Breakthroughs on the Brink: Turning the Tide on MS

By Patrick Perry

Richard Burt, M.D., chief of immunotherapy for autoimmune diseases at Northwestern University’s Feinberg School of Medicine, and his research team appear to have reversed the neurological dysfunction of early-stage multiple sclerosis patients by using the patients’ own adult stem cells, thereby “resetting” their immune systems.

In May one of the study participants, Edwin McClure, walked across the stage to receive his degree after completing a rigorous graduate program at Virginia Commonwealth University. The young man appeared strong, healthy, and confident.

The scene was in stark contrast to four years earlier when the high school star football player was battling a severe cold, fatigue, and inexplicable visual changes.

“It was like someone turned down a dimmer switch,” he recalls. “My mom thought the problems were due to sinus pressure and would eventually go away, but when I got over the cold and still had difficulty seeing, she took me to an optometrist.”

When nothing surfaced during visits to an optometrist and an ophthalmologist, McClure was referred to a neurologist for follow-up.

After a series of tests and an MRI scan, the doctor delivered the diagnosis - multiple sclerosis (MS). The visual changes the young man was experiencing were due to optic neuritis, an inflammation of the optic nerve that occurs in approximately 50 percent of patients with the disease.

McClure was placed on steroids and interferon injections?-?a regimen that successfully controlled symptoms for two years. But when the MS started to break through, his physician switched to another medication.

“Over the course of four months, I started to develop an allergic reaction to the drug,” McClure says. “Meanwhile, my disease was still progressing.”

McClure was at a crossroads: begin medications with significantly greater risk of side effects or, as his neurologist suggested, investigate a promising clinical trial underway at Northwestern University in Chicago.

He chose the latter, qualified, and enrolled in Dr. Burt’s study. McClure was one of the 21 patients in the trial, ages 20 to 53, who had relapsing-remitting MS for an average of five years and had not responded to at least six months of treatment with interferon beta. After an average follow-up of three years posttreatment, 17 patients (81 percent) improved and none got worse, according to Dr. Burt, whose findings were published in the March issue of The Lancet Neurology.

Resetting the Immune System

Dr. Richard Burt, M.D.
Courtesy Dr. Richard Burt, M.D.

“The concept is that your immune stem cells - your blood stem cells - could be used to regenerate a new immune system in virtually any autoimmune disease,” Dr. Burt tells the Post. “If we treated patients in the early relapsing-remitting phase of MS who were experiencing frequent acute attacks despite the use of interferon, patients got better. Six months after the procedure, they were even better. By two years, they seemed to have reached their peak improvement in neurological function. Most people tend to be early- to mid-range in their disability, and that’s when this therapy is really effective. But if you treat MS in a later stage, called secondary progressive MS, it doesn’t really help. In this stage, patients experience a steady worsening of irreversible neurological damage.”

In the procedure, Dr. Burt and colleagues first push immune stem cells from the bone marrow into the blood by using a growth factor and a drug called Cytoxan (cyclophosphamide). Ten days later, they harvest cells from the blood via catheter. The cells are then separated, frozen, and cultured to ensure that none are contaminated with bacteria during the process. Next, the patients are treated with drugs to inhibit the old immune system, and then the frozen stem cells are thawed and infused back into the patients to make a new immune system.

Reversing the Tide

Edwin McClure
Courtesy Edwin McClure

“I started to feel improvement while I was in the hospital,” McClure says. “I realized that I didn’t need my glasses to see. At home my parents noticed that my balance was improving and that I didn’t seem as fatigued as before. Honestly, these changes started within the first month after coming home. My life continued to improve. By the third month, I was actually going to the YMCA to exercise.”

Three years after treatment, McClure remains off medication and now experiences no symptoms of MS.

Like McClure, the majority of trial participants experienced benefits.

“We’ve seen patients who have had marked improvement in symptoms,” notes Dr. Burt, principal investigator of the clinical trial. “Your nervous system controls everything, so the part of the brain attacked by MS determines where you have a problem. Some patients had trouble walking - falling down and having to hold on to things - but after the procedure, they had marked improvement. Others had issues with incontinence, and that’s gone away. If you’re worried about incontinence, that’s quite remarkable. Numbness, tingling, inability to feel things, visual problems - blurred and double vision - can all reverse. Basically, any type of deficit can reverse.

In some patients, we actually had complete reversal - everything went away, and they were completely normal in all functional exams. In others, symptoms never completely reversed, but improved dramatically.”

