The effects of recombinant human alpha tumor necrosis factor (alpha-TNF) were compared with those of cytotoxic serum from patients with the acute Guillain-Barr6 syndrome (GBS) in myelinated cultures containing only rat Schwann cells and dorsal root ganglion neurons. Alpha-TNF did not damage rat peripheral nervous system tissue in culture. These observations suggest that alpha-TNF is not responsible for the cytotoxic activity of acute GBS serum in culture.
The acute Guillain-Barr6 syndrome (GBS) is a demyelinating peripheral neuropathy of humans 2. Although the GBS is considered to be mediated by immunological factors, exact pathogenic mechanisms are not known 2. The observation that severely affected GBS patients usually lose a significant percentage of body weight, in addition to suffering peripheral nerve damage, is also unexplained 2
Tumor necrosis factor, a cytokine secreted by activated monocytes and macrophages, is responsible for weight loss in many patients with malignancies or chronic infections 3 The observation that recombinant human alpha tumor necrosis factor (alpha-TNF) caused lysis of central nervous system (CNS) myelin and oligodendroglia in explant cultures of mouse spinal cord led Selmaj and Raine a4 to postulate a role for alpha-TNF in the CNS myelin damage which occurs in patients with multiple sclerosis. It is therefore conceivable that TNF might be responsible for both the peripheral nervous system (PNS) myelin damage and weight loss which occur in acute GBS patients.
Serum from approximately 40% of patients with the acute GBS produced lysis of PNS myelin and myelin-related Schwann cells (MRSCs) in culture 4. The factors responsible for the cytotoxicity of GBS serum are not known. The ionophore A23187 and acute GBS
* Present address: Department of Biology, Penn State University, McKeesport, PA 15132, U.S.A. Correspondence: Dr. Francis A. Mithen, Department of Neurology, St. Louis University School of Medicine, 3635 Vista Avenue at Grand, P.O. Box 15250, St. Louis, MO 63110-0250, U.S.A.
serum produce virtually identical ultrastructural damage to PNS myelin and MRSCs This observation 9 suggests that the cytotoxic effects of GBS serum in culture might be calcium-mediated. The purpose of the current study was to determine if recombinant human alpha-TNF could duplicate the cytotoxic effects of GBS serum in culture.
Cultures containing only dissociated rat sensory neurons and Schwann cells were prepared 8-10. The thoracic, lumbar and sacral dorsal root ganglia were removed from Sprague-Dawley rat embryos at 19-21 d of gestation. The ganglia were rinsed with calcium- and magnesium-free Hanks' balanced salt solution (CMFHBSS, GIBCO) and then incubated for 90 min at 34.5°C in a 0.25% (w/v) solution of 3X-recrystallized trypsin (Worthington) in CMFHBSS. The ganglia were mechanically dissociated by gentle trituration through a narrow bore pipette (opening diameter approx. 1 mm).
The dissociated ganglia were plated onto collagen-coated, Aclar plastic (Allied-Signal) 'minidishes' (2.5 cm diameter) at a density of two dissociated ganglia per dish. The cultures were incubated in nutrient medium for 18-22 h at 34.5°C in a humidified, 5% CO2-in-air atmosphere to allow cells to adhere to the collagen substrate. The cultures were briefly rinsed with Earle's balanced salt solution at room temperature to remove non-ad- herent cells and cellular debris.
The dissociated ganglia were incubated for 7 d in nutrient medium containing 5'-fluorodeoyuridine (5'-FU, Sigma) at a concentration of 10-5 M at 34.5°C in a humidified, 7.5% CO2-in-air atmosphere to kill rapidly dividing, non-neuronal cells. Although a substantial proportion o f Schwann cells are also killed during exposure to 5 '-FU, enough Schwann cells usually survive to repopulate the cultures upon return to serum-containing medium.
