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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cameron, L. Articles by Hamid, Q. Search for Related Content PubMed PubMed Citation Articles by Cameron, L. Articles by Hamid, Q.

Evidence for Local Eosinophil Differentiation Within Allergic Nasal Mucosa: Inhibition with Soluble IL-5 Receptor¹

Lisa Cameron^*, Pota Christodoulopoulos^*, Francois Lavigne, Yutaka Nakamura^*, David Eidelman^*, Alan McEuen, Andrew Walls, Jan Tavernier^§, Eleanor Minshall^*, Redwan Moqbel^¶ and Qutayba Hamid^2,*

^* Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; Notre-Dame Hospital, Montreal, Quebec, Canada; School of Medicine, University of Southampton, Southampton, United Kingdom; ^§ Flanders Interuniversity Institute for Biotechnology, University of Ghent, Ghent, Belgium; and ^¶ Pulmonary Research Group, University of Alberta, Edmonton, Alberta, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Eosinophil differentiation occurs within the bone marrow in response to eosinopoietic cytokines, particularly IL-5. Recently, however, eosinophil precursors (CD34/IL-5R⁺ cells) and IL-5 mRNA⁺ cells have been identified within the lungs of asthmatics, indicating that a population of eosinophils may differentiate in situ. In this report, we examined the presence of eosinophil precursors within allergic nasal mucosa and examined whether they undergo local differentiation following ex vivo stimulation. We cultured human nasal mucosa obtained from individuals with seasonal allergic rhinitis with either specific allergen, recombinant human IL-5 (rhIL-5), or allergen + soluble IL-5R (sIL-5R), shown to antagonize IL-5 function. Simultaneous immunocytochemistry and in situ hybridization demonstrated that there were fewer cells coexpressing CD34 immunoreactivity and IL-5R mRNA following culture with allergen or rhIL-5, compared with medium alone. Immunostaining revealed that the number of major basic protein (MBP) immunoreactive cells (eosinophils) was higher within tissue stimulated with allergen or rhIL-5, compared with unstimulated tissue. In situ hybridization detected an increase in IL-5 mRNA⁺ cells in sections from tissue cultured with allergen, compared with medium alone. These effects were not observed in tissue cultured with a combination of allergen and sIL-5R. Colocalization analysis indicated this expression to be mainly, but not exclusively, T cell (44%) and eosinophil (10%) derived. Our findings suggest that a subset of eosinophils may differentiate locally within allergic nasal mucosa, in what appears to be a highly IL-5-dependent fashion, and imply that this process might be regulated in vivo by endogenous production of sIL-5R.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
A cardinal feature of allergic disorders such as asthma and rhinitis is the elevated number of inflammatory cells, particularly eosinophils (1), within the respiratory mucosa. Activated eosinophils release a number of cytotoxic granule proteins, the most prominent being major basic protein (MBP)³ (2). MBP has been reported to damage respiratory epithelium (3) and to induce degranulation of other inflammatory cells (4, 5). Furthermore, eosinophils are a source of proinflammatory (IL-4, IL-6) (6, 7, 8), profibrotic (TGF-ß) (9, 10), and hemopoietic cytokines (IL-5, IL-3, and GM-CSF) (11, 12).

IL-5 is critical for eosinophil development. In vitro, addition of this cytokine to progenitor cell cultures gives rise to eosinophil colonies (13, 14). Intravenous injection of rIL-5 into guinea pigs resulted in rapid blood eosinophilia (15). Furthermore, pulmonary eosinophilia was not observed within Ag-challenged IL-5-knockout mice and was diminished in wild-type mice pretreated with anti-IL-5 Abs (16, 17). IL-5 acts through a hetero(di)meric receptor (IL-5R) composed of a ligand-specific -chain and the signal-transducing ß-chain (18). Transcripts coding for the -chain are differentially spliced, giving rise to either soluble or membrane-bound isoforms, which antagonize or mediate IL-5 function, respectively (19). IL-5 is thought to exert its effects at a relatively late period during eosinopoiesis, influencing terminal differentiation of CD34^-/CD33⁺ progenitors (20). However, a recent report by Sehmi et al. has shown the expression of the -subunit of the IL-5 receptor (IL-5R) on CD34⁺ cells and suggested that colocalization of these two markers may be indicative of eosinophil lineage-committed progenitors (CD34/IL-5R⁺) (21).

Tissue eosinophilia within the lungs and nose of individuals with allergic asthma and rhinitis has primarily been attributed to the influx of mature cells. Recent reports now suggest, however, that parallel mechanisms may also be at work. CD34⁺ progenitor cells have been detected within peripheral blood and lungs and are increased in number in atopics compared with normals (22, 23). Furthermore, elevated numbers of cells producing the eosinopoietic cytokines IL-5, IL-3, and GM-CSF have been observed within the nasal mucosa of individuals with allergic rhinitis following allergen challenge (24). Collectively, these studies suggested the possibility of local eosinophil differentiation within respiratory mucosa. Robinson et al. have recently demonstrated the presence of eosinophil precursors (CD34/IL-5R⁺) within the lungs and that their number was increased, as well as MBP immunoreactivity, in atopic asthmatics compared with normal controls (23). However, whether the change in eosinophil number was due to local differentiation of precursor cells or infiltration from systemic circulation remains unknown.

To study the prospect of local eosinophil differentiation, we employed an explant system to exclude the possibility of cell infiltration and enable the examination of events occurring solely within the tissue. Human nasal mucosal tissue, obtained from patients with allergic rhinitis, exhibited fewer CD34/IL-5R⁺ cells, but more MBP-immunoreactive and IL-5 mRNA⁺ cells, following ex vivo stimulation with specific allergen or recombinant human (rh)IL-5. Soluble IL-5R (sIL-5R) was seen to inhibit the allergen-induced changes in the number of CD34/IL-5R⁺ cells and MBP⁺ cells, but not IL-5 mRNA⁺ cells. Our results show the presence of eosinophil precursors within allergic nasal mucosa and provide evidence for their local differentiation following stimulation, ex vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue Culture

Nasal mucosal tissue was obtained from the inferior turbinate of ragweed-sensitive individuals with seasonal allergic rhinitis, outside the ragweed season, that were not receiving any glucocorticosteriods. Tissue was resected from patients undergoing sinus surgery, who had given informed consent before the procedure, and was rinsed in medium before culture. The time frame between tissue removal and the commencement of culture was consistently less than 1 h, and tissue was in culture medium at all times during this transition period. Serial sections of tissue were placed on 0.4-µm well inserts (Millipore, Bedford, MA) in 2 ml of defined medium (25, 26) with 1) 250 µl of ragweed allergen (10–2000 protein nitrogen units (PNU); Hollister-Stier, Spokane, Washington), 2) rhIL-5 (12.5 ng/ml; Dako), or 3) ragweed + rh sIL-5R (5 µg/ml) and incubated in 5% CO₂/95% air. Following culture, tissue was fixed in 4% paraformaldehyde, washed in a solution of 15% sucrose/PBS and blocked in optimal cutting temperature (OCT) medium by snap freezing in isopentane cooled in liquid nitrogen.

