By
From the * Department of Internal Medicine, Interleukin (IL)-5 has been shown to activate many signaling molecules in eosinophils, but
their functional relevance remains unknown. We have examined the functional relevance of
Lyn, Jak2, and Raf-1 kinases in eosinophil survival, upregulation of adhesion molecules and
degranulation. To this goal we used Lyn and Raf-1 antisense (AS) oligodeoxynucleotides
(ODN) to inhibit the expression of these proteins and tyrphostin AG490 to specifically block
the activation of Jak2. We have demonstrated that all three kinases are important for IL-5-
induced suppression of eosinophil apoptosis. However, Lyn and Jak2 tyrosine kinases are not
important for the upregulation of CD11b and the secretion of eosinophil cationic protein. In
contrast, Raf-1 kinase is critical for both these functions. This is the first identification of specific
signaling molecules responsible for three important functions of eosinophils. We have established a central role for Raf-1 kinase in regulating eosinophil survival, expression of Eosinophils are major inflammatory effector cells, with
blood and tissue eosinophilia being a feature of many
allergic disorders (1). Activated eosinophils can perform a
variety of specialized cellular functions, including cellular
adhesion, chemotaxis, generation of reactive oxygen metabolites, and secretion of cytotoxic enzymes (2). During an
allergic response, after exposure to a wide range of activators and chemotactic factors, eosinophils attach to the endothelial wall of the blood vessel at the site of the allergic
reaction. The process of cell attachment and tissue localization is mediated by families of cell adhesion molecules that
are expressed on both eosinophils and vascular endothelium (3). Eosinophils then migrate out of the bloodstream
into tissue and degranulate readily, releasing cytotoxic
products such as eosinophil cationic protein (ECP)1 and
major basic protein. Interleukin-5 is the principal regulatory cytokine that modulates all described functions of
eosinophils. First, it stimulates differentiation of eosinophils
from bone marrow cells leading to blood eosinophilia (4).
Second, it upregulates the expression of The exact mechanism of how IL-5 stimulates functions of
eosinophils is poorly understood. The earliest signaling
events to occur after stimulation of the IL-5 receptor by the
ligand involve the phosphorylation and activation of series of
protein tyrosine kinases, including Lyn, Syk, Jak2, and Fyn
(8). The consequence of activation of tyrosine kinases is
the propagation of signal through the Ras-Raf-1-mitogen-activated protein (MAP) kinase pathway and the Jak-STAT
pathway. Recently, Yosefi et al. (11) have shown that Lyn
and Syk tyrosine kinases are necessary for IL-5-induced inhibition of apoptosis in eosinophils. We have reported the requirement for SHP-2 tyrosine phosphatase in IL-5-stimulated prolongation of eosinophil survival (12). To date, these
are the only reports showing requirement of signaling molecules for IL-5-induced function of eosinophils.
The role of Jak2, Raf-1, MEK, and MAP kinases in mediating diverse cellular functions such as prolongation of
eosinophil survival and activation is not known. An approach using a dominant negative mutant indicated a role
for Jak2 in GM-CSF-induced cell proliferation (13). Further, depletion of Raf-1 kinase by antisense oligodeoxynucleotides resulted in inhibition of GM-CSF and IL-3-induced
proliferation and colony formation in leukemic cell lines
(14). The latter findings indicate that Raf-1 kinase is necessary for activation of different cellular functions. In this
study we took advantage of newly synthesized specific inhibitors and/or antisense oligodeoxynucleotides to alter activation or expression of the tyrosine kinases Jak2 and Lyn
and the serine/threonine kinase Raf-1 in human eosinophils. Having such altered cells, we asked about their ability
to respond to IL-5 with prolongation of survival, upregulation of CD11b adhesion molecule and priming for ECP release. Our findings revealed different requirements of the
studied kinases for mediating IL-5-induced survival and activation of eosinophils.
Reagents.
