Stem Cell Factor‐Mediated Activation Pathways Promote

Stem cell factor-mediated activation pathways promote murine eosinophil CCL6 production and survival Vladislav Dolgachev,1 Molly Thomas, Aaron Berlin, and Nicholas W. Lukacs Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA Abstract: Eosinophil activation during allergic The role of SCF on eosinophil biology has been identified. diseases has a detrimental role in the generation of Although it has been established that SCF has a function in pathophysiologic responses. Stem cell factor (SCF) eosinophil maturation within the bone marrow, its role in has recently shown an inflammatory, gene-activat- peripheral tissue has not been elucidated thoroughly. Evidence ing role on eosinophils and contributes to the gen- has identified that SCF up-regulates VLA-4 on eosinophils and eration of pathophysiologic changes in the airways augments adherence events to fibronectin and VCAM-1 [12]. during allergic responses. The data in the present Previous data from our laboratory have demonstrated that SCF study outline the signal transduction events that are can increase eosinophil activation and degranulation signifi- induced by SCF in eosinophils and further demon- cantly. This includes the release of eosinophil peroxidase, strate that MEK-mediated signaling pathways are generation and release of leukotriene C4, and production of crucial for SCF-induced CCL6 chemokine activa- chemokines [13]. Blocking of c-kit signaling with anti-SCF tion and eosinophil survival. SCF-mediated eosin- antibodies or c-kit intrinsic tyrosine kinase inhibitor (imatinib) ophil activation was demonstrated to include reduces lung tissue inflammation, eosinophilia, and mucus PI-3K activation as well as MEK/MAPK phosphor- production significantly [14–16]. In addition, other studies ylation pathways. Subsequent analysis of CCL6 have identified that substantial levels of SCF can be found in gene activation and production induced by SCF in chronic diseases, including fibrotic diseases and asthma, where the presence or absence of rather specific inhibi- eosinophils have been implicated in the pathogenesis and tors for certain pathways demonstrated that the severity of the responses [17–20]. MEK/MAPK pathway but not the PI-3K pathway The signal transduction events involved in SCF-induced was crucial for the SCF-induced CCL6 gene acti- c-kit activation have been investigated in the context of pro- vation. These same signaling pathways were shown genitor cell and mast cell maturation/activation pathways [21]. to initiate antiapoptotic events and promote eosin- c-Kit is a receptor tyrosine kinase, which constitutes a Type III ophil survival, including up-regulation of BCL2 subfamily that includes platelet-derived growth factor, CSF-1, and BCL3. Altogether, SCF appears to be a potent and fetal liver tyrosine kinase 3 ligand. Upon SCF stimulation, eosinophil activation and survival factor. J. Leu- c-kit dimerizes and activates its intrinsic tyrosine kinase and koc. Biol. 81: 1111–1119; 2007. autophosphorylates. This allows the initiation of specific signal transduction pathways [22, 23]. Downstream of c-kit, multiple Key Words: SCF ⅐ cell signaling signal transduction components are activated, including PI-3K, Src family members, the JAK/STAT pathway, and the Ras-Raf- MAPK cascade [22]. Activation of the PI-3K pathway involves INTRODUCTION association of the Src homology 2 (SH2) domain of the regu- latory subunit with phosphorylated receptors for the recruit- Activated eosinophils in allergic disease contribute to the ment of the p85 subunit of PI-3K. Downstream targets for progression of inflammation by producing cytokines and lipid 3Ј-phospholipids generated by PI-3K include serine/threonine mediators leading to tissue damage by release of toxic proteins kinases Akt and protein kinase C. PI-3K has been implicated and reactive oxygen species [1, 2]. Evidence suggests that in many aspects of cellular signaling, including DNA synthe- eosinophils are associated with development of lung dysfunc- sis, cell survival, membrane ruffling, chemotaxis, as well as tion and the consequent immunopathology in allergic diseases receptor and vascular trafficking [22, 24, 25]. [3–6]. The ability to promote eosinophil activation for release Another aspect of SCF-induced signaling is the activation of of inflammatory mediators has been a focus for a number of the Ras-Raf-MAPK cascade. This pathway is induced by phos- years and has helped to define how eosinophils might participate in chronic inflammatory diseases. Although a number of factors have been demonstrated to promote eosinophil survival, including IL-3, IL-5, and GM-CSF, there are likely other 1 Correspondence: University of Michigan Medical School, Department of mediators that also participate in long-term survival