The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stafford, S.
Right arrow Articles by Alam, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stafford, S.
Right arrow Articles by Alam, R.
The Journal of Immunology, 2002, 168: 1978-1983.
Copyright © 2002 by The American Association of Immunologists

Lyn Tyrosine Kinase Is Important for IL-5-Stimulated Eosinophil Differentiation1

Susan Stafford*, Clifford Lowell{dagger}, Sanjiv Sur* and Rafeul Alam2,*

* Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Medical Branch, Galveston, TX 77555; and {dagger} Department of Laboratory Medicine, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5R{alpha} is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5R{alpha} in eosinophils. To identify IL-5R{alpha}-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5R{alpha} under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5R{alpha}. Lyn kinase phosphorylates both IL-5R{alpha} and {beta}cR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn-/- mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5R{alpha} signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The pathologic hallmark of active asthma is eosinophilic inflammation of the airway (1). The increased number of eosinophils in the airways is most likely the result of increased differentiation (2) and prolonged survival of eosinophils (3). Indeed, there is evidence that differentiation of eosinophils is accelerated in subjects following allergic sensitization (2). The differentiation of eosinophils is primarily regulated by IL-5, although other cytokines also contribute to this process (1, 4). In addition, IL-5 causes prolongation of eosinophil survival and primes them for activation (5). Because of these activities, IL-5 is considered to play an important role in the pathogenesis of eosinophilic inflammation in various diseases, including asthma. Indeed, there are reports of increased production of IL-5 in airways from asthmatic patients (6, 7). Experiments with animal models pointed to a pivotal role for IL-5 in eosinophilic inflammation and airway hyperreactivity (8). However, a recent human study with an anti-IL-5 Ab failed to demonstrate clinical efficacy in asthma, although the treatment reduced the eosinophil count in the airway secretion (9). The results, albeit preliminary, suggest that multiple redundant mechanisms are operative in the pathogenesis of asthma. Nonetheless, the role of IL-5 in eosinophilic inflammation remains undisputed.

The IL-5R has two subunits, the ligand-specific {alpha} subunit and the {beta}c subunit, which is common to receptors for IL-3 and GM-CSF (10). The signal transduction mechanism of the {beta}cR has previously been studied. {beta}c is associated with Lyn (11, 12), Fes (13), Janus kinase (Jak)31 (14), and Jak2 (15, 16, 17) tyrosine kinases. Following receptor oligomerization, the tyrosine kinases are rapidly activated, which leads to receptor phosphorylation and recruitment of cytosolic signaling, including Syk (18), the adapter protein Shc (19), phosphatidylinositol-3 kinase (20), and STAT transcription factors (15, 16, 21, 22). The cytosolic signal is transduced via the Jak-STAT (15, 16) and Ras-mitogen-activated protein (MAP) kinase (12) pathway. Both pathways have been shown to play important roles in IL-5 signaling. The signal transduction mechanism of IL-5R{alpha} subunit is largely unknown. Since IL-5 plays an essential and nonredundant role in eosinophil differentiation, the foregoing function must be attributable to the signaling via the IL-5R{alpha}. Indeed, mice with null mutation for IL-5R{alpha} are unable to increase eosinophil differentiation in response to IL-5 (23, 24). Tyrosine kinases of the Src family are frequently associated with cytokine receptors and play an important role in generating cytosolic signals (25). The association of Src-type kinases with IL-5R{alpha} has not been previously reported. We investigated the physical association of Src-type kinases with IL-5R{alpha} and examined their biological relevance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Percoll was purchased from Pharmacia (Piscataway, NJ). The mAb against anti-phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-IL-5R {alpha} and {beta}, anti-Jak2, and Abs against the Src family of tyrosine kinases, Lyn, Hck, Fyn, Fgr, Blk, and Lck, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Jak2 inhibitor tyrphostin AG490 was purchased from Calbiochem (Carlsbad, CA) and resuspended in DMSO. ECL detection system was purchased from Amersham (Arlington Heights, IL).

Eosinophil purification

Peripheral blood for eosinophil purification was obtained from subjects with mild to moderate eosinophilia (6–12%). Eosinophils were isolated by sedimentation with 3% hydroxyethyl starch, followed by centrifugation on discontinuous Percoll gradients and by negative selection using anti-CD16 immunomagnetic beads (Miltenyi Biotec, Sunnyvale, CA), according to the method described previously (12). Eosinophils (>99% purity) were then suspended in RPMI 1640 in tubes coated with 3% human serum albumin.

