Epigallocatechin-3-Gallate Attenuates Leukocyte Infiltration in 67-kDa Laminin Receptor-Dependent and -Independent Pathways in the Rat Frontoparietal Cortex following Status Epilepticus

Status epilepticus (SE) evokes leukocyte infiltration in the frontoparietal cortex (FPC) without the blood-brain barrier disruption. Monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) regulate leukocyte recruitments into the brain parenchyma. Epigallocatechin-3-gallate (EGCG) is an antioxidant and a ligand for non-integrin 67-kDa laminin receptor (67LR). However, it is unknown whether EGCG and/or 67LR affect SE-induced leukocyte infiltrations in the FPC. In the present study, SE infiltrated myeloperoxidase (MPO)-positive neutrophils, as well as cluster of differentiation 68 (CD68)-positive monocytes in the FPC are investigated. Following SE, MCP-1 was upregulated in microglia, which was abrogated by EGCG treatment. The C–C motif chemokine receptor 2 (CCR2, MCP-1 receptor) and MIP-2 expressions were increased in astrocytes, which were attenuated by MCP-1 neutralization and EGCG treatment. SE reduced 67LR expression in astrocytes, but not endothelial cells. Under physiological conditions, 67LR neutralization did not lead to MCP-1 induction in microglia. However, it induced MIP-2 expression and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in astrocytes and leukocyte infiltration in the FPC. Co-treatment of EGCG or U0126 (an ERK1/2 inhibitor) attenuated these events induced by 67LR neutralization. These findings indicate that the EGCG may ameliorate leukocyte infiltration in the FPC by inhibiting microglial MCP-1 induction independent of 67LR, as well as 67LR-ERK1/2-MIP-2 signaling pathway in astrocytes.


Introduction
The brain is in part isolated from the systemic immune system by the blood-brain barrier (BBB). Therefore, microglia generally act as the primary immune cells in the brain parenchyma. Under pathophysiological conditions, activated microglia lead to bloodderived leukocyte infiltration by releasing various cytokines and chemokines. Infiltrating leukocytes further exacerbate secondary local inflammation by generating reactive oxygen species, proteolytic enzymes and cytokines/chemokines [1][2][3][4][5].
BBB disruption is a crucial step in the pathogenesis of several neuroinflammatory diseases in the brain [6]. Indeed, status epilepticus (SE), prolonged and uncontrolled seizures, results in the infiltration of neutrophil and monocyte in the rat piriform cortex (PC), accompanied by severe vasogenic edema [7]. Unlike the PC, SE evokes leukocyte infiltration in the frontoparietal cortex (FPC) without BBB breakdown. In FPC, SE induces leukocyte infiltration through inductions of monocyte chemotactic protein-1 (MCP-1) in microglia and macrophage inflammatory protein-2 (MIP-2) in astrocytes, in an interleukin-1β (IL-1β)-independent manner [8][9][10]. Therefore, the FPC is a suitable region to investigate the underlying mechanisms of leukocyte infiltration unaffected by altered vascular permeability following SE.

Tissue Preparation and Immunohistochemistry
Since the neutrophil and monocyte infiltrations peaked in the PFC at 2-3 days and 3-4 days after SE, respectively [8][9][10], we chose 3 days after SE as the ideal timepoint to evaluate the effect of EGCG on leukocyte infiltration. Three days after SE or 67LR infusion, animals were administered urethane anesthesia (1.5 g/kg, i.p.) and perfused with normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were collected in the same fixative overnight and 30 µm thick coronal sections were made using a cryostat. Sections were blocked with 3% bovine serum albumin and subsequently incubated with a cocktail solution containing isolectin B4 (IB4) or primary antibodies (Table 1) overnight at room temperature. Thereafter, sections were reacted with Brilliant Violet-, Cy2-or Cy3-conjugated secondary antibodies (for anti-sera) or streptavidin (for IB4). A negative control test was performed with pre-immune serum in place of the primary antibody. Experimental procedures in this study were carried out under the same conditions and in parallel. The random-selected areas (1 × 10 5 µm 2 ), approximately −3.0-3.6 mm from the bregma, were selected based on the rat brain in stereotaxic coordinates [25]. Thereafter, the number of infiltrating neutrophils and monocytes was counted, and SMI-71 fluorescent intensity was measured using AxioVision Rel. 4.8 and the ImageJ software (n = 7 rats in each group). The fluorescent intensity of MCP-1, MIP-2, 67LR or p-ERK1/2 was also measured in randomly selected 5-6 cells from each animal. Briefly, images were captured (gain value = 1) and digitally separated into green, red or blue panels. Each image was converted to black and white, and the background staining was subtracted automatically. Thereafter, each signal was normalized by setting the threshold level and represented as the number of 256 grayscale. Manipulation of the microscope was restricted to automatic exposure time and threshold adjustments.

