Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Jan 25.
Published in final edited form as: Allergy. 2015 Apr 16;70(7):805–812. doi: 10.1111/all.12624

Notch signaling mediates granulocyte-macrophage colony-stimulating factor priming-induced transendothelial migration of human eosinophils

LY Liu 1,*, H Wang 1,*, J J Xenakis 1, L A Spencer 1
PMCID: PMC9875669  NIHMSID: NIHMS1863812  PMID: 25846339

Abstract

Background:

Priming with cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances eosinophil migration and exacerbates the excessive accumulation of eosinophils within the bronchial mucosa of asthmatics. However, mechanisms that drive GM-CSF priming are incompletely understood. Notch signaling is an evolutionarily conserved pathway that regulates cellular processes, including migration, by integrating exogenous and cell-intrinsic cues. This study investigates the hypothesis that the priming-induced enhanced migration of human eosinophils requires the Notch signaling pathway.

Methods:

Using pan Notch inhibitors and newly developed human antibodies that specifically neutralize Notch receptor 1 activation, we investigated a role for Notch signaling in GM-CSF-primed transmigration of human blood eosinophils in vitro and in the airway accumulation of mouse eosinophils in vivo.

Results:

Notch receptor 1 was constitutively active in freshly isolated human blood eosinophils, and inhibition of Notch signaling or specific blockade of Notch receptor 1 activation during GM-CSF priming impaired priming-enhanced eosinophil transendothelial migration in vitro. Inclusion of Notch signaling inhibitors during priming was associated with diminished ERK phosphorylation, and ERK-MAPK activation was required for GM-CSF priming-induced transmigration. In vivo in mice, eosinophil accumulation within allergic airways was impaired following systemic treatment with Notch inhibitor, or adoptive transfer of eosinophils treated ex vivo with Notch inhibitor.

Conclusions:

These data identify Notch signaling as an intrinsic pathway central to GM-CSF priming-induced eosinophil tissue migration.

Keywords: asthma, cell migration, cytokine priming, γ-secretase, Notch pathway

A characteristic attribute of some phenotypes of asthma is eosinophil accumulation within the bronchial mucosa, associated with mucus production and tissue remodeling in acute and chronic disease, respectively. Current treatments for tissue eosinophilia are limited to glucocorticosteroids. Therefore, delineating cell-intrinsic pathways that control eosinophil migration into inflamed tissues is a central goal in developing targeted therapeutics.

Concentrations of IL-5 family cytokines (i.e., GM-CSF, IL-5, and IL-3) are increased in asthmatic airways and serum, and prime eosinophils by enhancing their basal migratory capacity (1). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is central to the recruitment in vitro and in vivo of human eosinophils (2-6), a phenomenon recapitulated in mouse models (7-10), and the in vivo primed phenotype of eosinophils from asthmatics can be induced in eosinophils from healthy donors in vitro by incubation with GM-CSF (2, 3). However, molecular mechanisms that drive GM-CSF priming effects on eosinophils are incompletely defined.

Notch signaling is an evolutionarily conserved pathway that regulates cell fate decisions throughout organogenesis and hematopoiesis (11), and aberrant Notch signaling is implicated in tumor pathophysiology (12-14). In the canonical pathway, ligand binding to any of the four mammalian Notch receptors (Notch 1–4) induces sequential α- and γ-secretase-dependent cleavages that release a fragment of the receptor intracellular domain (NICD) within the cytoplasm. Freed NICD undergoes nuclear translocation and turns on transcription of a number of Notch-responsive genes (11). Inhibition of Notch signaling is most commonly achieved, both in experimental studies and in human therapeutic approaches (i.e., in cancer clinical trials (15, 16)), by blockade of the final cleavage using γ-secretase inhibitors (GSIs), simultaneously inhibiting all four Notch receptors, and producing the pharmacological equivalent of a loss of Notch function.

We previously demonstrated that mature human blood eosinophils express Notch receptors and ligands (17). Here, we build on these studies to investigate a requirement for Notch signaling in GM-CSF priming-induced transendothelial migration of human eosinophils in vitro and the bronchoalveolar accumulation of mouse eosinophils in allergic airway inflammation in vivo.

Materials and methods

Human eosinophils

Blood eosinophils were isolated from mild allergic or normal donors by negative selection as described (17). Eosinophil purity (as determined by cytospin analysis of ≥350 cells from random fields) was >98% and viability >98%. Informed written consent was obtained in accordance with the Declaration of Helsinki, and Institutional Review Board approval was obtained from Beth Israel Deaconess Medical Center.