Preparation of cytosolic cell extracts and immunoprecipitation

Eosinophils (1–4 x 106/ml) were incubated with IL-5 (10-10 M) or medium at 37°C for the indicated period of time. The stimulation was terminated by addition of 1 vol of ice-cold PBS containing 1 mM Na3VO4. The cells were pelleted by centrifugation, washed rapidly with PBS, and lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 0.25% sodium deoxycholate, 1 µM PMSF, 1 µM Na3VO4, 1 mM NaF, 0.7% Triton X-100, and 1 µg/ml aprotinin, leupeptin, and pepstatin. After incubation on ice for 10 min, the lysates were passaged several times through a 26-gauge needle and detergent-insoluble materials were removed by centrifugation at 4°C at 12,000 x g. The protein concentration was determined using bicinchoninic acid assay (Pierce, Rockford, IL). Cell lysates were then resolved on SDS-PAGE and subjected to Western blotting using appropriate Abs according to the method described previously (12). In other experiments, cell lysates were immunoprecipitated with Abs against anti-IL-5R{alpha} and {beta}c and Lyn to study phosphorylation. For this purpose, the cell lysates were precleared by incubation with 20 µl Protein A/G Agarose Plus (Santa Cruz Biotechnology) for 2 h. After removal of the beads, the lysates were incubated with an appropriate Ab and Protein A/G Agarose Plus for 4 h at 4°C. The immunoprecipitates were washed three times with the cold lysis buffer and boiled in the Laemmli sample buffer.

Gel electrophoresis and immunoblotting

SDS-polyacrylamide gels were prepared according to the Laemmli protocol and used for immunoblotting. The concentration of polyacrylamide was 7 or 12% depending on the m.w. range of the proteins studied. Gels were blotted onto Hybond membranes (Amersham) for Western blotting using the ECL system. Blots were incubated in a blocking buffer containing 5% BSA in TBST buffer (20 mM Tris-base, 137 mM NaCl, made to pH 7.6, and 0.05% Tween 20) for 1 h, followed by incubation in the primary Ab (0.1 µg/ml) for 1 h. After washing five times in TBST buffer, blots were incubated for 30 min with a HRP-conjugated secondary Ab (0.1 µg/ml) directed against primary Ab. The blots were developed with the ECL substrate according to manufacturer’s protocol. In some experiments, blots were reprobed with another Ab after stripping in a buffer of 62.5 mM Tris-HCl (pH 6.7), 100 µM 2-ME, and 2% SDS at 50°C for 30 min.

In vitro kinase assay

Lyn, IL-5R{alpha}, and {beta}c were immunoprecipitated from eosinophils with respective Abs (Santa Cruz Biotechnology). The kinase assay was performed in a buffer containing 20 mM Tris (pH 7.4) 2 mM MgCl2, 0.5 µM cold ATP, and 2 µCi [{gamma}-32P]ATP for 20 min. The assay was stopped by addition of 6x Laemmli’s buffer. The reactions then were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and autoradiographed.

Murine bone marrow cell culture

In vitro liquid culture was performed as described elsewhere (26). OVA-sensitized BALB/c mice were sacrificed, and the femurs were removed. The bone marrow cavity was flushed with saline to obtain cells. The bone marrow cells (5 x 105 cells/ml) were suspended in IMDM. These cells were incubated with and without the inhibitors for 30 min at 37°C, followed by further culture in the presence of 1 ng/ml murine IL-3 and 6 ng/ml murine IL-5 plus 10% FCS for 1 wk. After harvesting, the total cell count was obtained and the remaining cells were used for cytospin preparations. These preparations were stained with Wright-Giemsa stain for counting the number of eosinophils.

Antisense ODNs

Two 15-mer Lyn sense and antisense oligodeoxynucleotides (ODN) were synthesized by Operon Technologies (Alameda, CA) based on previously published sequence information (18). These ODN do not match any other cDNA in the GenBank Database by basic local alignment search tool analyses. They have been shown to decrease the expression of Lyn in human eosinophils by one group (18), and this observation was confirmed in our laboratory (27). Sequences used were as follows: antisense Lyn (CATATTTCCCGCTCG) and sense Lyn (CGAGCGGGAAATATG). The ODNs were phosphorothioate modified and resuspended in sterile H2O at 100 µM concentration.

Statistical analysis

Results were expressed as mean ± SD. Data were analyzed for statistical significance using ANOVA and Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tyrosine phosphorylation of IL-5R{alpha}

The signal transduction mechanism of IL-5R{alpha} is largely unknown. IL-5R{alpha} has a short cytoplasmic tail, which consists of ~55 amino acid residues. The cytoplasmic tail has one tyrosine residue. We investigated whether IL-5R{alpha} underwent tyrosine phosphorylation upon ligand binding. Eosinophils were stimulated with IL-5, and the lysate was Western blotted with an anti-phosphotyrosine Ab. IL-5 induced tyrosine phosphorylation of the {alpha} receptor (Fig. 1Go). IL-5R{alpha} has previously been reported to be differentially glycosylated and appears as multiple bands on Western blotting in the molecular mass range of 60–85 kDa. We have observed predominantly two bands (Fig. 1Go) upon Western blotting of eosinophil lysates.



View larger version (53K):
[in this window]
[in a new window]
  		FIGURE 1. Phosphorylation of IL-5R{alpha} by IL-5 stimulation. Purified eosinophils were incubated with IL-5 (10-10 M) for various periods of time, and then cells were lysed and immunoprecipitated with an anti-IL-5R{alpha} Ab. The immunoprecipitate was Western blotted with the anti-phosphotyrosine (4G10) Ab (n = 3).