Abs and probes

Anti-human mAbs derived from hybridoma supernatants were used to detect MBP (BMK-13), an eosinophil marker (27); CD34 (QBEND/10, Serotec Kidlington, Oxford, England), to identify hemopoietic progenitors (28); CD3 (Dako), a pan T cell marker; and BB1, a newly described Ab specific for basophil granule protein (29). Each Ab was diluted in a standardized diluting buffer (Dako): BMK-13 at 1/30, CD34 at 1/50, CD3 at 1/100, and BB1 at 1/10. IL-5 and IL-5R cDNAs were inserted into pGEM vectors and linearized with the restriction enzymes BamHI or PstI, for anti-sense templates, and HindIII for sense templates, respectively. With these fragments, in vitro transcription was initiated with the RNA polymerase Sp6 and T7 in the presence of ³⁵S-labeled UTP to generate radio-labeled antisense probes recognizing RNA message encoding IL-5 or IL-5R and sense probes (30).

Immunocytochemistry

MBP immunoreactivity was detected by the avidin-biotin complex (ABC) method, as previously described (31). Anti-MBP was visualized using diaminobenzidine, with which positive cells appeared brown. BB1⁺ cells were detected using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique (32) with the fast red chromogen; positive cells were therefore red. The secondary and tertiary layer were repeated for maximum detection of BB1 immunoreactivity. Negative control experiments were performed by replacing the primary Ab with an isotype-matched control.

In situ hybridization

This was conducted as previously described (30). Briefly, sections were permeabilized with proteinase K (1 µg/ml) and Triton X-100. Prehybridization was performed in 50% formamide in 2x SSC solution. For hybridization, ³⁵S-labeled cRNA probes were applied and left overnight in a humid chamber at 42°C. Posthybridization washes of decreasing concentrations of SSC solution (4x to 0.1x SSC) were conducted at 42°C. Excess probe was destroyed with RNase A (20 µg/ml). Slides were dipped in Amersham (Oakville, Ontario, Canada) LM-2 emulsion and exposed for 11–15 days. Autoradiographs were developed in Kodak D-19 and counterstained with hematoxylin. Positive signal was identified as a collection of silver grains overlying the cells. Negative control experiments using sense probes and RNase treatment before antisense probe application were performed to confirm probe specificity.

Simultaneous immunocytochemistry and in situ hybridization

Eosinophil precursors, progenitor cells expressing receptors for IL-5, were identified as previously described (21). Briefly, alkaline phosphatase anti-alkaline phosphatase immunocytochemistry with Ab directed against the progenitor cell marker CD34 and the fast red chromogen was performed. Subsequently, sections underwent in situ hybridization with ³⁵S-labeled cRNA probes for IL-5R mRNA. Eosinophil precursors were identified as those cells exhibiting both a red staining for CD34 and an accumulation of silver grains overlying the cells. Colocalization of IL-5 to T cells and eosinophils was also performed using this protocol with Abs to detect CD3 and MBP immunoreactivity and cRNA probes for IL-5 mRNA.

Colocalization of CD34 and carbol chromotrope 2R

CD34 immunoreactivity was detected using the streptavidin peroxidase method (7) and the chromogen diaminobenzidine. Subsequently, slides were stained with 1% carbol chromotrope 2R (Sigma, St. Louis, MO) for 5 min, which identifies eosinophil granules. With this technique, cells double positive for CD34 and chromotrope stained reddish-brown.

Quantification and statistics

Using an Olympus light microscope (Carson Group, Markam, Ontario, Canada) at x200 magnification, slides were analyzed for positive signal in a blinded fashion by two independent examiners. CD34 immunoreactivity associated with hemopoietic progenitor cells was counted, i.e. vessel wall cells and fibroblasts were excluded. For in situ hybridization, positive signal was determined as a discrete cluster of silver grains overlying the cell, which could be seen to encompass the nucleus under dark field illumination. By placing the graticule under the basement membrane, the number of positive cells was counted and reported as the mean of at least 6–8 fields (0.2 mm²). Data are represented within the text and figures as the mean ± SD. Significance was assessed using a Dunnett’s test for the comparison of multiple groups to one control. A paired Student t test was applied to the values (i.e., ragweed minus medium alone) to determine the statistical difference between the various conditions ( ragweed vs rhIL-5). Correlational analysis was applied using Pearson’s correlation coefficient, and a Bonferroni post hoc test was used. Values of p < 0.05 were considered significant (SyStat version 7.1; SyStat, Evanston, IL).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Response of nasal mucosal explant tissue to ragweed allergen

Although previous work has demonstrated no difference in symptom scores when doses of 10–1000 PNU of ragweed allergen were used in nasal provocation studies (33), the conditions for ex vivo challenge of nasal mucosal tissue with ragweed allergen had not been determined. We therefore performed a dose response with 10, 100, 1000, and 2000 PNU of ragweed allergen (n = 3). The number of MBP and IL-5 mRNA⁺ cells in sections of nasal mucosal tissue cultured for 24 h with ragweed allergen increased progressively from 10 PNU to 1000 PNU; however, no further elevation was noted at 2000 PNU. Experiments for this study were therefore conducted with 1000 PNU of ragweed allergen.

Progenitor cells within nasal mucosal explant tissue

CD34 is expressed by hemopoietic progenitor cells as well as endothelial cells and fibroblasts (34, 35). As such, when counting the CD34⁺ cells, the vessels and fibroblasts were excluded as much as possible. A number of these CD34⁺ progenitor cells were seen scattered throughout the submucosal layer (Fig. 1a) in unstimulated tissue at 6 (Table I; n = 7, 19.9 ± 6.4) and 24 h (Table I; n = 11, 18.7 ± 4.2), and there were fewer following ex vivo exposure to ragweed allergen (13 ± 3.4, 11.7 ± 2.6; p < 0.01) as well as rhIL-5 (n = 5, 12.2 ± 1.9; n = 6, 14.4 ± 3.0; p < 0.05). sIL-5R molecules were used in this explant system to inhibit the action of IL-5 (18). We observed that culture with both ragweed and sIL-5R was associated with the presence of a similar number of CD34⁺ progenitor cells as unstimulated tissue (n = 6, 19.3 ± 3.7; p > 0.05), significantly higher than in sections of explant tissue cultured with ragweed alone (Table I; p < 0.01).

