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PHAGOCYTES
From the Department of Medicine, Mayo Clinic,
Rochester, MN.
Neurotrophins, such as nerve growth factor (NGF) and
neurotrophin-3 (NT-3), are essential for development, function, and
survival of peripheral sympathetic and sensory neurons. Most
eosinophilic leukocytes in the human body are localized in mucosal
tissues; however, the roles of eosinophils in human diseases are not
fully understood. We found that human eosinophils constitutively
express messenger RNA for NGF and NT-3, synthesize and store these
proteins intracellularly, and continuously replenish them. Incubation
of eosinophils with a transcription inhibitor, actinomycin D, for 8 hours completely depletes intracellular NGF and NT-3. New synthesis of
NGF is enhanced by Fc-receptor-mediated stimuli, such as
immunoglobulin (Ig)A and IgG immune complexes; in contrast, production
of NT-3 is not affected by these stimuli. Notably, supernatants of
eosinophils stimulated with IgA immune complex and interleukin 5 promote neurite extension of the PC-12 pheochromocytoma cell line; this
effect is abolished by pretreatment of the supernatants with
anti-NGF-neutralizing antibody. By enzyme-linked immunosorbent assay,
substantial amounts of NGF protein are also detected in the
supernatants of stimulated eosinophils. Furthermore, in patients with
seasonal allergic rhinitis, the concentrations of NGF in nasal
secretions correlate with the magnitudes of eosinophilic inflammation
in the airway, suggesting a potential clinical implication of
eosinophil NGF. Our observations propose a new pathologic mechanism by
which eosinophils may contribute to enhanced neurologic responses in
patients with allergic diseases and other eosinophilic disorders.
Alternatively, eosinophils may play important roles in maintenance and
restoration of homeostatic functions of mucosal tissues through the
pleitropic activities of NGF.
(Blood. 2002;99:2214-2220) Eosinophils are leukocytes associated with helminth
infection and allergic diseases (reviewed in Kita et al1).
Although the eosinophil is a formed element of the peripheral
circulation, it primarily resides in the tissues. More than 99% of
eosinophils in the human body are distributed in the mucosal tissues in
which the epithelial surfaces are exposed to the external
environment.1 However, the physiologic or pathologic roles
of tissue-resident eosinophils in host defense and human diseases are
not fully understood. Eosinophils release a number of proinflammatory
mediators, such as cytotoxic cationic proteins and lipid mediators,
which could be important in the pathophysiology of asthma and allergic
disease.1 Several lines of evidence, many from correlative
studies, have also implicated eosinophils in the mechanisms of
heightened neurologic responses in diseases, especially in bronchial
asthma2-4 and atopic dermatitis.5 Indeed,
airway eosinophilic inflammation and increased bronchial responsiveness
to bronchoconstrictive stimuli are hallmarks of human bronchial
asthma.2-4 Eosinophils are also potential sources of
cytokines and growth factors.6 These factors include
autocrine cytokines, such as granulocyte-macrophage colony-stimulating
factor7 and interleukin-5 (IL-5);8
immunomodulatory cytokines, such as IL-49,10 and
IL-10;10 chemokines and chemotactic cytokines, such as
regulated on activation, normal T cell-expressed and
-secreted11 and IL-16;12 and factors involved
in fibrosis and tissue repair, such as tumor growth factor (TGF)- In this study, we investigated whether eosinophils secrete the
neurotrophic cytokines, nerve growth factor (NGF) and neurotrophin-3 (NT-3). NGF is essential for survival, development, and function of
peripheral sympathetic and sensory neurons (reviewed in
Levi-Montalcini16). Indeed, overexpression of NGF in the
lungs of mice promotes innervation of tachykinin-containing sensory
neurons, hyperreactivity to capsaicin, and thickening of the basement
membrane in the airways.17 Increased expression and
release of NGF were also found in patients with allergic
rhinitis18 and are implicated in the development of neural
hyperresponsiveness in patients.19 In addition to its neurotrophic activity, NGF exerts broad biologic activities on nonneuroral cells involved in innate and adaptive immune systems, such
as lymphocytes,20 mast cells,21
basophils,22 and eosinophils themselves.23
Furthermore, NGF also promotes hemopoietic colony growth24
and may participate in wound healing and the tissue repair
process.25,26 NGF was shown to be expressed by various cells.23,27,28 However, little is currently known about
any cell type that could produce NGF or NT-3 on immunologically
specific activation. Furthermore, our knowledge about the regulatory
mechanisms that produce these essential NGFs is limited. In this study,
we found that human eosinophils constitutively synthesize NGF and NT-3.
