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Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Medical Branch, Galveston, TX 77555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two tyrosine kinases, Lyn and Janus kinase 2 (Jak2),4 are known to be physically associated with the common ß (ßc) receptor of IL-3/GM-CSF/IL-5 (14, 15). These kinases are phosphorylated on tyrosine residues after stimulation of eosinophils with IL-5 (14, 15, 16, 17). They subsequently transduce signals through the Ras-Raf-1-mitogen-activated protein kinase pathway and the Jak-STAT pathway. A few studies that have been performed with eosinophils show that Lyn, Syk, and Jak2 tyrosine kinases are important for the antiapoptotic activity of IL-5 or GM-CSF (17, 18, 19). Lyn and Jak2 are additionally important for eosinophil differentiation from stem cells (S.S. and R.A., unpublished observations); however, they are not critical for eosinophil degranulation or for the up-regulation of adhesion molecules (18).
Receptors are the primary target for pharmacologic intervention in
modern medicine, and receptor antagonists are the mainstay of therapy
for many diseases. Receptors are frequently coupled to multiple
signaling pathways. Receptor antagonists block all of these pathways
and, as a result, are prone to develop undesired side effects. To
circumvent this problem, we introduce a novel class of peptide
inhibitors that is designated to block only specific signaling pathways
of a receptor (Fig. 1
). To this goal, we
have developed a general strategy as follows: 1) identify a critical
signaling molecule that associates with the receptor, 2) map the
signaling molecule-binding site of the receptor, 3) design small
peptide(s) based upon the binding site and study their in vitro binding
activity, 4) N-acylate the peptide for cellular internalization and
examine the specificity of signaling inhibition and biologic effects in
vitro, and 5) study the biologic effects of the peptides in
vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A TF-1 cell line was purchased from the American Type Culture Collection (Manassas, VA). RPMI medium 1640, IMDM, and antibiotic-antimycotic were obtained from Life Technologies (Grand Island, NY), and FCS was supplied by Atlanta Biologicals (Norcross, GA). Percoll and an RIA kit for eosinophil cationic protein (ECP) were purchased from Pharmacia (Piscataway, NJ). Histopaque-1077, Con A, platelet-activating factor (PAF), propidium iodide (PI), OVA, rabbit polyclonal anti-human IgM and IgE Abs, and peroxidase-conjugated anti-mouse IgG Ab were obtained from Sigma (St. Louis, MO). The mAb against antiphosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-Lyn and anti-Jak2 Abs, mouse anti-IL-3/IL-5/GM-CSFRß mAb, HRP-conjugated goat anti-rabbit IgG Ab, and protein A/G Plus agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CD16 immunomagnetic beads were supplied by Miltenyi Biotec (Bergisch Gladbach, Germany). M-450 Pan-B and Pan-T Dynabeads were obtained from Dynal (Great Neck, NY). Human IL-5, human GM-CSF, and murine IL-3 were purchased from PeproTech (Rocky Hill, NJ). Murine IL-5 was obtained from R&D Systems (Minneapolis, MN). An enhanced chemiluminescence (ECL) detection system, Hybond ECL nitrocellulose membrane, and methyl [3H]thymidine were obtained from Amersham (Arlington Heights, IL).
Peptides and antisense (AS) oligodeoxynucleotides (ODNs)
The synthesis of peptides and their biotinylation or stearation
were performed by Quality Controlled Biochemicals (Hopkinton, MA). A
peptide (ßc 450–465: YGYRLRRKWEEKIPNP-NH2) was
synthesized based on the Lyn-binding sequence and modified by
biotinylation or N-stearation (20). As a control
N-stearated peptide, we obtained a peptide corresponding to amino acids
316–335 of IL-5
receptor (CREAGLWSEWSQPIYVGFSR-NH2).
All peptides were purified to >95% by HPLC. The purity of the
peptides and their modification were judged by mass spectrometry. A
15-mer Lyn AS ODN was synthesized by Operon Technologies (Alameda, CA)
based on previously published sequence information (17, 18). The sequence used is as follows: Lyn AS ODN
(CATATTTCCCGCTCG).
