Samia Ragheb

The Role of Cytokines in Schwann Cell Damage, Protection, and Repair

Cytokines, proteins that are secreted by many cells, including inflammatory and glial cells, mediate interactions between cells, generally through paracrine and autocrine networks. Their effects are highly pleiotropic, with overlap of some activities. The pathogenesis of Guillain-Barré syndrome (GBS), especially the classic inflammatory demyelinating polyneuropathy form, seems to involve lymphocytes and macrophages, which are rich sources of cytokines. Macrophages likely have a role in the pathogenesis of the primarily axonal, less inflammatory forms of GBS. Cytokines appear to be involved in damage to Schwann cells, myelin, and axons, although the exact roles of the different cytokines is uncertain. There is increasing evidence that cytokines, including some proinflammatory cytokines that ordinarily cause damage, may also protect the cells of the peripheral nervous system and aid in its repair. The evolution of inflammatory and demyelinating disorders, including the degree of recovery, is probably dependent on the interactions of the different cytokines.

Autoantibodies to Agrin in Myasthenia Gravis Patients

To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ.

Secretory products of multiple sclerosis B cells are cytotoxic to oligodendroglia in vitro

B cells are important in the pathogenesis of multiple sclerosis (MS) and some of the effects are not dependent on maturation of B cells into immunoglobulin (Ig) producing plasmablasts and plasma cells. B cells present antigen, activate T cells, and are involved in immunoregulation and cytokine secretion. To determine if B cells from MS patients secrete products that have deleterious effects on glial cells not mediated by Ig, and to compare effects with secretory products of normal controls (NC), we isolated B cells from 7 patients with relapsing remitting MS (RRMS) and 4 NC. B cells were cultured alone or after stimulation with CD40 ligand (CD40L), CD40L+cross-linking of the B cell antigen receptor (xBCR) and CD40L+xBCR+stimulation of toll like receptor 9 (TLR9). Supernatants were harvested and incubated with mixed central nervous system (CNS) neonatal rat glial cells. Supernatants from unstimulated NC B cells induced on average death of 7% (range 0-24%) of differentiated oligodendrocytes (OL); in contrast, supernatants from unstimulated B cells from RRMS patients induced death of 57% (range 35-74%) of OL. Supernatants of stimulated B cells from NC did not increase the minimal OL death whereas stimulation of B cells from RRMS had variable results compared to unstimulated B cells. Supernatants from both NC and RRMS induced microglial enlargement and loss of normal resting bipolar morphology. OL death did not correlate with levels of tumor necrosis alpha (TNF-α), lymphotoxin alpha (LT-α), interleukin 6 (IL-6), IL-10, transforming growth factor beta 1 (TGF-β1) or any combination or ratio of these cytokines. Analysis of 26 supernatants from NC and RRMS patients failed to detect IgM. There were very low levels of IgG in 8 of the 26 supernatants, and no correlation between of OL death and presence or absence of IgG. Sera used in both the B cell and glial cell cultures were heated, which inactivates complement. The effects of B cell supernatants on OL could be direct and/or indirect involving either microglia and/or astrocytes. The identity of the toxic factor(s) is as yet unknown. Thus we have demonstrated that B cells from patients with RRMS but not NC secrete one or more factors toxic to OL. It is possible that such factors produced by peripheral blood B cells when within the CNS could contribute to demyelination in MS patients.

Ectopic germinal centers, BAFF and anti-B-cell therapy in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by antibodies directed to molecules of the endplate of the neuromuscular junction. B cells play a major role in MG disease since they produce the pathogenic antibodies and therapies targeting B cells are effective. The aim of this article was to review the role of B cells in myasthenia gravis. We will first describe what we know about B cells in this disease and examine the involvement of the B cells in the thymus of MG patients. We will detail the role of factors associated with B-cell function such as BAFF. Finally, we will discuss the effects of therapy targeting B cells.

Multiple sclerosis: BAFF and CXCL13 in cerebrospinal fluid.

Background: There is increasing evidence of B-cell involvement in the pathogenesis of multiple sclerosis (MS). B-cell activating factor (BAFF) has an essential role in B-cell homeostasis. The chemokine CXCL13 has an important role in the formation and maintenance of B-cell follicles. Objective: To measure BAFF and CXCL13 levels in the cerebrospinal fluid (CSF) of patients with MS compared to patients with other neurological diseases. Methods: Cytokine/chemokine levels were measured by an enzyme-linked immunosorbent assay (ELISA). Results: In MS patients, BAFF levels were highest in patients with secondary progressive disease, and were higher during relapse in patients with relapsing–remitting and secondary progressive disease. CXCL13 levels were also higher during relapse. There was a positive correlation between CXCL13 and the IgG index, and an inverse correlation between BAFF and the IgG index. The implications of this finding are discussed. Conclusion: During relapse, we found various positive correlations between BAFF, CXCL13 and the cytokines IL-6 and IL-10. These findings show that molecules that are essential for B-cell recruitment, survival, maturation and function may be working in concert to affect B-cell homeostasis in MS and contribute to the pathophysiology of the disease.

