Analysis of B-cell activation of cerebrospinal fluid lymphocytes in multiple sclerosis

We have developed a microculture system to study pokeweed mitogen (PWM)-induced B-cell differentiation responses of CSF lymphocytes from patients with multiple sclerosis (MS) and other neurologic diseases (OND). B-cell differentiation was assessed by (1) enumeration of immunoglobulin-secreting cells (IgSC) by a protein A reverse hemolytic plaque assay; and (2) quantitation of supernatant IgG by ELISA. Cultures of MS CSF cells and OND CSF cells responded to PWM with a similar frequency, with responses in CSF cell cultures exceeding responses in corresponding blood cell cultures in several instances in both groups of patients. Numbers of IgSC in unstimulated cultures of MS CSF cells exceeded numbers in cultures of autologous peripheral blood mononuclear cells (PBM). Results suggest that CSF cells may be a particularly reactive population compared with PBM.     

Estradiol potentiates poke-weed mitogen-induced B cell stimulation in multiple sclerosis and healthy subjects

Female preponderance in many diseases suggested with autoimmune pathogenesis, multiple sclerosis (MS) being classified as one of them, indicates a role for hormonal factors such as estrogen in disease development. To bypass monthly hormonal fluctuations in females, we evaluated in male patients with MS and male blood donors the effect of 17-beta-estradiol on numbers of IgG, IgA and IgM producing cells in cultures of peripheral blood lymphocytes. While estradiol alone had no effect, estradiol in combination with poke-weed mitogen (PWM) yielded in both groups higher numbers of IgG and IgA producing cells when compared with numbers obtained by PWM stimulation alone, indicating an additory effect of estradiol to that of PWM on B cell maturation. This effect was less pronounced in MS than in blood donors, especially for IgG producing cells, probably reflecting higher B cell activation in vivo taking place in MS. On the contrary, cells producing IgG, IgA and IgM antibodies against myelin, myelin basic protein and measles virus were not detectable after stimulation with PWM, nor with PWM and estradiol. Estradiol can in many patients with MS and in blood donors be considered a potent co-activator of B cells in presence of B cell stimulating factor in the form of PWM.     

T cells from multiple sclerosis patients recognize immunoglobulin G from cerebrospinal fluid

Idiotopic sequences are created after V, D and J recombinations and by somatic mutations during affinity maturation of immunoglobulin (Ig) molecules, and may therefore be potential immunogenic epitopes. Idiotope-specific T cells are able to activate and sustain the B cells producing such idiotopes. It is therefore possible that idiotope-specific intrathecal T cells could help maintain the persisting intrathecal synthesis of oligoclonal IgG observed in patients with multiple sclerosis (MS). This study was undertaken to examine T-cell responses to cerebrospinal fluid (CSF) IgG. Peripheral blood mononuclear cells (PBMC) from 14 of 21 MS patients and four of 17 control patients with other neurological diseases proliferated upon stimulation with autologous CSF IgG, while five and three, respectively, responded to serum IgG. By comparison, responses to myelin basic protein were recorded in only four MS and three control patients. Data from a limited number of patients indicate that the CSF IgG responsive cells were CD4+ and human leucocyte antigen DR restricted, that PBMC also respond to CSF IgG from other MS patients and that the CSF may contain T cells responding to autologous CSF IgG. This suggests that CSF IgG, or substances bound to this IgG, may represent T-cell immunogens, which could contribute to the intrathecal immune response in MS.     