Transmigration

Human eosinophils were primed for 18 h in complete culture medium alone or supplemented with 10 pM recombinant GM-CSF in the presence of pan Notch signaling inhibitors or Notch receptor 1 neutralizing Abs as described in Data S1. Primed eosinophils were overlaid onto confluent monolayers of resting or preactivated human umbilical vein endothelial cells (HUVECs) grown on transwell inserts. Transmigrated eosinophils collected from lower wells are expressed as follows: (number of migrated eosinophils/total input eosinophils) × 100.

Western blotting

Freshly isolated human eosinophils were lysed and probed with Abs against Notch 1 intracellular domains or activated Notch 1 intracellular domains as detailed in Data S1.

Flow cytometry

As detailed in Data S1, cleaved Notch 1 intracellular domains were detected in saponin-permeabilized human eosinophils. Surface-expressed Notch receptors were detected on nonpermeabilized mouse eosinophils purified from spleens of IL-5 transgenic BALB/c mice as described (18), using Abs against extracellular regions of Notch receptor 1 or Notch receptor 2, or appropriate isotype controls.

ERK phosphorylation

Human eosinophils were incubated with complete cell culture medium alone or containing 100 pM GM-CSF, with or without GSI II, clone A6, or appropriate vehicle or isotype Ab control. At the indicated time points, cell lysates or intact cells were assayed for levels of phosphorylated ERK by multiplex analysis or flow cytometry, respectively, as described in Data S1.

In vivo model of eosinophil recruitment

Wild-type female BALB/c mice (6–8 weeks old) were sensitized intraperitoneally (i.p.) with OVA mixed with alum adjuvant and challenged with aerosolized OVA, as detailed in Data S1. Prior to each aerosolized OVA challenge, mice were treated i.p. with either GSI II or vehicle control. Twenty-four hours after the final airway challenge, BAL fluid was obtained from euthanized mice and assessed for cellular composition, as described in Data S1.

Eosinophil adoptive transfer

Mouse eosinophils were purified from spleens of IL-5 transgenic BALB/c mice as described (18) and treated with GSI II or vehicle control. Treated and control cells were labeled with Dye eFluor 670 or CFSE, respectively. Equal mixtures of labeled GSI or vehicle control-treated eosinophils were injected i.v. into OVA-sensitized and OVA-challenged eosinophil-deficient ΔdblGATA mice immediately before the final OVA aerosol challenge, as detailed in Data S1.

Statistical analysis

Statistical analyses were performed and graphs created with GraphPad Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA). One-way anova with Bonferroni post hoc test was used for selected pairs comparison unless otherwise stated. A P-value of <0.05 was considered significant.

Results

γ-secretase activity during GM-CSF priming is required for the priming-induced transendothelial migration of human eosinophils

Our initial strategy to silence Notch signaling was to utilize GSIs. Human blood eosinophils were isolated and primed ex vivo with GM-CSF in the presence or absence of GSI II. Following overnight priming, eosinophils were washed and overlaid onto the apical surface of confluent HUVEC monolayers. Granulocyte-macrophage colony-stimulating factor priming enhanced eosinophil transmigration, and inclusion of GSI during priming reversed the priming effect on eosinophil migration (Fig. 1A).

Figure 1.

Figure 1

Open in a new tab

γ-secretase activity is required during granulocyte-macrophage colony-stimulating factor (GM-CSF) priming for enhanced transendothelial migration of human eosinophils. Eosinophils were primed with or without 10 pM GM-CSF in the presence of GSI II, DAPT, or DMSO vehicle control before being overlaid onto nonprimed (A), IL-4/TNF-α-(B, D), or IL-1β-(C) activated human umbilical vein endothelial cell (HUVEC) monolayers. Data are expressed as the combined mean ± SEM of N = 7 independent experiments (A–C) or N = 5 independent experiments (D), using different eosinophil donors (*P < 0.05; **P < 0.01). EC, endothelial cells; GSI, γ-secretase inhibitor.