The study participants are also off all conventional disease-modifying medications now used to slow the rate of disease progression.

While the small trial is only a first step, the results offer a completely new way to treat MS. “This is the first time in the history of any therapy used to treat MS where it actually reverses neurological deficit,” stresses Dr. Burt.

“All other therapies were studied or approved for their ability to slow the rate of progression - in terms of clinical deficits or MRI load of lesion burden - but nothing has, up to this time, reversed deficit. That’s what’s exciting. However, I want to stress that we cannot say it is a cure and current results with three years of follow-up are encouraging.”

Dr. Burt and colleagues are enrolling patients in a larger trial to test the procedure in a randomized setting. “If the results of the trial hold up, I believe it will help open the door for it to be accepted as standard therapy,” adds Dr. Burt.

At present, clinical trials are underway at the University of Calgary in Canada, the University of Sao Paulo in Brazil, and at Northwestern University. If interested in learning more about the trial, e-mail d-spahovic@northwester.edu.

A Different Approach

Sergeant Preston Walker
Courtesy Sergeant Preston Walker

After undergoing conventional therapy for MS for several years, Fort Worth police sergeant Preston Walker learned about a new treatment for autoimmune disorders. Researchers were utilizing adult stem cells derived from cord blood at The Institute of Cellular Medicine in Costa Rica. Walker inquired about the potential of the treatment for multiple sclerosis.

“We knew that if the treatment worked, the potential benefits for multiple sclerosis patients could be limitless,” says Walker.

Dr. Neil Riordan, CEO of the Institute, suggested a therapy under consideration - using stem cells derived from a patient’s fat tissue. In May 2008, Walker flew to the clinic where doctors removed samples of his abdominal fat through a mini-liposuction, drawing out stem cells, which were later re-injected. According to Dr. Riordan, Walker and a colleague were the first to undergo this treatment protocol. “My quality of life has improved significantly,” Walker told the Post. “The problems with depression, fatigue, and balance have been corrected. I feel really good.”

In June 2009, Walker, who continues to take Avonex as a maintenance drug, plans a return trip to Costa Rica for a “tune-up,” as he puts it. “I’m curious to see if they can further improve my cognitive abilities.”

In this video we discuss the paper Bai et al. in which the immunological and regenerative activities of human bone marrow derived mesenchymal stem cells were assessed in two different animal models of multiple sclerosis.

Control of pathological immunity in multiple sclerosis may be accomplished (at least in part) by antigen-specific vaccination, by administration of immune modulators such as Interferon Beta, or by depletion of activated effector T cells using antibodies. 

Immune modulation by hormones offers a new method of addressing multiple sclerosis.  For example, it is known that mesenchymal stem cells have therapeutic effects in animal models of multiple sclerosis, and that these effects seem to be mediated both by immune modulaton but also by stimulation of regeneration.  Interestingly, hormones such as progesterone have been demonstrated to stimulate immune modulatory activities of mesenchymal stem cells.

A recent paper (Theil et al. Suppression of Experimental Autoimmune Encephalomyelitis by Ghrelin. J Immunol 2009 Jul 20) described the ability of the “hunger hormone” ghrelin to inhibit the mouse model of multiple sclerosis, experimental allergic encephalomyelitis (EAE).

Ghrelin is a hormone made by the pancreas and stomach cells that stimulates the feeling of hunger.  It is also known to stimulate growth hormone release.  Some have compared ghrelin as the opposite of leptin, a hormone known to inhibit hunger.  Interestingly leptin has been associated with induction of inflammation of autoimmunity.  For example, administration of leptin has been demonstrated to augment mouse multiple sclerosis (Matarese et al. Leptin potentiates experimental autoimmune encephalomyelitis in SJL female mice and confers susceptibility to males. Eur J Immunol. 2001 May;31(5):1324-32).

In the paper we are discussing, EAE was induced in B6 mice by administration of MOG peptide (myelin oligodendrocyte glycoprotein 35-55) and treated groups were administered ghrelin after immunization with the autoantigen.  As compared to vehicle-controls, the treated groups had a profound inhibition of EAE induction as assessed by the disease severity index.  Additionally, suppression of the inflammatory triad of TNF, IL-1, and IL-6 was observed at the mRNA level in cells that have infiltrated the spinal cord, as well as resident microglial cells.  In vitro treatment of microglial cells by ghrelin resulted in suppressed ability to produce inflammatory trial cytokines after stimulation with lps.

