INVITED REVIEW
The mechanism of IL-5 signal transduction

Tetsuya Adachi and Rafeul Alam

The University of Texas Medical Branch, Division of Allergy and Immunology, Department of Internal Medicine, Galveston, Texas 77555-0762

    ABSTRACT
Top
Abstract
Introduction
Concluding Remarks
References

Cytokines are important regulators of hematopoiesis. They exert their actions by binding to specific receptors on the cell surface. Interleukin-5 (IL-5) is a critical cytokine that regulates the growth, activation, and survival of eosinophils. Because eosinophils play a seminal role in the pathogenesis of asthma and allergic diseases, an understanding of the signal transduction mechanism of IL-5 is of paramount importance. The IL-5 receptor is a heterodimer of alpha - and beta -subunits. The alpha -subunit is specific, whereas the beta -subunit is common to IL-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) receptors and is crucial for signal transduction. It has been shown that there are two major signaling pathways of IL-5 in eosinophils. IL-5 activates Lyn, Syk, and JAK2 and propagates signals through the Ras-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2 tyrosine kinases and SHP-2 tyrosine phosphatase are important for eosinophil survival. In contrast to their survival-promoting activity, Lyn and JAK2 appear to have no role in eosinophil degranulation or expression of surface adhesion molecules. Raf-1 kinase, on the other hand, is critical for eosinophil degranulation and adhesion molecule expression. Btk is involved in IL-5 stimulation of B cell function. However, it does not appear to be important for eosinophil function. Thus a clear segregation of signaling molecules based on their functional importance is emerging. This review describes the signal transduction mechanism of the IL-3/GM-CSF/IL-5 receptor system and compares and contrasts IL-5 signaling between eosinophils and B cells.

interleukin-5; eosinophil; kinase; granulocyte-macrophage colony-stimulating factor

    INTRODUCTION
Top
Abstract
Introduction
Concluding Remarks
References

INTERLEUKIN-5 (IL-5) was initially found as a T cell-replacing factor (TRF) that induced differentiation of B cells into immunoglobulin-secreting cells (29, 111). It was subsequently shown that TRF had B cell growth-promoting activity (termed BCGF II) on BCL1 cells (116) and dextran sulfate-stimulated B cells (41). Takatsu et al. (120) further demonstrated that induction of cytotoxic T lymphocytes from thymocytes required T cell-derived soluble mediators referred to as killer helper factors (KHF) and found that KHF was identical to TRF (121). Because TRF had pleiotropic activities on multiple cells, it was renamed IL-5 in 1987. Murine IL-5 is known to enhance several functions of murine B cells, including immunoglobulin production, growth, and differentiation (122). The response of human B cells to human IL-5 has been a subject of controversy. Several recent publications tend to indicate that human B cells are capable of responding to human IL-5 under certain conditions. Human IL-5 augments immunoglobulin production of Staphylococcus aureus Cowan I (SAC) or pokeweed mitogen-stimulated human B blasts in the presence of specific cytokines such as IL-2 or IL-4 (9, 73). Huston et al. (46) have recently demonstrated that human IL-5 directly induces responsiveness of B cells stimulated with Moraxella catarrhalis (MCat) but not with SAC. They have also shown that freshly isolated human B cells express mRNA for IL-5Ralpha , IL-5Rbeta , and soluble IL-5Ralpha and that stimulation by either MCat or SAC increases IL-5Ralpha and IL-5Rbeta mRNA and decreases soluble IL-5Ralpha mRNA. Besides the effect on B cells, human IL-5 has also been shown to act on T cells. Human IL-5 increases the expression of IL-2Ralpha on human T cells (84) and augments cytotoxic T cell generation (78).

