Critical Appraisal of Review ArticleBRIAN G. HUTCHISON, MD, CCFPCanadian Family Physician VOL 39: May 1993
Frontiers in Bioscience 14, 2522-2545, January 1, 2009]Endothelial signaling in paracellular and transcellular leukocyte transmigrationErika S. WittchenDepartment of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-7295, USAhttp://www.bioscience.org/2009/v14/af/3395/fulltext.asp?bframe=figures.htm&doi=yes
How can microbial interactions with the blood–brain barrier modulate astroglial and neuronal function?Grab DJ, Chakravorty SJ, van der Heyde H, Stins MFCellular Microbiology (2011) 13(10), 1470–1478 Journal Reading dr. Ersifa Fatimah dr. Paulus Sugianto, SpS(K)
SummaryThe emerging of NVU concept• Homeostatic signalling : Neuronal – Glial – vascular Endothelial maintaining normal brain function• Observed in various neurological diseases: infection, degenerative dis., seizure, stroke, etcBBB endothelial activation: Role & impact is unclear• Beneficial vs Detrimental ?BBB as relay station in modulate astroneuronalfunctioning (scenarios & factors)• Penetrate vs Sequestered in vasculature (Cerebral Malaria) 2
The BBB endothelium & the NVUBBB: ‘just’ a lining for vessels? • Highly selective barrier between blood – CNS • To allow blood to flow & nutrients to pass into underlying tissue • Tight junction characteristic (compare: liver – fenestrae – plasma exchange)Endothelium in different tissues is very heterogeneous inexpression of surface rec. & response to stimuli • Interaction with underlying tissues: Extracellular matrix, astrocytes, pericytes • Brain-trophic BBB-derived factors can promote differentiation of neuroprecursor cells. 3
Endothelial-activating components (incl. microbialinvolvement) initiate certain brain diseases• Gut-derived LPS (HIV-1, alcohol abuse, depression) activate brain endothelium astroglial activation signals / modulate neurological functioning sickness-related behavioral changes• Modulation of microglial function by peripheral inflammation also depends on the prior history & immune statusThe role of BBB-EC activation in the development ofneurological diseases is still an understudied area ofresearch 4
In vitro BBB endothelial models to study its involvement in neurological diseasesIn vitro model• Single- / multi-cellCulture of human brain EC• Primary / ImmortalizedNon-human / Non-Brain ECElicitation BBB-like characteristic• Cultured on brain spesific extracellular matrix• In combination multi-cell co-cultures• Physical factors (vascular sheer stress, RBC interaction, TEER) complex models (available) 5
Microbes, BBB & neurological dysfunction; to cross or not to cross?High plasma concentration of neurotropic microbes BBB crossing mechanism: transcellular, paracellular, Trojan Horse Neurons, microglia, astrocytes can be infected / activated by microbial toxins Innate immunity: microbial ligands + TLR signal transduction via NFKB Release of mediators by activated astroglia alter neurological function, affect BBB function & integrity 6
Not all pathogens that causeneurological disease breach the vascular barrier and enter the CNS• In low numbers, circulating microbes & toxins might not penetrate into CNS but can still activate BBB-EC to release signals• Mild stimulation increase expression of cell adhesion molecules, release cytokine/ chemokine• Stronger stimulation opening intercellular junction & increases barrier permeability exposing brain to neurotoxic plasma substances, or antibody to ion channel affecting neurological function• How brain EC activation contributes in neuropathogenesis? 7
Plasmodium falciparum• Parasite lives in an infected RBC (PRBC) remodels the PRBC surface with parasite- encoded proteins (PfEMP-1) bind to endothelial receptor (ICAM-1) sequestration of PRBCs in vascular lumen ??? neurological symptoms 9
PRBC-mediated activation of the host BBB-EC increase expression of luminalICAM-1 & polarized release of cytokines to both luminal side & basal side.Traditionally cytokines are thought to be released in luminal side in order toalarm the immune system. However, circulating cytokines can also shuttleacross the BBB into CNS.Add the conditioned BBB medium to astrocyte & neuronal culture concentration dependent activation of astrocytes & neurons disruption ofaxonal transport & retraction of neuronal extensions. (~ axonal damage inautopsies of px with CM) 10
Toxoplasma gondii BALB/c & C57BL/6 Retinal EC & BUVEC murine models• ↑ induction of MHC I • ↑ transcripts for E-, & II Ag P-selectin, VCAM-1,• ↑ Cell adhesion ICAM-1 within 1-4 h molecules • ↑ inflammatory expression mediators: RANTES,• ↑ ALCAM expression IL-8, GM-CSF, COX2,• ↑ BBB permeability iNOS, MCP-1, Fractaline, GRO-1, T. gondii crosses BBB IP10 persist at least microglial infection & up to 72 h post astrocytes activation infection activation & loss BBB integrity 11
