fraction of eosinophils in the polyps compared with untreated counterparts (Fig. 2). Moreover, steroids dramatically reduced (>95%) the number of eosinophils in peripheral blood, but at the same time increased (>90%) the number of circulating neutrophils (Table I); the latter effect accounted for the total increase of peripheral blood leukocytes (Table I).

Effect of glucocorticosteroids on chemokine mRNA expression

To examine whether glucocorticoids affected mRNA levels for CCR3-binding chemokines in polyp tissue, we performed quantitative RT-PCR on RNA isolated from tissue specimens of patients treated for 7 days with systemic steroids. Levels of mRNA for eotaxin, eotaxin-2, and MCP-4 in polyps after treatment were significantly reduced compared with untreated counterparts, being similar to those found in lower turbinate mucosa (Fig. 3). Also, mRNA levels for RANTES were significantly reduced in treated polyps compared with untreated counterparts (Fig. 3).

Chemotactic activity of polyp tissue fluid

Because nasal polyps from untreated patients expressed high mRNA levels for CCR3-binding chemokines, we wanted to examine whether polyp tissue fluid could activate this receptor. Tissue fluid isolated from polyps with eosinophilia demonstrated significant chemotactic activity toward purified eosinophils, and initial dose-response experiments showed that eosinophils migrated most effectively in response to undiluted fluid (data not shown). Interestingly, eosinophils migrated 3- to 5-fold more efficiently in response to undiluted polyp tissue fluid than to an optimal concentration (200 ng/ml) of recombinant human eotaxin (Fig. 4). This effect was significantly (p = 0.01), but not completely, inhibited by mAb 7B11 directed against CCR3, while the chemotactic effect of recombinant human eotaxin was entirely inhibited by this mAb (Fig. 4). This finding suggested that the polyp fluid also contained eosinophil chemotactic factors acting independently of CCR3. However, mAb 7B11 completely abrogated eosinophil migration in response to an optimal concentration (100 ng/ml) of recombinant human RANTES (data not shown), suggesting that the chemotactic activity of that chemokine was mediated only through CCR3 and not CCR1, although RANTES can bind to both receptors.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Stimulation of eosinophil chemotaxis by nasal polyp fluid and its inhibition by blocking mAb against CCR3 (mAb 7B11). Tissue fluid obtained from nasal polyps with eosinophilia was used as attractant in microchemotaxis chamber. Purified labeled eosinophils, either untreated or pretreated with anti-CCR3, were examined for migration toward the supernatants. An optimal concentration of recombinant human eotaxin (200 ng/ml) served as positive control. The magnitude of migrated cells was expressed as arbitrary units measured in a fluorescence multiwell reader. Basal migration values are indicated by dotted line. *, p = 0.01. Representative results from two independent experiments (mean ± SD of triplicate wells).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The remarkable diversity of chemokines implies that they have a key role in controlling the characteristics of an inflammatory infiltrate, such as preferential accumulation of eosinophils in nasal polyps. Several chemokines bind CCR3 and therefore attract eosinophils, but only eotaxin and eotaxin-2 show selectivity for this receptor. Interestingly, mRNA levels for both these chemokines as well as MCP-4 were significantly increased in untreated nasal polyps compared with turbinate mucosa. Increased mRNA and protein expression of eotaxin has recently been detected in various human inflammatory lesions with increased eosinophil recruitment (15, 34, 35, 36), but to our knowledge this is the first study to report in situ mRNA expression for eotaxin-2. Notably, among the chemokines examined, eotaxin-2 showed the highest mRNA levels in polyp tissue (eight times higher than eotaxin), and it was 500-fold increased compared with the levels in turbinate mucosa of normal controls. In contrast to most other chemokines, eotaxin-2 mRNA expression has not been detected in normal organ blots (20). Therefore, this chemokine most likely plays a crucial role in eosinophil-associated inflammation.

