Johns Hopkins Researchers Discover Key Protein Linked To Transverse Myelitis: Il-6 Induces Regionally Selective Spinal Cord Injury 

Adam I. Kaplin, M.D., Ph.D., Deepa M. Deshpande, M.S., Erick Scott, B.S., Chitra Krishnan, M.S., Jessica S. Carmen, B.S., Irma Shats, M.S., Tara Martinez, B.S., Jennifer Drummond, B.S., Sonny Dike, M.D., Mikhail Pletnikov, M.D., Ph.D., Sanjay C. Keswani, M.B., Timothy H. Moran, Ph.D., Carlos A. Pardo, M.D., Peter A. Calabresi, M.D., and Douglas A. Kerr, M.D., Ph.D.

Johns Hopkins researchers from Project RESTORE (the TM Center and the MS Center) have discovered a single molecule that is a cause of transverse myelitis (TM), an autoimmune disease in the central nervous system.  TM is a focal inflammatory disorder of the spinal cord and exists on a spectrum of neuroinflammatory conditions characterized by abrupt neurologic deficits associated with inflammation, demyelination, and axonal damage. TM can exist as part of a multifocal central nervous system disease (e.g., Multiple Sclerosis), a multi-system disease (e.g., systemic lupus erythematosus), or as an isolated idiopathic entity.  Although the majority of TM patients suffer a single attack, 15 percent to 30 percent of patients go on to develop full-blown MS. TM evolves rapidly and without warning and usually results in permanent impairment, including weakness to paralysis of the legs and arms, bowel, bladder and sexual dysfunction, sensory dysfunction to pain, spasticity, fatigue and depression.  Our study focused on idiopathic TM patients, as defined according to the criteria developed by the TM Consortium Working Group and published in 2002.  (Transverse Myelitis Consortium Working Group. 2002. Proposed diagnostic criteria and nosology of acute transverse myelitis [review]. Neurology. 59:499). 

We investigated the diffusible immune derangements present in the spinal fluid of idiopathic TM patients and identified the role of the protein, IL-6, in the pathogenesis (cause) of this disease.   We also demonstrated that the levels of IL-6 are dramatically elevated in the spinal fluid of TM patients. The study is published in the October issue of The Journal of Clinical Investigation (J. Clin. Invest. doi:10.1172/JCI25141. http://www.jci.org).

The Johns Hopkins TM Center is the only center of excellence in the world dedicated to offering medical care and treatment to people who have TM and to research on TM.  We treat both pediatric and adult cases of TM.  The JHTMC also employs a multi-disciplinary approach to the treatment and study of TM.  By doing so, we have marshaled the resources and perspectives across medical specializations to better understand and treat this disease.  Finally, the TM Center has joined forces with the MS Center at Johns Hopkins to form Project RESTORE.  We are focused on identifying the relationships between the different neuroimmunologic diseases of the central nervous system.  There is much to learn by attempting to better understand the similarities and differences between these disorders.  Our focus on both research and clinical care and our comprehensive and multi-disciplinary approach to treatment and science offered us the most fertile environment for gaining the important insights that resulted from this study.

The JHTMC offers care to more TM patients than any other medical center in the world.  Through our experience in treating people with TM we regularly listened to people who described difficulties with concentration and memory.  Through our clinical care practice, we have also learned that depression is a common symptom of TM.  IL-6 has been implicated in mood and concentration disorders.  There is also indirect evidence that elevated IL-6 potentiates neural injury in Alzheimer disease, Parkinson disease, HIV encephalopathy, MS, depression, and cognitive impairment.  We, thus, became interested in whether IL-6 could be playing a role in the disease process of TM. 
 
IL-6 is a chemical messenger that cells of the immune system use to communicate with one another.  IL-6 is a glycoprotein cytokine (a protein secreted by cells of the lymph system that affects the activity of other cells and is important in controlling inflammatory responses). We hypothesized that cytokines play an important role in the pathogenesis (cause) of TM and examined the diffusible derangements within the cerebrospinal fluid (CSF) of a group of TM patients with a cytokine antibody array.

Our research began by analyzing 42 inflammatory proteins (cytokines) in the cerebrospinal fluid (CSF) of both TM and healthy patients.  The six TM patients in our study had not been started on immunomodulatory (steroid) therapy prior to CSF sampling.  There were eight patients in the control group. We found that IL-6 levels are selectively, consistently and dramatically elevated in the CSF of TM patients during the acute onset phase of the disease. The CSF IL-6 levels reported in our study are among the highest reported in any human disease (up to 4,209 pg/ml).  We also found that the levels of IL-6 among the TM population directly correlated with markers of tissue injury and the severity of paralysis.  Finally, and consistent with the indications of tissue injury, there was a direct correlation of IL-6 levels and sustained disability as measured by EDSS, expanded disability status scale at 6-month follow-up.  Functional status (acute and follow-up EDSS scores) were assessed by neurologists who were blinded to the immunologic assay results. The EDSS is a widely used neurological rating scale.  Our research demonstrated that the more severe the attack, the higher the amounts of IL-6, and the greater the chance of long-term and sustained clinical disability from acute neuronal injury.

