The protease inhibitor, Bowman-Birk Inhibitor, suppresses experimental autoimmune encephalomyelitis: a potential oral therapy for multiple sclerosis.

Available treatments for multiple sclerosis (MS) require frequent injections and have significant side effects. Proteases generated during inflammation are involved in the induction of tissue damage during inflammatory demyelination in the central nervous system (CNS). The Bowman-Birk Inhibitor (BBI), a soy-derived protease inhibitor with anti-carcinogenic and anti-inflammatory properties, has been shown to be well tolerated in clinical trials for pre-cancerous conditions, such as oral leukoplakia and the inflammatory disease, ulcerative colitis. We hypothesized that BBI may modulate experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The BBI concentrate (BBIC), a soybean extract enriched in BBI, was administered to myelin basic protein (MBP)-immunized Lewis rats by gastric gavage in different treatment regimens, during the induction or the effector phase of disease. BBIC significantly delayed disease onset and suppressed disease severity, clinically and pathologically, in all treatment protocols. Both in vitro and ex vivo, BBIC inhibited MBP-specific proliferation of lymph node cells. BBIC reduced the activity of matrix metalloproteinase (MMP)-2 and -9 in spleen cell supernatants and was detected in the CNS of treated rats. BBIC suppresses EAE, it can be administered orally, and it is safe and relatively inexpensive. It may have a therapeutic role in patients with MS.

Cutting edge: C3, a key component of complement activation, is not required for the development of myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis in mice.

Experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating disease of the CNS, is regarded as an experimental model for multiple sclerosis. The complement has been implicated in the pathogenesis of multiple sclerosis. To clarify the role of C in mouse EAE, we immunized mice deficient in C3 (C3(-/-)) and their wild-type (C3(+/+)) littermates with myelin oligodendrocyte glycoprotein peptide 35-55. C3(-/-) mice were susceptible to EAE as much as the C3(+/+) mice were. No differences were found for the production of IL-2, IL-4, IL-12, TNF-alpha, and IFN-gamma between C3(+/+) and C3(-/-) mice. This finding shows that C3, a key component in C activation, is not essential in myelin oligodendrocyte glycoprotein peptide-induced EAE in mice.

Flow cytometric analysis of infiltrating cells in the peripheral nerves in experimental allergic neuritis.

Experimental autoimmune neuritis (EAN) is an animal model that shares clinical, pathological and electrophysiological features with the human disease Guillain-Barré syndrome (GBS). In this study, we isolated and characterized by fluorescent activated cell sorter (FACS) phenotype of the inflammatory cells infiltrating cauda equina (CE) of Lewis rats at the active stage of the disease. We found that at this stage of EAN macrophages (Mphi) and alphabeta T cells were two major populations isolated from CE. We also found that among total cell population isolated from CE, gammadelta T and NK cells composed two small but distinct populations, while B cells were negligible. We characterized phenotype of alphabeta T cells in CE as CD45RC(+)CD8(+) (activated cytotoxic lymphocytes) and CD45RC(-)CD4(+) (memory Th cells). The phenotype of gammadelta T cells was found to be consisted of only CD45RC(+)CD8(+) cells. Both alphabeta and gammadelta T cells in CE expressed a higher level of CD25, CD44 and CD54 activation markers compared to the other tissues. Immunohistochemistry demonstrated that gammadelta T cells existed apart from the intense cellular infiltrate. This is the first report on the isolation and FACS analysis of CE-infiltrating cells, contributing a new and alternative approach to study the inflammatory lesions in EAN. We conclude that both alphabeta and gammadelta T cells have a unique activation/inflammatory phenotype required to traffic through and be retained in the peripheral nerves during EAN.

IL-12 reverses the suppressive effect of the CD40 ligand blockade on experimental autoimmune encephalomyelitis (EAE).

