Yonatan Ganor

Human T cells express a functional ionotropic glutamate receptor GluR3, and glutamate by itself triggers integrin-mediated adhesion to laminin and fibronectin and chemotactic migration.

T cells may encounter glutamate, the major excitatory neurotransmitter in the nervous system, when patrolling the brain and in glutamate-rich peripheral organs. Moreover, glutamate levels increase in the CNS in many pathological conditions in which T cells exert either beneficial or detrimental effects. We discovered that normal human T cells, human T leukemia cells, and mouse anti-myelin basic protein T cells express high levels of glutamate ion channel receptor (ionotropic) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3). The evidence for GluR3 on T cells includes GluR3-specific RT-PCR, Western blot, immunocytochemical staining and flow cytometry. Sequencing showed that the T cell-expressed GluR3 is identical with the brain GluR3. Glutamate (10 nM), in the absence of any additional molecule, triggered T cell function: integrin-mediated T cell adhesion to laminin and fibronectin, a function normally performed by activated T cells only. The effect of glutamate was mimicked by AMPA receptor-agonists and blocked specifically by the selective receptor-antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulfamoylbenzo[f]quinoxalin-2,3-dione (NBQX), and by relevant anti-integrin mAbs. Glutamate also increased the CXCR4-mediated T cell chemotactic migration toward the key chemokine CXCL12/stromal cell-derived factor-1. GluR3 expression on normal, cancer and autoimmune-associated T cells and the ability of glutamate to directly activate T cell function could be of substantial scientific and clinical importance to normal neuroimmune dialogues and to CNS diseases and injury, and especially to: 1) T cell transmigration to the CNS and patrolling in the brain, 2) T cell-mediated multiple sclerosis, and 3) autoimmune epilepsy, as neurotoxic anti-GluR3 Abs are found and suspected to cause/potentiate seizures and neuropathology in several types of human epilepsies. Thus far, GluR3 was found only on neurons and glia cells; our results reveal a novel peripheral source of this antigenic receptor.

The neuropeptides GnRH-II and GnRH-I are produced by human T cells and trigger laminin receptor gene expression, adhesion, chemotaxis and homing to specific organs

Can T cells be directly activated to de novo gene expression by gonadotropin-releasing hormone-II (GnRH-II), a unique 10-amino-acid neuropeptide conserved through 500 million years of evolution? GnRH-II, which has been identified in mammals, shares 70% homology with the mammalian hypothalamic neurohormone GnRH (GnRH-I), the primary regulator of reproduction, but is encoded by a different gene. Although both neuropeptides are produced mainly in brain, their localization and promoter regulation differ, suggestive of distinct functions. Indeed, GnRH-II barely affects reproduction and its role in mammalian physiology is unknown. We find here that human normal and leukemic T cells produce GnRH-II and GnRH-I. Further, exposure of normal or cancerous human or mouse T cells to GnRH-II or GnRH-I triggered de novo gene transcription and cell-surface expression of a 67-kD non-integrin laminin receptor that is involved in cellular adhesion and migration and in tumor invasion and metastasis. GnRH-II or GnRH-I also induced adhesion to laminin and chemotaxis toward SDF-1α, and augmented entry in vivo of metastatic T-lymphoma into the spleen and bone marrow. Homing of normal T cells into specific organs was reduced in mice lacking GnRH-I. A specific GnRH-I-receptor antagonist blocked GnRH-I- but not GnRH-II-induced effects, which is suggestive of signaling through distinct receptors. We suggest that GnRH-II and GnRH-I, secreted from nerves or autocrine or paracrine sources, interact directly with T cells and trigger gene transcription, adhesion, chemotaxis and homing to specific organs, which may be of clinical relevance.

