The effect of sea star coelomocyte extract on cell-mediated resistance to Listeria monocytogenes in mice.

Mice treated with sea star factor (SSF), a protein extracted from sea star coelomocytes, became highly susceptible to infection with a normally sublethal dose of Listeria monocytogenes. This was in contrast to the expected result of increased resistance originally postulated because of the macrophage-activating properties of SSF. Enhanced susceptibility was seen when SSF was given from 96 h before to 48 h after infection with Listeria. Mice pretreated with SSF failed to develop immunity to Listeria when given a dose of organisms capable of immunizing nontreated mice. Treatment of immune mice with SSF did not alter their immune status. In addition, incubation of immune lymphocytes with SSF in vitro did not alter their ability to adoptively transfer immunity to normal recipients. Immune lymphocytes treated with SSF and then incubated with anti-SSF and C did, however, lose the ability to transfer immunity. These results suggest that SSF enhances infection by binding to T lymphocytes, inhibiting their replication upon contact with Listeria antigen and thus preventing the generation of a population of sensitized lymphocytes capable of effecting anti-Listeria immunity.

Elevated secretion of reactive nitrogen and oxygen intermediates by inflammatory leukocytes in hyperacute experimental autoimmune encephalomyelitis: enhancement by the soluble products of encephalitogenic T cells.

Perivascular lesions within the central nervous system (CNS) of rats with hyperacute experimental autoimmune encephalomyelitis (HEAE) contained large numbers of peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN), cells enzymatically capable of producing reactive nitrogen and oxygen intermediates (RNI and ROI), which, in excess, are mediators of tissue damage. PBMC and PMN isolated from the CNS and periphery of HEAE-affected rats secreted significantly (p less than 0.01-0.0001) elevated levels of ROI and RNI compared with that of similar cell populations from pertussis- and saline-treated control animals. Coincubation of systemically derived PBMC and PMN with antigen-stimulated myelin basic protein-specific T cell lines led to further increases in ROI and RNI output of between 15.3 and 83.1%, an effect that could be largely attributed to heat-labile, soluble products released by these T cell lines. Our studies suggest a putative neuropathological role for ROI and RNI in HEAE, which may be mediated via cytokines emanating from autoreactive T lymphocytes.

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.

Immunoglobulin deficient rats as donors and recipients of effector cells of allergic encephalomyelitis.

Lewis rats, treated from birth with a rabbit anti-rat IgM antiserum are B-cell and immunoglobulin deficient. When sensitized with myelin basic protein (BP) and complete Freund's adjuvant (CFA), these rats do not make detectable antibodies to BP, nor do they develop clinical or histopathological evidence of allergic encephalomyelitis (EAE). We show here that transfer of anti-BP antibody containing serum to BP sensitized Ig deficient rats results in subsequent development of EAE. We also demonstrate that BP sensitized Ig deficient rats which do not develop EAE, nevertheless generate effector cells capable of transferring disease, and thus specific T-cell function is not inhibited by the anti-Ig treatment. Finally, Ig deficient rats were shown to be perfectly adequate recipients of passively induced EAE.

ICAM-1-dependent pathway is not critically involved in the inflammatory process of autoimmune encephalomyelitis or in cytokine-induced inflammation of the central nervous system.

To determine whether the rat homolog of intercellular adhesion molecule 1 (ICAM-1) plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE) we examined the effect of anti-ICAM-1 mAb, 1A29, on both active and passive EAE. We also examined its effect on a model of cytokine-induced inflammation in the central nervous system. Treatment of recipients of EAE effector cells with anti-ICAM-1 had no inhibitory activity, and in fact at high doses, treatment enhanced disease as evidenced by an earlier onset of symptoms. Treatment of active EAE with anti-ICAM-1 beginning on the day of sensitization did protect a proportion of animals from development of disease as well as reduce the severity of clinical signs in those which developed symptoms. Lymphocytes from both the draining lymph nodes and spleens of myelin basic protein (MBP)-immunized rats treated with anti-ICAM-1 failed to proliferate in response to MBP in vitro, suggesting that the antibody had prevented the animals from becoming sensitized to the antigen. Microinjection of tumor necrosis factor (TNF)-alpha into the spinal cords of rats led to the expression of ICAM-1 on vascular endothelium, and to the accumulation of leukocytes at sites of injection. The peak expression of ICAM-1 by endothelium and the peak accumulation of leukocytes following TNF alpha injection were not positively correlated. Furthermore, treatment of TNF alpha injected rats with anti-ICAM-1 did not inhibit the accumulation of leukocytes at the site of cytokine injection.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Demyelination and early remyelination in experimental allergic encephalomyelitis passively transferred with myelin basic protein-sensitized lymphocytes in the Lewis rat.