In humans, the biological effects of IL-5 are best characterized for eosinophils. IL-5 is not only essential for the terminal differentiation of eosinophils but is also important for the activation of mature eosinophils. These cells are the most important effector cells of allergic reactions. Allergic diseases were originally regarded as a manifestation of immediate hypersensitivity reactions, mostly mediated by mast cells. There is now clear evidence that the late-phase allergic reaction is an integral part of allergic disorders. The late-phase allergic reaction is characterized by the influx of huge numbers of eosinophils and, to a lesser extent, lymphocytes, monocytes, basophils, and neutrophils. In accordance with the foregoing statement, bronchial mucosa of asthmatic patients who die of severe bronchoconstriction show a marked eosinophilic infiltration (34). Eosinophils show signs of activation, e.g., increased release of eosinophil granular proteins and production of leukotriene C4 (LTC4) and superoxide. Eosinophil granular proteins, such as major basic protein (MBP), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO) combined with H2O2 and halide, cause damage to respiratory epithelium in guinea pigs (74). LTC4 has a potential role in the induction of smooth muscle contraction, mucus production, and edema formation in the pathogenesis of asthma (96). IL-5 may be the most important cytokine that primes eosinophil function. T cells from allergic patients are mostly of Th2 functional subtype and produce large quantities of IL-4 and IL-5 (56). Once eosinophils infiltrate airways, they produce IL-5, establishing a positive feedback mechanism (12, 26). The major functions of IL-5 on eosinophils are shown in Table 1. IL-5 also acts on basophils to enhance the release of mediators such as histamine and LTC4 (10, 43, 66).

                              
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Table 1.   The effect of IL-5 on eosinophils and signaling molecules

IL-5 belongs to the hematopoietic growth factor family of cytokines. It has significant functional homology with IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF). The reason for this homology is that the beta -receptor subunit is common to all three cytokines (Fig. 1) (72). The ligand specificity is preserved by distinct alpha -receptor subunits. This is similar to the common gamma -receptor subunit for IL-2, IL-4, IL-7, IL-9, and IL-15. The alpha -subunits are specific for each cytokine and bind their specific ligand with low affinity. The beta c-subunit forms a high-affinity receptor with all three alpha -subunits, despite its lack of capacity to bind the cytokines by itself. The beta c-subunit is not only required for the formation of the high-affinity receptor complex but is also crucial for signal transduction. Most signal transduction studies of the beta c receptor have been performed with IL-3- or GM-CSF-dependent cell lines. There are relatively few publications using primary cells such as eosinophils or B cells. Studies with primary cell types are, however, extremely important because many important granulocytic functions such as degranulation, chemotaxis, or cytotoxicity cannot be performed using cell lines.


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Fig. 1.   Structure of human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and IL-5 receptors. The alpha -subunits are specific for their respective ligands, whereas the beta -subunit is common among these receptors.

In this review, we discuss recent findings concerning IL-3/GM-CSF/IL-5 signaling in cell lines. We then analyze the findings of IL-5 signal transduction in eosinophils and B cells.

    A CLASSIFCATION OF INTRACELLULAR SIGNALING EVENTS

Receptor-driven signaling processes typically originate from the juxtamembranous region of the receptor. Some signaling molecules are physically associated with the receptor under basal conditions. Furthermore, many of them are N-acylated. N-acylation results in anchoring the signaling molecule into the lipid membrane (e.g., tyrosine kinases of the src family). The activation of these membrane-anchored tyrosine kinases and lipid kinases occur in the juxtamembranous hydrophobic milieu. Thus this initial step can be called the juxtamembranous signaling step (Fig. 2). In addition to membrane-anchored signaling molecules, there are cytosolic signaling molecules that frequently translocate from the receptor site to other cellular compartments (e.g., nucleus, cytoskeleton, mitochondria). These mobile signaling molecules mostly operate in a hydrophilic milieu. For the juxtamembranous signaling molecules to transduce signals to cytosolic signaling molecules, various interfacing molecules become necessary. The interfacing molecules either serve as docking sites for cytosolic signaling molecules or form multimolecular complexes with them and then translocate to other cellular compartments. Thus the second step can be called the signal interfacing step (Fig. 2). The third step of signaling involves the activation of mobile cytosolic signaling molecules. The latter molecules translocate into their target sites by unknown mechanisms. Subsequently, the signal is transduced and translated into biological functions. Thus this step can be called the mobile signaling step (Fig. 2). One possible outcome of the mobile signaling step is the activation of transcription factors and their migration to the nucleus, leading to initiation of gene transcription. This step can be called the transcription-activating step (Fig. 2).