Department
of Pediatrics, The University of Texas Medical Branch, Galveston, Texas 77555-0762
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2 integrins
and degranulation. Further, there appears to be a dissociation between two receptor-associated tyrosine kinases, i.e., Lyn and Jak2, and the activation of Raf-1 kinase. The delineation of the
functional relevance of signaling molecules will help design therapeutic approaches targeting
specific eosinophil function.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
2 integrins, leading to increased recruitment of eosinophils from circulation
(5). By inhibiting apoptosis, IL-5 prolongs survival of eosinophils resulting both in blood and tissue eosinophilia (6).
Finally, IL-5 primes eosinophils for degranulation and release of reactive oxygen metabolites (7).
Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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 according to the method of Gartner (15) as described previously. The cells were further purified by negative selection using anti-CD16 immunomagnetic beads (Miltenyi Biotec, Sunnyvale, CA). 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 × 106) were incubated with IL-5 at a concentration of 10Gel 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 molecular mass range of the proteins studied. Gels were blotted onto Hybond membranes for Western blotting using the enhanced chemiluminescence system. Blots were incubated in a blocking buffer containing 5% 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 h. After washing five times in TBST buffer, blots were incubated for 30 min with a HRP-conjugated secondary antibody (0.1 µg/ml) directed against primary Ab. The blots were developed with the enhanced chemiluminescence 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-mercaptoethanol, and 2% SDS at 50°C for 30 min.Antisense Oligodeoxynucleotides.
Two 15-mer Lyn sense (SS) and antisense (AS) oligodeoxynucleotides (ODN) were synthesized by Operon Technologies (Alameda, CA) based on previously published sequence information (11). These oligodeoxynucleotides have been reported to decrease the expression of Lyn in human eosinophils (11). Sequences used were as follows: AS Lyn (CATATTTCCCGCTCG) and SS Lyn (CGAGCGGGAAATATG). Two 18-mer Raf-1 ODN, AS (TCCCTGTATGTGCTCCAT) and SS (ATGGAGCACATACAGGGA), were synthesized according to a published report that has proven to block the expression of Raf-1, but not A-Raf, and inhibit GM-CSF-dependent cell proliferation (14). The ODNs were phosphorothioate modified and resuspended in sterile H2O at 100 µM concentration.Stimulation of Eosinophils.
Purified eosinophils were suspended at 106/ml in RPMI 1640 and cultured in duplicate in the presence of ODNs or inhibitors at appropriate concentrations. All cultures and assays were performed in wells or tubes coated with 3% HSA (Sigma Chemical Co.). After a 6-h incubation, eosinophils were counted and cultured with IL-5 (10Survival Assay.
For survival studies, eosinophils (0.5 × 106 cells/ml) were incubated with ODNs or inhibitors for 6 h or as indicated in the text and then stimulated with IL-5 (10Flow Cytometry.
Eosinophils (0.5 × 106 cells/ml) were incubated in the presence or absence of IL-5. After 1.5 h of stimulation, eosinophils were washed in FACS buffer (HBSS containing 1% BSA and 0.1% NaN3) and stained with an anti-human CD11b FITC-conjugated mAb or with isotype control for 1 h at 4°C. The cells were finally washed in the FACS® buffer, fixed with 1% paraformaldehyde and 10,000 cells were analyzed by single-color flow cytometry on a FACScan® (Becton Dickinson, San Jose, CA).Degranulation Assay.
Eosinophils were primed with IL-5 and then stimulated with a suboptimal dose of PMA or platelet-activating factor (PAF) that did not cause degranulation on its own. In brief, after stimulation with IL-5 or buffer for 1.5 h, eosinophils were incubated in RPMI 1640 (0.2 × 106 cells/ml) with PMA (0.1 ng/ml) or PAF (10Statistical Analyses.
