Adhesion of Leukocytes to Cerebral Venules Precedes Neuronal Cell Death and Is Sufficient to Trigger Tissue Damage After Cerebral Ischemia

So you described a problem, hinted at a solution, but didn't tell us where we can find that solution so we can get our doctors to use it. Useless.

Adhesion of Leukocytes to Cerebral Venules Precedes Neuronal Cell Death and Is Sufficient to Trigger Tissue Damage After Cerebral Ischemia

Rebecca Isabella Sienel^1,2†, Hiroharu Kataoka^3†, Seong-Woong Kim⁴, Fatma Burcu Seker^1,2 and Nikolaus Plesnila^1,2*

• ¹Laboratory of Experimental Stroke Research, Institute for Stroke and Dementia Research (ISD), University of Munich Medical Center, Munich, Germany
• ²Munich Cluster of Systems Neurology (Synergy), Munich, Germany
• ³Department of Neurosurgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
• ⁴Department of Neurosurgery, University of Giessen, Giessen, Germany

Background: Leukocytes contribute to tissue damage after cerebral ischemia; however, the mechanisms underlying this process are still unclear. This study investigates the temporal and spatial relationship between vascular leukocyte recruitment and tissue damage and aims to uncover which step of the leukocyte recruitment cascade is involved in ischemic brain injury.

Methods: Male wild-type, ICAM-1-deficient, anti-CD18 antibody treated, or selectin-deficient [fucusyltransferase (FucT IV/VII^−/−)] mice were subjected to 60 min of middle cerebral artery occlusion (MCAo). The interaction between leukocytes and the cerebrovascular endothelium was quantified by in vivo fluorescence microscopy up to 15 h thereafter. Temporal dynamics of neuronal cell death and leukocyte migration were assessed at the same time points and in the same tissue volume by histology.

Results: In wild-type mice, leukocytes started to firmly adhere to the wall of pial postcapillary venules two hours after reperfusion. Three hours later, neuronal loss started and 13 h later, leukocytes transmigrated into brain tissue. Loss of selectin function did not influence this process. Application of an anti-CD18 antibody or genetic deletion of ICAM-1, however, significantly reduced tight adhesion of leukocytes to the cerebrovascular endothelium (-60%; p < 0.01) and increased the number of viable neurons in the ischemic penumbra by 5-fold (p < 0.01); the number of intraparenchymal leukocytes was not affected.

Conclusions: Our findings suggest that ischemia triggers only a transient adhesion of leukocytes to the venous endothelium and that inhibition of this process is sufficient to partly prevent ischemic tissue damage.

Introduction

Ischemic stroke is one of the most frequent causes of death and disability worldwide (1, 2). Current therapies include thrombolysis with rtPA or rtPA in combination with mechanical thrombectomy (3–5); however, only up to 30% of stroke patients are eligible for these interventions (5). Hence, there is an ongoing and urgent need for the development of novel therapeutic options for the 70% of stroke patients who do not receive any causal treatment.

For more than four decades, inflammation has been recognized as a major pathomechanism, which is responsible for brain injury following ischemic stroke (6–11). A plethora of elegant experimental and clinical studies discovered that ischemia triggers an acute innate immune response within the brain parenchyma which results in the production of inflammatory cytokines, the upregulation of adhesion molecules on endothelial cells, and the subsequent recruitment of granulocytes and monocytes into ischemic tissue within the first few hours and days after vessel occlusion (12–19). Later on, T-lymphocytes invade the infarcted tissue and may cause further damage (20–22). Despite these impressive steps forward in our understanding of postischemic inflammation, none of the above-mentioned mechanisms translated into a viable therapeutic approach for stroke patients (14). Hence, reevaluation of previous experimental findings and identification of significant knowledge gaps may help identify so far unexplored or neglected therapeutic principles related to postischemic leukocyte recruitment.

Histopathological studies in human tissue, nonhuman primates, and rodents and investigations using radioactively labeled leukocytes in humans and experimental animal models univocally demonstrate that cerebral ischemia is associated with accumulation of polymorphonuclear leukocytes (PMNs) or granulocytes in the brain (10, 11, 23–31). According to experimental studies using radioactively labeled leukocytes or direct visualization of leukocytes by intravital microscopy, recruitment of leukocytes to the ischemic brain starts within the first 2 h after the onset of ischemia (9, 32–35), a time course also supported by investigations in stroke patients (30, 36). The nature of this accumulation seems to follow two different mechanisms: leukocytes may plug capillaries and arterioles during ischemia, thus participating in the so-called no reflow phenomenon (33, 37, 38), and/or they may adhere to the endothelium of postcapillary venules due to upregulation of adhesion molecules (27, 32, 34, 35). No matter which concept of accumulation is followed, most laboratories report that depletion of granulocytes or inhibition or deletion of adhesion molecules reduces ischemic brain damage and improves outcome following experimental stroke (27, 33, 37, 39–50). Hence, there is general agreement that leukocytes accumulate in the brain within the first few hours after cerebral ischemia and inhibition or deletion of adhesion molecules reduce ischemic brain damage. Beyond this generally accepted concept, however, many crucial issues on the role of leukocytes for ischemic tissue damage are still unsolved or highly debated (17, 27, 28). One of the main reasons for this discussion is that most of the above-cited studies used static, histopathological techniques to investigate leukocytes after stroke. Therefore, our knowledge about the dynamics of adhesion, transmigration, and accumulation of leukocytes in the brain after a stroke and how these processes are related to tissue damage is still surprisingly limited.

Technically, leukocyte dynamics after stroke can be addressed by longitudinal in vivo imaging; however, the few studies using this approach either focused on the very first hours after cerebral ischemia, a time when the neuronal injury was not yet present, or on time points later than 24 h after stroke, when the ischemic injury had already occurred (27, 32–35, 51). Consequently, we still do not know whether leukocytes are present in the brain when ischemic damage occurs or whether leukocytes are present at the site of injury (6, 7, 17, 19–21, 52–57). To answer these two important issues, we investigated the full-time course and sequence of leukocyte accumulation to the ischemic brain in parallel with neuronal cell death and tried to decipher which part of the leukocyte adhesion cascade may be involved in ischemic tissue damage.

More at link.

 

Acute Routine Leukocyte and Neutrophil Counts Are Predictive of Poststroke Recovery at 3 and 12 Months Poststroke: An Exploratory Study

And just WHY THE FUCK are you predicting recovery rather than producing recovery? Your mentors and senior researchers incompetently didn't tell you that the only goal in stroke is 100% recovery? Current predictions are totally useless since they are based on the 10% full recovery rate. Of course your doctor doesn't bother telling you how fucking bad the failures are in stroke recovery, that might lead to firings.

Predicting to a 10% full recovery rate is the height of stupidity. So first you need to create 100% recovery protocols, THEN you can do predictions. You are doing the order of research wrong, If we had stroke leadership we could fix this problem.