Histological studies were performed on Lewis rats with experimental allergic encephalomyelitis (EAE) passively transferred by myelin basic protein (MBP)-sensitized syngeneic spleen cells in order to determine the relationship between demyelination and neurological signs. Neither inflammation nor demyelination was present on the day prior to the onset of neurological signs but both were present in the spinal roots and spinal cord on the day of onset of tail weakness (4 days after passive transfer). Demyelination and the neurological signs both increased over the next 48 h. There was evidence that the caudal roots were more severely affected than the rostral roots. The peripheral nerves were spared. Demyelination in the spinal cord was concentrated in the dorsal root entry and ventral root exit zones. The initial stages of repair of demyelinated spinal root fibres by Schwann cells were observed on the earliest day that clinical recovery commenced (day 7). At this time some demyelinated fibres were closely associated with debris-free Schwann cells, and occasional fibres were completely invested by 1-2 layers of Schwann cell cytoplasm. Remyelination (compact myelin lamellae formation) by Schwann cells was first observed in the spinal roots on day 9. By the time of complete clinical recovery (day 11) the majority of affected spinal root cores had thin new myelin sheaths. Repair of central nervous system myelin by oligodendrocytes was slower than peripheral nervous system myelin repair. Investment of demyelinated spinal cord axons by oligodendrocytes was observed on day 9, and remyelination by these cells was seen on day 10. We conclude that the neurological signs of passively induced MBP-EAE can be accounted for by demyelination of the lumbar, sacral and coccygeal spinal roots and spinal cord root entry and exit zones, and that the subsequent clinical recovery can be explained by investment and remyelination of demyelinated peripheral and central nervous system fibres by Schwann cells and oligodendrocytes respectively.

Suppression of hyperacute and passively transferred experimental autoimmune encephalomyelitis by the anti-oxidant, butylated hydroxyanisole.

Butylated hydroxyanisole (BHA) was used to treat hyperacute, ordinary passive, and hyperacute passive experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. The anti-oxidant, delivered via mini-osmotic pumps, reduced the incidence, severity and mortality in hyperacute EAE and also reduced the incidence, severity and duration of disease in passively induced EAE and hyperacute passive EAE. In all cases, cellular infiltration by both mononuclear and polymorphonuclear leukocytes were significantly reduced in treated rats. BHA appears therefore to act at the effector stage of EAE, reducing cellular infiltration in the brain and spinal cord and minimising clinical signs without blocking sensitisation or activation. This was supported by the finding that spleen cells from BHA-treated donors immunised for hyperacute EAE transferred disease at least as well as cells recovered from untreated donors.

Selective localisation of neuro-specific T lymphocytes in the central nervous system.

Using experimental autoimmune encephalomyelitis (EAE) in the rat as a model of central nervous system (CNS) inflammation, activated and quiescent T lymphocytes with different antigen specificities were labelled with the fluorescent dye Hoechst 33342 and tested by fluorescence microscopy for their ability to accumulate in different regions of the spinal cord and in other organs at varying times post inoculation. With this highly sensitive assay it was found that activated myelin basic protein (MBP)-specific T cell lines accumulated in the spinal cord (a 1000-fold increase in the lumbar/sacral region by day 4) and caused clinical signs of EAE. In contrast, interleukin-2 (IL-2)-maintained (quiescent) MBP-specific T cell lines failed to accumulate in the CNS and cause disease. Activated ovalbumin (OA)-specific and purified protein derivative of tuberculin (PPD)-specific T cell lines were also found at significantly higher levels in the spinal cord than non-activated cells although they failed to accumulate to a substantial degree when injected alone. When injected with activated MBP-specific T cells the activated OA- and PPD-specific cell lines accumulated in the spinal cord following initial accumulation of the MBP-specific cells, demonstrating that during the inflammatory process there is considerable non-specific recruitment of cells into the inflammatory site. CNS accumulation of activated MBP-specific T cell lines occurred 1-2 days later in irradiated animals than in non-irradiated recipients. This was consistent with irradiated animals also exhibiting a later onset of disease and suggests that irradiation may directly affect the endothelium in a way that makes it less adhesive. In conclusion, this study demonstrates that activated lymphocytes of any specificity enter the spinal cord, and that the neuro-antigen specific cells accumulate there and lead to the recruitment of other cells. Non-activated cells, even those with neural antigen specificity fail to enter the cord. Understanding the nature of what an 'activated' lymphocyte is may allow us to design strategies to inhibit such immune-mediated inflammation.