Cell culture
TF-1 cells were maintained in RPMI 1640 with 10% FCS and 1 ng/ml human GM-CSF. RPMI 1640 was supplemented with antibiotic-antimycotic (100 U/ml penicillin G, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B).
Eosinophil purification
Peripheral blood was obtained from subjects with mild to moderate eosinophilia (6–12%). Eosinophils were isolated by sedimentation with 6% hydroxyethyl starch followed by centrifugation on a 1.088 Percoll density gradient according to the method of Hansel et al. (23). The cells were further purified by negative selection using anti-CD16 immunomagnetic beads and the magnetic cell separation system (Miltenyi Biotec). Eosinophils (>99% purity) were then suspended in RPMI 1640 in tubes coated with 3% human serum albumin.
B cell and T cell purification
The purification of B cells and T cells was done as described previously with slight modifications (24). Briefly, peripheral blood was obtained from normal subjects. PBMCs were isolated using Histopaque-1077 according to the manufacturer’s instructions. After the depletion of monocytes adhering to plastic petri dishes, the cells were further purified by negative selection using M-450 Pan-B Dynabeads (anti-CD19) for T cells and M-450 Pan-T Dynabeads (anti-CD2) for B cells. The purified T cells and B cells were then suspended in RPMI 1640. The purity of the B cells and T cells was >95%.
Preparation of cytosolic cell extracts and immunoprecipitation
Purified eosinophils (1–2 x 106 cells) were incubated with the N-stearated peptide for 2 h at 37°C followed by stimulation with 10 ng/ml of human IL-5 for 1–3 min. The reaction was terminated by the addition of 5 volumes of ice-cold 1.2x lysis buffer (60 mM Tris-HCl (pH 7.4), 180 mM NaCl, 1.2 mM Na3VO4, 1.2 mM NaF, 1.2 mM EDTA, 1.2 mM EGTA, 1.2 mM PMSF, 1.2% Nonidet P-40, 0.3% sodium deoxycholate, and 1.2 µg/ml of aprotinin, leupeptin, and pepstatin). After 20 min on ice, detergent insoluble materials were removed by centrifugation at 4°C at 12,000 x g. The protein concentration was determined using the bicinchoninic acid assay (Pierce, Rockford, IL).
For immunoprecipitation, the cell lysates were precleared by incubation with 20 µl of the protein A/G Plus agarose for 30 min. After removal of the beads, the lysates were incubated with the appropriate Ab (1–2 µg for each sample) for 1 h followed by incubation with 20 µl of protein A/G Plus agarose for 2 h at 4°C. The beads were washed three times with the cold 1x lysis buffer. Whole cell lysates or immunoprecipitates were boiled in 2-fold concentrated Laemmli reducing buffer for 2 min.
Gel electrophoresis and Western blotting
SDS-polyacrylamide gels were prepared according to the Laemmli protocol and used for Western blotting. The concentration of polyacrylamide was 8%. Gels were blotted onto Hybond membranes for Western blotting using the ECL system. Blots were incubated in a blocking buffer containing 10% BSA in TBST buffer (20 mM Tris-base, 137 mM NaCl (pH 7.6), and 0.05% Tween 20) for 1 h followed by incubation in the primary Ab (0.1 µg/ml) for 1–2 h. After washing three times in TBST buffer, blots were incubated for 30 min with an HRP-conjugated secondary Ab (0.05 µg/ml) directed against the primary Ab. The blots were developed with the ECL substrate according to the manufacturer’s instructions. In some experiments, blots were reprobed with another Ab after stripping in a buffer of 62.5 mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS at 50°C for 30 min.
Murine bone marrow (BM) cell culture
In vitro liquid culture was performed as described elsewhere with modifications (25). OVA-sensitized BALB/c mice were sacrificed, and femurs were removed. The BM cavity was flushed with saline to obtain cells. The BM cells (5 x 105 cells/ml) were suspended in IMDM supplemented with antibiotic-antimycotic. These cells were incubated with the N-stearated peptide for 2 h at 37°C followed by further culture in the presence of 1 ng/ml of murine IL-3 and 6 ng/ml of murine IL-5 plus 10% FCS for the indicated days. After harvesting, the total cell count was obtained; the remaining cells were used for cytospin preparations. These preparations were stained with Wright’s stain to count the number of eosinophils.