Western Blot and Quatitative Real-Time PCR (qRT-PCR)
Three days after SE and 67LR IgG infusion, animals were decapitated and the FPC was rapidly obtained. Western blot was performed by the standard methods. Briefly, the proteins were separated by electrophoresis and transferred to nitrocellulose membranes. After the blocking, membranes were incubated with primary antibody (Table 1). After further reaction with peroxidase-conjugated secondary antibody followed by ECL solution, immunobands were detected and quantified with the ImageQuant LAS4000 system (GE Healthcare Korea, Seoul, South Korea). The density of the immunobands was calibrated with with β-actin. The ratio of phospho-protein to total protein was also measured. For qRT-PCR, brain tissues were homogenized, and the total RNA was extracted using Trizol Reagents (Thermo Fisher Scientific Korea, Seoul, South Korea). Total RNA was reverse-transcribed into first-strand cDNA using the PrimerScript 1st strand cDNA synthesis kit (Takara, Shiga, Japan). Quantification of mRNA expression was performed in triplicate using a SYBR Green SuperMix (Bioneer, Taejon, South Korea) and with the MyiQ Single-Color Real-Time PCR Detection System (Bioneer, Taejon, South Korea). Primer sequences were 5 -GTGCTGACCCCAATAAGGAA-3 (forward primer for rat MCP-1) and 5 -TGAGGTGGTTGTGGAAAAGA-3 (reverse primer for rat MCP-1); 5 -TGAAGTTTGTCTCAACCCTGAAGCC-3 (forward primer for rat MIP-2) and 5 -AGGTCAGTTAGCCTTGCCTTTGTTC-3 (reverse primer for rat MIP-2); and 5 -TGGAG TCTACTGGCGTCTT-3 (forward primer for rat GAPDH) and 5 -TGTCATATTTCTCGTG GTTCA-3 (reverse primer for rat GAPDH). All primers were purchased from Bioneer (Taejon, South Korea). After initial denaturation at 95 • C for 10 min, 50 cycles of primer annealing and elongation were conducted at 55 • C for 45 s, followed by denaturation at 95 • C for 1 s. qRT-PCR data for MCP-1 and MIP-2 were normalized to GAPDH determined from the same experiment.

Data Analysis
Data were analyzed using a Mann-Whitney test or Kruskal-Wallis test, followed by Dunn-Bonferroni post hoc comparison. A p-value of less than 0.05 was considered significant.

