Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Apr 15;180(8):5645-52.
doi: 10.4049/jimmunol.180.8.5645.

Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo

Jonathan K Alder  1 , Robert W Georgantas 3rd, Richard L Hildreth, Ian M Kaplan, Sebastien Morisot, Xiaobing Yu, Michael McDevitt, Curt I Civin
Affiliations

Kruppel-like factor 4 is essential for inflammatory monocyte differentiation in vivo

Jonathan K Alder et al. J Immunol. .

Abstract

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4(-/-) fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4(+/+) cells in competitive repopulation assays. However, hematopoietic "KLF4(-/-) chimeras" generated by transplantation of KLF4(-/-) fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115(+)Gr1(+)) monocytes, and had reduced numbers of resident (CD115(+)Gr1(-)) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4(-/-) chimeras, bone marrow monocytic cells from KLF4(-/-) chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
KLF4 was expressed at high levels in BM monocytic cells and activated macrophages. A–C, Committed B cells (CD45+B220+), CD4 T cells (CD45+CD3+CD4+), CD8 T cells (CD45+CD3+CD8+), and NK cells (CD45+NK1.1+) were FACS-sorted from C57BL/6 splenocytes. Erythroid (CD45−Ter119+) and monocytic cells (CD45+CD115+Gr1dimF4/80+) were FACS-sorted from BM. RNA was harvested, and qRT-PCR was performed in triplicate and normalized to hydroxymethylbilane synthase. D, Peritoneal macrophages were exposed to LPS for 24 h. Data are representative of n = 3 experiments, and error bars represent SEM. E, Western blots of cell lysates from whole BM, spleen, thymus, and purified CD115+ BM monocytic cells. Cells were cultured for 5 h in the presence of the proteasome inhibitor MG132 before harvesting for Western blot. F, Densitometric analysis of blot in E; KLF4 expression was normalized to GAPDH expression in each cell type.
FIGURE 2
FIGURE 2
Loss of KLF4 did not affect HPC or HSC function. A, Five × 104 E14.5 FL cells were plated in methylcellulose CFC assay cultures containing hematopoietic cytokines (see Materials and Methods). Colonies were counted 7 days after plating. Data is representative of n = 6 for KLF4+/+ or KLF4+/− and n = 3 for KLF4−/− FL cells. In several experiments, we observed only a slight difference between KLF4+/+ and KLF4+/− chimeras, so we have grouped these in several (indicated) experiments. Error bars, SEM; *, p = 0.031. B, Competitive repopulation assay comparing donor cell repopulating capacity of 1) KLF4+/+ CD45.2+ FL vs CD45.1+KLF4+/+ BM (●, solid line) to that of 2) CD45.2+KLF4−/− FL vs CD45.1+KLF4+/+ BM (□, dotted line). Percents FL-derived (CD45.2+) granulocytes are plotted over 40 wk. C, Complete blood cell counts were performed on blood from KLF4+/+ (n = 5), KLF4+/− (n = 5), and KLF4−/− (n = 10) chimeras. Before performing complete blood cell counts, donor chimerism of >95% was confirmed by flow cytometry.
FIGURE 3
FIGURE 3
Loss of KLF4 disrupted late monocytic differentiation. A, Representative FACS plots of BM, blood, and peritoneal exudates (PE) isolated from KLF4+/+ or KLF4+/− and KLF4−/− chimeras 10 wk after transplant. Cells were stained with indicated mAbs. Cells are morphologically gated on total viable cells except for peripheral blood, which is gated based on monocytic light scattering. In all plots, cells are gated on CD45.2+ cells (donor-derived). Arrow added to emphasize lack of inflammatory monocytes in KLF4−/− chimeras’ blood. B, Percent donor-derived monocytic cells (CD45.2+CD115+Gr1+ or CD45.2+CD115+Gr1−) or macrophages (CD45.2+CD115+F4/80+) found in BM, PB, and PE from KLF4+/+ or KLF4+/− and KLF4−/− chimeras. The graphs shown are the combined results of ≥ three independent FL transplants with n ≥ 5 in all experiments. * and **, p = 0.0009 and p < 0.0001, respectively. C, Similar to B except total number of cells is plotted. *** and ## indicate p = 0.0012 and p = 0.0129, respectively. D, Numbers of Gr1+ and Gr1− monocytes in peripheral blood from KLF4+/+ or KLF4+/− and KLF4−/− chimeras. Total white blood cell count × percent monocytes by flow cytometry determined the total numbers of monocytes. ** and #, p < 0.0001 and p = 0.0016, respectively. E, Percent Annexin V-positive BM monocytic cells (CD45.2+CD115+Gr1+AnnexinV+). ###, p = 0.0008.