Blockade of the CD40 ligand (CD40L)-CD40 interaction suppresses experimental autoimmune encephalomyelitis (EAE). Since this interaction induces IL-12, an essential cytokine for EAE induction, we hypothesized that CD40L blockade may suppress EAE through IL-12 inhibition. Here we show that exogenous IL-12 abolishes the ability of anti-CD40L monoclonal antibodies to prevent EAE. Anti-IL-12 antibodies prevent this reversal and protect from EAE. These results show that IL-12 is sufficient to overcome CD40L blockade and suggest that, of the multiple consequences of the CD40L-CD40 interaction, IL-12 induction is an essential one for induction of EAE.

Effect of timing of intravenous administration of myelin basic protein on the induction of tolerance in experimental allergic encephalomyelitis.

Immunological tolerance and suppression of clinical and histological experimental allergic encaphalomyelitis (EAE) can be induced by the intravenous (i.v.) administration of myelin basic protein (MBP). In this report we have characterized the effect of the time of i.v. administration of MBP on the course of EAE in Lewis rats. Rats were treated with the i.v. administration of one or two 500 micrograms doses of MBP either before or after active immunization. Results indicated that i.v. administration of MBP in rats before active immunization with MBP/CFA (naïve rats) was most effective when given 14 days before active immunization, but treatment of rats actively immunized with MBP (immunized rats) was most effective at the onset of disease. Treatment at other times was less effective. The i.v. administration of the peptide MBP 68-88 (p68-88) containing the dominant encephalitogenic epitope could also suppress MBP-induced EAE in a dose dependent manner. Intravenous administration of two injections of p68-88 to naïve rats on days 10 and 3 before, or on days 0 and 7 after, active immunization with MBP suppressed the development of EAE in a dose dependent manner. Treatment of rats with i.v. MBP after, but not before, the transfer of MBP-reactive EAE effector cells suppressed the development of EAE in the recipient rats. Transfer of lymphoid cells from tolerized naïve rats failed to protect recipient rats against development of active or passive EAE. These results indicate the importance of timing and dose of the antigen on the induction of tolerance and suggests different mechanisms of tolerance induction by intravenous MBP in immunized and naïve rats. They also emphasize the importance of timing in designing efficient treatment strategies when i.v. tolerance is contemplated in EAE and possibly multiple sclerosis.

Reversal of spontaneous progressive autoimmune encephalomyelitis by myelin basic protein-induced clonal deletion.

Autoimmune encephalomyelitis can be initiated spontaneously and developed progressively in TCR transgenic mice specific for myelin basic protein when exposed to non-sterile environment, thus more closely mimicking human multiple sclerosis. By intravenous administration of myelin basic protein, we succeeded in reversing the clinical and pathological signs of progressive spontaneous disease in these mice. Flow cytometry showed that the majority of transgenic T cells in lymph nodes and spleen as well as spinal cords of treated mice were deleted. Dramatically increased numbers of apoptotic cells were found in peripheral immune organs of treated animals. Proliferative responses of single transgenic T cell to autoantigen were significantly decreased in treated mice, indicating that the remaining T cells were anergic. Moreover, production of both Th1 and Th2 cytokines was suppressed. This study is the first demonstration of reversal of progressive, spontaneous autoimmune disease of the central nervous system, and provides direct evidence that apoptosis-induced clonal deletion, along with anergy of remaining cells, but not Th2 switch, play a major part in the reversal of this disease by intravenous administration of autoantigen.

Antibodies against IL-12 prevent superantigen-induced and spontaneous relapses of experimental autoimmune encephalomyelitis.

Immunization of (PL/J x SJL/J)F1 mice with myelin basic protein (MBP) induces relapsing experimental autoimmune encephalomyelitis (EAE). Relapses occur 7 to 10 days after recovery from the initial paralysis. Staphylococcal enterotoxins (SE) A or B, administered after recovery from the initial paralysis, induce immediate relapses. IL-12 is involved in the induction of EAE. Here, we show that SEA and SEB induce IL-12 in splenocytes from (PL/J x SJL/J)F1 mice in vitro and increase the level of IL-12 in the sera of mice treated with these superantigens. IL-12 administration mimics SE in inducing spontaneous relapses and in enhancing the severity and frequency of spontaneous relapses. IL-12 neutralization blocks SE-induced and subsequent relapses of EAE, and, when instituted after recovery from the initial attack, prevents spontaneous relapse. This is the first report of prevention of relapses of EAE with anti-IL-12 Ab, an approach which may prove useful in the prevention of exacerbations in multiple sclerosis.