Show all authors

Vinh A. Nguyen, Sheila G. Crewther, PhD, David W. Howells, PhD, ...

First Published September 17, 2020 Research Article 

https://doi.org/10.1177/1545968320948607

Article information

 

Abstract

 

 

Background and Aims. 

White blood cell (WBC) and neutrophil counts (NC) are common markers of inflammation and neurological stroke damage and could be expected to predict poststroke outcomes.  

Objective. 

The aim of this study was to explore the prognostic value of early poststroke WBC and NC to predict cognition, mood, and disability outcomes at 3 and 12 months poststroke.  

Methods. 

Routine clinical analyses WBC and NC were collected at 3 time points in the first 4 days of hospitalization from 156 acute stroke patients. Correlations using hierarchical or ordinal regressions were explored between acute WBC and NC and functional recovery, depression, and cognition at 3 and 12 months poststroke, after covarying for age and baseline stroke severity.  

Results. 

We found significant increases in NC between <12 hours and 24 to 48 hours time points (P = .05). Hierarchical regressions, covaried for age and baseline stroke severity, found that 24 to 48 hours WBC (P = .05) and NC (P = .04) significantly predicted 3-month cognition scores. Similarly, 24 to 48 hours WBC (P = .05) and NC (P = .02) predicted cognition scores at 12 months. Increases in WBC and NC were predictive of increased cognition scores at both 3 and 12 months (positive recovery) though there were no significant associations between WBC and NC and disability or depression scores.  

Conclusions. 

 Routine acute stroke clinical laboratory tests such as WBC and NC taken between 24 and 48 hours poststroke are predictive of cognition poststroke. It is interpreted that higher rapid immunological activation in the acute phase is an indicator for the trajectory of positive stroke recovery.

Keywords blood biomarkers, poststroke recovery, cognition, white blood cell, neutrophil, leukocyte

Access Options

 

The Inflammatory Response After Ischemic Stroke: Targeting β2 and β1 Integrins

Sounds like this is describing the neuronal cascade of death. But I see nothing here that suggests that the stroke strategy will be updated and followup occur. 

The Inflammatory Response After Ischemic Stroke: Targeting β₂ and β₁ Integrins

Danielle N. Edwards^1,2 and Gregory J. Bix^3,4*

• ¹Sanders–Brown Center on Aging, University of Kentucky, Lexington, KY, United States
• ²Department of Neuroscience, University of Kentucky, Lexington, KY, United States
• ³Department of Neurology, University of Kentucky, Lexington, KY, United States
• ⁴Department of Neurosurgery, University of Kentucky, Lexington, KY, United States

Ischemic stroke is a leading cause of death and disability with limited therapeutic options. Resulting inflammatory mechanisms after reperfusion (removal of the thrombus) result in cytokine activation, calcium influx, and leukocytic infiltration to the area of ischemia. In particular, leukocytes migrate toward areas of inflammation by use of integrins, particularly integrins β₁ and β₂. Integrins have been shown to be necessary for leukocyte adhesion and migration, and thus are of immediate interest in many inflammatory diseases, including ischemic stroke. In this review, we identify the main integrins involved in leukocytic migration following stroke (α_Lβ₂, α_Dβ₂, α₄β₁, and α₅β₁) and targeted clinical therapeutic interventions.

Introduction

Ischemic stroke is a leading cause of death and disability in the United States with limited therapeutic interventions available, including tissue plasminogen activator (t-PA) and endovascular mechanical thrombectomy (Rao et al., 2014; Benjamin et al., 2017; Rai et al., 2017). These interventions are focused on the removal of the thrombus, restoring blood flow, oxygen and glucose to hypoperfused areas, but are unable to affect the inflammatory, necrotic, and blood-brain barrier (BBB) mechanism that follow. In particular, the initial inflammatory cascade is initiated by the decrease in ATP production, release of cytokines, influx of intracellular calcium, reactive oxygen species, etc., that develops during occlusion and continues for days afterward (Sandoval and Witt, 2008). Using shear forces from cerebral blood flow, marrow-derived leukocytes (including polymorphonuclear leukocytes (PMNs), neutrophils, lymphocytes and monocytes) are recruited to the site of injury (Dereski et al., 1993; del Zoppo, 1994; Stefanidakis and Koivunen, 2006).

For the purpose of this review, we will focus on the recruitment and rolling of leukocytes under the direction of integrins, as well as some of their ligands following reperfusion after ischemic stroke. We will then introduce recent β₂ and β₁ integrin-specific stroke clinical trials, and, finally, discuss potential future directions for the field.

Role of Integrins Post-Stroke: an Overview

Integrins are a diverse group of heterodimers composed of 18 different α and β subunits, creating 24 unique combinations. Integrins exist on every cell type, while exhibiting a high diversity of ligands and grouped into four different receptor groups: RGD (Arg-Gly-Asp), laminin receptors, collagen receptors, and leukocyte-specific receptors. Within these groups, integrins can have a variety of ligands and roles following ischemic stroke (reviewed in Edwards and Bix, 2019). Under normal cerebrovascular conditions, integrins are in a highly inactive state, typically in a bent conformation (Takagi et al., 2002; Nishida et al., 2006). Following ischemic stroke, activation signals are sent. Chemokines are translocated to the lumen, on the apical side of endothelial cells, to induce “inside-out” signaling (Chavakis, 2012). Integrins then undergo a conformational change to increase integrin affinity for potential ligands while enhancing detection by localizing to the leading or rear-facing edge of the leukocyte’s cell wall for ligand detection (Ridley et al., 2003; Hyun et al., 2009). Activated integrins then bind to available ligands, permitting leukocytic rolling and intracellular signaling. This is termed “outside-in” signaling (Hato et al., 1998; Tominaga et al., 1998; Ley et al., 2007). Leukocytes continue movement to the site of injury, looking for areas to cross the endothelial cell barrier, and eventually coming to a halt. Aggregation/clustering of integrins increases binding avidity (strength of binding), preventing flow conditions from detaching leukocytes from the endothelial cells (Ley et al., 2007). Using transmigration, leukocytes will infiltrate into the cerebral parenchyma using these integrin-ligand connections.

