COVID-19 vaccine-readiness for anti-CD20-depleting therapy in autoimmune diseases.

Although most autoimmune diseases are considered to be CD4 T cell- or antibody-mediated, many respond to CD20-depleting antibodies that have limited influence on CD4 and plasma cells. This includes rituximab, oblinutuzumab and ofatumumab that are used in cancer, rheumatoid arthritis and off-label in a large number of other autoimmunities and ocrelizumab in multiple sclerosis. Recently, the COVID-19 pandemic created concerns about immunosuppression in autoimmunity, leading to cessation or a delay in immunotherapy treatments. However, based on the known and emerging biology of autoimmunity and COVID-19, it was hypothesised that while B cell depletion should not necessarily expose people to severe SARS-CoV-2-related issues, it may inhibit protective immunity following infection and vaccination. As such, drug-induced B cell subset inhibition, that controls at least some autoimmunities, would not influence innate and CD8 T cell responses, which are central to SARS-CoV-2 elimination, nor the hypercoagulation and innate inflammation causing severe morbidity. This is supported clinically, as the majority of SARS-CoV-2-infected, CD20-depleted people with autoimmunity have recovered. However, protective neutralizing antibody and vaccination responses are predicted to be blunted until naive B cells repopulate, based on B cell repopulation kinetics and vaccination responses, from published rituximab and unpublished ocrelizumab (NCT00676715, NCT02545868) trial data, shown here. This suggests that it may be possible to undertake dose interruption to maintain inflammatory disease control, while allowing effective vaccination against SARS-CoV-29, if and when an effective vaccine is available.

Failed B cell survival factor trials support the importance of memory B cells in multiple sclerosis.

Clinical trials are probably the most informative experiments to help an understanding of multiple sclerosis (MS) biology. Recent successes with CD20-depleting antibodies have focused attention towards B cell subsets as important mediators in MS. The trial of tabalumab (NTC00882999), which inhibits B cell activation factor (BAFF), is reported and reviewed and this trial is contrasted with the trial on the inhibition of a proliferation-inducing ligand (APRIL) and BAFF using atacicept (NCT00642902). Both tabalumab and atacicept induce depletion of mature B cells and inhibit antibody formation, but they fail to deplete memory B cells and do not inhibit relapsing MS. Atacicept is reported to augment memory B cell responses and may precipitate relapse, suggesting the importance of APRIL. However, BAFF inhibition can enhance peripheral blood memory B cell responses, which was not associated with augmented relapse. Although other interpretations are possible, these data further support the hypothesis that memory B cells may be of central importance in relapsing MS, as they are the major CD20+ B cell subset expressing APRIL receptors. They also suggest that quantitative and/or qualitative differences in B cell responses or other factors, such as an immune-regulatory effect associated with APRIL, may be important in determining whether MS reactivates following neutralization of peripheral B cell maturation and survival factors.

Control of experimental spasticity by targeting the degradation of endocannabinoids using selective fatty acid amide hydrolase inhibitors.

BACKGROUND: It has been previously shown that CB1 cannabinoid receptor agonism using cannabis extracts alleviates spasticity in both a mouse experimental autoimmune encephalomyelitis (EAE) model and multiple sclerosis (MS) in humans. However, this action can be associated with dose-limiting side effects. OBJECTIVE: We hypothesised that blockade of anandamide (endocannabinoid) degradation would inhibit spasticity, whilst avoiding overt cannabimimetic effects. METHODS: Spasticity eventually developed following the induction of EAE in either wild-type or congenic fatty acid amide hydrolase (FAAH)-deficient Biozzi ABH mice. These animals were treated with a variety of different FAAH inhibitors and the effect on the degree of limb stiffness was assessed using a strain gauge. RESULTS: Control of spasticity was achieved using FAAH inhibitors CAY100400, CAY100402 and URB597, which was sustained following repeated administrations. Therapeutic activity occurred in the absence of overt cannabimimetic effects. Importantly, the therapeutic value of the target could be definitively validated as the treatment activity was lost in FAAH-deficient mice. Spasticity was also controlled by a selective monoacyl glycerol lipase inhibitor, JZL184. CONCLUSIONS: This study demonstrates definitively that FAAH inhibitors provide a new class of anti-spastic agents that may have utility in treating spasticity in MS and avoid the dose-limiting side effects associated with cannabis use.

