Elevation of neutrophil carcinoembryonic antigen‐related cell adhesion molecule 1 associated with multiple inflammatory mediators was related to different clinical stages in ischemic stroke patients

Abstract Background We aimed to analyze the level of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) in neutrophils of ischemic stroke (IS) patients at different stages, together with its roles in neutrophils. Patients and methods Sixty‐seven patients were classified into acute phase group (n = 19), subacute phase group (n = 28), and stable phase group (n = 20), and 20 healthy individuals who had received physical examination at the same time period as healthy control. We then analyzed the expression level of CEACAM1 and cell viability in CEACAM1 positive and CEACAM1 negative neutrophils by flow cytometry and the content of plasma CEACAM1, neutrophil gelatinase‐associated lipocalin (NGAL), matrix metalloproteinases‐9 (MMP‐9) was measured using enzyme‐linked immunosorbent assay (ELISA), while that of interleukin‐10 (IL‐10) and tumor necrosis factor (TNF) was determined using a Human Enhanced Sensitivity Flex set. Results Compared with healthy control, the percentage of CEACAM1 positive neutrophils in IS patients showed a significant increase, and a significant increase was also noticed in the content of plasma CEACAM1 at the subacute stage. Reduction in cell viability was observed in CEACAM1 positive neutrophils compared with CEACAM1 negative counterparts. There was a positive correlation between CEACAM1 expression rate in neutrophils and plasma CEACAM1 and IL‐10 content in the subacute group. Compared with acute group and healthy control group, there was an instinct increase in the level of plasma MMP‐9 and NGAL in subacute group. Conclusions Our data showed that there was a rapid increase of CEACAM1 in neutrophils at the acute stage of IS. We speculated that CEACAM1 may serve as an inhibitory regulator involving in the progression of IS.


| INTRODUC TI ON
Stroke, with an estimated global lifetime risk of 24.9%, presents an increasing trend worldwide. 1 As the third major cause for disabilityadjusted life-years, 2 it shows a high mortality and morbidity. 3 Ischemic stroke (IS), accounting for 80%-85% of the stroke population, is mainly associated with several factors, especially the thromboembolic occlusion in the cerebral artery. Upon onset of IS, many patients present hypoxic damages and excitotoxicity. Subsequently, a majority would show massive accumulation and infiltration of inflammatory cells during ischemic reperfusion that may trigger further brain injuries. 4,5 Meanwhile, human body would persistently release the anti-inflammatory factors to defend the excessive inflammation to maintain the homeostasis. Thereafter, the immune system showed gradual failure, which finally entered into systemic immunosuppression, [6][7][8] possibly leading to stroke-associated infection (SAI). 9,10 Susceptibility to infection may severely hamper the prognosis and is mainly responsible for the increased mortality among IS patients. 6,11 On this basis, patients with cerebral ischemia may present immunological function disorders, especially the functional disorder of the innate immunocytes associated with the early-stage blood-brain barrier (BBB) injury and late-stage SAI. 12 Nevertheless, little is known about the exact mechanisms in this process.
Neutrophils are the major effector cells of the innate immunity system. Upon IS onset, the neutrophils were the first type of cells adhered to the cerebral endothelium and infiltrated to the ischemic brain in the presence of cell adhesion molecules and chemotactic factors. 13 Besides, it would lead to BBB and nervous system injuries through releasing the inflammatory mediators including proinflammatory cytokines, matrix metalloproteinases, and reactive oxygen species. 14 The circulating leukocytes would infiltrate into the lesion sites after onset of IS in the presence of proinflammatory factors and chemotactic factors, which then further led to BBB injury through releasing proinflammatory cytokines, reactive oxygen species and matrix metalloproteinases (MMPs), together with formation of the vasogenic edema. [15][16][17] In the previous study, MMP-9 was considered to play an important role in the BBB injury after IS, while the MMP-9 inhibitor provided protection against potential alternations in BBB permeability. Furthermore, increased serum IL-10 and MMP-9 was observed in patients with IS, among which IL-10 was found to play an important anti-inflammatory role in responding to brain injury. [18][19][20][21] Previous study showed that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) played a crucial role in the immune regulation. 22,23 As a member of the immunoglobulin superfamily, CEACAM1 is known as an inhibitory immune co-receptor suppressing the signal transmission via two immunreceptor tyrosine-based inhibition motifs. 23,24 Moreover, it is closely related to the proliferation and apoptosis of neutrophils and lymphocytes, [25][26][27] together with the secretion of cytokines. 28,29 Our previous study indicated that there was significant increase in T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) in CD8 + T cell in the IS patients, 30 and CEACAM1 was also co-localized with TIM-3. 31 Neutrophil gelatinase-associated lipocalin (NGAL) is reported to involve in the innate immunity, cellular differentiation and proliferation, and iron homeostasis. 32 Besides, it may form a complex with MMP-9. 33 Thus, CEACAM1 could directly or indirectly involve in the innate and regulatory immunity. In this study, we aimed to analyze the counts of neutrophils and the level of the serum CEACAM1, MMP-9, and NGAL in the IS patients and analyze their possible roles in the IS process.

