Polyinosinic-Polycytidylic Acid Induces CXCL1 Expression in Cultured hCMEC/D3 Human Cerebral Microvascular Endothelial Cells
Yuchen Lia, b Tadaatsu Imaizumib Tomoh Matsumiyab Kazuhiko Seyab
Shogo Kawaguchib Jiangli Dingb Hiroki Ohkumaa
a Department of Neurosurgery, Hirosaki University School of Medicine, Hirosaki, Japan; b Department of Vascular Biology, Hirosaki University School of Medicine, Hirosaki, Japan
Keywords
Blood-brain barrier · Brain capillary vascular endothelial cells · CXCL1 · Poly IC · TLR3
Abstract
Objective: Brain microvascular endothelial cells are integral components of the blood-brain barrier and play a role in pro- tecting the brain from invading microbes. CXC motif chemo- kine 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). The aim of this study was to investigate the effect of an TLR3 agonist on the expression of CXCL1 in brain vascular endo- thelial cells. Methods: hCMEC/D3 human cerebral microvas- cular endothelial cells were cultured and treated with poly- inosinic-polycytidylic acid (poly IC), a potent synthetic dsRNA agonist for TLR3. The production of CXCL1 mRNA and protein was assessed by real-time RT-PCR and ELISA. The ex- pression of CXCL1 was compared with that of CXCL8. The effect of pretreatment of cells with a NF-κB inhibitor (SN50), a p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), an interferon (IFN) regulatory factor 3 inhibitor
(MRT67307), and an anti-type I IFN-neutralizing antibody mixture was examined. Phosphorylation of p38 was exam- ined 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 in- duces the expression of CXCL1 in hCMEC/D3 cells. NF-κB, p38 MAPK, and JNK are involved in this reaction. There is a cross-talk between NF-κB and p38, and NF-κB partially regu- lates phosphorylation of p38. CXCL1 produced by brain mi- crovascular endothelial cells may contribute to the brain’s defense against viral infection and various neurological dis- eases associated with neutrophil accumulation.
Introduction
Chemokines play important roles in inflammatory re- actions by attracting leukocytes to infected sites. CXC motif chemokine ligand 1 (CXCL1) is a member of the CXC subfamily of chemokines [1]. CXCL1 was originally identified as a factor that promotes the growth of mela- noma and was named growth-related oncogene α [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 the CXCL1/
CXCR2 axis induces chemotaxis of neutrophils [3]. In ad- dition, the CXCL1/CXCR2 axis is associated with bone marrow development, inflammation, angiogenesis, and wound healing [4].
In the brain, CXCR2 is expressed in neurons [5], mi- croglia [6] and oligodendrocyte progenitor cells [7]. The CXCL1/CXCR2 axis may be associated with the patho- genesis of various diseases in the brain, e.g., the accumu- lation of CXCL1 and CXCR2 in the hippocampi of mon- keys after ischemia-reperfusion [8], and an increase of CXCL1 in brains extracted from mice after infecting them with herpes simplex encephalitis [9]. CXCL1 produced by astrocytes may contribute to 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 the microglia in Alzheimer’s disease [6]. A high level of CXCL1 protein was detected in cerebral microdialysis samples from patients with a se- vere traumatic brain injury [12].
Pathogen-associated molecular patterns are recog- nized by pattern recognition receptors once the patho- gens invade into the tissues. Pattern recognition receptors also recognize danger-associated molecular patterns re- leased from damaged or injured cells. The binding of li- gands to pattern recognition receptors trigger the innate immune reactions, and Toll-like receptors (TLRs) are members of the pattern recognition receptors [13]. Among the TLRs, TLR2, TLR3, TLR4, and TLR6 have been reported to be expressed in human cerebral endo- thelial 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 an- tiviral innate immune reactions via a type I interferon (IFN)-dependent or -independent manner [15]. Nuclear factor (NF)-κB 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 the brain from in- vading microbes [17]. In brain microvascular endothelial cells, the activation of TLR3 with polyinosinic-polycyti- dylic 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 whether brain endothelial cells express CXCL1 in response to TLR3 activation.
Poly IC is a synthetic dsRNA and 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 endo- thelial cells [22]. We also compared the expression pat- tern of CXCL1 with that of CXCL8, which also induces the chemotaxis of neutrophils. The roles of NF-κB, IRF3, type-I IFN, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) in this reac- tion were also investigated. In addition, cross-talk of these signaling molecules was examined.