Evaluation of the Effects of Sativex (THC BDS: CBD BDS) on Inhibition of Spasticity in a Chronic Relapsing Experimental Allergic Autoimmune Encephalomyelitis: A Model of Multiple Sclerosis.

This study investigated the antispasticity potential of Sativex in mice. Chronic relapsing experimental allergic encephalomyelitis was induced in adult ABH mice resulting in hind limb spasticity development. Vehicle, Sativex, and baclofen (as a positive control) were injected intravenously and the "stiffness" of limbs assessed by the resistance force against hind limb flexion. Vehicle alone caused no significant change in spasticity. Baclofen (5 mg/kg) induced approximately a 40% peak reduction in spasticity. Sativex dose dependently reduced spasticity; 5 mg/kg THC + 5 mg/kg CBD induced approximately a 20% peak reduction; 10 mg/kg THC + 10 mg/kg CBD produced approximately a 40% peak reduction in spasticity. Sativex has the potential to reduce spasticity in an experimental mouse model of multiple sclerosis (MS). Baclofen reduced spasticity and served as a positive control. Sativex (10 mg/kg) was just as effective as baclofen, providing supportive evidence for Sativex use in the treatment of spasticity in MS.

Evaluating potential therapies for bladder dysfunction in a mouse model of multiple sclerosis with high-resolution ultrasonography.

BACKGROUND: In multiple sclerosis (MS), demyelinating and neurodegenerative lesions develop throughout the central nervous system, which result in a loss of neurotransmission. As a result, people with MS exhibit a wide range of symptoms including dysfunction of the bladder, which can lead to urinary incontinence or retention. Such signs can develop in animal models of MS. Current assessments of bladder properties in animal models are generally invasive, electrophysiological techniques. OBJECTIVE: The use of a non-invasive, ultrasound system for measuring bladder volume in a mouse model of MS. METHODS: Chronic relapsing experimental autoimmune encephalomyelitis was induced in mice. The bladder volume was assessed using ultrasonography, during the disease course and following therapy with bethanechol chloride. RESULTS: It was demonstrated that volumes obtained ultrasonically positively-correlated (r = 0.960) with the urine volumes obtained by manual expression. It was also shown for the first time that bladder size increased significantly in mice with residual neurological deficit. Indeed, this increase in bladder size showed a strong, positive-correlation (r = 0.951) with the hind limb spasticity. Following treatment with bethanechol chloride, bladder volume significantly decreased in mice with chronic experimental autoimmune encephalomyelitis. CONCLUSION: This study demonstrates a novel outcome measure in experimental MS that allows; repeated, non-invasive, high resolution ultrasonic monitoring of bladder function.

Abnormally phosphorylated tau is associated with neuronal and axonal loss in experimental autoimmune encephalomyelitis and multiple sclerosis.

The pathological correlate of clinical disability and progression in multiple sclerosis is neuronal and axonal loss; however, the underlying mechanisms are unknown. Abnormal phosphorylation of tau is a common feature of some neurodegenerative disorders, such as Alzheimer's disease. We investigated the presence of tau hyperphosphorylation and its relationship with neuronal and axonal loss in chronic experimental autoimmune encephalomyelitis (CEAE) and in brain samples from patients with secondary progressive multiple sclerosis. We report the novel finding of abnormal tau phosphorylation in CEAE. We further show that accumulation of insoluble tau is associated with both neuronal and axonal loss that correlates with progression from relapsing-remitting to chronic stages of EAE. Significantly, analysis of secondary progressive multiple sclerosis brain tissue also revealed abnormally phosphorylated tau and the formation of insoluble tau. Together, these observations provide the first evidence implicating abnormal tau in the neurodegenerative phase of tissue injury in experimental and human demyelinating disease.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) in plasminogen activator inhibitor-1 knockout mice: the effect of fibrinolysis during neuroinflammation.