Results are expressed as mean ± SD. All statistical analyses were performed using Student's paired t test. P <0.05 was considered significant. ![]() |
Results |
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One of the earliest events that occurs after IL-5 stimulation is the activation and phosphorylation of the receptor-bound Lyn tyrosine kinase. This kinase has been already shown to be essential for mediating IL-5-dependent inhibition of apoptosis in human eosinophils. To determine the role of Lyn kinase in IL-5-induced function of eosinophils, first we evaluated the effect of Lyn AS ODN on the tyrosine kinase expression. Since eosinophils are terminally differentiated cells with a 3-4-d life span, the use of AS ODN is the most practical method to specifically alter expression of Lyn kinase. As demonstrated in Fig. 1, eosinophils exposed to 10 µM AS ODN for 6 h expressed little or no detectable p53/p56 Lyn kinase, whereas SS ODN did not alter Lyn level. The AS ODN used in our assay did not alter expression of a downstream signaling molecule MAP/ERK2 kinase (Fig. 1) and another tyrosine kinase Jak2 (data not shown). Higher concentrations (15 µM) of both AS and SS ODN nonspecifically inhibited both Lyn and ERK2 expression. For this reason all experiments were performed with 10 µM concentration of Lyn ODN. The viability of eosinophils assessed at this time (immediately before stimulation with IL-5) always exceeded 90% and was not different from control samples, indicating that at 10 µM concentration the ODNs were not toxic to the cells.
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We examined the biological relevance of activation of Jak2 by IL-5. For this purpose eosinophils were treated with AG490 (25 and 50 µM) for 6 h (19). The inhibitor at a concentration <50 µM was not toxic to eosinophils. More than 90% of eosinophils were viable after 6 h of incubation with AG490. At 25 µM concentration, AG490 completely blocked IL-5-induced tyrosine phosphorylation of Jak2 (Fig. 2 A). In contrast, preincubation of eosinophils with AG490 at 25-50 µM concentrations had no effect on IL-5-induced tyrosine phosphorylation of p53 and p56 Lyn kinases (Fig. 2 B). We also studied the effect of AG490 on Lyn kinase activation. Activated Lyn kinase has previously been shown to undergo autophosphorylation. AG490 at 25 µM concentration did not affect autophosphorylation of Lyn kinase (Fig. 2 B, right), confirming its specificity for Jak2 kinase at low concentrations (19). Interestingly, at 50 µM concentration of AG490 some inhibition of Lyn autophosphorylation was noted. In subsequent experiments we used a 25 µM concentration of AG490.
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We previously reported that IL-5 stimulated the phosphorylation and activation of Raf-1 kinase in eosinophils. To assess the role of Raf-1 kinase in transducing signals for eosinophil function, we performed several sets of experiments using Raf-1 AS ODN. First, we asked, whether the treatment with AS ODN had any effect on the expression of Raf-1 and a downstream signaling molecule, ERK2. As shown on Fig. 3 a, Raf-1 AS, at 7.5 µM concentration, almost completely inhibited the expression of Raf-1 without affecting ERK2 expression. The SS ODN did not inhibit Raf-1 expression. Next, we studied the effect of Raf-1 AS ODN on ERK2 activation. Eosinophils treated with medium or SS ODN showed tyrosine phosphorylation of ERK2 upon stimulation with IL-5. However, this tyrosine phosphorylation was completely inhibited by the AS ODN (Fig. 3 b, left). When the membrane was reprobed with the anti-ERK-2 antibody, we found that there was no change in ERK-2 expression after ODN treatment (Fig. 3 b, right). The results suggest that Raf-1 is necessary for phosphorylation and perhaps activation of ERK-2 kinase. In a separate experiment, the necrotic and apoptotic effects of various treatments were studied by flow cytometric analysis of annexin V and 7-amino actinomycin D (7-AAD)-stained cells. The percentage of cells showing necrotic death was <1% in AS ODN- and AG490-treated cells and was not different from control buffer-treated cells. Both Lyn and Raf-1 AS ODN as well as AG490 reversed the antiapoptotic effect of IL-5 on eosinophils (data not shown).