β₂ Integrins

β₂ integrins are the only group of integrins exclusively expressed on leukocytes (derived from hematopoietic cells) (Schenkel et al., 2004), and like most integrins, are highly conserved across species (Schittenhelm et al., 2017). They are also the most highly expressed integrin on circulating blood leukocytes, tending to cluster at the retraction area of the cell (the rear), in both an active and inactive state, compared to other β₁, β₄, β₃, and β₇ integrins found on circulating leukocytes (Pierini et al., 2000; Lindbom and Werr, 2002). Genetic leukocyte adhesion changes (LAD-1, as discussed above) has been attributed to mutations in the β₂ subunit, reducing β₂ expression. Thus, leukocytic movement is reduced on the cell surface with less movement toward the site of inflammation (Arnaout, 1990; Scharffetter-Kochanek et al., 1998). Importantly, in β₂ inhibited mice, there is not total arrest of leukocytic recruitment or infiltration (Pierini et al., 2000), suggesting that other factors likely play a role. There are 4 identified heterodimers of β₂ integrins, and of these, the most highly studied are α_Lβ₂ and α_Mβ₂ in ischemic stroke, and will be reviewed in more detail below. The other β₂ integrins, α_Xβ₂ and α_Dβ₂, have not been individually studied in the context of stroke as have α_Lβ₂ and α_Mβ₂ integrins, though CD18 (β₂) inhibition in addition to t-PA has been shown to increase the time window of t-PA administration without an increase in hemorrhagic transformation in a rat embolic stroke model (Zhang et al., 1999). Furthermore, Figure 2 summarizes the results in this section.

FIGURE 2

Figure 2. Representative image of the β₂ integrin response following experimental stroke and inhibitory antibody treatment in preclinical trials. Inhibition of (A) α_Lβ₂ and (B) α_Mβ₂ integrins post-stroke responses and effects.

α_Lβ₂ Integrin

Integrin α_Lβ₂ is also referred to as CD11a/CD18 and LFA-1 (lymphocyte functional-associating antigen-1). α_Lβ2 integrin acutely increases in ischemic stroke patients, with detectable amounts through 72 h associated around the area of ischemia (Gerhard et al., 2000; Zhao et al., 2002). This suggests a correlation between α_Lβ₂ integrin expression and inflammatory damage following ischemia. α_Lβ₂ is expressed on all leukocytes (Soriano et al., 1999), though at particularly high levels on T-lymphocytes (Hammond et al., 2014; Walling and Kim, 2018). In healthy individuals, extracted blood analysis revealed that α_Lβ₂ activation requires leukocytic rolling on P- or E-selectins, inducing an active conformational change (Kuwano et al., 2010), but it is the binding of chemokines g-protein coupled receptors (GPCR) and Rap-1 activation that induces the high-affinity conformational state of α_Lβ₂ (Steffen et al., 1994; Greenwood et al., 1995; Ghandour et al., 2007). In this state, α_Lβ₂ has many possible ligands, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, and junctional adhesion molecule-1 (JAM-1) (Marlin and Springer, 1987; de Fougerolles et al., 1991, 1994; Tian et al., 2000), though ICAM-1 is preferentially bound (Walling and Kim, 2018). The high avidity α_Lβ₂-ICAM-1 complex, once formed, allows t-lymphocytes to move against circulatory flow and the shear forces, resulting in the high-speed movement of leukocytes (Katakai et al., 2013; Dominguez et al., 2015).

In an intraluminal model of experimental ischemic stroke, α_Lβ₂ inhibition with the use of transgenic mice results in reduced infarct volume, edema volume and mortality. However, this phenomenon is evident in transient, but not permanent middle cerebral artery occlusion (Arumugam et al., 2004). This may be due to the previously mentioned high avidity of α_Lβ₂-ICAM-1 bonds, and is evident in an in vitro study using α_Lβ₂ (LFA-1) knock-in mice that experience high avidity through binding of lymphocytes mediated through ICAM-1 binding, but are unable to continue movement due to a non-polarized uropod (Park et al., 2010). An explanation for this phenomenon may be that the recycling process within the leukocyte is overwhelmed (Shaw et al., 2004). By enhancing α_Lβ₂ expression, recycling may not be able to allow for dislocation of α_Lβ₂-ICAM-1 complexes, preventing movement from the loss of high adhesion bonds. Enhanced α_Lβ₂ expression could be a potential new avenue for therapy, especially if no enhanced mortality, infection, etc., are observed.

Independently, ICAMs play a significant role in inflammation following ischemic stroke. ICAM-1, in particular, is acutely increased in both cultured human endothelial cells undergoing hypoxia and following intraluminal suture middle cerebral artery occlusion, while expression remains sustained for up to a week post-injury (Hess et al., 1994a,b; Zhang et al., 1995). ICAM-2, another possible ligand, does not change in expression following cytokine stimulation (de Fougerolles et al., 1991; Nortamo et al., 1991a,b). Furthermore, serum of ischemic stroke patients contains soluble ICAM-1, but not ICAM-2 in addition to being a risk factor (Kaplanski et al., 1994; Shyu et al., 1997). Antibodies targeting ICAM-1 in rodents and humans have shown contradictory results. An intraluminal suture middle cerebral artery occlusion model in mice and rats showed a decrease in leukocyte infiltration and infarct volume (Connolly et al., 1996; Kitagawa et al., 1998; Vemuganti et al., 2004), while one study reported opposing effects (Furuya et al., 2001). ICAM-1 inhibition was translated to the clinic through testing of the murine ICAM-1 antibody, Enlimomab in ischemic stroke. Unfortunately, the study was halted early due to increased rate of infection, infarct volumes, neurological scores and mortality for patients (Furuya et al., 2001).

α_Mβ₂ Integrin

Integrin α_Mβ₂, also known as CD11b/CD18 and Mac-1 (macrophage-1 antigen)_, exhibits many similarities to α_Lβ₂ through its expression on all leukocytes (Springer et al., 1979), and common ligand binding partners such as the family of ICAMs and JAMs (von Andrian et al., 1991). Additional ligands are fibrinogen, heparin (von Andrian et al., 1991), elastase (Cai and Wright, 1996), complement C3 fragment (C3bi) (Micklem and Sim, 1985), kinogen components, and urokinase and its receptor (Chavakis et al., 1999). Just as α_Lβ₂, hypoxia induced factors (cytokines, chemokines, etc.) induce conformational change of α_Mβ₂ to a high affinity ligand-binding state (Stanimirovic et al., 1997). Binding assays with ICAM-1 as a ligand and both α_Lβ₂ and α_Mβ₂ as receptors show α_Lβ₂ integrin is preferably bound (Lub et al., 1996). This suggests that the binding sites on both α_Lβ₂ and α_Mβ₂ compete for ICAM-1 binding.

Following experimental ischemic stroke in rats, integrin α_Mβ₂ is upregulated (Campanella et al., 2002), and has shown benefit when inhibited. Antibodies against both CD11b/CD18 reduce infarct volume and reestablish cerebral blood flow as a result of decreased neutrophil infiltration following intraluminal stroke surgery (Chen et al., 1994; Bowes et al., 1995; Zhang et al., 1995). In a different approach, the addition of recombinant neutrophil inhibitory factor (rNIF) inhibits a binding domain on Mac-1 and yields similar results in the same intraluminal occlusion model (Jiang et al., 1998). Furthermore, and similarly to α_Lβ₂ integrin inhibition, inhibition of α_Mβ₂ is also effective in transient, but not permanent experimental ischemic stroke in an embolic occlusion model (Zhang et al., 2003).