EGCG Attenuates SE-Induced Leukocyte Infiltration in the FPC
First, we investigated whether EGCG treatment affects leukocyte infiltration in the FPC following SE. SE did not alter the BBB (SMI-71) integrity in the FPC (Z = 0.513, p = 0.608, n = 7 rats, respectively, Mann-Whitney test; Figure 1A,B). In control (non-SE) animals, MPO-positive neutrophils were undetectable in the parenchyma of the FPC ( Figure 1A). Following SE, the number of MPO-positive neutrophils was~41 cells/10 5 µm 2 in the FPC of vehicle-treated rats. EGCG attenuated neutrophil infiltration to~17 cells/10 5 µm 2 in this region (Z = 3.130, p = 0.002, n = 7 rats, respectively, Mann-Whitney test; Figure 1A,C). Similar to MPO-positive neutrophils, CD68-positive monocytes were rarely observed in the FPC of control animals ( Figure 1A). Following SE, round-, spheroid-and ramifiedshaped CD68-positive monocytes were detected in the FPC ( Figure 1A). The number of CD68-positive monocytes was~51 cells/10 5 µm 2 in the FPC of vehicle-treated rats. EGCG attenuated monocyte infiltration to~23 cells/10 5 µm 2 in this region (Z = 3.137, p = 0.002, n = 7 rats, respectively, Mann-Whitney test; Figure 1A,C). Furthermore, the shape of the infiltrating monocytes was round or spheroid, rather than ramified ( Figure 1A). These findings indicate that EGCG may ameliorate leukocyte infiltration in the FPC following SE. Considering that blood-derived monocytes replace the resident microglia [8,26,27], our findings also suggest that EGCG may inhibit monocyte transformation to microglia.
In control animals, MCP-1 expression was rarely detected in the FPC (Figure 2A). Following SE, MCP-1 expression was observed in most hypertrophic and amoeboid isolectin B4 (IB4)-positive microglia (an indicative of activated microglia, Figure 2A). CCR2 expression was mainly detected in CD68-positive monocytes, as well as astrocytes ( Figure  2B). In EGCG-treated animals, microglia showed hyper-ramified processes that were covered by thorny spines, indicating the inhibition of microglial transformation to hypertrophic and amoeboid shapes ( Figure 2A). EGCG also reduced MCP-1 expression in microglia (Z = 6.228, p < 0.001, n = 40 cells in 7 rats, respectively, Mann-Whitney test; Figure  2A,C). Compatible with immunohistochemistry, the qRT-PCR date revealed that EGCG On the other hand, MIP-2 expressions were observed in most astrocytes and a few neurons following SE, although its MIP-2 expression was undetected in the FPC of control rats ( Figure 3A,B). EGCG abolished SE-induced MIP-2 induction in these cell populations (Z = 6.237, p < 0.001, n = 40 cells in 7 rats, respectively, Mann-Whitney test; Figure 3A,C). EGCG also inhibited the SE-induced MIP-2 mRNA upregulation in the hippocampus (Z = 2.309, p = 0.021, n = 4 rats, respectively, Mann-Whitney test; Figure 3D). Taken together, our findings indicate that EGCG may diminish NF-κB-mediated MCP-1 induction in microglia following SE, which abolished CCR2-mediated MIP-2 expression in astrocytes.

SE Reduces 67LR Expression in Astrocytes, but Not in Endothelial Cells, in the FPC
Since EGCG is a 67LR ligand [16,17], we investigated whether SE alters 67LR expression in the FPC. Compatible with previous studies [17,19,20,24], 67LR expression was observed in astrocytes and endothelial cells in the FPC ( Figure 4A). Following SE, 67LR expression was diminished in astrocytes, which was ameliorated by EGCG (Z = 5.771, p < 0.001, n = 40 cells in 7 rats, respectively, Mann-Whitney test; Figure 4A,B). However, SE did not change 67LR expression in endothelial cells, which was unaffected by EGCG (Z = 0.583, p = 0.56, n = 40 cells in 7 rats, respectively, Mann-Whitney test; Figure 4A,B). Therefore, it is likely that EGCG may attenuate SE-induced 67LR downregulation in astrocytes.

SE Reduces 67LR Expression in Astrocytes, but Not in Endothelial Cells, in the FPC
Since EGCG is a 67LR ligand [16,17], we investigated whether SE alters 67LR expression in the FPC. Compatible with previous studies [17,19,20,24], 67LR expression was observed in astrocytes and endothelial cells in the FPC ( Figure 4A). Following SE, 67LR expression was diminished in astrocytes, which was ameliorated by EGCG (Z = 5.771, p < 0.001, n = 40 cells in 7 rats, respectively, Mann-Whitney test; Figure 4A,B). However, SE did not change 67LR expression in endothelial cells, which was unaffected by EGCG (Z = 0.583, p = 0.56, n = 40 cells in 7 rats, respectively, Mann-Whitney test; Figure 4A,B). Therefore, it is likely that EGCG may attenuate SE-induced 67LR downregulation in astrocytes.