View larger version (104K): [in this window] [in a new window]   FIGURE 1. Representative photos of nasal mucosal explant tissue 24 h following culture. a, The presence of CD34+ progenitor cells within tissue cultured in medium alone. Under dark field illumination, cells coexpressing both CD34 immunoreactivity and IL-5R mRNA are also seen within unstimulated tissue; these cells appear both orange and illuminated (b, note the arrow). c, The considerable number of MBP+ cells within tissue cultured with ragweed allergen.

View this table: [in this window] [in a new window]   Table I. CD34+ progenitor cells and IL-5R mRNA+ cells within nasal mucosal explant tissue1

Colocalization of CD34 and IL-5R mRNA has been suggested as a marker of eosinophil precursors (21). Fig. 2 illustrates the presence of CD34/IL-5R mRNA⁺ cells within nasal mucosa cultured for either 6 or 24 h in medium alone (11.1 ± 4.0, 12.2 ± 3.2; Fig. 1b). At both time points, the number of these cells was significantly less in tissue cultured with ragweed allergen (5.1 ± 1.5, 5.0 ± 1.8; p < 0.01) or rhIL-5 (5.2 ± 1.6, 7.6 ± 2.7; p < 0.01). When ragweed and sIL-5R were added together, more CD34/IL-5R mRNA⁺ cells were observed (11.8 ± 2.4) than nasal tissue cultured with ragweed alone (p < 0.01); the latter exhibited similar numbers as those in medium alone (12.2 ± 3.2). In fact, there were 60% fewer CD34/IL-5R mRNA⁺ cells within ragweed-stimulated than unstimulated tissue. In contrast, culture with ragweed allergen and sIL-5R resulted in only a 3% change (Fig. 2). When the number of CD34⁺/IL-5R mRNA⁺ cells was examined as a percentage of total CD34⁺ progenitor cells, we observed that 58% ± 0.1 and 65% ± 0.7 (6 and 24 h) of CD34⁺ progenitor cells were expressing IL-5R mRNA⁺ within unstimulated tissue. Following culture with ragweed allergen alone, these percentages were substantially lowered, 39% ± 0.1 and 41% ± 0.1 (p < 0.01). Incubation with rhIL-5 was also associated with a smaller percentage of progenitor cells expressing IL-5R mRNA at both 6 (42% ± 0.1) and 24 h (51% ± 0.1; p < 0.01), as compared with unstimulated tissue (Table I).

View larger version (52K): [in this window] [in a new window]   FIGURE 2. Number of CD34/IL-5R mRNA+ cells within nasal mucosal explant tissue. Fewer cells were observed following both 6 and 24 h of culture with ragweed allergen or rhIL-5 (*, p < 0.01). At 24 h, there were more CD34/IL-5R mRNA+ cells within rhIL-5-stimulated than ragweed-stimulated tissue (*, p < 0.01). The combination of ragweed + sIL-5R was associated with more CD34/IL-5R mRNA+ cells than ragweed alone (*, p < 0.01).

There were similar numbers of MBP immunoreactive cells within the submucosa of unstimulated tissue following either 6 (n = 7, 3.0 ± 2.3) or 24 h of culture (n = 10, 2.1 ± 1.1; Fig. 3 and Fig. 4a). Furthermore, these numbers were not significantly different from within tissue blocked immediately following resection (n = 3, 2.2 ± 0.46; p > 0.05). This baseline level of eosinophils may be attributed to the fact that most of these patients were also allergic to house dust mite; however, significant increases were observed within nasal tissue that was cultured for 6 (n = 7, 10.1 ± 5.2) or 24 h with ragweed allergen (n = 10, 9.4 ± 3.5; p < 0.01), the majority of which were found just beneath the basement membrane (Fig. 1c and 4b). An inverse correlation was observed between the increase in the number of MBP-immunoreactive cells and the reduction in CD34/IL-5R⁺ cells in ragweed-stimulated vs unstimulated tissue (r² = 0.64, p < 0.01). At both 6 and 24 h, the numbers of MBP⁺ cells was also greater in explant tissue cultured with rhIL-5 (n = 6, 8.2 ± 4.8; n = 9, 6.2 ± 1.7) than in medium alone (p < 0.01, Fig. 4c), which was less than what we observed following culture with ragweed allergen (p < 0.01, Fig. 4b). Combination of ragweed and sIL-5R molecules was associated with fewer MBP⁺ cells (n = 6, 1.9 ± 1.6; Figs. 2 and 4d) than ragweed alone (p < 0.01, Fig. 4b), numbers similar to unstimulated tissue (Fig. 4a).

View larger version (39K): [in this window] [in a new window]   FIGURE 3. The number of MBP-immunoreactive cells within nasal mucosal explant tissue. More MBP+ cells were observed following culture with allergen or rIL-5 than medium alone at both 6 and 24 h (*, p < 0.01). Significantly more MBP+ cells were observed following culture with ragweed compared with rhIL-5 (*, p < 0.01). Combination of ragweed + sIL-5R inhibited the increase in MBP+ cells seen following culture with ragweed alone (*, p < 0.01).

View larger version (138K): [in this window] [in a new window]   FIGURE 4. Representative MBP immunostaining 24 h following culture in the four different conditions. Note the presence of MBP+ cells within unstimulated tissue (a), but that there were substantially more with tissue cultured with ragweed Ag (b) or rhIL-5 (c). Furthermore, d demonstrates that culturing tissue with ragweed + sIL-5R inhibited the increase in MBP+ cells.

Since the Abs employed to detect CD34 and MBP were both mouse monoclonals, they could not be used in colocalization studies to identify immature eosinophils. Instead, this was achieved by colocalizing CD34 immunoreactivity to chromotrope 2R, which identifies the eosinophil granules. The number of chromotrope⁺ cells was increased, similar to what was observed for MBP immunoreactivity, within tissue cultured with ragweed allergen (n = 5, 8.6 ± 1.7) compared with medium alone (2.0 ± 1.6, p < 0.01). There was a population of double positive (CD34 and chromotrope) cells, but their numbers were not different in stimulated (1.4 ± 0.9) compared with unstimulated tissue (2.6 ± 0.9, p > 0.05; Fig. 5). However, when the CD34⁺/chromotrope⁺ cells were examined as a percentage of total cell population, we observed a 14.6% increase per total CD34⁺ progenitor cells and a 27.2% reduction per total chromotrope⁺ cells. Since the absolute number of double positive cells remained relatively constant, these results suggest the progression from CD34⁺/chromotrope^-, to CD34⁺/chromotrope⁺ and finally to the CD34^-/chromotrope⁺ cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Percentage of immature eosinophils within nasal mucosal explant tissue. An increased proportion of CD34+/chromotrope+ cells per total CD34+ progenitor cells was observed within ragweed-stimulated compared with unstimulated tissue (*, p < 0.05). Although there seemed to be fewer CD34+/chromotrope+ per total chromotrope+ cells within tissue cultured with ragweed as compared with medium alone, this did not reach significance.

