Transforming growth factor-β1 suppresses autoantigen-induced expression of pro-inflammatory cytokines but not of interleukin-10 in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is associated with high levels of circulating T lymphocytes that respond to the myelin antigens myelin basic protein (MBP) and proteolipid protein (PLP) by producing various cytokines including interferon-γ (IFN-γ) that makes MS worse and transforming growth factor-β (TGF-β), an endogenously produced immunosuppressant that might act beneficially. To further define the role of TGF-β in MS, we examined the effects of recombinant TGF-β1 (rTGF-β1) on autoantigen-mediated regulation of cytokines in MS and myasthenia gravis (MG). Blood mononuclear cells (MNC) were cultivated with or without rTGF-β1, and with or without autoantigen or the recall antigen PPD. MNC expressing cytokine mRNA were detected after in situ hybridization with radiolabeled cDNA oligonucleotide probes. Femtogram concentrations of rTGF-β1 suppressed MBP-, PLP- and PPD-induced upregulation of IFN-γ, IL-4, IL-6, tumor necrosis factor-α (TNF-α), TNF-α and perforin in MS, and acetylcholine receptor (AChR)-induced augmentation of these pro-inflammatory cytokines in MG, but had no effects on autoantigen- or PPD-induced expression of IL-10 or TGF-β itself. rTGF-β1 also suppressed numbers of myelin antigen-reactive IFN-γ- and IL-4-secreting cells in MS and AChR-reactive IFN-γ and IL-4 secreting cells in MG. The selective suppressive effects of TGF-β1 on autoantigen-induced upregulation of pro-inflammatory cytokines makes TGF-β1 attractive as a treatment alternative in MS and MG.     

Increased transforming growth factor-β, interleukin-4, and interferon-γ in multiple sclerosis

The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the T-helper type 1 (Th1) cell–associated interferon-σ (IFN-σ), the Th2 cell–related interleukin-4 (IL-4), and the immune response–downregulating cytokine transforming growth factor-β (TGF-β), but proof for their involvement in MS has been lacking. By adopting in situ hybridization with complementary DNA oligonucleotide probes for human IFN- IL-4, and TGF-β, the expression of mRNA for these cytokines was detected in mononuclear cells (MNC) from blood and cerebrospinal fluids. Strongly elevated levels of MNC expressing all three cytokines were found in peripheral blood and at even higher frequencies in cerebrospinal fluid from untreated patients with MS and optic neuritis, i.e., a common first manifestation of MS, compared with patients with other neurological diseases and healthy subjects. In MS and optic neuritis, IL-4 mRNA expressing cells predominated, followed by TGF-β– and IFN-σ–positive cells. Control patients with myasthenia gravis had similarly elevated levels of IFN-σ and TGF-β mRNA expressing blood MNC but lower numbers of IL-4–positive cells. No or slight disability of MS was associated with high levels of TGF-β mRNA expressing cells, while MS patients with moderate or severe disability had high levels of IFN-σ–positive cells. IFN-σ and TGF-β may have opposing effects in MS, and treatments inhibiting IFN-σ and/or promoting TGF-β might ameliorate MS.     

Cytokine phenotype of human autoreactive T cell clones specific for the immunodominant myelin basic protein peptide (83–99)

Experimental allergic encephalomyelitis (EAE), an animal model resembling multiple sclerosis (MS), is mediated by myelin antigen-specific CD4+ T cells secreting cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-β (TNF-β), and the proinflammatory cytokine TNF-α—all associated with the T-helper-1 (Th1) T cell subset. Based on numerous similarities between MS and EAE, it has been postulated that Th1-like T cells are involved in the pathogenesis of MS. Production of proinflammatory cytokines such as IFN-γ and, in particular, TNF-α/β by autoreactive T cells is considered crucial for the initiation and amplification of inflammatory brain lesions and possibly also for direct myelin damage. In contrast, regulatory cytokines such as interleukin-4 (IL-4), IL-10, and IL-13, which are associated with the Th2-like phenotype, may play a role in the resolution of relapses. