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The Impact of Integrin β2 on Granulocyte/Macrophage Progenitor Proliferation

Abstract Previously, we reported that although the HSPC frequency in bone marrow cells (BMC) was comparable between β2−/− and β2+/+ mice, transplantation of β2−/− BMC into lethally irradiated CD45.1 recipient resulted in more myeloid cell production than β2+/+ BMC. The objective of this study is to address if integrin β2 deficiency skews granulocyte/macrophage progenitor (GMP) proliferation. FACS analysis demonstrated that GMP frequency and cell number were higher and megakaryocyte/erythrocyte progenitor frequency and cell number were lower in β2−/− mice than β2+/+ mice. However, the common myeloid progenitors (CMP) frequency and cell number were similar between the two groups. The increased GMP number was due to GMP proliferation as evidenced by the percentage of BrdU‐incorporating GMP. Whole genome transcriptome analysis identified increased FcεRIα expression in β2−/− CMP compared to β2+/+ CMP. FcεRIα expression on β2−/− GMP was detected increased in β2−/− mice by qRT‐PCR and FACS. Although transplantation of FcεRIαhi GMP or FcεRIαlo GMP into lethally irradiated CD45.1 recipient resulted in comparable myeloid cell production, transplantation of β2 deficient FcεRIαhi GMP generated more myeloid cells than β2+/+ FcεRIαhi GMP. GATA2 expression was increased in β2−/− GMP. Using a luciferase reporter assay, we demonstrated that mutation of the GATA2 binding site in the FcεRIα promoter region diminished FcεRIα transcription. In vitro, the addition of IgE, the ligand of FcεRIα, promoted GMP expansion, which was abrogated by inhibition of JNK phosphorylation. Integrin β2 deficiency promoted GMP proliferation and myeloid cell production, which was mediated via FcεRIα/IgE‐induced JNK phosphorylation in GMP. Stem Cells 2019;37:430–440


INTRODUCTION
Hematopoietic stem cells (HSC) are the only cell source that generates all types of blood cells in one's life. On the way to blood cell production, HSC will differentiate into common lymphoid progenitors and common myeloid progenitors (CMP). CMP further give rise to megakaryocyte/erythrocyte (MEP) and granulocyte/macrophage progenitors (GMP) [1]. Each of these steps is tightly controlled to generate the appropriate number of terminally differentiated cells that could carry out physical function for immune defense, oxygen transportation, and coagulation.
Myeloid progenitors express a series of adhesion receptors including CD44, integrin β1, and CD41 which enable them to adhere to bone marrow (BM) stromal cells [2,3]. Interestingly, blocking of CD44 using anti-CD44 antibody not only reduced hematopoietic cell adhesion to hyaluronate but also abrogated myelopoiesis, indicating the regulatory role of adhesion receptors in hematopoietic progenitor cell proliferation and/or differentiation [2]. In line with these findings, we previously identified integrin β2 expression in hematopoietic stem/progenitor cells (HSPCs) which regulated cell adhesion and migration toward intracellular adhesion molecule-1. Although the HSPC frequency in BM cells (BMC) was comparable in integrin-β2 knockout (β2−/−) mice compared with wild-type mice, transplantation of integrin β2−/− BMC into lethally irradiated CD45.1 recipient resulted in greater white blood cell production compared with transplantation of integrin β2+/+ BMC [4]. In unpublished data, we found that the GMP frequency in total BMCs was higher in integrin β2−/− mice than their β2+/+ littermate controls. We thus postulated that integrin β2 deficiency could skew myeloid progenitor proliferation, aside from affecting cell adhesion in BM niche.
To test the hypothesis, we performed genome wide transcriptome studies using microarrays on FACS-sorted CMPs isolated from integrin β2−/− and integrin β2+/+ mice. We identified that the expression of the Fc epsilon receptor I (FcεRIα), a high affinity receptor for IgE, was increased in β2−/− GMP compared with β2+/+ GMPs. Here, we also describe how the FcεRIα/IgE axis regulated GMP proliferation.
β2 Deficiency Enhanced Myeloid Lineage Production Next, we characterized the impact of β2 deficiency on myeloid lineage reconstitution. Equal amounts of integrin β2+/+ BMC or integrin β2−/− BMC were mixed with the same amount of CD45.1 BMC and injected into lethally irradiated CD45.1 recipient. By chimerism analysis, transplantation of integrin β2−/− BMC resulted in greater CD8+ T cells, B220+ B cells, and CD11b+ myeloid cell production after 16 weeks of transplantation (Fig. 3A).