Replication of donor lymphocytes in recipients is not essential for the passive transfer of allergic encephalomyelitis.

Gliotoxin is a fungal metabolite belonging to the class of epipolythiodioxopiperazines which possesses both immunomodulating and anti-phagocytic activities. We have examined the effect of gliotoxin on passively induced allergic encephalomyelitis and report here that pulse treating activated experimental allergic encephalomyelitis (EAE) effector lymphocytes with gliotoxin inhibits, in a dose-dependent manner, their ability to transfer disease. Cells treated with 300 ng/ml are unable to replicate in vitro in response to concanavalin A stimulation, nor did they produce interleukin-2 (IL-2) following stimulation. Furthermore this concentration of gliotoxin also causes complete fragmentation of genomic DNA in treated cells, yet these cells are still capable of transferring clinical EAE. These data suggest that replication of donor lymphocytes in the recipient is not essential for the development of EAE.

Our shifting understanding of the role of nitric oxide in autoimmune encephalomyelitis: a review.

Nitric oxide was first described being produced in inflammatory cells involved in experimental autoimmune encephalomyelitis in 1992. Since then some 45 papers have appeared examining the role of NO in this central nervous system autoimmune inflammatory disease. Of the first 10 papers published all resulted in the interpretation that NO was a pathologic or "bad" molecule in the context of EAE. A few papers then began to appear suggesting that NO may not in fact always be a harmful molecule and by the end of 1997 early 1998, 22 papers suggested a destructive role for the molecule while three suggested it was protective. The past two years have seen a significant increase in reports supporting a protective mechanism for NO in EAE such that as of July 1999, 27 papers suggest a destructive and 15 a protective role for NO with a few uncommitted. This review sets out in a more or less chronological order the studies examining the role of NO in EAE and maps our changing understanding of the molecules role in this CNS inflammatory disease and by inference perhaps multiple sclerosis.

Astrocytic hypertrophy: an important pathological feature of chronic experimental autoimmune encephalitis in aged rats.

Experimental autoimmune encephalomyelitis (EAE) was induced in young (2-3 month old), middle-aged (12-13 month old) and geriatric (24-26 month old) Lewis (JC) rats by active immunisation with myelin basic protein (MBP) in complete Freund's adjuvant (CFA). It was found that aged Lewis (JC) rats developed a more chronic form of EAE than younger rats of the same strain, a phenomenon observed in both male and female rats despite males developing more severe disease than females at all ages. Middle-aged recipients also developed more severe disease than young recipients when EAE was induced by the adoptive transfer of lymphocytes from actively immunised young donors, suggesting that disease chronicity in middle-aged animals is a property of the central nervous system (CNS) milieu. Histological studies demonstrated that disease chronicity did not correlate with the number of inflammatory lesions in the CNS, young animals containing substantial numbers of CNS lesions following recovery and lesions being largely absent from middle-aged animals which still exhibited signs of disease. No significant differences were found in the degree of fibrin deposition or demyelination between young and middle-aged or symptomatic and asymptomatic animals. However, astrocytic hypertrophy was found to correlate with manifestation of disease in both young and middle-aged animals and in particular with disease chronicity in middle-aged animals. In parallel studies, no significant differences were found in the levels of the inflammatory mediators tumor necrosis factor (TNF)-alpha, prostaglandin E (PGE)2, reactive nitrogen intermediates (RNI) and corticosterone in young and middle-aged animals. However, markedly elevated corticosterone levels were found in both young and middle-aged animals with the development of clinical signs which returned to baseline levels with the resolution of clinical signs. Elevated levels of RNI were evident in animals immediately prior to and during the early stages of symptomatic EAE. Although these results suggest that nitric oxide may play a role in the pathogenesis of disease, whereas corticosterone may play a role in the immunoregulation of the disease, these factors cannot explain differences in disease chronicity evident in middle-aged animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitric oxide is a potential down-regulating molecule in autoimmune disease: inhibition of nitric oxide production renders PVG rats highly susceptible to EAE.

