Breaching the Great Wall: Attack of the Immune Cells on the Central Nervous System

The brain is considered the most complex living structure known in the universe. This organ is responsible for controlling all bodily functions ranging from heart rate to motor functions. Given the complexity and importance of the brain, it is vital that proper defenses are maintained, one of which is the blood brain barrier. The blood brain barrier (BBB), as the name implies, serves as an anatomical barrier against invading pathogens by blocking their entry into the brain. Think of it as the Great Wall of China that was built in order to protect the Chinese empire against invasion by nomadic tribes. In this analogy, the brain would be considered the Chinese empire. Similar to the bricks and stones making up the Great Wall, tight junctions present between the endothelial cells of the blood vessels make up the BBB and they function to allow passage of only certain molecules such as oxygen. Even molecules such as glucose must undergo an active transport mechanism in order to pass through the BBB. The central nervous system (CNS) is considered an immunoprivileged organ in that CNS antigens are not accessible to immune cells in the periphery. Likewise, peripheral immune cells and pathogens cannot easily penetrate the BBB.

During the course of various neurological diseases such as multiple sclerosis (MS), vascular dementia, and stroke, this great wall becomes compromised thereby triggering the infiltration of immune cells into the CNS. This process in turn causes inflammation. Studies on animal models of these diseases aim to unravel the mechanisms by which certain immune cells breach the BBB (Simka, 2009). Unraveling such mechanisms provides therapeutic targets for future investigations in the hope of finding an effective treatment.

Leukocytes or white blood cells directly engage with antigens in order to exert their immunologic effects. This process requires the navigation of leukocytes through the bloodstream and their recruitment into the target organ. The process by which leukocytes exit the bloodstream and enter the target tissue is known as leukocyte extravasation. When tissues become infected with a pathogen, infected cells secrete cytokines, which act as signals to activate the endothelial cells of the surrounding blood vessels. This endothelial activation leads to the upregulation of particular proteins on endothelial cell surfaces, which in turn act as receptors for circulating leukocytes. Examples of these proteins include intercellular adhesion molecule (ICAM) and E-selectins. The leukocytes then start to accumulate by the blood vessel linings and a lose interaction between the leukocyte and the blood vessel endothelium begins to take place. This lose interaction causes the leukocytes to express adhesion molecules on their cell surfaces thereby providing them a way to create stronger interactions with ICAMs on endothelial cells. Once direct contact is initiated between the adhesion molecules on leukocytes and the cell surface proteins on endothelial cells, the leukocytes arrest and they become stuck to the lining of the blood vessels. At this stage, diapedesis, occurs in which leukocytes flatten themselves as they pass between endothelial cells into the tissues. Simply stated, cytokines released by infected cells inform the lining of the blood vessels that a pathogen is present. This signal alerts circulating leukocytes to pass through the blood vessels and exert their effects onto infected tissues.

The mechanism by which immune cells enter the CNS closely resembles the way immune cells enter peripheral tissues. When CNS tissue becomes infected, activated leukocytes circulating through blood vessels infiltrate the brain and spinal cord by adhering to adhesion molecules present on the endothelial cells of the BBB. Past studies reveal the role of ICAM-1 in the disruption of the BBB (Dietrich et al. 2001). Furthermore, leukocyte trafficking across the BBB depends on the interaction between ICAM-1 expressed by BBB endothelial cells and integrin α_Lβ₂ found on leukocytes. The resulting interaction in turn triggers the migration of leukocytes through the BBB into the brain via G-protein mediated signaling (Adamson et al. 2002).