We found a virtually identical pattern in the CSF from all the TM patients examined in this manner, that is, dramatic elevations in IL-6 levels. This uniformity in the pattern was surprising in that TM has widely been considered to be a heterogeneous disorder, and one may have expected the cytokine derangements to reflect this heterogeneity. However, it should be noted that recent nosologic strategies have attempted to categorize TM patients into various classifications, including monophasic vs. recurrent and idiopathic vs. those associated with systemic disease. For this study, we have limited the analysis to patients with idiopathic TM and have excluded those with identified systemic inflammatory disease. Therefore, this classification scheme may have resulted in a more uniform patient population with relatively homogenous immune system derangements.

The next phase of our research focused on identifying the specific immune process involved in a TM attack.  We were able to isolate the various proteins involved in this process, and the sequential activation of these proteins which ultimately results in spinal cord neural injury and cell death.  IL-6 was necessary and sufficient to mediate cellular injury in spinal cord tissue culture sections through activation of the cascading progression of the immune process. To our knowledge, this work provides evidence for the first time that a single signaling protein (IL-6) is the central mediator of tissue injury in an autoimmune CNS disease. We also describe the signaling pathway of IL-6 mediated spinal cord neural injury through the various steps of the inflammatory attack to the resulting cell death.

We found that the predominant source of IL-6 production was from astrocytes in and around the area of inflammation.  Astrocytes are a part of the central nervous system (brain and spinal cord).  Astrocytes have been shown to produce IL-6 in response to direct stimulation by proinflammatory cytokines, viral and bacterial pathogens, and neurotransmitters. What triggers the initial biosynthesis of IL-6 in astrocytes is currently being investigated, but potential candidates include an immune response following vaccination or an antecedent infection that could involve mechanisms such as molecular mimicry or superantigen-mediated inflammation. Why some individuals mount a dramatic elevation of their IL-6 levels that results in the pathophysiological injury seen in TM is still unknown, but the potential contribution of genetic differences to CNS IL-6 production has been previously described.

The primary targets of IL-6 mediated cytotoxicity (cell destruction) are oligodendrocytes and axons.  Oligodendrocytes help to produce the protective myelin sheath coating around nerve cells and axons. Thus, by our explaining a cause of both demyelination and axonal degeneration, our study offers one possible mechanism responsible for autoimmune demyelinating disorders, such as TM. Related disorders have also been found to have elevated IL-6 within the central nervous system. Acute disseminated encephalomyelitis, like TM, is a monophasic, inflammatory disorder of the central nervous system that is often post-infectious. Similarly, several reports have suggested that IL-6 is involved in the pathogenesis of MS.

There has been recent awareness about the dual role of IL-6 as both protective and injurious. In contrast to the view that IL-6 may be purely injurious to the nervous system, several studies have shown that IL-6 may be neuroprotective.  It is possible that in low doses, IL-6 serves as a beneficial neuroprotector and in high doses, can be destructive. IL-6 levels in adult CNS are usually low or undetectable under baseline conditions (a healthy individual) but increase dramatically in response to injury, inflammation, and CNS disease.

The next phase of our study was to conduct a number of experiments in order to verify what we had learned from our observations and analyses on the role of IL-6 in the inflammatory and demyelinating process which takes place during an acute TM attack.  We performed experiments to determine whether we could replicate our observations in vitro (experiments carried out in an artificial environment, such as cell cultures) and in vivo (experiments carried out inside a living organism, such as studies of rats or mice).  Through both the cell culture and animal studies, we confirmed that elevated IL-6 levels were directly injurious to the spinal cord.   We confirmed each of the sequential steps in the cascading inflammatory process, as well as the central role of IL-6 in both mediating the process and causing the ultimate damage to oligodendrocytes, and thus causing demyelination.  We were able to demonstrate that spinal fluid from TM patients induced death of spinal cord cells when cultured in a dish and that IL-6, when infused in adult rats, induced paralysis. Rats intrathecally infused with IL-6 developed progressive weakness and spinal cord inflammation, demyelination, and axonal damage.
Under the microscope, tissue from IL-6-infused rats showed demyelination and injury of axons, pathology that was nearly identical to that seen in human patients with TM.

We demonstrated that IL-6 is both necessary and sufficient to mediate the kind of spinal cord injury found in patients with TM. We provide evidence that the targets of this IL-6 -mediated injury are oligodendrocytes and axons, which result in demyelination and axonal injury.