Materials and Methods
Reagents
A poly IC and an IRF3 inhibitor (MRT67307) were purchased from Sigma (St. Louis, MO, USA). An NF-κB translocation inhib- itor (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 obtained from BIOMOL (Ham- burg, Germany), and a JNK inhibitor (SP600125) was purchased from Merk Millipore (Temecula, CA, USA). An M-MLV reverse transcriptase was obtained from Thermo Fisher Science (Ashe- ville, NC, USA). The illustra RNA spin kin was purchased from GE Healthcare (Buckinghamshire, UK). Oligo (dT)18 for reverse tran- scription and oligonucleotide primers for polymerase chain reac- tion (PCR) were synthesized by Fasmac (Atsugi, Japan). SsoAd- vanced Universal SYBR Green Supermix was obtained from Bio- Rad (Hercules, CA, USA), and the enzyme-linked immunosorbent assay (ELISA) kits for CXCL1 (human CXCL1/GRO alpha quan- tikine ELISA kit, DGR00B) and CXCL8 (human IL-8/CXCL8 quantikine ELISA kit, D8000C) were purchased from R&D sys- tems (Minneapolis, MN, USA). Rabbit antibodies against phos- phorylated p38 (9215), p38 (9212), phosphorylated NF-κB p65 (3033), p65 (4764), phosphorylated JNK (9251), and JNK (9252) were from Cell Signaling Technologies (Danvers, MA, USA).
Cell Culture and Treatment
hCMEC/D3 human cerebral microvascular endothelial cells were obtained from Merk Millipore. The cells were cultured in en- dothelial growth medium-2 (Lonza, Walkersville, MD, USA) [20]
and were treated with 0.4–50 µg/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 µM SN50, 1 µM MRT67307, a human type I IFN-neutralizing antibody mixture (1:20 dilution), 10 µM SB203580, or 2 µM SP600125 before adding 30 µg/mL poly IC.
ELISA
The conditioned medium was collected after incubation and centrifuged. The concentration of CXCL1 and CXCL8 in the su- pernatant was measured using commercially available ELISA kits.
Western Blotting
The cells were pretreated with inhibitors for 1 h, as described above, and were treated with 30 μg/mL poly IC for an additional 1h. The cells were lysed using Laemmli’s buffer and subjected to 5–20% polyacrylamide gel electrophoresis. Proteins were trans- ferred to a polyvinylidene difluoride membrane, and the mem- brane was incubated with a rabbit antibody against phosphory-(1:1,000), p65 (1:1,000), phosphorylated JNK (1:1,000), or JNK (1:1,000). 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 hCMEC/D3 cells in comparison with those of poly IC- treated cells. The treatment of cells with 0.4–50 µg/mL poly IC upregulated the expression of CXCL1 mRNA (Fig. 1a) and the secretion of CXCL1 protein (Fig. 1b) in a concentration-dependent manner. Concentration-de- pendent expression of CXCL1 was similar to that of CXCL8 (Fig. 1). The time course of the expression of CXCL1 and CXCL8 is shown in Figure 2. The CXCL1 mRNA level rapidly increased after treatment. It reached lated p38 (1:1,000), p38 (1:1,000), phosphorylated NF-κB p65 its 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 me- dium reached its maximal level at 16 h and then pla- teaued, while the level of CXCL8 protein gradually in- creased up to 24 h (Fig. 2b).
The pretreatment of cells with SN50 resulted in a sig- nificant decrease in the expression of CXCL1 and CXCL8 mRNA (Fig. 3a) and protein (Fig. 3b) induced by poly IC. The poly IC-induced expression of CXCL1 and CXCL8 mRNA was not affected by pretreatment with MRT67307 (Fig. 4) nor with an anti-type I IFN antibody mixture (Fig. 5). The induction of CXCL10 by poly IC was de- creased by these pretreatments.
The poly IC-induced expression of CXCL1 and CXCL8 mRNA and protein was partially, but significantly, de- creased by the pretreatment of cells with SB203580 (Fig. 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 (Fig. 7). Conversely, SP600125 inhibited poly IC-induced CXCL8 mRNA at 2 h and at 24 h, and CXCL8 protein secretion also decreased (Fig. 7).
In order to examine the cross-talk between p38, JNK, and NF-κB signaling pathways, we next examined the ef- fect of inhibitors on the phosphorylation of these signal- ing 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-κB p65, and phosphorylation of JNK was not changed by SN50 or SB203580 (data not shown).