Basophils also arise from a CD34⁺ progenitor, express the IL-5R, and produce MBP, although at much lower levels than the eosinophil (36, 37, 38). For this reason, we employed a basophil-specific Ab, BB1 (29), to identify these cells within nasal mucosal tissue. Our observations demonstrate that there are basophils within unstimulated tissue (n = 5; 5.8 ± 0.8), but that the number of these cells was not significantly different following ragweed stimulation (6.8 ± 2.2, p > 0.05).

IL-5 gene expression

The number of IL-5 mRNA⁺ cells within nasal mucosal tissue was greater following both 6 and 24 h culture with ragweed allergen (n = 7, 10.1 ± 5.1; n = 9, 10.6 ± 3.2) compared with medium alone (3.8 ± 2.3, 3.5 ± 1.6; p < 0.001). There was no significant difference in the number of these cells within tissue cultured with rhIL-5 for either 6 h (n = 4, 4.6 ± 5.2) or 24 h (n = 8, 3.2 ± 2.6). Furthermore, there were similar numbers of IL-5 mRNA⁺ cells in tissue cultured with ragweed and sIL-5R molecules (n = 5, 8.6 ± 2.4), compared with ragweed only (10.0 ± 3.3, p > 0.05). Colocalization studies to phenotype the cells producing IL-5 mRNA demonstrated that, within sections from nasal mucosal tissue cultured with ragweed allergen, 44.0% ± 2.0 were associated with CD3⁺ cells (T cells) and 10.0% ± 8.3 with MBP⁺ cells (eosinophils). Furthermore, we also determined the proportion of these cell populations undergoing IL-5 gene transcription; 13% ± 0.8 of CD3⁺ cells were IL-5 mRNA⁺, whereas 5% ± 4.0 of the MBP⁺ cells were IL-5 mRNA⁺ (n = 4, Table II).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We show that both CD34⁺ progenitor and CD34/IL-5R⁺ cells are present within allergic nasal mucosa, the latter accounting for over 58% of the total progenitor cell population. Whether these eosinophil precursors arise from the bone marrow, previously associated with a population of CD34/IL-5R⁺ cells (21), and accumulate in the tissue or undergo lineage commitment locally could not be determined here. However, CD34⁺ progenitor cells have been demonstrated within the peripheral blood and lungs of both atopic and nonatopic control subjects (22, 23), and higher numbers of CD34/IL-5R⁺ cells have been detected in the lungs, but not the blood, of atopic asthmatics compared with atopic as well as nonatopic controls (23). Furthermore, it has recently been suggested that IL-5 may induce the expression of its own membrane-bound receptor (41). Together with the present finding that IL-5 mRNA⁺ cells are increased following ex vivo stimulation, these studies suggest that the presence of CD34⁺ progenitor cells are a characteristic feature of both normal and allergic mucosal tissue and that the local environment may indeed provide the necessary stimuli for eosinophil lineage commitment within the tissue itself. A further implication of this work is that the migration of progenitor cells, like mature cells, to the inflammatory lesion may be a controlled mechanism. Progenitor cell chemokines such as stromal cell-derived factor (SDF)-1 (42) and their expression within the tissue are potentially interesting areas of investigation.

The purpose of studying the nasal mucosa in an explant system was to eliminate the possibility that changes in cell numbers may be due to their infiltration, an issue inherent to most in vivo studies. Fewer eosinophil precursors and more MBP⁺ cells (r² = 0.64) within stimulated, compared with unstimulated, tissue supports the notion that CD34/IL-5R⁺ cells may undergo local differentiation, giving rise to the observed increase in MBP⁺ cells. This is in line with previous work by Eidelman et al. demonstrating increased MBP immunoreactivity in explanted slices of rat lung following culture with specific allergen (43). The fact that there were similar numbers of MBP⁺ cells within unstimulated tissue after 24 h of culture as that blocked immediately following resection indicates that the difference in eosinophil number was not merely due to cell death within unstimulated tissue. Furthermore, the increased proportion of immature eosinophils (CD34⁺/chromotrope⁺ cells) per total number of progenitor cells, with the accompanying reduction per total number of eosinophils, in stimulated compared with unstimulated tissue, suggests a progression along the differentiation pathway. We cannot rule out the possibility that a proportion of the CD34/IL-5R⁺ or MBP⁺ cells may be partly associated with tissue basophils. Until recently, no reagents were available to us for the immunodetection of these cells, and, as such, their presence within allergic tissue has not been well documented. Using a newly developed basophil-specific Ab (29), we demonstrated no change in cell number in stimulated compared with unstimulated tissue, indicating that the observed increase in MBP immunoreactivity was eosinophil derived. Although we have not examined chemokine expression, the consistent observation that MBP⁺ cells migrate toward and infiltrate the epithelial layer within stimulated tissue indicates the likelihood that the epithelium is producing eotaxin and/or MCP-4, which occurs in vivo following intranasal challenge with specific allergen (44, 45). Although these data strongly suggest local eosinophil differentiation, they do not in any way exclude a role for eosinophil infiltration. Elevated numbers of peripheral blood (46, 47) as well as tissue (27, 48) eosinophils in subjects with allergic disorders are well documented. Our present work indicates, however, that, in addition to the infiltration of mature eosinophils, a population may originate from within the target organ itself.

Culture with rhIL-5 protein was associated with similar, albeit less pronounced, effects on the number of CD34/IL-5R mRNA⁺ and MBP⁺ cells as observed following culture with specific allergen. The more striking reduction in CD34/IL-5R mRNA⁺ cells in response to rhIL-5 at 6 (rather than 24 h) could be due to protein degradation. Alternatively, since the total number of IL-5R mRNA⁺ cells was higher within rhIL-5- than ragweed-stimulated tissue at 24 h (but not 6 h), it is possible that IL-5 may induce synthesis of IL-5R mRNA, as has been previously suggested (41). Although it appeared that there were more IL-5 mRNA⁺ cells following 6 h culture with rhIL-5 compared with control, it was not statistically different, indicating that this cytokine has no or little effect on the expression of its own mRNA. These results imply that allergen induces a more broad-spectrum response than rhIL-5 alone. Presumably, activation of mast cells and release of IL-4, likely to induce T cell production of IL-5 (49), is the predominant pathway involved. In fact, in a concurrent study, we have observed increased numbers of mast cells and T cells producing IL-4 mRNA within allergen-stimulated compared with unstimulated tissue (50).

