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Original Article

Central nervous system inflammation

Soriano, S. G.*; Piva, S.

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European Journal of Anaesthesiology: February 2008 - Volume 25 - Issue - p 154-159
doi: 10.1017/S0265021507003390
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Abstract

Introduction

The central nervous system (CNS) has long been regarded as an immune-privileged organ with minimal interaction with the immune system during physiologic states [1]. However, insults to the brain and spinal cord, in the form of trauma, hypoxia, ischaemia or toxicity, activate a multifaceted cascade of inflammatory mediators. The primary injury to the CNS results in cellular dysfunction, which in turn triggers other pathologic processes that initiate delayed secondary injury. Accumulating evidence suggests that CNS injury elicits an inflammatory response that is augmented by reperfusion. The aim of this article is to review the role of neuroinflammation mediated by the activation of adhesion molecules in the acute setting of neuroanaesthesia and critical care. We will use data obtained from our own work on experimental models of stroke in rodents as a paradigm for examining the inflammatory process that evolves after acute CNS injury. Chronic neurological disorders have also been linked to neuroinflammatory processes and have been extensively reviewed elsewhere [2].

Components of the adhesion cascade

Early leucocyte influx follows an ischaemic insult and may contribute to ischaemia-related neuronal damage [3]. Leucocytes are recruited to tissues by chemokines and are critically involved in mediating inflammatory injury to the brain parenchyma by liberating reactive oxygen species (ROS), proteases, eicosanoids and cytokine [4]. Leucocyte adhesion and extravasation are controlled by adhesion molecules present on leucocytes and the endothelial cells. Transmigation of circulating leucocytes evolves by multiple process: (1) rolling on the endothelial surface; (2) slowing; (3) activation; (4) flattening; (5) adherence to the endothelium; and (6) intercellular and transcellular migration into the perivascular tissue (Fig. 1) There are three major families of adhesion molecules: integrins, immunoglobulins and selectins (Fig. 2).

Figure 1.
Figure 1.:
Adhesion cascade: leucocytes marginate on to the endothelial surface and make initial slowing by selectin interactions. Firm binding is mediated by ICAM-1-Mac-1/LFA-1 complexes. Leucocytes then migrate via trans-cellular and trans-junctional routes following gradient created by chemokines.
Figure 2.
Figure 2.:
The three superfamilies of the adhesion cascade and their primary location on the endothelium and leucocyte.

Adhesion molecules

Integrins.

Members of the integrin family of adhesion molecules are heterodimers that mediate cell-cell, cell-extracellular matrix and cell-pathogen interactions by binding to distinct, but often overlapping, combinations of ligands. Structurally, integrins are transmembrane proteins composed of heterodimers of alpha and beta subunits. The β2 integrins (αLβ2 or lymphocyte function-associated protein-1 (LFA-1) and αMβ2 or Mac-1 are expressed exclusively on leucocytes. LFA-1 and Mac-1 are crucial in leucocyte migration into sites of inflammation, and, in the case of αLβ2, into lymphoid tissues αLβ2 is required for a wide variety of cell-cell interactions

The leucocyte adhesion receptor, Mac-1 (CD11b/CD18), is a β2 integrin, which is constitutively expressed on the surface of leucocytes but is transformed to an active conformation, as well as quantitatively upregulated on the cell surface, by inflammatory mediators [5-7]. Mac-1 mediates firm adhesion of leucocytes to the blood vessel by binding to its endothelial ligand, intercellular adhesion molecule-1 (ICAM-1) [8]. It has several other ligands including complement and plays a pivotal role in leucocyte chemotaxis, aggregation, phagocytosis and respiratory burst.

Immunoglobulins.

The vascular endothelium is lined with membrane-bound immunoglobulin structures that serve as counter-ligands for integrins. Immunoglobulin gene superfamily (IgSF) mediates strong attachment and transendothelial migration of leucocytes, and include ICAM-1, vascular cell adhesion molecule-1 (VCAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1). ICAM-1 is constitutively expressed in low levels but are rapidly upregulated by cytokines. VCAM-1 binds primarily to the β1 integrin, VLA-4, which is expressed on monocytes and lymphocytes. Soluble fractions of these ligands detach from the cellular membrane and can be detected both in plasma and in cerebrospinal fluid (CSF). Concentrations of sICAM-1 and sVCAM-1 serum and CSF levels increase after stroke and traumatic brain injury (TBI), indirectly suggesting the involvement of these molecules.