TCR Activation Eliminates Glutamate Receptor GluR3 from the Cell Surface of Normal Human T Cells, via an Autocrine/Paracrine Granzyme B-Mediated Proteolytic Cleavage

The majority of resting normal human T cells, like neuronal cells, express functional receptors for glutamate (the major excitatory neurotransmitter in the CNS) of the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor subtype 3 (GluR3). Glutamate by itself (∼10 nM) activates key T cell functions, including adhesion to fibronectin and laminin and chemotactic migration toward CXCL12/stromal cell-derived factor 1. In this study, we found by GluR3-specific immunostaining, flow cytometry, and Western blots that GluR3 cell surface expression decreases dramatically following TCR activation of human T cells. CXCR4, VLA-4, and VLA-6 also decrease substantially, whereas CD147 increases as expected, after TCR activation. Media of TCR-activated cells “eliminates” intact GluR3 (but not CXCR4 and VLA-6) from the cell surface of resting T cells, suggesting GluR3 cleavage by a soluble factor. We found that this factor is granzyme B (GB), a serine protease released by TCR-activated cells, because the extent of GluR3 elimination correlated with the active GB levels, and because three highly specific GB inhibitors blocked GluR3 down-regulation. Media of TCR-activated cells, presumably containing cleaved GluR3B peptide (GluR3 aa 372–388), inhibited the specific binding of anti-GluR3B mAb to synthetic GluR3B peptide. In parallel to losing intact GluR3, TCR-activated cells lost glutamate-induced adhesion to laminin. Taken together, our study shows that “classical immunological” TCR activation, via autocrine/paracrine GB, down-regulates substantially the expression of specific neurotransmitter receptors. Accordingly, glutamate T cell neuroimmune interactions are influenced by the T cell activation state, and glutamate, via AMPA-GluR3, may activate only resting, but not TCR-activated, T cells. Finally, the cleavage and release to the extracellular milieu of the GluR3B peptide may in principle increase its antigenicity, and thus the production, of anti-self GluR3B autoantibodies, which activate and kill neurons, found in patients with various types of epilepsy.

Autoantibodies to glutamate receptors can damage the brain in epilepsy, systemic lupus erythematosus and encephalitis

Glutamate is the major excitatory CNS neurotransmitter. Glutamate receptor autoantibodies have now been called to our attention, as they are found in many patients with epilepsy, systemic lupus erythematosus (SLE) and encephalitis, and can unquestionably cause brain damage. AMPA GluR3 autoantibodies have been found thus far in 27% of patients with different epilepsies, while NMDA NR2A or NR2B autoantibodies, some of which cross-react with double-stranded DNA, have been detected in 30% of SLE patients, with or without neuropsychiatric impairments. NR2 autoantibodies were also found in patients with epilepsy (33%), encephalitis and stroke. NR2 and GluR3 autoantibodies do not cross-react in patients with epilepsy. Human and animal studies show that both types of glutamate receptor autoantibodies can certainly damage the brain. GluR3 autoantibodies bind to neurons, posses a unique ability to activate their glutamate-receptor antigen, and cause neuronal death (either by excitotoxicity or by complement fixation independent of receptor activation), multiple brain damage and neurobehavioral/cognitive impairments. In animal models (mice, rats or rabbits) GluR3 autoantibodies may cause seizures, augment their severity or modulate their threshold. NR2/dsDNA autoantibodies, once present in the CNS, can bind and subsequently kill hippocampal and cortical neurons by an excitotoxic complement-independent mechanism. Herein, we discuss epilepsy, autoimmune epilepsy, SLE and neuropsychiatric SLE in general; summarize the up-to-date in vivo and in vitro evidence concerning the presence of glutamate receptor autoantibodies in human diseases; discuss the activity and pathogenicity of different glutamate receptor autoantibodies; and end with our conclusions, recommendations and suggested future directions.

Autoimmune Epilepsy: Some Epilepsy Patients Harbor Autoantibodies to Glutamate Receptors and dsDNA on both Sides of the Blood-brain Barrier, which may Kill Neurons and Decrease in Brain Fluids after Hemispherotomy