in tissues Pathology, 109 Zina Pitcher Place, Room 4618, Ann Arbor, MI 48109-2200, altered by chronic disease [7–10]. In particular, studies have USA. E-mail: [email protected] Received September 29, 2006; revised November 29, 2006; accepted De- suggested recently that stem cell factors (SCF) may play a role cember 18, 2006. in eosinophil activation during chronic disease [11]. doi: 10.1189/jlb.0906595 0741-5400/07/0081-1111 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 81, April 2007 1111 phorylated tyrosine residues on the SCF-stimulated c-kit re- phospho-Akt (Ser473), phospho-MEK1/2 (Ser217/221), phospho-p44/42 ceptor, which recruits SH2-containing proteins to the receptor MAPK (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182; 1:1000), and complex that couples the c-kit-associated receptor tyrosine corresponding HRP-conjugated secondary IgG antibodies (1:2000). All the antibodies were from Cell Signaling (Beverly, MA, USA). Proteins were visu- kinase to Ras [22]. The SH2-containing proteins include Grb2, alized by chemiluminiscense (Super Signal West Pico chemiluminescent sub- Shc, SH2-containing tyrosine phosphatase 2, and Grap. Sub- strate, Pierce, Rockford, IL, USA) using a Kodak photo imager. ␤-Actin was sequently, Grb2-Sos colocalizes with SH2-containing proteins detected as a loading control. and increases in Ras activity. The next activation step is when Quantitative PCR analysis of CCL6 Ras interacts with c-Raf-1, which activates MEK, a MAPK [22, 23, 26, 27]. MEK phosphorylates p38 MAPK and ERK1/2 and RNA was purified from isolated eosinophils using TRIzol reagent (Gibco, leads to activation of the downstream component S6 kinase Grand Island, NY, USA) and chloroform. RNA was quantified by measuring ␮ pp90rsk. The MAPK activation pathways have been shown to absorbance at 260 nm. Samples were then standardized to 5 g/ml with diethylpyrocarbonate water. RNA was then reverse-transcribed to cDNA, and induce a number of inflammatory and immune mediators via 1 ␮l of this cDNA was used in the TaqMan reaction mixture as described NF-␬B activation [28]. None of these pathways has been ex- previously [15]. The specific primer/probe sets for real-time PCR were prede- amined in eosinophils, but as our data demonstrate, they are veloped by Applied Biosystems (Foster City, CA, USA). differentially involved in eosinophil activation and survival. Apoptosis/survival assays Apoptotic cells were characterized by the inability to exclude trypan blue MATERIALS AND METHODS stain, loss of mitochondrial membrane potential, DNA degradation, and caspase-3 activation. Animals Viability assays Dr. Fred Lewis at the Biomedical Research Laboratory (Rockville, MD, USA) Eosinophils were cultured for 24 h with different treatments. Then, cells were supplied Swiss Webster mice heavily infected with the Schistosoma mansoni resuspended carefully and incubated with trypan blue for 2 min at room helminth parasite. These mice displayed severe infection and significant temperature. Then, cell samples were counted using a hemocytometer. Dead eosinophilia, and Ͼ50% of circulating granulocytes were eosinophils. They cells were determined as trypan blue-positive. were used throughout our studies for elicitation and isolation of eosinophils. Mitochondrial membrane potential Antigen-elicited peritoneal eosinophil purification Following treatments, eosinophil cells were stained with JC-1 (10 ␮g/ml) at 37°C for 20 min. Following harvesting and washing with PBS, cells were Eosinophils were elicited by injection of thioglycolate plus soluble egg antigen resuspended in prewarmed (37°C) complete serum medium and analyzed. JC-1 (SEA) into the peritoneum of S. mansoni-infected mice. SEA was prepared in aggregates were detectable in the fluorescence 2 (FL2) channel (red fluores- our laboratory by grinding isolated eggs from heavily infected S. mansoni mice cence, emission at 590 nm), and JC-1 monomers were detectable in the FL1 as described previously [29]. The injection of SEA into infected mice induces channel (green fluorescence, emission at 527 nm) [32]. The incorporation of a pool of circulating eosinophils recruited into the peritoneum in an antigen- JC-1 can be affected by factors other than mitochondria (e.g., cell size, plasma specific manner. Using these mice, we could elicit eosinophils that were from membrane permeability) [33]. To ensure that staining patterns were similar, an antigen-induced, Th2-associated environment. After 48 h, the mice were cells (untreated and treated) were exposed to the Kϩ ionophore valinomycin peritoneal-lavaged, and the cells were collected. The initial population, which (100 nM) [34] for 15 min at room temperature and then analyzed again. was isolated from the peritoneum, was ϳ50% eosinophils,

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