Selectins.

Selectins are a family of transmembrane molecules, expressed on the surface of leucocytes and activated endothelial cells. The initial attachment of leucocytes during inflammation, by rolling of leucocytes along the endothelium via transient, reversible, adhesive interactions, is called leucocyte rolling.

L-selectin, the smallest of the vascular selectins, is constitutively expressed on granulocytes, monocytes and a vast array of circulating lymphocytes. It captures leucocytes during the early phases of the adhesion cascade. Following capture, L-selectin is shed from the leucocyte surface after chemoattractant stimulation.

P-selectin is expressed in a-granules of activated platelets and granules of endothelial cells. Within minutes of stimulation of the endothelial cells by inflammatory mediators such as histamine, thrombin or phorbol esters, P-selectin is expressed. The primary ligand for P-selectin is PSGL-1 (P-selectin glycoprotein ligand-1), which is constitutively found on all leucocytes. The transient interactions between P-selectin and PSGL-1 allow leucocytes to roll along the venular endothelium. Accordingly, P-selectin is largely responsible for the rolling phase of the leucocyte adhesion cascade.

Cytokines and chemokines.

Cytokines are low-molecular-weight proteins that serve as activators and attractants, respectively, of the inflammatory cascade. Interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α) are secreted by a variety of cell types, have both pro- and anti-inflammatory effects on leucocytes and endothelial cells, and trigger ROS and arachidonic acids, which leads to tissue destruction. Chemokines are a subset of cytokines that mediate the migration and activation of cells, especially phagocytic cells and lymphocytes.

Inflammatory processes during CNS injury

Inflammation plays a major role in the evolution of cerebral injury after cerebral ischaemia and TBI. Within hours, transcription factors are activated locally in brain tissue, and upregulate proinflammatory genes, including the cytokines, TNF-α, IL-1β and chemokines such as IL-8, interferon inducible protein-10, monocyte chemoattractant protein-1 and fractalkine. The production of these inflammatory cytokines promotes the transmigration of the inflammatory cells in the brain parenchyma. Experimental studies in animal models of focal ischaemic stroke have suggested that leucocytes play an important role in the development of secondary injury after acute CNS injury.

Inflammatory processes in the brain have been associated with increased expression of different classes of adhesion molecules and chemokines, which mediate leucocyte trafficking and microglial activation. The trafficking of cells into the brain depends on the expression of adhesion molecules and their counter-receptors on migrating cells and on the cerebrovascular endothelium. Animal studies using antibodies to adhesion molecules and knockout mice demonstrated the importance of these molecules in the pathophysiology of stroke. Microglia are the resident ‘macrophage' of the CNS, and distinct microglia activation occurs immediately after transient focal ischaemia and peaks between 16 and 24 h after middle cerebral artery occlusion (MCAO) [9]. The pivotal role of these inflammatory cells on the expansion of the injury pattern after insults to the CNS makes them an ideal therapeutic target.

Inactivation of adhesion molecules ICAM-1 and Mac-1 by monoclonal antibodies in an experimental rodent stroke model resulted in a significant reduction of ischaemic damage [10,11]. Furthermore, neutralizing antibody to CD11a, CD18 and ICAM-1 results in a reduction in brain oedema, infarct size and leucocyte accumulation after transient MCAO in rats. However, administration of this antibody also led to partial peripheral white blood cell depletion. Antibody-antigen interactions can lead to complex responses, including triggering of signal transduction events and incomplete inactivation of functional binding sites on the target molecule.

An alternative in vivo approach to understand the role of leucocyte adhesion receptors in the pathogenesis of stroke is to use genetically altered (knockout) mice with specific deficiencies in the ligand in question. We exploited this approach by subjecting a variety of knockout mice deficient in Mac-1, ICAM-1, endothelial selectins and fractalkine.