 
Association of Src-type tyrosine kinases with IL-5R{alpha}

The members of the Src family tyrosine kinases are frequently associated with cytokine receptors. For this reason, we have examined the expression of Src family tyrosine kinases in eosinophils by Western blotting. Eosinophils express Hck, Lyn, Lck, and Fgr, but not Fyn (Fig. 2Go). We are also unable to detect Src and Blk (data not shown) in eosinophils. The results are in agreement with a recent report on Src-type kinases in eosinophils (28). We have previously reported that IL-5 activates Lyn kinase in eosinophils. For this reason, we have initially examined the association of Lyn with IL-5{alpha}. Coprecipitation studies reveal that Lyn kinase is physically associated with IL-5R{alpha} under basal conditions (Fig. 3Go). Stimulation of eosinophils with IL-5 for a short period of time (3 min) did not increase the association of Lyn with IL-5R{alpha}.



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 2. Expression of Src family kinases in eosinophils. Purified eosinophils were Western blotted for the presence of Hck, Lyn, Lck, Fgr, and Fyn using polyclonal Abs. Hck, Lyn, Lck, and Fgr were readily detectable. Fyn was not detectable in eosinophils (Eos) but was present in mononuclear cells (MNC) (n = 2).

 


View larger version (63K):
[in this window]
[in a new window]
  		FIGURE 3. Coprecipitation of IL-5R{alpha} with Lyn tyrosine kinase. Purified eosinophils were stimulated with IL-5 (10-10 M) for 3 min and then lysed and immunoprecipitated separately with Abs against IL-5R{alpha} and Lyn. The immunoprecipitates were then Western blotted with Abs against Lyn and IL-5R{alpha}, respectively (n = 3).

 
We have previously reported that Lyn is also associated with {beta}cR (12). Thus, coprecipitation of Lyn with IL-5R{alpha} could be the result of coprecipitation of IL-5R{alpha} with {beta}c. To address this concern, we first performed immunoprecipitation of stimulated and nonstimulated eosinophils with an anti-{beta}c Ab. The immunoprecipitating pellet (pellet I) was separated from the supernatant (supernatant I), and both were Western blotted for the presence of {beta}c. As expected, the pellet, but not the supernatant, showed the presence of the {beta}cR (Fig. 4Go). This supernatant, which does not contain {beta}c, was then immunoprecipitated with an anti-IL-5R{alpha} Ab. The immunoprecipitate (pellet II) was separated from the supernatant (supernatant II). Pellet I, pellet II, and supernatant II were Western blotted with the anti-Lyn Ab. The Western blot of pellet I confirms our previous report that a fraction of Lyn is associated with {beta}c, and this association is increased following IL-5 stimulation of cells. The Western blot of pellet II suggests that IL-5R{alpha} is associated with Lyn under basal conditions in the absence of {beta}c. This physical association is modestly reduced after IL-5 stimulation. The Western blot of supernatant II suggests that a fraction of Lyn is not associated with either receptor subunit and is most likely associated with other receptors. In a next step, we examined the association of Hck with IL-5R{alpha} and {beta}c using a similar protocol. Our results show that IL-5R{alpha} is not associated with Hck (Fig. 5Go). Modest amounts of Hck are associated with {beta}c, but the majority of Hck is in the supernatant fraction. Similar experiments with Fgr and Lck do not show any association with IL-5R subunits (data not shown).



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 4. Interaction of Lyn with IL-5R{alpha} independent of {beta}c. Eosinophils were stimulated with (+) and without (-) IL-5 (10-10 M) for 3 min and then lysed. The lysates were first immunoprecipitated with an anti-{beta}c Ab, and the supernatant was separated from the precipitated pellet. The pellet and an aliquot of the supernatant were Western blotted with the anti-{beta}c Ab. The membrane containing the {beta}c immunoprecipitate was then reprobed with the anti-Lyn Ab. The supernatant was subject to a second immunoprecipitation with the anti-IL-5R{alpha} Ab. The immunoprecipitated pellet was separated from the supernatant, and both were Western blotted with the anti-Lyn Ab (n = 2).

 


View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 5. Hck does not associate with IL-5R{alpha} and {beta}c. Eosinophils were stimulated with (+) and without (-) IL-5 (10-10 M) for 3 min and then lysed. The supernatant was separated from the immunoprecipitate (I) and subjected to a second immunoprecipitation (II) with the polyclonal rabbit anti-IL-5R{alpha} Ab and control rabbit serum (C). The immunoprecipitate was separated from the supernatant (III). All samples (I–III) were Western blotted with the anti-Hck Ab (n = 2).

 
Lyn phosphorylates IL-5R{alpha} and {beta}c subunits

We have shown that IL-5R{alpha} undergoes phosphorylation upon stimulation of eosinophils with IL-5. Next, we examined whether Lyn phosphorylates IL-5R in vitro. To this goal, we immunoprecipitated Lyn kinase and performed kinase assay in the presence of IL-5R{alpha} and {beta}c immunoprecipitates. Lyn kinase phosphorylated both {alpha} and {beta}c subunits of IL-5R (Fig. 6Go).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 6. Phosphorylation of IL-5R{alpha} and {beta}c by Lyn kinase. Lyn was immunoprecipitated from IL-5-stimulated eosinophils, washed extensively with the lysis buffer, and used in the immune complex kinase assay using washed IL-5R{alpha} and {beta}c immunoprecipitates as substrates. The kinase reactions were performed in the presence of [{gamma}-32P]ATP to detect in vitro phosphorylated products by autoradiography. In control experiments, the receptor immunoprecipitates were incubated alone in the kinase buffer without the Lyn immunoprecipitate.