Culturing tissue with sIL-5R almost completely attenuated the ragweed-induced change in eosinophil precursor and MBP-immunoreactive cell numbers, indicating the obligatory nature of IL-5 for eosinophil differentiation. Although this finding is contrary to work by Clutterbuck (13) and Shalit (14) et al. demonstrating the IL-5-independent ability of IL-3 and GM-SCF to induce the development of eosinophil colonies, it is in agreement with recent work by Popken-Harris et al. illustrating that rhIL-5 is sufficient for the formation of both pro- and mature MBP (51). Furthermore, Robinson et al. have now suggested that the effects of GM-CSF and IL-3 may be mediated through the production of IL-5 itself (41), which would be in agreement with the present findings. However, sIL-5R did not inhibit ragweed-induced IL-5 gene expression, further substantiating the observation that this cytokine does not seem to control its own synthesis. The drastic effect of sIL-5R on the ragweed-induced eosinophil differentiation leads one to consider whether endogenous production of soluble receptors may regulate IL-5 function, in vivo. Indeed, eosinophil-differentiated HL-60 cells and eosinophils derived from CD34⁺ cord blood cells have been shown to produce mainly IL-5R mRNA coding for the soluble isoform of the -chain (18) and Yasruel et al. (52) have shown that the expression of sIL-5R mRNA in bronchial biopsies of atopic and nonatopic asthmatics directly correlated with the patients’ FEV₁. As such, factors that induce production of the soluble isoform of the IL-5R may prove highly useful for abrogating eosinophil development as well as other IL-5-mediated events.

An increased number of IL-5 mRNA⁺ cells is a well recognized characteristic of allergic respiratory mucosa (48, 53) and has been considered to be the consequence of inflammatory cell infiltration. Here, we demonstrate the production of IL-5 mRNA following ex vivo stimulation, providing direct evidence that local inflammatory cells increase their cytokine production following allergen exposure. Although we show that a significant proportion of IL-5 mRNA is T cell derived, eosinophils were also seen to produce this cytokine. However, these two cell types accounted for only 54% of the total IL-5 mRNA⁺ cells, in contrast to previous in vivo findings demonstrating that 88% of IL-5 mRNA⁺ cells were T cells and eosinophils (54). This underlines the importance of other sources of IL-5, particularly mast cells (54), within allergic nasal mucosa. Regardless of the source, it appears that enough IL-5 is made locally to induce changes in the number of eosinophil precursors and MBP-immunoreactive cells, lending support to the idea that in vivo circulating cells may enter the tissue and rely on the local cytokine environment for phenotype acquisition.

Studies have shown that MBP immunoreactivity (BMK-13) is absent in CD34⁺ cells before IL-5 stimulation (55), that MBP message is produced as early as day 3 (56), and MBP protein, or at least pro-MBP, is produced after 6 days of culture with IL-5 (51). Here, we demonstrate an increase in the number of MBP-immunoreactive cells after only 6 h of culture with allergen or rhIL-5. At present, we cannot explain these rapid kinetics; however, they are consistent with the work of Eidelman et al. demonstrating an increased number of MBP⁺ cells after 6 h of culturing slices of rat lung with specific allergen (43). Furthermore, the fact that we identified immature eosinophils (CD34⁺/chromotrope⁺) within sections of unstimulated tissue implies that the nasal mucosa of these individuals was sensitized and as such may be primed for rapid MBP production. Recent in vitro studies of eosinophil progenitor differentiation have revealed that there is a pattern of granulogenesis and cationic protein condensation, including the proteolytic processing of pro-MBP to MBP, within the secondary granules (55, 51). Whether BMK-13 binds to pro-MBP as well as the mature protein is not known. Consequently, the observed increase in MBP immunoreactivity at 6 h could be attributed to cleavage of pro-MBP, rather than de novo protein synthesis. Although we did not see eosinophils that were clearly intravascular, the possibility that the airway microvasculature retains a population of marginated eosinophils that transmigrate toward the airway mucosa following allergen challenge cannot be ruled out. Considering the relatively few MBP⁺ cells detected within unstimulated tissue, however, it is unlikely that the increase observed here could be accounted for in this way.

Cell culture, while greatly advancing our understanding of cell and cytokine function, is a limited system lacking the important elements of cell-cell and cell-matrix interactions, as well as complex intercytokine networking. The strength of the explant technique is two-fold. First, it side-steps the difficulty of recreating an "in vivo-like" environment, and, second, it allows for the delineation of local vs systemic events. Using nasal mucosal explant tissue, we have demonstrated a concomitant decrease in eosinophil precursors and increase in MBP-immunoreactive cells following ex vivo stimulation and that sIL-5R inhibits these changes. This study provides strong evidence to support the concept that a subset of eosinophils may differentiate locally within allergic tissue and indicates that this event is highly IL-5 dependent. In addition, these findings suggests that endogenous production of sIL-5R functions as a regulator of this process.

	Acknowledgments

 
We thank Ms. Elsa Schotman and Ms. Cathy Fragiskatos for their invaluable technical expertise and assistance.

	Footnotes

 
¹ This work was supported by the Medical Research Council of Canada, the Inspiraplex Network Centers of Excellence, and the J. T. Costello Memorial Research Foundation. L.C. is a recipient of a Canadian Society of Allergy and Clinical Immunology/Merck Frosst fellowship. Q.H. is a Research Scholar of the Fonds de la Recherche en Santé du Québec. R.M. is an Alberta Heritage Senior Medical Scholar.

² Address correspondence and reprint requests to Dr. Qutayba Hamid, Meakins-Christie Laboratories, McGill University, 3626 Saint Urbain Street, Montreal, Québec, Canada H2X 2P2. E-mail address:

³ Abbreviations used in this paper: MBP, major basic protein; rhIL-5, recombinant human IL-5 protein; sIL-5R, soluble IL-5R; PNU, protein nitrogen unit.

