David O. Willenborg

Immunoglobulin-deficient rats fail to develop experimental allergic encephalomyelitis

Lewis rats were treated from day of birth with a rabbit anti-rat IgM antiserum. As adults these animals were found to have no detectable serum IgM and greatly reduced levels of IgG. They failed to respond to the B-cell mitogen LPS, or to make antibodies to sheep red blood cells (SRBC) or myelin basic protein (BP). These B-lymphocyte and immunoglobulin-deficient rats failed to develop clinical or histological evidence of EAE when sensitized with either whole spinal cord or purified BP. That some T-cell functions of these suppressed animals were not altered was seen by their ability to respond normally to PHA and to reject tissue allografts. The results would suggest that B-cell function (immunoglobulin-antibody production) is essential for the induction of EAE.

Cytokines and murine autoimmune encephalomyelitis: Inhibition or enhancement of disease with antibodies to select cytokines, or by delivery of exogenous cytokines using a recombinant vaccinia virus system

To examine the complex role of cytokines in the pathogenesis of actively induced murine EAE we measured the levels of a number of cytokines (IL‐6, IFN7 and TNF) in the spinal cord and CSF of mice with active experimental autoimmune encephalomyelitis (EAE) and found them all to be elevated. We next treated mice with antibodies to these three cytokines, which were over expressed in the CNS, to determine if they would alter disease and found the following: anti‐IL‐6 had no significant effect on disease, anti‐IFNγ exacerbated disease, and anti‐TNF either enhanced, had no effect or inhibited EAE depending on the antibody used. We then treated mice with exogenous cytokines, delivered using a recombinant vaccinia virus system, and found that the IL‐6 and TNF virus constructs inhibited EAE whereas the IFN1β construct had no effect on disease. Other cytokine recombinant viruses were also tested and it was found that the IL‐1β, IL‐2 and IL‐10 viruses inhibited EAE while an IL‐4 virus either had no effect or enhanced disease. We do not know the mechanism of action of the various cytokines in this system, but irrespective of the mechanism(s), this work clearly demonstrates that delivery of select cytokines using recombinant virus‐cytokine constructs can provide a powerful means of downregulating experimental organ‐specific autoimmune disease.

Renal Glomeruli and Tubular Injury Following Indomethacin, Ibuprofen, and Gentamicin Exposure in a Neonatal Rat Model

Indomethacin, ibuprofen, and gentamicin are commonly administered to neonates between 24 and 28 wk gestation when glomerulogenesis is still occurring. Indomethacin is known to cause renal failure in up to 25% of infants treated. Possible morphologic effects of these drugs are largely unknown. The purpose of this study was to determine the type of renal changes found on light (LM) and electron microscopy (EM) following administration of indomethacin, ibuprofen, and gentamicin in a neonatal rat model. Rat pups were exposed to indomethacin or ibuprofen and/or gentamicin antenatally for 5 d before birth or postnatally for 5 d from d 1 of life. Pups were killed at 14 d of age. LM examination in all indomethacin- and ibuprofen-treated pups both antenatally and postnatally showed vacuolization of the epithelial proximal tubules, interstitial edema, intratubular protein deposition but no significant glomerular changes. EM examination showed pleomorphic mitochondria and loss of microvilli in the tubules. The glomeruli showed extensive foot process effacement and irregularities of the glomerular basement membrane. EM changes were most marked in pups treated antenatally with ibuprofen, and indomethacin with gentamicin postnatally. Indomethacin, ibuprofen, and gentamicin cause significant change in glomerular and tubular structure in the neonatal rat model.