Increased FcεRIα Expression in GMP By Microarray Analysis
To further dissect the mechanism underlying the increased GMP proliferation in β2−/− mice, CMP were sorted by FACS and whole genome transcriptome profiles were obtained by microarray analysis. Differentially expressed genes were filtered out by gene-set analysis. Pathways enriched in β2−/− CMP as compared with β2+/+ CMP were assessed by KEGG pathway analysis (Fig. 3C). By qPCR, the different expression levels of FcεRIα were confirmed in CMP as well as GMP ( Fig. 3D and 3E).
When we performed competitive BM transplantation using FcεRIα hi GMP and FcεRIα lo GMP, both types of GMP yielded comparable production of granulocytes, monocytes, and cKit+ FcεRIα+CD45 + mast cells (Fig. 4D-4F). But when FcεRIα hi GMP isolated from β2+/+ and β2−/− mice were transplanted into irradiated recipients, β2 deficient FcεRIα hi GMP generated more granulocytes and monocytes but reduced mast cells than β2+/+FcεRIα hi GMP (Fig. 4G-4I). However, transplantation of β2+/+FcεRIα lo GMP and β2−/−FcεRIα lo GMP resulted in similar granulocyte and monocyte production (Fig. 4J, 4K). Mast cell  Collectively, these data suggest that FcεRIα hi GMP and FcεRIα lo GMP have similar capacity in granulocyte and monocyte production. In the absence of β2, both FcεRIα hi GMP and FcεRIα lo GMP were increased. And β2 deficient FcεRIα hi GMP could differentiate into more granulocytes and monocytes compared with β2+/+ FcεRIα hi GMP.

FcεRIα/IgE-Induced GMP Proliferation via JNK Phosphorylation
IgE is a well-known high affinity ligand of FcεRIα. By ELISA, we found that the levels of IgE in plasma and BM fluid were elevated  (Fig. 5A, 5B). When lineage −/low or sorted GMP cells were allowed to differentiate in the presence or absence of IgE for 5 days in vitro, GMP number was increased by the addition of IgE (Fig. 5C, 5D). To dissect whether IgE acts through FcεRIα in GMP proliferation, FcεRIα hi lineage −/low , and FcεRIα lo lineage −/low cells were sorted out by FACS and cultivated with or without 100 ng/ml IgE for 5 days. FACS analysis confirmed that IgE increased GMP number more dramatically in FcεRIα hi lineage −/low than in FcεRIα lo lineage −/low cells (Fig. 5E).
To investigate how IgE/FcεRIα induced GMP proliferation, fresh BMC of β2+/+ and β2−/− mice were permeabilized and stained with GMP markers together with antibody against phospho-ERK, phospho-Akt, or phosphor-JNK. FACS analysis did not reveal any difference in the percentage of pERK+ GMP between β2+/+ and β2−/− GMP cells (1.77% ± 0.43% vs. 2.91% ± 0.62%, p = .14). However, the percentages of pAkt+ GMP and pJNK+ GMP were both 2.1-fold higher in β2−/− GMP compared with β2+/+ GMP (Fig. 5F). To further testify whether Akt or JNK phosphorylation was required for IgE-induced GMP proliferation, lineage −/low cells were stimulated with 100 ng/ml IgE in the presence or absence of Akt inhibitor VIII or JNK inhibitor SP600125 for 5 days. Inhibition of pJNK significantly abrogated the elevated GMP number induced by IgE without affecting GMP levels at basal condition. Nevertheless, inhibition of Akt phosphorylation reduced GMP number at both basal and IgE condition (Fig. 5G, 5H). Put together, these data hint that the elevation of GMP proliferation induced by IgE is mediated via phospho-JNK.