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Fig. 2.   A model of IL-5 signaling. Signaling events are classified in 4 steps, i.e., juxtamembranous signaling step, signal-interfacing step, mobile signaling step, and transcription-activating step. JAK2, Janus kinase 2; MAPK, mitogen-activated protein kinase; MEK, MAP or extracellular signal-regulated kinase; PI-3 kinase, phosphatidylinositol 3-kinase.

    IL-3/GM-CSF/IL-5 SIGNALING IN CELL LINES

The Juxtamembranous Signaling Step

Specific cytoplasmic regions of alpha  and beta  receptors activate tyrosine kinases. When IL-3, IL-5, and GM-CSF bind to corresponding alpha  receptors, tyrosine phosphorylation of several cytoplasmic proteins and the beta c receptor occurs. Because neither alpha - nor beta -receptor subunits contain a kinase domain in their cytoplasmic region, juxtamembranous tyrosine kinases are involved in this process. IL-3, GM-CSF, and IL-5 induce tyrosine phosphorylation of cellular proteins in factor-dependent myeloid cell lines (49, 54, 114). Tyrosine kinase inhibitors, such as herbimycin A, inhibit this phosphorylation (110). Multiple tyrosine kinases including Lyn (3, 17, 128), Yes (17), Fyn (3), Fes (39), Hck (3, 64), and JAK2 (Janus kinase 2) (98, 113, 133) have been shown to be activated rapidly by IL-3 and GM-CSF in myeloid cell lines, suggesting that activation of these kinases are the initial signaling events. Previous studies have shown that Lyn (63), Fes (11), and JAK2 (11, 98) tyrosine kinases are physically associated with the beta c receptor. These tyrosine kinases bind to a membrane proximal region between amino acids 451 and 517 (102); this site is also essential for induction of c-myc and pim-1 (Fig. 3) (107). It is likely that the receptor dimerization activates one of the receptor-bound tyrosine kinases first. Subsequently, a cascade of tyrosine phosphorylation of other molecules occurs.


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Fig. 3.   Association of IL-5 receptor with signaling molecules. Lyn is constitutively associated with the beta c receptor. Both the membrane-proximal proline-rich regions in the alpha  and beta c receptor (the latter is so-called box 1) are critical for JAK2 binding and activation. Shc and SHP-2 recognize their specific phosphorylated tyrosine residues. Dotted lines, 2 critical regions of the beta c, which are essential for induction of specific signaling molecules.

The binding of JAK2 kinase to receptors has been studied by Murakami et al. (77). For this purpose they initially have studied the gp130 subunit of the IL-6 receptor. They have postulated that the so-called box 1 region is important for signaling via the gp130 receptor subunit. The proline-rich motif (PXP motif) in the box 1 region is conserved among many cytokine receptors, e.g., beta c receptor of IL-3/GM-CSF/IL-5, IL-2 receptor beta  chain, granulocyte colony-stimulating factor receptor, and erythropoietin receptor. The mutation of the two proline residues in the PXP motif of gp 130 results in complete loss of IL-6 signaling activity. In accordance with this observation, Watanabe et al. (133) have reported that the box 1 region is essential for GM-CSF-dependent JAK2 activation. The deletion of box 1 from the receptors for growth hormone and erythropoietin abolishes their JAK2-binding property (123). Interestingly, however, Ogata et al. (86) have recently shown that a GST fusion protein with the cytoplasmic region (amino acid residues 456-544) of the beta c, which contains the box 1 motif, cannot bind JAK2. These results indicate that the box 1 region is essential but not sufficient for JAK2 binding.

The IL-5 receptor alpha -subunit is also known to be important for IL-5 signaling. A murine IL-3-dependent FDC-P1 cell line, into which mutant mIL-5Ralpha lacking its whole cytoplasmic domain was transfected, does not respond to IL-5, indicating that the cytoplasmic region of the alpha -subunit is critical for IL-5 signaling (118). A study using the COOH-terminal truncated cytoplasmic domain of IL-5Ralpha has revealed that JAK2 is constitutively associated with the IL-5Ralpha and that the region between amino acids 346 and 387 is responsible for this association (86). Like the beta c, at least two sites are indispensable for JAK2 binding, the regions of amino acids 351-356 and 366-387 (Fig. 3). The former region contains the box 1-like PPXP motif. The deletion or mutation of the proline-rich motif from IL-5Ralpha causes loss of growth, stimulatory effects, induction of c-fos, c-jun, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 (119).