These data suggest that ghrelin itself may be useful for the treatment of multiple sclerosis, as well as the possibility of using it in combination with other agents that block microglial activation.  For example, the endocannabinoid anandamide has previously been demonstrated to inhibit microglial inflammatory activity.

Suppression of microglial-based inflammation is important because the microglia are activated by cytokine producing T cells and are critical components of multiple sclerosis neurodegeneration, not only by inflammatory mediators, but also by glutamate excitotoxicity.

Conceptually the best way to treat multiple sclerosis is to specifically inhibit the immune response against the myelin sheath, while leaving the immune response against other proteins intact.  Additionally, the best way to treat it, is also to induce regeneration of neurons and central nervous system components that have already been damaged, perhaps through the use of stem cells.

Successful use of stem cell therapy in multiple sclerosis is believed to occur because the fat contains numerous cell populations that inhibit pathological immune responses, such as T regulatory cells (whose function is suppressed in multiple sclerosis), while at the same time containing stem cells, especially mesenchymal stem cells, which can repair damaged tissue.

The reprogramming of the immune system to selectively stop attacking one protein, or a series of specific proteins is called “immunological tolerance”.  Originally the concept of tolerance came from experiments by the scientists Billingham-Brent-Medawar decades ago who demonstrated that if two genetically distinct animals had shared circulation during embryonic development, when the animals reached adulthood they would readily accept tissue grafts from each other but reject grafts from others.  In other words the animals were made “tolerant” to each other. 

This concept of selectively “teaching” the immune system that it should not attack a specific antigen is how approaches such as the myelin basic protein DNA vaccine developed by Bayhill Therapeutics seems to work.  This vaccine, called BHT-3009, was demonstrated to induce antigen-specific immune modulation in multiple sclerosis patients in a Phase I/II study, and was subsequently demonstrated to be capable of causing a 50-61% reduction in new lesion formation as detected by MRI and profound reduction of anti-myelin antibodies in a subsequent Phase II study in multiple sclerosis patients.  DNA vaccines seem to work in part through inducing interferon beta, which seems to be involved in shifting of the T cell cytokine production profile away from Th1 and Th17, although this is controversial.

Another way to “trick” the immune system into selectively not attacking an antigen is to provide the antigen orally.  A published study fed multiple sclerosis patients cow myelin (which contains both myelin basic protein and proteolipid protein) and examined whether this affected immune response to myelin (Hafler, DA et al. Oral administration of myelin induces antigen-specific TGF-beta 1 secreting T cells in patients with multiple sclerosis. Ann NY Acad Sci 1997 Dec 19;835:120-31).

The investigators took blood from 34 patients with relapse remitting multiple sclerosis that were either fed cow myelin (17 patients) or not fed it (17 control patients) and generated T cell lines that recognized either myelin basic protein (MBP), proteolipid protein (PLP), or tetanus toxin (TT). 

A profound increase in the number of cells secreting the antiinflammatory cytokine TGF-b on stimulation with MBP or PLP was seen in patients who ate the cow myelin as opposed to controls.  Interestingly, there was no increase in production of the inflammatory cytokine interferon gamma, nor were there alterations in response to tetanus toxin. 

These data suggest that at least at an immunological level administration of oral cow myelin is helpful in patients with multiple sclerosis.  The question now because, can one increase therapeutic effects by combining the cow myelin with something like lithium, or with fat stem cells?  Furthermore, clinically used drugs such as metformin, which may conceptually increase Treg generation by suppressing IL-17 may be useful to expand the overall tolerogenic profile of oral tolerance induction.

A recent study (Rossi et al. Exercise attenuates the clinical, synaptic and dendritic abnormalities of experimental autoimmune encephalomyelitis. Neurobiol Dis 2009 Jul 7) seems to suggest that exercise may be beneficial in slowing progression of multiple sclerosis. 

The investigators induced a multiple sclerosis-like disease in mice by administration of peptides derived from the myelin protein called myelin oligodendrocyte glycoprotein (MOG) and induced the mice to undergo voluntary running in a wheel.

Voluntary exercise decreased progression of disease, and overall severity, as compared to control animals.  Furthermore, the inflammation-associated suppression of GABA synapse stimulation by cannabinoid CB1 receptors, that is associated with the animal model of multiple sclerosis was inhibited as a result of exercise.  Additionally, exercise effectively reduced dendritic spine loss induced by by the multiple sclerosis-like disease in striatal neurons.

