Akt cross-links IL-4 priming, stem cell factor signaling, and IgE-dependent activation in mature human mast cells

Challenge of human mast cells with both stem cell factor (SCF) and IL-4 enhances antigen-dependent mediator release raising the assumption of intracellular crosstalk between the IL-4, SCF, and Fc?RI signaling pathways. Here, we analyzed the intracellular crosstalk of IL-4-, SCF-, and IgE-dependent activation pathways in mucosal mast cells isolated from human intestine. The release of β-hexosaminidase, leukotriene C₄, and IL-8, but not IL-6, was strongly enhanced in response to sequential challenge of mast cells with IL-4, SCF and Fc?RI cross-linking compared to stimulation by Fc?RI cross-linking alone. Previous studies revealed that MAPK and other serine/threonine kinases are involved in mast cell activation processes. Here we found that activation of mast cells by Fc?RI cross-linking alone results in phosphorylation of ERK and p38, but not of Akt. Stimulation with SCF alone also induced phosphorylation of ERK and p38, and additionally of Akt. IL-4 priming enhanced activation of ERK, but blocked activation of p38. Activation of p38 was required for IL-6 production explaining the inhibitory effect of IL-4 on IL-6 expression in human mast cells. Moreover, IL-4 priming that anteceded Fc?RI cross-linking induced activation of Akt. The combined challenge of mast cells with IL-4, SCF and Fc?RI cross-linking substantially up-regulated activation of Akt, whereas blocking of Akt inhibited the pronounced production and release of IL-8 in response to the three mast cell agonists. In summary, our data demonstrate that ERK, p38, and especially Akt play an important role in cross-linking IL-4 priming, SCF signaling, and IgE-dependent activation of mature human mast cells.     

The tyrosine kinase network regulating mast cell activation

Summary:  Mast cell mediator release represents a pivotal event in the initiation of inflammatory reactions associated with allergic disorders. These responses follow antigen-mediated aggregation of immunoglobulin E (IgE)-occupied high-affinity receptors for IgE (FcεRI) on the mast cell surface, a response which can be further enhanced following stem cell factor-induced ligation of the mast cell growth factor receptor KIT (CD117). Activation of tyrosine kinases is central to the ability of both FcεRI and KIT to transmit downstream signaling events required for the regulation of mast cell activation. Whereas KIT possesses inherent tyrosine kinase activity, FcεRI requires the recruitment of Src family tyrosine kinases and Syk to control the early receptor-proximal signaling events. The signaling pathways propagated by these tyrosine kinases can be further upregulated by the Tec kinase Bruton's tyrosine kinase and downregulated by the actions of the tyrosine Src homology 2 domain-containing phosphatase 1 (SHP-1) and SHP-2. In this review, we discuss the regulation and role of specific members of this tyrosine kinase network in KIT and FcεRI-mediated mast cell activation.     

Human mast cells express stem cell factor

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT-PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase-only (MC_T) and tryptase/chymase (MC_TC) subsets. SCF protein product was found in conjunctival MC_T and MC_TC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross-linkage of IgE receptor (Fcε–RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor-mediated pathways. © 1998 John Wiley & Sons, Ltd.     

Role of phosphoinositide 3-kinase signaling in mast cells: new insights from knockout mouse studies

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases essential for diverse physiological reactions. In recent years a series of gene-targeted mice lacking different types of PI3Ks and related molecules have been generated which enable us to understand the role of PI3K pathways, particularly class I members, in vivo. Analyses of such gene-targeted mice have led to major discoveries in the physiological roles of PI3K signaling in mast cell biology. In particular the role of PI3Ks has been extensively studied in signaling through the high-affinity IgE receptor (FcRI), since mast cells are the main effector cells in type I allergic reaction associated with IgE-dependent mechanisms. Furthermore, the knockout mice have provided significant information concerning the role of PI3K signals in mast cell differentiation. This review presents several new insights into mast cell biology, which have been elucidated by the analyses of these knockout mice.Abbreviations BH Bcr homology - FcRI Fc receptor for IgE - Gab2 Grb2-associated binder 2 - GPCR G-protein coupled receptor - IL Interleukin - ITAM Immunoreceptor tyrosine-based activation motif - JNK Jun N-terminal kinase - LAT Linker for activation of T cells - PI3K Phosphoinositide 3-kinase - PKC Protein kinase C - PtdIns Phosphatidylinositol - SCF Stem cell factor - SH2 Src homology 2 - Sl Steel locus - Th2 Type 2 helper T - W White spotting locus     

