IL-15 mRNA expression is up-regulated in blood and cerebrospinal fluid mononuclear cells in multiple sclerosis (MS)

IL-15, produced by monocytes and epithelial cells, is a novel cytokine with actions similar to IL-2. IL-15 induces T cell proliferation, B cell maturation and natural killer (NK) cell cytotoxicity, and is a chemoattractant for T cells. We investigated the expression of IL-15 mRNA in blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) in MS, an inflammatory disease of the central nervous system where cytokines are involved. MS patients had higher numbers of IL-15 mRNA-expressing blood MNC than patients with aseptic meningo-encephalitis (AM) and healthy controls. In CSF, MS patients had even higher numbers of IL-15 mRNA-expressing cells than in blood. This discrepancy between IL-15 mRNA expression between blood and CSF MNC was not seen in AM patients. Patients examined during the secondary chronic-progressive phase of MS had higher numbers of IL-15 mRNA-expressing blood MNC compared with patients examined during the relapsing-remitting phase. Levels of IL-15 mRNA-positive blood MNC were similar in patients with AM, myasthenia gravis, non-inflammatory neurological diseases and healthy controls. Taken together these data indicate that IL-15 mRNA expression is up-regulated in MS, further suggesting a role for proinflammatory cytokines in the pathogenesis of MS.     

Blood levels of transforming growth factor-beta 1 (TGF-β1) are elevated in both relapsing remitting and chronic progressive multiple sclerosis (MS) patients and are further augmented by treatment with interferon-beta 1b (IFN-β1b)

The serum levels of TGF-β1, measured by solid-phase ELISA, were determined to be significantly augmented in patients with both relapsing remitting (RR) and secondary chronic progressive (CP) MS compared with sex- and age-matched healthy controls. Moreover, in RR MS patients, the blood levels of the cytokine were further augmented either during relapses or, in a rapid but reversible fashion, by s.c. injection with 8 million International Units (MIU) IFN-β1b. Because TGF-β1 possesses multiple anti-inflammatory activities, we hypothesize that the increase in its circulating levels in RR and CP MS patients might represent an endogenous anti-inflammatory mechanism aimed at counteracting ongoing immunoinflammatory events, and that IFN-β may further potentiate this natural defensive apparatus.     

Reconstitution of immune cell populations in multiple sclerosis patients after autologous stem cell transplantation

Multiple sclerosis is an inflammatory T cell‐mediated autoimmune disease. In a Phase II clinical trial, high‐dose immunosuppressive therapy combined with autologous CD34⁺ haematopoietic stem cell transplant resulted in 69·2% of subjects remaining disease‐free without evidence of relapse, loss of neurological function or new magnetic resonance imaging (MRI) lesions to year 5 post‐treatment. A combination of CyTOF mass cytometry and multi‐parameter flow cytometry was used to explore the reconstitution kinetics of immune cell subsets in the periphery post‐haematopoietic cell transplant (HSCT) and the impact of treatment on the phenotype of circulating T cells in this study population. Repopulation of immune cell subsets progressed similarly for all patients studied 2 years post‐therapy, regardless of clinical outcome. At month 2, monocytes and natural killer (NK) cells were proportionally more abundant, while CD4 T cells and B cells were reduced, relative to baseline. In contrast to the changes observed at earlier time‐points in the T cell compartment, B cells were proportionally more abundant and expansion in the proportion of naive B cells was observed 1 and 2 years post‐therapy. Within the T cell compartment, the proportion of effector memory and late effector subsets of CD4 and CD8 T cells was increased, together with transient increases in proportions of CD45RA‐regulatory T cells (T_regs) and T helper type 1 (Th1 cells) and a decrease in Th17·1 cells. While none of the treatment effects studied correlated with clinical outcome, patients who remained healthy throughout the 5‐year study had significantly higher absolute numbers of memory CD4 and CD8 T cells in the periphery prior to stem cell transplantation.     

Cannabinoid influence on cytokine profile in multiple sclerosis

Cannabinoids have been suggested as possessing immunomodulatory properties, and cannabinoid receptors are present on leucocytes. Clinically, there is some evidence that cannabinoids may be therapeutically useful in treating multiple sclerosis, which is generally believed to be an autoimmune condition. This paper reports data derived from the Cannabinoids in MS (CAMS) study, which was the largest randomized controlled trial yet conducted to evaluate the therapeutic efficacy of cannabinoids. We found no evidence for cannabinoid influence on serum levels of interferon (IFN)-gamma, interleukin (IL)-10, IL-12 or C-reactive protein as measured using enzyme-linked immunosorbent assay (ELISA), in comparison to control values. Mitogenic stimulation experiments also failed to demonstrate any significant reduction in percentage of CD3+, IFN-gamma producing cells after exposure to cannabinoids in vivo, although numbers were small. Further work is needed to establish the functional significance of cannabinoid receptors on immune cells.     

Atorvastatin does not alter serum levels of sCD95 and sCD95L in multiple sclerosis

Elimination of autoreactive T cells by apoptosis is critical for restricting immune responses to self-antigens. An errant lytic interaction between the CD95 death receptor and its ligand CD95L is presumed to be involved in the pathogenesis of multiple sclerosis (MS). Statins are promising agents for the treatment of MS and were shown to modulate levels of soluble death receptors. Here, we evaluated the in vivo effects by interferon (IFN)-beta and atorvastatin on soluble CD95 (sCD95) and sCD95L in serum of patients with MS. Concentrations of sCD95 and sCD95L did not show any differences between MS and healthy control subjects. In patients with MS, treatment with IFN-beta increased serum levels of sCD95 and sCD95L significantly (P < 0.01 and P < 0.05 respectively). Addition of atorvastatin to IFN-beta did not alter serum levels of sCD95 and sCD95L significantly. Our study suggests that atorvastatin does not affect IFN-beta-induced increases of the soluble death receptors in the serum of patients with MS.     

多发性硬化患者外周血淋巴细胞CD56的表达

目的 探讨CD56^ 淋巴细胞在多发性硬化（MS）的变化。方法 流式细胞仪测定32例复发缓解型MS患者（复发发期23例,缓解期9例）及13例复发期MS患者予糖皮质激素治疗后外周血淋巴细胞CD56表达的阳性百分率。结果 复发期和缓解期MS患者CD56的表达均高于对照组,15例复发期MS患者CD56的阳性百分率与血脑屏障受损呈正相关,复发期MS患者CD56的水平与距发作时间、整个病程、EDSS伤残评分无关。缓解期MS CD56的水平与病程无关,激素对CD56的表达我影响。结论 CD56^ 淋巴细胞涉及MS的发病机制。     

A restricted T cell response to myelin basic protein (MBP) is stable in multiple sclerosis (MS) patients

The close resemblance of MS to the animal model experimental autoimmune encephalomyelitis (EAE) has provided compelling data sustaining a pathogenic role of circulating T cells reactive against MBP. T cell antigen receptor (TCR) usage in EAE is commonly considered restricted; nevertheless, dynamic changes of TCR usage correlate with the course of EAE, resulting in a limited repertoire during early stages of disease activity followed by the recruitment of other T cells reactive against new determinants. Although a broader TCR repertoire mediates the response to MBP in humans, a restricted intra-individual heterogeneity may occur in some MS patients. In the present study we characterize the response to MBP in MS subjects with relapsing remitting disease from two sampling time points 12 months apart. MBP-specific T cell lines (TCL) were first generated from eight MS individuals and two healthy subjects. New TCL were obtained after 12 months from one control and three MS patients whose response, at the first time point, was directed against a single epitope. Interestingly, these three subjects had a stable and mild disease. Few TCL obtained at two time points from the MS individuals recognized the same immunodominant epitope and shared identical TCR Vβ sequences. In the control we could not detect a restriction of the repertoire. These findings suggest that in some MS patients with benign disease a predominant T cell response to a single determinant may be detectable at different moments and is mediated by clonally expanded populations.     

Monocyte-derived dendritic cells: a potential target for therapy in multiple sclerosis (MS)

Monocytes can differentiate into dendritic cells (DC), cells with a pivotal role in both protective immunity and tolerance. Defects in the maturation or function of DC may be important in the development of autoimmune disease. We sought to establish if there were differences in the cytokine (granulocyte-macrophage colony-stimulating factor and IL-4)-driven maturation of monocytes to DC in patients with MS and whether drugs used to treat MS affected this process in vitro. We have demonstrated that there is no defect in the ability of magnetic activated cell sorting (MACS)-purified monocytes from patients with MS to differentiate to DC, but equally they show no tendency to acquire a DC phenotype without exogenous cytokines. Interferon-beta1a prevents the acquisition of a full DC phenotype as determined by light and electron microscopy and by flow cytometry. Methylprednisolone not only prevents the development of monocyte-derived DC but totally redirects monocyte differentiation towards a macrophage phenotype. Evidence is evolving for a role for DC in central nervous system immunity, either within the brain or in cervical lymph nodes. The demonstrated effect of both drugs on monocyte differentiation may represent an important site for immune therapy in MS.     

Artemisinin therapeutic efficacy in the experimental model of multiple sclerosis

Context: The immune system through T-helper 1 (Th1) and Th17 cells play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), whereas the Th2 responses inhibit myelin degeneration. Artemisinin, as an anti-malaria as its agent, has been used widely in the treatment of malaria, shifts the lymphocyte responses from Th1 to Th2.