Moreover, when the cells are activated with inflammatory stimuli, they
secrete substantial amounts of NGF and promote the development of
neuronal cells by NGF activity.
Isolation of eosinophils, neutrophils, and mononuclear
cells
Detection of NGF and NT-3 mRNA by RT-PCR
The specificities of the amplified products were confirmed by dot blot analyses using internal oligonucleotide probes (data not shown). PCR was performed in a thermal cycler for 35 cycles (94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds), followed by a 7-minute extension at 72°C. Five microliters of the PCR products with 1 µL 6× DNA loading buffer was subject to electrophoresis on 1.5% agarose gels and stained with 0.5 µg/mL ethidium bromide. Activation of eosinophils Eosinophils were suspended in RPMI 1640 medium supplemented with 25 mM HEPES and L-glutamine (Celox Laboratories, Hopkins, MN) containing 10% (vol/vol) 56°C heat-inactivated defined calf serum (RPMI-DCS; HyClone Laboratories, Logan, UT). Soluble immune complex in the presence or absence of interleukin-5 (IL-5) was used to stimulate eosinophils as previously described.8,9,33 Briefly, eosinophils suspended in RPMI-DCS at 1 × 106 cells/mL were incubated in 48-well tissue culture plates with or without human secretory immunoglobulin A (sIgA; ICN Pharmaceutical, Aurora, OH), human serum IgA (ICN Pharmaceutical), or human serum IgG (ICN Pharmaceutical) at a final concentration of 20 µg/mL. After a 1-hour incubation at 4°C and without washing, eosinophils were stimulated with corresponding antihuman sIgA monoclonal antibody (diluted 1:1000; Sigma), goat antihuman IgA (20 µg/mL; ICN Pharmaceutical), or goat antihuman IgG (20 µg/mL; ICN Pharmaceutical) and cultured in the presence or absence of 25 ng/mL IL-5 at 37°C and 5% CO2 for 24 hours. After incubation, cell-free supernatants were collected and stored at 20°C until used for
bioassay of NGF activity and for NGF protein assay (see below). To
quantitate intracellular contents of NGF, NT-3, and an eosinophil
granule protein, eosinophil-derived neurotoxin (EDN), eosinophil cell
pellets were lysed with 1 mL 1% Triton-X in phosphate-buffered saline
(PBS) and 3 freeze-thaw cycles. To quantitate intracellular NGF, NT-3,
and EDN, eosinophils were cultured without stimuli and either no
inhibitor or with the transcription inhibitor, actinomycin D (1 µM;
Sigma), for up to 8 hours and lysed. By trypan blue dye exclusion,
eosinophil viability after 8 hours of treatment with actinomycin D was
more than 96%.