STI571 inhibits growth and adhesion of human mast cells in culture

Stem cell factor (SCF)/c-kit system is critical for human mast cell development. We thus examined the effects of STI571, an inhibitor of the c-kit tyrosine kinase receptor, on the proliferation and function of human mast cells. STI571 at concentrations of 10(-6) M or higher almost completely abolished the SCF-dependent progeny generation from cord blood-derived cultured mast cells through an inhibition of the tyrosine phosphorylation of c-kit. The compound also suppressed the early phase of mast cell development. The extinction of mast cell growth induced by STI571 may be due largely to apoptosis according to the flow cytometric analysis and gel electrophoresis. Two-hour exposure to STI571 that failed to influence the total viable cell number suppressed adhesion of the cells to fibronectin in the presence of SCF without altering the expressions of integrin molecules. Our results may provide a fundamental insight for the clinical application of STI571 in allergic disorders.     

Role of phospholipase A2 and G-proteins in the IgE-dependent activation of mast cells and macrophages

The effect of para-bromophenacyl bromide (a selective inhibitor of phospholipase A₂) and pertussis toxin has been investigated on IgE-dependent histamine release and on IgE-dependent macrophage-mediated cytotoxicity. Para-bromophenacyl bromide, inhibited dose-dependently IgE-dependent stimulation of mast cells and macrophages (IC₅₀'s of 5.0×10^–7 M and 2.5×10^–7 M, respectively). In contrast, pertussis toxin only inhibited the IgE-dependent stimulation of macrophages, whereas the IgE-dependent activation of mast cells was not affected. These results suggest that the transducing mechanisms following the activation of the high affinity receptor for IgE (FcRI on mast cells) as well as the low affinity receptor for IgE (FcRII on macrophages) induce the activation of phospholipase A₂. FcRII might be coupled to a pertussis toxin sensitive G-protein.     

IL‐6, but not IL‐4, stimulates chemokinesis and TNF stimulates chemotaxis of tissue mast cells: involvement of both mitogen‐activated protein kinases and phosphatidylinositol 3‐kinase signalling pathways

Misiak‐T?oczek A, Brzezińska‐B?aszczyk E. IL‐6, but not IL‐4, stimulates chemokinesis and TNF stimulates chemotaxis of tissue mast cells: Involvement of both mitogen‐activated protein kinases and phosphatidylinositol 3‐kinase signalling pathways. APMIS 2009; 117: 558–67. An increase in the number of mast cells within tissues is observed in many pathophysiological conditions. Current data indicate that migration of mature mast cells might be one of the key mechanisms responsible for rapid local accumulation of these cells. Considering that interleukin (IL)‐6 and IL‐4, as well as tumour necrosis factor (TNF), influence mast cell activity in various ways, the purpose of the current study was to examine whether these cytokines function as rat peritoneal mast cell chemoattractants. We showed that IL‐4, in the concentration range from 10^?6 to 10^?3 ng/ml, did not induce a mast cell migratory response, even in the presence of laminin and fibronectin. Under the same experimental conditions, mast cells were shown to migrate in response to IL‐6 stimulation in the presence of laminin. The optimal concentration of IL‐6 for maximal migration of mast cells was 10^?4 ng/ml (i.e. ～5 nM). In comparison, the optimal concentration of TNF for maximal migration of mast cells was 5 × 10^?5 ng/ml (i.e. ～3 fM). IL‐6‐stimulated mast cell migration was the result of chemokinesis, whereas TNF‐induced migration was the result of chemotaxis. Mast cell migratory responses to IL‐6 and TNF were entirely blocked by specific anti‐IL‐6R and anti‐TNFR1 antibodies. We also documented that the migration response of mast cells to stimulation with IL‐6 and TNF was mediated through signal transduction pathways involving mitogen‐activated protein kinases and phosphatidylinositol 3‐kinase. Taken together, our results indicate that IL‐6, as well as TNF, induces tissue mast cell migration. Thus, these proinflammatory cytokines can be responsible for mast cell accumulation at the site of diverse conditions accompanied by inflammation.     