Levels of mRNA for eotaxin-2 and MCP-4 were also significantly higher in the lower turbinate of untreated patients compared with normal controls. Moreover, in the patient group, 6 of 12 specimens showed histologically slight to moderate inflammation. However, only two of these lower turbinate specimens showed accumulation of eosinophils. This suggested that the levels of these chemokines, although elevated, were below a critical threshold concentration necessary to attract eosinophils. Simultaneous expression of several CCR3-activating chemokines might be crucial to achieve the proper cues for eosinophil extravasation. Alternatively, this redundancy could ensure a threshold concentration of chemokines that is necessary to engage CCR3 on eosinophils. Interestingly in this respect, it has been shown that a subset of T cells bearing CCR3 accumulates in nasal polyps (37), supporting the notion that CCR3-binding chemokines are important in the pathogenesis.

Altogether, based on this study and previous reports (28, 38), accumulation of eosinophils in nasal polyps appears to involve multiple leukocyte adhesion and chemoattractant receptors and their ligands, as well as eosinophil survival factors such as IL-5 (39). The lack of significant correlation between chemokine mRNA levels and the percentage of tissue eosinophils is therefore not incompatible with the notion that CCR3-binding chemokines play an important role in this process; it rather reflects a complex biological situation in which leukocyte emigration and retention depend on an array of diverse factors.

Because Abs to eotaxin-2 and MCP-4 were unavailable in our laboratory, we could not examine protein expression of these chemokines in situ. Recent studies have detected mRNA and protein expression for eotaxin, MCP-4, and RANTES in many different cell types at sites of inflammation (15, 17, 34, 36, 40, 41, 42). Notably, resident cells such as epithelial cells, fibroblasts, and endothelial cells have been shown to produce significant levels of such chemokines after stimulation in vitro (17, 18, 36, 41, 42, 43, 44, 45). It is therefore likely that several cell types within nasal polyps are responsible for the increased transcript levels for eotaxin, eotaxin-2, and MCP-4. In agreement with previous studies, we found high levels of RANTES mRNA in untreated polyps (46). However, these levels were not different from those found in normal control samples, suggesting that RANTES plays a role in normal homeostasis rather than in the pathogenesis of nasal polyps.

Systemic glucocorticoids have prominent anti-inflammatory effects and are the most effective drugs in the treatment of eosinophil-related inflammatory diseases such as asthma and nasal polyps. In this study, we observed that such treatment often reduced the size of nasal polyps by more than 50% concurrent with a significant reduction of tissue eosinophils. The significant decrease of tissue eosinophils in polyps of steroid-treated patients could partly be caused by the dramatic reduction (>95%) of blood eosinophils after treatment. However, it is well documented that glucocorticoids act locally in peripheral tissues, and it has been shown that topical administration of these drugs has a profound effect on the accumulation of eosinophils in both asthma and nasal polyposis (24, 25, 26, 47). Interestingly in this context, we found that mRNA levels for all tested chemokines were reduced in polyps of treated patients to levels similar to those detected in turbinate mucosa. Whether this result was caused indirectly via extracellular mediators that regulate chemokine production or by a direct effect of glucocorticoids on this production could not be determined. In favor of the latter possibility, glucocorticoids have been shown to inhibit directly cytokine-induced production of eotaxin, MCP-4, and RANTES in epithelial cells in vitro (41, 42, 45). Anyhow, the observed pronounced reduction of chemokine expression in nasal polyps after steroid treatment apparently defines an important mechanism for how steroids inhibit tissue accumulation of eosinophils.

We found that supernatants from polyp tissue expressed strong chemotactic activity for eosinophils. This effect was significantly inhibited by a blocking mAb specific for CCR3, demonstrating that polyps with eosinophilia contained high levels of CCR3-binding ligands. This finding was consistent with our mRNA data and strongly suggests that transcripts for CCR3-binding chemokines were translated into functional proteins. Interestingly, tissue fluid demonstrated 3–5 times more chemotactic activity than an optimal concentration of recombinant human eotaxin, implying the involvement of additional factors. Thus, engagement of multiple distinct receptors most likely has an additive or synergistic effect on eosinophil extravasation. The fact that anti-CCR3 did not completely abrogate the chemotactic activity supports the possibility that such additional chemotactic factors act independently of CCR3. In agreement with these observations, it was recently shown that classical chemoattractants such as C5a and FMLP had an additive effect on CCR-3-mediated transendothelial chemotaxis of eosinophils (48). It would have been interesting to test the chemotactic activity of tissue fluid also from untreated polyps with only small numbers of eosinophils, as well as from steroid-treated polyps. However, the limited size of the tissue samples, especially after steroid treatment, and also the fact that polyps with few eosinophils are infrequent (3 of 15 in our study), rendered it impossible to obtain sufficient material for such experiments.