Biased expression of T cell receptor genes characterizes activated T cells in multiple sclerosis cerebrospinal fluid

To better characterize the inflammatory response that occurs in the nervous system in multiple sclerosis (MS), T-cell receptor (TCR) gene expression was quantified from cerebrospinal fluid (CSF) cells of 21 patients with active disease. Unstimulated CSF cells expressed each of 22 different TCR beta chain variable region (Vβ) gene families in proportion to their expression in simultaneously sampled peripheral blood. When CSF cells from individuals with MS were expanded by in vitro culture in T-cell growth factor/interleukin 2 and 4-containing medium (TCGF/IL2/IL4), restricted numbers of Vβ genes were expressed. In many subjects, expanded CSF cells expressed predominantly Vβ2. In contrast to CSF, expansion of corresponding peripheral blood mononuclear cells (PBMC) in TCGF/IL2/IL4 resulted in persistent expression of an Vβ gene families. Within individuals, different Vβ genes were overexpressed by PBMC compared with CSF cells. No effect of the HLA haplotype of the individual on CSF Vβ gene expression was observed. Expanded CSF cells retained their capacity to respond to mitogen stimulation, but the proliferative response to myelin basic protein (MBP) was not enhanced. Finally, freshly obtained CSF cells stimulated directly with MBP also expressed a limited number of Vβ genes, although these were generally different from patterns observed following stimulation with TCGF/IL2/IL4. Thus, restricted populations of T cells capable of responding to TCGF/IL2/IL4, presumably reflecting in vivo activated cells, are compartmentalized in the nervous system in MS. © 1996 Wiley-Liss, Inc.     

Reduction of both pro- and anti-inflammatory cytokines after 6 months of interferon beta-1a treatment of multiple sclerosis

Treatment of multiple sclerosis (MS) with interferon beta (IFNbeta) reduces relapse rate, magnetic resonance imaging (MRI) activity and progression of disability. It has been suggested that this beneficial effect is paralleled by an inhibition of proinflammatory cytokines such as interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) and an induction of anti-inflammatory cytokines such as interleukin-4 (IL-4) and interleukin-10 (IL-10). In this study, we record a reduced number of spontaneously IFNgamma mRNA-expressing cerebrospinal fluid mononuclear cells (CSF-MC) and IFNgamma, TNFalpha and IL-10 mRNA-expressing peripheral blood mononuclear cells (PBMC) after 6 months of IFNbeta-1a treatment, paralleled by a decreased purified protein derivate (PPD)-stimulated and unstimulated IFNgamma secretion by PBMC. These effects were not apparent after 2 weeks of treatment, and IFNbeta-1a induced IFNgamma production by naive PBMC in vitro. We did not record increased numbers of IL-4 mRNA-expressing CSF-MC or PBMC, increased plasma IL-10 levels, increased numbers of IgG, A or M secreting plasma cells or in vitro induction of IL-10 production by IFNbeta-1a. We conclude that long-term cytokine modulation by IFNbeta-1a differs from acute effects and that downregulation of both pro- and anti-inflammatory cytokines, rather than a shift in the cytokine profile, is apparent after 6 months of IFNbeta-1a treatment of MS patients.     

Multiple sclerosis: relation of in vitro IgG secretion to T suppressor cell number and function

The proportion of MS patients whose pokeweed mitogen-stimulated mononuclear cells (MNCs) secreted greater than 1,000 ng/ml IgG per 10(6) cells (ie, "high responders") was increased compared with controls. The suppressor effect mediated by a constant number of T8+ cells from high responders, both MS and control, on IgG secretion by standard T helper (T4+) plus B-cell cultures was significantly lower than that for the same number of T8+ cells from "low responders." The proportion of T8+ cells within MNCs from MS patients did not correlate with levels of IgG secretion. Our results indicate that high levels of IgG secretion by MNCs, an occurrence overrepresented in the MS population, is significantly influenced by functional properties of T suppressor cells.     

Spontaneous proliferation of cerebrospinal fluid mononuclear cells in multiple sclerosis : A longitudinal study

Cerebrospinal fluid (CSF) and peripheral blood (PB) specimens from 32 MS patients longitudinally followed for up to 24 months and from a group of control patients without intrathecal inflammation were studied for the occurrence of activated lymphocytes with an autoradiography method. MS patients had higher numbers of proliferating mononuclear cells in CSF than did the controls, both during remission and exacerbation phases, whereas this difference was not found in the PB. ACTH treatment decreased the number of proliferating cells in CSF but had no effect on those of the PB of MS patients. A large variation in spontaneous proliferation of CSF cells was evident during the follow-up of individual patients, and there seemed to be no uniform correlation to the clinical fluctuations.     