Received for publication August 9, 1999. Accepted for publication November 17, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Varney, V. A., M. R. Jacobson, R. M. Sudderick, D. S. Robinson, A. M. A. Irani, L. B. Schwartz, I. S. MacKay, A. B. Kay, S. R. Durham. 1992. Immunohistology of the nasal mucosa following allergen-induced rhinitis: identification of activated T lymphocytes, eosinophils and neutrophils. Am. Rev. Resp. Dis. 146:170.[Medline]
2. Bascom, R., U. Pipkorn, D. Proud, S. Dunnette, G. J. Gleich, L. M. Lichtenstein, R. M. Naclerio. 1989. Major basic protein and eosinophil-derived neurotoxin concentrations in nasal-lavage fluid after antigen challenge: effect of systemic corticosteroids and relationship to eosinophil influx. J. Allergy Clin. Immunol. 84:338.[Medline]
3. Hisamatsu, K., T. Ganbo, T. Nakazawa, Y. Murakami, G. J. Gleich, K. Makiyama, H. Koyama. 1990. Cytotoxicity of human eosinophil granule major basic protein to human nasal sinus mucosa in vitro. J. Allergy Clin. Immunol. 86:52.[Medline]
4. Thomas, L. L., L. M. Zheutlin, G. J. Gleich. 1989. Pharmacological control of human basophil histamine release stimulated by eosinophil granule major basic protein. Immunology 66:611.[Medline]
5. Moy, J. N., G. J. Gleich, L. L. Thomas. 1990. Noncytotoxic activation of neutrophils by eosinophil granule major basic protein: effect on superoxide anion generation and lysosomal enzyme release. J. Immumol. 145:2626.[Abstract]
6. Nonaka, M., R. Nonaka, K. Woolley, E. Adelroth, K. Miura, Y. Okhawara, M. Glibectic, K. Nakano, P. O’Byrne, J. Dolovich, M. Jordana. 1995. Distinct immunohistochemical localization of IL-4 in human inflamed airway tissues: IL-4 is localized to eosinophils in vivo and is released by peripheral blood eosinophils. J. Immunol. 155:3234.[Abstract]
7. Moqbel, R., S. Ying, J. Barkans, T. W. Newman, P. Kimmitt, M. Wakelin, L. Taborda-Barata, Q. Meng, C. J. Corrigan, S. R. Durham, A. B. Kay. 1995. Identification of messenger RNA for IL-4 in human eosinophils with granule localization and release of the translated product. J. Immunol. 155:4939.[Abstract]
8. Hamid, Q., J. Barkans, Q. Meng, J. Abrams, A. B. Kay, R. Moqbel. 1992. Human eosinophils synthesize and secrete interleukin-6, in vitro. Blood 80:1496.[Abstract/Free Full Text]
9. Elovic, A., D. T. Wong, P. F. Weller, K. Matossian, S. J. Galli. 1994. Expression of transforming growth factors- and ß₁ messenger RNA and product by eosinophils in nasal polyps. J. Allergy Clin. Immunol. 93:864.[Medline]
10. Minshall, E. M., D. Y. M. Leung, R. J. Martin, Y. L. Song, L. Cameron, P. Ernst, Q. Hamid. 1997. Eosinophil-associated TGF-ß₁ mRNA expression and airways fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 17:326.[Abstract/Free Full Text]
11. Broide, D. H., M. M. Paine, G. S. Firestein. 1992. Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics. J. Clin. Invest. 90:1414.
12. Kita, H., T. Ohnishi, Y. Okubo, D. Weiler, J. S. Abrams, G. J. Gleich. 1991. Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils. J. Exp. Med. 174:745.[Abstract/Free Full Text]
13. Clutterbuck, E. J., E. M. A. Herst, C. J. Sanderson. 1989. Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interactions with IL-1, IL-3, IL-6 and GM-CSF. Blood 73:1504.[Abstract/Free Full Text]
14. Shalit, M., S. Sekhsaria, H. L. Malech. 1995. Modulation of growth and differentiation of eosinophils from human peripheral blood CD34⁺ cells by IL-5 and other growth factors. Cell. Immunol. 160:50.[Medline]
15. Palframan, R. T., P. D. Collins, R. J. Williams, S. M. Rankin. 1998. Eotaxin induces a rapid release of eosinophils and their progenitors from the bone marrow. Blood 91:2240.[Abstract/Free Full Text]
16. Foster, P. S., S. P. Hogan, A. J. Ramsay, K. I. Matthaei, I. G. Younge. 1996. Interleukin-5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med. 183:195.[Abstract/Free Full Text]
17. Egan, R. W., D. Athwahl, C. Chou, S. Emtage, C. Jehn, T. T. Kung, P. J. Mauser, N. J. Murgolo, M. W. Bodmer. 1995. Inhibition of pulmonary eosinophilia and hyperreactivity by antibodies to interleukin-5. Int. Arch. Allergy Immunol. 107:321.[Medline]
18. Tavernier, J., R. Devos, S. Cornelis, T. Tuypens, J. Van der Heyden, W. Fiers, G. Plaetinck. 1991. A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific chain and a ß chain shared with the receptor for GM-CSF. Cell 66:1175.[Medline]
19. Tavernier, J., T. Tuypens, G. Plaetinck, A. Verhee, W. Fiers, R. Devos. 1992. Molecular basis of the membrane-anchored and two soluble isoforms of the human interleukin 5 receptor a subunit. Proc. Natl. Acad. Sci. USA 89:7041.[Abstract/Free Full Text]
20. Ema, H., T. Suda., K. Nagayoshi, Y. Muira, C. I. Civin, H. Nakauchi. 1990. Target cells for granulocyte macrophage colony stimulating factor, interleukin-3 and interleukin-5 in the differentiation pathways of neutrophils and eosinophils. Blood 76:1956.[Abstract/Free Full Text]
21. Sehmi, R., L. J. Wood, R. Watson, R. Foley, Q. Hamid, P. M. O’Byrne, J. A. Denburg. 1997. Allergen-induced increase in IL-5R subunit expression on bone marrow derived CD34⁺ cells from asthmatic subjects; a novel marker of progenitor cell commitment toward eosinophil differentiation. J. Clin. Invest. 100:2466.[Medline]
22. Sehmi, R., K. Howie, D. R. Sutherland, W. Schragge, P. M. O’Byrne, J. A. Denburg. 1996. Increased levels of CD34⁺ hemopoietic progenitors in atopic subjects. Am. J. Respir. Cell Mol. Biol. 15:645.[Abstract]
23. Robinson, D. R., R. Damia, K. Zeibecoglou, S. Molet, J. North, T. Yamada, A. B. Kay, Q. Hamid. 1999. CD34⁺/interleukin-5R messenger RNA⁺ cells in the bronchial mucosa of asthma: potential airway eosinophil progenitors. Am. J. Respir. Cell Mol. Biol. 20:9.[Abstract/Free Full Text]
24. Durham, S. R., S. Ying, V. Varney, M. Jacobson, R. Sudderick, I. Mackay, A. B. Kay, Q. Hamid. 1992. Cytokine messenger RNA expression for IL-3, IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor in the nasal mucosa after local allergen provocation: relationship to tissue eosinophilia. J. Immunol. 148:2390.[Abstract]
25. Dandurand, R. J., C. G. Wang, S. Laberge, J. G. Martin, D. H. Eidelman. 1994. In vitro allergic bronchoconstriction in the Brown Norway rat. Am. J. Respir. Crit. Care Med. 149:1499.[Abstract]
26. Minshall, E., C. G. Wang, R. Dandurand, D. Eidelman. 1997. Heterogeneity of responsiveness of individual airways in cultured lung explants. Can. J. Physiol. Pharmacol. 75:911.[Medline]
27. Moqbel, R., J. Barkans, B. L. Bradely, S. R. Durham, A. B. Kay. 1992. Application of monoclonal antibodies against major basic protein (BMK-13) and eosinophil cationic protein (EG1 and EG2) for quantifying eosinophils in bronchial biopsies from atopic asthma. Clin. Exp. Allergy 22:265.[Medline]