Together, our findings accord with a combinatory role played by several CCR3-binding chemokines in tissue recruitment of eosinophils, and strengthen the supposed importance of these chemokines in the pathogenesis of eosinophil-related disorders such as nasal polyps and bronchial asthma.

	Acknowledgments

 
We thank Dr. Charles Mackay for providing mAb 7B11, and the technical staff at LIIPAT for excellent assistance.

	Footnotes

 
¹ This work was supported by the Norwegian Cancer Society, the Research Council of Norway, the Research Fund for Asthma and Allergy, and the Red Cross Research Fund for Children with Asthma and Allergy.

² Address correspondence and reprint requests to Dr. Frode L. Jahnsen, LIIPAT, Rikshospitalet, N-0027 Oslo, Norway. E-mail address:

³ Abbreviation used in this paper: MCP, monocyte-chemotactic protein.

Received for publication February 2, 1999. Accepted for publication May 17, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Wardlaw, A. J., R. Moqbel, A. B. Kay. 1995. Eosinophils: biology and role in disease. Adv. Immunol. 60:151.[Medline]
2. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301.[Medline]
3. Foxman, E. F., J. J. Campbell, E. C. Butcher. 1997. Multistep navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139:1349.[Abstract/Free Full Text]
4. Ebnet, K., E. P. Kaldjian, A. O. Anderson, S. Shaw. 1996. Orchestrated information transfer underlying leukocyte endothelial interactions. Annu. Rev. Immunol. 14:155.[Medline]
5. Butcher, E. C., L. J. Picker. 1996. Lymphocyte homing and homeostasis. Science 272:60.[Abstract]
6. Baggiolini, M., B. Dewald, B. Moser. 1997. Human chemokines: an update. Annu. Rev. Immunol. 15:675.[Medline]
7. Piali, L., C. Weber, G. LaRosa, C. R. Mackay, T. A. Springer, I. Clark Lewis, B. Moser. 1998. The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig. Eur. J. Immunol. 28:961.[Medline]
8. Kitayama, J., C. R. Mackay, P. D. Ponath, T. A. Springer. 1998. The C-C chemokine receptor CCR3 participates in stimulation of eosinophil arrest on inflammatory endothelium in shear flow. J. Clin. Invest. 101:2017.[Medline]
9. Luster, A. D.. 1998. Mechanisms of disease: chemokines-chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338:436.[Free Full Text]
10. Ponath, P. D., S. X. Qin, T. W. Post, J. Wang, L. Wu, N. P. Gerard, W. Newman, C. Gerard, C. R. Mackay. 1996. Molecular cloning and characterization of a human eotaxin receptor expressed selectively on eosinophils. J. Exp. Med. 183:2437.[Abstract/Free Full Text]
11. Daugherty, B. L., S. J. Siciliano, J. A. Demartino, L. Malkowitz, A. Sirotina, M. S. Springer. 1996. Cloning, expression, and characterization of the human eosinophil eotaxin receptor. J. Exp. Med. 183:2349.[Abstract/Free Full Text]
12. Uguccioni, M., C. R. Mackay, B. Ochensberger, P. Loetscher, S. Rhis, G. J. LaRosa, P. Rao, P. D. Ponath, M. Baggiolini, C. A. Dahinden. 1997. High expression of the chemokine receptor CCR3 in human blood basophils: role in activation by eotaxin, MCP-4, and other chemokines. J. Clin. Invest. 100:1137.[Medline]
13. Sallusto, F., C. R. Mackay, A. Lanzavecchia. 1997. Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005.[Abstract/Free Full Text]
14. Uguccioni, M., P. Loetscher, U. Forssmann, B. Dewld, H. Li, S. H. Lima, Y. Li, B. Kreider, G. Garotta, M. Thelen, M. Baggiolini. 1996. Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin. J. Exp. Med. 183:2379.[Abstract/Free Full Text]
15. Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, H. Smith, X. Shi, J.-A. Gonzalo, W. Newman, J.-C. Gutierrez-Ramos, C. R. Mackay. 1996. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J. Clin. Invest. 97:604.[Medline]
16. Kitaura, M., T. Nakajima, T. Imai, S. Harada, C. Combadiere, H. L. Tiffany, P. M. Murphy, O. Yoshie. 1996. Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. J. Biol. Chem. 271:7725.[Abstract/Free Full Text]