A deficiency in the leucocyte adhesion receptor ICAM-1 led to a >75% reduction in infarct volume in the ischaemia-reperfusion model [10,12] and was associated with a 40-80% reduction in tissue leucocyte accumulation in the infarcted area. A 26% reduction in infarct volume in Mac-1-deficient mice was associated with a 50%, although not significant, reduction in the number of leucocytes present in the periphery of the infarcted area. The qualitatively similar effect of ICAM-1 and Mac-1 may arise, because Mac-1 on leucocytes binds to ICAM-1 on the endothelial surface, thereby enhancing the migration of leucocytes into the brain during stroke. Alternatively, it is possible that the interaction of Mac-1 with additional ligands, such as fibrinogen or heparin, is important in Mac-1-mediated leucocyte recruitment [13,14].

Despite the similar decrease in leucocyte accumulation in infarcted tissue, ICAM-1-deficient mice have considerably smaller infarct volumes than the Mac-1-deficient mice. Thus, ICAM-1 deficiency may confer protection by mechanisms other than the reduction of leucocyte accumulation in tissues. It is known that infarcts are initiated during the ischaemic period due to deprivation of oxygen, so any mechanism that reduces the ischaemic time, such as a decrease in the no-reflow phenomenon during the reperfusion period, would be protective. In fact, ICAM-1-deficient mice and leucocyte-depleted animals were shown to have an increase in rCBF after transient cerebral ischaemia [12]. The decrease in the no-reflow phenomenon in these animals may be the result of a decrease in leucocyte-leucocyte or leucocyte-platelet interactions and subsequent vessel occlusion. The relevant receptors in this phenomenon may be ICAM-1 and LFA-1, a sister β2 integrin of Mac-1, which is present on leucocytes and platelets and is an important ligand for ICAM-1. LFA-1-mediated leucocyte adherence and transendothelial migration can partially compensate for the impairment of the Mac-1-mediated adhesion. Certainly, the redundancy of Mac-1 and LFA-1 function as well as other integrin subtypes may all contribute to the ischaemic injury detected in this study [15]. The selectins are overexpressed during rat cerebral ischaemia [16,17]. However, mice deficient in P- and E-selectin are not less susceptible to cerebral ischaemia than the wild-type mice [18]. This is another important demonstration of the discrepancy between the expression of the molecules and their functions.

In both the ICAM-1- and Mac-1-deficient mice, the effect of reperfusion-induced injury at time points >24 h after the onset of ischaemia was not assessed. It is possible that the lack of Mac-1 or ICAM-1 may only delay the conversion of the ischaemic lesion to infarction. On the other hand, leucocyte subtypes differ at 1 and 7 days after the onset of MCAO, with leucocyte predominance in the former and monocytes and macrophages in the latter [19]. Both monocytes and activated microglia resident in the brain may be critical in further worsening cerebral ischaemic injury [20]. Notably, Mac-1 and ICAM-1 are expressed and upregulated on activated microglia [21,22] and may play a role in microglia- and monocyte-mediated maturation of ischaemic lesions. Therefore, the role of the microglia in the development of stroke injury is a fertile area for further investigation.

Both monocytes and activated microglia residing in the brain may play a role in the progression of cerebral ischaemic injury. In the setting of cerebral ischaemia and reperfusion, fractalkine mediates inflammation through its secreted and membrane-bound form. Secreted fractalkine facilitates leucocyte migration along directional concentration gradients [16]. Fractalkine-mediated chemotaxis of microglia and monocytes was inhibited by neutralization of the CX3CR1 receptor [17]. The role of the membrane-bound form of fractalkine in cerebral injury is more intriguing. The membrane-bound form of fractalkine serves as an adhesion molecule and may play a role in direct neuron to microglia signalling [18,23]. CX3CR1 is the receptor for fractalkine and is predominantly expressed on activated microglia. Fractalkine expressed on neurons bind directly to its receptor CX3CR1 on microglia. This interaction has been demonstrated after facial motor nerve axotomy models and in neuronal and glial cell cultures [18]. However, targeted deletion of the fractalkine receptor CX3CR1 did not interfere with microglial activation, monocyte extravasation or dendritic cell activation [24]. Furthermore, microglial activation in models of inflammatory peripheral nerve injury was not suppressed. This finding may be explained by physiological compensation by other cell types that are activated and result in the maintenance of the inflammatory response.