Discussion
Neutrophils release antimicrobial and pro-inflam- matory molecules and play an essential role in physio- logical and pathological inflammatory reactions. Al- though neutrophils are thought to mediate central pro- cesses 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 hepati- tis virus exhibited increased levels of viral replication in the brain compared with control mice [23]. Addition- ally, there was a decrease in the BBB permeability and mononuclear leukocyte infiltration into the brain in these neutropenic mice [23]. Matrix metalloproteinases 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 the 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 in- cluding encephalitis, inflammation, autoimmune dis- orders, and neurodegenerative diseases. Sustained CXCL1 expression amplifies the severity of white mat- ter damage in mice infected with neurotropic JHM strain of mouse hepatitis virus [10].
In the present study, we found that CXCL1 was synthe- sized and secreted when cultured hCMEC/D3 cells were treated with a TLR3 agonist poly IC. CXCL1 secreted from brain microvascular endothelial cells may induce chemo- taxis 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 the CXCL1 protein was different from that of CXCL8.
When brain vascular endothelial cells are appropriate- ly stimulated with viral dsRNA, CXCL1 may function to protect the brain. However, the dysregulation of CXCL1 may lead to various pathological inflammations in the brain. In order to maintain the homeostasis of the brain, the expression of CXCL1 in brain microvascular endo- thelial cells should be tightly regulated. Therefore, we next examined the molecular mechanisms by which CXCL1 expression induced by poly IC was regulated. NF- κB and IRF3 are major transcriptional factors in the sig- naling pathways mediated by TLR3 [16], and NF-κB is known to be a key molecule in CXCL1 expression in- duced by other stimuli [24]. SN50, an inhibitor of NF-κB 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 cyto- kine in immune response in the downstream of TLR3 sig- naling, pretreatment with an anti-type I IFN-neutralizing antibody mixture did not affect the poly IC-induced CXCL1 and CXCL8 expression. The poly IC-induced ex- pression of CXCL10, another member of the CXC che- mokines, was inhibited by either MRT67307 or by an an- ti-type I IFN-neutralizing antibody mixture. p38 MAPK has been reported to be involved in the expression of in- terleukin-6, CXCL8, and CXCL10 induced by poly IC in human neonatal dermal microvascular endothelial cells [25]. However, it is unknown whether p38 MAPK is in- volved in TLR3 signaling in brain microvascular endo- thelial 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 suggests that p38 MAPK is involved in the poly IC-induced CXCL1 and CXCL8 expression in brain microvascular endothelial cells. JNK is another sig- naling molecule involved in inflammatory reactions, and TLR3 signaling induces interleukin-6 via JNK in brain vascular endothelial cells [26]. In the present study, the pretreatment of hCMEC/D3 cells with a JNK inhibitor (SP600125) resulted in a decrease of CXCL1 and CXCL8 protein expression induced by poly IC. SP600125 also de- creased both CXCL1 and CXCL8 mRNA expression in cells treated with poly IC for 24 h. However, in cells treat- ed with poly IC for 2 h, SP600125 inhibited the expression of CXCL8 mRNA, but not that 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 expres- sion of these similar chemokines.
Intracellular signaling pathways after inflammatory stimuli are complex, and there is cross-talk between NF- κB and the mitogen-activated protein kinase family, in- cluding p38 and JNK [27]. However, this cross-talk be- tween these signaling pathways in human cerebral endo- thelial cells after TLR3 activation is not fully understood. In the present study, we found that poly IC-induced phosphorylation of p38 was partially inhibited by pre- treatment of cells with SN50. This indicates that there is cross-talk between NF-κB and p38, and the activation of p38 is at least partly induced via an NF-κB activation.
CXCR2 is expressed in brain endothelial cells, and CXCL1 induces the expression of adhesion molecules in brain endothelial cells [28] and angiogenesis [29] in a 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-κB, p38 MAPK, and JNK are involved in this reaction. The activation of NF-κB signaling may partially contrib- ute to p38 signaling. CXCL1 produced by brain microvas- cular endothelial cells may be involved in innate antiviral immunity in the brain.
Statement of Ethics
No human participants were involved in this study.
Disclosure Statement
The authors have no conflicts of interest to declare.
Funding Sources
This research was supported by Hirosaki University Institu- tional Research.
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 contrib- uted to cell culture. Kazuhiko Seya contributed to cell culture. Sho- go Kawaguchi contributed to real-time PCR and ELISA. Jiangli Ding contributed to cell culture. Hiroki Okuma contributed to study design.
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