17. Garcia, Z. E., C. Combadiere, M. E. Rothenberg, M. N. Sarafi, F. Lavigne, Q. Hamid, P. M. Murphy, A. D. Luster. 1996. Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J. Immunol. 157:5613.[Abstract]
18. Elsner, J., R. Hochstetter, D. Kimmig, A. Kapp. 1996. Human eotaxin represents a potent activator of the respiratory burst of human eosinophils. Eur. J. Immunol. 26:1919.[Medline]
19. Jose, P. J., D. A. Griffithsjohnson, P. D. Collins, D. T. Walsh, R. Moqbel, N. F. Totty, O. Truong, J. J. Hsuan, T. J. Williams. 1994. Eotaxin: a potent eosinophil chemoattractant cytokine detected in a guinea pig model of allergic airways inflammation. J. Exp. Med. 179:881.[Abstract/Free Full Text]
20. Patel, V. P., B. L. Kreider, Y. Li, H. Li, K. Leung, T. Salcedo, B. Nardelli, V. Pippalla, S. Gentz, R. Thotakura, et al 1997. Molecular and functional characterization of two novel human C-C chemokines as inhibitors of two distinct classes of myeloid progenitors. J. Exp. Med. 185:1163.[Abstract/Free Full Text]
21. Forssmann, U., M. Uguccioni, P. Loetscher, C. A. Dahinden, H. Langen, M. Thelen, M. Baggiolini. 1997. Eotaxin-2, a novel CC chemokine that is selective for the chemokine receptor CCR3, and acts like eotaxin on human eosinophil and basophil leukocytes. J. Exp. Med. 185:2171.[Abstract/Free Full Text]
22. Elsner, J., H. Petering, C. Kluthe, D. Kimmig, R. Smolarski, P. Ponath, A. Kapp. 1998. Eotaxin-2 activates chemotaxis-related events and release of reactive oxygen species via pertussis toxin-sensitive G proteins in human eosinophils. Eur. J. Immunol. 28:2152.[Medline]
23. Luster, A. D., M. E. Rothenberg. 1997. Role of the monocyte chemoattractant protein and eotaxin subfamily of chemokines in allergic inflammation. J. Leukocyte Biol. 62:620.[Abstract]
24. Schwiebert, L. A., L. A. Beck, C. Stellato, C. A. Bickel, B. S. Bochner, R. P. Schleimer. 1996. Glucocorticosteroid inhibition of cytokine production: relevance to antiallergic actions. J. Allergy Clin. Immunol. 97:143.[Medline]
25. Laitinen, L. A., A. Laitinen. 1996. Remodeling of asthmatic airways by glucocorticosteroids. J. Allergy Clin. Immunol. 97:153.[Medline]
26. Barnes, P. J.. 1995. Inhaled glucocorticoids for asthma. N. Engl. J. Med. 332:868.[Free Full Text]
27. Jahnsen, F. L., I. N. Farstad, J. P. Aanesen, P. Brandtzaeg. 1998. Phenotypic distribution of T cells in human nasal mucosa differs from that in the gut. Am. J. Respir. Cell Mol. Biol. 18:392.[Abstract/Free Full Text]
28. Jahnsen, F. L., G. Haraldsen, J. P. Aanesen, R. Haye, P. Brandtzaeg. 1995. Eosinophil infiltration is related to increased expression of vascular cell adhesion molecule-1 in nasal polyps. Am. J. Respir. Cell Mol. Biol. 12:624.[Abstract]
29. Benavides, G. R., B. Hubby, W. M. Grosse, R. A. McGraw, R. L. Tarleton. 1995. Construction and use of a multi-competitor gene for quantitative RT-PCR using existing primer sets. J. Immunol. Methods 181:145.[Medline]
30. Jung, H. C., L. Eckmann, S. K. Yang, A. Panja, J. Fierer, W. E. Morzycka, M. F. Kagnoff. 1995. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J. Clin. Invest. 95:55.
31. Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, W. J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294.[Medline]
32. Jahnsen, F. L., P. Brandtzaeg, T. S. Halstensen. 1994. Monoclonal antibody EG2 does not provide reliable immunohistochemical discrimination between resting and activated eosinophils. J. Immunol. Methods 175:23.[Medline]
33. Heath, H., S. X. Qin, P. Rao, L. J. Wu, G. LaRosa, N. Kassam, P. D. Ponath, C. R. Mackay. 1997. Chemokine receptor usage by human eosinophils: the importance of CCR3 demonstrated using an antagonistic monoclonal antibody. J. Clin. Invest. 99:178.[Medline]