FIGURE 4
FIGURE 4
BM monocytic cells from KLF4−/− chimeras had dysregulated differentiation and cell surface molecules. A, Representative histograms of CD45.2+CD115+Gr1+ BM monocytic cells from KLF4+/+ (shaded) and KLF4−/− (empty) chimeras. B, Normalized mean fluorescence intensity of indicated molecules from CD115+Gr1+CD45.2+ BM monocytic cells. n = 8 from ≥ three independent FL transplants from KLF4+/+ or KLF4+/− and KLF4−/− chimeras; *, p < 0.0001. C, qRT-PCR of mRNA from KLF4+/+ or KLF4+/− or KLF4−/− BM monocytes. n = 3 from three independent FL transplants; ** and ***, p = 0.03 and p = 0.02, respectively.
FIGURE 5
FIGURE 5
Loss of KLF4 did not reduce iNos activity or TNF-α mRNA levels. Thioglycolate-stimulated peritoneal macrophages were harvested from KLF4+/+ or KLF4+/− and KLF4−/− chimeras and allowed to adhere to tissue culture plates overnight. Cells were washed three times and medium was added with the indicated amounts of LPS. A, After 24 h of culture in the presence of LPS, Greiss assays were performed on supernatants from each well and cells were harvested for RNA analysis. B, TNF-α mRNA levels, measured by qRT-PCR (n = 2).
FIGURE 6
FIGURE 6
Induced expression of KLF4 in HL60 cells induced proliferative arrest, p21, and features of monocytic differentiation. In brief, 105 FUGW-transduced and KLF4-ER-transduced HL60 cells were plated, and viable cells were counted each day using trypan blue. 4HT or ethanol was added to each well on day 0, and cells were split 1/2 when they reached 106 cells/ml. Shown are representative results from one of three independent clones. B, Day 2 HL60 cells were stained for DNA content with PI. C, Results are from a single experiment in which KLF4-ER-transduced HL60 cells were treated with 4HT and harvested at 6, 24, and 48 h after treatment. qRT-PCR results were normalized to KLF4-ER cells treated with ethanol. D, KLF4-ER-transduced HL60 cells were treated with ethanol (shaded) or 4HT (empty) for 48 h and then stained with the indicated mAbs. E and F, Cytospins of KLF4-ER-transduced HL60 cells cultured for 5 days in the presence of vehicle (ethanol; E) or 4HT (F).
FIGURE 7
FIGURE 7
KLF4 expression enhanced macrophage differentiation and blocked granulocytic differentiation. A, HL60 cells were cultured in the presence of TPA (50 ng/ml) and/or 4HT for 48 h. Photomicrographs were taken at 60× magnification and are representative of three independent experiments. B, HL60 cells were cultured for 5 days in the presence or absence of RA (1 μM) and/or 4HT. Cells were immunostained with CD66 and CD14. The plots shown are representative of three independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bieker JJ. Kruppel-like factors: three fingers in many pies. J Biol Chem. 2001;276:34355–34358. - PubMed
    1. Safe S, Abdelrahim M. Sp transcription factor family and its role in cancer. Eur J Cancer. 2005;41:2438–2448. - PubMed
    1. Carlson CM, Endrizzi BT, Wu J, Ding X, Weinreich MA, Walsh ER, Wani MA, Lingrel JB, Hogquist KA, Jameson SC. Kruppel-like factor 2 regulates thymocyte and T-cell migration. Nature. 2006;442:299–302. - PubMed
    1. Bai A, Hu H, Yeung M, Chen J. Kruppel-like factor 2 controls T cell trafficking by activating L-selectin (CD62L) and sphingosine-1-phosphate receptor 1 transcription. J Immunol. 2007;178:7632–7639. - PubMed
    1. Nuez B, Michalovich D, Bygrave A, Ploemacher R, Grosveld F. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature. 1995;375:316–318. - PubMed

Publication types

MeSH terms