1
Polyinosinic-polycytidylic acid induces CXCL1 expression in cultured hCMEC/D3 human cerebral microvascular endothelial cells.
㸦ᇵ㣴
hCMEC/D3
ࣄࢺ⬻ᚤᑠ⾑⟶ෆ⓶⣽⬊࠾࠸࡚polyinosinic-
polycytidylic acid
ࡣCXCL1
ࡢⓎ⌧ࢆㄏᑟࡍࡿ㸧⏦ㄳ⪅ᘯ๓ᏛᏛ㝔་Ꮫ◊✲⛉
⬻⚄⤒⛉Ꮫ㡿ᇦ⬻⚄⤒እ⛉Ꮫᩍ⫱◊✲ศ㔝
Ặྡᮤ㞵㎮
ᣦᑟᩍᤵ⇃ὒ
2
Abstract
Objective: Brain microvascular endothelial cells are integral components of blood-brain barrier (BBB),
and play a role to protect brain from invading microbes. CXC motif chemokine ligand 1 (CXCL1) induces the chemotaxis of neutrophils, and neutrophils are important in host defense in the brain.
However, dysregulated neutrophil infiltration leads to brain diseases. Toll-like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double-stranded RNA (dsRNA), and the aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endothelial cells. Methods: The hCMEC/D3 human cerebral microvascular endothelial cells were cultured and treated with polyinosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. Production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The expression of CXCL1 was compared with that of CXCL8. Effect of pretreatment of cells with a NF-NB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N- terminal kinase (JNK) inhibitor (SP600125), an IRF3 inhibitor (MRT67307) and an anti-type I IFN neutralizing antibody mixture was examined. Phosphorylation of p38 was examined using western blotting. Results: Treating cultured hCMEC/D3 human cells with poly IC induced the expression of CXCL1 as well as another chemokine CXCL8. Pretreatment of cells with SN50, SB203580, and SP600125 decreased the induction of CXCL1 by poly IC. However, it was not affected by MRT67307 or by an anti-type I IFN neutralizing antibody mixture. Pretreatment of cells with SN50 decreased the poly IC-induced phosphorylation of p38. Conclusions:Poly IC induces the expression of CXCL1 in hCMEC/D3 cells. NF-NB, p38 MAPK and JNK are involved in this reaction. There is a cross-talk between NF-NB and p38, and NF-NB partially regulates phosphorylation of p38. CXCL1 produced by brain microvascular endothelial cells may contribute to the brain’s defense against viral infection and various neurological diseases associated with neutrophil accumulation.
3 Introduction
Chemokines play important roles in the inflammatory reactions by attracting leukocytes to infected sites. CXC motif chemokine ligand 1 (CXCL1) is a member of CXC subfamily of chemokines [1]. CXCL1 was originally identified as a factor that promotes the growth of melanoma and was named as growth-related oncogene-D [2]. The biological activities of CXCL1 on target cells are revealed through the activation of CXC receptor (CXCR) 2, a G protein-coupled seven-transmembrane receptor [3]. CXCR2 is expressed in neutrophils and CXCL1/CXCR2 axis induces chemotaxis of
neutrophils [3]. In addition, CXCL1/CXCR2 axis is associated with bone marrow development, inflammation, angiogenesis, and wound healing [4].
In the brain, CXCR2 is expressed in neurons [5], microglia [6] and oligodendrocyte progenitor cells [7]. The CXCL1/CXCR2 axis may be associated with the pathogenesis of various diseases in brain.
For example, accumulation of CXCL1 and CXCR2 in the hippocampi of monkeys after ischemia- reperfusion [8], and an increase of CXCL1 in the brains extracted from mice after infecting them with herpes simplex encephalitis [9]. CXCL1 produced by astrocytes may contribute to the neutrophil infiltration and demyelination induced by mouse hepatitis virus [10]. Moreover, CXCL1 and CXCR2 contribute to chronic stress-induced depression in mice [11]. The expression of CXCR2 is upregulated in microglia in Alzheimer’s disease [6]. A high level of CXCL1 protein was detected in a cerebral microdialysis sample from patients with a severe traumatic brain injury [12].
Pathogen-associated molecular patterns are recognized by pattern recognition receptors once pathogens invade into the tissues. Pattern recognition receptors also recfognize danger-associated molecular patterns released from damaged or injured cells. The binding of ligands to pattern recognition receptors trigger the innate immune reactions and Toll-like receptors (TLRs) are
members of pattern recognition receptors [13]. Among TLRs, TLR2, TLR3, TLR4 and TLR6 have been
4 reported to be expressed in human cerebral endothelial cells [14]. TLR3 is a receptor for double- stranded RNA (dsRNA), which is produced by most viruses during their replication. Binding of dsRNA to TLR3 activates antiviral innate immune reactions via type I interferon (IFN)-dependent or -
independent manner [15]. The nuclear factor (NF)-NB and IFN regulatory factor 3 (IRF3) are major transcriptional factors in the downstream of TLR3 signaling [16].