Objective: In this study, we have investigated the therapeutic effects of artemisinin on the EAE treatment.

Materials and methods: EAE was induced in the inbred C57BL6 mice. High and low doses of prednisolone and artemisinin were injected daily with the control and test groups, respectively. The spleen and the brain of the mice were removed and used for ELISA and histological studies.

Results: The mean weight of mice was significantly (p value?p value p value?p value?Conclusions: Since, artemisinin can shift the immune responses from Th1 to Th2, therefore, it can be helpful in the treatment of MS after more investigation.     

Cellular immune responses in multiple sclerosis patients treated with interferon‐beta

We investigated cellular immune responses at baseline in peripheral blood mononuclear cells (PBMC) of patients with multiple sclerosis (MS) treated with interferon (IFN)‐β and classified into responders and non‐responders according to clinical response criteria. Levels for IFN‐γ, interleukin (IL)‐17A, IL‐17F, IL‐10 and IL‐4 were determined in activated PBMC of 10 responders, 10 non‐responders and 10 healthy controls by cytometric bead arrays. Cytokine levels in cell culture supernatants were similar between responders and non‐responders, and comparable to those obtained in healthy controls. These findings do not support differential cellular immune responses in PBMC at baseline between IFN‐β responders and non‐responders.     

Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry

Acute RSV infection in infancy may produce some asthma-like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type-2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV-infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL-4 and interferon-gamma (IFN-gamma) were measured by flow cytometry. The cells from RSV-infected infants produced more IL-4 and less IFN-gamma than those from healthy controls. IL-4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL-4 production was greatest in infants with mild infections, whereas IFN-gamma production increased with disease severity. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells.     

DNA flow cytometry of thymomas

Forty-seven thymomas have been examined by DNA flow cytometry and results correlated with histology, stage, associated clinical features and survival. Twenty-five cases were DNA diploid, 14 were aneuploid and no results could be obtained for eight cases. The presence of aneuploidy correlated with more advanced (stage II and III) disease and the presence of myasthenia gravis and was more frequent in epithelial predominant thymomas. Tumour recurrence was more frequent in DNA aneuploid tumours, stage II/III disease and epithelial predominant neoplasms. Multifactorial analysis showed that DNA aneuploidy was predictive of tumour recurrence independent of the effects of stage and histology.     

Aberrant T cell responses to myelin antigens during clinical exacerbation in patients with multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of presumed T cell autoimmunity against self myelin. We hypothesized that if myelin-reactive T cells are associated with the disease processes, they may undergo activation and expansion during acute exacerbation. In this study, we examined the precursor frequency, epitope recognition and cytokine profile of myelin-reactive T cells in 14 relapsing/remitting MS patients during exacerbation and remission. The study revealed that T cells recognizing the immunodominant peptides of candidate myelin antigens, including myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein, occurred at increased precursor frequency during acute exacerbation. The T cell responses to MBP focused on the immunodominant regions (residues 83-99 and 151-170) during exacerbation and shifted toward other epitopes of MBP at the time of remission. Furthermore, there was a marked increase in the production of T(h)1 cytokines among T cell lines obtained during exacerbation compared to those obtained during remission. The study demonstrated that myelin-reactive T cells underwent selective activation and expansion during acute MS exacerbation. In contrast, myelin-reactive T cells found during remission in the same patients generally resembled those identified in healthy controls with some discrepancies. The findings suggest potential association of aberrant myelin-reactive T cell responses with acute exacerbation in MS, which may reflect transient activation of myelin-reactive T cell populations of pathogenic potential.     

Multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (TNF-alpha)- and IL-10-secreting blood cells that is corrected by interferon-beta (IFN-beta) treatment

The up-regulated B cell responses detectable in cerebrospinal fluid (CSF) and the augmented myelin antigen-specific T cell responses observed in the CSF as well as systematically in patients with multiple sclerosis (MS) suggest the involvement of cytokines in disease development and perpetuation. Here we report on the parallel involvement of TNF-alpha, IL-6, IFN-gamma and IL-10 in MS and controls, using enzyme-linked immunospot (ELISPOT) assays to detect and enumerate cytokine-secreting mononuclear cells (MNC) prepared from blood and, for IL-6 and IL-10, from CSF without in vitro stimulation. MS is associated with elevated levels of TNF-alpha-secreting blood MNC when compared with levels in groups of control patients with myasthenia gravis (MG) and other neurological diseases (OND) or healthy subjects. This elevation was confined to patients with untreated MS and not present in those examined during ongoing treatment with IFN-beta. Untreated patients with MS had lower numbers of IL-10-secreting blood MNC compared with the three control groups. In patients undergoing treatment with IFN-beta, numbers of IL-10-secreting cells were in the same range as in controls. Normalization of TNF-alpha from elevated, and of IL-10 from decreased levels could be one reason for the beneficial effects of IFN-beta in MS, although it remains to be shown whether these changes reflect phenomena primarily involved in MS pathogenesis or secondary changes. In CSF, levels of IL-10-secreting cells were higher than in blood in both MS and OND, with no difference between these groups. Systemic aberrations of IL-6 and IFN-gamma and of IL-6 in CSF in MS versus controls were only minor, irrespective of treatment with IFN-beta.     