 
Jak2 associates with IL-5R{alpha}

Previously, IL-5R{alpha} has been shown to be associated with Jak2 (14). We have confirmed this finding in coprecipitation studies (Fig. 7Go). Like Lyn kinase, Jak2 appears to be associated with IL-5R{alpha} under basal conditions. There is only modest increase in IL-5R{alpha} binding to Jak2 following IL-5 stimulation of eosinophils.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 7. Coprecipitation of Jak2 with IL-5R{alpha}. Eosinophils were stimulated with IL-5 (10-10 M) for increasing periods of time and then lysed. The lysates were immunoprecipitated with an anti-Jak2 Ab followed by immunoprecipitation with the anti-IL-5R{alpha} Ab (n = 3).

 
Lyn kinase and Jak2 are important for eosinophil differentiation

The differentiation of eosinophils from stem cells occurs stepwise. Lineage-committed stem cells initially require IL-3 for their proliferation. Subsequent stimulation with IL-5 leads to the differentiation of eosinophils. Previous studies have shown that a combination of IL-3 and IL-5 stimulates eosinophilopoiesis in vitro (29). We have used an in vitro liquid culture system using bone marrow cells from mouse according to the method described previously (26). In this model, allergic sensitization of mice significantly increases the sensitivity of bone marrow stem cells to IL-5. The percentage of in vitro differentiated eosinophils increases from ~10% in nonsensitized mice to 30% in sensitized mice. We have studied the importance of Lyn kinase and Jak2 for eosinophil differentiation using bone marrow cells from sensitized mice. Murine bone marrow cells were incubated with IL-3 and IL-5 for 1 wk, followed by cytospin preparations for Wright-Giemsa staining. Murine eosinophils were recognized by the ring-shaped nucleus and the presence of eosinophilic granules. The presence of major basic protein-containing granules in bone marrow-derived eosinophils was confirmed by immunocytochemical staining in a previous publication (26). The total cell and eosinophil counts after 1 wk were 46 ± 6 and 14 ± 2 x 104 cells, respectively (n = 3). Approximately 32% of the total cells were of eosinophilic lineage.

To block Lyn kinase, we used two strategies. First, we used a Lyn antisense ODN, which blocks the expression of Lyn kinase, but not Jak2, in eosinophils (27). Tyrphostin AG490 was used to block Jak2 (30). The Lyn antisense ODN and AG490 blocked eosinophil differentiation from stem cells in a dose-dependent manner (Fig. 8Go, A and B). The Lyn antisense ODN did not affect proliferation of other cells in the culture (Fig. 8GoC). However, AG490 showed a tendency to inhibit growth of all lineages in the culture, which is in agreement with the previous finding that Jak2 is essential for hemopoiesis in general (31, 32).



View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 8. Effect of inhibition of Lyn kinase and Jak2 on eosinophil differentiation. Murine bone marrow cells were isolated from immunized mice and then cultured with IL-3 and IL-5 for 8 days, as described previously. For Lyn inhibition, we used Lyn antisense and sense ODN that were previously reported to block Lyn expression without altering Jak2. Jak2 was inhibited using the pharmacologic inhibitor AG490.

 
Eosinophil differentiation is impaired in Lyn knockout mice

Next we have examined the differentiation of eosinophils in Lyn knockout mice. Lyn knockout mice do not have any developmental abnormalities (33). However, they show some dysregulation of Ig synthesis, implying a regulatory role of Lyn kinase in this process (34, 35). Furthermore, they have impaired mast cell degranulation. We have immunized Lyn knockout mice and control mice (C57/B6) with OVA to stimulate eosinophilopoiesis in vivo. Bone marrow cells were then cultured in vitro in the presence of IL-3 and IL-5, as described above. The IL-5-induced differentiation of eosinophils was strikingly inhibited in Lyn-/- mice as compared with that in control mice (10 ± 2 vs 27 ± 2, p < 0.01, Fig. 9Go). The total cell count in bone marrow cultures from knockout and control mice was similar. The noneosinophilic cells are mostly of monocytic lineage, indicating that Lyn is not essential for their growth.