Eosinophil survival assay and measurement of ECP
Purified eosinophils (5 x 105 cells/ml) were suspended in RPMI 1640 with 5% FCS and treated with the stearated peptides at incremental concentrations for 2 h at 37°C. After the incubation, the cells were cultured with 1 ng/ml of human IL-5 for 3 days. The viability of the cultured eosinophils was assessed by counting PI-stained dead cells. For ECP release experiments, the stearated peptide-treated eosinophils were primed with GM-CSF (1 ng/ml) for 2 h and subsequently stimulated with PAF (5 x 10-8 M) for 20 min. The supernatants were separated by centrifugation, and the concentration of ECP was measured by RIA.
Allergen inhalation challenge and bronchoalveolar lavage (BAL)
Allergen sensitization of mice was done as described previously
with slight modifications (26). Briefly, mice were
sensitized by an i.p. injection of 75 µl (1 mg/ml) of chicken OVA
(grade V) with 25 µl of Imject Alum (Pierce). After 1 wk, the mice
received a second injection. After an additional 1 wk, mice were
exposed to OVA (10 mg/ml) via inhalation for 1 h daily for 5 days.
At 2 wk after the last allergen inhalation, the mice were pretreated
intrabronchially with one of the peptides (2 µmol, 
0.5 mg) or
saline and subsequently challenged with OVA inhalation 1 h later.
BAL was performed after 24 h. After the total cell count of the
BAL fluid had been obtained, the remaining cells were used for cytospin
preparations. These preparations were stained with Wright’s stain to
count the number of eosinophils.
Histamine release assay
Histamine release from basophils was studied as described previously (24). Briefly, peripheral blood leukocytes were separated from venous blood by sedimentation with 6% hydroxyethyl starch and suspended in HACM buffer (HEPES-buffered saline (pH 7.4), 0.03% human serum albumin, 2 mM CaCl2, and 1 mM MgCl2). The cells were incubated with the N-stearated peptide for 1 h at 37°C and subsequently stimulated with a 1/1000 dilution of a rabbit polyclonal anti-IgE Ab. Supernatants were collected, and the histamine content was measured with an automated fluorometric analyzer.
[3H]Thymidine incorporation
Purified T cells and B cells (5 x 106 cells/ml) were suspended in RPMI 1640 with 5% FCS and cultured with the N-stearated peptide for 2 h at 37°C followed by stimulation of T cells with 5 µg/ml of Con A and B cells with 10 µg/ml of immobilized anti-IgM Ab. The cells were cultured for 96 h, with the addition of 5 µCi/ml of [3H]thymidine (5 Ci/mmol) during the last 6 h. The cells were harvested, and the incorporation of [3H]thymidine was counted using a scintillation counter (Packard, Downers Grove, IL).
Statistical analysis
Results are expressed as mean ± SD. Data were analyzed for statistical significance using ANOVA, Student’s paired t test, and the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-5R has two subunits: the ligand-specific subunit and
the ßc subunit common to IL-3, IL-5, and GM-CSF (27).
The ßc subunit physically associates with Lyn and Jak2 kinases
(14, 15). We have previously mapped the Lyn-binding site
of the ßc. Three overlapping peptides from this region (ßc
450–465, ßc 457–465, and ßc 462–481) bound Lyn kinase in vitro
(20). To enable cellular internalization, the Lyn-binding
peptides were modified by N-stearation. We performed many experiments
with all three lipopeptides. The lipopeptides produced nearly identical
results. For simplicity, we will present the data obtained with one of
the peptides, ßc 450–465. To determine whether the N-stearated
peptide binds Lyn in situ and competitively blocks its association with
ßc, we incubated eosinophils with the N-stearated ßc 450–465
peptide or control peptides for 2 h. The control peptides were
biotinylated ßc 450–465 peptide and N-stearated IL-5 316–335
peptide. The cell lysates were immunoprecipitated with an anti-ßc
Ab followed by electrophoresis and Western blotting with an
anti-Lyn or anti-Jak2 Ab. The N-stearated ßc 450–465
peptide, but not the control peptides, abrogated the coprecipitation of
Lyn with ßc (Fig. 2A). We
also performed anti-Jak2 Western blotting using the same ßc
immunoprecipitates. In contrast to the anti-Lyn blot, the
coprecipitation of Jak2 with ßc was not inhibited by the peptide
(Fig. 2B). We have observed a similar effect of the
lipopeptide when the lysates were immunoprecipitated with anti-Lyn
or anti-Jak2 Ab and immunoblotted with anti-ßc Ab
(20). Our results indicate a specific binding of the
lipopeptide to Lyn kinase in situ.