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Having established specific inhibition of Lyn, Jak2, and Raf-1 kinases, we studied the effect of the AS ODN and AG490 on eosinophil survival, upregulation of CD11b adhesion molecule and the release of eosinophil cationic protein. In all subsequent experiments, eosinophils were preincubated with the ODN or AG490 for 6 h and then stimulated with IL-5 for 1.5 h. The cells were then aliquoted for three separate and parallel experiments: (a) survival assay after 32 h; (b) measurement of CD11b upregulation by flow cytometry; and (c) ECP secretion after stimulation with a subthreshold concentration of PMA.
The Effect of AS ODN and AG490 on Eosinophil Survival.After preincubation with the inhibitors (ODN and AG490)
and stimulation with IL-5, eosinophils were resuspended in
RPMI 1640 without IL-5, inhibitors, and FCS. We excluded FCS from this stage of experiment because serum
has been shown to alleviate the requirement for the Ras
pathway for GM-CSF dependent viability and proliferation in mutant cells with c receptor lacking amino acid residues 626-763 (17). The viability of the cells cultured without serum was 69 ± 5% when assessed 32 h after stimulation with IL-5. In contrast to IL-5-stimulated cells, there
was 33 ± 7% viable cells in samples incubated with medium alone in lieu of IL-5 (Fig. 4 A). Lyn AS ODN
blocked the ability of IL-5 to prevent eosinophil death (35 ± 5% vs. 74 ± 7% vs. 69 ± 5% for AS-, SS-, and medium-treated cells, respectively, P <0.01). The results indicate a
critical role of Lyn kinase in IL-5-induced survival of eosinophils and confirm a previously published report (11).
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The incubation of eosinophils with AG490 blocked the IL-5-induced prolongation of eosinophil survival (Fig. 4 B). There were 31 ± 6% viable eosinophils 32 h after stimulation with IL-5 in samples pretreated with AG490. The dead cells had characteristics of apoptosis by microscopic examination. Similar results were obtained with a broad spectrum tyrosine kinase inhibitor, herbimycin A (Fig. 4 B). At 2 µM concentration, herbimycin A blocked both Lyn and Jak2 tyrosine phosphorylation in eosinophils in our control immunoblottings. As expected, this inhibitor blocked IL-5-induced prolongation of viability to a degree similar to that observed with AG490 and Lyn antisense ODN. Like Lyn AS ODN and AG490, Raf-1 AS ODN abrogated the IL-5-induced prolongation of eosinophil survival (34 ± 9% vs. 74 ± 7% in control cells; Fig. 4 C).
The Effect of AS ODN and AG490 on CD11b Expression.We examined the effect of the AS ODN and AG490 on IL-5-induced upregulation of CD11b adhesion molecule. CD11b/CD18 complex is constitutively expressed on eosinophils and its surface density can be upregulated by stimulation with IL-5 and GM-CSF. Indeed, a considerable upregulation of CD11b expression was found as early as 60 min after stimulation with IL-5, reaching the peak at 1.5 h. This increase in CD11b was not inhibited in cells treated with Lyn AS or SS ODN indicating that Lyn was not important in this process (Fig. 5 A). Similarly, AG490 did not affect the expression of CD11b (Fig. 5 B). Eosinophils treated with AG490 were still able to upregulate CD11b expression in response to IL-5. However, herbimycin, at 2 µM concentration, inhibited the upregulation of CD11b expression in response to IL-5. Thus, we conclude that protein tyrosine phosphorylation is important for CD11b upregulation, but Lyn and Jak2 kinases are not essential for this process. However, unlike Lyn AS ODN and AG490, Raf-1 AS ODN blocked the IL-5- stimulated upregulation of CD11b on eosinophils (Fig. 5 C). The SS ODN had no effects.