β₁ Integrins

β₁ integrins are a diverse set of integrins, with laminin-binding, collagen-binding, RGD-binding and leukocyte heterodimers. β₁ integrins are not as highly expressed on leukocytes as β₂ integrins, but they do play a major role in leukocyte adhesion and migration following ischemic stroke. The activity of β₁ integrins is similar to β₂ integrins. They undergo a conformational change to induce “inside-out” and “outside-in” cellular signaling (Campanero et al., 1994). As the cells migrate, the β₁ integrins are most commonly clustered around the uropod, but will be located in any area of the leukocyte that is in contact with the endothelial cell or extracellular matrix (Campanero et al., 1994; Caimi et al., 2001). Inhibition of the β₁ integrin, just as with β₂ integrin inhibition, does not fully stop leukocyte rolling. However, when both β₁ and β₂ integrins are inhibited, complete leukocyte arrest occurs (Lobb and Hemler, 1994; Pierini et al., 2000). This suggests that both β₁ and β₂ integrins are necessary for leukocyte migration, regardless of expression load. Of all the β₁ integrins, both α₄β₁ and α₅β₁ appear to be the most highly expressed and the most studied in post-stroke inflammation. The other β₁ integrin expressed on leukocytes, α₉β₁, has not been studied in the context of stroke as its expression and role has not yet been fully elucidated in the brain. Figure 3 summarizes the results discussed in this section.

FIGURE 3

Figure 3. Representative image of the β₁ integrin response following experimental stroke and inhibitory antibody treatment in preclinical trials. Inhibition of (A) α₄β₁ and (B) α₅β₁ integrins post-stroke responses and effects.

α₄β₁ Integrin

α₄β₁, also known as CD49d/CD29 VLA-4 (very late antigen-4)_, is localized primarily to leukocytes (neutrophils, monocytes, lymphocytes, macrophages, etc.) and microglia as a leukocyte-specific receptor. Additionally, α₄ will also dimerize with β₄, which is found in gut endothelium (Hammond et al., 2014). Activation of α₄β₁ integrin results from the binding of upregulated chemokines to GPCRs in the same manner as α_Lβ₂ as discussed above (Vajkoczy et al., 2001). This stimulates binding to α₄β₁’s preferred ligand, VCAM-1, but experiments have shown some preference for paxillin ICAM-1 (Steffen et al., 1994; Ghandour et al., 2007), and fibronectin (Hart and Greaves, 2010) as well. Interestingly, instead of using the β₁ submit of the heterodimer for binding, integrin α₄β₁ uses its α subunit of α₄β₁ to mediate binding to VCAM-1 (Luo et al., 2007).

Preclinical ischemic stroke studies targeting α₄β₁ have shown increasingly varied results. Most researchers reported a decrease in VCAM-1 expression, cytokine production, and infiltrating leukocytes (Liesz et al., 2011; Langhauser et al., 2014; Llovera et al., 2015), but this reduction in inflammation did not result in reduced infarct volumes or functional deficit following analysis of a randomized preclinical trial involving six different centers (Llovera et al., 2015). Langhauser et al went one step further and found that no treatment paradigm (prophylactic or therapeutic) and no model (transient or permanent) showed efficacy (Langhauser et al., 2014). On the other hand, both Becker, 2002 and Relton et al., 2000 found that inhibition of α₄ improved both infarct volumes and functional deficits. When a preclinical randomized control trial was implemented at multiple centers, researchers found efficacy only in patients with small infarct volumes (Llovera et al., 2015). Collectively, these contradictory results may be caused by a couple of scenarios, 1) the varying expression of integrin α₄β₁ expression following ischemic stroke resulting in continued leukocyte infiltration, or 2) integrin α₄β₁ is not a primary driver of post-stroke pathophysiology, but other factors, including other integrins, promote leukocyte migration (Hammond et al., 2014).

α₅β₁ Integrin

α₅β_1, also known as CD49e/CD29 and VLA-5, plays an as yet largely undetermined role in inflammation, with studies primarily limited to cell culture. What is known is that leukocytes express different β₁ integrins with α₅β₁ composing around 50% of all β₁ –integrins expressed on neutrophils (Pierini et al., 2000) and monocytes (Pacifici et al., 1994). Additionally, α₅β₁ is necessary for leukocyte adhesion. Only inhibition of both α₅β₁ and β₂ integrins completely blocks adhesion in vitro (Pierini et al., 2000), while inhibition of α₅β₁ alone prevents transmigration across the BBB (Labus et al., 2018) in vitro. As an RGD receptor, fibronectin has been shown to be the primary and preferred [over other potential ligands such as fibrinogen (Suehiro et al., 1997)] ligand for α₅β₁ on endothelial cells and leukocytes (Schaffner et al., 2013; Bharadwaj et al., 2017). Importantly, in the presence of activated α_Lβ_2, leukocyte α₅β₁ binding to fibronectin is enhanced (Bohnsack, 1992; Loike et al., 1999; Gronholm et al., 2016). α₅β₁ integrin expression is induced by cytokines, particularly TNFα (Li et al., 2011) toward the leading edge of the cell in contrast with other integrins at the uropod (Pierini et al., 2000). Furthermore, α₅β₁ integrin appears to be highly sensitive to calcium (Pierini et al., 2000), an ion that is increased rapidly following reperfusion (Sandoval and Witt, 2008). Upon calcium buffering, α₅β₁ expression moves from the front of the cell to the uropod and the leukocyte becomes elongated. The change in expression localization and morphology is attributed to non-movement as the leukocyte cannot detach α₅β₁ from the vascular wall (Pierini et al., 2000). Recently, Edwards et al. (2019) found that inhibition of α₅β₁ integrin by the small peptide ATN-161 prevented CD45+ leukocytes from infiltrating the brain parenchyma following the tandem/transient common carotid artery/middle cerebral artery occlusion model. Additionally, mice were observed to have reduced BBB permeability, functional deficits, edema, and infarct volume following middle cerebral artery occlusion (Roberts et al., 2015; Edwards et al., 2019). Thus, targeting α₅β₁ after ischemic stroke could be a new avenue for reduction of inflammation following ischemic stroke.

Clinical Implications

The preclinical studies discussed here point towards the potential of targeting β₂ and β₁ integrins in the treatment of post-stroke inflammation. Though their potential has not fully been elucidated, many clinical trials, not just limited to stroke, have been approved in the last 10 years targeting these integrins.

The most common target for post-stroke inflammation are the β₂ integrins. Though some efficacy has been reported, no clinical stroke trials to date have targeted the α_L subunit in stroke patients. However, one clinical trial with the monoclonal antibody, Efalizumab, has shown promise in decreasing T-lymphocyte rolling in patients with moderate-severe plaque psoriasis (Lebwhohl et al., 2003).