Captopril and lisinopril suppress production of interleukin-12 by human peripheral blood mononuclear cells.

Angiotensin converting enzyme (ACE) inhibitors have immunomodulatory functions and can suppress a number of proinflammatory, monocyte/macrophage-derived cytokines. Interleukin-12 is a cytokine produced primarily by monocytes and macrophages, which plays an essential role in cell mediated immunity and stimulates the development of T helper type 1 immune responses. In this study, we investigated the ability of ACE inhibitors, captopril and lisinopril, to suppress IL-12 production by human peripheral blood mononuclear cells (PBMC). We show that both ACE inhibitors significantly inhibit production of IL-12 by PBMC stimulated with bacterial lipopolysaccharide (LPS) or Staphylococcus aureus Cowan (SAC). Although both ACE inhibitors also suppressed IFN-gamma production by human anti-CD3/anti-CD28-stimulated T-cells, the addition of exogenous IFN-gamma to the PBMC stimulation medium does not abrogate the ability of ACE inhibitors to suppress IL-12 production. Inhibition of IL-12 was not associated with inhibition of IL-1beta, but correlated with the suppression of ACE. Therefore, suppression of IL-12 may contribute to the immunomodulatory effect of ACE inhibitors and may be responsible for the beneficial effect of captopril and other ACE inhibitors in inflammatory or autoimmune conditions in which IL-12 is involved.

The expression of cytokine mRNA in the cauda equina of Lewis rats with experimental allergic neuritis.

Autoreactive CD4+ T cells can transfer experimental allergic neuritis (EAN) to naive recipients. In order to further analyze the role of these T cells and their corresponding cytokines in EAN, we studied the expression of mRNA for IFN-gamma, IL-4, and IL-10 in the cauda equina of rats with EAN using a quantitative competitive reverse transcriptase PCR method. Nerves were studied on days 0 (pre-immunization), 10 (disease onset), 13 (clinical progression), 16 (disease peak), as well as 20, 24, and 34 post immunization (recovery). IFN-gamma messages increased at disease onset and peaked at day 13 p.i. IL-10 message remained at a very low level at disease onset and surged at day 16. Both messages were low in recovery stage. IL-4 message was undetectable at any time point. These data suggest a pro-inflammatory role of IFN-gamma and anti-inflammatory role of IL-10 in EAN lesions. It is also possible that a clonal switch from Th1 to Th2 occurs in EAN lesions during the disease course.

Propranolol antagonizes the anti-inflammatory effect of alcohol and improves survival of infected intoxicated rabbits.

We studied the effects of alcohol and propranolol on the course of peritonitis in rabbits. Induction of sterile peritonitis with normal saline led to a 50% augmentation of granulocyte adherence in normal rabbits, and a mean cumulative granulocyte count of 27,000/mm(3) in peritoneal exudate by 8 h. Rabbits intoxicated with alcohol at the time of peritonitis induction maintained a granulocyte adherence below pretreatment values, and only delivered a cumulative mean of 12,000 granulocytes/mm(3) into the peritoneal fluid. When intoxicated rabbits received propranolol intravenously at the time of intoxication, adherence increased above preperitonitis levels, and stayed significantly above values for animals given alcohol alone. In addition, the defect in granulocyte delivery was prevented by propranolol, resulting in a mean cumulative granulocyte count in peritoneal fluid of 24,000/mm(3).When peritonitis was induced with live pneumococci instead of a sterile inflammatory stimulus, 14/18 normal animals survived the infection and were culture-negative when sacrificed at 2 wk. In contrast, 17/18 intoxicated animals died of the infection, in a mean of 2.8 days. 9 of 18 intoxicated animals who also received propranolol survived, and those who died lived a mean of 7.5 days. The survival rates and the time-to-death among the nonsurvivors given propranolol were both significantly greater than in the animals intoxicated without propranolol. Thus, propranolol prevents the granulocyte adherence and delivery defects induced by alcohol intoxication, and significantly improves survival from infection.