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It has been previously reported that tyrosine phosphorylation is involved in cytokine priming of the respiratory burst and degranulation (18). In our study, we asked whether Lyn kinase participated in the priming of eosinophils for ECP release. As a degranulating stimulus we used phorbol 12-myristate 13-acetate (PMA) at the suboptimal concentration of 0.1 ng/ml. This dose of PMA did not induce ECP release above the background level (data not shown). However, when eosinophils were preincubated with IL-5 for 1.5 h, they became more susceptible to stimulation with PMA and released three times more ECP (122 ± 13 ng/ml vs. 38 ± 5 ng/ml of ECP for unprimed eosinophils, P <0.01). Neither Lyn AS nor SS ODN modified IL-5 priming of ECP secretion (Fig. 6 A). Similar to Lyn AS ODN, AG490 did not block ECP secretion (Fig. 6 B). The data indicate that Lyn and Jak2 are not required for IL-5 priming for ECP release.
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In contrast to Lyn AS ODN and AG490, Raf-1 AS ODN almost completely blocked ECP secretion from eosinophils (Fig. 6 C). The SS ODN had no effects. There are some suggestions from the literature that PMA signaling may involve Raf-1 kinase. Thus, the PMA stimulation of cells may be more susceptible to RAF-1 AS inhibition. To address this issue, we performed experiments using a different eosinophil degranulator, PAF, at a subthreshold concentration. We observed a similar inhibition of ECP release from Raf-1 AS ODN-treated cells (132 ± 22 ng/ml for medium-pretreated cells vs. 42 ± 6 ng/ml for AS-treated cells, n = 3). Thus, although we cannot rule out that Raf-1 is involved in PAF signaling, our results suggest that this kinase is required for transducing signals for ECP release.
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Discussion |
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In this study, we have prepared eosinophils lacking active forms of Lyn, Jak2, and Raf-1 kinases and analyzed
their physiologic responses to IL-5. We have made the following observations. (a) IL-5 is unable to inhibit apoptosis
of cells not expressing active Lyn, Jak2, and Raf-1 kinases.
(b) The induction of CD11b expression and ECP secretion
by IL-5 are intact in the absence of Lyn and Jak2 activation.
However, other tyrosine kinases are important for CD11b
upregulation. (c) The IL-5-mediated suppression of eosinophil apoptosis, upregulation of CD11b, and ECP secretion
are impaired in eosinophils lacking Raf-1 kinase. Based on
these results, we conclude that Lyn, Jak2, and Raf-1 kinases
are necessary for the antiapoptotic effect of IL-5, whereas
Raf-1 is additionally essential for eosinophil activation and
degranulation. Previously, Lyn kinase was found to be important for eosinophil survival (11). However, the role of
Lyn in 2 integrin expression and eosinophil degranulation has been unknown. This is the first report on the importance of Lyn, Jak2, and Raf-1 kinase in three important
functions of eosinophils. We have established a central role
for Raf-1 kinase in the multifaceted regulation of eosinophil function. Further, our work reveals a differential requirement of signaling molecules for survival, adhesion,
and exocytosis of eosinophils.