Most cytokines exist in dimers under physiological conditions. Consequently, receptors are likely to dimerize on ligand binding. The same principle may also apply to IL-5, since it also exists as a dimer. In this model, the dimerization brings the beta c receptor-associated JAK2 kinases to close proximity and promotes cross-phosphorylation. However, dimerization may not be an absolute requirement for signaling, since a modified monomeric form of IL-5 has been shown to activate cells (24). Alternatively, the IL-5Ralpha -associated JAK2 may activate the beta c-bound JAK2. This possibility is supported by the signaling capability of a chimeric receptor in which the cytoplasmic domain of IL-5Ralpha is substituted by that of beta c (119). However, IL-5Ralpha is essential for IL-5 signaling because the beta c receptor cannot bind the ligand by itself.

The importance of JAK2 activation has been studied in cell lines. In BA/F3 cells, a dominant negative JAK2 mutant, which lacks the kinase domain, suppresses the activation of beta c, Shc (src homologues and collagen), SHP-2 (SH2-containing phosphatase-2), and c-fos as well as c-myc (133). The kinase domain of JAK2 also seems to be important for the induction of Bcl-2 (B cells lymphoma/leukemia-2) and the maintenance of cell viability (106). JAK2 and other members of the Janus kinase family are known to transduce signals via the signal transducers and activators of transcription (STAT) transcription factors. Recently, JAK2 has been shown to activate the Ras-mitogen-activated protein kinase (Ras-MAPK) pathway not only through the receptor subunit but also directly. For example, Vav associates with JAK2 after GM-CSF stimulation in MO7e cells (70). Furthermore, tyrosine phosphorylation of Vav is significantly increased when JAK2 is coexpressed with Vav in the insect cell line, Sf21. In interferon-gamma (IFN-gamma )-stimulated cells, Raf-1 is activated by JAK2 in the presence of Ras, and a ternary complex of Ras, JAK2, and Raf-1 is observed (137). In this condition Raf-1 is phosphorylated on tyrosine-340 and tyrosine-341 by v-Src, whereas JAK2 does not phosphorylate these residues, suggesting that Raf-1 is regulated by JAK2 and v-Src through a different mechanism.

Signal-Interfacing Step

Adapter proteins. The next signaling step for many tyrosine kinases is the interfacing step with adapter proteins. The tyrosine phosphorylation of beta c by tyrosine kinases creates docking sites for many signaling molecules including adapter proteins. Shc, an adapter protein, binds to beta c. It contains a COOH-terminal Src homology 2 (SH2) domain and a novel non-SH2 phosphotyrosine-binding (PTB) domain that specifically recognizes a phosphorylated NPXpY motif in target proteins. At this point it is not clear which of these domains is responsible for binding to the beta c receptor. Lanfrancone et al. (61) have shown that Shc associates with the beta c through its SH2 domain in GM-CSF-stimulated TF-1 cells. In contrast, Pratt et al. (97) claim that the NH2-terminal PTB domain of Shc binds to tyrosine-577 of the beta c receptor (Fig. 3). The importance of various tyrosine residues of the beta c receptor is also unclear at this time. In BA/F3 cells, all detectable tyrosine phosphorylation of the beta c receptor is eliminated by a substitution of tyrosine-750 with phenylalanine (48). The mutation inhibits cellular viability and reduces Shc tyrosine phosphorylation, whereas tyrosine phosphorylation of JAK2, SHP-2, and Vav is intact. However, other studies suggest that the former effect may be indirect, because only tyrosine-577 is actually required for phosphorylation of Shc and the above-mentioned mutation possibly impairs phosphorylation of tyrosine-577 (28, 50). Although the role of these tyrosine residues remains to be solved completely, tyrosine-577 appears to be important for propagation of signals through Ras-MAPK and activation of the c-fos promoter (Fig. 3) (50). Shc is likely to have an important role in the maintenance of cell viability. BA/F3 cells expressing Shc with mutations of tyrosine-239 and tyrosine-240 to phenylalanine, although able to propagate signals through the Ras pathway, cannot maintain IL-3-induced cell survival (36). In this condition c-myc gene induction is reduced, suggesting a critical role of c-myc activation for inhibition of apoptosis in BA/F3 cells.