Brain-derived neurotrophic factor in patients with multiple sclerosis

The aim of the present research was to verify the production of BDNF by peripheral blood mononuclear cells (PBMCs), unstimulated and stimulated with phytohemagglutinin (PHA), anti-OKT3 Ab and myelin basic protein (MBP), in 35 patients affected by multiple sclerosis (MS), 20 with relapsing-remitting (R-R) MS and 15 with secondary progressive (SP) MS. Seven R-R MS patients were assessed during the attack, in the subsequent recovery phase and also 3 months after relapse. The production of BDNF by PBMCs was also evaluated in 20 age- and sex-matched control subjects. Levels of BDNF were also determined in CSF of both patient groups and 20 control subjects. RESULTS: Levels of BDNF (pg/ml) in the supernatants of unstimulated and PHA-, anti-OKT3 Ab- and MBP-stimulated PBMCs in patients with R-R MS were significantly higher during relapse and in the recovery phase compared with values detected in the stable phase of the disease. Significantly lower BDNF values were found in unstimulated and stimulated PBMC supernatants of patients with SP MS compared to control subjects. This reduction was greater in patients with a 1-point increase in the EDSS score in the last 6 months compared with that in patients without a progression of the disability score. Reduction in the levels of BDNF was also confirmed in the CSF of SP MS patients compared with R-R MS patients assessed during a stable phase of the disease and control subjects. DISCUSSION: On the basis of recent experimental findings, a neuroprotective effect of BDNF produced by inflammatory cells can be hypothesized during relapses in MS. This can favor remyelination. The reduced production of BDNF by PBMCs of patients with SP MS can contribute to the progression of demyelinating disease and axonal loss in this form.     

Analysis of peripheral blood lymphocyte phenotype and function during dexamethazone treatment of progressive multiple sclerosis

Five patients with chronic progressive multiple sclerosis (MS) and three control patients with lumbar disc herniation were treated with dexamethazone during 14 days. The effect on peripheral blood T-cell subsets and on the proliferative response of peripheral blood mononuclear cells (PBMC) to pokeweed mitogen (PWM) and anti-mu antibody was analyzed. Before treatment, the proportion of CD3+ and CD4+ PBMC was similar in MS and control patients, but the proportion of CD8+ and DR+ PBMC was lower and the PBMC were less responsive to anti-mu stimulation in MS patients compared to controls. Steroid treatment induced reversible granulocytosis and lymphocytosis. CD3+ and CD4+ cells increased and DR+ cells decreased in MS patients but not in controls. Proliferation of anti-mu stimulated PBMC increased in MS-patients during the two weeks of treatment, but decreased in controls. The enhancement in the MS patients of pre-existing immune abnormalities suggests that a cautious attitude is warranted in the use of steroid treatment in chronic progressive MS.     

Immunoglobulin-producing cells in CSF and blood from patients with multiple sclerosis and other inflammatory neurological diseases enumerated by Protein-A plaque assay

Using the Protein-A plaque assay, numbers of IgG + IgA + IgM producing cells determined in patients with multiple sclerosis (MS) were 0.1–5% in CSF and 0.1–0.7% in peripheral blood; interestingly, 7 of 11 MS patients had IgM producing cells in CSF. In patients with aseptic meningitis (AM), the corresponding values were 0.04–7.5% in CSF and 0.4–2.4% in peripheral blood. There were more Ig producing cells in peripheral blood from patients with AM and MS than in healthy subjects. cocorrelation between numbers of IgG producing cells in CSF and the concentrations of intrathecally produced IgG (CSF IgG index) was registered in patients with AM: the same was true for IgA. The Protein-A plaque method, adopted for 20 × 10³ lymphocytes, makes possible enumeration of Ig-producing cells in CSF and discrimination among cells secreting different Ig classes, thereby being a powerful tool for studying immune reactions in the CNS-CSF compartment.     