Eosinophils are terminally differentiated cells that die rapidly in the tissue or culture due to apoptosis. They exhibit the classical changes in morphology including cytoplasmic and nuclear condensation resulting in cell shrinkage, DNA fragmentation, and loss of nucleoli (20). It is an important mechanism by which eosinophils are cleared from the tissue without eliciting inflammation (21). It has been known for many years that IL-5, GM-CSF, and IL-3 enhance their survival in culture for up to two weeks. The inhibition of eosinophil apoptosis by interleukins is considered an important mechanism for the development of blood and tissue eosinophilia in diseases such as asthma and other allergic disorders. However, an understanding of the induction of apoptosis and its inhibition is still fragmentary and mainly originates from cell lines that may not directly represent mature hematopoietic cells. Several genes have been identified that participate either as inducers or repressors of the programmed cell death (22). Among these, Bcl-2 and Bax are homologous proteins that have opposing effects on apoptosis. Bcl-2 serves to prolong cell survival, whereas Bax acts as an accelerator of apoptosis (23). These proteins act as ion channels and adaptor proteins regulating the release of mitochondrial proteins into the cytosol in order to activate the cysteine proteases (caspases) that are the terminal effectors of apoptosis. Overexpression of Bcl-2 inhibits mitochondrial permeability transition, whereas Bax overexpression induces it. One mechanism of inhibition of apoptosis by Bcl-2 is its ability to associate with Raf-1, translocating this kinase from the cytosol to the mitochondrial membrane (24). Once there, Raf-1 induces phosphorylation of BAD, a proapoptotic Bcl-2 family protein that abrogates the cytoprotective functions of Bcl-2 by heterodimerizing with it (25). This mechanism could explain our findings on the requirement of Raf-1 kinase for antiapoptotic effect of IL-5. In this regard, IL-3 has previously been shown to induce Bcl-2 in myeloid cell precursors (26).
Another mechanism by which Raf-1 could transduce
the antiapoptotic signal is through activation of downstream signaling molecules such as MEK and MAP kinases
and/or NFB nuclear factor. As we have shown in this
work, inhibition of Raf-1 expression prevents tyrosine
phosphorylation of ERK-2 MAP kinase. MAP kinases are known to induce c-fos and c-jun (27). The truncation of
the
c receptor at 516 or 626 amino acid residues abolishes
activation of Ras-Raf-MAP, c-fos and c-jun and inhibits
the antiapoptotic effect of GM-CSF (28). Moreover, the
contribution of MAP kinases in regulation of STAT1/3
nuclear factors has recently been reported (29). Because
Raf-1 is essential for both MAP activation and prolongation of survival, the viability signaling may be linked to
molecules and genes activated by MAP kinases.
There are reports of NFB activation by Raf-1 kinase
(30). NF
B consists of two subunits (p50 and p65) and exists in a complex with I
B in resting cells. Activated Raf-1
phosphorylates I
B releasing NF
B that enters the nucleus
and activates various genes carrying the NF
B response element. Cells lacking NF
B are more sensitive to TNF-
-induced cytotoxicity, confirming that one of the target
genes for NF
B is a gene encoding a survival factor (31).
Although Raf-1 activation by antiapoptotic cytokines such as IL-3, GM-CSF, and IL-5 is well documented, the activation of NF
B in eosinophils has not been investigated.
In this work we confirmed the essential role of Lyn kinase in the antiapoptotic effect of IL-5 as previously reported (11). Lyn kinase is one of the earliest activated kinases after IL-5 stimulation. It is constitutively associated
with the c receptor for IL-5. It may serve to activate the
cytosolic tyrosine kinase Syk (8, 11). Other potential substrates for Lyn kinase are Raf-1 kinase and SHP2 tyrosine
phosphatase. Our results suggest a dissociation of Lyn from
Raf-1 activation since they have nonoverlapping functions.
This is the first report on the importance of Jak2 kinase
in eosinophil survival. Jak2 kinase is constitutively associated with the c subunit of the IL-5 receptor and is activated upon ligand binding. There are several possible target
molecules for Jak2 kinase: IL-5
c receptor, STAT nuclear
factors, SHP2 tyrosine phosphatase, and Raf-1. Jak2 is essential for phosphorylation of the receptor as IL-5R
c
phosphorylation by GM-CSF is abolished in mutant Jak2-negative BA/F3 cells (13). Interestingly, one of the tyrosine
residues phosphorylated by Jak2 is Y750, which is considered a viability domain of the IL-5
c receptor. This tyrosine residue was found to be necessary for activation of
Shc, p21 ras, Raf-1, and MAP kinases. STAT nuclear factors are the first described downstream molecules for the
Janus kinase family. The Jak-STAT pathway is considered
critical for cytokine-stimulated proliferation of cells. Their
function in nonproliferating cells such as eosinophils is unknown. Our results define a role for Jak2 in survival but
not for eosinophil activation.