Evaluation of a radiolabelled peripheral benzodiazepine receptor ligand in the central nervous system inflammation of experimental autoimmune encephalomyelitis: a possible probe for imaging multiple sclerosis

PurposePeripheral benzodiazepine receptors (PBRs) are upregulated on macrophages and activated microglia, and radioligands for the PBRs can be used to detect in vivo neuroinflammatory changes in a variety of neurological insults, including multiple sclerosis. Substituted 2-phenyl imidazopyridine-3-acetamides with high affinity and selectivity for PBRs have been prepared that are suitable for radiolabelling with a number of positron emission tomography and single-photon emission computed tomography (SPECT) isotopes. In this investigation, the newly developed high-affinity PBR ligand 6-chloro-2-(4′-iodophenyl)-3-(N,N-diethyl)imidazo[1,2-a]pyridine-3-acetamide, or CLINDE, was radiolabelled with 123I and its biodistribution in the central nervous system (CNS) of rats with experimental autoimmune encephalomyelitis (EAE) evaluated.MethodsEAE was induced in male Lewis rats by injection of an emulsion of myelin basic protein and incomplete Freund’s adjuvant containing Mycobacterium butyricum. Biodistribution studies with 123I-CLINDE were undertaken on EAE rats exhibiting different clinical disease severity and compared with results in controls. Disease severity was confirmed by histopathology in the spinal cord of rats. The relationship between inflammatory lesions and PBR ligand binding was investigated using ex vivo autoradiography and immunohistochemistry on rats with various clinical scores.Results123I-CLINDE uptake was enhanced in the CNS of all rats exhibiting EAE when compared to controls. Binding reflected the ascending nature of EAE inflammation, with lumbar/sacral cord > thoracic cord > cervical cord > medulla. The amount of ligand binding also reflected the clinical severity of disease. Ex vivo autoradiography and immunohistochemistry revealed a good spatial correspondence between radioligand signal and foci of inflammation and in particular ED-1+ cells representing macrophages and microglia.ConclusionThese results demonstrate the ability of 123I-CLINDE to measure in vivo inflammatory events represented by increased density of PBRs and suggest that 123I-CLINDE warrants further investigation as a potential SPECT marker for imaging of CNS inflammation.

Nitric Oxide Contributes to Resistance of the Brown Norway Rat to Experimental Autoimmune Encephalomyelitis

The Brown Norway (BN) rat is reported to be resistant to the induction of experimental autoimmune encephalomyelitis (EAE) and a number of mechanisms have been suggested to explain this resistance. In work reported here we provide evidence that such resistance in the BN rat can be accounted for, at least in part, by their ability to produce higher levels of nitric oxide (NO) than susceptible strains of rats. Spleen cells from the BN rat make significantly more NO following in vitro stimulation than do cells from the Lewis or PVG rat and following in vivo immunization using complete Freunds adjuvant (CFA) the BN rat makes substantially more NO than either susceptible strain. If carbonyl iron is used as adjuvant in vivo there is no increase in NO levels in the BN rat and they are rendered highly susceptible to EAE. Immunizing with CFA simultaneously with neuroantigen and carbonyl iron drives up NO levels and the resistance is restored. EAE produced using carbonyl iron is characterized by extensive macrophage/microglia presence in the central nervous system lesions of the BN rat yet the cytokine profile in the lymph nodes does not differ from that in the EAE Lewis rats.

Phosphosugars are potent inhibitors of central nervous system inflammation.

Adoptively transferred allergic encephalomyelitis can be inhibited by various phosphosugars, particularly mannose‐6‐phosphate. The sugar specificity suggests that inhibition may be due to depletion of lymphocyte cell‐surface lysosomal enzymes, which are essential for the passage of lymphocytes across the vascular endothelium and the entry of lymphocytes into the central nervous system parenchyma.—Willenborg, D. O.; Parish, C. R.; Cowden, W. B. Phosphosugars are potent inhibitors of central nervous system inflammation. FASEB J. 3: 1968‐1971; 1989.