Measurement of NGF, NT-3, and EDN proteins To measure NGF and NT-3 proteins in cell lysates and supernatants, we used commercial enzyme-linked immunosorbent assay (ELISA) kits (Promega Corporation, Madison, WI) according to the manufacturer's recommended procedure with the standard curve slightly modified. The specificities of the assay were confirmed by the manufacturer, and the low end of the standard curve was 3.9 pg/mL. Eosinophil granule protein, EDN, in cell lysates was quantitated by radioimmunoassay (RIA). The RIA is a double antibody competition assay performed by using radioiodinated EDN, rabbit anti-EDN antibody, and burro antirabbit IgG.34 The minimum detection limit of this EDN assay was 2 ng/mL. All assays were performed in duplicate.Biologic assay of NGF activity The bioassay using the PC-12 pheochromocytoma cell line for NGF activity is commonly used to detect and to measure biologically active NGF, which stimulates neurite outgrowth of PC-12 cells.35 This cell line does not respond to NT-3.35 The PC-12 cell line (ATCC, Manassas, VA) was expanded and maintained in complete medium consisting of 85% F-12 medium, 10% heat-inactivated horse serum, and 5% fetal calf serum as previously described.36 To detect and quantitate the biologic activity of NGF in eosinophil supernatants, PC-12 cells were replated onto collagen-coated 24-well tissue culture plates at 2 × 104 cells/well, and eosinophil culture supernatants or serial dilutions of NGF as controls were added to the wells. After 24 hours, the numbers of PC-12 cells with neurite outgrowth were counted by using the dark field inverted microscope (Nikon). Cells with at least 2 neurites and each neurite more than about 50 µm long were judged as neurite outgrowth-positive cells. All the samples were tested in duplicate, and the enumeration was performed in a blinded manner. The specificity of the assay was confirmed by adding neutralizing goat antihuman NGF antibody (100 ng/mL; R&D Systems) or goat IgG (Sigma) to the replicate samples.Immunocytochemistry Immunocytochemistry for NGF and NT-3 in eosinophil sections was performed with goat antibodies for these proteins and fluorescein isothiocyanate (FITC)-conjugated rabbit antigoat IgG. Briefly, freshly isolated eosinophils were collected and mixed with 1% agar in PBS and then fixed in 4% freshly prepared paraformaldehyde, processed, and embedded in paraffin as previously described.37 Five-micrometer sections were mounted on microscope slides, deparaffinized with xylene, and rehydrated. After partial digestion with 0.1% trypsin (Sigma), the slides were blocked with 10% normal rabbit serum overnight at 4°C. Serial sections were incubated with goat antihuman NGF antibody (5 µg/mL; R&D Systems), goat antihuman NT-3 antibody (5 µg/mL; R&D Systems), or normal goat IgG (5 µg/mL) at 37°C for 30 minutes. The slides were washed and blocked with 1% chromotrope 2R (EM Science, Cherry Hill, NJ). Sections were then incubated with affinity-purified FITC-conjugated rabbit antigoat IgG (Southern Biotechnology Associates, Birmingham, AL) at 37°C for 30 minutes. Sections were washed and mounted with 10% PBS:90% glycerol solution containing p-phenylenediamine, coverslipped, and sealed. The fluorescence images were captured with a confocal laser-scanning microscope (LSM 310; Carl Zeiss, Oberkochen, Germany) at a pinhole setting of 50. The excitation wavelength from an argon/krypton laser was set to 488 nm, and the emission wavelength was set to 530 ± 15 nm. Transmission images were also captured from the same field to visualize the morphology of the cells.NGF and NT-3 levels in nasal lavage fluids from patients with ragweed hay fever during allergy season Fifteen patients, aged 18 to 60 years, with moderate to severe ragweed allergic rhinitis were enrolled in the study. Patients had histories of ragweed hay fever within the previous 2 years, positive skin prick tests to ragweed extract (403 000 AU/mL) with wheal more than 8 mm, and elevated levels of ragweed-specific IgE antibody. Patients were not taking any form of glucocorticoid treatment within 1 month before the study. Nasal lavages were obtained from patients at the beginning of September when the ragweed hay fever season reaches a peak in Rochester, MN. Lavage was performed with the head tilted back by instilling 5 mL normal saline into one nostril, followed by bringing the head forward to collect the fluid in a 150-mL beaker. This procedure was performed twice in each nostril using a total of 20 mL normal saline, and the recovery was regularly more than 80%. The nasal lavage fluid was passed through a 42-µm nylon mesh filter (Nitex; Tetko, Briarcliff Manor, NY), and the filtrate was centrifuged at 420g for 12 minutes at 4°C. The supernatant fluids were recovered and stored at 4°C for analysis of NGF, NT-3, and EDN by using ELISA or RIA as described above. The Institutional Review Board at the Mayo Clinic reviewed and approved this study.Statistical analyses All results are presented as mean ± SEM from the number of experiments indicated. Statistical significance of the differences was assessed with Student paired or unpaired t test. Correlations were assessed by using the Pearson or Spearman correlation of the InStat statistics package (GraphPad Software, San Diego, CA).