Development, migration, and survival of mast cells

Mast cells play a pivotal role in immediate hypersensitivity and chronic allergic reactions that can contribute to asthma, atopic dermatitis, and other allergic diseases. Because mast cell numbers are increased at sites of inflammation in allergic diseases, pharmacologic intervention into the proliferation, migration, and survival (or apoptosis) of mast cells could be a promising strategy for the management of allergic diseases. Mast cells differentiate from multipotent hematopoietic progenitors in the bone marrow. Stem cell factor (SCF) is a major chemotactic factor for mast cells and their progenitors. SCF also elicits cell-cell and cell-substratum adhesion, facilitates the proliferation, and sustains the survival, differentiation, and maturation, of mast cells. Therefore, many aspects of mast cell biology can be understood as interactions of mast cells and their precursors with SCF and factors that modulate their responses to SCF and its signaling pathways. Numerous factors known to have such a capacity include cytokines that are secreted from activated T cells and other immune cells including mast cells themselves. Recent studies also demonstrated that monomeric IgE binding to FcωRI can enhance mast-cell survival. In this review we discuss the factors that regulate mast cell development, migration, and survival.     

A glycolipid-specific monoclonal antibody modulates Fc receptor stimulation of mast cells

In an effort to identify membrane components participating in coupling stimulus to secretion in mast cells, monoclonal antibodies were produced from spleen cells of mice immunized with plasma membranes isolated from rat mast cells of the RBL-2H3 line. The resultant mAbs were screened by their capacity to modulate the secretory response of these cells to crosslinking of their type 1 Fc receptor (FcRI). Following this scheme, we obtained a hybridoma designated B17, which secretes an IgM-class mAb (B17) that binds to and modulates secretion from RBL-2H3 cells. By immunoblotting, B17 was shown to bind to a membrane component of low molecular weight, later identified as a glycolipid. While B17 partially inhibits IgE binding to RBL-2H3 cells, no noticeable inhibition of B17 binding by IgE was observed. mAb B17 does not cause any secretory response on its own, and its modulatory effect on FcRI-mediated secretion is bimodal: it either enhances or inhibits secretion, depending on the B17 dose and also on the nature and dose of the agent used for crosslinking the FcRI. When secretion was induced by IgE and suboptimal or optimal doses of multivalent antigen, B17 (2–80 nM) caused an increase in secretion. However, higher doses of B17 (>150nM) inhibited secretion. Secretion induced by supraoptimal doses of antigen, or by the FcRI-specific mAb F4 was inhibited by B17 at all the dose range tested (2–200 nM). In contrast, B17 had no effect on secretion induced by Ca²⁺ ionophores. These results demonstrate that FcRI function is modulated by a mAb binding to a membrane glycolipid.     

The c-kit ligand, stem cell factor, promotes mast cell survival by suppressing apoptosis.

Stem cell factor (SCF) and its receptor (SCFR), a member of the receptor tyrosine kinase III family that is encoded by the c-kit gene, critically regulate several complex biological programs including hematopoiesis, mast cell development, cutaneous pigmentation, and gametogenesis. We show herein that mouse mast cells die rapidly after the withdrawal of SCF in vivo or in vitro, and provide morphological evidence that such mast cells undergo programmed cell death or apoptosis. We also show that when in vitro-derived mouse mast cells maintained in SCF are removed from SCF-containing medium for only 5 or 6 hours, the cells' genomic DNA exhibits the ladder-like pattern of oligonucleosome-sized fragments typical of apoptosis. These findings demonstrate that SCF can regulate the survival of a cellular lineage which expresses the SCFR by suppressing apoptosis. They also identify a mechanism that can result in striking and rapid reductions in the size of tissue mast cell populations without histological evidence of the concomitant induction of a significant inflammatory response.     