34. Minshall, E. M., L. Cameron, F. Lavigne, D. Leung, D. Hamilos, E. A. Garcia Zepada, M. Rothenberg, A. D. Luster, Q. Hamid. 1997. Eotaxin mRNA and protein expression in chronic sinusitis and allergen-induced nasal responses in seasonal allergic rhinitis. Am. J. Respir. Cell Mol. Biol. 17:683.[Abstract/Free Full Text]
35. Lamkhioued, B., P. M. Renzi, Y. S. Abi, Z. E. Garcia, Z. Allakhverdi, O. Ghaffar, M. D. Rothenberg, A. D. Luster, Q. Hamid. 1997. Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation. J. Immunol. 159:4593.[Abstract]
36. Garciazepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, A. D. Luster. 1996. Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2:449.[Medline]
37. Gerber, B. O., M. P. Zanni, M. Uguccioni, M. Loetscher, C. R. Mackay, W. J. Pichler, N. Yawalkar, M. Baggiolini, B. Moser. 1997. Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils. Curr. Biol. 7:836.[Medline]
38. Symon, F. A., G. M. Walsh, S. R. Watson, A. J. Wardlaw. 1994. Eosinophil adhesion to nasal polyp endothelium is P-selectin-dependent. J. Exp. Med. 180:371.[Abstract/Free Full Text]
39. Simon, H. U., S. Yousefi, C. Schranz, A. Schapowal, C. Bachert, K. Blaser. 1997. Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia. J. Immunol. 158:3902.[Abstract]
40. Ying, S., D. S. Robinson, Q. Meng, J. Rottman, R. Kennedy, D. J. Ringler, C. R. Mackay, B. L. Daugherty, M. S. Springer, S. R. Durham, et al 1997. Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma: association with airway hyperresponsiveness and predominant colocalization of eotaxin mRNA to bronchial epithelial and endothelial cells. Eur. J. Immunol. 27:3507.[Medline]
41. Stellato, C., L. A. Beck, G. A. Gorgone, D. Proud, T. J. Schall, S. J. Ono, L. M. Lichtenstein, R. P. Schleimer. 1995. Expression of the chemokine RANTES by a human bronchial epithelial cell line: modulation by cytokines and glucocorticoids. J. Immunol. 155:410.[Abstract]
42. Stellato, C., P. Collins, P. D. Ponath, D. Soler, W. Newman, G. LaRosa, H. D. Li, J. White, L. M. Schwiebert, C. Bickel, et al 1997. Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines. J. Clin. Invest. 99:926.[Medline]
43. Bartels, J., C. Schluter, E. Richter, N. Noso, R. Kulke, E. Christophers, J. M. Schroder. 1996. Human dermal fibroblasts express eotaxin: molecular cloning, mRNA expression, and identification of eotaxin sequence variants. Biochem. Biophys. Res. Commun. 225:1045.[Medline]
44. Marfaing-Koka, A., O. Devergne, G. Gorgone, A. Portier, T. J. Schall, P. Galanaud, D. Emilie. 1995. Regulation of the production of the RANTES chemokine by endothelial cells: synergistic induction by IFN- plus TNF- and inhibition by IL-4 and IL-13. J. Immunol. 154:1870.[Abstract]
45. Lilly, C. M., H. Nakamura, H. Kesselman, A. C. Nagler, K. Asano, Z. E. Garcia, M. E. Rothenberg, J. M. Drazen, A. D. Luster. 1997. Expression of eotaxin by human lung epithelial cells: induction by cytokines and inhibition by glucocorticoids. J. Clin. Invest. 99:1767.[Medline]
46. Teran, L. M., H. S. Park, R. Djukanovic, K. Roberts, S. Holgate. 1997. Cultured nasal polyps from nonatopic and atopic patients release RANTES spontaneously and after stimulation with phytohemagglutinin. J. Allergy Clin. Immunol. 100:499.[Medline]
47. Hamilos, D. L., S. E. Thawley, M. A. Kramper, A. Kamil, Q. A. Hamid. 1999. Effect of intranasal fluticasone on cellular infiltration, endothelial adhesion molecule expression, and proinflammatory cytokine mRNA in nasal polyp disease. J. Allergy Clin. Immunol. 103:79.[Medline]
48. Kitayama, J., M. W. Carr, S. J. Roth, J. Buccola, T. A. Springer. 1997. Contrasting responses to multiple chemotactic stimuli in transendothelial migration: heterologous desensitization in neutrophils and augmentation of migration in eosinophils. J. Immunol. 158:2340.[Abstract]