28. Fina, L., H. V. Molgaard, D. Robertson, N. J. Bradely, P. Mongan, D. Delia, D. R. Sutherland, M. A. Baker, M. F. Greaves. 1990. Expression of the CD34 gene in vascular endothelial cells. Blood 75:2417.[Abstract/Free Full Text]
29. McEuen, A. R., M. G. Buckley, S. J. Compton, A. F. Walls. 1999. Development and characterization of a monoclonal antibody specific for human basophils and the identification of a unique secretory product of basophil activation. Lab. Invest. 79:27.[Medline]
30. Hamid, Q., J. Wharton, G. Terenghi, C. J. S. Hassall, J. Aimi, K. M. Taylor, J. Nakazato, J. I. Dixon, G. Burnstock, J. Polak. 1987. Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage. Proc. Natl. Acad. Sci. USA 84:6760.[Abstract/Free Full Text]
31. Giaid, A., R. P. Michel, D. J. Stewart, M. Sheppard, B. Corrin, Q. Hamid. 1993. Expression of endothelin-1 in lungs of patients with cryptogenic fibrosing alveolitis. Lancet 341:1550.[Medline]
32. Bentley, A. M., Q. Meng, D. S. Robinson, Q. Hamid, A. B. Kay, S. R. Durham. 1993. Increases in activated T lymphocytes, eosinophils, and cytokine mRNA expression in interleukin-5 and granulocyte/macrophage colony-stimulating factor in bronchial biopsies after allergen challenge in atopic asthmatics. Am. J. Respir. Cell Mol. Biol. 93:35.
33. Doyle, W. J., D. P. Skoner, J. T. Seroky, P. Fireman. 1995. Reproducibility of the effects of intranasal ragweed challenges in allergic subjects. Ann. Allergy Asthma Immunol. 74:171.[Medline]
34. Puri, K. D., E. B. Finger, G. Gaudernack, T. A. Springer. 1995. Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules. J. Cell Biol. 131:261.[Abstract/Free Full Text]
35. Simmons, P. J., B. Torok-Storb. 1991. CD34 expression by stromal precursors in normal human adult bone marrow. Blood 78:2848.[Abstract/Free Full Text]
36. Ackerman, D. J., G. M. Kephart, T. M. Habermann, P. R. Greipp, G. J. Gleich. 1983. Localization of eosinophil granule major basic protein in human basophils. J. Exp. Med. 158:946.[Abstract/Free Full Text]
37. Kirshenbaum, A. S., J. P. Goff, S. W. Kessler, J. M. Mican, K. M. Zsebo, D. D. Metcalfe. 1992. Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34⁺ pluripotent progenitor cells. J. Immunol. 148:772.[Abstract]
38. Yamada, T., Q. Sun, K. Zeibecoglou, J. Bungre, J. North, A. B. Kay, A. F. Lopez, D. S. Robinson. 1998. Interleukin-3, interleukin-5 and granulocyte-macrophage colony stimulating factor receptor subunit and common ß subunit expression by peripheral leukocytes and blood dendritic cells. J. Allergy Clin. Immunol. 101:677.[Medline]
39. Denburg, J. A., J. Dolovich, D. Harnish. 1989. Basophil mast cell and eosinophil growth and differentiation factors in human allergic disease. Clin. Exp. Allergy. 19:249.[Medline]
40. Denburg, J. A.. 1998. The origins of basophils and eosinophils in allergic inflammation. J. Allergy Clin. Immunol. 102:S74.[Medline]
41. Robinson, D. S., J. North, S. Rankin, A. B. Kay, X. Van ostade, J. Van der Heyden, G. Brusselle, J. Vanderkerckhove, A. Verhee, J. Tavernier. 1999. Interleukin-5 regulates the isoform expression of its own receptor -subunit. J. Allergy Clin. Immunol. 103:A917.
42. Aiuti, A., I. J. Webb, C. Bleul, T. Springer, J. C. Gutierrez-Ramos. 1997. The chemokine SDF-1 is a chemoattractant for human CD34⁺ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34⁺ progenitors to peripheral blood. J. Exp. Med. 185:111.[Abstract/Free Full Text]
43. Eidelman, D. H., E. M. Minshall, R. J. Dandurand, E. Schotman, Y. L. Song, Z. Yasruel, R. Moqbel, Q. Hamid. 1996. Evidence for major basic protein immunoreactivity and interleukin-5 gene activation during the late phase response in explanted airways. Am. J. Respir. Cell Mol. Biol. 15:582.[Abstract]
44. Minshall, E. M., L. Cameron, F. Lavigne, D. Y. 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]
45. Christodoulopoulos, P., E. Wright, S. Frenkiel, A. Luster, Q. Hamid. 1999. Monocyte chemotactic proteins in allergen-induced inflammation in the nasal mucosa: effect of topical corticosteroids. J. Allergy Clin. Immunol. 103:1036.[Medline]
46. Durham, S. R., D. A. Loegering, S. Dunnette, G. J. Gleich, A. B. Kay. 1989. Eosinophils and eosinophil-derived proteins in allergic asthma. J. Allergy Clin. Immunol. 84:931.[Medline]
47. Frick, W. E., J. B. Sedgwick, W. W. Busse. 1988. Hypodense eosinophils in allergic rhinitis. J. Allergy Clin. Immunol. 82:119.[Medline]
48. Masuyama, K., S. J. Till, M. R. Jacobson, A. Kamil, L. Cameron, S. Juliusson, O. Lowhagen, A. B. Kay, Q. Hamid, S. R. Durham. 1998. Nasal eosinophilia and IL-5 mRNA expression in seasonal allergic rhinitis induced by natural allergen exposure: effect of topical corticosteroids. J. Allergy Clin. Immunol. 102:610.[Medline]
49. Swain, S. L., A. D. Weinberg, M. English, G. Huston. 1990. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145:3796.[Abstract]
50. Cameron, L., S. Durham, E. Wright, L. Smurthwaite, E. Minshall, S. Frenkiel, Q. Hamid, H. Gould. 1998. Evidence for local IgE RNA transcript synthesis in allergic nasal mucosa following in vitro antigen challenge. J. Allergy Clin. Immunol. 101:A667.
51. Popken-Harris, P., J. Checkel, D. Loegering, B. Madden, M. Springett, G. Kephart, G. J. Gleich. 1998. Regulation and processing of a precursor form of eosinophil granule major basic protein (pro-MBP) in differentiating eosinophils. Blood 92:623.[Abstract/Free Full Text]
52. Yasruel, Z., M. Humbert, T. C. Kotsimbos, Y. Ploysongsang, E. Minshall, S. R. Durham, R. Pfister, G. Menz, J. Tavernier, A. B. Kay, Q. Hamid. 1997. Membrane-bound and soluble IL-5R mRNA in the bronchial mucosa of atopic and nonatopic asthmatics. Am. J. Respir. Crit. Care Med. 155:1413.[Abstract]
53. Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A. J. Wardlaw, C. J. Corrigan, B. Bradley, S. R. Durham, J. V. Collins, P. K. Jeffery, D. Quint, A. B. Kay. 1991. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J. Clin. Invest. 87:1541.
54. Ying, S., S. R. Durham, J. Barkans, K. Masuyama, M. Jacobson, S. Rak, O. Lowhagen, R. Moqbel, A. B. Kay, Q. A. Hamid. 1993. T cells are the principal source of interleukin-5 mRNA in allergen-induced rhinitis. Am. J. Resp. Cell Mol. Biol. 9:356.
55. Makmudi-Azer, S., J. R. Velazque, P. Lacy, R. Moqbel. 1999. Evidence for differential transport of granule proteins during IL-5-dependent differentiation of eosinophil colonies from human CD34. J. Allergy Clin. Immunol. 103:A428.
56. Shalit, M., S. Sekhsaria, F. Li, S. Mauhorter, S. Mahanti, H. Malech. 1996. Early commitment to the eosinophil lineage by cultured human peripheral blood CD34⁺ cells: messenger RNA analysis. J. Allergy Clin. Immunol. 98:344.[Medline]