Exercise has been demonstrated to induce insulin like growth factor (IGF)-1 expression in certain cell types in response to mechanical motion, as well as growth hormone administration.  It is interesting to note that IGF-1 stimulates production of new myelin, as well protects the animal model of multiple sclerosis from disease (Yao et al. Insulin-like growth factor-I given subcutaneously reduces clinical deficits, decreases lesion severity and upregulates synthesis of myelin proteins in experimental autoimmune encephalomyelitis. Life Sci 1996;58(16):1301-6).  So it would be interesting to see if exercise, along with vitamins and stem cells may be syngergistic in treatment of multiple sclerosis.

Stem cell therapy of multiple sclerosis is associated with immune modulation, as well as the possibility of inducing regeneration of damaged neural tissue.  In the quest to figure out novel agents that may be useful in combination with stem cell therapy, scientists assess various drugs.  One class of interesting drugs to evaluate are drugs that are already on the market for different diseases.  For example, erythropoietin was previously demonstrated to inhibit multiple sclerosis in animal models.  Erythropoietin is a hormone made by the kidneys that normally stimulates red blood cell production from the bone marrow hematpoietic stem cell.  Erythropoietin is administered as a drug in patients with anemia to increase red blood cells.  Interestingly, erythropoietin is also associated with suppression of inflammatory Th1 and Th17 responses, upregulation of antiinflammatory Th2 responses, and stimulation of endogenous stem cells, including stem cells in the brain. 

The video above describes the effects of lithium on the animal model of multiple sclerosis called experimental allergic encephalomyelitis (EAE).  It demonstrates that administration of lithium suppresses autoreactive T cells but not overall T cell responses.  Furthermore, the paper demonstrated that lithium administration not only suppressed disease onset, but also reversed established disease. 

It appears that lithium mediates its effects through the suppression of the GSK-3 enzyme, which is involved not only in inflammation but also self-renewal of stem cells. 

The above video is provided for educational purposes only and is not suggesting the use of lithium in treatment of multiple sclerosis patients, it is only providing some scientific information that may be useful in future clinical trials and scientific experiments.

Treatment of multiple sclerosis patients with their own fat derived stem cells has generated promising results which we recently published.  There are several mechanisms by which this treatment may mediate therapeutic effects.  One particular mechanism that we will discuss here is production of soluble factors by fat derived mesenchymal stem cells that may be protective to neurons.

In the paper (Wei et al. Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis. Neurosciences Letters. 2009 Jun 21) conditioned media from fat derived mesenchymal stem cells was assessed for therapeutic activity in terms of ability to protect neurons from cell death.  The idea being that the fat stem cells produce factors that can inhibit apoptosis of brain cells.

The investigators used a model of neuronal cell death by culturing cerebellar granule neurons in absence of serum and potassium in order to induce apoptosis.  A dose-dependent inhibition of neuronal apoptosis was observed when adipose stem cell conditioned media was added to the cultures.  Inhibition of caspase-3 activity was observed in the protected neurons (which would make sense since caspase-3 is involved in the vast majority of apoptotic signalling).  Additionally, the adipose stem cell conditioned media was capable of stimulating the anti-apoptotic akt signalling pathway. 

In order to investigate what specific molecules were in the adipose stem cell conditioned media that induced protective effects on the neurons, it was found that neutralization of insulin growth factor (IGF)-1 resulted in loss of anti-apoptotic activity.

This study brings up several interesting questions. 

Firstly, would the supernatant from the adipose derived stem cells also have effects on oligodendrocytes?  In multiple sclerosis there is eventual neuronal death, however, activation of oligodendrocytes is very important in terms of re-myelinating the injured tissue.  We do know that bone marrow cells are capable of inducing remyelination under specific conditions, so the question would be if adipose stem cell supernatant may also mediate such an effect.  This would be relatively easy to test in an in vitro system, or using the experimental allergic encephalomyelitis model.  However the issue would be whether the supernatant would also mediate immune modulatory properties that may mask the effects of potential remyelinating activity.

The second question is whether the adipose stem cell supernatant would have effects on other models of neuronal apoptosis.  In the context of multiple sclerosis, neuronal apoptosis mediated by excess glutamate seems to be an important factor.  Alternatively, the adipose stem cell supernatant may increase astrocyte uptake of glutamate.