Elimination of this fractalkine-mediated inflammation may minimize the role of leucocyte recruitment and microglial activation; therefore, we generated fractalkine-deficient mice to study its role in postischaemic brain injury. After transient focal cerebral ischaemia, fractalkine-deficient mice had a 28% reduction in infarction size and lower mortality rate, when compared with wild-type littermates. The findings of this study indicate a possible role for fractalkine in augmenting postischaemic injury and mortality after transient focal cerebral ischaemia. Fractalkine can mediate both chemotaxis and increased levels of intracellular calcium in microglia and leucocytes [16,24]. Neuronal release of soluble fractalkine after an excitotoxic insult facilitates chemotaxis of microglial and monocytic cells [17]. Therefore, it is logical that we see a protective effect of fractalkine deficiency in an inflammatory situation, such as stroke. Fractalkine may also signal through other yet unidentified inflammatory pathways. Notably, Mac-1 and ICAM-1 are also expressed and upregulated on activated microglia and may play a role in microglia and monocyte-mediated maturation of ischaemic lesions. Inactivation of these adhesion molecules attenuates tissue injury in experimental stroke models.

In summary, our cumulative data confirm that inactivation of ICAM-1, Mac-1 and fractalkine in an experimental rodent model of stroke reduces infarct volume after temporary focal cerebral ischaemia. These findings demonstrate the utility of inactivating specific adhesion molecules and cytokines for cerebral protection during ischaemia-reperfusion injury and pose as another therapeutic target for the management of stroke.

Therapeutic avenues

Attenuation of the inflammatory response during CNS injury has the potential to reduce the progression of adverse secondary processes that follows acute brain insults. Application of this concept to the bedside has been the focus of several clinical trials.

Pharmacological blockade

Laboratory reports on non-human subjects have convincingly demonstrated the efficacy of the functional blockade of adhesion molecules in reducing secondary injury after acute CNS injury. Emlimomab is a commercially produced murine immunoglobulin G monoclonal antibody against ICAM-1. The US Food and Drug Administration (FDA) approved a clinical trial of Enlimomab administered to 625 stroke patients. Surprisingly, significantly more adverse events, primarily infections and fever, poor neurological scores and death occurred in the enlimomab-treated patients [25]. These findings halted the trial and subsequent monoclonal antibody blockade studies in the treatment of stroke. Ensuing laboratory investigations in rodents demonstrated that anti-ICAM-1 rat monoclonal antibody (1A29) produced host antibodies against 1A29, activation of neutrophils and complement [26].

Hypothermia

Mild hypothermia provides significant protection in laboratory models of acute CNS injury. The reasons for this protective effect are likely to be multifactorial. Decreases in brain temperature to 30-34°C result in reduced inflammatory cell infiltrate, less microglial activation and reduction of a variety of inflammatory mediators such as nitric oxide, inflammatory cytokines and superoxide. Hypothermia under thiopental anaesthesia suppresses natural killer cell activity, mitogen-induced activation of lymphocytes and reduces the production of certain cytokines, IL-1β and IL-2. In this way, it may contribute to the immune alterations observed in the perioperative period [27].

Anaesthetic drugs

Previous investigators have demonstrated that volatile anaesthetic drugs (isoflurane and sevoflurane) diminish this inflammatory response [28]. Subsequent reports show that this blunted response is partially attributable to the reduced expression of CD11a and CD11b, both components of the β2 integrins [29,30]. These studies measured the degree of activation by fluorescence-activated cell sorter (FACS) analysis of monoclonal antibody binding to known epitopes on the β2 integrin protein and adhesion assays. Isoflurane have been shown to improve long-term neurologic and histologic outcome from temporary focal ischaemia independent of ischaemic duration, temperature and blood pressure [31]. Perhaps the neuroprotective effect of volatile anaesthetics may also be due to attenuation of the inflammatory process.

Future directions

There is unequivocal evidence that neutralizing the adhesion cascade minimizes the resultant injury after cerebral ischaemia-reperfusion injury. Each individual component of adhesion molecule-induced injury provides a potential pharmacological target and can propose a specific therapy. Once the molecular targets are identified, the logical direction to take is to investigate the effect of anaesthetic drugs and techniques on the adhesion cascade.

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Keywords:

SEPSIS; INFLAMMATION; CYTOKINES; CENTRAL NERVOUS SYSTEM; CELL ADHESION MOLECULES

© 2008 European Society of Anaesthesiology