In response to IL-3, GM-CSF, and IL-5 stimulation, Shc not only binds to beta c but also undergoes tyrosine phosphorylation (18, 25, 71). IL-3 and GM-CSF also induce tyrosine phosphorylation of a 140-kDa protein that is constitutively bound to growth factor receptor-bound protein-2 (Grb2) through Shc activation (53, 61, 71). Liu et al. (65) identified a 145-kDa protein that associates with Shc through its SH2 domain after cytokine stimulation and competes with Grb2 for the same tyrosine-phosphorylated site on Shc. This 145-kDa protein exhibits inositol polyphosphate 5-phosphatase activity (19). Furthermore, it has been shown that a 135-kDa protein having phosphatase activity is constitutively associated with Grb2 and inducibly associated with Shc in GM-CSF-stimulated UT-7 cells (85). These 140-kDa, 145-kDa, and 135-kDa proteins are now considered to be identical and are called SH2-containing inositol-5'-phosphatase (SHIP). The interaction with SHIP suggests that Shc is likely to propagate signals through the Ras and inositol pathways.

The tyrosine phosphatase, SHP-2 (previously known as Syp, PTP1D, and SHPTP2), is also known to associate with the beta c receptor via its SH2 domain and is activated after stimulation with IL-3 or GM-CSF (135). Once phosphorylated on tyrosine residues, SHP-2 generates a binding site for Grb2, which in turn leads to activation of the Ras signaling pathway. SHP-2 tyrosine phosphorylation is mediated through multiple sites of beta c (Fig. 3). It has been shown that beta c with a mutation of tyrosine-577 to phenylalanine and a COOH-terminal truncated mutant at position-589 are equally capable of inducing tyrosine phosphorylation of SHP-2. This is in contrast to Shc, because its phosphorylation is dependent on tyrosine-577 (Fig. 3). A guanine nucleotide-exchanging protein, Vav, is known to regulate the Ras pathway. It is constitutively associated with the SH3 domain of Grb2 and phosphorylated on tyrosine residues by IL-3, GM-CSF, and IL-5 (40, 68, 70, 108). In addition, the involvement of JAK2 and Tec kinases for the activation of Vav has been suggested (68, 70).

Activation of phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase (PI3K) has been implicated in various cell functions including mitogenic signals, membrane ruffling, oxidative burst, and glucose transport. IL-3, GM-CSF, and IL-5 activate PI3K (35, 107, 108). Because the beta c-subunit does not have a consensus motif for PI3K binding, it is necessary for beta c to use a distinct mechanism for its association with and activation of PI3K. Several groups have identified molecules that intermediate between beta c and PI3K. One possible molecule is SHP-2, which associates with beta c and with the p85 subunit of PI3K after treatment with IL-3 or GM-CSF (135). Jücker and Feldman (52) have described an unknown p80 protein that is tightly associated with Src/Yes and Lyn, as well as beta c and the p85 subunit of PI3K in GM-CSF-stimulated TF-1 cells (52). This report suggests that the src family kinases may be involved in tyrosine phosphorylation of PI3K. Recently, Tec tyrosine kinase has been shown to link beta c to PI3K through JAK2 kinase in IL-3 signaling (117). PI3K transduces signals to a number of serine/threonine kinases, such as novel/atypical protein kinase C (nPKC/aPKC) (83, 127), Akt, c-jun NH2-terminal kinase (JNK), and p70 S6 kinase (60). Although the functional role of PI3K in IL-3/GM-CSF/IL-5 signaling is not well known, del Peso et al. (22) have recently shown that IL-3 propagates signals via the PI3K-Akt-BAD pathway, indicating that PI3K may participate in cell survival through the phosphorylation of BAD.