The third question would be whether various conditions can be applied to the adipose stem cells so as to enhance secretion of protective factors in the conditioned media.  For example, there are publications demonstrating that mesenchymal stem cells cultured in the presence of low oxygen actually increase secretion of various therapeutic factors, including IGF-1.  This may be because the mesenchymal stem cells “feel” the lack of oxygen and as a natural reparative mechanism start secreting factors that protect other cells.  This may be possible given that mesenchymal stem cells have been demonstrated to inhibit neuronal cell death in vivo in models of stroke.  Alternatively, another way to “stress” mesenchymal stem cells in vitro may be treatment with inflammatory cytokines such as TNF-alpha.  At the recent FOCIS meeting in San Francisco, data was presented demonstrating that mesenchymal stem cells secrete higher levels of antiinflammatory factors after exposure to immunological stress, as in the form of a mixed lymphocyte reaction.  Collection of supernatant of stem cells under various conditions of stress may be one interesting way to increase efficacy of the approach described in the current paper.

The macrophages in the brain, called microglia, play an important role in multiple sclerosis.  On the one hand activated microglia can generate free radicals and glutamate, which is neurotoxic, on the other hand, microglia may be involved in neural remodeling and brain repair.  It may be that microglia have inflammatory and antiinflammatory properties in a similar way that macrophages have M1 or M2 phenotypes.  Stromal vascular fraction cells contain high proportions of M2 antiinflammatory macrophages, which may be one of the reasons for the effects of autologous fat cells in treatment of multiple sclerosis.  There are some reports that neurotransmitters such as anandamine may modulate microglia production of inflammatory cytokines.  It is therefore interesting to see what type of other agents may modulate microglial activity.

In a recent paper (Russo et al. Involvement of mTOR kinase in cytokine dependent microglial activation and cell proliferation. Biochem Pharmacol. 2009 Jun 30) the effects of modulating a protein called mammalian target of rapamycin (mTOR) was examined in microglial cytokine production.

mTOR is a kinase that is associated with cell multiplication, activation, and survival.  It is downstream of several biological pathways and its activation is associated with many cancers.  Rapamycin, an immune suppressive drug that has been shown to be tolerogenic in some situations, is an inhibitor of mTOR. 

The investigators demonstrated that activation of microglial cells isolated from the cortical area of rats had activated mTOR after treatment with lipopolysaccharide (an activator of macrophages), as well as after treatment with inflammatory cytokines.  Nitric oxide release by microglial cells causes damage to neurons.  The study found out that treatment with mTOR inhibitors resulted in the inhibition of cytokine induced nitric oxide production.  Treatment of the microglial cells with mTOR inhibitors also resulted in the inhibition of proliferation and suppression of cyclooxygenase, an enzyme that causes formation of prostaglandins, which are also associated with inflammation. 

Thus the study suggests that administration of mTOR inhibitors may be a method of inhibiting microglial activation.  In fact, there is a publication combining vitamin D3 and rapamycine for the inhibition of the mouse model of multiple sclerosis (Branisteanu et al. Synergism between sirolimus and 1,25-dihydroxyvitamin D3 in vitro and in vivo. J Neuroimmunol 1997 Nov;79(2):138-47).

We have discuss previously that numerous nervous system molecules have immune modulatory effects.  For example, the endocannabinoid anandamide is capable of converting microglial cells from secreting inflammatory compounds to producing antiinflammatory cytokines such as interleukin-10.  Microglial cells generally cause pathology in multiple sclerosis through production of glutamate, as well as release of inflammatory agents.  By inducing microglial cells to produce interleukin-10, mechanisms similar to those that mesenchymal stem cells use to control inflammation may be activated.  Neurologically-associated molecules also may play a role in homing of stem cells.  For example, the neurotransmitter Substance P has recently been shown to act as attractant of stem cells. 

Here we discuss an interesting new way of modulating T cells so as to prevent onset and progression of an animal model of multiple sclerosis.  The publication (Bittner et al. TASK1 modulates inflammation and neurodegeneration in autoimmune inflammation of the central nervous system.  Brain 2009 Jul 1) discusses the role of TASK1 in T cell activity in the experimental allergic encephalomyelitis model. 

What is TASK1?

TASK1 stands for “TWIK-related acid-sensitive potassium channel 1″, which is a the two-pore domain potassium channel family that is important for maintaining resting membrane potential and balancing neuronal excitability.  TASK1 activity is inhibited by low pH and is activated by certain anesthetics… and It is also known as OAT1; TASK; TBAK1; K2p3.1; and KCNK3, with the official name being KCNK3 potassium channel, subfamily K, member 3. 