Regulation of FcεRIα Expression By GATA2
Previous data have shown that GATA1 and GATA2 are required for regulating FcεRIα mRNA and protein expression in mast cells [16]. We then tested if FcεRIα expression in GMP was also regulated by GATA2. β2+/+ and β2−/− GMP were sorted by FACS. qPCR data demonstrated that GATA2 was more highly expressed while GATA1 was less expressed in β2−/− GMP compared with β2+/+ GMP (Fig. 6A, 6B). When protein lysates of CMP or GMP were subjected for capillary western blot, GATA1 expression was reduced in both β2−/− CMP and β2−/− GMP compared with their correspondent controls (Fig. 6C, 6D). GATA2 expression was increased in β2−/− GMP but not in β2−/ CMP when compared with their correspondent controls (Fig. 6E).
To elucidate the transcriptional regulation of GATA2, HEK293 cells were co-transfected with a plasmid containing GATA2 cDNA and plasmid containing the FcεRIα promoter with luciferase as a reporter. Quantified by luciferase assay, FcεRIα promoter activity was increased. By contrast, when the binding sequences of GATA2 in the FcεRIα promoter region were mutated, transcription activity was dramatically decreased, suggesting direct regulation of GATA2 on FcεRIα (Fig. 6F). Figure 6. Transcriptional regulation of FcεRIα by GATA2. GMP were sorted out by FACS. After extracting RNA, qRT-PCR analysis the change of GATA1 (A, n = 3-5) and GATA2 (B, n = 6-9) in GMP cells. Gene expression was normalized to β-actin. (C-E): β2+/+ and β2−/− CMP and GMP were isolated by FACS sorting. They were lysed for protein extraction. After protein concentration determination, equal amount of proteins was separated by capillary western blot. GATA1, GATA2, and GAPDH expression were studied. Each sample was pooled from 2 to 4 mice. n = 3-6. (F): Dual luciferase reporter gene assay to analysis that whether GATA2 could be combined with FcεRIα. For FcεRIα promoter-mutant (mt) plasmid, the binding sequence of GATA2 on FcεRIα promoter region was mutated. Firefly luciferase activity was normalized to Renilla luciferase activity. n