Rat strains vary in their susceptibility to experimental autoimmune encephalomyelitis (EAE) and in many cases, factors other than MHC antigens are thought to play a role in this. We found that PVG rats, which have a very low susceptibility to EAE, were rendered highly susceptible to clinical disease when treated with N-methylarginine (NMA) an inhibitor of nitric oxide synthase (NOS). The clinical course of the ensuing disease in NMA-treated PVG rats was in most cases fulminating in nature and accompanied by some mortality. Following immunisation with myelin basic protein (MBP)-complete Freund's adjuvant (CFA), PVG rats developed higher serum levels of the surrogate markers of nitric oxide production, reactive nitrogen intermediates (RNI; nitrite and nitrate), than did their Lewis counterparts. This in vivo finding was reflected in vitro, where the levels of RNI produced in 24, 48 and 72 h IFN-gamma-stimulated spleen cell cultures for PVG rats were significantly higher than those for Lewis rats. A mechanism by which increased NO production might protect PVG rats against clinical EAE was suggested by the finding that lymph node cells, isolated from NMA-treated MBP-immunised PVG rats, proliferated in response to MBP at a rate approximately 3 x greater than those from MBP-immunised, saline treated rats. Thus, the greater number of MBP-specific T cells generated in the NOS inhibitor-treated vs. untreated rats could account for their increased susceptibility to developing clinical EAE. The findings in this study suggest that NO plays a role in protecting PVG rats against developing EAE.

Lymphocytes utilise sialylated surface molecules to accumulate in developing lesions of autoimmune encephalomyelitis.

The accumulation of desialylated radiolabelled normal spleen cells and non-neuroantigen specific CD4 T-lymphocytes was measured in the lumbosacral spinal cord of Lewis rats with autoimmune encephalomyelitis (EAE) induced with myelin basic protein in Freund's adjuvant. The labelled cells were preincubated with sialidase and thoroughly washed prior to intravenous injection into rats exhibiting early clinical signs of EAE. Four hours later, the rats were killed and blood and spinal cord samples were radioassayed. Compared with untreated cells, desialylation markedly reduced the accumulation of both normal spleen cells and memory T-lymphocytes in the spinal cord, despite similar levels of cells being present in the blood. In another experiment, the accumulation of desialylated, macrophage-depleted spleen lymphocytes was measured during the onset, recovery and short-term "relapse" phases of acute EAE. Again, compared with controls the accumulation of desialylated lymphocytes was always significantly less, despite similar numbers of cells in the circulation. Lastly, intravenous injections of sialidase produced delayed onset of both clinical and histological signs in rats with passively-transferred EAE. These data confirm and extend previous findings, using a different animal model, that sialyl residues on the lymphocyte surface are important to the accumulation of such cells at inflammatory sites in the central nervous system. The possible relevance of these findings to human demyelinating disease is discussed.

Castanospermine, an oligosaccharide processing inhibitor, reduces membrane expression of adhesion molecules and prolongs heart allograft survival in rats.