Mobile Signaling Step

Ras-MAPK Pathway. The outcome of activation of various adapter proteins and GTPases in the interfacing step is the stimulation of p21 G proteins. One of the most extensively studied p21 G proteins is Ras. Ras translocates Raf-1 kinase to the juxtamembranous compartment, where the latter is activated by tyrosine kinases, protein 14-3-3, and possibly by PKC. Raf-1 regulates extracellular signal-regulated kinase (ERK), a member of the MAPK family, through activation of MAP or ERK kinase (MEK). ERK is a positive regulator of c-fos induction. IL-3, GM-CSF, and IL-5 are known to stimulate this signaling cascade, i.e., to activate Ras (27, 109), Raf (14, 55), and ERK (87, 100, 134). These cytokines also activate downstream targets of MAPK, p90 S6 kinase [ribosomal S6 kinase (RSK)] (51), and MAPK-activated protein (MAPKAP) kinase 2 (1). The characteristic feature of this step is that the involved signaling molecules easily translocate to various cellular compartments on activation. For example, a significant pool of Raf-1 translocates to mitochondrial membranes, whereas ERK migrates to the cytoskeleton, nucleus, and possibly other compartments.

The Ras-ERK pathway is involved in the prevention of cell death in certain cell types. BA/F3 cells expressing the truncated mutant of beta c at position 544, which is unable to activate Ras, undergo apoptosis despite GM-CSF stimulation (57). The expression of activated Ras restores the capacity of the cells to survive. In agreement with this finding, expression of an activated mutant of Ras prevents BA/F3 cells from apoptosis caused by deprivation of IL-3, whereas the dominant negative mutant of Ras exhibits no inhibitory effect on IL-3-dependent cell proliferation (124). The results from these studies indicate that the signaling pathway for DNA synthesis appears to be different from the anti-apoptotic pathway. Several proteins of the Bcl-2 family have been identified that participate either as inducers or repressors of programmed cell death (104). The expression of Bcl-2 or Bcl-XL genes is likely to be involved in the inhibition of apoptosis following IL-3 stimulation (58, 62, 95). One possible 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 (131). Once there, Raf-1 phosphorylates BAD, a pro-apoptotic Bcl-2 family protein that abrogates the cytoprotective functions of Bcl-2 and Bcl-XL by heterodimerizing with them. Only the nonphosphorylated BAD is able to heterodimerize with Bcl-2/Bcl-XL to promote cell death (33). Two recently published papers have shown that BAD is phosphorylated on serine following IL-3 stimulation, indicating its activation and possible participation in the inhibition of apoptosis (22, 139).

The MAPK family. The MAPK family consists of a growing number of serine/threonine kinases that are characteristically activated by threonine and tyrosine phosphorylation. The members include ERK1, ERK2, ERK5, and ERK6, JNK/stress-activated protein kinase (SAPK) and various isoforms of p38 (p38, p38beta , p38beta -2, p38gamma , and p38delta ). JNK, activated by stimuli such as tumor necrosis factor, ultraviolet radiation, or protein synthesis inhibitors, binds to the c-jun transactivation domain and phosphorylates serine-63 and serine-73 (23). The p38 has an important role in the regulation of actin microfilament dynamics through the activation of MAPKAP kinase 2/3 and heat shock protein 27 (37). Recent studies have shown that major members of the MAPK family, i.e., ERK1/2, JNK/SAPK, and p38 are involved in beta c receptor signaling. We have already discussed the activation of ERK. In murine hematopoietic BA/F3-derived cell lines, a significant increase of JNK1 activity is observed after IL-3 or GM-CSF stimulation (125). Dominant-negative Ras fails to induce JNK1 activation by IL-3, and the constitutively active form of Ras causes no increase in JNK1 activity, suggesting that Ras is essential but not sufficient for IL-3-induced JNK1 activation. IL-5 also induces JNK activation, which seems to be responsible for 12-O-tetradecanoylphorbol 13-acetate response element- and serum responsive element (SRE)-dependent transcription (21). Nagata et al. (79, 80) have shown that IL-3 also activates p38 MAPK as well as JNK. In this system, IL-3 does not activate their upstream signaling molecules, SEK1 (SAPK/ERK kinase 1)/MKK4 (MAPK kinase 4), and MKK3/MKK6, respectively. Taken together, signaling pathway(s) other than the Ras-MAPK, e.g., the PI3K pathway (60) or the Ca2+ signaling pathway (115), may be involved in the regulation of JNK and p38.