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

Fc epsilon RI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that Fc epsilon RI also uses a Fyn kinase-dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, Fc epsilon RI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.     

The protein tyrosine kinase Tec regulates mast cell function

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon FcεRI stimulation of BM‐derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C₄ levels were reduced in Tec^?/? BMMC. Furthermore, the production of IL‐4 was severely impaired, and GM‐CSF, TNF‐α and IL‐13 levels were also diminished. Finally, a comparison of WT, Tec^?/?, Btk^?/? and Tec^?/?Btk^?/? BMMC revealed a negative role for Btk in the regulation of IL‐4 production, while for the efficient production of TNF‐α, IL‐13 and GM‐CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well‐characterized family member Btk.     

Downstream signals initiated in mast cells by FcεRI and other receptors

The significant contributions this past year to our understanding of IgE receptor (FcεRI) signaling in mast cells include studies with truncated Syk in a vaccinia expression system and Syk-negative variants of rat basophilic (RBL-2H3) cells. These studies demonstrate an essential role for Syk in initiating signals for secretion and release of arachidonic acid via phospholipase A₂ and mitogen-activated protein kinase. A newly recognized addition to the repertoire of FcεRI-mediated signaling systems is the activation of sphingosine kinase, which contributes to calcium mobilization in mast cells. Advances have been made in our understanding of other receptors that regulate proliferation and differentiation of mast cells, and in our understanding of the ability of mast cells to mount acquired and acute responses to antigenic and bacterial challenge.     

Human Mast Cell Apoptosis Is Regulated Through Bcl-2 and Bcl-XL

It is well established that human mast cell proliferation and maturation are regulated by kit ligand (stem cell factor). Little is known, however, about how these two processes are negatively regulated and thus, how mast cell number is controlled in normal and pathologic conditions. We therefore first hypothesized that SCF-dependent human mast cells would undergo programmed cell death (apoptosis) on removal of SCF as has been shown for growth factor-dependent rodent mast cells. We then examined whether SCF acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. As hypothesized, elimination of SCF from primary peripheral blood-derived human mast cell cultures resulted in a significant apoptotic process. During apoptosis, down-regulation of the two apoptosis-regulatory proteins Bcl-2 and Bcl-X_Lwas observed. Moreover, a deregulated expression of these two proteins was found in two human mast cell lines which are SCF-independent. Thus, SCF functions as a survival factor by repressing apoptosis of human mast cells through Bcl-2 and Bcl-X_L. Deregulated expression of these antiapoptotic proteins may contribute to proliferation and accumulation of mast cells in certain forms of systemic mast cell disorders.     

Targeted elimination of cells expressing the high-affinity receptor for IgE (FcϵRI) by a Pseudomonas exotoxin-based chimeric protein

The interaction between IgE and its high-affinity receptor Fc?RI found on mast cells and basophils is the primary effector pathway in allergic response. To achieve a targeted elimination of cells expressing Fc?RI receptors, we constructed a chimeric protein in which a Fc fragment of mouse IgE is attached to a truncated form of Pseudomonas exotoxin (PE). To prepare the targeting moiety, we used a DNA sequence corresponding to amino acids 301–437, representing 30 residues of domain 2 and domain 3 of the mouse IgE constant region. This sequence was fused at the 5′ of a cDNA encoding PE₄₀, a truncated form of PE lacking the cell binding domain. The chimeric protein, termed Fc_2′?3?PE₄₀, was expressed in Escherichia coli and partially purified. The protein is highly cytotoxic to mouse mast cell lines and bone marrow-derived primary mast cells. This cytotoxicity is specific, as it could be blocked upon addition of whole IgE. Moreover, the protein had no effect on other cell lines of hemopoietic origin. The Fc_2′?3?PE₄₀ chimeric protein offers a new approach to the treatment of allergic disorders.     