Human eosinophils constitutively express NGF and NT-3 mRNA and synthesize these proteins We used RT-PCR to investigate whether eosinophils express mRNA for interstitial cell growth factors. Eosinophils from all 6 donors constitutively and reproducibly expressed mRNA for TGF- 1,10 TGF-2 (data not shown), NGF, and NT-3 (this
report). In Figure 1A, human mast cell
line, HMC-1 cells, expressed NT-3 and NGF mRNA, as shown
previously.27 Furthermore, consistent with previous findings,23 freshly isolated human eosinophils
constitutively expressed mRNA for NT-3 and NGF. The control, PCR of
eosinophil RNA preparations without RT reaction, showed no detectable
signals (data not shown). As shown in Figure 1B, these mRNAs were
expressed in other eosinophil donors.
Eosinophils characteristically store many eosinophil-derived cytokines and growth factors, providing a preformed pool of cytokines available for release (reviewed in Lacy and Moqbel38). Therefore, we examined eosinophil storage of NGF and NT-3 proteins and compared these stored amounts with those in neutrophils and PBMCs. Freshly isolated eosinophils contained 127.3 ± 40 pg/106 cells and 112 ± 21.6 pg/106 cells of NGF and NT-3, respectively, within or associated with the cells (mean ± SEM, n = 4). In contrast, both NGF and NT-3 were undetectable in neutrophils. PBMCs contained a small amount (5.8 ± 3.2 pg/106 cells) of NGF, but NT-3 was undetectable, suggesting that NGF and NT-3 are uniquely stored in eosinophils. Immunocytochemistry and confocal microscopy were used to localize
stored NGF in eosinophils. As shown in Figure
2, eosinophil sections stained with
anti-NGF antibody revealed a diffusely labeled cytoplasm, vesicular
staining within the cells, and intense staining on or beneath the cell
membrane. Sections stained with control immunoglobulin (goat IgG)
yielded no or barely visible staining (data not shown).
To examine whether stored NGF and NT-3 proteins have been phagocytized or newly synthesized by blood eosinophils, isolated eosinophils were cultured in vitro in the presence of the transcription inhibitor, actinomycin D,39 for up to 8 hours. A 4-hour treatment with actinomycin D reduced NGF and NT-3 by 86% and 80%, respectively, compared with cells without actinomycin D. Furthermore, an 8-hour treatment with actinomycin D almost depleted intracellular NGF and NT-3 (95% reduction of both NGF and NT-3). The estimated half-lives of NGF and NT-3 in the presence of actinomycin D were short, 1.5 hours and 1.8 hours, respectively. Thus, these experiments suggest that mature peripheral blood eosinophils continuously transcribe mRNA for and synthesize and store NGF and NT-3 proteins. Synthesis of NGF protein is enhanced by immunologic stimuli We further investigated whether this synthesis and storage of NGF and NT-3 proteins are regulated by immunologic stimuli. IL-5 has multiple effects on eosinophils, including maturation, survival, and activation, and it is considered an important cytokine in eosinophilic inflammation and allergic disorders (reviewed in Costa et al40 and Cuss41). Eosinophils express receptors for IgA and IgG, namely Fc R42 and
Fc RII43; the sIgA immune complex is a potent agonist for
eosinophil cytokine synthesis.8,9,33 As shown in Figure
3A, eosinophils incubated for 24 hours
with sIgA-anti-sIgA immune complexes, but without IL-5, showed a
3-fold increase in intracellular NGF. IgA-anti-IgA and IgG-anti-IgG
immune complexes also doubled intracellular NGF, suggesting that immune complexes stimulate production and accumulation of NGF. IL-5 by itself
slightly increased intracellular NGF. Interestingly, combinations of
IL-5 and immune complexes significantly reduced the intracellular NGF
compared with immune complexes alone (P < .05 in sIgA and IgG), suggesting that these combinations either induce extracellular secretion of NGF protein or inhibit the production of NGF. This question was addressed subsequently with a PC-12 cell bioassay and
protein ELISA (see below). In contrast to NGF, the amount of NT-3 was
neither increased nor decreased by immune complexes, IL-5, or their
combinations (Figure 3B). Furthermore, the intracellular content of
preformed granule protein, EDN, was not affected by immune complexes
(Figure 3C).