This article has been cited by other articles:

				N. Hashida, N. Ohguro, K. Nakai, M. Kobashi-Hashida, S.-i. Hashimoto, K. Matsushima, and Y. Tano Microarray Analysis of Cytokine and Chemokine Gene Expression after Prednisolone Treatment in Murine Experimental Autoimmune Uveoretinitis Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4224 - 4234. [Abstract] [Full Text] [PDF]

				H. Schjerven, P. Brandtzaeg, and F.-E. Johansen Hepatocyte NF-1 and STAT6 Cooperate with Additional DNA-Binding Factors to Activate Transcription of the Human Polymeric Ig Receptor Gene in Response to IL-4 J. Immunol., June 15, 2003; 170(12): 6048 - 6056. [Abstract] [Full Text] [PDF]

				A. Kibe, H. Inoue, S. Fukuyama, K. Machida, K. Matsumoto, H. Koto, T. Ikegami, H. Aizawa, and N. Hara Differential Regulation by Glucocorticoid of Interleukin-13-induced Eosinophilia, Hyperresponsiveness, and Goblet Cell Hyperplasia in Mouse Airways Am. J. Respir. Crit. Care Med., January 1, 2003; 167(1): 50 - 56. [Abstract] [Full Text] [PDF]

				J. GALON, D. FRANCHIMONT, N. HIROI, G. FREY, A. BOETTNER, M. EHRHART-BORNSTEIN, J. J. O'SHEA, G. P. CHROUSOS, and S. R. BORNSTEIN Gene profiling reveals unknown enhancing and suppressive actions of glucocorticoids on immune cells FASEB J, January 1, 2002; 16(1): 61 - 71. [Abstract] [Full Text] [PDF]

				C. Bandeira-Melo, A. Herbst, and P. F. Weller Eotaxins . Contributing to the Diversity of Eosinophil Recruitment and Activation Am. J. Respir. Cell Mol. Biol., June 1, 2001; 24(6): 653 - 657. [Full Text] [PDF]

				H. Schjerven, P. Brandtzaeg, and F.-E. Johansen Mechanism of IL-4-Mediated Up-Regulation of the Polymeric Ig Receptor: Role of STAT6 in Cell Type-Specific Delayed Transcriptional Response J. Immunol., October 1, 2000; 165(7): 3898 - 3906. [Abstract] [Full Text] [PDF]

				B. LAMKHIOUED, E. A. GARCIA-ZEPEDA, S. ABI-YOUNES, H. NAKAMURA, S. JEDRZKIEWICZ, L. WAGNER, P. M. RENZI, Z. ALLAKHVERDI, C. LILLY, Q. HAMID, et al. Monocyte Chemoattractant Protein (MCP)-4 Expression in the Airways of Patients with Asthma . Induction in Epithelial Cells and Mononuclear Cells by Proinflammatory Cytokines Am. J. Respir. Crit. Care Med., August 1, 2000; 162(2): 723 - 732. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jahnsen, F. L. Articles by Johansen, F.-E. Search for Related Content PubMed PubMed Citation Articles by Jahnsen, F. L. Articles by Johansen, F.-E.