Brain microvascular endothelial cells are an integral component of the blood-brain barrier (BBB) and have multiple barrier functions to protect brain from invading microbes [17]. In brain
microvascular endothelial cells, activation of TLR3 with polyinosinic-polycytidylic acid (poly IC) induces the expression of several CC and CXC chemokines including CCL2 [18, 19], CCL5 [18, 20], CXCL8 [18] and CXCL10 [21]. However, it is not known if brain endothelial cells express CXCL1 in response to TLR3 activation.
Poly IC is a synthetic dsRNA and is a potent agonist for TLR3. In this study, we examined the effect of poly IC on the expression of CXCL1 in cultured hCMEC/D3 cells, a cell line derived from human brain microvascular endothelial cells [22]. We also compared the expression pattern of CXCL1 with that of CXCL8, which also induces the chemotaxis of neutrophils. In this reaction, the roles of NF-NB, IRF3, type-I IFN, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in this reaction were also investigated. In addition, cross-talk of these signaling molecules was examined.
Materials and Methods
Reagents
5 A poly IC and an IRF3 inhibitor MRT67307 were purchased from Sigma (St. Louis, MO, USA). An NF-NB translocation inhibitor SN50 was obtained from ENZO Life Science (Farmingdale, NY, USA). A human type I IFN neutralizing antibody mixture was purchased from PBL assay science (Piscataway, NJ, USA). A p38 MAPK inhibitor SB203580 was from BIOMOL (Hamburg, Germany), and a JNK inhibitor SP600125 was obtained from Merk Millipore (Temecula, CA, USA). An M-MLV reverse transcriptase was from Thermo Fisher Science (Asheville, NC, USA). The illustra RNA spin kin was purchased from GE Healthcare (Buckinghamshire, UK). Oligo (dT)18 for reverse transcription and oligonucleotide primers for polymerase chain reaction (PCR) were synthesized by Fasmac (Atsugi, Japan). Ssoadvanced Universal SYBR Green Supermix was from Bio-Rad (Hercules, CA, USA), and the enzyme-linked immunosorbent assay (ELISA) kits for CXCL1 (human CXCL1/GRO alpha quantikine ELISA kit, DGR00B) and CXCL8 (human IL-8/CXCL8 quantikine ELISA kit, D8000C) were purchased from R&D systems (Minneapolis, MN, USA). Rabbit antibodies against phosphorylated p38 (9215), p38 (9212), phosphorylated NF-NB p65 (3033), p65 (4764), phosphorylated JNK (9251) and JNK (9252) were from Cell Signaling Technologies (Danvers, MA, USA).
Cell culture and treatment
The hCMEC/D3 human cerebral microvascular endothelial cells were obtained from Merk Millipore. The cells were cultured in the endothelial growth medium-2 (Lonza, Walkersville, MD, USA) [20], and were treated with 0.4 - 50 Pg/mL poly IC for up to 24 h. In the experiments to examine the effect of inhibitors, the cells were pre-incubated for 1 h with 10 PM SN50, 1 PM MRT67307, a human type I IFN neutralizing antibody mixture (1:20 dilution), 10 PM SB203580 or 2 PM SP600125 before adding 30 Pg/mL poly IC.
6 RNA extraction and real-time RT-PCR
RNA was extracted from the cells using an Illustra RNAspin kit, and reverse-transcription was performed using oligo (dT)18 primer and M-MLV reverse transcriptase. The cDNA for CXCL1, CXCL8, CXCL10 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified with 40 cycles using specific primers and SsoAdvanced Universal SYBR Green Supermix. The primers used were as follows. CXCL1-F: 5’-ATGGCCCGCGCTGCTCTCTCC-3’, CXCL1-R: 5’-GTTGGATTTGTCACTGTTCAG-3’, CXCL8-F: 5’-AGGAGTGCTAAAGAACTTCGA-3’, CXCL8-R: 5'-TGAATTCTCAGCCCTCTTCAA-3’, CXCL10-F:
5’-TTCAAGGAGTACCTCTCTCTAG-3’, CXCL10-R: 5’-CTGGATTCAGACATCTCTTCTC-3’, GAPDH-F: 5’- GCACCGTCAAGGCTGAGAAC-3’ and GAPDH-R: 5’-ATGGTGGTGAAGACGCCAGT-3’.
ELISA
The conditioned medium was collected after the incubation and centrifuged. The
concentration of CXCL1 and CXCL8 in the supernatant was measured using commercially available ELISA kits.
Western blotting
The cells were pretreated with inhibitors for 1 h as above, and were treated with 30 ʅg/mL poly IC for an additional 1 h. The cells were lysed using Laemmli’s buffer and subjected to 5-20 % polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane, and the membrane was incubated with a rabbit antibody against phosphorylated p38 (1:1000), p38 (1:1000), phosphorylated NF-NB p65 (1:1000), p65 (1:1000), phosphorylated JNK
7 (1:1000) or JNK (1:1000). A horseradish peroxidase-conjugated anti-rabbit IgG antibody was used as a secondary antibody, and the bands were detected using a chemiluminescence substrate.
Results
In the absence of an agonist, the mRNA and protein expression levels of CXCL1 were low in cultured hCMCE/D3 cells in comparison with poly IC-treated cells. The treatment of cells with 0.4–50 Pg/mL poly IC upregulated the expression of CXCL1 mRNA (Figure 1a) and the secretion of CXCL1
protein (fig. 1b) in a concentration-dependent manner. Concentration-dependent expression of CXCL1 was similar to that of CXCL8 (fig. 1ab). The time course of the expression of CXCL1 and CXCL8 was shown in Figure 2. The CXCL1 mRNA level rapidly increased after treatment. It reached the maximal level at 2 h, decreased at 4 h and almost plateaued thereafter (fig. 2a). The time course of CXCL8 mRNA expression was similar to that of CXCL1 (fig. 2a). The secretion of CXCL1 protein in the conditioned medium reached maximal level at 16 h and then plateaued, while the level of CXCL8 protein gradually increased up to 24 h (fig. 2b).
Pretreatment of cells with SN50 resulted in significant decrease in the expression of CXCL1 and CXCL8 mRNA (ILJ 3a) and protein (ILJ 3b) induced by poly IC. The poly IC-induced expression of CXCL1 and CXCL8 mRNA was not affected by pretreatment with MRT67307 (ILJ 4) nor with an anti- type I IFN antibody mixture (ILJ 5). The induction of CXCL10 by poly IC was decreased by these pretreatments.
The poly IC-induced expression of CXCL1 and CXCL8 mRNA and protein was partially, but significantly, decreased by the pretreatment of cells with SB203580 (ILJ 6). The pretreatment of cells with SP600125 partially inhibited the induction of CXCL1 mRNA and protein in cells treated with poly IC for 24 h, while CXCL1 mRNA expression in cells treated with poly IC for 2 h was not changed
8 (ILJ 7). Conversely, SP600125 inhibited poly IC-induced CXCL8 mRNA at 2 h and at 24 h, and CXCL8 protein secretion also decreased (ILJ 7).
In order to examine the cross-talk between p38, JNK and NF-NB signaling pathways, we next examined the effect of inhibitors on the phosphorylation of these signaling molecules. Treatment of cells with poly IC for 1 h significantly induced the phosphorylation of p38, and pretreatment of cells with SN50 partially inhibited the increase of phosphorylated p38 (fig. 8). Pretreatment with
SP600125 did not affect p38 phosphorylation (fig. 8). Neither SB203580 nor SP600125 inhibited the phosphorylation of NF-NB p65, and phosphorylation of JNK was not changed by SN50 or SB203580 (data not shown).
Discussion
Neutrophils release antimicrobial and pro-inflammatory molecules, and play an essential role in physiological and pathological inflammatory reactions. Although neutrophils are thought to mediate central processes in host defense against bacterial infections, neutrophils are also involved in anti-viral reactions. A previous study reported that neutrophil-depleted mice infected with neurotropic JHM strain of mouse hepatitis virus exhibited increased levels of viral replication in the brain compared with the control mice [23]. Additionally, there was a decrease in the BBB
permeability and mononuclear leukocyte infiltration into the brain in these neutropenic mice [23].
Matrix metalloproteinases (MMPs) secreted from neutrophils may degrade extracellular matrix proteins in the basal lamina of the BBB, and the BBB integrity can decline [23]. Loosening of BBB may be followed by infiltration of virus-specific CD8+ T cells across the BBB and migration into the
parenchyma of brain [23]. However, there is a possibility that the loss of BBB integrity and a decline in brain homeostasis increase the risk for various neurological diseases including encephalitis, inflammation, autoimmune disorders, and neurodegenerative diseases. Sustained CXCL1 expression
9 amplifies the severity of white matter damage in mice infected with neurotopic JHM strain of mouse hepatitis virus [24].
In the present study, we found that CXCL1 was synthesized and secreted when cultured hCMEC/D3 cells were treated with a TLR3 agonist poly IC. The CXCL1 secreted from brain
microvascular endothelial cells may induce chemotaxis of neutrophils, which may take a protective role against viral encephalitis in the mechanisms mentioned above. The time course of the CXCL1 mRNA expression was similar to that of CXCL8, while the time course of secretion of CXCL1 protein was different from that of CXCL8.
When brain vascular endothelial cells are appropriately stimulated with viral dsRNA, CXCL1 may function to protect brain. However, dysregulation of CXCL1 may lead to various pathologic
inflammations in brain. In order to maintain the homeostasis of brain, the expression of CXCL1 in brain microvascular endothelial cells should be tightly regulated. Therefore, we next examined the molecular mechanisms by which CXCL1 expression induced by poly IC was regulated. The NF-NB and IRF3 are the major transcriptional factors in the signaling pathways mediated by TLR3 [16], and NF- NB is known to be a key molecule in CXCL1 expression induced by other stimuli [25]. SN50, an
inhibitor of NF-NB translocation, inhibited the poly IC-induced expression of CXCL1 and CXCL8, while an IRF3 inhibitor MRT67307 did not. Although type I IFN is a key cytokine in immune response in the downstream of TLR3 signaling, pretreatment with anti-type I IFN neutralizing antibody mixture did not affect the poly IC-induced CXCL1 and CXCL8 expression. The poly IC-induced expression of CXCL10, another member of CXC chemokines, was inhibited by either MRT67307 or by an anti-type I IFN neutralizing antibody mixture. The P38 MAPK is reported to be involved in the expression of interleukin (IL)-6, CXCL8 and CXCL10 induced by poly IC in human neonatal dermal microvascular endothelial cells [26]. However, it is unknown if p38 MAPK is involved in TLR3 signaling in brain microvascular endothelial cells. In the present study, the pretreatment of hCMEC/D3 cells with a p38 MAPK inhibitor SB203580 decreased the expression of CXCL1 and CXCL8 induced by poly IC. This
10 suggests that p38 MAPK is involved in the poly IC-induced CXCL1 and CXCL8 expression in brain microvascular endothelial cells. JNK is another signaling molecule involved in inflammatory reactions and TLR3 signaling induces IL-6 via JNK in brain vascular endothelial cells [27]. In the present study, the pretreatment of hCDEC/D3 cells with a JNK inhibitor SP600125 resulted in a decrease of CXCL1 and CXCL8 protein expression induced by poly IC. SP600125 also decreased both of CXCL1 and CXCL8 mRNA expression in cells treated with poly IC for 24 h. However, in cells treated with poly IC for 2 h, SP600125 inhibited the expression of CXCL8 mRNA, but not of CXCL1 mRNA. This suggests that JNK is involved in the expression of CXCL1 and CXCL8 induced by poly IC, but there may be some complex mechanisms by which JNK regulates the expression of these similar chemokines.
Intracellular signaling pathways after inflammatory stimuli are complex, and there are cross-talk among NF-NB and mitogen-activated protein kinase family including p38 and JNK [28]. However, the cross-talk among these signalings in human cerebral endothelial cells after TLR3 activation is not known. We found in the present study that poly IC-induced phosphorylation of p38 was partially inhibited by pretreatment of cells with SN50. This indicates that there is a crosstalk between NF-NB and p38, and activation of p38 is at least partly induced via NF-NB activation.
CXCR2 is expressed in brain endothelial cells, and CXCL1 induces the expression of adhesion molecules in brain endothelial cells [29] and angiogenesis [30] in CXCR2-dependent manner.
Therefore, CXCL1 produced by brain endothelial cells via TLR3 signaling may function to activate endothelial cells in an autocrine fashion.
In summary, poly IC induces the expression of CXCL1 in hCMEC/D3 cells in a similar manner to CXCL8, and NF-NB, p38 MAPK and JNK are involved in this reaction. Activation of NF-NB signaling may partially contribute to p38 signaling. The CXCL1 produced by brain microvascular endothelial cells may be involved in innate antiviral immunity in the brain.
11 Statement of Ethics
No human participant was involved in this study.
Disclosure Statement
The authors have no conflicts of interest to declare.
Funding Sources
This research was supported by Hirosaki University Institutional Research.