Purpose: Elucidating the potential contribution of specific autoantibodies (Abs) to the etiology and/or pathology of some human epilepsies. Methods: Six epilepsy patients with Rasmussens encephalitis (RE) and 71 patients with other epilepsies were tested for Abs to the –B— peptide (amino acids 372-395) of the glutamate/AMPA subtype 3 receptor (GluR3B peptide), double-stranded DNA (dsDNA), and additional autoimmune disease-associated autoantigens, and for the ability of their serum and cerebrospinal-fluid (CSF) to kill neurons. Results: Elevated anti-GluR3B Abs were found in serum and CSF of most RE patients, and in serum of 17/71 (24%) patients with other epilepsies. In two RE patients, anti-GluR3B Abs decreased drastically in CSF following functional-hemispherotomy, in association with seizure cessation and neurological improvement. Serum and CSF of two RE patients, and serum of 12/71 (17%) patients with other epilepsies, contained elevated anti-dsDNA Abs, the hallmark of systemic-lupus-erythematosus. The sera (but not the CSF) of some RE patients contained also clinically elevated levels of –classical— autoimmune Abs to glutamic-acid-decarboxylase, cardiolipin, β2-glycoprotein-I and nuclear-antigens SS-A and RNP-70. Sera and CSF of some RE patients caused substantial death of hippocampal neurons. Conclusions: Some epilepsy patients harbor Abs to GluR3 and dsDNA on both sides of the blood-brain barrier, and additional autoimmune Abs only in serum. Since all these Abs may be detrimental to the nervous system and/or peripheral organs, we recommend testing for their presence in epilepsy, and silencing their activity in Ab-positive patients.

Human T-leukemia and T-lymphoma express glutamate receptor AMPA GluR3, and the neurotransmitter glutamate elevates the cancer-related matrix-metalloproteinases inducer CD147/EMMPRIN, MMP-9 secretion and engraftment of T-leukemia in vivo.

Glutamate is the major excitatory neurotransmitter of the nervous system. We previously found that glutamate activates normal human T-cells, inducing their adhesion and chemotaxis, via its glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype 3 (GluR3) expressed in these cells. Here, we discovered that human T-leukemia (Jurkat) and cutaneous sezary T-lymphoma (HuT-78) cells also express high levels of GluR3. Furthermore, glutamate (10 nM) elevates CD147/EMMPRIN, a cancer-associated matrix metalloproteinases (MMPs) inducer, promoting spread of many tumors. Glutamate-induced CD147 elevation in both cancerous and normal human T-cells was mimicked by AMPA (glutamate/AMPA-receptor agonist) and blocked by CNQX (glutamate/AMPA-receptor antagonist). Importantly, glutamate also increased gelatinase MMP-9 secretion by T-lymphoma. Finally, ex vivo pre-treatment of T-leukemia with glutamate enhanced their subsequent in vivo engraftment into chick embryo liver and chorioallantoic membrane. Together, these findings reveal that glutamate elevates cancer associated proteins and activity in T-cell cancers and by doing so may facilitate their growth and spread, especially to and within the nervous system. If so, glutamate receptors in T-cell malignancies should be blocked.

Autoantibodies against an extracellular peptide of the GluR3 subtype of AMPA receptors activate both homomeric and heteromeric AMPA receptor channels

Autoantibodies to the GluR3-subtype of AMPA/glutamate receptors are found in the sera and cerebrospinal fluid of some individuals with epilepsy. They could possibly play a role in the pathophysiology of epilepsy since anti-GluR3 sera display glutamatergic agonist activity. We have investigated here the ability of affinity-purified antibodies (Abs) directed against the immunogenic peptide GluR3B (amino-acid 372–395) to interact with and activate recombinant GluR3-receptor channels expressed by Xenopus oocytes. We report here that the affinity-purified anti-GluR3B Abs directly activate GluR3-containing homomeric and heteromeric AMPA receptor complexes without the requirement of neuronal, glial or blood ancillary molecules. We present some of the properties of the purified anti-GluR3B Abs and discuss the possible physiological or pathological consequences of their activation of glutamate receptors.

Immunization with the glutamate receptor-derived peptide GluR3B induces neuronal death and reactive gliosis, but confers partial protection from pentylenetetrazole-induced seizures.

Do autoantibodies (Abs) against glutamate/AMPA receptor subtype 3 affect the severity of seizures? Rats immunized with the GluR3B-peptide (amino acids (aa) 372-395) or with the control GluR3A-peptide (aa 245-274) produced the respective anti-GluR3B and anti-GluR3A Abs (both types of Abs found in some epilepsy patients). The GluR3B-immunized rats exhibited neuronal death and reactive gliosis in the brain, but not overt spontaneous seizures. Surprisingly, in response to the chemoconvulsant pentylenetetrazole, the GluR3B-immunized rats displayed fewer jerks, a lower percentage of generalized seizures, and a lower overall seizure-severity score than GluR3A-immunized, scrambled GluR3B-immunized or non-immunized control rats. These findings, combined with the previously demonstrated ability of anti-GluR3B Abs to bind, activate, and kill neurons and glia, suggest that if these Abs are present in the brain they may cause neuronal death, which by itself may be pro-epileptic, but they may also decrease the excitability of seizure-related neural circuits, thereby conferring partial protection from seizures induced by other exogenously applied epileptogenic stimuli. The present results could have clinical implications for epilepsy.