Inhibition of allergic encephalomyelitis by the iron chelating agent desferrioxamine: differential effect depending on type of sensitizing encephalitogen

Induction of experimental allergic encephalomyelitis (EAE) in Lewis rats by injection of guinea pig (GP) spinal cord homogenate (SCH) plus adjuvant (SCH-CFA) can be inhibited by treatment with the iron chelating agent desferrioxamine (DFOM). Interestingly, induction of EAE with purified myelin basic protein (BP-CFA) is not inhibited with DFOM. This dichotomy does not appear to be due to any quantitative differences in the two inocula since minimal clinical EAE produced by threshold levels of BP is not inhibited with DFOM. Passive EAE is not inhibited irrespective of the type of encephalitogen used to sensitize the donors. This suggests that the inhibitory effect of DFOM is acting on the afferent limb of the immune response to SCH-CFA. Injection of BP-CFA and SCH-CFA into the same site, mixing BP with central nervous system (CNS) lipids, or incorporating BP into liposomes, all induce EAE which can be partially inhibited by treatment with DFOM. These results support the hypothesis that the close association of lipids with the encephalitogen (i.e. BP) in SCH required extensive lipid breakdown before adequate antigen presentation can occur, and it is at this level that DFOM exerts its inhibitory effect.

Indomethacin, ibuprofen and gentamicin administered during late stages of glomerulogenesis do not reduce glomerular number at 14 days of age in the neonatal rat

Premature neonates are frequently administered indomethacin, ibuprofen and gentamicin during the period of active glomerulogenesis. These drugs are known to have nephrotoxic effects, but the morphological effect of these drugs is unknown. The purpose of this study was to determine whether administration of these drugs during the late stages of glomerulogenesis in the rat has an effect on glomerular endowment. Rat pups were given, intraperitoneally, indomethacin, ibuprofen or indomethacin and gentamicin for the first 5xa0days of their postnatal life. The pups were killed at 14xa0days of age at completion of glomerulogenesis. The total number of glomeruli in the left kidney was determined by the physical disector/fractionator stereological technique. There was no difference between treatment groups in total number of glomeruli per kidney (Pu2009=u20090.45). There were significantly fewer glomeruli per gram of kidney in those rat pups that had received indomethacin or ibuprofen (Pu2009<u20090.0001). The reduction in the number of glomeruli per gram of kidney may indicate augmented growth of nephron tubules and/or collecting ducts, and/or be a consequence of oedema secondary to drug exposure. Further study is required to determine whether reduced glomerular number is seen in older animals or following exposure to these drugs at different time-points in kidney development.

Inhibition of experimental allergic encephalomyelitis by the α-glucosidase inhibitor castanospermine

The alkaloid castanospermine is a potent inhibitor of oligosaccharide processing in vitro. Our recent findings indicating the importance of carbohydrate moieties in some critical step of the neuro-immunologic inflammatory process of allergic encephalomyelitis prompted us to investigate the effect of castanospermine on this disease process. The alkaloid inhibited passively induced allergic encephalomyelitis in a dose-dependent manner when administered continuously for 7 days beginning at the time of lymphocyte transfer. Although clinical disease was totally inhibited, treated animals did have inflammatory lesions in the central nervous system. These lesions were qualitatively different from those seen in untreated animals in that the inflammatory cells were tightly packed around the vessels and showed little migration into surrounding tissues. Castanospermine also effectively inhibited clinical disease in recipient animals which had had a previous episode of allergic encephalomyelitis. Castanospermine did not alter the disease when treatment was started after the onset of clinical symptoms.

Total Body Irradiation Allows Reinduction of Allergic Encephalomyelitis in Convalescent Lewis Rats

The ability of total body irradiation to alter recovery from and development of acquired resistance to allergic encephalomyelitis has been studied in Lewis rats. Irradiation before sensitization inhibits disease production, whereas irradiation after sensitization has no effect. In the latter situation animals recover from disease in a normal fashion. The generation of resistance to reinduction of disease is not affected by irradiation given either before or after the primary sensitization. However, irradiation given after both the primary and secondary challenges allows the development of a secondary disease episode. Studies on antibodies against basic protein in such treated animals suggest that antibodies play a role in resistance to reinduction of disease.