DISCUSSION
Maintenance of the blood system is a tightly controlled process which involves differentiation from HSC to progenitors and from progenitors to terminal differentiated blood cells [17]. In response to infection, HSC become activated to generate immune cells and myeloid cells to enhance the immune defense in the body. Once the acute infection is resolved, HSC returns to a quiescent status to retain stemness [18]. However, prolonged myelocytosis can be caused by sustained GMP proliferation even if HSC are quiescent [19,20]. Compared with the vast number of studies exploring the regulation of HSC and HSPC biology, our knowledge of how myeloid progenitors are activated and differentiated is limited.
Integrin β2 mediates cell adhesion to endothelial cells and stromal cells. Intriguingly, we demonstrated previously that β2 deficient mice display increased GMP frequency and myelocytosis. Therefore, we initiated this study to examine the role of integrin β2 in GMP proliferation. The key findings are summarized as follow: (a) β2 deficiency increased GMP proliferation, leading to myeloid cell expansion; (b) β2 deficiency enhanced GATA2 expression which could transcriptionally activate FcεRIα expression. Binding of IgE to its receptor, FcεRIα, potently induced GMP proliferation which was mediated via JNK phosphorylation; (c) transplantation of FcεRIα hi and FcεRIα lo GMP resulted in comparable granulocyte and monocyte production; and (d) β2 deficient FcεRIα hi GMP had greater capacity in giving rise to granulocytes and monocytes compared with wide type FcεRIα hi GMP. Thus, GMP proliferation is delicately regulated by integrins on cell membrane and cytokines from the microenvironment [21,22]. Our proposed model demonstrating how β2 deficiency promoted GMP proliferation is presented in Figure 7.
This study provides an example stating of how GMP proliferation could cause exuberant myeloid cell expansion. We demonstrate that myelocytosis does not depend on HSC and HSPC activation but can result solely from myeloid progenitor proliferation. Nagareddy et al. reported that hyperglycemia stimulated GMP proliferation which was the core for monocytosis and neutrophilia in type 1 diabetes mellitus, even if HSC and HSPC frequency was not affected by the hyperglycemia in mice [13]. Mechanistically, hyperglycemia increased reactive oxygen species production and promoted S100A8 and S100A9 secretion from neutrophils in BM. Binding of S100A8 and S100A9 to RAGE on CMP triggered M-CSF production. In conjunction with GM-CSF, they induced CMP and GMP proliferation [13]. Here, we observed increased S100A9 levels in BM fluid of β2−/− mice but RAGE expression in GMP did not differ between the two groups.
Accumulative evidence has shown that IL-3, IL-6, GM-CSF, TNF-α, and S100A8/A9, by acting through their receptors on GMP, are able to promote myeloid cell production [6][7][8][9][10][11][12]. Except these conventional stimulators, our study verified that integrin β2 could control GMP proliferation. Leukocyte adhesion deficiency (LAD) is a human disease caused by β2 integrin deficiency [23] and mutations in the protein Kindlin-3 also causes a LAD (type III) syndrome [24]. It is well known that Talin and Kindlin bind to the cytoplasmic domains of integrin β2 and mediate downstream signal transduction of the integrin upon ligand binding [25]. Recently, the role of Kindlin-3 in hematopoiesis was uncovered. Using Kindlin-3-deficient mice, they showed that Kindlin-3-deficient HSC were quiescence and remained in the BM but became hyperactivated and lost in the circulation when they were transplanted into irradiated recipients [26]. These observations together with our findings expand our understanding of integrin β2 from cell adhesion to regulation of hematopoiesis and myelopoiesis. GATA1 is required for the maturation of red blood cells, megakaryocytes, and mast cells [27] whereas GATA2 is a mediator of hematopoiesis, especially myeloid lineage differentiation [28]. Haploinsufficiency of GATA2 is implied in myelodysplastic syndrome and acute myeloid leukemia [29]. GATA1 and GATA2 have been identified transcription factors to FcεRI transcription in mast cells [16]. FcεRI consists of α-, β-, and γ-chains.
Although transfection of small interfering RNAs (siRNAs) against GATA1 and GATA2 inhibit FcεRI expression in mast cell line, they regulate FcεR1 transcription in different manners. GATA2 siRNA suppresses both α and β transcript but GATA1 siRNA only suppresses the α transcript [16]. In our study, we observed increased mast cell number in blood and BM of β2−/− mice and increased GATA2 expression in β2−/− GMP, compared with wide type controls. In line with previous reports [15,16], we confirmed that GATA2 could regulate FcεRIα transcription by luciferase reporter assay. These data indicate that GATA2 rather than GATA1 could be the driven force for mast cell production in β2−/− mice. In addition, in vitro, we demonstrated that the addition of IgE induced GMP expansion FcεRIα + lineage −/low cells rather than FcεRIα − lineage −/low cells. Although we do not know how β2 deficiency increased GATA2 expression, transcriptional activation of the GATA2 target gene, FcεRIα, was the core for GMP proliferation in β2−/− mice.
The present study must be interpreted within the context of some potential limitations. FcεRIα hi GMP are generally considered as mast progenitors [30]. First, we observed greater myeloid cell production in recipients transplanted with β2−/− BMC, which could be resulted from both impaired myeloid cell retention in BM niche and skewed GMP proliferation; second, we could only quantify FcεRIα + CD45 + cKit + mast cells to assess their production. Alternatively, histamine levels were elevated in the blood and BM fluid in β2−/− mice compared with their littermate controls. Third, we do not know how IgE levels were increased in the PB and BM fluid in β2−/− mice. Even though, a recent study reported that patients with type I LAD have increased risk of autoimmune complications [31]. And fourth, we do not know which partner of β2 participated in GMP proliferation in the study. It is well known that integrin β2 has four partners: CD11a, CD11b, CD11c, and CD11d. As reported in the literature, subjects with LAD disease were absence of CD18, CD11b, CD11c but CD11a expression and function was present [32][33][34]. Therefore, how β2 integrins participate in immunity and which partner involves in skewed GMP proliferation need further investigation.
In conclusion, β2 deficiency upregulated GATA2/FcεRIα expression in GMP. Binding of IgE to FcεRIα stimulated GMP proliferation which was abrogated by the inhibition of JNK phosphorylation. Therefore, GMP became sustained proliferation for enhanced myeloid cell production.

CONCLUSION
HSC activation and sustained GMP proliferation both contribute to myeloid lineage production. Herein, we demonstrated a novel function for the GATA2/ FcεRIα/pJNK axis in GMP proliferation. In the absence of integrin β2, GATA2 expression was increased which could transcriptionally activate its target gene, FcεRIα. Binding IgE to FcεRIα induced JNK phosphorylation leading to GMP expansion. Ultimately, GMP become constitutively activated for proliferation, leading to myelocytosis.