Murine mast cells attach to and migrate on laminin-, fibronectin-, and matrigel-coated surfaces in response to FcεRI-mediated signals

We have examined the possibility that mouse bone marrow-derived cultured mast cells (BMCMC) have the capacity to attach to and migrate on extracellular matrix components in vitro through the use of time lapse videography. Unactivated mast cells did not display significant interaction with slide flasks coated with either 3% BSA or collagen IV, and Fc_εRI-mediated activation of BMCMC did not appreciably increase their attachment and migratory characteristics. Both activated and unactivaled BMCMC adhered to surfaces coated with a synthetic IKVAV laminin polypeptide, but this association resulted in the immobilization of the cells to the substrate. BMCMC did not adhere to surfaces coated with laminin, fibronectin or matrigel until Fc_ε RI-mediated activation, after which they displayed rapid, random movement on these surfaces. Cells continually interacted with laminin. fibronectin or matrigcl by flattening, interspaced by periods of movement as rounded cells with small pseudopodia. The mean velocity of BMCMC on laminin, fibronectin or matrigel was similar and averaged approximately 180 μm/hr. The mean velocity of BMCMC on these three substrates was not significantly different from the mean velocity of monocytes on laminin. The movement of BMCMC on these substrates demonstrated a directional tendency. In summary. these results demonstrate that mast cells activated through Fc_εRI are capable of attachment to and motion on components of extracellular matrix, and demonstrate one mechanism by which mast cells may migrate to areas of inflammation and wound repair.     

Downstream signals initiated in mast cells by FcRI and other receptors

The significant contributions this past year to our understanding of IgE receptor (FcRI) signaling in mast cells include studies with truncated Syk in a vaccinia expression system and Syk-negative variants of rat basophilic (RBL-2H3) cells. These studies demonstrate an essential role for Syk in initiating signals for secretion and release of arachidonic acid via phospholipase A₂ and mitogen-activated protein kinase. A newly recognized addition to the repertoire of FcRI-mediated signaling systems is the activation of sphingosine kinase, which contributes to calcium mobilization in mast cells. Advances have been made in our understanding of other receptors that regulate proliferation and differentiation of mast cells, and in our understanding of the ability of mast cells to mount acquired and acute responses to antigenic and bacterial challenge.     

Distinct patterns of early response gene expression and proliferation in mouse mast cells stimulated by stem cell factor,interleukin-3, or IgE and antigen

Stem cell factor (SCF) is encoded at the Sl locus of the mouse and is the ligand for the c-kit receptor. Recombinant rat SCF¹⁶⁴ (rrSCF¹⁶⁴) induces proliferation and promotes maturation of mouse mast cells in vitro and in vivo and can also induce c-kit receptor-dependent mouse mast cell degranulation. We now report that in both quiescent and non-quiescent mouse bone marrow-derived cultured mast cells (BMCMC) rrSCF¹⁶⁴ induces increased mRNA levels for the “early response genes” c-fos, c-jun and junB but has only slight effects on the expression of junD. Recombinant mouse interleukin-3 (IL-3) also promotes proliferation of both quiescent and non-quiescent BMCMC. However, IL-3 induces increased expression of c-fos and juriB only in quiescent BMCMC. Cross-linking of Fee receptor type I (Fc?RI) on BMCMC by IgE and specific antigen induces a pattern of early gene expression very similar to that induced by rrSCF¹⁶⁴. However, BMCMC stimulated through the Fc?RI did not proliferate and, in comparison to control BMCMC, exhibited significantly decreased proliferation in response to rrSCF¹⁶⁴ or IL-3. These results indicate that stimulation of BMCMC proliferation by IL-3 or rrSCF¹⁶⁴ induces distinct patterns of early response gene expression and suggest that the proliferative effects of these growth factors may be mediated through distinct signal transduction pathways. Our data also point to previously unappreciated similarities between the effects of signaling through the c-kit receptor or the Fc?RI on mast cell expression of fos and jun genes.