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 9. Eosinophil differentiation in Lyn-/- mice. Lyn knockout mice and C57/B6 control mice were immunized with OVA, and bone marrow cells were collected for eosinophil differentiation studies according to the method described previously. Eosinophil differentiation was stimulated by culturing them in IL-3 and IL-5 for 8 days. Eosinophil differentiation was significantly reduced (10.2 ± 2 vs 27 ± 2.2, n = 6, p < 0.01) in Lyn knockout mice as compared with that in control mice (A). The total number of cells (mostly of monocytic lineage) in the culture was not affected (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IL-5R{alpha} subunit plays an essential role in IL-5 signaling and eosinophil differentiation. The significance of Src family kinases in IL-5R{alpha} signaling has not been previously investigated. We show that eosinophils express four different Src family kinases, Hck, Lyn, Fgr, and Lck. Among these four tyrosine kinases, only Lyn is physically associated with IL-5R{alpha}, and this association occurs independent of its association with {beta}c following IL-5 stimulation. Lyn kinase is able to phosphorylate IL-5R{alpha} in vitro. Lyn is important for IL-5-induced eosinophil differentiation from bone marrow stem cells. We have confirmed a previous report that Jak2 is associated with IL-5R{alpha}. Jak2 is also important for eosinophil differentiation.

Tyrosine kinases of the Src family are associated with many cytokine and growth factor receptors. Previous studies have implied an important role of Src-type kinases in a variety of cellular functions, including cell growth, differentiation, proliferation, adhesion, locomotion, and apoptosis (reviewed in Ref. 25). Specifically, in vitro studies have implicated an important role of the kinases in myeloid cell growth and differentiation. For example, Lyn is associated with receptors for B cell Ag receptor (36), erythropoietin receptor (37), Fc{gamma}RI (38), Fc{alpha}R (39), Fc{epsilon}RI (40), and c-Kit (41). In Lyn-/- mice, differentiation of erythrocytes, granulocytes, macrophages, and mast cells is not impaired (33, 34, 35), suggesting that erythropoietin, GM-CSF, and c-Kit signaling are unaltered. In Lyn-/- mice, basal production of neutrophils is mildly increased, whereas the B cell number is decreased by 50% (34). Basal eosinophilopoiesis appears normal in Lyn-/- mice, as reflected by the normal eosinophil count in the peripheral blood (~3% in both Lyn-/- and control mice). However, no specific information regarding IL-5-induced differentiation of eosinophils in Lyn-/- mice is available in the published literature.

Our results suggest that Lyn-/- mice have impaired IL-5-induced eosinophilopoiesis. The results imply that basal eosinophilopoiesis is regulated by multiple factors. It has been shown that the commitment of stem cells to granulocytic lineages is stochastic and independent of growth factors. Once committed, eosinophil progenitors are regulated by a multitude of positive and negative factors, including IL-3, IL-5, GM-CSF, IL-6, TGF-{beta}, corticosteroids, CC chemokines, and others (1). This is confirmed by the observation that basal eosinophil differentiation in IL-5R{alpha} (21) and {beta}c (42) knockout mice is reduced but not eliminated. For example, peripheral blood eosinophil count in IL-5R{alpha}-/- mice is reduced by <50% (23).

Lyn exerts a positive signaling effect by phosphorylating the immunoreceptor tyrosine-based activation motif of the B cell Ag receptor and a negative signaling effect by phosphorylating the immunoreceptor tyrosine-based inhibitory motif of the Fc{gamma}RII (38). In Lyn-/- mice, the negative signaling in B cells is affected more than the positive signaling, which results in increased Ab synthesis (32, 33, 34, 35). c-Kit signaling is unimpaired, and mast cell differentiation is normal. However, mast cell activation through Fc{epsilon}RI is attenuated (33, 35). These observations suggest that the downstream signaling effect of Lyn is cell and receptor specific. Lyn is redundant for downstream signaling by erythropoietin, IL-3, and GM-CSF receptors but is important for signaling through Fc{gamma}RII and Fc{epsilon}RI. Our results suggest that Lyn is also important for IL-5R{alpha} signaling in eosinophil progenitors. Interestingly, a subpopulation of mouse B cells, the so-called B1 cells, responds to CD38 ligation and IL-5 with cell proliferation and IgG1 synthesis. This effect is significantly abrogated in Lyn-/- mice, suggesting a pivotal role of Lyn in IL-5R signaling in B cells (43).

The signaling pathways downstream of Lyn kinase that lead to eosinophil differentiation are unknown. We have previously shown that extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are important for eosinophil differentiation from stem cells (26). Antisense inhibition of Lyn blocks the activation of ERK1/2 in eosinophils (27), indicating that the latter kinases are coupled to the Lyn signaling pathway. Whether p38 MAP kinases are linked to Lyn kinase in eosinophils is unknown. The activation of ERK1/2 in Lyn knockout mice has previously been examined. ERK1/2 activation is impaired in mast cells but enhanced in B cells from Lyn-/- mice (34). We were unable to study the activation of MAP kinases in purified eosinophils from Lyn-/- mice because of severely impaired production of eosinophils. Given that antisense Lyn ODN block ERK1/2 activation in eosinophils (27), it is likely that ERK1/2 activation is impaired in Lyn-/- eosinophil progenitor cells and results in reduced eosinophil production.