UNLABELLED: During neuroinflammation in multiple sclerosis (MS) fibrinogen, not normally present in the brain or spinal cord, enters the central nervous system through a compromised blood-brain barrier. Fibrin deposited on axons is ineffectively removed by tissue plasminogen activator (tPA), a key contributory factor being the upregulation of plasminogen activator inhibitor-1 (PAI-1). AIMS: This study investigated the role of PAI-1 during experimental neuroinflammatory disease. METHODS: Chronic relapsing experimental allergic encephalomyelitis (CREAE), a model of MS, was induced with spinal cord homogenate in PAI-1 knockout (PAI-1(-/-)) and wild type (WT) mice, backcrossed onto the Biozzi background. RESULTS: Disease incidence and clinical severity were reduced in PAI-1(-/-) mice, with animals developing clinical signs significantly later than WTs. Clinical relapses were absent in PAI-1(-/-) mice and the subsequent reduction in neuroinflammation was coupled with a higher capacity for fibrinolysis in spinal cord samples from PAI-1(-/-) mice, in association with increased tPA activity. Axonal damage was less apparent in PAI-1(-/-) mice than in WTs, implicating fibrin in both inflammatory and degenerative events during CREAE. CONCLUSIONS: PAI-1 is a potential target for therapy in neuroinflammatory degenerative diseases, allowing effective fibrin removal and potentially reducing relapse rate and axonal damage.

Cannabinoid control of neuroinflammation related to multiple sclerosis.

The cannabis plant (Cannabis sativa) has been known by many names but the question remains 'Can we call it medicine?' There has been renewed interest in the value of cannabis for the control of neuroinflammatory conditions such as multiple sclerosis, where it has been shown to have some effect on spasticity and pain both experimentally and in clinical trials in humans. However, in addition to symptom control potential, the question remains whether cannabinoids can modify the neuroinflammatory element which drives relapsing neurological attacks and the accumulation of progressive disability. In experimental studies it has been recently shown that synthetic cannabinoids can affect the immune response both indirectly via CB1 receptor-mediated signalling nerve centres controlling the systemic release of immunosuppressive molecules and directly by CB2 receptor-mediated inhibition of lymphocyte and macrophage/microglial cell function. However, these immunosuppressive possibilities that would limit the frequency of relapsing attacks will probably not be realized clinically, following use of medical cannabis, due to dose constraints. However, cannabinoids may still affect the glial response within the damaged central nervous system, which facilitate the slow, neurodegenerative processes that account for progressive neurodegeneration, and therefore may have utility in addition to value of cannabis-related drugs for symptom control.

Control of spasticity in a multiple sclerosis model is mediated by CB1, not CB2, cannabinoid receptors.

BACKGROUND AND PURPOSE: There is increasing evidence to suggest that cannabis can ameliorate muscle-spasticity in multiple sclerosis, as was objectively shown in experimental autoimmune encephalomyelitis models. The purpose of this study was to investigate further the involvement of CB1 and CB2)cannabinoid receptors in the control of experimental spasticity. EXPERIMENTAL APPROACH: Spasticity was induced in wildtype and CB1-deficient mice following the development of relapsing, experimental autoimmune encephalomyelitis. Spastic-hindlimb stiffness was measured by the resistance to flexion against a strain gauge following the administration of CB1 and CB2 agonists. KEY RESULTS: As previously suggested, some CB2-selective agonists (RWJ400065) could inhibit spasticity. Importantly, however, the anti-spastic activity of RWJ400065 and the therapeutic effect of non-selective CB1/CB2 agonists (R(+)WIN55,212-2 and CP55, 940) was lost in spastic, CB1-deficit mice. CONCLUSIONS AND IMPLICATIONS: The CB1 receptor controls spasticity and cross-reactivity to this receptor appears to account for the therapeutic action of some CB2 agonists. As cannabinoid-induced psychoactivity is also mediated by the CB1 receptor, it will be difficult to truly dissociate the therapeutic effects from the well-known, adverse effects of cannabinoids when using cannabis as a medicine. The lack of knowledge on the true diversity of the cannabinoid system coupled with the lack of total specificity of current cannabinoid reagents makes interpretation of in vivo results difficult, if using a purely pharmacological approach. Gene knockout technology provides an important tool in target validation and indicates that the CB1 receptor is the main cannabinoid target for an anti-spastic effect.

Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain.

Cannabinoid type 1 receptors and transient receptor potential vanilloid type 1 channels have been proposed to act as metabotropic and ionotropic receptors, respectively, for two classes of endogenous polyunsaturated fatty acid amides, the acylethanolamides and the acyldopamides. Furthermore, we and others have shown that functional crosstalk occurs between these two receptors when they are expressed in the same cell. Although demonstrated in sensory neurons of the dorsal root ganglia, spinal cord and myenteric neurons, co-expression of cannabinoid type 1 and transient receptor potential vanilloid type 1 has not yet been studied in the brain. In the present study, we addressed this issue by using commercially available specific antibodies whose specificity was confirmed by data obtained with brains from cannabinoid type 1(-/-) and transient receptor potential vanilloid type 1(-/-) mice. Double cannabinoid type 1/transient receptor potential vanilloid type 1 immunofluorescence and single cannabinoid type 1 or transient receptor potential vanilloid type 1 avidin-biotin complex immunohistochemistry techniques were performed and both methods used point to the same results. Cannabinoid type 1/transient receptor potential vanilloid type 1 expression was observed in the hippocampus, basal ganglia, thalamus, hypothalamus, cerebral peduncle, pontine nuclei, periaqueductal gray matter, cerebellar cortex and dentate cerebellar nucleus. In particular, in the hippocampus, cannabinoid type 1/transient receptor potential vanilloid type 1 expression was detected on cell bodies of many pyramidal neurons throughout the CA1-CA3 subfields and in the molecular layer of dentate gyrus. In the cerebellar cortex, expression of cannabinoid type 1/transient receptor potential vanilloid type 1 receptors was found surrounding soma and axons of the vast majority of Purkinje cell bodies, whose cytoplasm was found unstained for both receptors. Cannabinoid type 1 and transient receptor potential vanilloid type 1 immunoreactivity was also detected in: a) the globus pallidus and substantia nigra, in which some intensely transient receptor potential vanilloid type 1 immunopositive cell bodies were found in dense and fine cannabinoid type 1/transient receptor potential vanilloid type 1 positive and cannabinoid type 1 positive nerve fiber meshworks, respectively; b) the cytoplasm of thalamic and hypothalamic neurons; and c) some neurons of the ventral periaqueductal gray. These data support the hypothesis of a functional relationship between the two receptor types in the CNS.

Cannabinoid-receptor 1 null mice are susceptible to neurofilament damage and caspase 3 activation.

Administered cannabinoids have been shown to ameliorate signs of CNS inflammatory disease in a number of animal models, including allergic encephalomyelitis. More recently, neuroprotective actions have been attributed to activation of the cannabinoid 1 receptor in a number of in vitro and in vivo models. One of these, chronic relapsing experimental allergic encephalomyelitis, is considered a robust analog of multiple sclerosis. In this study, spinal cord tissue from cannabinoid receptor 1 knockout mice was analyzed for neurofilament H and myelin basic protein content, as markers of neurons/axons and myelin respectively, during the course of chronic relapsing experimental allergic encephalomyelitis. Dephosphorylation of a neurofilament H epitope, immunoreactive to the SMI32 antibody, was assessed as a marker of axonal damage and levels of the endpoint cell death mediator caspase 3 were evaluated. It was found that both neurofilament and myelin basic protein levels decrease over the course of disease, indicating concomitant neuronal/axonal loss and demyelination. Loss of each marker was more severe in cannabinoid receptor 1 knockout animals. Increased SMI32 reactivity was observed as disease progressed. SMI32 reactivity was significantly increased in knockout animals over wildtype counterparts, an indication of greater axonal dephosphorylation and injury. Active caspase 3 levels were increased in all animals during disease, with knockout animals displaying highest levels, even in knockout animals prior to disease induction. These results indicate that lack of the cannabinoid receptor 1 is associated with increased caspase activation and greater loss and/or compromise of myelin and axonal/neuronal proteins. The increase of caspase 3 in knockout mice prior to disease induction indicates a latent physiological effect of the missing receptor. The data presented further strengthen the hypothesis of neuroprotection elicited via cannabinoid receptor 1 signaling.