| Flow cytometry
The gating strategy of different immune cell populations is shown in Figure 1A and Figure S1. Briefly, the frequency of lymphocytes, neutrophils, and monocytes in the leukocyte population was determined based on their CD45 (APC BD Bioscience)-SSC profile.

| Cell viability
The heparinized blood was lysed using TQ-PREP before the neu-

| Intracellular MMP-9 assays
The neutrophils were labelled with CEACAM1-PE (R&D) at room temperature for 30 min and then were resuspended with 1 ml PBS.
Finally, flow cytometry was conducted to analyze the intracellular MMP-9.

| Plasma cytokine assays
The concentrations of plasma TNF and IL-10 were determined using a Human Enhanced Sensitivity Flex set (BD Bioscience). The lower limits of the test for the detection of the cytokines were 67.3 fg/ml for TNF and 13.7 fg/ml for IL-10.

| Statistical Analysis
We

| Apoptotic phenotype of CEACAM1positive and negative neutrophils
To confirm the correlation between CEACAM1 expression and the apoptosis of neutrophils, flow cytometry was utilized to determine the positive rate of Annexin-V in the CEACAM1 positive and negative neutrophils. As shown in Figure 2A

| Determination of plasma soluble CEACAM1
In this part, we determined plasma CEACAM1 levels among IS groups and the control group. The level of plasma CEACAM1 in the subacute group showed a significant increase compared with that in the healthy control group (p = 0.016, Figure 3A). No statistical differences were noticed between subacute group and the other groups (p > 0.05). Moreover, the expression of CEACAM1 on the surface of neutrophils in the subacute group was closely correlated with the plasma CEACAM1 content (p = 0.001, r = 0.614, Figure 3B).

| Determination of poor-plate plasma MMP-9 and NGAL
We determined the plasma MMP-9 concentration in different groups. Significant increase was noticed in the plasma MMP-9 concentration in the subacute group when comparing with that of the control group (p = 0.005) and acute group (p = 0.020, Figure 3C). There were no statistical differences among the other groups (p > 0.05). We also determined the level of plasma NAGL in different groups.
Compared with the healthy control individuals, a significant elevation was noticed in the plasma NAGL in the subacute group (p = 0.001) and stable group (p < 0.001, Figure 3D). There were no statistical differences in the plasma NAGL between the other groups (p > 0.05).

| Determination of plasma soluble TNF and IL-10
We also determined the level of plasma TNF and IL-10 in different groups. A significant increase was noticed in the plasma IL-

| MMP-9 production by CEACAM1positive and negative neutrophils
To confirm the correlation between expression of CEACAM1 and the production of MMP-9 of neutrophils, the neutrophils were sub-