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To study the effect of the lipopeptide on cytokine signaling, we
examined the IL-5-induced tyrosine phosphorylation of cellular proteins
in eosinophils. The cells were incubated in the presence or absence of
the N-stearated peptide for 2 h and subsequently stimulated with
or without IL-5 for 3 min. A stearated peptide derived from IL-5R
receptor was used as a control. After lysing the cells, the lysates
were subjected to electrophoresis and Western blotting with an
antiphosphotyrosine Ab. The stearated ßc 450–465 peptide, but not
the stearated IL-5R 316–335 peptide, inhibited the IL-5-induced
tyrosine phosphorylation of a number of cellular proteins in a
dose-dependent manner in eosinophils (Fig. 3).
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316–335
peptide (Fig. 4A). We also
examined the effect of the lipopeptide on Jak2 activation. The
IL-5-stimulated tyrosine phosphorylation of Jak2 kinase was not
affected by pretreatment with the stearated peptide in eosinophils
(Fig. 4B).
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The results of our Lyn-binding lipopeptide suggest that Lyn is not
necessary for Jak2 activation. To further confirm our data, we
performed similar experiments with Lyn AS ODNs using the
GM-CSF-dependent cell line TF-1. As shown in Fig. 5, pretreating TF-1 cells with Lyn AS
ODNs inhibited the GM-CSF-induced activation of Lyn but not Jak2. Taken
together, these results indicate that Lyn is not essential for Jak2
activation in IL-5/GM-CSF signaling. It is possible that other
src family kinases are involved in this process.
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The differentiation of eosinophils from progenitor cells is
dependent upon IL-5 (2). Previous studies have shown that
a combination of IL-3 and IL-5 stimulates eosinopoiesis in vitro
(25). IL-3 is required as a pluripotent stem cell growth
factor. To investigate the role of Lyn kinase in eosinophil
differentiation, we studied the effect of the N-stearated peptide on
murine BM cells. The cells (5 x 105
cells/ml) were cultured with IL-3 and IL-5 for 1 wk. The total number
of cells without peptides, with stearated ßc 450–465 peptide, with
biotinylated ßc 450–465 peptide, and with stearated IL-5R
316–335 peptide were 39 ± 1, 31 ± 6, 39 ± 6, and
38 ± 4 x 104 cells after 1 wk,
respectively (n = 3). The eosinophil number was
significantly reduced in the presence of the N-stearated 450–465
peptide but not in the presence of the control peptides (Fig. 6A). Next, we studied the
kinetics of eosinophil differentiation. We obtained 5 ± 1x
104 cells of mature eosinophils and 22 ±
5 x 104 cells of eosinophilic myelocytes
before culture (n = 3). As shown in Fig. 6B,
IL-5 induced eosinophilic differentiation from stem cells within 3
days. The inhibitory effect of the stearated ßc 450–465 peptide
could be observed after 3 days and lasted 
10 days.
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We and others have shown that Lyn is important for eosinophil
growth and survival (17, 18). We reasoned that the
N-stearated Lyn-binding peptide would have inhibitory effects on
eosinophil survival. As shown in Fig. 7,
most of the eosinophils underwent apoptosis after 3 days without IL-5
(18% viable cells). In contrast, eosinophil viability was prolonged to
82 ± 3% after stimulation with IL-5 (n = 3).