This article has been cited by other articles:

				J. H. Kang, D. H. Lee, H. Seo, J. S. Park, K. H. Nam, S. Y. Shin, C.-S. Park, and I. Y. Chung Regulation of Functional Phenotypes of Cord Blood Derived Eosinophils by {gamma}-Secretase Inhibitor Am. J. Respir. Cell Mol. Biol., November 1, 2007; 37(5): 571 - 577. [Abstract] [Full Text] [PDF]

				J. A. Denburg and S. F. van Eeden Bone marrow progenitors in inflammation and repair: new vistas in respiratory biology and pathophysiology. Eur. Respir. J., March 1, 2006; 27(3): 441 - 445. [Full Text] [PDF]

				P. J. Sterk and P. S. Hiemstra Eosinophil Progenitors in Sputum: Throwing out the Baby with the Bath Water? Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 549 - 550. [Full Text] [PDF]

				B. Sitkauskiene, A.-K. Johansson, S. Sergejeva, S. Lundin, M. Sjostrand, and J. Lotvall Regulation of Bone Marrow and Airway CD34+ Eosinophils by Interleukin-5 Am. J. Respir. Cell Mol. Biol., March 1, 2004; 30(3): 367 - 378. [Abstract] [Full Text] [PDF]

				S. C. Dorman, A. Efthimiadis, I. Babirad, R. M. Watson, J. A. Denburg, F. E. Hargreave, P. M. O'Byrne, and R. Sehmi Sputum CD34+IL-5R{alpha}+ Cells Increase after Allergen: Evidence for In Situ Eosinophilopoiesis Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 573 - 577. [Abstract] [Full Text] [PDF]

				S. Sergejeva, A.-K. Johansson, C. Malmhall, and J. Lotvall Allergen exposure-induced differences in CD34+ cell phenotype: relationship to eosinophilopoietic responses in different compartments Blood, February 15, 2004; 103(4): 1270 - 1277. [Abstract] [Full Text] [PDF]

				L. Cameron, A. S. Gounni, S. Frenkiel, F. Lavigne, D. Vercelli, and Q. Hamid S{epsilon}S{micro} and S{epsilon}S{gamma} Switch Circles in Human Nasal Mucosa Following Ex Vivo Allergen Challenge: Evidence for Direct as Well as Sequential Class Switch Recombination J. Immunol., October 1, 2003; 171(7): 3816 - 3822. [Abstract] [Full Text] [PDF]

				B. Lamkhioued, S. G. Abdelilah, Q. Hamid, N. Mansour, G. Delespesse, and P. M. Renzi The CCR3 Receptor Is Involved in Eosinophil Differentiation and Is Up-Regulated by Th2 Cytokines in CD34+ Progenitor Cells J. Immunol., January 1, 2003; 170(1): 537 - 547. [Abstract] [Full Text] [PDF]

				L. Y. Liu, J. B. Sedgwick, M. E. Bates, R. F. Vrtis, J. E. Gern, H. Kita, N. N. Jarjour, W. W. Busse, and E. A. B. Kelly Decreased Expression of Membrane IL-5 Receptor {alpha} on Human Eosinophils: I. Loss of Membrane IL-5 Receptor {alpha} on Airway Eosinophils and Increased Soluble IL-5 Receptor {alpha} in the Airway After Allergen Challenge J. Immunol., December 1, 2002; 169(11): 6452 - 6458. [Abstract] [Full Text] [PDF]

				L. Y. Liu, J. B. Sedgwick, M. E. Bates, R. F. Vrtis, J. E. Gern, H. Kita, N. N. Jarjour, W. W. Busse, and E. A. B. Kelly Decreased Expression of Membrane IL-5 Receptor {alpha} on Human Eosinophils: II. IL-5 Down-Modulates Its Receptor Via a Proteinase-Mediated Process J. Immunol., December 1, 2002; 169(11): 6459 - 6466. [Abstract] [Full Text] [PDF]

M.