In preclinical studies targeting α_Mβ₂, a hookworm isolated recombinant glycoprotein targeting rNIF (UK279276) (Zhang et al., 2003) and humanized Hu23F2G (Leukarrest) (Yenari et al., 1998), were both shown to decrease infarct volume and increase functional recovery following reperfusion. Both therapies had negligible side effects in Phase 1 studies and thus were continued to a Phase II study, respectively, before the trials were halted due to no observed efficacy (Becker, 2002; Krams et al., 2003). The failure to target α_Mβ₂ integrin may be due to the observation that human ischemic stroke patients do not experience the increase in α_Mβ₂ expression as seen in rodent stroke models (Caimi et al., 2001). Interestingly, when given in conjunction with United Kingdom279276, patients experienced a slight improvement (Krams et al., 2003), but no follow-up has been conducted. This interesting effect may be worth additional investigation in future clinical trials.

Clinical inhibition of β₁ integrins, on the other hand, is small and varied. Of the current clinical trials, one trial has emerged targeting α₄β₁ in the context of ischemic stroke. The monoclonal antibody targeting the α₄ subunit (Natalizumab) has been successful in protecting patients from relapses in multiple sclerosis (Polman et al., 2006) and Crohn’s disease (Sandborn et al., 2015). However, in a Phase II ischemic stroke study, patients receiving Natalizumab showed no improvement in infarct growth or neurological scores over 30 days. Furthermore, two patients (out of 79) died from serious infections attributed to Natalizumab treatment (Elkins et al., 2017). At this time, there are no further clinical trials planned.

Future Considerations

As discussed in this review, targeting leukocytic integrins has had limited to no efficacy in clinical trials. Importantly, these studies have collectively employed only three different therapeutics and two targets; there are still significant areas that can be investigated. Though not discussed here, most preclinical investigations have focused on the ligands themselves rather than the integrin as the therapeutic target, highlighting the continued importance of integrins in stroke.

It is also important to note that preclinical studies carried out in rodents inadequately model the post-stroke pathophysiology that patients experience. Preclinical stroke research is also typically limited, focusing on one species, sex, and age that do not necessarily match the demographic of stroke patients (see Kahle and Bix, 2012 for a review of this topic). Furthermore, as the changes following stroke and/or reperfusion are inadequately understood, identifying appropriate therapeutic targets that translate from the lab to clinical trials, has been particularly challenging.

However, this does not suggest abandoning therapeutic trials for ischemic stroke. As mentioned above, stroke is a leading cause of death and disability, separate from cardiovascular disease. This will not improve without intervention with our aging and obese population. Fortunately, with the advent of stroke mortality-altering therapies, i.e., t-PA and endovascular mechanical thrombectomy, our financial burden has shifted to aftercare. When we review the amount of trials performed for thrombolytic agents (Multicentre Acute Stroke Trial–Italy (MAST-I) Group, 1995; National Institute of Neurological Disorders and Stroke rt-Pa Stroke Study Group, 1995; The Multicenter Acute Stroke Trial–Europe Study Group, 1996) and endovascular thrombectomy [MR CLEAN (Berkhemer et al., 2015), ESCAPE (Goyal et al., 2015), EXTEND IA (Campbell et al., 2015), SWIFT PRIME (Saver et al., 2015), and REVASCAT (Jovin et al., 2015)] as potential treatments of ischemic stroke, it is obvious that the complexities of stroke affect the outcome of the clinical trial. This includes, but is not limited to, the time a patient takes to arrive at an ER, time to treatment, location of the stroke, amount of surrounding collaterals, current medications and co-morbidities (diabetes, cancer, etc.), and if the patient has experienced multiple strokes.

Based on current advances, as well as previous failures, a focus on integrins as a therapeutic target for stroke is emerging. A significant reason for this focus may be the complex, multi-dimensional role that integrins appear to play in brain pathophysiology. Integrins are diverse, existing on all cell types with varying roles depending upon expression and activation. This complexity can represent a significant challenge to integrin-targeted therapies inasmuch as such therapies could have diverse, even unintended off-target effects. However, we believe that this can be overcome by a better understanding of how integrin function and expression is altered after stroke, with the potential to exploit stroke-dependent integrin changes to therapeutic effect. For example, identifying a specific integrin to be upregulated in select cells in the post-stroke brain or brain-targeting cells, but not in other organs, could render it a viable therapeutic target. This emphasizes the need and importance of preclinical stroke research to discover and unravel the complexities of integrin biology. We are confident that such studies will result in viable new stroke therapies.

Conclusion

In this review, we have implicated integrins as an area of research for limiting inflammation following ischemic stroke. To date, therapeutic inhibition of α_Lβ₂, α_Mβ₂, and α₄β₁ has shown promising results in preclinical studies, but translation to the clinic has been disappointing. Going forward, more targeted antibodies to all reactive β₁ and β₂ integrins after ischemic stroke may prove more beneficial, but more research needs to be done to completely understand the human inflammatory response and how that relates to changes in preclinical models.

Role of Immune Cells Migrating to the Ischemic Brain

I got nothing out of this that could help any survivor recover better. But since I'm not medically trained I obviously know nothing. 

Role of Immune Cells Migrating to the Ischemic Brain

Anna M. Planas

Originally published27 Aug 2018Stroke. 2018;49:2261-2267

The central nervous system and the immune system are tightly interconnected through complex communicating networks.¹ Immune cells are distributed in specific central nervous system compartments. Microglia are the innate immune cells resident in the brain parenchyma. Macrophages surround the blood vessels and also line the leptomeninges and the choroid plexus together with dendritic cells and lymphocytes, among other immune cells, where they play immunosurveillance functions. Therefore, the immune system keeps a close watch on brain function and reacts when brain homeostasis is lost because of injuries or diseases. Sterile organ damage may turn immune cells into harmful agents and for this reason they are regarded as targets for therapeutic intervention in acute stroke.² Stroke induces strong inflammatory reactions involving the local production of cytokines, such as TNF-α (tumor necrosis factor-α) by various brain cells, including human neurons,³ activation of glial and endothelial cells, blood-brain barrier damage, and infiltration of different types of leukocytes after an orchestrated time course.⁴ Given the variety of leukocyte subsets trafficking to the ischemic brain tissue, this review will focus on neutrophils, monocyte/macrophages, and T and NK (natural killer) lymphocytes. For further information, the readers are addressed to previous reviews on dendritic cells⁵ and B lymphocytes.⁶ The different immune cells are considered separately in the next sections, but leukocyte infiltration surely comprises intercellular crosstalks by mechanisms that are not entirely known.