多发性硬化症髓鞘素组分特异性抗体分泌细胞

本文用酶联免疫斑点法（Ｅｌｉｓｐｏｔ）检测了２３例临床确诊多发性硬化症（ＭＳ）和１２例无菌性脑膜炎（ＡＭ）患者外周血（ＰＢ）和脑脊液（ＣＳＦ）中髓鞘素碱性蛋白（ＭＢＰ）、髓鞘素结合糖蛋白（ＭＡＧ）和含脂质蛋白（ＰＬＰ）特异性ＩｇＧ抗体分泌细胞。两组患者ＣＳＦ中该３种抗体分泌细胞均呈明显增多趋势，ＭＳ组尤著，但两组ＰＢ中该类细胞数均很少。指示对髓鞘素组分的Ｂ细胞免疫应答主要局限于与中枢神经系统（ＣＮ     

Cytokine secretion by peripheral blood mononuclear cells in myasthenia gravis.

We studied spontaneous secretion of anti-acetylcholine receptor antibody (AChRAb), IgG and cytokines by peripheral blood mononuclear cells (PBMC) from 19 MG patients without therapy and 10 normal controls. IgG secretion was higher in the culture medium of MG than in that of normal controls. AChRAb secretion was correlated with IgG secretion in MG. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha secreted by PBMC from MG patients were not different from those produced by those from normal controls. IgG secretion was, however, correlated with the secretions of IL-5 and IL-6 in MG. Spontaneous B cell activation was suspected in patients with MG.     

HTLV-I-associated myelopathy endemic in Texas-born residents and isolation of virus from CSF cells

We report three Texas-born patients with spastic paraparesis and well-documented infection with HTLV-I. CSF examination showed moderate pleocytosis, protein elevation, and elevated IgG index. Oligoclonal bands were present in two patients. On MRI, one patient had frontal lobe lesions that were low intensity on T1- and high intensity on T2-weighted images. HTLV-I immunoblot studies of serum and CSF revealed reactivity to p19, p24, p53, gp46, or gp68 from all three patients. Titration studies of serum and CSF antibodies on ELISA and immunoblot assays indicated an intrathecal virus-specific response. HTLV-I-specific p19 antigen capture assay and polymerase chain reaction (PCR) demonstrated HTLV-I in lymphocyte cultures derived from each patient's peripheral blood mononuclear cells (PBMC) or CSF cells. Using HTLV-I- and HTLV-II-specific pol and gag primers, PCR studies of PBMC cells obtained directly from the patients demonstrated that the patients were infected with HTLV-I and not HTLV-II. These three cases are to our knowledge the only US cases in whom virus isolation from the CSF has been accomplished. Importantly, two patients may be the first US cases of myelopathy arising from endemic infection.     

Analysis of immunoglobulin secretion by lymph organs with myasthenia gravis

OBJECTIVES: To investigate in vitro anti-acetylcholine receptor (AChR) antibody secretion by lymph organs with myasthenia gravis (MG) with special attention to clinical status and stages. MATERIALS AND METHODS: Twenty MG patients before therapy were included in this study. Four patients were negative for serum anti-AChR antibodies (AChRAb). All patients received extended thymectomy. Thymic cells, bone marrow (BM) cells and peripheral blood mononuclear cells (PBMC) were cultured for 1 week. AChRAb secretion and IgG secretion in the culture medium were determined by immunoprecipitation assays and ELISA respectively. RESULTS: PBMC secreted AChRAb and IgG most efficiently, followed by BM cells, and then thymic cells. Even in patients whose diseases were of short duration (less than 2 months), the tendency was not changed. AChRAb secretion by BM cells and PBMC significantly correlated with serum AChRAb levels. Thymectomy did not change AChRAb or IgG secretion by PBMC within 3 months. AChRAb secretion by PBMC paralleled the clinical status. Some seronegative patients turned positive by culturing PBMC. CONCLUSION: PBMC is the most efficient site at producing AChRAb, even in patients of short duration (equal to, or less than, 2 months). Monitoring AChRAb secretion by PBMC is useful to estimate the autoimmune activity of MG.     