Trypanosoma bruceiUpregulate brain endothelial Release IL-6, CXCL-8, CCL-2,expression of ICAM-1, VCAM- TNF-α 1, E-selectin Trypanosoma transmigration requires cystein proteases Brain endothelial potentially Gαq-mediated Ca & Protease-contribute to ↑ MMP2 in CSF Activated Receptor-2 (in vitro study) signalling (in vitro & gene- profiling studies) 12
Borrelia burgdorferiB. burgdorferi (/+ co-infection Anaplasmaphygocytophilum-infected neutrophils) activationof BBB-EC↑ expression of host cell adhession molecules,plasminogen activator & its receptor↑ release of cytokines, chemokines, metalloprotease 13
HIV• Loss of junctional molecules BBB breakdown (biopsy & post mortem samples in vivo)• High viral titres + secreted viral protein activate BBB-EC expression cell adhesion molecule, release cytokines (brain side) affect neurological functioning• Indirect pathogen-induced peripheral stimulus can also alter neurological function, doesn’t readily enter the CNS, but accumulates in BBB-EC but still astroglial activation is observed. (ie: LPS) 14
Mechanism for BBB-related neurological ModulationReduced release of neuroprotective secretions vsIncreased release of cytokine/chemokine, MMPBBB-derived cytokines glial cell migration, angiogenesis,tumorigenesis, wound healing, modulate astrocyte-neuronal interaction neurological dysfunctionWhich particular cytokines are involved? How they willaffect neuronal functioning? Their targets brain cell? Unclear 15
Astrocyte secreteIn vitro BBB-CM models CXCL1,2,3• ↑ CCL20 transcript & • CXCR2 present in • Alteration of the release neuron regulate chemokine/ receptor• ↑ CCR6 in cell APMA type glutamate ratio in the brain may resembling neuroglia receptor function be a mechanism to (oligodendrocyte) ↑ receptor modulate neuronal neuronal signal cooperativety & function, but when modulation/ postsynaptic the BBB is transmission transmission overactivated, this amplitude may lead to neuronal • CXCL1 modulate dysfunction Purkinje neuron activity & ↑ ERK phosphorylation in cortical neuron 16
Reactive Nitrogen MMP & NO NO Species• Released from BBB • Alters connexin • Trigger lipid EC modulate reduction of inter- peroxidation matrix – BBB EC astrocytic gap- affect brain interaction ~ junction parenchyma & barrier integrity communications vasculature • synaptic function & could also affect neurocognitive long term astrocyte–BBB-EC sequelae (ie: potentiation ~ or astrocyte– sepsis) cleavage NR1 neurons subunit of NMDA communications receptor & ICAM-5 17
ConclusionsAn alteration in the ‘resting’ status of theBBB-Ec by neurotropic microbes can lead tomodulation of astroglial/neuronal function An activated BBB could function as a communication-relay station between peripheral signals to the brain can contribute to symptoms ranging from mild symptom to serious neurological dysfunction 18
• It is not clear at what point the altered release of these factors would tip the balance from playing a beneﬁcial role in astroneuronal homeostasis and repair to a detrimental one leading to neurological dysfunction• Many questions still remain and further clariﬁcation of interactions between the BBB-Ec and underlying CNS components are needed lead to novel therapeutic targets for treating neurological dysfunction in a wide variety of disorders & be beneﬁcial in the repair of astroneuronal function 20
Critical Appraisal1. Focus on a specific question2. Clearly defines the questions being addressed3. Methods of conducting search for relevant primary studies are described4. Primary studies are critically appraised, preferably in relation to explicit methodologic criteria5. When results of primary studies are being presented, research design and population studied are described6. Quantitative data from primary studies are summarized, preferably with CI or P values7. Author obviously biased (-)8. No references or a scanty list of references (-)9. Summary statements regarding important issues are merely followed by one or more references (or no references) without further description of the studies or their results (-)10. Magnitude of effect is discussed 21
How can microbial interactions withthe blood–brain barriermodulate astroglial and neuronal function?--Review of the Concepts Ersifa Fatimah, dr PPDS Neurologi Universitas Airlangga – RSUD dr Soetomo Surabaya, 2012
Tight Junction• Specific structures present between cells such as brain microvascular cells that ‘seal’ the monolayer and prevent passage of macromolecules between the luminal and abluminal sides of the cells.• It has a crucial role in maintaining the integrity of the BBB.• It is composed of molecules such as vascular endothelial (VE)-cadherin, zona occludens 1, junction adhesion molecules 1 to 3, or occludin.