Neutrophils

Neutrophils are among the first cells attracted to the brain after ischemic stroke where they are detected in the microvessels within the first hour⁷ and peak at 1 to 3 days.^4,7,8 Neutrophils are short-lived innate immune cells containing different types of granules with antimicrobial pro-oxidant and proteolytic enzymes that can damage the tissues. Accordingly, neutrophils are regarded as detrimental following compelling evidence associating these cells with blood-brain barrier breakdown and brain injury.^7,9 Also, higher blood neutrophil counts are associated with larger infarct volumes in acute ischemic stroke patients.¹⁰ Nevertheless, the pathogenic role of neutrophils in ischemic stroke is still not conclusive. For instance, there are conflicting results in the literature on the potential benefit of neutrophil depletion in experimental ischemia models.^9,11 Furthermore, we lack entire demonstration that neutrophils reach the ischemic tissue before substantial neuronal death has occurred.¹¹ Nonetheless, neutrophils can exert detrimental effects already from the vessel wall. Adhesion of neutrophils to the inflamed endothelium after ischemia/reperfusion is involved in the no-reflow phenomenon, obstructing blood flow in precapillary arterioles, postcapillary venules, and the capillary bed.^7,12 In addition, neutrophils in the vessel lumen and at perivascular locations can damage the blood-brain barrier by releasing proteolytic enzymes and pro-oxidant molecules (Figure).⁹ Moreover, neutrophils can produce NETs (neutrophil extracellular traps) promoting clot formation.¹³ NETs can precipitate thrombotic events and impair tPA (tissue-type plasminogen activator)-induced thrombolysis.¹⁴ In turn, thrombolysis may exacerbate detrimental effects of neutrophils because tPA promotes neutrophil transmigration to the reperfused tissue by proteolytic activation of plasmin and matrix metalloproteinases.¹⁵ These effects might contribute to explain why neutrophilia and high neutrophil-to-lymphocyte ratio are associated with the risk of hemorrhagic transformation in ischemic stroke patients treated with tPA.¹⁶ After permanent middle cerebral artery occlusion (MCAo) in mice, we observed the formation of intravascular NETs and found NETs in perivascular locations and in the brain parenchyma.¹⁷

• Download figure
• Download PowerPoint

Figure. Schematic representation of leukocyte infiltration to the ischemic brain tissue. Neutrophils are attracted to the activated endothelium and reach perivascular spaces after extravasation from intracerebral venules and leptomeningeal vessels. Activated neutrophils are prothrombotic and can damage the blood-brain barrier (BBB). The presence of neutrophils in the brain parenchyma is observed only under certain circumstances, but the conditions determining that neutrophils remain in perivascular spaces or reach the parenchyma are still poorly defined. Attracted by certain chemokines, immature proinflammatory monocytes infiltrate the ischemic tissue where they mature to macrophages, acquire signs of alternative polarization, and seem to be involved in tissue repair. Current experimental evidence suggests that lymphocytes, in particular T cells and γδ T cells, play detrimental roles in the acute phase of stroke by promoting thromboinflammation and tissue damage. Natural killer (NK) cells are attracted to the ischemic tissue, but their function is not fully clear. RBC indicates red blood cells.

There is also some controversy on whether neutrophils actually reach the ischemic brain parenchyma at all. An elegant study by Enzmann et al¹⁸ noticed the massive accumulation of neutrophils in perivascular spaces surrounding venules within the ischemic tissue after ischemia/reperfusion in mice. Most neutrophils remained perivascular, and only a few were detected in the brain parenchyma.¹⁸ This is an important observation because it highlights that perivascular spaces are a niche for neutrophils where they accumulate after transient MCAo. We also detected neutrophils in perivascular locations and leptomeningeal spaces in the mouse after permanent MCAo.¹⁷ These results suggest that, besides extravasating from intracerebral venules, neutrophils extravasate from leptomeningeal vessels and migrate from the subpial space along the vessels penetrating the cortex.¹⁷ However, our study¹⁷ and previous studies⁷ found neutrophils in the ischemic brain parenchyma using models of permanent MCAo. Ischemic conditions involving severe endothelial damage, vessel rupture, and microbleeds or hemorrhagic transformation, are expected to facilitate the presence of neutrophils in the brain parenchyma. Other conditions, such as high blood glucose, also promote neutrophil infiltration.¹⁹ A recent study²⁰ analyzed the postmortem brain of 16 ischemic stroke patients and confirmed the presence of neutrophils in the leptomeninges and perivascular spaces, but neutrophils were rare in the infarcted parenchyma with the exception of 1 patient deceased 3 days after stroke with no signs of infection. Interestingly, the time to death of this series of patients was 1 day in 2 cases, 3 days in the case above mentioned, and then times ranged from 8 days to 240 days poststroke.²⁰ Neutrophils display a specific time-window of attraction to the damaged tissues after acute injuries, and they have a short life in tissues. Therefore, the time to death of ischemic stroke patients is critical to look for the presence of neutrophils in the brain parenchyma. More studies of human tissue within the first days poststroke are necessary to understand under which conditions neutrophils might gain access to the infarcted brain parenchyma.
Despite many advances, there are aspects of neutrophil behavior in stroke that are still difficult to interpret. For instance, neutrophils with anti-inflammatory and repair phenotypes were found in the ischemic brain tissue of experimental animals,²¹ neutrophils of ischemic stroke patients show a reduced oxidative burst and NET formation,²² and microglia surrounding blood vessels phagocyte neutrophils.²³ The possibility that neutrophils were passive bystanders under some circumstances but active players in others depending on specific features of the ischemic lesion needs further consideration.