Activated endothelium at the site of inflammation upregulates adhesion molecules that promote leukocyte tethering, adhesion, and diapedesis via interactions with leukocyte-expressed integrins. To determine whether Notch-signaling-dependent control of GM-CSF-primed eosinophil transmigration would be overcome by an activated endothelium, eosinophils primed with GM-CSF in the presence or absence of GSI were washed, and layered over endothelial monolayers preactivated with either IL-4 and TNF-α, or IL-1β. Endothelial cell activation by itself induced eosinophil transmigration above baseline, and the GM-CSF priming effect on eosinophil transendothelial migration remained susceptible to treatment with GSI II (Fig. 1B,C), or another GSI, DAPT (Fig. 1D), in the presence of an activated endothelium. Viability of eosinophils after overnight GM-CSF priming in the presence of vehicle control or GSIs, as measured by exclusion of propidium iodide, was as follows: vehicle = 95 ± 1%; GSI II = 95 ± 2%; and DAPT = 95 ± 1%, suggesting the impaired transmigration of eosinophils primed in the presence of GSI II or DAPT was not caused by differences in eosinophil viability. These data directly implicate γ-secretase activity in GM-CSF priming-induced eosinophil migration.

Notch receptor 1 is constitutively active in isolated human blood eosinophils and mediates GM-CSF priming-induced eosinophil transendothelial migration

Although GSIs are the conventional inhibitors of Notch currently utilized in experimental and clinical settings, approximately 50 cellular substrates exist for γ-secretase in addition to Notch receptors. We previously demonstrated that human blood eosinophils express Notch receptors 1 and 2 (17). Using a combination of antibodies recognizing intracellular epitopes of intact and/or activated Notch 1 (experimental design shown in Fig. 2A), both full-length receptors and cleaved intracellular domains of activated Notch receptor 1 (NICDs) were detected within freshly isolated blood eosinophils, suggestive of constitutive activation of Notch 1 (Fig. 2B,C). Constitutive expression of cleaved Notch receptor 1 was also detected in eosinophils from whole blood, suggesting Notch 1 activation was not an artifact of eosinophil isolation (Fig. S1).

Figure 2.

Figure 2

Open in a new tab

Notch receptor 1 is constitutively active in freshly isolated human blood eosinophils and is required for granulocyte-macrophage colony-stimulating factor (GM-CSF) priming-induced transendothelial migration. (A) Upon binding to its ligand, an α-secretase-dependent cleavage event releases the extracellular portion of Notch receptor 1 (1), exposing a γ-secretase-sensitive cleavage site. Subsequent cleavage by γ-secretase (2) frees a 95-kD Notch intracellular domain (NICD) fragment. A polyclonal antibody recognizing an intracellular epitope of Notch receptor 1 unaffected by receptor activation (pICD) is shown in blue, while VAL1744 antibodies (shown in red) recognize an epitope that is exposed only after γ-secretase-dependent cleavage of activated Notch receptor 1. Lysates were prepared of freshly isolated human eosinophils and probed by Western blot with pICD (B) or VAL1744 (C, inset). In C, freshly isolated eosinophils were permeabilized and expression of the cleaved NICD assessed by flow cytometry with VAL1744. Shaded histogram = isotype control. Data shown are representative of N ≥ 3 eosinophil donors. (D) Notch receptor 1 activation was inhibited by blocking ligand binding (Clone A6, green) or by preventing exposure of the α-secretase-sensitive site (NRR1, orange). (E, F) Eosinophils were primed with or without GM-CSF in the presence of clone A6 (E), NRR1 (F), or their respective isotype controls before being overlaid onto IL-4 and TNF-α-activated human umbilical vein endothelial cell (HUVEC) monolayers and assessed for their transmigratory ability. Data shown are the combined mean ± SEM of N = 5 independent experiments, using different eosinophil donors (**P < 0.01).

To determine whether Notch receptor 1 activation contributes to GM-CSF-induced priming effects on eosinophil transmigration, GM-CSF priming was carried out in the presence of two different anti-Notch receptor 1 neutralizing antibodies. Clone A6 has been reported to neutralize Notch receptor 1 activation, presumably by interfering with ligand binding (19). NRR1 antibodies bind an epitope within the negative regulatory region of Notch 1 and inhibit Notch signaling by stabilizing the receptor in a manner that prevents the ligand binding-induced change in structural conformation required to expose the α-secretase-dependent cleavage site (20) (illustrated in Fig. 2D). Consonant with data generated using the pan Notch inhibitors, eosinophil transmigration was inhibited when either clone A6 or NRR1 anti-Notch 1-blocking Abs were present during GM-CSF priming (Fig. 2E,F). Viability of eosinophils after overnight priming with GM-CSF was as follows: IgG2b = 94 ± 3%; clone A6 = 89 ± 5%; IgG1 = 96 ± 3%; and NRR1 = 95 ± 3%, suggesting the impaired transmigration of eosinophils primed in the presence of Notch 1 neutralizing antibodies was not caused by overt differences in eosinophil viability. Antibodies targeting eosinophil cell surface-expressed MHC I had no effect on GM-CSF-primed eosinophil migration, indicating Notch 1 neutralizing antibody-dependent inhibition of eosinophil migration was not caused by a bystander effect of Fc receptor cross-linking (Fig. S2).

Inhibition of Notch signaling depresses ERK-MAPK signaling

Granulocyte-macrophage colony-stimulating factor priming of eosinophils induces a long-lived response that persists despite receptor desensitization (21). Activation of ERK-MAPK signal transduction pathways are implicated in both early transient events and subsequently in the prolonged effects of GM-CSF priming. Preliminary kinetics experiments using multiplex analysis revealed two peaks (at approximately 5 and 60 min) of GM-CSF-induced ERK phosphorylation. In the presence of GSI II, GM-CSF-induced ERK phosphorylation was diminished at 60 min of stimulation (Fig. 3A). Anti-Notch 1 neutralizing antibodies (clone A6) were included in parallel with some experiments and yielded a similar trend (Fig. 3B). In a complementary approach, intracellular flow cytometry was used to assess ERK phosphorylation on a single cell basis. In support of multiplex analyses, GM-CSF-stimulated eosinophils exhibited a decrease in ERK phosphorylation at 60 min in the presence of either GSI or anti-Notch 1 Abs when compared to vehicle or irrelevant Ab controls, respectively (Fig. 3C,D).

Figure 3.

Figure 3

Open in a new tab

Inhibition of Notch signaling impairs granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced ERK phosphorylation. Eosinophils were cultured in medium alone or containing GM-CSF with γ-secretase inhibitor (GSI), clone A6, or vehicle or isotype control for 60 min. (A, B) ERK phosphorylation was detected from eosinophil lysates by multiplex analysis. Phosphorylated ERK (normalized to total ERK) is expressed as fold change in phospho-ERK expression relative to baseline control cells from the same experiment. Each data point represents an average from duplicate wells, and each line connects vehicle control- and GSI-treated data points from a single donor. In C and D, GM-CSF-induced phosphorylated ERK was detected by intracellular flow cytometry. Upper panels show representative histograms from one representative donor: shaded histogram, nonstimulated eosinophils; solid black line, GM-CSF stimulated in the presence of vehicle or isotype control; red line, GM-CSF stimulated in the presence of GSI (C) or clone A6 (D). Lower panels show data from seven independent experiments, expressed as the delta MFI of phosphorylated ERK over isotype control. Each line connects control- and GSI- or clone A6-treated data points from a single donor. Red lines identify the representative data shown in the respective upper panels. *P < 0.05, paired, two-tailed Student’s t-test. (E, F) Eosinophils were primed with or without GM-CSF in the presence or absence of GSI or DMSO vehicle control, and +/− MEK inhibitor U0126 (N = 10), its inactive analogue control U0124 (N = 9) (E), or PMA (N = 3) (F). Following priming, eosinophils were overlaid onto endothelial cell monolayers preactivated with IL-4 and TNF-α. Data shown are mean ± SEM of data from all independent experiments combined (*P < 0.05 vs nonstimulated vehicle control; †P < 0.05 vs GM-CSF stimulated vehicle control).

To determine whether diminished ERK phosphorylation in the presence of Notch signaling inhibitors is sufficient to cause the observed defect in transendothelial migration, eosinophils were primed with GM-CSF in the presence of the MEK1/2 inhibitor U0126 or the nonfunctional peptide mimic U0124. Inhibition of MEK1/2 during GM-CSF priming attenuated eosinophil migration, even at suboptimal concentrations (i.e., 2.5 μM, Fig. 3E). Using the reverse approach, PMA, a robust activator of ERK-MAPK pathways through protein kinase C activation of Raf (22), rescued the GSI-induced phenotype of impaired GM-CSF priming-induced eosinophil transmigration (Fig. 3F).

In vivo administration of GSI attenuates accumulation of eosinophils in allergic airways

Eosinophils isolated from the spleens of naïve IL-5 transgenic mice expressed Notch receptors 1 and 2 (Fig. 4A). Of note, the low surface expression observed for Notch 1 was replicated using NRR1 antibodies (not shown). Upon confirmation that mouse eosinophils expressed Notch receptors, we investigated a requirement for Notch signaling for eosinophil transmigration in vivo. OVA-sensitized mice were exposed to aerosolized OVA as described in Materials and methods and Fig. 4B. This method induces robust eosinophil accumulation within bronchoalveolar lavage fluid (BALF), readily detected by a combination of forward-side scatter characteristics and expression of surface Siglec-F (Fig. 4C). Systemic administration of GSI II 30 min prior to each airway challenge reduced the accumulation of eosinophils within BALF (Fig. 4D). Differences in total BAL cells recovered from GSI- and vehicle-treated mice did not reach statistical significance, and the loss of BAL eosinophils fully accounted for the discrepancy in total numbers of BAL cells recovered, indicating eosinophils were the only or at least the quantitatively dominant cellular population impacted by GSI treatment (compare Fig. 4D and E). Attenuation of BAL eosinophilia was apparent despite recoveries of statistically similar percentages of bone marrow eosinophils (7.81 ± 1.34 and 8.87 ± 0.18 for control- and GSI-treated mice, respectively, N = 3), suggesting the deficiency in BAL eosinophilia was not a consequence of GSI-induced hematopoietic defects.

Figure 4.

Figure 4

Open in a new tab

In vivo administration of γ-secretase inhibitor (GSI) impairs eosinophil accumulation within allergic airways. (A) Surface expression of Notch receptors 1 and 2 on mouse eosinophils. (B) Experimental design. (C–E) Total BAL cells were counted and stained with PE-labeled rat anti-mouse Siglec-F mAb. BAL eosinophils were gated on their forward and side scatter characteristics and further identified as Siglec-F+/SSChigh eosinophils (C). (D) BAL eosinophils were quantified from six mice per group in two independent experiments and expressed as percentage of total BAL cells (top panel), or total cell numbers (bottom panel). (E) Total BAL cells. Data are presented as mean ± SD. **P < 0.01 (unpaired two-tailed Student’s t-test). Eos, eosinophils; NS, not significant.

Ex vivo exposure to GSI impairs the migration of adoptively transferred eosinophils into allergic airways

Notch signaling is implicated in immune polarization of T lymphocytes (23-26). To verify a direct effect of GSI on eosinophil recruitment in vivo, distinct from a GSI-dependent effect on T-cell-derived eosinophil growth or chemotactic factors, donor eosinophils were treated with GSI or DMSO vehicle control ex vivo, mixed together in equal parts, and instilled into eosinophil-deficient recipient mice prior to the final OVA airway challenge (Fig. 5A). Analysis of BALF 24 h after the final OVA airway challenge revealed a nearly threefold reduction in the number of GSI-pretreated donor eosinophils present in the BALF, in comparison with donor eosinophils pretreated with DMSO vehicle control (Fig. 5B).

Figure 5.

Figure 5

Open in a new tab

Ex vivo treatment with γ-secretase inhibitor (GSI) impairs eosinophil accumulation within allergic airways. (A) Experimental design. (B, left panel) Representative FACS plot is shown of BAL eosinophils recovered from recipient mice 24 h after final airway challenge. Numbers above boxes indicate percentage of labeled eosinophils out of the total BAL eosinophils. (B, right panel) Data from three mice are combined and expressed as mean ± SD. ***P < 0.002, Student’s two-tailed t-test. Eos, eosinophils.

Discussion

Granulocyte-macrophage colony-stimulating factor priming-enhanced migration of eosinophils plays a key role in the accumulation of eosinophils within inflamed bronchi in allergic asthma. However, cell-intrinsic mechanisms that regulate priming-induced eosinophil migration are poorly defined. Here, we demonstrate that Notch signaling, an evolutionarily conserved pathway that integrates intrinsic and extrinsic cues to direct pleiotropic cellular functions, is required for GM-CSF-primed eosinophil transendothelial migration and airways accumulation in allergic airway disease. Inclusion of GSIs during GM-CSF priming inhibited eosinophil migration across nonactivated or cytokine-activated endothelial monolayers, directly implicating γ-secretase activity in GM-CSF priming-induced eosinophil transendothelial migration. Freshly isolated human blood eosinophils constitutively express the cleaved fragment of Notch receptor 1, indicating that Notch 1 is constitutively activated in circulating eosinophils. It is unknown how constitutive Notch 1 activation is maintained in circulating eosinophils, but may be due to interactions with Notch ligands expressed on vascular endothelium, myeloid cells, or serum-derived Notch ligand-expressing exosomes. Eosinophils exposed during GM-CSF priming to antibodies that inhibit ligand binding or α-secretase cleavage of Notch 1 exhibited impaired transendothelial migration, implicating Notch signaling, specifically through surface-expressed Notch 1, in the GM-CSF priming effects on transendothelial migration of human eosinophils. Further studies are needed to determine whether or not signaling through Notch receptor 2 might also contribute to GM-CSF-primed eosinophil transmigration and whether combined inhibition of Notch receptors 1 and 2 might be necessary to fully eradicate the priming effects of GM-CSF.

Inhibition of Notch signaling was associated with diminished ERK phosphorylation measured at 60 min of GM-CSF stimulation. ERK-MAPK pathway activation was required for GM-CSF-primed eosinophil transmigration, and partial inhibition of MEK phosphorylation was sufficient to impair migration. Moreover, PMA, a strong stimulus for PKC-dependent activation of ERK-MAPK signaling at the level of Raf (thus bypassing Ras) (22), rescued transmigration in GSI-treated eosinophils. Taken together, these data suggest one mechanism whereby Notch signaling may promote GM-CSF-primed transmigration is through enhancing Ras-Raf-MEK-ERK-MAPK signaling and suggest the nodal point between ERK and Notch pathways may be upstream of Raf. Complementary to our findings, Chung et al. found eosinophils differentiated from cord blood progenitors in the presence of GSI to be unable to phosphorylate ERK upon subsequent exposure to the chemokine eotaxin (27), although in that system, eosinophils derived in the presence of GSI exhibited higher basal levels of ERK phosphorylation than their vehicle-treated controls (28). Notch and ERK-MAPK signaling pathways are frequently interdependent throughout development (29). In Drosophila, the majority of Ras-responsive genes are also targets of Notch, and Notch directly regulates the transcription of genes encoding proteins of the Ras pathway, and vice versa (30). Noncanonical, cytoplasmic functions for NICDs independent of nuclear translocation are also implicated in Notch–Ras pathway interactions (31, 32). However, it is also plausible that Notch signaling impacts ERK phosphorylation of GM-CSF-primed eosinophils indirectly through modulating another GM-CSF-induced, intermediary step.

To investigate the physiological significance of Notch signaling to eosinophil recruitment in vivo, we utilized a standard mouse model of allergic airway inflammation. Our data show, to our knowledge for the first time, that mouse eosinophils express Notch receptors 1 and 2. Our data further demonstrate impaired recruitment of mouse eosinophils into allergic lungs following whole-body administration of GSI II. Notch signaling has been proposed as a therapeutic target for asthma, and in a mouse model of allergic airway inflammation, intranasally administered GSI attenuated pathological outcomes, including levels of serum IgE and airway hyper-responsiveness (33). In that study, the authors attributed improved clinical outcomes to known effects of Notch signaling on CD4+ T-cell differentiation (23-26), while acknowledging Notch-dependent effects on additional cell types could not be excluded. Data presented here would suggest eosinophil recruitment may also have been directly impacted and may have contributed to the attenuation of symptoms in those studies. Importantly, failure of GSI-treated eosinophils adoptively transferred into eosinophil-deficient recipients to migrate into inflamed lungs (Fig. 5) establishes the direct effect of γ-secretase activity on eosinophil recruitment and confirms the biological significance of Notch signaling specifically by mature eosinophils in airway disease.

These data suggest targeting Notch signaling pathways may provide a multipronged approach to asthma therapy. Further studies designed to elucidate specific Notch receptor–ligand pairs and cell-intrinsic molecular mechanisms that differentially regulate Notch signaling outcomes in eosinophils and T cells may provide customizable strategies that manipulate clinical outcomes and limit potential for collateral effects. Moreover, with the growing interest in GSIs for the treatment of cancers and other diseases (12-14), these data are also more broadly relevant, as they anticipate functional consequences of such treatments on eosinophils that might directly or indirectly impact prognoses.

Supplementary Material

Supplemental Files

Data S1. Supplemental materials and methods.

Figure S1. NICD detection within eosinophils from unfractionated whole blood indicates NICD detection in isolated eosinophils is not due to Notch 1 activation through the process of eosinophil purification.

Figure S2. Antibodies targeting eosinophil surface-expressed MHC I or CD4 had no impact on GM-CSF-primed eosinophil migration.

Acknowledgments

The authors acknowledge and thank Drs. Chris Siebel and Amy Shelton, and Genentech for providing NRR1 anti-Notch 1 neutralizing antibodies. Funding for this work was provided by NIH R01 HL095699, an AHA Grant-in-Aid, and NIH R37 AI020241.

Footnotes

Conflicts of interest

The authors declare that they have no conflicts of interest.

References

  • 1.Warringa RA, Mengelers HJ, Kuijper PH, Raaijmakers JA, Bruijnzeel PL, Koenderman L. In vivo priming of platelet-activating factor-induced eosinophil chemotaxis in allergic asthmatic individuals. Blood 1992;79:1836–1841. [PubMed] [Google Scholar]
  • 2.Ebisawa M, Liu MC, Yamada T, Kato M, Lichtenstein LM, Bochner BS et al. Eosinophil transendothelial migration induced by cytokines. II. Potentiation of eosinophil transendothelial migration by eosinophil-active cytokines. J Immunol 1994;152:4590–4596. [PubMed] [Google Scholar]
  • 3.Warringa RA, Koenderman L, Kok PT, Kreukniet J, Bruijnzeel PL. Modulation and induction of eosinophil chemotaxis by granulocyte-macrophage colony-stimulating factor and interleukin-3. Blood 1991;77:2694–2700. [PubMed] [Google Scholar]
  • 4.Robinson DS, Hamid Q, Ying S, Tsicopoulos A, Barkans J, Bentley AM et al. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992;326:298–304. [DOI] [PubMed] [Google Scholar]
  • 5.Woolley KL, Adelroth E, Woolley MJ, Ellis R, Jordana M, O’Byrne PM. Effects of allergen challenge on eosinophils, eosinophil cationic protein, and granulocyte-macrophage colony-stimulating factor in mild asthma. Am J Respir Crit Care Med 1995;151:1915–1924. [DOI] [PubMed] [Google Scholar]
  • 6.Boomars KA, Schweizer RC, Zanen P, van den Bosch JM, Lammers JW, Koenderman L. Eosinophil chemotactic activity in bronchoalveolar lavage from idiopathic pulmonary fibrosis is dependent on cytokine priming of eosinophils. Eur Respir J 1998;11:1009–1014. [DOI] [PubMed] [Google Scholar]
  • 7.Stampfli MR, Wiley RE, Neigh GS, Gajewska BU, Lei XF, Snider DP et al. GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice. J Clin Invest 1998;102:1704–1714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Su YC, Rolph MS, Hansbro NG, Mackay CR, Sewell WA. Granulocyte-macrophage colony-stimulating factor is required for bronchial eosinophilia in a murine model of allergic airway inflammation. J Immunol 2008;180:2600–2607. [DOI] [PubMed] [Google Scholar]
  • 9.Yamashita N, Tashimo H, Ishida H, Kaneko F, Nakano J, Kato H et al. Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF). Cell Immunol 2002;219:92–97. [DOI] [PubMed] [Google Scholar]
  • 10.Cates EC, Fattouh R, Wattie J, Inman MD, Goncharova S, Coyle AJ et al. Intranasal exposure of mice to house dust mite elicits allergic airway inflammation via a GM-CSF-mediated mechanism. J Immunol 2004;173:6384–6392. [DOI] [PubMed] [Google Scholar]
  • 11.Maillard I, Adler SH, Pear WS. Notch and the immune system. Immunity 2003;19:781–791. [DOI] [PubMed] [Google Scholar]
  • 12.Miele L, Miao H, Nickoloff BJ. NOTCH signaling as a novel cancer therapeutic target. Curr Cancer Drug Targets 2006;6:313–323. [DOI] [PubMed] [Google Scholar]
  • 13.Nam Y, Aster JC, Blacklow SC. Notch signaling as a therapeutic target. Curr Opin Chem Biol 2002;6:501–509. [DOI] [PubMed] [Google Scholar]
  • 14.Shih Ie M, Wang TL. Notch signaling, gamma-secretase inhibitors, and cancer therapy. Cancer Res 2007;67:1879–1882. [DOI] [PubMed] [Google Scholar]
  • 15.Krop I, Demuth T, Guthrie T, Wen PY, Mason WP, Chinnaiyan P et al. Phase I pharmacologic and pharmacodynamic study of the gamma secretase (Notch) inhibitor MK-0752 in adult patients with advanced solid tumors. J Clin Oncol 2012;30:2307–2313. [DOI] [PubMed] [Google Scholar]
  • 16.Tolcher AW, Messersmith WA, Mikulski SM, Papadopoulos KP, Kwak EL, Gibbon DG et al. Phase I study of RO4929097, a gamma secretase inhibitor of Notch signaling, in patients with refractory metastatic or locally advanced solid tumors. J Clin Oncol 2012;30:2348–2353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Radke AL, Reynolds LE, Melo RCN, Dvorak AM, Weller PF, Spencer LA. Mature human eosinophils express functional Notch ligands mediating eosinophil autocrine regulation. Blood 2009;113:3092–3101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Wang HB, Ghiran I, Matthaei K, Weller PF. Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells. J Immunol 2007;179:7585–7592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Asano N, Watanabe T, Kitani A, Fuss IJ, Strober W. Notch1 signaling and regulatory T cell function. J Immunol 2008;180:2796–2804. [DOI] [PubMed] [Google Scholar]
  • 20.Wu Y, Cain-Hom C, Choy L, Hagenbeek TJ, de Leon GP, Chen Y et al. Therapeutic antibody targeting of individual Notch receptors. Nature 2010;464:1052–1057. [DOI] [PubMed] [Google Scholar]
  • 21.Pazdrak K, Young TW, Stafford S, Olszewska-Pazdrak B, Straub C, Starosta V et al. Cross-talk between ICAM-1 and granulocyte-macrophage colony-stimulating factor receptor signaling modulates eosinophil survival and activation. J Immunol 2008;180:4182–4190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ueda Y, Hirai S, Osada S, Suzuki A, Mizuno K, Ohno S. Protein kinase C activates the MEK-ERK pathway in a manner independent of Ras and dependent on Raf. J Biol Chem 1996;271:23512–23519. [DOI] [PubMed] [Google Scholar]
  • 23.Maekawa Y, Tsukumo S, Chiba S, Hirai H, Hayashi Y, Okada H et al. Delta1-Notch3 interactions bias the functional differentiation of activated CD4 + T cells. Immunity 2003;19:549–559. [DOI] [PubMed] [Google Scholar]
  • 24.Amsen D, Blander JM, Lee GR, Tanigaki K, Honjo T, Flavell RA. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 2004;117:515–526. [DOI] [PubMed] [Google Scholar]
  • 25.Minter LM, Turley DM, Das P, Shin HM, Joshi I, Lawlor RG et al. Inhibitors of gamma-secretase block in vivo and in vitro T helper type 1 polarization by preventing Notch upregulation of Tbx21. Nat Immunol 2005;6:680–688. [PubMed] [Google Scholar]
  • 26.Tu L, Fang TC, Artis D, Shestova O, Pross SE, Maillard I et al. Notch signaling is an important regulator of type 2 immunity. J Exp Med 2005;202:1037–1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kang JH, da Lee H, Seo H, Park JS, Nam KH, Shin SY et al. Regulation of functional phenotypes of cord blood derived eosinophils by gamma-secretase inhibitor. Am J Respir Cell Mol Biol 2007;37:571–577. [DOI] [PubMed] [Google Scholar]
  • 28.Kang JH, da Lee H, Lee JS, Kim HJ, Shin JW, Lee YH et al. Eosinophilic differentiation is promoted by blockage of Notch signaling with a gamma-secretase inhibitor. Eur J Immunol 2005;35:2982–2990. [DOI] [PubMed] [Google Scholar]
  • 29.Sundaram MV. The love-hate relationship between Ras and Notch. Genes Dev 2005;19:1825–1839. [DOI] [PubMed] [Google Scholar]
  • 30.Hurlbut GD, Kankel MW, Artavanis-Tsakonas S. Nodal points and complexity of Notch-Ras signal integration. Proc Natl Acad Sci U S A 2009;106:2218–2223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Hodkinson PS, Elliott PA, Lad Y, McHugh BJ, MacKinnon AC, Haslett C et al. Mammalian NOTCH-1 activates beta1 integrins via the small GTPase R-Ras. J Biol Chem 2007;282:28991–29001. [DOI] [PubMed] [Google Scholar]
  • 32.Carmena A, Speicher S, Baylies M. The PDZ protein Canoe/AF-6 links Ras-MAPK, Notch and Wingless/Wnt signaling pathways by directly interacting with Ras, Notch and Dishevelled. PLoS ONE 2006;1:e66. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Kang JH, Kim BS, Uhm TG, Lee SH, Lee GR, Park CS et al. Gamma-secretase inhibitor reduces allergic pulmonary inflammation by modulating Th1 and Th2 responses. Am J Respir Crit Care Med 2009;179:875–882. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Files

Data S1. Supplemental materials and methods.

Figure S1. NICD detection within eosinophils from unfractionated whole blood indicates NICD detection in isolated eosinophils is not due to Notch 1 activation through the process of eosinophil purification.

Figure S2. Antibodies targeting eosinophil surface-expressed MHC I or CD4 had no impact on GM-CSF-primed eosinophil migration.

NIHMS1863812-supplement-Supplemental_Files.pdf (125KB, pdf)

RESOURCES