The inhibition of intracellular oligosaccharide processing is a new approach to immunosuppression in allotransplantation. The net effect of such inhibition is reduction in the membrane expression of certain glycoproteins. Hence cell-cell interaction in allorejection may be impaired in the presence of glycoprotein processing inhibitors because the expression of key ligand-receptor pairs of N-linked glycoproteins including adhesion molecules is inhibited. The aims of this study were to measure the immunosuppressive ability of castanospermine (CAST) in a rat heart allograft model, to measure its effect on membrane expression of adhesion molecules (LFA-1 alpha, LFA-1 beta, ICAM-1), class I and class II MHC antigens and on other T cell associated molecules (CD4, CD8, CD39, CD45, W3/13), to test its tolerogenic potential and its toxicity. Membrane expression of these molecules was measured by flow cytometry for single cells and by immunoperoxidase staining for the allograft. In grafted rats CAST significantly reduced the expression of LFA-1 alpha on lymphoid cells in the thymus, lymph node, spleen and heart allografts. ICAM-1 expression on endothelial cells of the allograft vasculature, class I and class II MHC expression on lymphoid cells in the thymus, class II MHC expression on lymphoid cells in the allograft; and CD4, CD8, CD45 and W3/13 expression on lymphoid cells in some organs. By contrast, in non-grafted rats CAST significantly upregulated expression of class I MHC and CD45 in the thymus, lymph node and spleen, ICAM-1 and CD4 on lymphoid cells in the spleen, but reduced expression of LFA-1 alpha on lymphoid cells in the thymus. It also prolonged rat heart allograft survival in a dose-dependent manner and with limited testing was relatively non-toxic. In conclusion, CAST is an immunosuppressive molecule which may work by downregulation of the ligand-receptor adhesion molecule pair, LFA-1 alpha-ICAM-1 although subtle downregulation of class I and II MHC, CD4 and CD8 molecules could also contribute to its immunosuppressive activity. Hence, both lymphocyte-endothelial cell binding and lymphocyte activation may be inhibited by CAST. This work suggests that CAST may hold significant potential as a transplant immunosuppressant probably as an adjuvant agent to inhibitors of interleukin 2 secretion.

Protection against allergic encephalomyelitis by recruitment of myelin basic protein reactive lymphocytes into a single lymph node.

The efferent popliteal lymphatic of sheep was cannulated and sheep myelin basic protein (BP) was repeatedly injected into the drainage site of that popliteal node. All the recirculating lymphocytes leaving the node were removed from the sheep via the efferent cannula. Sheep undergoing such chronic stimulation and drainage failed to develop allergic encephalomyelitis when challenged with an encephalitogenic inoculum in the contralateral leg. Chronic stimulation without subsequent removal of lymphocytes did not protect animals against challenge. These results suggest that protection is due to the stimulation, recruitment and removal of specific BP reactive cells from the recirculating lymphocyte pool. Possible therapeutic applications in multiple sclerosis are discussed.

Direct injection of cytokines into the spinal cord causes autoimmune encephalomyelitis-like inflammation.

A single micro-injection of Tumour Necrosis Factor alpha (TNF) or gamma Interferon (IFN-gamma) into the lumbosacral spinal cord of the rat produced meningitis and mononuclear cuffs within the cord, an inflammatory response remarkably similar in pattern to that observed during experimental autoimmune encephalomyelitis (EAE), a research analog of multiple sclerosis. Rats injected with saline or heat-inactivated cytokine exhibited no such inflammatory response. In other experiments, the accumulation of radiolabeled spleen cells into spinal cord was measured after the injection of various doses of TNF and IFN-gamma, results indicated that both cytokines elicited accumulation of spleen cells in an additive but not synergistic manner. Potentially, the direct injection model offers a new and simplified way of examining mechanisms of early inflammation in the central nervous system, without the need for immunisation with neuroantigen or passive transfer of sensitised cells.

Inhibition of experimental allergic encephalomyelitis by the alpha-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.

Successful isolation, cultivation and partial characterization of naturally occurring ovine squamous cell carcinoma.

Seventeen epithelial cell lines have been successfully established from naturally occurring ovine squamous cell carcinomata. Culture establishment was most successful when tumor tissue was directly explanted rather than treated enzymatically. Success in establishing cultures also appeared to be related to the site on the body from which the tumor biopsy was taken, with tumors derived from the nose being most readily cultured. Several of the cell lines were successfully transplanted to nude mice where the growth patterns observed in the original host, i.e. expansive or invasive, were maintained. All cell lines assumed one of two distinct morphological types; however, no association could be seen between morphology and pattern of in vivo growth.

Immune reactivity to autochthonous ovine squamous cell carcinomata.

The immune reactivity of tumor-bearing sheep to autochthonous tumor cells was investigated in the efferent lymph of a lymph node distant from the primary tumor site. This was done while the primary tumor was in situ and after its resection. The immune response to challenge with tumor cells while the primary tumor was in situ was associated with the detection of antibodies which were specific for the tumor cells but did not cause their demise. However, both humoral and cellular cytotoxic reactivities were detected in the lymph following removal of the primary tumor and rechallenge with tumor cells. This response had the characteristic of a secondary immune response which indicates sensitization to tumor antigens by the host. Thus the presence of the primary tumor has interfered with pre-existing host immunity.