In the publication it was demonstrated that mice made genetically deficient for the TASK1 gene were substantially resistant to induction of experimental allergic encephalomyelitis.  Interestingly, T cells from the TASK1 knockout mice had an inhibited proliferative and cytokine response in vitro, suggesting the resistance to EAE may be associated with alterations on the T cell side and not just on the neuronal side.  Conceptually, one may expect neurons from TASK deficient animals to be more resistant to damage due to possible role of TASK1 in induction of apoptosis.  Indeed, the authors did demonstrate using in vitro isolated neurons from TASK1 deficient and wild-type neurons that a protective effect was observed associated with TASK1 deficiency.

Anandamide, which we previously described as having potentially beneficial effects on the mouse model of multiple sclerosis (EAE) by virtue of its ability to alter microglial production of inflammatory agents was demonstrated to inhibit TASK1 activity.  In vitro administration of anandamide was shown to inhibit T cell production of inflammatory cytokines.

Perhaps most exciting from the pubication was that administration of anandamide was capable of inhibiting progression of EAE after disease onset was initiated. 

It will be interesting in the future to try to tease out the effects of anandamide between activities on the T cells and activities on the microglia, which do not necessarily need to be exclusive.  For example, one could envision a system where T regulatory cells may be selectively “reprogramming” microglia to reduce inflammatory activities.  Or conversely, T regulatory cells may be generated in the central nervous system as a result of microglial presentation of self-antigen in the presence of interleukin-10 generated by the anandamide-reprogrammed microglia.

In conclusion, the current work supports further investigation into the TASK1 channel as a possible target for drug development in multiple sclerosis.  Given that mesenchymal stem cells already have demonstrated therapeutic effects in multiple sclerosis but in mice and man, it will be interesting to see if co-administration of anandamide may enhance mesenchymal stem cell regenerative activity by modulating the local microenvironment.

While we all know that multiple sclersosis is an immunologically mediated disease, it is interesting to learn about some of the non-immunological mechanisms that are associated with this condition. 

For example, we generally think of T cells as being the main effectors of demyelination and damage to the central nervous system because agents that suppress T cell infiltration into the central nervous system or their activation seem to be useful in the treatment of multiple sclerosis.  However other indirect mechanisms are also involved.  For example, microglial cells which secrete inflammatory cytokines also release glutamate, which causes excitotoxicity to neurons.

Astrocytes are the major “glial” component of the central nervous system.  The primary function of astrocytes is to support neurons in their activities.  Astrocytes do this in many ways, including; a) providing control of blood flow in neuronal areas; b) maintaining a proper nutrient environment locally, for example, astrocytes produce lactate and other nutrients that are needed for neuronal function; c) telling the oligodendrocytes when to start stimulating production of myelin; d) acting in repair of damaged tissue; and e) cleaning up left over neurotransmitters.

Based on the numerous functions of astrocytes, the authors of the paper we will be discussing (Tilleux et al. Selective up-regulation of GLT-1 in cultured astrocytes exposed to soluble mediators released by activated microglia.  Neurochem Int 2009 Jul-Aug;55(1-3):35-40), sought to determine whether astrocytes may uptake excess glutamate, and how astrocyte uptake of glutamate may be initiated.

As a model of injury, rat microglial cells were stimulated with LPS in vitro.  LPS is the component of Gram Negative Bacteria that causes endotoxic shock and is one of the most potent activators of cells of the myeloid lineage.  Specifically, LPS is known to induce production of many cytokines from macrophages and microglial cells including TNF-alpha, IL-12, and IL-18. 

Conditioned media from the LPS stimulated microglial culture was added to cultures of rat astrocytes and expression of the type 1 glutamate transporter was assessed.  The Type 1 glutamate transporter is an important protein found in glial and neurons that sucks up the glutamate from the synaptic cleft in order to prevent excitotoxicity.  The conditioned media, which represented numerous inflammatory stimuli was capable of upregulating expression of the type 1 glutamate transporter. 

Upregulation of astrocyte expression of the type 1 glutamate transporter could also be achieved by direct addition of TNF-alpha to the astrocytes.

Treatment of astrocytes with dibutyryl cAMP, which activates astrocytes, was also capable of upregulating expression of the type 1 glutamate transporter. 

These data suggest that astrocytes are an important protective mechanisms against glutamate toxicity and that astrocytes can actually “feel” when inflammation will occur and respond accordingly by upregulating glutamate transporters.