The influences of cytokines as a possible substrate for the psychological effects of immunomodulation therapy in multiple sclerosis

Changes in the emotional background make significant contributions to the clinical picture of multiple sclerosis (MS), seriously reducing patients' quality of life and hindering adaptation during the process of rehabilitation. The relationship between mental state and disease activity has been discussed in the world literature in recent years and the opinion that mental disorders in MS are pathogenetically based is increasingly widespread. The present article discusses the potential role of substances mediating immune responses-cytokines-in the effects of changes in the functional activity of the nervous system and psychoemotional status. The question of their likely involvement in the pathogenesis of the side effects of immunomodulatory therapy of MS with beta-interferons and glatiramer acetate is discussed. Translated from Zhurnal Nevrologii i Psikhiatrii imeni S. S. Korsakova, Multiple Sclerosis, Supplement, No. 4, pp. 36–41, 2007.     

Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4: detection by flow cytometry

Background IgE-stimulated cultured basophils from atopic subjects are capable of secreting interleukin-4 (IL-4). We describe a flow-cytometric technique which identified intracellular IL-4 in unstimulated basophils unseparated from peripheral blood mononuclear cells (PBMC) in both atopic (AT) and nonatopic (NC) volunteers.
Methods Freshly isolated PBMC were fixed in 4% paraformaldehyde (PFA). Surface staining with 22E7, a noncompetitive anti-Fc-a antibody, allowed identification of basophils. Permeabilization by 0.1 % saponin allowed staining of intracellular cytokines with specific monoclonal antibodies (mAbs). Two series of experiments utilizing different protocols and anticytokine mAbs were performed. The first protocol required a two-stage fluorochrome staining technique. The availability of fluorochrome-conjugated mAbs allowed a simpler, one-stage labelling procedure for the second protocol.
Results With the first protocol, IL-4 (but not IFN-y), immunoreactivity was detectable in a majority (median 77%) of peripheral blood basophils from both AT and NC subjects (N = 8). Basophil IL-4 immunoreactivity was again evident in experiment 2 but did not differ significantly between AT and NC subjects - either evaluated as percentage of IL-4+ basophils (AT median=66%, NC median=38.4%, P=0.41) or lL-4-specific mean fluorescence (AT median=0.85, NC median=0.3, P=0.07).
Conclusions This simple technique allowed the study of intracellular cytokine expression in unstimulated blood basophils. It demonstrated constitutive basophil expression of lL-4 (but not IFN-y) in all subjects, with no significant increases in atopies.     

Circulating gamma/delta T lymphocytes from systemic sclerosis (SSc) patients display a T helper (Th) 1 polarization

Systemic sclerosis (SSc) is a connective tissue disease in which immune system activation is evidenced by high levels of different cytokines in the sera and/or in the supernatants of cultured peripheral blood mononuclear cells (PBMC) and by the presence of specific autoantibodies. gamma/delta T cells accumulate in the lung and the skin of SSc patients suggesting their potential role in the development and maintenance of the disease. The aim of this study was to assess cytokine production and cytotoxic activity of circulating gamma/delta T lymphocytes obtained from SSc patients and to evaluate their potential role during this disorder. Our results showed that both the proportion and the absolute number of IFN-gamma gamma/delta-producing cells (i.e. displaying a Th1 polarization) in SSc was significantly higher than either the proportion and the absolute number of IL-4 gamma/delta-producing cells in SSc or the proportion and the absolute number of IFN-gamma gamma/delta-producing cells in healthy controls (P < 0.05 for both groups). Furthermore, the cytotoxic activity of enriched gamma/delta T cells was significantly increased in SSc patients compared with controls. The results concerning the Vdelta1+ T cell subset paralleled those of total gamma/delta T lymphocytes. In contrast, alpha/beta T cells from SSc and control subjects displayed Th2 cytokine production. All these findings were independent of both disease subset and clinical status. Our data demonstrate that, although SSc is generally considered a Th2 autoimmune disease, Th1 polarization of gamma/delta T cells and an increase in their cytotoxic activity is observed in SSc, suggesting that gamma/delta T cells could have a relatively autonomous role in the pathogenesis in this disease.