Human eosinophils constitutively express CD11b/CD18
adhesion molecule. In vitro CD11b/CD18 can be upregulated by a number of stimuli including IL-5, GM-CSF, and
PAF (32, 33). CD11b upregulation on eosinophils is probably analogous to that on neutrophils, in which CD11b is
rapidly mobilized from cytoplasmic stores and upregulation
does not require de novo protein synthesis (34). Little is
known on signaling events leading to increased expression of CD11b/CD18. It has been shown that antisense oligodeoxynucleotides to the p65 subunit of NFB block
CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes (35). In our work, we could
not upregulate CD11b expression in eosinophils lacking
Raf-1 kinase. It is possible that the CD11b upregulation through Raf-1 is linked to NF
B activation.
The suppression of Lyn and Jak2 did not block CD11b upregulation by IL-5. The result does not imply that tyrosine phosphorylation is dispensable for the function, because treatment of eosinophils with herbimycin A completely inhibited IL-5-induced upregulation of CD11b. The data are in agreement with the study of Nishizumi et al. (36) who found that Lyn-deficient mast cells had impaired tyrosine phosphorylation and Ca2+ mobilization but retained capability for adhesion and degranulation. There are two possible interpretations of our results. When Lyn is inhibited, Jak2 induces Raf-1 activation. Likewise, when Jak2 is inhibited, Lyn may signal for Raf-1 activation. Alternatively, a third tyrosine kinase (or kinases) is responsible for Raf-1 signaling. Among the candidates are Fyn, Btk, Fes, and Syk, all of which have been shown to be activated by IL-5 (10, 37, 38). Additional pathways may include protein kinase C.
Recently, it has become evident that CD11b/CD18 is not simply an adhesion site on the cell surface, but may modulate a number of cellular processes such as eosinophil degranulation and superoxide production. For example, superoxide production and release of EDN induced by GM-CSF is abolished by the treatment of eosinophils with anti-CD11b and anti-CD18 mAbs (39). Our finding that similar signaling processes are involved in both CD11b upregulation and degranulation may indicate that these events are closely linked. We have used IL-5 for eosinophil priming and PMA or PAF for the induction of ECP release. Raf-1 appears to control the signaling processes originating from all these diverse stimuli, leading to ECP secretion. Based on these observations we believe that Raf-1 is a critical signal for eosinophil degranulation.
In summary, we have defined specific functions of Lyn, Jak2, and Raf-1 kinases in eosinophil activation. Eosinophils play a pivotal role in asthma and other allergic diseases. The identification of the critical signaling molecules for eosinophil survival, adhesion, and degranulation will help develop specific inhibitors for use in allergic and other eosinophilic disorders.
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Footnotes |
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Address correspondence to Rafeul Alam, The University of Texas Medical Branch, Department of Internal Medicine, Rt-0762, Galveston, TX 77555-0762. Phone: 409-772-3411; Fax: 409-772-5841; E-mail: ralam{at}utmb.edu
Received for publication 24 October 1997 and in revised form 21 April 1998.
This work was supported by grants from the National Institutes of Health (RO1 35713 and RO1 15939) and the John Sealy Memorial Foundation. K. Pazdrak was supported by a McLaughlin Foundation Postdoctoral Fellowship, Texas Allergy and Immunology Society Memorial Foundation, and the President's Grant-In-Aid from the American Academy of Allergy, Asthma, and Immunology.
Abbreviations used in this paper AS, antisense; ECP, eosinophil cationic protein; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; ODN, oligodeoxynucleotide; PAF, platelet-activating factor; SS, sense.
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