Discussion
Infiltrating leukocytes develop detrimental inflammatory responses by releasing proinflammatory cytokines [37,38]. They also produce chemoattractants for the subsequent monocyte chemotaxis, such as human cationic antimicrobial protein (hCAP18, also known as LL-37) and cathepsin G [39,40]. Therefore, inhibition of leukocyte infiltration prevents or reduces the secondary brain damage induced by neuroinflammation [37].
MCP-1 and its receptor CCR2 is the first characterized chemokine system in humans [41]. MCP-1 activates monocyte recruitment by itself, and also promotes neutrophil infiltration [30,31,42,43]. In the brain, microglia rapidly induce MCP-1 expression in response to harmful stresses via NF-κB-and p38 MAPK signaling pathways that are also involved in microglial activation (transformation) [9,10,13,14,44]. Indeed, NF-κB p65 protein level increases in the brain following acute seizures [45][46][47]. Furthermore, p65 S276 phosphorylation of NF-κB p65 subunit is required for microglial activation and MCP-1 induction following SE, which plays an important role in leukocyte infiltration in the FPC [9,10]. Consistent with these reports, the present data showed that SE elevated total p65 protein and its S276 phosphorylation levels. Since SE enhanced p65 S276 phosphorylation more than the total p65 protein upregulation, the p65 phosphorylation ratio was increased as compared to the control animals. In addition, EGCG attenuated SE-induced leukocyte infiltration in the FPC by diminishing the total p65 protein, its S276 phosphorylation and MCP-1 expression in microglia. EGCG also inhibited the transformation of resident microglia and infiltrating monocytes in the brain parenchyma. Considering that EGCG abrogates p38 MAPK and NF-κB signaling pathways [11,12], the present data indicate that EGCG may inhibit microglial activation, and in turn, ameliorate MCP-1 transcription following SE.
The roles of 67LR in EGCG-mediated MCP-1 regulation in peripheral macrophages in response to LPS are controversial: EGCG induces MCP-1 expression through p38 MAPK-c-Jun NH2-terminal kinase (JNK) signaling axis following LPS treatment, which is abrogated by 67LR neutralization [48]. In contrast, EGCG suppresses MCP-1 expression in response to LPS via the ERK1/2, p38 MAPK, JNK and NF-κB pathways, which is abolished by 67LR neutralization [12]. In the present study, 67LR expression was mainly observed in astrocytes and endothelial cells, but not in microglia and neurons, in the intact brain. SE reduced 67LR expression in astrocytes. Furthermore, 67LR neutralization led to leukocyte infiltration without microglial MCP-1 induction under physiological conditions. Of note, EGCG diminished leukocyte infiltration following SE and 67LR neutralization. Therefore, our findings suggest that in the brain, EGCG may inhibit MCP-1 induction through NF-κB-dependent and 67LR-independent pathways in microglia following SE, unlike fully differentiated peripheral macrophages in response to LPS.
As aforementioned, MCP-1 also recruits neutrophils in MIP-2-dependent and -independent manners [30,42,43]. In a previous study [48], EGCG did not directly induce leukocyte migration, but induced MIP-2 in peripheral macrophages mediated by 67LR following LPS treatment. In contrast to this report, the present data demonstrate that SE resulted in astroglial MIP-2 induction with 67LR downregulation, which was mitigated by EGCG treatment and MCP-1 neutralization. These findings indicate that MCP-1 released from activated microglia may cause 67LR downregulation and the subsequent MIP-2 induction in astrocytes following SE. The present study also reveals that 67LR neutralization led to astroglial MIP-2 induction and leukocyte infiltration without microglial MCP-1 induction under physiological conditions, which was attenuated by EGCG co-treatment. These findings suggest that 67LR may play an inhibitory role in MIP-2-mediated leukocyte infiltration in the FPC.
Exogenous soluble laminin and EGCG inhibit ERK1/2 phosphorylation mediated by 67LR [23,51]. Ku et al. [52,53] reported that 67LR neutralization abrogates the inhibitory effects of EGCG on ERK1/2 phosphorylation. Thus, they speculated that 67LR neutralization might prevent the EGCG-67LR interaction through sterical hindrance [52,53]. However, the present data demonstrate that 67LR neutralization increased ERK1/2 phosphorylation, which was attenuated by EGCG or U0126 co-treatment. Furthermore, both EGCG and U0126 attenuated leukocyte infiltration and ERK1/2 and MIP-2 upregulation induced by 67LR neutralization. In the present study, we used the antibody recognizing the amino acid 250-350 regions on 67LR for neutralization. Since amino acid 272-280 regions on 67LR are the inhibition site of its functions by neutralization [54] and 161-170 regions are the EGCG binding site, respectively [55], it is likely that 67LR neutralization may not affect EGCG-67LR binding in the present study. Adversely, EGCG and 67LR antiserum could competitively bind to 67LR in the present study.
On the other hand, the cortical insults caused by trauma, bleeding and infection result in SE and acquired epilepsy [58,59]. Indeed, post-traumatic injury (TBI) is one of the causes of acquired epilepsy [58]. TBI leads to a subsequent neuronal damage resulting from neuroinflammation, which contributes to synchronized hyperexcitability and robust spontaneous seizures [58]. Furthermore, cobalt-induced neocortical injury leads to focal seizures, which are developed into SE induced by homocysteine (a N-methyl-D-aspartate receptor agonist) administration [59]. Cobalt-induced lesions render the cerebral cortex sensitive to BBB disruption induced by homocysteine [59]. Considering these reports, it is likely that SE-induced leukocyte infiltration into the FPC may affect epileptogenic events without vasogenic edema. Further studies are needed to elucidate the roles of leukocyte infiltration and/or vasogenic edema in the neocortex in epileptogenesis.
In the present study, EGCG effectively attenuated SE-induced NF-κB S276 phosphorylation. Under resting condition, NF-κB is sequestered in the cytoplasm through direct binding with the inhibitor of the κB (IκB) family. IκB kinase (IKK) activation phosphorylates IκB, which leads to IκB degradation, liberates NF-κB from the NF-κB-IκB complex and evokes nuclear NF-κB translocation [60]. Interestingly, N-acetylcysteine (NAC, an antioxidant) abolishes nuclear NF-κB translocation by directly inhibiting NF-κB p65 phosphorylation without affecting IκB degradation [61]. Furthermore, CDDO-Me abrogates NF-κB-mediated signaling pathways by direct inhibition of IKK [62]. Considering that EGCG decreases IκB phosphorylation [63][64][65], it is likely that inhibition of NF-κB canonical pathway by regulating IKK activity may also be relevant to the anti-inflammatory properties of EGCG, which would regulate chemokine and cytokine syntheses. With respect to these previous reports. Furthermore, it is postulated that a general mechanism of antioxidants against inflammation may regulate the early step of NF-κB signaling pathway (inhibi-tions of IKK activation, IκB phosphorylation, IκB degradation and p65 phosphorylation), although their specific targets would be distinct and unidentified.