The JAK-STAT pathway and its cross talk with the Ras-MAPK pathway. The JAK-STAT pathway is important for signaling of many cytokine receptors, including IL-3/GM-CSF/IL-5 receptors (47). The activated JAK kinases phosphorylate members of the STAT family of nuclear factors on tyrosine residues. Thus far, seven members of the STAT family have been identified: STAT1 (STAT1alpha , STAT1beta ), STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. The tyrosine-phosphorylated STATs form homo- or heterodimers, which cause their translocation to the nucleus and binding to the gamma -activating sequences (GAS) motif of the promoter region of various genes. IL-3, GM-CSF, and IL-5 mainly utilize STAT5 homologues, currently known as STAT5A and STAT5B, in myeloid cell lines (5, 75, 89). Although the importance of STAT5 activation in IL-3/IL-5/GM-CSF action is not fully clear, GM-CSF-stimulated cell proliferation and gene transcription are impaired in STAT5A knockout mice (31). Besides STAT5, these cytokines also activate STAT1 (11, 81), STAT3 (11, 13, 81), and STAT6 (99).

The Ras-MAPK and the JAK-STAT pathways converge at various levels. The c-fos promoter contains SRE and SIE (c-sis-inducible element) sites, and the former is regulated by MAPK, whereas the latter carries the GAS sequence, which is the DNA binding site for STAT (32, 69). Mui et al. (76) have shown that activated Ras alone can induce c-fos expression but that the maximal expression requires the action of both Ras and STAT5. Another converging point is the gene transcription by STAT proteins. STAT1 and STAT3 have been shown to be serine phosphorylated in response to cytokine stimulation (136, 140). For STAT1, the site of phosphorylation is serine-727, which is a consensus phosphorylation site for a proline-directed serine kinase such as MAPK and is likely to be important for transcriptional activation. Interestingly, MAPK appears to be associated with the alpha -subunit of IFN-alpha /beta receptor and modifies STAT proteins to activate early response genes (20). A study using a dominant negative MAPK has shown that STAT1 and STAT3 are regulated not only by tyrosine kinases but also partially by MAPK in GM-CSF signaling (101). However, it is controversial whether MAPK actually regulates serine phosphorylation of all STAT proteins. For example, STAT5 activation requires a distinct serine/threonine kinase other than MAPK in IL-2 signaling (8).

    IL-5 SIGNALING IN PRIMARY CELLS

IL-5 Signaling in Eosinophils

The results of studies with human eosinophils agree in many respects with the findings using cell lines. IL-5 activates Lyn (93, 138), Syk (138), JAK2 (6, 94, 129) tyrosine kinases, and PI3K (16), which transduce signals to downstream molecules. It has been suggested that these tyrosine kinases have a critical role in the survival of eosinophils (Table 1). Lyn knockout mice are eosinopenic and have reduced eosinopoiesis in bone marrow, further confirming the role of Lyn kinase in eosinophil growth and differentiation (42). In addition to tyrosine kinases, SHP-2 tyrosine phosphatase has an important function in IL-5 signaling in eosinophils. SHP-2 is physically associated with the beta c receptor and transduces signals to Grb2/Sos as a positive regulator. The SHP-2 antisense oligodeoxynucleotides inhibit eosinophil survival as well as tyrosine phosphorylation of MAPK (Table 1) (90). Shc is also activated by IL-5 (7). The activation and importance of other tyrosine kinases (Fyn, Fes, etc.) or other molecules (Vav, etc.) in IL-5 signaling of eosinophils are unclear at this time.

Transforming growth factor-beta (TGF-beta ) is a broadly active anti-inflammatory and immunomodulatory cytokine. It antagonizes the majority of the effect of cytokines on eosinophils (2). The molecular targets of TGF-beta action on eosinophils appear to involve Lyn, MAPK, JAK2, and STAT1 (91). TGF-beta inhibits IL-5-induced tyrosine phosphorylation of these proteins. The mechanism of this inhibition may be due to the interception of initial signaling molecules such as Lyn and JAK2. However, TGF-beta does not modulate the function of tyrosine phosphatases such as SHP-2.