Quantification of neurodegeneration by measurement of brain-specific proteins.

Quantification of neurodegeneration in animal models is typically assessed by time-consuming and observer-dependent immunocytochemistry. This study aimed to investigate if newly developed ELISA techniques could provide an observer-independent, cost-effective and time-saving tool for this purpose. Neurofilament heavy chain (NfH(SM135)), astrocytic glial fibrillary acidic protein (GFAP), S100B and ferritin, markers of axonal loss, gliosis, astrocyte activation and microglial activation, respectively, were quantified in the spinal cord homogenates of mice with chronic relapsing experimental allergic encephalomyelitis (CREAE, n=8) and controls (n=7). Levels of GFAP were found to be threefold elevated in CREAE (13 ng/mg protein) when compared to control animals (4.5 ng/mg protein, p<0.001). The inverse was observed for NfH(SM135) (21 ng/mg protein vs. 63 ng/mg protein, p<0.001), ferritin (542 ng/mg protein vs. 858 ng/mg protein, p<0.001) and S100B (786 ng/mg protein vs. 2080 ng/mg protein, N.S.). These findings were confirmed by immunocytochemistry, which demonstrated intense staining for GFAP and decreased staining for NfH(SM135) in CREAE compared to control animals. These findings indicate that axonal loss and gliosis can be estimated biochemically using the newly developed ELISA assays for NfH(SM135) and GFAP. These assays may facilitate the quantification of pathological features involved in neurodegeneration.

Medicinal cannabis: is delta9-tetrahydrocannabinol necessary for all its effects?

Cannabis is under clinical investigation to assess its potential for medicinal use, but the question arises as to whether there is any advantage in using cannabis extracts compared with isolated Delta9-trans-tetrahydrocannabinol (Delta9THC), the major psychoactive component. We have compared the effect of a standardized cannabis extract (SCE) with pure Delta9THC, at matched concentrations of Delta9THC, and also with a Delta9THC-free extract (Delta9THC-free SCE), using two cannabinoid-sensitive models, a mouse model of multiple sclerosis (MS), and an in-vitro rat brain slice model of epilepsy. Whilst SCE inhibited spasticity in the mouse model of MS to a comparable level, it caused a more rapid onset of muscle relaxation, and a reduction in the time to maximum effect compared with Delta9THC alone. The Delta9THC-free extract or cannabidiol (CBD) caused no inhibition of spasticity. However, in the in-vitro epilepsy model, in which sustained epileptiform seizures were induced by the muscarinic receptor agonist oxotremorine-M in immature rat piriform cortical brain slices, SCE was a more potent and again more rapidly-acting anticonvulsant than isolated Delta9THC, but in this model, the Delta9THC-free extract also exhibited anticonvulsant activity. Cannabidiol did not inhibit seizures, nor did it modulate the activity of Delta9THC in this model. Therefore, as far as some actions of cannabis were concerned (e.g. antispasticity), Delta9THC was the active constituent, which might be modified by the presence of other components. However, for other effects (e.g. anticonvulsant properties) Delta9THC, although active, might not be necessary for the observed effect. Above all, these results demonstrated that not all of the therapeutic actions of cannabis herb might be due to the Delta9THC content.

Myelin/axonal pathology in interleukin-12 induced serial relapses of experimental allergic encephalomyelitis in the Lewis rat.