To test whether IL-6 is simply correlated with or is causative of cellular injury in the spinal cord, we carried out studies using rat culture spinal cord sections. We added CSF from a TM patient (with IL-6 of 1,997 pg/ml) or a control patient onto spinal cord culture sections and evaluated cell death. We found that CSF from the TM patient induced death of spinal cord cells, while CSF from a control patient did not. We concluded that IL-6 was necessary for this death.

Transverse Myelitis involves an inflammatory attack in the spinal cord; there is no brain or optic nerve involvement.  We postulated that the reason IL-6 elevations injure only the spinal cord and not other regions of the nervous system was because distinct regions of the nervous system have different responses to IL-6.  The next step in our study set out to test this hypothesis.  We previously identified that oligodendrocytes and axons were preferentially susceptible to IL-6 - induced injury.  The target cells injured by IL-6 resulted in neural injury to spinal cord cultures. 

We next performed animal studies to corroborate our findings.  We found that when IL-6 was introduced into the cerebral ventricles (brain) of adult rats, the cascading inflammatory response was not activated as it had been in the spinal cord studies.  When IL-6 was introduced into the spinal cords of adult rates, over an eight-day period, their hind limb grip strength progressively weakened.  By the completion of the study, the IL-6 infused rats had lost nearly 50% of their baseline hind limb strength.  We performed analyses of the spinal nerve tissues and determined that there was demyelination and axonal degeneration, and that there was white matter disruption in the spinal cords, while the grey matter was largely spared.  This pathology in IL-6-infused rats was similar to the axonal degeneration and demyelination seen in the spinal cord of a patient with severe fatal TM.

We also performed experiments using cell cultures to test our hypothesis regarding the regional vulnerability of the spinal cord to IL-6 relative to the brain. Low doses of IL-6 appeared to be protective against cellular injury, while higher doses were only slightly injurious to brain tissues.  This contrasts dramatically with what we observed for spinal cord cultures.  Even small doses of IL-6 injured spinal cord tissues.  The cell culture experiments supported our finding that IL-6 is not universally injurious to the nervous system, but rather is selectively injurious to the spinal cord.

Previous studies have implicated IL-6 in preventing cell death as well as potentially playing a causative role in neurodegenerative diseases. The protective or destructive actions of IL-6 may result from selective dose and regional effects. We observed that IL-6 causes preferential cytotoxicity (cell injury and death) in white matter compared to gray matter in the spinal cord. We also found that low doses of IL-6 prevented cell death in cell cultures of sections from the brain, whereas higher doses had little effect on cell death. In contrast, no IL-6 dose tested in spinal cord sections was found to be protective, and higher doses were extremely cytotoxic.

Our study has led us to the conclusion that the regional inflammatory responses found among the various neuroimmunologic diseases of the central nervous system (MS, TM, neuromyelitis optica, and optic neuritis) may be explained by spatially restricted responses to cytokines, including IL-6.  Regional vulnerability of different parts of the central nervous system (brain, spinal cord and optic nerve) may be explained by spatially distinct responses to IL-6. These different types of responses might be a part of why different autoimmune disorders of the nervous system affect distinct regions and cause distinct symptoms.

We have found in this study that a single signaling molecule (IL-6) is a critical determinant of patient outcome in TM. The implications of these findings are that therapeutic strategies capable of modulating this pathway may improve outcomes in TM patients. Our work provides direct evidence for a signaling inflammatory cascade involving proteins that accounts for the damage to axons and oligodendrocytes, and thus demyelination in the spinal cord. Since spinal cord dysfunction is a major determinant of disability in several neurologic disorders including TM and MS, the description and explanation of this pathway identifies important therapeutic targets for preventing this disability in the future.

By understanding this pathway, we can work on identifying therapies with more refined targets that can be used to disrupt or stop the inflammatory attack before significant damage is done to the spinal cord.  These results reinforce the importance of early diagnosis and rapid therapeutic treatment of TM and the other neuroimmunologic disorders.  Our study suggests that the more quickly therapies can be administered to modulate the inflammatory response, the greater the possibility for a more positive outcome. 

TM is related to other autoimmune disorders of the nervous system, including Guillain-Barré syndrome, MS, neuromyelitis optica, optic neuritis, and acute disseminated encephalomyelitis. This study may give us a foothold in understanding all of these disorders; how they may be related to each other, and how they may be distinguished from each other. The benefits from our findings will not only be to those who are paralyzed by TM, but to those who have disabilities due to a variety of autoimmune disorders. We are actively using these findings to aid in developing future diagnostic, prognostic and therapeutic advancements.

Acknowledgments
This research was supported by The Transverse Myelitis Association, the Noel P. Rahn Fellowship, the Dana Foundation, the Miriam and Peter Haas Foundation, the Katie Sandler Fund for Research at Johns Hopkins University, Bruce Downey, and Barr Laboratories and the National Institutes of Health.