When eosinophils were incubated with the N-stearated ßc 450–465
peptide, the IL-5-induced prolongation of eosinophil survival was
reduced in a dose-dependent manner (Fig. 7). The control biotinylated
peptide or the stearated IL-5R peptide did not affect IL-5-induced
eosinophil survival. When eosinophils were incubated at a 10-fold
higher concentration of IL-5 (10 ng/ml), the majority of the inhibitory
effect of the peptide could be overcome (data not shown). Thus, the
data suggest that the effect of the stearated peptides is not
toxic.
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We also studied the effect of the N-stearated peptide on eosinophil degranulation. Eosinophils secrete a number of granular proteins during degranulation. One of the important granular proteins is ECP. Both IL-5 and GM-CSF prime eosinophils for degranulation at low concentrations. However, GM-CSF has been shown to be a bit stronger than IL-5 in this regard. In our experiments, we primed eosinophils with a low concentration of GM-CSF (1 ng/ml) and subsequently stimulated with PAF for ECP release. ECP release without and with GM-CSF priming was 243 ± 42 and 549 ± 79 ng/106 cells, respectively (n = 4). As predicted from the function of Lyn, ECP release from eosinophils was not affected by the N-stearated Lyn-binding peptide (data not shown).
Effect of the N-stearated Lyn-binding peptide on airway eosinophilic inflammation
IL-5 plays a critical role in allergen-induced airway eosinophilic
inflammation. In the next step, we examined the effect of the peptide
on airway eosinophilic inflammation in a murine model of asthma.
OVA-sensitized mice were pretreated intrabronchially with one of the
peptides (2 µmol) or saline and subsequently challenged with OVA
inhalation 1 h later. After an additional 24 h, BAL was
performed and the number of eosinophils was counted. In the mice with
saline pretreatment, 70% of the cells in the BAL fluid were
eosinophils (the absolute value was 4.3 ± 0.4 x
105 eosinophils/BAL, n = 6).
Administration of the stearated ßc 450–465 peptide, but not of the
biotinylated ßc peptide or the stearated IL-5R peptide,
significantly inhibited the eosinophil influx in the BAL fluid (Fig. 8). We did not see any significant
difference in the number of lymphocytes and macrophages following the
stearated peptide treatment as compared with control peptides or
saline.
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Lyn kinase is associated with Fc
RIß and activated after a
ligand binds to the receptor (28, 29). For this reason, we
examined the effect of the Lyn-binding peptide on basophil histamine
release. The anti-IgE-induced histamine release from leukocytes was
21 ± 7% (n = 3) of the total histamine content.
The stearated peptide at a concentration of 
20 µM did not affect
histamine release from basophils (data not shown).
Lyn kinase has been shown previously to be involved in B cell activation (30). For this reason, we studied the effect of the N-stearated Lyn-binding peptide on anti-IgM-stimulated B cell proliferation. The thymidine uptake of unstimulated and anti-IgM-stimulated B cells was 3806 ± 1534 cpm and 5992 ± 1485 cpm, respectively (n = 7). The N-stearated peptide showed a tendency for increased B cell proliferation, but the result was not statistically significant (p > 0.05). The thymidine uptake of B cells was 9096 ± 4216 cpm at a 10 µM concentration of the peptide. The peptide induced an enhanced proliferation of B cells in four donors but had no effects on the other three donors. Therefore, the effect seems to be variable and donor-dependent.