   Footnotes
 
1 This work was supported by grants from National Institutes of Health (PO1 AI46004 and RO1 AI50179) and the John Sealy Memorial Endowment Fund. Back

2 Address correspondence and reprint requests to Dr. Rafeul Alam, Department of Internal Medicine, Division of Allergy and Immunology, University of Texas Medical Branch, Rt-1083, Galveston, TX 77555-1083. E-mail address: ralam{at}utmb.edu Back

3 Abbreviations used in this paper: Jak, Janus kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; ODN, oligodeoxynucleotide. Back

Received for publication October 5, 2001. Accepted for publication December 14, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gleich, G. J.. 2000. Mechanisms of eosinophil-associated inflammation. J. Allergy Clin. Immunol. 105:651.[Medline]
  2. Denburg, J. A., R. Sehmi, H. Saito, J. Pil-Seob, M. D. Inman, P. M. O’Byrne. 2000. Systemic aspects of allergic disease: bone marrow responses. J. Allergy Clin. Immunol. 106:(Suppl. 5):S242.[Medline]
  3. Vignola, A. M., P. Chanez, G. Chiappara, L. Siena, A. Merendino, C. Reina, R. Gagliardo, M. Profita, J. Bousquet, G. Bonsignore. 1999. Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis. J. Allergy Clin. Immunol. 103:563.[Medline]
  4. Yamaguchi, Y., T. Suda, J. Suda, M. Eguchi, Y. Miura, N. Harada, A. Tominaga, K. Takatsu. 1988. Purified interleukin-5 supports the terminal differentiation and proliferation of murine eosinophilic precursors. J. Exp. Med. 167:43.[Abstract/Free Full Text]
  5. Yamaguchi, Y., T. Sudo, S. Ohta, K. Tominaga, Y. Miura, T. Kasahara. 1991. Analysis of the survival of mature eosinophils: interleukin-5 prevents apoptosis in mature human eosinophils. Blood 78:2542.[Abstract/Free Full Text]
  6. Sur, S., G. J. Gleich, M. C. Swanson, K. R. Bartemes, D. H. Broide. 1995. Eosinophilic inflammation is associated with elevation of interleukin-5 in the airways of patients with spontaneous symptomatic asthma. J. Allergy Clin. Immunol. 96:661.[Medline]
  7. 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.
  8. Foster, P. S., S. P. Hogan, A. J. Ramsay, K. I. Matthaei, I. G. Young. 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]
  9. Leckie, M. J., A. ten Brinke, J. Khan, Z. Diamant, B. J. O’Conner, C. M. Walls, A. K. Mathur, H. C. Cawley, K. F. Chung, R. Djukanovic, et al 2000. Effects of an IL-5 blocking monoclonal antibody on eosinophils, airway hyperresponsiveness, and the late asthmatic response. Lancet 356:2144.[Medline]
  10. Takatsu, K.. 1991. Interleukin 5 (IL-5) and its receptor. Microbiol. Immunol. 35:593.[Medline]
  11. Torigoe, T., R. O’Conner, D. Santoli, J. C. Reed. 1992. IL-3 regulates the activity of Lyn protein tyrosine kinase in myeloid-committed leukemic cell lines. Blood 80:617.[Abstract/Free Full Text]
  12. Pazdrak, K., D. S. Schreiber, P. A. Forsythe, L. Justement, R. Alam. 1995. The intracellular signal transduction mechanism of IL-5 in eosinophils: the involvement of lyn tyrosine kinase and the ras-raf 1-MEK-MAP kinase pathway. J. Exp. Med. 181:1827.[Abstract/Free Full Text]
  13. Hanazono, Y., S. Chiba, K. Sasaki, H. Mano, A. Miyajima, K. Arai, Y. Yazaki, H. Hirai. 1993. c-fps/fes protein-tyrosine kinase is implicated in a signaling pathway triggered by granulocyte-macrophage colony-stimulating factor and interleukin-3. EMBO J. 12:1641.[Medline]
  14. Ogata, N., T. Kouro, A. Yamada, M. Koike, N. Hanai, T. Ishikawa, K. Takatsu. 1998. Jak2 and Jak1 constitutively associate with an IL-5 receptor {alpha} and {beta}c subunit, respectively, and are activated upon IL-5 stimulation. Blood 91:2264.[Abstract/Free Full Text]
  15. Pazdrak, K., S. Stafford, R. Alam. 1995. The activation of the Jak2-STAT1 signaling pathway by IL-5 in eosinophils. J. Immunol. 155:397.[Abstract]
  16. Van der Bruggen, T., E. Caldenhoven, D. Kanters, P. Coffer, J. A. Raaijmakers, J. W. Lammers, L. Koenderman. 1995. Interleukin-5 signaling in human eosinophils involves JAK2 tyrosine kinase and STAT1{alpha}. Blood 85:1442.[Abstract/Free Full Text]
  17. Sato, S., T. Katagiri, S. Takaki, Y. Kikuchi, Y. Hitoshi, S. Yonehara, S. Tsukada, D. Kitamura, T. Watanabe, O. Witte, K. Takatsu. 1994. IL-5 receptor-mediated tyrosine phosphorylation of SH2/SH3-containing proteins and activation of Bruton’s tyrosine and Janus 2 kinases. J. Exp. Med. 180:2101.[Abstract/Free Full Text]