Monocyte/Macrophages

After brain ischemia, microglia acquire a reactive morphology resembling macrophages. Classically, immunohistochemical studies have described the presence of reactive microglia/macrophages peaking at ≈4 days postischemia in rats or mice, but it was not possible to distinguish whether these cells derived from resident microglia or they infiltrated from the periphery. Nowadays, flow cytometry, cell type-specific fluorescent reporter mice, adoptive transfer of fluorescent cells, generation of chimeras, and recently identified specific microglia markers, allow differentiating resident reactive microglia from infiltrating macrophages. Monocyte infiltration is detected within the first 24 hours postischemia, peak at 4 days, and some of these cells persist for weeks and acquire features of tissue macrophages. Immature CCR2⁺Ly6C^hi proinflammatory monocytes are the subset of monocytes first attracted to the ischemic brain tissue.^24–26 These cells might be released by the bone marrow, but a study reported that monocytes reaching the ischemic brain originate in the spleen.²⁵
Infiltrating macrophages were classically associated with inflammation and brain damage after ischemic stroke. In mice, monocyte infiltration is largely dependent on CCR2 (C-C motif chemokine receptor type 2), the receptor of the chemokine CCL2 (C-C motif chemokine ligand 2), also known as MCP1 (monocyte chemoattractant protein 1). To investigate the role of monocytes, several studies used CCR2-deficient mice or CCR2 inhibitors, with the limitation that besides the subset of Ly6C^hi monocytes other cells, like some T cells, also express CCR2. CCR2-deficiency reduced the ischemic brain lesions in mice.²⁷ Challenging this view, CCR2 drug inhibitors exacerbated the brain lesion.²⁸ Furthermore, anti-CCR2 blocking antibodies impaired spontaneous long-term functional recovery,²⁹ depletion of monocytes/macrophages worsened the ischemic lesion,³⁰ and infiltrating macrophages prevented hemorrhagic transformation of the ischemic lesion.²⁴ By systemic injection of fluorescent monocytes after brain ischemia, we observed fluorescent cells in the subpial space, and along the vessels penetrating the cortex,²⁶ supporting the view that a subset of infiltrating macrophages establish persistent interactions with the blood vessels (Figure).
The phenotype of activated macrophages depends on the environmental stimuli. The M1 and M2 phenotypes are prototypical states of macrophage polarization achieved in culture after exposure to certain cytokines. The M1 phenotype is proinflammatory whereas the M2 phenotype promotes resolution of inflammation and repair. Macrophages infiltrating the ischemic tissue, including the Ly6C^lo population and some of the Ly6C^hi monocytes, acquire features of alternatively polarized M2 macrophages during the first week postischemia.^26,28–30 Studies of human ischemic infarcts reported that macrophages initially showed proinflammatory features that with lesion maturation transformed into anti-inflammatory phenotypes.²⁰ Interestingly, a study noticed that after ischemia in mice, the expression of M2 markers increased within the first week but then decreased, whereas proinflammatory markers persisted and predominated at week 2, suggesting a long-lasting inflammatory status.³¹
The factors that contribute to the time-dependent changes in macrophage phenotypes in the ischemic brain tissue are not entirely identified. Increased anaerobic glycolysis and activation of the hypoxia-inducible factor-1 are associated with proinflammatory M1 phenotypes, whereas energy production in M2 phenotypes rather relies on fatty acid oxidation.³² In M1 activated macrophages, arginine metabolism occurs through inducible nitric oxide synthase leading to generation of reactive oxygen and nitrogen species that damage proteins, lipids, and DNA. In contrast, M2 macrophages metabolize arginine through arginase-1 generating polyamines involved in cell division and collagen synthesis, among other functions.³² However, under pathological conditions 1 single phenotypic feature may not be sufficient to attribute any specific phenotype to the cells. Likely, cellular metabolic adaptations to ischemia and reperfusion together with the cytokine environment and phagocytic activity have an impact on the phenotype and function of macrophages and microglia.

Lymphocytes

T Cells

Severe stroke reduces the numbers of lymphocytes in the circulation and lymphoid organs.³³ In contrast, T-cell numbers increase in the ischemic brain within the first 24 hours and can persist for long times.⁴ During the first hours after ischemia/reperfusion, T cells facilitate adhesion of platelets and leukocytes to the vascular endothelium³⁴ causing a phenomenon called thromboinflammation³⁵ by which molecular and cellular players in thrombosis and coagulation promote proinflammatory pathways exacerbating the brain lesion.³⁶ However, the interaction of T cells with platelets may also have hemostatic effects preventing hemorrhagic transformation after severe ischemic stroke.³⁷ T cells are found in subpial and cortical vessels and infiltrating the ischemic lesion.^38,39 In addition, the choroid plexus is a gateway for T cells migrating to the periphery of cortical infarction.⁴⁰ Importantly, CD8⁺ cytotoxic T cells were detected in human ischemic infarcts.²⁰ Also, ischemic stroke patients show increased frequency of CD4⁺CD28^null cells in blood associated with stroke severity and serum levels of proinflammatory cytokines.⁴¹ CD4⁺CD28^null T cells are an interesting subset of T cells because they have enhanced effector functions, are associated with senescent T cells, and expand under inflammatory conditions.⁴² At later phases, CD4⁺ cells accumulate in the brain of mice peaking at day 14 and persisting at day 30 after ischemia/reperfusion.⁴³ Furthermore, emerging evidence suggests that antigen-mediated T-cell responses take place in subacute or chronic stages after stroke and may worsen stroke outcome.^43–47 However, in central nervous system trauma, protective autoimmunity mediated by T-cell responses is involved in promoting recovery.⁴⁸ Overall, T cells seem to play innate functions and interact with players in thrombosis and hemostasis in the acute phase of stroke, whereas at later stages they exert adaptive functions that could affect stroke outcome in the long term.

γδ T Cells

Subsets of unconventional innate T cells with invariant T-cell receptor could play a role in acute ischemic brain damage. Growing evidence supports that γδ T cells are pathogenic in experimental brain ischemia/reperfusion by secreting IL (interleukin)-17 and exacerbating the inflammatory response.^49–51 Moreover, IL-17A⁺ lymphocytes were detected in the postmortem brain of stroke patients.⁵⁰ Interestingly, γδ T cells are abundant in the gut from where they seem to traffic to the leptomeninges after brain ischemia.⁵² T helper 17 cells (Th17) and γδ T cells increase in the blood of stroke patients in association with increased levels of IL-17A, IL-23, IL-6, and IL-1β.⁵³ In spite of the fact that IL-17 producing cells are a small subset of cells, they seem to play a prominent role in orchestrating the inflammatory response in acute stroke and exacerbating the lesion (Figure).

Regulatory Lymphocytes

Regulatory lymphocytes exert immunomodulatory and immunosuppressor functions. Several lines of evidence support beneficial effects of regulatory T cells (Treg)⁵⁴ and regulatory B cells⁵⁵ in experimental brain ischemia. However, other studies found acute detrimental effects of Treg in brain ischemia/reperfusion⁵⁶ as previously reviewed.⁵⁷ Although the number of Treg found in the ischemic brain parenchyma during the first days poststroke is low, Treg strongly accumulate in the ischemic lesion 15 days poststroke where potentially they could inhibit autoimmune responses.⁴³ Increased apoptosis of Tregs, loss of Tregs in peripheral blood, and impaired suppressive function of the remaining Treg population has been reported in ischemic stroke patients.^53,58,59 However, other studies reported upregulation of Tregs in stroke patients in spite that decreased Treg function was observed, particularly in female patients.⁶⁰ Notably, an increased proportion of Treg cells was reported in the spleen of mice 4 days after transient MCAo.⁶¹

NK Cells

NK innate lymphocytes show a rapid and transient increase in the ischemic brain tissue.^4,8 A study reported no benefits of depleting NK cells in permanent or transient MCAo.⁴⁴ In contrast, another study suggested pathogenic actions of NK cells by promoting inflammation and neuronal cytotoxicity.⁶² This study reported infiltration of NK cells in the ischemic brain tissue of humans and mice where NK cell numbers peaked as soon as 3 hours postischemia and then declined.⁶² Interestingly, the β2-nACh-R (nicotinic acetylcholine) receptor seems to be involved in the NK cell decline observed in the ischemic tissue from 3 hours postischemia.⁶³ Induced-persistence of NK cells in the ischemic tissue achieved by interfering with this cholinergic receptor did not modify lesion size but increased systemic IFNγ (interferon γ), protected from bacterial infection, and enhanced poststroke survival.⁶³ More studies are needed to validate the putative capacity of brain infiltrating NK cells to prevent poststroke infection, the role of central acetylcholine in this process, as well as the suggested rapid pathogenic effect of NK cells worsening the acute ischemic brain lesion.