IL-3, GM-CSF, and IL-5 promote growth and survival of eosinophils and prime their function such as degranulation, chemotaxis, and cytotoxicity. Coffer et al. (16) have shown that PI3K, but not Ras-MAPK, has a role in zymosan-stimulated superoxide production in IL-5-primed eosinophils. We have demonstrated that Lyn and JAK2 are important for eosinophil survival but not for degranulation and upregulation of CD11b (92). In contrast, Raf-1 appears to be critically involved in all of the above functions. Antisense inhibition of Raf-1 blocks IL-5-induced eosinophil survival, ECP release, and expression of CD11b. This paper tends to suggest a central role for Raf-1 kinase in controlling eosinophil function (Table 1).

IL-5 Signaling in B cells

The IL-5-induced B cell proliferation is defective in a Fyn knockout mouse model, indicating the involvement of Fyn in IL-5 signaling in mice (4). IL-5 activates JAK2 and Btk tyrosine kinases as well as PI3K, Shc, Vav, and HS1 in an IL-5-dependent murine B cell line, Y16 (108). The importance of Btk has been shown in the model of X-linked immunodeficient (XID) mice. The B cells from XID mice that carry the xid gene with a point mutation in the pleckstrin homology domain of Btk exhibit impaired responsiveness to murine IL-5 (103, 126). The XID B cells express fewer alpha - and beta -subunits of IL-5 receptor than normal B cells, resulting in impaired proliferation of and IgM production by B cells after IL-5 stimulation (44). In contrast, Btk is unlikely to be critical for eosinophil growth and function. The XID mice show IL-5-induced eosinophilia to the same degree as normal mice. Eosinophils from IL-5-treated XID mice respond to IL-5 with prolonged survival (44). Therefore, Btk plays a critical role in IL-5 stimulation of B cells, but not eosinophils.

    CONCLUDING REMARKS
Top
Abstract
Introduction
Concluding Remarks
References

In summary, IL-3/GM-CSF/IL-5 activate multiple tyrosine kinases and other signaling molecules, leading to the propagation of signals via the JAK-STAT and the Ras-MAPK pathways. Both pathways are important for cell proliferation in factor-dependent cell lines. Thus far, only signaling similarities but not differences among the three hematopoietins have been found. The signaling similarities may explain the functional homology among the cytokines. However, they do not explain why the three cytokines have distinct roles in growth and differentiation of eosinophils, neutrophils, and monocytes. Particularly, the critical role of IL-5 in eosinophil differentiation cannot be explained with the current knowledge of the signaling pathways. One possibility is that the ligand-specific alpha  receptor generates distinct signaling pathways that result in specific biological actions. From this standpoint, the elucidation of the signaling mechanism of IL-5Ralpha will be extremely useful.

Unlike cell lines, very few signal-function relationship studies have been performed with primary cells such as terminally differentiated eosinophils. However, signal transduction studies with primary cells are extremely important, because cell functions, such as chemotaxis and degranulation, cannot be investigated with cell lines. The few studies that have been performed with eosinophils have already produced interesting results. For example, despite their critical role in cell proliferation and eosinophil survival, Lyn and JAK2 do not appear to be important for eosinophil degranulation or regulation of adhesion molecules. In contrast, Raf-1 seems to play a central role in regulating most of the eosinophil functions, including survival, degranulation, and adhesion. The identification of critical signaling molecules that are responsible for important eosinophil functions will help develop specific inhibitors for therapeutic use in asthma and other allergic diseases.

    ACKNOWLEDGEMENTS

The work is supported by National Institute of Allergy and Infectious Diseases Grant AI-35713 and by the McLaughlin Fellowship Fund and the John Sealy Memorial Fund.

    FOOTNOTES

Address for reprint requests: R. Alam, The Univ. of Texas Medical Branch, Division of Allergy & Immunology, Dept. of Internal Medicine, 301 Univ. Blvd., Galveston, TX 77555-0762.

    REFERENCES
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Abstract
Introduction
Concluding Remarks
References

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