  18. Yousefi, S., D. C. Hoessli, K. Blaser, G. B. Mills, H. U. Simon. 1996. Requirement of Lyn and Syk tyrosine kinases for the prevention of apoptosis by cytokine in human eosinophils. J. Exp. Med. 183:1407.[Abstract/Free Full Text]
  19. Bates, M. E., W. W. Busse, P. J. Bertics. 1998. Interleukin 5 signals through Shc and Grb2 in human eosinophils. Am. J. Respir. Cell Mol. Biol. 18:75.[Abstract/Free Full Text]
  20. Coffer, P. J., R. C. Schweizer, G. R. Dubois, T. Maikoe, J. W. Lammers, L. Koenderman. 1998. Analysis of signal transduction pathways in human eosinophils activated by chemoattractants and the T-helper 2-derived cytokines interleukin-4 and interleukin-5. Blood 91:2547.[Abstract/Free Full Text]
  21. Caldenhoven, E., T. van Dijk, J. A. Raaijmakers, J. W. Lammers, L. Koenderman, R. P. de Groot. 1995. Activation of the STAT3/acute phase response factor transcription factor by interleukin-5. J. Biol. Chem. 270:25778.[Abstract/Free Full Text]
  22. Mui, A. L.-F., H. Wakao, A. M. O’Farrell, N. Harada, A. Miyajima. 1995. Interleukin-3, granulocyte-macrophage colony-stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J. 14:1166.[Medline]
  23. Yoshida, T., K. Ikuta, H. Sugaya, K. Maki, M. Takagi, H. Kanazawa, S. Sunaga, T. Kinashi, K. Yoshimura, J. Miyazaki, et al 1996. Defective B-1 cell development and impaired immunity against Angiostrongylus cantonensis in IL-5R{alpha}-deficient mice. Immunity 4:483.[Medline]
  24. Tanaka, H., N. Kawada, T. Yamada, K. Kawada, K. Takatsu, H. Nagai. 2000. Allergen-induced airway inflammation and bronchial responsiveness in interleukin-5 receptor {alpha} chain-deficient mice. Clin. Exp. Allergy 30:874.[Medline]
  25. Corey, S. J., S. M. Anderson. 1999. Src-related protein tyrosine kinases in hematopoiesis. Blood 93:1.[Free Full Text]
  26. Adachi, T., B. K. Choudhury, S. Stafford, S. Sur, R. Alam. 2000. The differential role of extracellular signal-regulated kinases and p38 mitogen-activated protein kinase in eosinophil functions. J. Immunol. 165:2198.[Abstract/Free Full Text]
  27. Pazdrak, P., B. Olszewska-Pazdrak, S. Stafford, R. Alam. 1998. Lyn, Jak2 and Raf-1 kinases are critical for the anti-apoptotic effect of interleukin-5 whereas only Raf-1 kinase is essential for eosinophil activation and degranulation. J. Exp. Med. 188:421.[Abstract/Free Full Text]
  28. Lynch, O. T., M. A. Giembycz, I. Daniels, P. J. Barnes, M. A. Lindsay. 2000. Pleiotropic role of lyn kinase in leukotriene B4-induced eosinophil activation. Blood 95:3541.[Abstract/Free Full Text]
  29. Takamoto, M., K. Sugane. 1995. Synergism of IL-3, IL-5, and GM-CSF on eosinophil differentiation and its application for an assay of murine IL-5 as an eosinophil differentiation factor. Immunol. Lett. 45:43.[Medline]
  30. Meydean, N. T., T. Grunberger, H. Dadi, M. Sahar, E. Arpaia, Z. Lapidot, J. S. Leeder, M. Freedman, A. Cohen, A. Gazit, et al 1996. Inhibition of acute lymphoblastic leukemia by a Jak-2 inhibitor. Nature 379:645.[Medline]
  31. Neubauer, H., A. Cumano, M. Muller, H. Wu, U. Huffstadt, K. Pfeffer. 1998. Jak2 deficiency defines an essential developmental checkpoint in definitive hematopoiesis. Cell 93:397.[Medline]
  32. Parganas, E., D. Wang, D. Stravopodis, D. J. Topham, J. C. Marine, S. Teglund, E. F. Vanin, S. Bodner, O. R. Colamonici, J. M. van Deursen, et al 1998. Jak2 is essential for signaling through a variety of cytokine receptors. Cell 93:385.[Medline]
  33. Hibbs, M. L., D. M. Tarlinton, J. Armes, D. Grail, G. Hodgson, R. Maglitto, S. A. Stacker, A. R. Dunn. 1995. Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease. Cell 83:301.[Medline]
  34. Chan, V. W., F. Meng, P. Soriano, A. L. DeFranco, C. A. Lowell. 1997. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity 7:69.[Medline]
  35. Nishizumi, H., K. Horikawa, I. Mlinaric-Rascan, T. Yamamoto. 1998. A double-edged kinase Lyn: a positive and negative regulator for antigen receptor-mediated signals. J. Exp. Med. 187:1343.[Abstract/Free Full Text]
  36. Pleiman, C. M., C. Abrams, L. Timson-Gauen, W. Bedzyl, J. Jongstra, A. S. Shaw, J. J. Cambier. 1994. Distinct p53/p56Lyn and p59Fyn domains associate with nonphosphorylated and phosphorylated Ig-{alpha}. Proc. Natl. Acad. Sci. USA 91:4268.[Abstract/Free Full Text]