T cells do not express Lyn kinase but do express a large number of other tyrosine kinases (e.g., Lck, Fyn, and ZAP-70). To determine the effect of the peptide on other tyrosine kinases in situ, we investigated its activity on Con A-stimulated T cell proliferation. The thymidine uptake of unstimulated and Con A-stimulated T cells was 18,934 ± 9,983 cpm and 227,191 ± 33,192 cpm, respectively (n = 3). The peptide had no effect on Con A-stimulated T cell proliferation. The thymidine uptake by T cells was 220,774 ± 11,819 cpm in the presence of a 10 µM concentration of the stearated peptide. The results suggest that the peptide does not interfere with other signaling molecules.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based upon the Lyn-binding sequence, we designed a novel inhibitor that blocks eosinophil differentiation and survival by interfering with IL-5-stimulated Lyn kinase signaling. The inhibitor also blocked eosinophil infiltration of the airways in a mouse model of asthma. To our knowledge, this is the first report of inhibition of eosinophil growth and differentiation by a peptide inhibitor. The peptide was N-stearated to enable cellular internalization. N-acylation of small peptides has recently been shown to cause their internalization through the lipid membranes as demonstrated by spin label electron spin resonance and 2H nuclear magnetic resonance (21). The N-myristoylation of a protein kinase C substrate analogue causes its internalization and specific inhibition of the kinase (22). Although we do not provide direct evidence of cellular internalization of our peptide, our experimental results clearly indicate that the peptide inhibits the binding of Lyn to the ßc. The binding of Jak2 to the ßc is not affected by the peptide. This type of specific inhibition is possible only if the peptide is internalized. If the peptide is to act extracellularly through the IL-5R, one would expect the inhibition of Jak2 as well as Lyn kinases. The coprecipitation of Lyn with ßc receptor is inhibited by the stearated peptide, suggesting that the inhibitor blocks the binding of Lyn to the IL-5R ßc subunit in the cytosol. Furthermore, the activation of Lyn, but not Jak2, was inhibited by the Lyn-binding peptide. In support of these findings, the results obtained with the Lyn-binding peptide are very similar to those observed with Lyn AS ODNs, which did not block Jak2 activation. The results imply that Jak2 is not a downstream molecule of Lyn in IL-5 signaling.
The selective action of our peptide can be explained by the molecular
interaction of Lyn and Jak2 with the ßc receptor. Both these tyrosine
kinases are physically associated with the membrane-proximal region of
the ßc. However, our Lyn-binding peptide did not bind to Jak2 both in
vitro and in situ (20). Thus, the difference between Lyn-
and Jak2-binding sites on the ßc accounts for the specificity of our
peptide. Another explanation for the specificity comes from the
domain-specific interaction of Lyn kinase with receptors. Lyn has an
N-terminal unique domain, followed by an Src homology 3 (SH3) domain,
SH2 domain, and the tyrosine kinase domain. The N-terminal 10-aa
residues of Lyn are required for binding to the Ig -chain Ag
receptor homology 1 motif of the B cell Ag receptor (31).
A direct interaction of Lyn with the FcRIß receptor through its
unique domain has also been detected using the two-hybrid system
(32). Using truncated Lyn GST fusion proteins, we have
found that the unique domain is responsible for the binding of Lyn to
the ßc (20). On the basis of these results, we predict
that our lipopeptide competes only with the receptors that bind Lyn via
the unique domain but not via the SH2 or SH3 domains.
Lyn kinase has been shown to be associated with the Ig receptor
(30, 31), CD14 (33), CD19 (34),
and CD22 (35) in B cells and FcRIß in mast cells
(28, 29). Lyn regulates B cell function both positively
(36, 37) and negatively (38, 39), whereas it
has an amplifying effect on mast cell degranulation (40).
Our Lyn-binding peptide had variable effects on B cell proliferation
and no effect on basophil histamine release. This negative result can
be explained as follows: Although Lyn binds to all ßc, Ig and
FcRIß via the unique domain, the binding sites on the receptors
have no sequence homology. Thus, it is likely that various regions
within the unique domain bind to different receptors. In this scenario,
the Lyn-binding peptide from the ßc receptor will not block the
binding site for other receptors.
IL-5 stimulates the growth and terminal differentiation of eosinophils from committed stem cells (2). In the present study, we found that eosinophil differentiation from stem cells was blocked by the stearated Lyn-binding peptide. These data are consistent with the results obtained with Lyn AS ODNs (S.S. and R.A., unpublished observations). We cultured murine BM cells with murine IL-3 and IL-5. In contrast to humans, the mouse has two homologous ß subunits, ßc and ßIL-3 (27). Because of the presence of ßIL-3, our peptide could block only IL-5 signaling while keeping IL-3 signaling intact. One would expect that the peptide may also block IL-3 signaling, which is essential for overall hemopoiesis in the human system, because the human has only one ß subunit, ßc. Interestingly, however, the ßc/ßIL-3 double-knockout mouse showed normal hemopoietic parameters except for a reduced number of peripheral eosinophils and impaired eosinophil function (41). This definitive study suggests that the ßc receptor is critical only for eosinopoiesis but not for other myeloid lineages. Perhaps other growth factors (e.g., G-CSF and M-CSF) compensate for the function of ßc/ßIL-3 receptors.