Antibodies to glutamate receptor subtype 3 (GluR3) are found in some patients suffering from epilepsy as the main disease, but not in patients whose epilepsy accompanies antiphospholipid syndrome or Sneddon's syndrome.

Autoantibodies (Abs) to the “B” peptide (amino acids 372–395) of glutamate/AMPA receptor subtype 3 (GluR3) are found in serum and cerebrospinal fluid of some patients with different types of epilepsy. Since such anti-GluR3B Abs can activate and/or kill neurons in vitro and in vivo, they may contribute to epilepsy. To investigate whether anti-GluR3B Abs may also be relevant to epilepsy when it accompanies some autoimmune-diseases, we tested for these Abs in patients suffering from epilepsy that accompanies anti-phospholipid syndrome (APS) or Sneddons syndrome (SNS), both being autoimmune-diseases with frequent neurological complications. We tested 77 pediatric patients whose epilepsy is their main disease; 31 adult patients whose epilepsy accompanies APS (primary or SLE-associated) or SNS; 45 epilepsy-free APS and SNS patients; and 90 healthy controls. Compared to the controls, significantly elevated anti-GluR3B Abs were found in 22/77 (29%) patients whose epilepsy is their main disease, but in none of the patients whose seizures accompany APS or SNS. Yet, all the APS and SNS patients harbored the characteristic anti-phospholipid Abs (aPL), directed against cardiolipin and β2-glycoprotein I, and had lupus anti-coagulant. Thus, anti-GluR3B Abs are not crossreactive with aPL, and not produced as a non-specific consequence of seizures on the one hand, or autoimmune-diseases on the other. Taken together with new findings accumulated recently in our lab, we suggest that anti-GluR3B Abs are produced primarily in the periphery due to specific/non-specific “irritation” of the immune system, and that once they reach the brain via a leaky blood–brain barrier they may cause neuronal/glial damage and facilitate the outburst of epilepsy and additional neurological abnormalities. In contrast, the presence of anti-GluR3B Abs does not seem to increase the probability of developing APS, SNS or the seizures that often accompany these autoimmune-diseases. These findings may have important diagnostic and therapeutic implications.

Monozygotic twins discordant for epilepsy differ in the levels of potentially pathogenic autoantibodies and cytokines

Can autoantibodies (Abs) and cytokines play a role in epilepsy? Monozygotic twins discordant for epilepsy (most probably Rasmussens encephalitis (RE)), compared to 49 neurologically intact controls, were both found to contain in their serum (at the time of epilepsy diagnosis) significantly elevated levels of specific Abs against peptide B (amino acids 372–395) of the ionotropic glutamate receptor of AMPA subtype 3 (i.e. GluR3B peptide). Interestingly, both twins also had clinically elevated levels of Abs to double-stranded (ds) DNA, glutamic acid decarboxylase, nuclear antigens, β2-glycoprotein I and cardiolipin, as in “classical” autoimmune diseases. Both twins also had significantly elevated levels of IFNγ, TNFα, IL-4 and IL-10 in the serum, compared to the controls. Comparing the twins revealed that the epileptic twin had significantly higher levels of five of the above anti-self Abs, but significantly lower levels of all four cytokines compared to her healthy sister. Importantly, the epileptic twin, alike three other RE patients tested herein, contained elevated levels of Abs to GluR3B and dsDNA also in cerebrospinal fluid (CSF) (unavailable of the healthy twin). Our results suggest that the various autoimmune Abs studied herein, all of which are known already to have a potential to be pathogenic in the nervous system and/or peripheral organs, may play a role in some types of epilepsy. The titer of such Abs and of key cytokines may be crucial for either facilitating or arresting the development of epilepsy. Our findings also show that anti-GluR3B Abs in serum are not necessarily detrimental (their presence in the CSF may be more dangerous), and that they are not a mere side effect of already existing epilepsy, as they were found herein in serum of a healthy individual. These findings and suggestions may be of clinical importance and call for further studies.