BBB-EC• Microvascular Ecs, one cell is found per cross-section of a vessel (in capillaries).• Not fenestrated, highly functional adherens, tight junctions [can be identified via the expression of zona occludens (ZO)-1, occludin, claudin, and junctional adhesion molecules (JAM-1,2,3) high transendothelial electrical resistance that helps reduce paracellular flux of ions and small charged molecules in vivo]• Brain ECs display higher numbers of mitochondria, have a low level of pinocytosis, and present higher numbers of microvilli at their surface.• Brain ECs have a specialised basement membrane that is essential in the maintenance of the BBB.
• Astrocytes participate in the upregulation of several transport systems : GLUT-1, the L- and A-amino acid carrier systems , P-glycoprotein.• Participate in the functioning of the metabolic BBB, whereby molecules that enter brain ECs due to their lipophilic nature or because of their affinity for some transport system can be converted by metabolic processes into chemical compounds that cannot cross their abluminal membrane. [Ex: L-dopa carried into the EC cytoplasm by LAT-1 transporter is enzymatically converted into dopamine and dihydroxyphenylacetic acid that are sequestered there]
Nomenclature & Principal Functions of Glial Cells
Astrocytes in BBB• The most numerous cell types in the brain• Astrocytes are central to the health of the CNS.• Modulate synaptic transmission & ionic composition of the brain, control metabolic processes & microvascular behaviour, produce neurotrophins. – In recent years, it has become clear that their activities are drastically modified by ischaemia/ hypoxia & by cytokines, both of which are involved in several neuroinflammatory diseases.• Provide physical support for neurons, control the extracellular milieu, act as a bioenergetic regulator, and influence vascular properties, for example, BBB integrity and blood flow.
• A glial syncytium with gap junctions role in maintaining homeostasis, response amplification, and rapid cell–cell signalling via calcium waves. – Astrocytes play an essential role in CNS homeostasis via the numerous cooperative metabolic processes they establish with neurons, including neurotransmitter recycling, supply of energy metabolites, and mediation of neurovascular coupling. – Many of these astrocytic functions are mediated via gap junction communication. The combination of hexameric connexin Cx proteins determines the types of ions and small molecules that are able to pass from the cytoplasm of one cell into the next and therefore have a direct influence over astrocyte syncytium communication. – Distinct astrocyte subpopulations establish different connexin expression patterns in the CNS depending on physiological requirements of the tissue.
• Modulation of the CNS microenvironment including extracellular K+ homeostasis and pH.• Astrocytes remove excess glutamate, the major excitatory neurotransmitter in the brain. When released in excess, glutamate is neurotoxic and can trigger neuronal cell death. Astrocytes remove excess glutamate from the extracellular space.• Astrocytes supply glutamine to maintain glutamatergic neurotransmission–astroglial glutamate transport is essential for neuronal glutamatergic transmission by operating the glutamate–glutamine shuttle.• Regulation of blood flow – subserving ‘neurovascular coupling’.• Regulation of water movement via aquaporin AQP-4 and AQP-9 on vascular endfeet.