Lewis rats, on recovery from monophasic clinical experimental allergic encephalomyelitis (EAE), can be induced to develop repeated paralytic relapses with a graded reduction in clinical severity following intraperitoneal administration of IL-12. By the time of the third relapse, the number and size of inflammatory cuffs in the spinal cord were reduced with the makeup of the cellular infiltrate shifting to a significantly increased number of B cells. Serum levels of myelin basic protein (MBP)-specific IgG1 and IgG2b were found to rise over time while MBP and MBP peptide-positive macrophages and microglia became evident in perivascular cuffs and in spinal cord parenchyma, indicative of myelin phagocytosis. Axonal death was observed in semithin and EM sections of spinal cord in third relapse animals in association with iNOS and tPA immunostaining throughout gray and white matter. These neurotoxic or excitotoxic agents may contribute to axonal damage directly or indirectly by activated microglia and macrophages, leading to limited damage of the axonal-myelin unit.

Endocannabinoids control spasticity in a multiple sclerosis model.

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.

Cannabinoids control spasticity and tremor in a multiple sclerosis model.

Chronic relapsing experimental allergic encephalomyelitis (CREAE) is an autoimmune model of multiple sclerosis. Although both these diseases are typified by relapsing-remitting paralytic episodes, after CREAE induction by sensitization to myelin antigens Biozzi ABH mice also develop spasticity and tremor. These symptoms also occur during multiple sclerosis and are difficult to control. This has prompted some patients to find alternative medicines, and to perceive benefit from cannabis use. Although this benefit has been backed up by small clinical studies, mainly with non-quantifiable outcomes, the value of cannabis use in multiple sclerosis remains anecdotal. Here we show that cannabinoid (CB) receptor agonism using R(+)-WIN 55,212, delta9-tetrahydrocannabinol, methanandamide and JWH-133 (ref. 8) quantitatively ameliorated both tremor and spasticity in diseased mice. The exacerbation of these signs after antagonism of the CB1 and CB2 receptors, notably the CB1 receptor, using SR141716A and SR144528 (ref. 8) indicate that the endogenous cannabinoid system may be tonically active in the control of tremor and spasticity. This provides a rationale for patients' indications of the therapeutic potential of cannabis in the control of the symptoms of multiple sclerosis, and provides a means of evaluating more selective cannabinoids in the future.

Factors controlling T-cell migration across rat cerebral endothelium in vitro.

The migration of lymphocytes through primary cultures of rat brain microvascular endothelial cell monolayers was examined in vitro by time-lapse videomicroscopy. Antigen-specific T cell line migration was dependent on the duration of culture (post-antigen stimulation) with exogenous interleukin-2 (IL-2). Peak migration (approximately 50% of T-cells during the 4 h migration assay) occurred after 4 days of culture with IL-2 but did not coincide with maximal expression of LFA-1, VLA-4 or the IL-2 receptor. On unstimulated endothelia antibody blockade of LFA-1 or ICAM-1 inhibited T-cell line migration to 8.0% and 6.8% of control values, respectively, whereas blocking VLA-4 and VCAM-1 had no effect. On IL-beta activated endothelium blocking LFA-1 and ICAM-1 was less effective (24.9% and 27.3% of control values, respectively) and blockade of VLA-4 and VCAM-1 brought about a reduction to 63.0% and 68.3% of controls respectively. Inhibition of IL-2-dependent proliferation with an IL-2 receptor blocking antibody also significantly inhibited T-cell migration to 22.2% of controls. Peripheral lymph node (PLN) lymphocytes could also be induced to migrate through untreated cerebral endothelial cell monolayers by cross-linking CD3 which was also time and IL-2-dependent with maximal migration (22.7%) occurring after three days in the presence of exogenous IL-2. Blocking LFA-or ICAM-1 resulted in a significant reduction in migration across IL-1 beta-activated endothelial cells to 17.4% and 20.9% of control values respectively although blocking the VLA-4/VCAM-1 interaction had no significant effect. Activation of PLN lymphocytes with concanavalin A for up to 5 days did not induce migration but when left in contact with the endothelial monolayer for 24 h migration reached 31.0%. These studies indicate that T-cells require a combination of signals to trigger the migratory phenotype which is necessary to enable them to penetrate the blood-brain barrier.

SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics.

In the central nervous system the blood-brain and blood-retinal barriers (BBB and BRB respectively) are instrumental in maintaining homeostasis of the neural parenchyma and controlling leucocyte traffic. These cellular barriers are formed primarily by the vascular endothelium of the brain and retina although in the latter the pigmented epithelial cells also form part of the barrier. From primary cultures of rat brain endothelium, retinal endothelium and retinal pigment epithelium (RPE) we have generated temperature sensitive SV40 large T immortalised cell lines. Clones of brain (GP8.3) and retinal (JG2.1) endothelia and RPE (LD7.4) have been derived from parent lines that express the large T antigen at the permissive temperature. The endothelial cell (EC) lines expressed P-glycoprotein, GLUT-1, the transferrin receptor, von Willebrand factor and the RECA-1 antigen and exhibited high affinity uptake of acetylated LDL and stained positive with the lectin Griffonia simplicifolia. The RPE cell line was positive for cytokeratins and for the rat RPE antigen RET-PE2. All the cell lines expressed major histocompatibility complex (MHC) class 1 and intercellular adhesion molecule (ICAM)-1 constitutively and could be induced to express MHC class II and vascular cell adhesion molecule (VCAM)-1 following cytokine activation. The EC also expressed platelet endothelial cell adhesion molecule (PECAM)-1. Monolayers of these cells could support the migration of antigen-specific T cell lines. The generation of immortalised cell lines derived from the rat BBB and BRB should prove to be useful tools for the study of these specialised cellular barriers.

Lymphocyte migration into the CNS modelled in vitro: roles of LFA-1, ICAM-1 and VLA-4.

We examined the changes in intercellular adhesion molecule-1 (ICAM-1) expression on brain endothelium in response to tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). ICAM-1 is normally present on these cells and is induced over 24 hr by both cytokines with a time-course which matches enhancement in lymphocyte adhesion. Anti-lymphocyte function-associated antigen-1 (anti-LFA-1) (CD11a), anti-very late antigen-4 (anti-VLA-4) (CD49d) and anti-CD18 block binding of mitogen-activated lymphocytes to brain endothelium and the effects of anti-LFA-1 and anti-VLA-4 are additive. Anti-ICAM-1 does not however block adhesion, nor does depletion of endothelial ICAM-1 reduce lymphocyte binding. Titration of the interacting cells indicated that the antibody blocking is due to interference in the endothelial/lymphocyte interaction. None of the antibodies affect the binding of non-activated lymphocytes, which is itself normally much lower than that of activated cells. The time at which lymphocyte adhesiveness is greatest for the endothelium corresponds with the time at which the lymphocytes express highest levels of LFA-1 and VLA-4. The data show a role for LFA-1 and VLA-4 in the early interaction of activated lymphocytes with brain endothelium. Kinetic studies indicate that the ligand for VLA-4 is VCAM-1. The ligand for LFA-1 could not be determined with certainty, but if it is ICAM-1, the levels of ICAM-1 on brain endothelium are not critical.

An assay for the analysis of lymphocyte migration across cerebral endothelium in vitro.

We describe a recently developed assay for the analysis of leukocyte migration across cerebral endothelium in vitro. The endothelium is grown as monolayers on Goretex or Cyclopore membranes coated with extracellular matrix proteins and supported on inserts. This system permits the recovery and phenotyping of cells which migrate down through the endothelium. Using labelled lymphocytes we were able to differentiate four populations of cells, with differing degrees of mobility in the migration assay. We have compared the results from this system with those from conventional adhesion assays. Binding of cells to the endothelium is rapid, but is confined to a particular subpopulation of the applied lymphocytes. We have followed cell migration over 24 h in the system using normal and cytokine-activated endothelium and have found that whereas adhesion depends both on the state of lymphocyte activation and on the condition of the endothelium, the level of migration of stimulated lymphocytes is largely independent of endothelial activation. Moreover, whereas CD8+ cells bind well to the endothelium, it is the CD4+ cells which migrate most effectively. Comparison of brain and epididymal fat endothelium showed similar migration levels over 2 h, but migration was greater across epididymal fat endothelium at 24 h.