Production of viral antibodies in vitro by CSF cells from mumps meningitis and multiple sclerosis patients

Cerebrospinal fluid (CSF) cells from 4 mumps meningitis and 11 multiple sclerosis (MS) patients were cultured in vitro for 7 days with and without pokeweed mitogen (PWM) stimulation. The cells produced varying amounts of IgG without stimulation and no significant increase of IgG synthesis was observed after PWM stimulation. Antibodies against mumps, measles, rubella, herpes simplex, and adeno viruses were measured in the supernatants of the cultures by a sensitive enzyme immunoassay. In the mumps meningitis patients, the largest amount of antibody was against mumps virus but low amounts of antibodies with other specificities were also synthesized by CSF cells of one patient. The most commonly detected specificities in MS patients were against measles and rubella viruses, whereas antibodies against adeno and mumps viruses were detected in only one CSF cell supernatant. No antibodies produced against herpes simplex virus in vitro were detected in any of the supernatants. The amounts of viral antibodies produced in vitro and intrathecally were only partially correlated.     

Interferon beta-1b modulates MCP-1 expression and production in relapsing-remitting multiple sclerosis

Monocyte chemoattractant protein-1 (MCP-1) seems to be involved in the pathogenesis of multiple sclerosis (MS). We found that in unstimulated (PHA(-)) and PHA-stimulated (PHA(+)) peripheral blood mononuclear cells (PBMC), MCP-1 and TNFalpha levels are higher in stable untreated MS patients. Interferon gamma (IFNgamma) is higher in relapsing patients in PHA(-) cultures and in stable patients in PHA(+) cultures. Chronic IFNbeta-1b treatment down-regulates TNFalpha, IFNgamma and MCP-1 production except for TNFalpha in relapsing patients. IFNbeta-1b, in vitro, increases MCP-1, TNFalpha and IFNgamma spontaneous production in all patients. Multivariate analysis suggests that MCP-1 production is dependent from clinical status and not from TNFalpha and IFNgamma production. Logistic regression analysis shows that MCP-1 production is significantly modified by treatment. Further studies are needed to clarify the role of MCP-1 in MS.     

In vitro synthesis of antibodies to acetylcholine receptor by peripheral blood mononuclear cells of patients with myasthenia gravis

We studied the in vitro synthesis of antibodies to acetylcholine receptor (anti-AChR) by peripheral blood mononuclear cells (PBM) of patients with myasthenia gravis (MG) and normal subjects (NS). PBM from three of eight patients with generalized MG (MG-G) synthesized anti-AChR in vitro in the absence of pokeweed mitogen (PWM), and seven of eight did so in the presence of PWM. In individual subjects with MG-G, the levels of anti-AChR secreted in vitro by PBM correlated with serum anti-AChR antibody levels (r = 0.77) but not with the amount of IgG secreted in vitro (r = 0.44). No anti-AChR secretion was seen in culture of PBM from a patient with ocular MG, a patient with thymoma without MG, or six NS.     

Tumor necrosis factor alpha but not lymphotoxin is overproduced by blood mononuclear cells in multiple sclerosis.

The cytotoxic cytokines tumor necrosis factor alpha (TNF-alpha) and lymphotoxin (LT) possess toxic activity against myelin and/or oligodendrocytes in vitro. Multiple sclerosis (MS) plaques within the central nervous system (CNS) are infiltrated by peripheral blood mononuclear cells (PBMC). In this study the production of TNF-alpha and LT by PBMC in active MS were measured. PBMC were isolated from the blood of MS patients in relapse and also patients with other neurological diseases (OND) and healthy controls (HC). Isolated cells were cultured unstimulated or stimulated with phytohemagglutinin A (PHA), lipopolysaccharide (LPS) and myelin basic protein (MBP) – a hypothetical autoantigen for MS. Cytokine production was assessed using ELISA method. In the MS group, PBMC without stimulation as well as after stimulation with MBP displayed a significantly increased production of TNF-alpha. LT production was similar in MS and control groups. These results suggest that TNF-alpha but not LT is overproduced by PBMC during MS relapse.