• Astrocytes both produce and respond to immunomodulators, for example, through cytokine (ciliary neurotrophic factor, CNTF; leukaemia inhibitory factor, LIF) and chemokine (chemokine receptor, CCR; C-X-C chemokine receptor, CXCR) receptors and signalling.• Formation of astrocytic scar – consisting of astrogliosis – which can have both beneficial and deleterious consequences on the brain parenchyma
Mechanisms by which pathogens cross the brain endothelial monolayer.The b2 adrenergic receptor (b2R) and the chemokine receptor, CCR5, are given as examples; several otherreceptors are known to serve as tools for central nervous system (CNS) invasion, as thoroughly reviewed in.Abbreviations: MF, monocyte–macrophage; MPs, microparticles. From Trends in Parasitology, 2012
Figure 1. Schematic diagram of the leukocyte adhesion and transmigrationcascade.Given an inflammatory stimulus, leukocytesinitially loosely adhere on the vascular ECs, rolling alongthe blood vessel wall via transient selectin-mediated interactions (1). During the activation stage, both theEC and leukocyte begin to upregulate expression and/or activity of adhesion receptors on the cellsurfaces(2), and this is required for initiating the firm adhesion stage (3). Finally, leukocytes exit the bloodstream,crossing theendothelium by the process known as transmigration or diapedesis (4). The mechanisms andpathways by which transmigration occurs are poorly understood ….
Modes of leukocyte TEM: paracellular versus transcellular.• Leukocytes can transmigrate across the endothelium via two independent routes. Use of the "paracellular" route requires transient junctional disruption as leukocytes migrate between adjacentECs. Paracellular TEM may involve a series of PECAM- enriched EC surface-connected membrane compartments that are located adjacent to the junctional region. Conversely, "transcellular" TEM involves leukocyte passage directly through an individual EC, bypassing the need to disassemble EC junctions. Local fusion of caveolae or vesiculo-vacuolar organelles may be a potential mechanism of transcellular pore formation. Recent data suggest that transcellular TEM is strongly dependent on the formation of "invasive podosomes" that are extended by the leukocyte, while EC apical cup structures may aid in both types of TEM.
Figure 2. Molecular components of EC and EC-leukocyte interactions.ECs contain both adherens junctions and tight junctions. Transmembrane proteins located along theparacellular cleft of two adjoining ECs interact, and thus provide the physical barrier to thetransmigrating leukocyte. Cytoplasmic junctional proteins provide a link between the transmembraneproteins and the cellcytoskeleton. Leukocytes interact via integrins to EC adhesion molecules present on theapical surface. In endothelial cells, adherens junctions and tight junctions can be interspersed along theentire length of the lateral membrane.
Figure 3. Low molecular weight GTPases, via downstream effectors, are key participantsof EC signaling pathways involved in leukocyte TEM. (A) Signals transduced by EC adhesion molecules downstream of leukocyte engagement regulate activity of lowmolecular weight GTPases. GEFs activate the GTPase, enabling it to interact with downstream effectors. In turn, GAPs aid thehydrolysis of GTP to GDP, inactivating the GTPase and inhibiting downstream signaling events. (B) Effector signaling downstream from GTPase activation can control cell adhesion, cytoskeleton remodeling, and membrane dynamics. In turn, this influences barrier function, membrane fusion events, and the formation of cytoskeleton-enriched structures such as apical cups that are involved inleukocyte TEM.
Figure 4. Schematic diagram of signaling events initiated downstream of ICAM- 1 engagement. Leukocyte binding to ICAM-1triggers diverse signaling pathways within the EC (highlighted inred). Phosphorylation of target proteins (Section 4.1.3.), particularly the VE-cadherin complex (Section 4.1.4.),production of ROS (Section 4.1.5.), activation of Rho family GTPases (Section 4.1.1.), and calciumsignaling (Section 4.1.2.) are centrally involved. These pathways all contribute to the junctional disruptionand/or actin remodeling that is permissive for leukocyte TEM to occur.
Figure 5. Schematic diagram of signaling pathways initiated downstream of VCAM-1engagement Leukocyte adhesion to VCAM-1 mainly signals via Rac1-mediated ROS generation. ROS inhibition ofphosphatases and activation of redox-sensitive kinases serve to increase phosphorylation of junctional proteins,and together with production of MMPs, leads to junctional disruption. The Rac effector PAK has also beenimplicated in actin remodeling via MLC-generated tension and contractility.