  37. Tilbrook, P. A., E. Ingley, J. H. Williams, M. L. Hibbs, S. P. Klinken. 1997. Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells. EMBO J. 16:1610.[Medline]
  38. Duchemin, A. M., C. L. Anderson. 1997. Association of non-receptor protein tyrosine kinases with the Fc{gamma}RI/{gamma}-chain complex in monocytic cells. J. Immunol. 158:865.[Abstract]
  39. Gulle, H., A. Samstag, M. M. Eibl, H. M. Wolf. 1998. Physical and functional association of Fc{alpha}R with protein tyrosine kinase Lyn. Blood 91:383.[Abstract/Free Full Text]
  40. Jouvin, M. H. E., M. Adamczewski, R. Numerof, O. Letourneur, A. Valle, J.-P. Kinet. 1994. Differential control of the tyrosine kinases Lyn and Syk by the two signaling chains of the high affinity immunoglobulin E receptor. J. Biol. Chem. 269:5918.[Abstract/Free Full Text]
  41. Linnekin, D., C. S. DeBerry, S. Mou. 1997. Lyn associates with the juxtamembrane region of c-Kit and is activated by stem cell factor in hematopoietic cell lines and normal progenitor cells. J. Biol. Chem. 272:27450.[Abstract/Free Full Text]
  42. Nishinakamura, R., A. Miyajima, P. J. Mee, V. L. Tybulewicz, R. Murray. 1996. Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions. Blood 88:2458.[Abstract/Free Full Text]
  43. Yasue, T., H. Nishizumi, S. Aizawa, T. Yamamoto, K. Miyake, C. Mizoguchi, S. Uehara, Y. Kikuchi, K. Takatsu. 1997. A critical role of Lyn and Fyn for B cell responses to CD38 ligation and interleukin 5. Proc. Natl. Acad. Sci. USA 94:10307.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
J. M. Beekman, L. P. Verhagen, N. Geijsen, and P. J. Coffer
Regulation of myelopoiesis through syntenin-mediated modulation of IL-5 receptor output
Blood, October 29, 2009; 114(18): 3917 - 3927.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Yoon, A. Terada, and H. Kita
CD66b Regulates Adhesion and Activation of Human Eosinophils
J. Immunol., December 15, 2007; 179(12): 8454 - 8462.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
M. Martinez-Moczygemba, D. P. Huston, and J. T. Lei
JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization
J. Leukoc. Biol., April 1, 2007; 81(4): 1137 - 1148.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C.-L. Chu and C. A. Lowell
The Lyn Tyrosine Kinase Differentially Regulates Dendritic Cell Generation and Maturation
J. Immunol., September 1, 2005; 175(5): 2880 - 2889.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S.-J. E. Beavitt, K. W. Harder, J. M. Kemp, J. Jones, C. Quilici, F. Casagranda, E. Lam, D. Turner, S. Brennan, P. D. Sly, et al.
Lyn-Deficient Mice Develop Severe, Persistent Asthma: Lyn Is a Critical Negative Regulator of Th2 Immunity
J. Immunol., August 1, 2005; 175(3): 1867 - 1875.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
H. Inoue, R. Kato, S. Fukuyama, A. Nonami, K. Taniguchi, K. Matsumoto, T. Nakano, M. Tsuda, M. Matsumura, M. Kubo, et al.
Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness
J. Exp. Med., January 3, 2005; 201(1): 73 - 82.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
B. J. Lannutti and J. G. Drachman
Lyn tyrosine kinase regulates thrombopoietin-induced proliferation of hematopoietic cell lines and primary megakaryocytic progenitors
Blood, May 15, 2004; 103(10): 3736 - 3743.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
O. Cen, M. M. Gorska, S. J. Stafford, S. Sur, and R. Alam
Identification of UNC119 as a Novel Activator of SRC-type Tyrosine Kinases
J. Biol. Chem., February 28, 2003; 278(10): 8837 - 8845.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
W. Duan, I. C. Kuo, S. Selvarajan, K. Y. Chua, B. H. Bay, and W. S. F. Wong
Antiinflammatory Effects of Genistein, a Tyrosine Kinase Inhibitor, on a Guinea Pig Model of Asthma
Am. J. Respir. Crit. Care Med., January 15, 2003; 167(2): 185 - 192.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Vicentini, P. Mazzi, E. Caveggion, S. Continolo, L. Fumagalli, J. A. Lapinet-Vera, C. A. Lowell, and G. Berton
Fgr Deficiency Results in Defective Eosinophil Recruitment to the Lung During Allergic Airway Inflammation
J. Immunol., June 15, 2002; 168(12): 6446 - 6454.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stafford, S.
Right arrow Articles by Alam, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stafford, S.
Right arrow Articles by Alam, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS