Retinoic acid modulates IL-5 receptor expression and selectively inhibits eosinophil-basophil differentiation of hemopoietic progenitor cells

doi:10.1067/mai.2002.121527

Journal of Allergy and Clinical Immunology

Volume 109, Issue 2, February 2002, Pages 307-313

https://doi.org/10.1067/mai.2002.121527 Get rights and content

Abstract

Background: IL-5 plays a central role in eosinophil and basophil differentiation, exerting its effects through the IL-5 receptor (IL-5Rα). Currently, little is known concerning regulation of IL-5Rα expression in the context of commitment of hemopoietic progenitor cells to the eosinophil and basophil lineages. Objective: Because all-trans retinoic acid (ATRA) is known to modulate some aspects of hemopoietic differentiation, we examined the effects of ATRA on eosinophil-basophil differentiation and IL-5Rα expression. Methods: Progenitor cells were obtained from bone marrow aspirates and cord blood samples. Enriched populations of CD34⁺ cells were isolated by means of positive immunomagnetic selection with MACS beads. Results: In semisolid methylcellulose cultures of normal human bone marrow, ATRA (10⁻⁶ mol/L) selectively suppressed eosinophil-basophil colony-forming units but had no effect on granulocyte-macrophage colony-forming units. Similarly, ATRA (10⁻⁶ mol/L) inhibited eosinophil-basophil differentiation of cord blood CD34⁺ cells in liquid culture, whereas neutrophil differentiation proceeded without impediment. Most importantly, these effects of ATRA (10⁻⁸ to 10⁻⁶ mol/L) on CD34⁺ cells were associated with a dose-dependent inhibition of IL-5Rα but no change in GM-CSF receptor expression, as detected with flow cytometry. Conclusions: These findings indicate that retinoids can differentially regulate expression of IL-5Rα, but not GM-CSF receptor, and that these effects have functional consequences in vitro on eosinophil and basophil differentiation. (J Allergy Clin Immunol 2002;109:307-13.)

Section snippets

Materials

Materials were purchased as follows: Lymphoprep was obtained from NycoMed Pharma AS; McCoys 5A medium, penicillin, streptomycin, and FCS were obtained from Gibco BRL; heparin, dextran (molecular weight, 260,000 kD), sodium azide, ATRA, and paraformaldehyde were obtained from Sigma Aldrich Canada; 2-mercaptoethanol was obtained from BDH Inc; recombinant IL-3, IL-5, and GM-CSF were obtained from Pharmingen Canada; and methylcellulose (Methocel A4M premium) was obtained from Dow Chemicals. The

ATRA selectively inhibits eosinophil-basophil CFUs

In initial experiments sternal bone marrow samples were obtained preoperatively from patients undergoing cardiac surgery, and CFUs were enumerated in 14-day methylcellulose-based assays, with Mo-conditioned medium as a source of GM-CSF. Data are expressed as CFUs per 2.5 × 10⁵ nonadherent bone marrow cells. As shown in Fig 1, ATRA (10⁻⁶ mol/L) reduced eosinophil-basophil CFUs from 21 ± 5.9 to 9.25 ± 2.7 colonies (P < .05), associated with no significant change in the proportion of

Discussion

Our findings demonstrate that ATRA has the ability to selectively inhibit eosinophil-basophil differentiation and surface expression of IL-5Rα by hemopoietic progenitor cells in vitro. First, ATRA suppressed bone marrow eosinophil-basophil CFUs but had no effect on granulocyte-macrophage CFUs (Fig 1). Second, ATRA suppressed eosinophil-basophil differentiation of CD34⁺ cord blood cells in long-term liquid culture, whereas neutrophil differentiation proceeded without impediment (Table I).

Acknowledgements

We thank Dr A. Lopez (Adelaide, Australia) for generous provision of antibodies, A. Verhee (Ghent, Belgium) for technical assistance, and Lynne Larocque for excellent help in manuscript preparation.

References (41)

A Miyajima et al.
Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5
Blood
(1993)
K Takatsu et al.
Interleukin-5 and its receptor system: implications in the immune system and inflammation
Adv Immunol
(1994)
JA Denburg et al.
Heterogeneity of human peripheral blood eosinophil-type colonies: evidence for a common basophil-eosinophil progenitor
Blood
(1985)
EJ Clutterbuck et al.
Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF
Blood
(1989)
DS Robinson et al.
Relationships among numbers of bronchoalveolar lavage cells expressing messenger ribonucleic acid for cytokines, asthma symptoms, and airway methacholine responsiveness in atopic asthma
J Allergy Clin Immunol
(1993)
H Ema et al.
Target cells for granulocyte colony-stimulating factor, interleukin-3, and interleukin-5 in differentiation pathways of neutrophils and eosinophils
Blood
(1990)
M Valtieri et al.
Two-step differentiation of AML-193 leukemic line: terminal maturation is induced by positive interaction of retinoic acid with granulocyte colony-stimulating factor (CSF) and vitamin D3 with monocyte CSF
Blood
(1991)
C Labbaye et al.
Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture
Blood
(1994)
A Tocci et al.
Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program
Blood
(1996)
H Nakajima et al.
Retinoids (all-trans and 9-cis retinoic acid) stimulate production of macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor by human bone marrow stromal cells
Blood
(1994)

T Kinoshita et al.

Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors

Blood

(2000)

E Ingley et al.

Characterization of a receptor for interleukin-5 on human eosinophils and the myeloid leukemia line HL-60

Blood

(1991)

J Tavernier et al.

Interleukin 5 regulates the isoform expression of its own receptor alpha-subunit

Blood

(2000)

J Tavernier et al.

A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific a chain and a b chain shared with the receptor for GM-CSF

Cell

(1991)

Z Sun et al.

Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene

J Biol Chem

(1995)

JA Denburg et al.

Basophil and eosinophil differentiation in allergic reactions

J Allergy Clin Immunol

(1994)

LA Dent et al.

Eosinophilia in transgenic mice expressing interleukin 5

J Exp Med

(1990)

H Shi et al.

Infiltration of eosinophils into the asthmatic airways caused by interleukin 5

Am J Respir Cell Mol Biol

(1997)

Q Hamid et al.

Expression of mRNA for interleukin-5 in mucosa bronchial biopsies from asthma

J Clin Invest

(1991)

PS Foster et al.

Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model

J Exp Med

(1996)

Cited by (24)

Targeting myeloid-derived suppressor cells using all-trans retinoic acid in melanoma patients treated with Ipilimumab
2018, International Immunopharmacology
Citation Excerpt :
We found that patients treated with ATRA and Ipilimumab had a significantly lower frequency of eosinophils in the periphery compared to people treated with Ipilimumab alone. This is consistent with previous literature showing that ATRA reduces the expression of IL-5 receptor on eosinophil progenitor cells, resulting in decreased differentiation of eosinophils [41]. There have been few reports in the literature in regard to the impact of eosinophils in patients being treated with Ipilimumab.
Immune checkpoint inhibitors have improved overall survival rates for many cancers, yet the majority of patients do not respond to treatment and succumb to disease progression. One tumor-related mechanism limiting the efficacy of immunotherapies in melanoma is the recruitment and expansion of myeloid-derived suppressor cells (MDSCs). Therefore, targeting MDSCs in combination with immunotherapies is an attractive strategy to improve response rates and effectiveness.
We tested this strategy by designing a randomized phase II clinical trial treating advanced melanoma patients with either Ipilimumab monotherapy or Ipilimumab plus all-trans retinoic acid (ATRA). Clinicaltrails.gov identifier (NCT02403778). The frequency of circulating MDSCs and the activation of CD8(+) T cells was measured by flow cytometry. Expression of immunosuppressive genes was measured with quantitative real time-PCR. T cell suppressive functions were measured by mixed lymphocyte reaction.
Here we show that in vitro treatment with ATRA decreases immunosuppressive function of MDSCs in mixed lymphocyte reactions. Additionally, ATRA reduces the expression of immunosuppressive genes including PD-L1, IL-10, and indoleamine 2,3‑dioxygenase by MDSCs. Furthermore, the addition of ATRA to standard of care Ipilimumab therapy appears safe, as ATRA did not increase the frequency of grade 3 or 4 adverse events. Finally, ATRA significantly decreased the frequency of circulating MDSCs compared to Ipilimumab treatment alone in advanced-stage melanoma patients.
These results illustrate the importance of MDSCs in immunotherapy resistance and provide evidence that targeting MDSCs in cancer patients may augment immunotherapeutic approaches.
Novel lineage- and stage-selective effects of retinoic acid on mouse granulopoiesis: Blockade by dexamethasone or inducible NO synthase inactivation
2017, International Immunopharmacology
Citation Excerpt :
The following aspects require further elaboration: Our findings differ strikingly from those of a single report of ATRA effects on normal (nonleukemic) human hemopoietic progenitors in eosinopoiesis [39], which describes a suppressive effect of ATRA on the expression of IL-5 receptor α chain, while GM-CSF receptor α chain expression was unaffected. In that study, IL-5-stimulated eosinophil colony formation was reduced in the presence of ATRA, while GM-CSF-stimulated myeloid colony formation was not (a major difference relative to our results).
Despite the close relationship of eosinophils and neutrophils, these granulocyte lineages respond to distinct cytokines and play unique roles in immune responses. They nevertheless respond to shared physiological/pharmacological regulators, including glucocorticoids and retinoids, and to ubiquitous mediators, including NO. Others showed that, in humans, all-trans retinoic acid (ATRA) suppresses eosinophil differentiation, but promotes neutrophil differentiation. Mechanisms of dual co-regulation of physiological granulopoiesis were here examined in murine bone-marrow, a model system suitable for exploration of immunopharmacological mechanisms, given the availability of experimental resources, including mutant/knockout mouse strains. We examined the effects of ATRA on mouse eosinophil and neutrophil production, using wild-type (BALB/c, C57BL/6) and mutant (iNOS-, CD95L-, or CD95-KO) bone-marrow cultures, further assessing the modification of ATRA activity by dexamethasone and iNOS blockade. ATRA (10^− 6–10^− 8 M) significantly decreased eosinophil production relative to IL-5 controls. This effect was iNOS-independent, but CD95L- and caspase-dependent, and prevented by dexamethasone (10^− 7 M in vitro; 1–20 mg·kg^− 1 in vivo). In myeloid colony formation assays, ATRA markedly suppressed GM-CSF-responsive progenitors, through an iNOS-dependent, CD95-independent, dexamethasone-sensitive mechanism. By contrast, ATRA potently enhanced GM-CSF-dependent neutropoiesis in liquid culture from BALB/c or C57BL/6 bone-marrow. This novel stimulatory effect was resistant to dexamethasone and abolished in iNOS-KO bone-marrow. ATRA injections also induced lineage- and stage-selective effects on granulopoiesis in vivo. ATRA therefore co-regulates eosinophil and neutrophil production in murine bone-marrow through multiple lineage- and stage-selective mechanisms.
Eliciting eosinophil CCR3 expression by synthetic retinoids
2014, Allergology International
Vitamin A reduces lung granulomatous inflammation with eosinophilic and neutrophilic infiltration in Sephadex-treated rats
2004, European Journal of Pharmacology
Vitamin A is known to suppress the activity of the transcription factors, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), as do glucocorticoids. The possibility that vitamin A exerts various anti-inflammatory effects therefore seems likely. Sephadex beads were administered intravenously to anesthesized rats pretreated with a subcutaneous injection of vitamin A (3000, 10,000, or 30,000 IU/kg) or vehicle once daily for 3 days. After 16 h, the leukocyte differential, tumor necrosis factor (TNF)-α and eotaxin, and the DNA-binding activity of NF-κB were measured in bronchoalveolar lavage fluid (BALF). Additionally, lung histology was assessed using preparations stained with May–Giemsa stain. Sephadex beads caused histological granulomatous changes and eosinophilic and neutrophilic infiltration into the lung, and markedly increased cell counts of eosinophils and neutrophils, concentrations of TNF-α and eotaxin, and NF-κB binding to DNA in BALF. Vitamin A significantly inhibited all responses. Vitamin A may inhibit Sephadex-induced lung granulomatous formation, and eosinophilic and neutrophilic infiltration due to its suppression of TNF-α and eotaxin production, and NF-κB activation.
Gene microarray analysis reveals interleukin-5-dependent transcriptional targets in mouse bone marrow
2004, Blood
Citation Excerpt :
Among the genes undergoing the most prominent alterations in expression are Syntaxin 3 (+8-fold), which has been identified as a component of the eosinophil exocytotic apparatus,33 the signal transduction elements phospholipase D1 (–34-/–2.1-fold), syk (–27-fold) which is intriguing, given the role of syk in IL-5–mediated eosinophil activation.34 Also undergoing transcriptional alteration is the retinoic acid receptor (–14-fold), an interesting parallel given that its ligand, retinoic acid, has been shown to modulate expression of the receptor for IL-5.35 Many genes that are traditionally associated with eosinophil development and differentiation were either not detected or did not respond within the patterns defined by profiles 1 or 2 in Figure 3 (Table 2).
Interleukin-5 (IL-5) is a hematopoietic differentiation factor that promotes the development of mature eosinophils from progenitors in bone marrow. We present a multifactorial microarray study documenting the transcriptional events in bone marrow of wild-type and IL-5–deficient mice at baseline and in response to infection with Schistosoma mansoni. The microarray data were analyzed by a 4-way subtractive algorithm that eliminated confounding non-IL-5–related sequelae of schistosome infection as well as alterations in gene expression among uninfected mice. Among the most prominent findings, we observed 7- to 40-fold increased expression of transcripts encoding the classic eosinophil granule proteins (eosinophil peroxidase, major basic protein, the ribonucleases) together with arachidonate-15-lipoxygenase and protease inhibitor plasminogen activator inhibitor 2 (PAI-2), in the IL-5–producing, infected wild-type mice only. This was accompanied by increased transcription of genes involved in secretory protein biosynthesis and granule-vesicle formation. Interestingly, we did not detect increased expression of genes encoding eosinophil-related chemokine receptors (CCR1, CCR3) or members of the GATA or CCAAT/enhancer binding protein (C/EBP) transcription factor families. These data suggest that the IL-5–responsive progenitors in the mouse bone marrow are already significantly committed to the eosinophil lineage and that IL-5 promotes differentiation of these committed progenitors into cells with recognizable and characteristic cytoplasmic granules and granule proteins.
Retinoic acid inhibits in vitro development of mast cells but has no marked effect on mature human skin tryptase- and chymase-positive mast cells
2003, Journal of Investigative Dermatology
Citation Excerpt :
The AML14 leukemic cell line, which can differentiate into eosinophils, is induced by RA to neutrophil differentiation (Paul et al, 1995). Furthermore, RA inhibits the development of eosinophils and basophils, but not neutrophils and macrophages, from human bone marrow or cord blood (Upham et al, 2002). Taken together, these results suggest that RA promotes the differentiation of neutrophils and monocytes whereas the development of erythrocytes, eosinophils, basophils, and mast cells is suppressed.
Stem cell factor plays a key role in the development of human mast cells via interaction with Kit receptor. We and other groups have previously shown that a number of cytokines can regulate the stem-cell-factor-dependent development of mast cells in vitro. In this study we investigated the effect of retinoic acid on human mast cells in vitro and in vivo. Retinoids are known to have strong modulatory effects on hematopoietic differentiation. We found that all-trans-retinoic acid, at concentrations as low as 1 nM, inhibits the stem-cell-factor-dependent differentiation of mast cells in vitro. This effect of retinoic acid was found to be on progenitor cells, whereas more mature mast cells were less affected. The use of specific agonists binding either to the RAR or the RXR nuclear receptors indicated involvement of both the RAR/RXR and RXR/RXR pathways in inhibiting mast cell differentiation. In contrast to the effects on mast cell progenitors, retinoic acid had no effect on the number of mature mast cells in skin organ cultures. Furthermore, topical treatment of normal skin with a retinoic-acid-containing cream caused an increase in the number of tryptase-positive mast cells, whereas the numbers of the major cutaneous mast cell type, tryptase- and chymase-positive mast cells, remained unaffected. Our results suggest that retinoic acid suppresses commitment of progenitor cells into the mast cell lineage and/or acts on early mast cell progenitors, whereas mature cutaneous mast cells are less susceptible to retinoic acid.

View all citing articles on Scopus

^☆: *Dr Upham is currently affiliated with the University Department of Medicine, QE2 Medical Centre, Nedlands, Australia.

^☆☆: Supported by grants from the Medical Research Council of Canada. J.W.U. is the recipient of a Neil Hamilton Fairley Fellowship (#967211) from the National Health & Medical Research Council of Australia.

^★: Reprint requests: Judah A. Denburg, MD, Department of Medicine, HSC 3V46, McMaster University, 1200 Main St West, Hamilton, Ontario L8N 3Z5, Canada.

View full text

Mechanisms of AllergyRetinoic acid modulates IL-5 receptor expression and selectively inhibits eosinophil-basophil differentiation of hemopoietic progenitor cells☆,☆☆,★

Abstract

Section snippets

Materials

ATRA selectively inhibits eosinophil-basophil CFUs

Discussion

Acknowledgements

Blood

Adv Immunol

Blood

Blood

J Allergy Clin Immunol

Blood

Blood

Blood

Blood

Blood

Blood

Blood

Blood

Cell

J Biol Chem

J Allergy Clin Immunol

Eosinophilia in transgenic mice expressing interleukin 5

J Exp Med

Infiltration of eosinophils into the asthmatic airways caused by interleukin 5

Am J Respir Cell Mol Biol

Expression of mRNA for interleukin-5 in mucosa bronchial biopsies from asthma

J Clin Invest

Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model

J Exp Med

Mechanisms of Allergy
Retinoic acid modulates IL-5 receptor expression and selectively inhibits eosinophil-basophil differentiation of hemopoietic progenitor cells☆,☆☆,★