A Potential Role for B-Cell Activating Factor in the Pathogenesis of Autoimmune Myasthenia Gravis

OBJECTIVE To compare serum B-cell activating factor (BAFF) levels in patients with myasthenia gravis (MG) with those in control subjects without MG. DESIGN Case-control study. Subjects Forty-three patients with MG were compared with control subjects without MG. These included 48 healthy subjects, 25 patients with multiple sclerosis, and 3 patients with amyotrophic lateral sclerosis. RESULTS In all subjects studied, there was no correlation between the serum BAFF level and the concentration of total IgG, IgA, or IgM. The BAFF levels in patients with multiple sclerosis or amyotrophic lateral sclerosis were not significantly different from those in healthy subjects. However, BAFF levels in patients with MG were significantly higher than those of all the control subjects. There was no correlation or dependence between the serum BAFF level and the extent or severity of disease. There was a trend for BAFF levels to be higher in patients who were seropositive for acetylcholine receptor-specific antibodies. CONCLUSIONS We report that BAFF levels are increased in patients with autoimmune MG. Our data suggest that BAFF is likely to play a role in the pathogenesis of MG by promoting the survival and maturation of autoreactive B cells.

Long-term therapy with glatiramer acetate in multiple sclerosis: effect on T-cells

Glatiramer acetate (GA) is an immunotherapeutic drug for multiple sclerosis (MS). Several mechanisms of action have been demonstrated which target and affect T-cells that are specific for myelin antigen epitopes. We measured the in vitro proliferation of GA-responsive T-cells from untreated MS patients and from normal healthy subjects; in addition, we determined the effect of prolonged GA therapy or interferon-b therapy on the in vitro proliferation of GA-responsive T-cells of MS patients. We found that GA induces the proliferation of T-cells isolated from individuals who have not been previously exposed to GA, and that long-term in vivo therapy of MS patients with GA abrogates the GA-induced proliferative response of T-cells. In GA-treated patients, there is no evidence of generalized immunosuppression; both tetanus toxoid and anti-CD3 induced proliferative responses remain unaffected. We propose that prolonged in vivo exposure to GA may result in the eventual induction of anergy or deletion of a population of GA-responsive cells that may also be T-cells that are pathogenic in MS. This mechanism of action, in addition to other mechanisms that have been demonstrated, suggests that GA has pleiotropic effects on the immune system in MS.

The frequency of CD5+ B lymphocytes in the peripheral blood of patients with myasthenia gravis

A subset of human B lymphocytes expresses Leu-1 (CD5), a pan-T cell marker, which is the equivalent of the murine Lyt-1 molecule. CD5+ B cells produce autoantibodies in vitro; therefore, they may play a role in the pathogenesis of autoimmune disorders. In myasthenia gravis (MG), autoantibodies are directed against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. We examined the peripheral blood leukocytes of MG patients (n = 21) and controls (n = 15) for the presence of Leu-1+ B lymphocytes. A fraction of B-1 (CD20)+ cells expressed Leu-1 at a low density. There was a statistically significant difference in the frequency of Leu-1+ B cells between patients and controls. We observed 2 frequency ranges of Leu-1+ B cells (0 to 30% and above 30%), which were not related to the total percentage of B-1+ cells in the blood. Fifty-seven percent of MG patients had a high frequency of Leu-1+ B cells compared with 13% of controls.

Interleukin-1α, but not interleukin-1β, is a co-mitogen for neonatal rat Schwann cells in vitro and acts via interleukin-1 receptors

Abstract The culture of neonatal rat Schwann cells (SC) with unfractionated cytokines induces an increase in SC proliferation. Previous studies demonstrated that while incubation of SC with interleukin-1(IL-1) does not result in enhanced SC mitogenesis, a mixture of antibodies to IL-1α plus IL-1β inhibits cytokine-induced proliferation. We undertook the current studies to: (i) confirm that neither isoform of IL-1 directly causes SC proliferation; (ii) determine if there is a difference in the effect of antibodies to IL-1α versus IL-1β; and (iii) determine if IL-1 contribution to cytokine-induced proliferation of SC is mediated via IL-1 receptors. IL-1α or IL-1β from several sources, over a wide range of concentrations, failed to induce SC proliferation. Polyclonal antibodies to IL-1α from several suppliers and a monoclonal antibody to IL-1α inhibited SC priliferation, whereas similar antibodies to IL-1β had no effect on cytokine-induced SC proliferation. Addition of excess IL-1α to an incubation mixture of unfractionated cytokines plus anti-IL-1α abolished the inhibitory effect of the antibodies. Addition of IL-1 receptor antagonist (IL-1 Ra) to unfractionated cytokines inhibited SC proliferation. Therefore, while neither IL-1α nor IL-1β is a solitary mitogen for neonatal rat SC, IL-1α but not IL-1β acts as a co-mitogen. Moreover, IL-1α seems to exert its co-mitogenic effect via receptors for IL-1.

The role of B cell-activating factor in autoimmune myasthenia gravis.

B cell–activating factor (BAFF) is important in the development and maturation of B cells and their progeny—plasma blasts and plasma cells. There is increasing evidence that BAFF is involved in the pathogenesis of several autoimmune diseases including myasthenia gravis. Increased expression of BAFF and receptors for BAFF have been demonstrated in thymus of patients with myasthenia gravis, and an increase in serum levels of BAFF have been reported in patients with myasthenia gravis. While the exact role of BAFF in the pathogenesis of myasthenia gravis is not clear, BAFF and its receptors may provide potential targets for therapy in patients with myasthenia gravis.