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 patients with stroke. However, the roles of neutrophils in the pathogenesis of stroke have not been well defined. In this study, we measured the expression of serum CEACAM1, MMP-9 and NGAL content, as well as the expression of CEACAM1 in the neutrophils. The serum CEACAM1, MMP-9 and NGAL in the IS patients showed significant increase, and there was also significant elevation in the counts of CEACAM1 positive neutrophils. These features of CEACAM1, MMP-9 and NGAL may represent an unknown mechanism associated with the pathogenesis of IS.
According to previous works, MMP-9 was implicated in the BBB breakdown and formation of vasogenic edema after stroke[38, 39]. Consistently, our results showed that the serum MMP-9 level in patients at subacute phases was significantly higher than that of the acute stage and the normal control. Interestingly, there were no statistical differences in the plasma MMP-9 between the normal 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 the subsequent brain edema and nervous injuries.
In an IS mice model, CEACAM1 was involved in controlling the secretion of MMP-9 by neutrophils in postischemic inflammation in the BBB after stroke[24]. 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 major source of CEACAM1 expression in the peripheral blood. CEACAM1 expression on the surface of peripheral neutrophils of IS patients at each stage was significantly higher than that of the normal control. Meanwhile, the plasma CEACAM1 and MMP-9 in IS patients also showed significant changes. Ludewig Peter and his colleges described a significant increase of the expression of MMP-9 in CEACAM1−/− IS mice model compared with that of the wild type mice and the results were validated with stimulation of neutrophils under in vitro conditions showing increased MMP-9 secretion in CEACAM1-deficient neutrophils 120 min afterwards[24]. These results suggested that elevation of CEACAM1 in stroke patients was possibly closely related to BBB injury mediated by MMP-9. Moreover, it has been reported that CEACAM1 inhibited the inflammatory response induced by formation of toll-like receptor 4-CEACAM1 complex on neutrophils responding to pathogen challenge, which resulted in reduction of proinflammatory cytokine (e.g. IL-1β) [30]. These results indicated that the expression of CEACAM1 in the neutrophils regulated the expression of MMP-9 and proinflammatory factors. Nevertheless, in this study, upon LPS stimulation, no statistical differences were noticed in the MMP-9 expression in CEACAM1 positive and negative neutrophils in IS patients. Our data showed that neutrophil CEACAM1 expression showed no influence or regulatory effect on viability or function of neutrophils. On this basis, we speculated that CEACAM1 may play a role in pathophysiology of IS through other pathways. For instance, Pan et al reported that CEACAM1 acted as a co-inhibitory receptor for G-CSFR regulation granulopoiesis[28]. CEACAM1 regulated the vascular homeostasis and integrity, which resulted in decrease of basal and acute vascular permeability[40, 41]. In other aspect, our study showed that neutrophil CEACAM1 expression was positively correlated with plasma IL-10 level in patients at IS acute and subacute phases. Previous in vitro and in vivo models of IS showed IL-10-mediated neuroprotection directly and indirectly[19], but the specific mechanism remained unclear. Related studies are needed to further illustrate the exact mechanism in the future.
Neutrophils, one of the main innate immunoreactive cells, play crucial roles in killing the bacteria and fungi. In a previous study, CEACAM1 was considered to be related to the proliferation of neutrophils and granulopoiesis[28], as well as the secretion and apoptosis of cytokines[27, 30]. Our data showed that the CEACAM1 on the surface of neutrophils in the IS patients was significantly higher compared to the normal control. Nevertheless, our data showed that there were no statistical differences in the apoptosis of CEACAM1 positive neutrophils compared with negative counterparts. On this basis, we sepculated that there might be other molecules involving 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[42]. Our data indicated that plasma CEACAM1 was positively correlated with the CEACAM1 on the neutrophils in the peripheral blood of subacute stage stoke 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[43]. As a ligand of Tim-3, CEACAM1 could mediate the function of Tim-3 modulated T lymphocytes[44, 45]. Thus, it could affect the Tim-3 positive T lymphocyte proliferation[29], function[46] and secretion of cytokines[31]. Our previous study indicated that there was elevation of Tim-3 on the peripheral blood T lymphocytes in the stroke[32]. 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.
Recent study indicated that stroke patients with increased plasma NGAL showed poorer prognosis[47]. Our data on the IS patients were in line with the study by Hochmeister Sonja et al[47], in which significant increase was noticed in the plasma NGAL at the subacute stage. 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[34]. Evidences indicated that NGAL, MMP-9/NGAL and MMP-9 were co-expressed in the activated monocytes and neutrophils. NGAL could regulate the activity of MMP-9. In the presence of NGAL, the degradation of MMP-9 was obviously inhibited, which led to the preserve of the MMP-9 activity[35]. In this study, despite the fact that MMP-9 elevation was merely noticed at the subacute stage, there was persistent elevation of 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 summary, CEACAM1 expression level in neutrophils was higher in stroke patients of different stages than that in normal individuals. Additionally, significant elevation was noticed in the serum CEACAM1, MMP-9 and NGAL in stroke patients. CEACAM1 may serve as an important inhibitory regulator for mediating the early-stage BBB injury induced by MMP-9. Nevertheless, persistent high-expression of serum NGAL would combine with MMP-9, which may contribute to the persistent injury of BBB. Moreover, as a ligand of Tim-3, CEACAM1 may involve in the negative immunoregulation of T lymphocytes, which may lead to severe immune dysfunction and subsequent infections. In the near 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 stroke, ischemia-reperfusion injury and secondary infection in the patients and animal models.