Therapeutic Intervention

Strategies designed to prevent negative actions of leukocytes have been taken to the clinic in acute ischemic stroke patients but with no success to date.⁶⁴ Several studies with drugs blocking the action of neutrophils were investigated.^11,64 As an example, the ASTIN trial (Acute Stroke Therapy by Inhibition of Neutrophils) investigated a compound known as UK-279 276, a recombinant neutrophil inhibitory factor that selectively binds the CD11b integrin of macrophage-1 antigen (CD11b/CD18).⁶⁵ The treatment did not improve recovery above placebo. This trial followed encouraging results of a few preclinical studies with UK-279 276 in experimental models of brain ischemia/reperfusion.⁶⁶ However, only a fifth of patients in the ASTIN trial received tPA.⁶⁵
Monocyte/macrophages may acutely exacerbate the inflammatory responses, but experimental studies have identified their involvement in resolution of inflammation, vascular protection, and recovery of function,^24,27–29 possibly linked to the phagocytic and vasculoprotective roles of these cells. These protective actions of monocytes are in line with the beneficial effects of administration of autologous bone marrow–derived mononuclear cells (MNC) after experimental ischemic stroke.⁶⁷ MNC contain myeloid and lymphoid cells, as well as hematopoietic and mesenchymal stem cells. MNC administration 24 hours after MCAo improved functional recovery, reduced lesion size and proinflammatory cytokines, and enhanced vessel density and neurogenesis,⁶⁸ and these benefits were long lasting.⁶⁹ Furthermore, MNC reduced blood-brain barrier permeability and decreased the severity of hemorrhagic transformation after tPA in an embolic stroke model.⁷⁰ However, MNC therapy did not improve outcome in hypertensive rats.⁷¹ MNC reach the periphery of brain infarction soon after administration,⁶⁸ and then the cells seem to differentiate into smooth muscle cells and endothelial cells, incorporate into vessel walls, and enhance the growth of leptomeningeal anastomoses, the circle of Willis, and basilar arteries.⁶⁹ Phase I trials administering MNC to ischemic stroke patients have shown safety, but the clinical efficacy of this cell therapy awaits demonstration.⁷²
Anti-inflammatory treatments, such as minocycline, have not been successful in the clinic.⁶⁴ Experimental evidences, including a cross-laboratory preclinical study in mouse models of brain ischemia,⁷³ support the therapeutic potential of the IL-1Ra (IL-1β receptor antagonist). A recent clinical trial with subcutaneous administration of IL-1Ra showed safety and reduction of plasma IL-6.⁷⁴ However, the analysis excluded a major clinical benefit of the treatment, and negative effects potentially attributable to interactions of IL-1Ra with tPA became apparent.⁷⁴
Experimental studies support damaging effects of T lymphocytes in the acute phase of stroke. Accordingly, fingolimod, a drug approved for remitting-relapsing multiple sclerosis that sequesters lymphocytes in the lymph nodes preventing lymphocyte access to the inflamed tissues, showed beneficial effects in preclinical studies and small clinical trials in acute ischemic stroke patients, including patients receiving thrombolysis.⁶⁴ By acting on S1P1 (sphingosine-1-phosphate receptor 1), fingolimod induces sustained lymphopenia, but current data do not show higher incidence of poststroke infection in patients receiving fingolimod. Fingolimod also acts on endothelial S1P1 receptor increasing vascular barrier function that might contribute to the observed benefits of this drug in ischemic stroke. Given that the benefits of fingolimod seem to be mediated by S1P1, whereas certain side effects are dependent on other S1P receptors, selective S1P1 agonists were studied in experimental stroke showing reduced lesion size after ischemia/reperfusion in mice.⁷⁵ In contrast to the benefits of blocking T-cell trafficking, systemic administration of regulatory T lymphocytes in rodent models of ischemic stroke reduced infarct size, ameliorated the neurological functions,⁷⁶ and reduced hemorrhagic transformation after tPA.⁷⁷
Leukocyte recruitment to inflammatory sites is attenuated by blocking α4β1 integrin (VLA-4 [very late antigen-4]) with natalizumab, an antibody in clinical use for multiple sclerosis treatment. Blockade of VLA-4 with CD49d antibody was investigated in a multicentric preclinical study in mice using 2 different models of cerebral ischemia.⁷⁸ CD49d antibody attenuated leukocyte infiltration and reduced infarct volume in small cortical lesions but not in large infarctions. Natalizumab was investigated in ischemic stroke patients in the ACTION trial (Effect of Natalizumab on Infarct Volume in Acute Ischemic Stroke).⁷⁹ Natalizumab did not meet the primary end point of the study, but secondary and exploratory end points suggested improvement of clinical outcomes,^64,79 encouraging the second ACTION2 trial. This phase-IIb trial was recently completed and the notes released by the sponsor (Biogen) state that natalizumab did not improve clinical outcomes compared with placebo.

Final Remarks

Experimental studies support detrimental effects of certain types of leukocytes in acute ischemic stroke. However, to date, this knowledge has not been translated into clinical treatments. No doubt immunomodulatory interventions in the acute phase of stroke need fine-tuning and long-term experimental studies to ensure that repair processes in subacute and chronic phases are not disturbed and the neurological deficits are attenuated. Cell therapies based on administration of autologous MNC have shown promising results in preclinical studies by promoting functional recovery, but clinical efficacy remains to be demonstrated. Results of various experimental models of brain ischemia suggest the possibility that the putative pathogenic contribution of certain leukocytes to the acute ischemic lesion might differ depending on lesion severity, regions affected, and degree of reperfusion. Importantly, most of the studies described above were obtained in young healthy male mice in spite of the fact that aging, sex, and comorbidities influence the phenotype and function of immune cells. Identification of the ischemic conditions where leukocytes might have a meaningful contribution to the brain lesion, the relevant subsets of leukocytes, and the time-window for intervention, requires more investigation. Combining immunomodulatory strategies with reperfusion therapies offer the opportunity to attenuate negative responses of the immune system that might impair reperfusion at the microvascular bed or trigger detrimental effects on the brain tissue.

Acknowledgments

I acknowledge relevant studies used to prepare this article that could not be cited because of word count restriction.

Sources of Funding

Supported by the Spanish Ministerio de Economía y Competitividad (SAF2017-87459-R).

Disclosures

None.

Footnotes

Correspondence to Anna M. Planas, PhD, Institut d’Investigacions Biomèdiques de Barcelona (IIBB), Consejo Superior de Investigaciones Científicas (CSIC), Rosselló 161, Planta 6, 08036-Barcelona, Spain. Email anna.planas@iibb.csic.es