A recent study by Reijerkerk et al. (2011) examines the role of the endothelin system in monocyte trafficking across the BBB. Monocytes are one type of leukocytes that primarily mediate the innate immune system. The purpose of the study was to investigate the role of different components of the endothelin system, such as endothelin 1 (ET-1), its type B receptor (ET_B), and endothelin-converting enzyme-1 (ECE-1) in monocyte diapedesis. ET-1 is a pro-inflammatory molecule produced by endothelial cells and ECE-1 serves as the enzyme responsible for the production of ET-1 from its precursor, preproendothelin. Past studies have implicated the role of ET-1 in upregulating the expression of ICAMs in brain endothelial cells, which in turn promoted the recruitment of leukocytes into the CNS tissue (McCarron et al. 1993). In the present study, the authors devised an in vitro model of the interaction between BBB endothelial cells and monocytes. Specifically, they generated co-cultures of human brain endothelial cell monolayers and human blood monocytes in order to assess monocyte migration through the endothelial cells. The authors used the human brain endothelial cell line hCMEC/D3, which has been widely implicated in studying the properties of the BBB. When used in culture, the hCMEC/D3 cell line closely resembles the BBB and expresses chemokine receptors as well as tight cell junctions. Monocyte migration through the endothelial cell monolayer was analyzed by counting the number of monocytes that have adhered or pass through the cell monolayer. The authors used synthetic inhibitors and gene-specific knockdown of ET-1, ET_B, and ECE-1 in order to assess the role of the different components of the endothelin system in monocyte migration. Specifically, the expression of ET-1, its receptor, as well as its synthesis in hCMEC/D3 cells were silenced by using shRNA. The method of using shRNA is a common technique in silencing the expression of target genes. The rationale behind inhibiting all 3 components of the endothelin system was to suppress the effects of ET-1 in every way possible. Knocking down the ET_B receptor prevents interaction with its ligand, ET-1. Likewise, silencing ECE-1 prevents the synthesis of ET-1 from its precursor. Results show that monocyte migration through the endothelial cell layer was reduced both in the presence of synthetic inhibitors as well as in gene-specific knockdown of the different components of the endothelin system. Furthermore, a staining experiment, which showed the location of the ET_B receptor and ECE-1 enzyme in these cells, showed that both the receptor and the enzyme were present in nuclear and cytosolic forms. Most of the fragments were localized to the nucleus, however, the authors did not specify which fragment was responsible for the suppression of monocyte trafficking.

These results implicate the role of the endothelin system in immune cell breaching of the BBB. Having studied an animal model of MS for 3 years, this study reveals exciting results regarding possible treatments for MS, in which one of the pathological hallmarks is a compromised BBB. This study shows that endothelin receptor antagonists and suppression of ET-1 synthesis may provide therapeutic means to reduce inflammation in neurological diseases such as MS. The next step for this study would be to create an in vivo model to determine whether silencing the different components of the endothelin system would indeed reduce inflammation and clinical severity of the disease in animals. Perhaps endothelin-1 transgenic mice could be utilized to study the course of MS and determine whether these mice have decreased inflammation compared to normal mice. An in vivo study to supplement these results would further strengthen the role of the endothelin system in monocyte diapedesis across the BBB. In addition, although the authors did not show which fragment was responsible for suppressing monocyte migration, the nuclear and cytoplasmic localization of the endothelin system components provide novel aspects for therapeutic targets in the future. The study indeed unraveled key components responsible for immune cell trafficking across the BBB.

Reference:Reijerkerk, A., Lakeman, K. A., Drexhage, J. A., van Het Hof, B., van Wijck, Y., van der Pol, S. M., et al. (2011). Brain endothelial barrier passage by monocytes is controlled by the endothelin system. Journal of Neurochemistry,

Other Citations:
Adamson, P., Wilbourn, B., Etienne-Manneville, S., Calder, V., Beraud, E., Milligan, G., et al. (2002). Lymphocyte trafficking through the blood-brain barrier is dependent on endothelial cell heterotrimeric G-protein signaling. The FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 16(10), 1185-1194.
Dietrich, J. B. (2002). The adhesion molecule ICAM-1 and its regulation in relation with the blood-brain barrier. Journal of Neuroimmunology, 128(1-2), 58-68.
McCarron, R. M., Wang, L., Stanimirovic, D. B., & Spatz, M. (1993). Endothelin induction of adhesion molecule expression on human brain microvascular endothelial cells. Neuroscience Letters, 156(1-2), 31-34.
Simka, M. (2009). Blood brain barrier compromise with endothelial inflammation may lead to autoimmune loss of myelin during multiple sclerosis. Current Neurovascular Research, 6(2), 132-139.