Conclusions
The present study reveals for the first time that MCP-1 regulated the 67LR-ERK1/2-MIP-2 signaling pathway in astrocytes following SE, which elicited leukocyte infiltration in the FPC independent of vascular permeability. In addition, 67LR neutralization led to leukocyte infiltration via an MCP-1-independent manner under physiological conditions. Furthermore, EGCG attenuated leukocyte infiltration by inhibiting microglial MCP-1 induction and astroglial MIP2 upregulation following SE and 67LR neutralization. Therefore, our findings suggest that the inhibition of MCP-1 induction (in microglia) and/or preservation of 67LR functions (in astrocytes) may be a strategy to mitigate neuroinflammation ( Figure 8). antioxidants against inflammation may regulate the early step of NF-κB signaling pathway (inhibitions of IKK activation, IκB phosphorylation, IκB degradation and p65 phosphorylation), although their specific targets would be distinct and unidentified.

Conclusions
The present study reveals for the first time that MCP-1 regulated the 67LR-ERK1/2-MIP-2 signaling pathway in astrocytes following SE, which elicited leukocyte infiltration in the FPC independent of vascular permeability. In addition, 67LR neutralization led to leukocyte infiltration via an MCP-1-independent manner under physiological conditions. Furthermore, EGCG attenuated leukocyte infiltration by inhibiting microglial MCP-1 induction and astroglial MIP2 upregulation following SE and 67LR neutralization. Therefore, our findings suggest that the inhibition of MCP-1 induction (in microglia) and/or preservation of 67LR functions (in astrocytes) may be a strategy to mitigate neuroinflammation ( Figure 8). Figure 8. A schematic diagram of the underlying mechanisms of the effect of EGCG on SE-induced leukocyte infiltration. SE led to NF-κB-mediated MCP-1 induction in microglia, which was attenuated by EGCG. Released MCP-1 from microglia recruited blood-derived monocytes and activated MIP-2 upregulation in astrocytes via the CCR2-67LR-ERK1/2 signaling pathway. EGCG and U0126 also attenuated MIP-2 induction in astrocytes by facilitating 67LR-mediated ERK1/2 inactivation, which ameliorated neutrophil/monocyte infiltration.

Supplementary Materials:
The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Full-length gel images of Western blot data.   A schematic diagram of the underlying mechanisms of the effect of EGCG on SE-induced leukocyte infiltration. SE led to NF-κB-mediated MCP-1 induction in microglia, which was attenuated by EGCG. Released MCP-1 from microglia recruited blood-derived monocytes and activated MIP-2 upregulation in astrocytes via the CCR2-67LR-ERK1/2 signaling pathway. EGCG and U0126 also attenuated MIP-2 induction in astrocytes by facilitating 67LR-mediated ERK1/2 inactivation, which ameliorated neutrophil/monocyte infiltration.