After 7 d of incubation in medium containing 5 '-FU, cultures were maintained at 34.5 °C in a humidified, 7.5% CO2-in-air atmosphere in nutrient medium containing 73% Eagle's MEM with Earle's salts and L-glutamine (GIBCO), 15% human placental cord serum, 5% chicken embryo extract, glucose at an added concentration of 324 mg/d l and nerve growth factor prepared from male mouse submaxillary glands as described 10
Cultures containing fibroblasts a n d / o r mononuclear phagocytes are identified with an inverted microscope and discarded. Schwann cells start to proliferate within 2-3 weeks of incubation in nutrient medium ~0. Peripheral myelin sheaths start to develop within 3-4 weeks of incubation 10 and continue to develop for several months 9
Test groups of 4 -6 well-myelinated culture specimens at least 6 months of age were each maintained in nutrient media containing: (A) 10000 uni t s /ml of recombinant human alpha-TNF which was certified to be cytotoxic for L-929 cells (Genzyme Corp.) or (B) 50% cytotoxic GBS serum 4. Trials were conducted with two different lots of alpha-TNF. The cultures were maintained at 34.5°C in a humidified, 5% CO2-in-air atmosphere for up to 8 d. Culture medium containing alpha-TNF was changed every 2-3 days. Representative specimens were fixed and processed for detailed examination by both light and electron microscopy 4.8-10
Alpha-TNF did not damage myelin, Schwann cells, or dorsal root ganglion neurons, even after 8 d of incubation. As previously reported 4, cytotoxic GBS serum caused lysis of myelin and MRSCs within several hours of incubation, but spared neurons and non-myelin-related Schwann cells.
The failure of human alpha-TNF to damage PNS tissue in culture does not, of course exclude a pathogenic role for alpha-TNF in the acute GBS. Since Robbins et al. 12 noted that recombinant human "I2",IF was cytotoxic for rat oligodendroglia in culture, it is unlikely that our results can be explained simply on the basis that human T N F cannot interact with rat tissue in culture. Although Selmaj and Raine 14 noted definite CNS
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myelin breakdown at alpha-TNF concentrations as low as 500 units/ml, PNS myelin might be more resistant to the cytotoxic effects of alpha-TNF. The extent of CNS myelin damage reported by Selmaj and Raine 14 correlated approximately with the added concentration of alpha-TNF. It seems unlikely therefore, that concentrations of alpha-TNF less than 10000 units/ml would be cytotoxic for PNS tissue in culture, if a concentration of 10000 units/ml was not, but this possibility was not investigated. Although it is conceivable that TNF might interact with other components of GBS serum to produce cytotoxic effects in culture, we did not explore this possibility by adding anti-TNF antibodies to culture media during cytotoxicity testing or pretreating cytotoxic GBS serum to remove whatever TNF might be present.
Franciotta et al. 6 reported that alpha-TNF concentrations were slightly elevated (0.2-0.8 units/ml) in the cerebrospinal fluid (CSF) of two acute GBS patients, but, unfortunately, did not report the individual serum concentrations for these patients. The concentrations of alpha°TNF used in the current study greatly exceeded the vast majority of serum levels measured in humans 6,7,1335. Concentrations of alpha-TNF higher than those used in the current study might be produced intraneurally in acute GBS patients by the activated macrophages which have been noted to invade peripheral nerves in patients with this disorder 11
A
Fig. 1. Bright-field photomicrographs from whole-mount, Sudan-Black-stained cultures which were exposed to: (A) recombinant human a lpha-TNF at a concentration of 10 000 u n i t s / m l for 8 d or (B) cytotoxic GBS serum at a 50% concentration for 10 h. The darkly stained myelin sheaths (arrows) in A are normal in appearance, while
the myelin segments (arrowheads) in B are fragmented. Calibration bar = 20 t tm in each micrograph.
Alternatively, alpha-TNF might not be responsible for peripheral nerve damage in humans. The observation that activated monocytes from patients with the acquired immunodeficiency syndrome (AIDS) and AIDS-related complex are deficient in TNF production 1, despite a higher than expected incidence of GBS among these individuals 5, supports the suggestion that TNF might not cause PNS dysfunction in humans. More- over, serum and CSF concentrations of alpha-TNF were significantly elevated (5-100 units/ml) in 3 of 5 patients with rheumatoid arthritis who had no reported PNS dysfunction 6. Although the role, if any, of TNF in the acute GBS will have to be defined in additional clinical studies, it seems unlikely that alpha-TNF is responsible for the observed cytotoxic activity of GBS serum in culture.
ACKNOWLEDGEMENTS
This work was supported by the Department of Veterans Affairs. We thank Mr. Jesse Urhahn and Mr. Byron Madkins for excellent technical assistance and Mrs. Mary Althage for expert secretarial help.
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