12 Author Contributions
Yuchen Li contributed to cell culture, real-time RT-PCR and ELISA
Tadaatsu Imaizumi contributed to cell culture, real-time RT-PCR, ELISA and study design
Tomoh Matsumiya contributed to cell culture
Kazuhiko Seya contributed to cell culture
Shogo Kawaguchi contributed to real-time PCR and ELISA
Hiroki Okuma contributed to study design
13 References
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17 Figure Legends
Fig. 1. Treatment of hCMEC/D3 cells with poly IC induces the expression of CXCL1 and CXCL8 in a concentration-dependent manner. (a) The hCMEC/D3 cells were treated with 0.4 - 50 Pg/mL poly IC for 2 h. RNA was extracted from the cells and reverse-transcribed to cDNA, which was used as a template for quantitative real-time RT–PCR analysis for CXCL1, CXCL8 and GAPDH. (b) The cells were treated with poly IC as in (a) and incubated for 24 h. The conditioned medium was collected from the cells and the concentration of CXCL1 and CXCL8 proteins in the medium was examined using ELISA kits.
Fig. 2. The treatment of hCMEC/D3 cells with poly IC results in the induction of CXCL1 and CXCL8 expression in a time-dependent manner. The cells were treated with 30 Pg/mL poly IC for up to 24 h.
(a) RNA was extracted from the cells and subjected to quantitative real-time RT–PCR analysis for CXCL1, CXCL8 and GAPDH expression as in Fig. 1. (b) The concentration of CXCL1 and CXCL8 proteins in the conditioned medium was examined using ELISA kits.
Fig. 3. The pretreatment of cells with SN50, an inhibitor of NF-NB translocation, results in the decrease in poly IC-induced expression of CXCL1 and CXCL8. The cells were pretreated with 10 ʅg/mL SN50 for 1 h, followed by treatment with 30 ʅg/mL poly IC. (a) After an additional 2 or 24 h
incubation, RNA was extracted from the cells and real-time RT-PCR was performed. (b) After an additional 24 h incubation, the conditioned medium was collected and concentration of CXCL1 and CXCL8 proteins in the medium was examined using ELISA kits. ** p < 0.01 by t-test.
18 Fig. 4. MRT67307 has no effect on the expression of CXCL1 and CXCL8 induced by poly IC but inhibits CXCL10 expression in hCMEC/D3 cells. The cells were pretreated with 10 Pg/mL MRT67307. After 1 h treatment, the cells were treated with 30 ʅg/mL poly IC. RNA was extracted from the cells after an additional 2 or 24 h incubation, and subjected to real-time RT-PCR for CXCL1, CXCL8, CXCL10 and GAPDH. ** p < 0.01 by t-test.
Fig. 5. Type I IFN is not involved in the expression of CXCL1 and CXCL8 induced by poly IC but involved in CXCL10 expression. The cells were pretreated with an anti-type I IFN antibody mixture (1:20) for 1 h, and followed by treatment with 30 Pg/mL poly IC for an additional 2 or 24 h. Real-time RT-PCR was performed as in Figure 4. ** p < 0.01 by t-test.
Fig. 6. P38 MAPK is involved in poly IC-induced expression of CXCL1 and CXCL8. The cells were pretreated with 10 PM SB203580, an inhibitor of p38 MAPK, for 1 h. (a) After the cells was treated with 30 Pg/mL poly IC for an additional 2 or 24 h, the cells were subjected to real-time RT–PCR for CXCL1, CXCL8 and GAPDH. (b) After an additional 24 h incubation with 30 Pg/mL poly IC, the medium was collected and subjected to ELISA for CXCL1 and CXCL8. ** p < 0.01 by t-test.
Fig. 7. JNK is involved in poly IC-induced expression of CXCL1 and CXCL8. The cells were pretreated for 1 h with 2 PM SP600125, an inhibitor of JNK, which was followed by treatment with 30 Pg/mL poly IC. (a) RNA was extracted from the cells after an additional 2 or 24 h incubation and subjected to real-time RT–PCR for CXCL1, CXCL8 and GAPDH. (b) After an additional 24 h incubation, the conditioned medium was collected and concentration of CXCL1 and CXCL8 proteins in the medium was examined using ELISA kits. * p < 0.05, ** p < 0.01 by t-test.
19 Fig. 8. SN50 partially inhibits phosphorylation of p38 induced by poly IC. The cells were pretreated with 10 ʅg/mL SN50 or 2 PM SP600125 for 1 h, followed by treatment with 30 ʅg/mL poly IC. After an additional 1 h incubation, the cells were lysed and western blotting for phosphorylated p38 (p- p38) and p38 was performed.
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63