Eosinophils secrete biologically active NGF on immunologic stimuli To address whether NGF is secreted by activated eosinophils and whether NGF release by eosinophils is biologically relevant, we cultured PC-12 cells with supernatants of eosinophils stimulated with IL-5 and sIgA immune complexes. As shown in Figures 4A and 5, 22% of PC-12 cells cultured with medium alone expressed at least 2 neurites 50 µm. NGF, at 2 ng/mL, strikingly promoted neurite
outgrowth of PC-12 cells, resulting in 76% of cells expressing neurites (Figures 4B, 5). When supernatants of eosinophils incubated with IL-5 and sIgA immune complex for 24 hours were added to PC-12 cells instead of NGF, they significantly increased the number of
neurite-positive cells (49% ± 4%, n = 5, P < .05;
Figures 4D, 5). In contrast, supernatants from eosinophils incubated
with medium alone (Figures 4C, 5) or sIgA immune complex plus IL-5 without eosinophils (Figure 5) did not promote neurite outgrowth. Furthermore, the effects of supernatants from eosinophils incubated with IL-5 and sIgA were abolished by anti-NGF antibody but not by
control antibody (Figure 5). Thus, supernatants from eosinophils incubated with IL-5 plus sIgA immune complex likely contain
biologically active NGF that can stimulate neurite elongation of
PC-12 cells.
The NGF activity in eosinophil supernatants was semiquantitated by
using a standard curve made by serial dilutions of recombinant NGF
included in each experiment. As shown in Figure
6, there was no detectable NGF activity
in medium or medium containing IL-5 plus sIgA immune complex. When
eosinophils were incubated without stimuli, only 1 of 6 eosinophil
supernatants contained detectable NGF activity. In contrast, all 6 supernatants from eosinophils incubated with IL-5 plus sIgA immune
complex contained strong NGF activity, varying from 150 to 1400 pg/mL
(490 ± 200 pg/mL, mean ± SEM, n = 6). We were unable to do
similar experiments for NT-3 because cell lines responding to NT-3 were
not easily available.
To confirm the results of the bioassay, we used ELISA and quantitated
NGF proteins in eosinophil supernatants. As shown in Figure
7, approximately 70 pg/mL NGF was
detected in the supernatants of eosinophils cultured with medium alone,
suggesting that some NGF protein is spontaneously secreted without
apparent stimuli. Incubation of eosinophils with sIgA immune complex
significantly increased the amounts of NGF in the supernatants
(P < .05). In contrast, IgG or IgA immune complexes by
themselves did not show significant effects. IL-5 by itself did not
induce NGF secretion; however, it increased the secretion of NGF by
eosinophils stimulated with sIgA or IgA immune complexes. A combination
of sIgA immune complex and IL-5 produced 335 ± 42 pg/mL NGF
(mean ± SEM, n = 4), which is roughly comparable to the value
obtained from the PC-12 bioassay. Thus, eosinophils stimulated with
sIgA immune complex secrete substantial amounts of NGF protein and
bioactivity.