IL-5 is a regulatory factor not only for eosinophilopoiesis but also for the prevention of eosinophil apoptosis (3). Our results using the Lyn-binding peptide suggest that Lyn is important for eosinophil survival. We further studied the effect of Lyn signaling inhibitor on ECP release and found that Lyn is not essential for eosinophil degranulation. These results are supported by the data obtained with Lyn AS ODNs (17, 18). It has also been shown that Jak2 is critical for survival but not for degranulation.
The stearated ßc peptide blocked not only in vitro eosinophil function but also eosinophil infiltration in vivo. The influx of eosinophils into the airways is a complex process and is likely to involve not only IL-5 but also CC chemokines and other factors. The efficacy of the stearated peptide in blocking airway eosinophilic influx is thus, remarkable. IL-5 has modest direct chemotactic effects on eosinophils, but, more importantly, it primes eosinophils for chemotaxis in response to other chemoattractants. i.v. IL-5 dramatically enhances the local accumulation of eosinophils induced by intradermal eotaxin or leukotriene B4 in guinea pigs (42). In support of the above observations, an s.c. injection of eotaxin is unable to develop tissue eosinophilia in IL-5-deficient mice (43). These results suggest a critical role for IL-5 in eosinophil locomotion in vivo, and our peptide may target this function.
In summary, we have developed a novel peptide inhibitor that blocks eosinophil differentiation, survival, and airway eosinophil influx. Excessive production of eosinophils and their subsequent invasion of the airways and other target organs are characteristic features of asthma and allergic diseases. In addition, there is evidence that eosinophil survival in these diseases is prolonged due to the action of IL-5. Therefore, the Lyn-binding peptide may have therapeutic applications in asthma, allergic diseases, and other eosinophilic disorders. Furthermore, the strategy to develop novel peptide inhibitors can be applied to other receptors, broadening its implication in medicine.
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Footnotes |
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2 U.S. patent pending (serial no. 60/059, 630).
3 Address correspondence and reprint requests to Dr. Rafeul Alam, University of Texas Medical Branch, Department of Internal Medicine, Division of Allergy and Immunology, Clinical Sciences Building 409, Galveston, TX 77555-0762. E-mail address:
4 Abbreviations used in this paper: Jak, Janus kinase; AS, antisense; BAL, bronchoalveolar lavage; ßc, common ß; ECP, eosinophil cationic protein; ODN, oligodeoxynucleotide; SH, Src homology; PAF, platelet-activating factor; ECL, enhanced chemiluminescence; PI, propidium iodide; BM, bone marrow.
Received for publication February 25, 1999. Accepted for publication April 30, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Proc. Natl. Acad. Sci. USA 91:4268.This article has been cited by other articles:
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E. Lach-Trifilieff, K. Menear, E. Schweighoffer, V. L. J. Tybulewicz, and C. Walker Syk-deficient eosinophils show normal interleukin-5-mediated differentiation, maturation, and survival but no longer respond to Fcgamma R activation Blood, October 1, 2000; 96(7): 2506 - 2510. [Abstract] [Full Text] [PDF]
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T. Adachi, B. K. Choudhury, S. Stafford, S. Sur, and R. Alam The Differential Role of Extracellular Signal-Regulated Kinases and p38 Mitogen-Activated Protein Kinase in Eosinophil Functions J. Immunol., August 15, 2000; 165(4): 2198 - 2204. [Abstract] [Full Text] [PDF]
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J. Du, Y. M. Alsayed, F. Xin, S. J. Ackerman, and L. C. Platanias Engagement of the CrkL Adapter in Interleukin-5 Signaling in Eosinophils J. Biol. Chem., October 13, 2000; 275(42): 33167 - 33175. [Abstract] [Full Text] [PDF]
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) of
the stearated ßc 450–465 peptide. The peptide significantly blocked
eosinophil differentiation. *, p < 0.05 vs
medium alone (Student’s paired t test).