| DISCUSS ION
Cerebral edema after BBB breakdown is considered an important cause for the severe morbidity and mortality after IS, serving as an important cause for the early stroke-related death. 36 Besides, SAI is the major cause for the late-stage mortality in IS. 37 Increasing evidence suggested that functional disorder of the immunocytes was an important cause for the early-stage BBB injury and subsequent SAI among IS patients. 12 However, the roles of neutrophils in the pathogenesis of IS have not been well defined. According to previous studies, MMP-9 was implicated in the BBB breakdown and formation of vasogenic edema after IS. 38,39 In an IS mice model, CEACAM1 was reported to involve in controlling the secretion of MMP-9 by neutrophils in post-ischemic inflammation in the BBB after IS. 22 This demonstrated that CEACAM1 may inhibit the neutrophil-mediated tissue damages and breakdown of BBB in focal cerebral ischemia. In this study, we determined the expression of CEACAM1 in leukocytes of IS patients. Our data confirmed that neutrophils were the major source of CEACAM1 in peripheral blood. CEACAM1 expression on the surface of peripheral neutrophils of IS patients at each stage was significantly higher than that of the healthy control. Then, we measured the expression of plasma CEACAM1, MMP-9, and NGAL content, which showed that the plasma MMP-9 level in patients at subacute phases was significantly higher than that of the acute stage and the healthy control. Interestingly, there were no statistical differences in the plasma MMP-9 between the healthy control and the acute group.
This implied that there was persistent elevation of MMP-9 within several days after onset of IS, which may be closely related to Neutrophils are main resource of MMP-9 after IS onset. 40 On this basis, we speculated that CEACAM1 may regulated neutrophil cell viability and finally influenced MMP-9 secretion.
Previous study indicated that IS patients with increased plasma NGAL showed poorer prognosis. 41 Our data on the IS patients were in line with the study by Hochmeister Sonja et al, 41 in which a significant increase was noticed in the plasma NGAL at the subacute stage.
Evidences indicated that NGAL, MMP-9/NGAL, and MMP-9 were co-expressed in the activated monocytes and neutrophils. Esidue in the PEX domain of MMP-9. 32 NGAL could regulate the activity of MMP-9. In a previous study, the Cys-87 in NGAL could form a disulphide bond with an as yet unidentified cysteine residue in the PEX domain of MMP-9. 32 In the presence of NGAL, the degradation of MMP-9 was obviously inhibited, which led to the preserve of the MMP-9 activity. 33 In this study, despite the fact that MMP-9 elevation was merely noticed at the subacute stage, there was persistent increase in plasma NGAL. We speculated from our data that high concentration of NGAL may activate or preserve the MMP-9 activity. This may contribute to the fact that the activity level of MMP-9 was higher than the normal level even its concentration was not higher in the stable period. On this basis, we speculated that NGAL and MMP-9 played important roles in BBB injury.
In this study, we examined plasma TNFα and IL-10, our study showed that expression evaluation of cytokines IL-10 and neutrophil CEACAM1 expression was positively correlated with plasma IL-10 level in patients at IS acute and subacute phases. The positive relation between CEACAM1 and IL-10 may suggest that CEACAM1 played roles in regulation of downstream cytokine secretion. 42 Previous in vitro and in vivo models of IS showed IL-10-mediated neuroprotection directly and indirectly, 43 but the specific mechanism remained unclear. Related studies are needed to further illustrate the exact mechanism in the future.
In a previous study, CEACAM1 was considered to be related to the proliferation of neutrophils and granulopoiesis 26 as well as the secretion of cytokines and apoptosis. 25,28 In this study, the level of CEACAM1 on the surface of neutrophils in IS patients was significantly higher compared with the healthy control. Meanwhile we also discovered that there was higher MFI of apoptosis markers in CEACAM1positive neutrophils compared with negative counterparts. On this basis, we speculated that CEACAM1 might involve in regulation of CEACAM1-induced neutrophil proliferation and granulopoiesis.
CEACAM1 on the surface of neutrophils in the peripheral blood is an important source of plasma CEACAM1. 44 Our data indicated that plasma CEACAM1 was positively correlated with the CEACAM1 on the neutrophils in the peripheral blood of subacute stage IS patients. To our best knowledge, TIM-3 serving as an important immune checkpoint factor of T lymphocytes is mainly responsible for the negative regulation of T lymphocytes. 45 As a ligand of TIM-3, CEACAM1 could mediate the function of TIM-3 modulated T lymphocytes. 46,47 Thus, it could affect the TIM-3 positive T lymphocyte proliferation, 27 function 48 and secretion of cytokines. 29 Our previous study indicated that there was increased TIM-3 on the peripheral blood T lymphocytes in the IS. 30 We speculated that CEACAM1 could bind with TIM-3 through the T lymphocyte surface, which may induce injury of immune function of lymphocytes at the late-stage IS patients and finally result in increased risk of infection. In the future, more studies are required to validate this speculation.
MMP-9 deficiency has been proved to increase in vitro apoptosis in multiple tissues. 49 MMP-9 induced increase in expression of FasL in diabetes wound repair. 50 In kidney development, MMP-9 was discovered to protect mesenchymal cells from apoptosis. 49,51 The process may involve various downstream molecules of MMP-9.
In a study on post-ischemic cerebral injury, evidence showed that reduction in expression of MMP-9 may lead to protective effects on BBB injury and neuronal apoptosis of adipocyte fatty acid-binding protein (A-FABP) deficiency. 52 MMP-9 may also play the role of apoptosis inhibition through hedghog pathway. 53 There has been evidence about CEACAM1-mediated apoptosis in exacerbates hypoxic cardiomyocyte injury and post-infarction cardiac remodeling, 54 and early tumor development. 55 In this study, we found significant reduction in cell viability in CEACAM1-positive neutrophils compared with CEACAM1-negative ones, and we also noticed elevation of MMP-9 at the subacute stage. It is speculated that after onset of cerebral ischemic stroke, increased CEACAM1 level induced more apoptosis of neutrophils. MMP-9 elevation may due to negative feed back mechanism in response to elevated apoptosis level. However, we lack evidence to support such speculation.
CEACAM1 has been proved to be implicated in vascular physiology and pathophysiology, which includes vessel formation, endothelial barrier function regulation, vascular remodeling, and stabilization. 56 In CEACAM1 −/− mice, multiple pathways involved in NADPH oxidases elevation and activation. 57 Increased signaling by VEGFR-2 and TNFα/ NF-κB pathway and the renin-angiotensin system activation was both observed in CEACAM1 −/− mice, suggesting that CEACAM1 played a significant role in endothelial oxidation. 56  In the future, attempts should be made to verify the interaction among CEACAM1, MMP-9, and NGAL, and to further illustrate the roles of CEACAM1 in the IS, ischemia-reperfusion injury and secondary infection in the patients and animal models.
However, there are some limitations in this study. The study failed to gather blood samples at different stages from each patient.
Though change in CEACAM1 and related markers has been classified, the pathophysiological significance of CEACAM1 elevation has not been confirmed. Besides, the small sample size in the study may interfere in the results and needs larger sample size to verify the discoveries.
In this study, rapid elevation of neutrophil CEACAM1 and change in levels of associated inflammatory mediators in the plasma were

ACK N OWLED G M ENT
We are grateful to Dr. Lingtong Huang and Dr. Yunbo Chen for generous help with the sample collection.

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PATI E NT CO N S E NT S TATE M E NT
Written informed consent was obtained from all the participants.