Eosinophils, NGF, and NT-3 in vivo The experiments described above show that eosinophils constitutively make and spontaneously secrete some NGF and that they produce and secrete increased amounts of NGF when exposed to immunologic stimuli in vitro. Can these in vitro phenomena be reproduced in vivo in disease? To address this question, we collected nasal lavage fluids from patients with allergic rhinitis during the peak of the ragweed hay fever season and measured the NGF concentrations by ELISA. To monitor the magnitude of eosinophil infiltration and activation, we also determined the EDN concentrations in the same lavage fluids.44 As shown in Figure 8, NGF was clearly detected in nasal secretions of patients with allergic rhinitis, and there was a significant correlation between EDN and NGF concentrations in these patients' lavage samples (P < .001). In contrast, there was no correlation between EDN and NT-3 concentrations. Thus, there is a strong association between eosinophilic inflammation and NGF levels, but not NT-3 levels, in the airways of patients with allergic disease.
One of the novel findings in this study is that eosinophils stimulated with immune complexes have an enhanced ability to produce and secrete NGF, suggesting that NGF can be expressed in an immunologically specific, FcR-dependent manner. Previous studies suggested that mast cells,45 T cells,28 and eosinophils23 could express NGF mRNA and protein. Although little is known about the regulatory mechanism of NGF secretion, one study suggests that perturbation of high-affinity IgE receptors on mast cells triggers NGF release.46 Our observations add to this knowledge and suggest that eosinophils interacting with sIgA and IL-5 secrete substantial and biologically significant amounts of NGF. Therefore, immunologic or inflammatory responses may affect the functions of the peripheral nervous system through increased production of NGF by immune cells. It is well known that neurogenic mediators, such as substance P and other neuropeptides, affect the functions of immune cells.47 Therefore, 2-way communication likely exists between the immune and the nervous systems. Furthermore, NGF may be a particularly important eosinophil-derived mediator at mucosal sites of chronic allergic inflammation where eosinophils may encounter large quantities of allergen-specific immunoglobulins and allergens.48 This concept was buttressed by the strong correlations between NGF levels and the magnitude of eosinophilic inflammation in the airways of patients with allergic rhinitis during hay fever season (Figure 8). Another original finding is the constitutive nature of NGF and NT-3 production and storage by human eosinophils. Human eosinophils, unlike lymphocytes, tend to store synthesized cytokines; so far, at least 9 eosinophil-derived cytokines are reported to be stored intracellularly as preformed mediators (reviewed in Lacy and Moqbel38). Immunochemical and subcellular fractionation studies suggest that these proteins are likely stored in crystalloid granules and small secretory vesicles.11,49 Our study shows that NGF and NT-3 may also be cytokines falling into this category. Although the exact locations of the NGF and NT-3 storage were not the major focus of this study, Figure 3 clearly indicates that the production and storage of NGF by eosinophils are regulated differently from those of the crystalloid granule proteins, such as EDN. Furthermore, synthesis and storage of NGF and NT-3 were constitutive and were quite sensitive to actinomycin D-induced depletion. In addition, a small but detectable amount of NGF was secreted into supernatants without exogenous stimuli. Previously, we showed that eosinophils constitutively produce and store IL-4 and IL-10 and that the process is inhibited by actinomycin D treatment.10 In contrast, in the same experiments, eosinophils stored minimal amounts of IL-8, but, once stimulated with immunoglobulins, they produced and secreted a large quantity of IL-8.10 Therefore, at least 2 classes of cytokines and growth factors are likely produced by eosinophils; one is constitutively produced and stored, and the other is produced mainly after cellular activation. In the future, it will be important to know which eosinophil mediators belong to which classes and to investigate the biologic relevance of these mediators in the context of the physiologic and pathologic functions of eosinophils. Perhaps some factors are associated with the homeostatic roles of eosinophils and some factors are associated with the proinflammatory roles of eosinophils. NGF increases nerve conductance and sensitivity in vitro.50 Increased expression of NGF in vivo in the airways of transgenic mice correlates positively with increased peribronchial nerve density.17 NGF transgenic mice show increased airway levels of tachykinins, such as substance P, heightened mucous production, and airway hyperreactivity to capsaicin17; these findings are distinctly reminiscent of findings in patients with asthma and allergic diseases.51 Furthermore, intravenous and intranasal administration of NGF induced airway hyperreactivity in guinea pigs and mice, respectively.52,53 Increased levels of NGF were observed in the airways of symptomatic patients with allergic rhinitis18 or patients with asthma 18 hours after segmental allergen challenge.54 Interestingly, histologic examination of airways from patients with asthma or from allergen-challenged guinea pigs revealed eosinophils physically associated with the nerve bundles.55,56 Therefore, given the NGF effects in vitro and in vivo, eosinophils, by production of NGF, could be involved in the increased sensitivity or amplitude or both of the sensory terminal neuron's responses to exogenous stimuli. In fact, we found significant correlations between the nasal levels of NGF and eosinophilic inflammation in patients with allergic rhinitis (P < .001; Figure 8). We recognize that this study does not exclude other potential sources of NGF, such as mast cells45,46 and T cells.28 However, our findings in vivo, as well as eosinophils' ability to produce NGF on immunologic stimuli, strongly suggest that eosinophils produce NGF in various human diseases. Thus, the degree of eosinophil activation may contribute to the neuronal hyperresponsiveness or pathologic pain states frequently accompanying chronic eosinophilic inflammation, such as asthma, allergic diseases, and several eosinophilic disorders (eg, eosinophilia myalgia syndrome).57 Our observations may also have important implications for the
physiologic roles of eosinophils in mucosal immunity and inflammation. Perhaps soluble immune complexes formed in vivo in mucosal tissues of
patients with allergic diseases48,58 induce cytokine and growth factor production in eosinophils, making eosinophils a "regulator" of inflammation and tissue homeostasis. This may be especially important because eosinophils can produce factors inhibiting inflammation and promoting wound healing, such as TGF-
We thank Ms Kay Bachman, Ms Judith Blomgren, Ms Sandra Dunnette, and Ms Kathleen Bartemes for their help in studies of patients with hay fever. We also thank Ms Cheryl Adolphson for editorial assistance, Ms Linda Arneson for secretarial assistance, Ms Gail Kephart for technical assistance, and Dr Douglas Plager for critical reading of the manuscript.
Submitted May 25, 2001; accepted November 6, 2001.
Supported by grants AI 34486 and AI 34577 from the National Institutes of Health and by the Mayo Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hirohito Kita, Department of Medicine, Guggenheim Bldg Rm 406, 200 First St SW, Mayo Clinic, Rochester, MN 55905; e-mail: kita.hirohito{at}mayo.edu.
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  M. Abram, M. Wegmann, V. Fokuhl, S. Sonar, E. O. Luger, S. Kerzel, A. Radbruch, H. Renz, and M. Zemlin Nerve Growth Factor and Neurotrophin-3 Mediate Survival of Pulmonary Plasma Cells during the Allergic Airway Inflammation J. Immunol., April 15, 2009; 182(8): 4705 - 4712. [Abstract] [Full Text] [PDF]
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D. R. Curran, R. K. Morgan, P. J. Kingham, N. Durcan, W. G. McLean, M. T. Walsh, and R. W. Costello Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L326 - L332. [Abstract] [Full Text] [PDF]
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P. J. Kingham, W. G. McLean, M.-T. Walsh, A. D. Fryer, G. J. Gleich, and R. W. Costello Effects of eosinophils on nerve cell morphology and development: the role of reactive oxygen species and p38 MAP kinase Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L915 - L924. [Abstract] [Full Text] [PDF]
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A. D. Klion, P. Noel, C. Akin, M. A. Law, D. G. Gilliland, J. Cools, D. D. Metcalfe, and T. B. Nutman Elevated serum tryptase levels identify a subset of patients with a myeloproliferative variant of idiopathic hypereosinophilic syndrome associated with tissue fibrosis, poor prognosis, and imatinib responsiveness Blood, June 15, 2003; 101(12): 4660 - 4666. [Abstract] [Full Text] [PDF]
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D. B. Jacoby Airway Neural Plasticity: The Nerves They Are A-Changin' Am. J. Respir. Cell Mol. Biol., February 1, 2003; 28(2): 138 - 141. [Full Text] [PDF]
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Abstract
Introduction
denotes
significant difference from the values cultured with the same stimuli
but without IL-5 (P < .05). ND, not determined.
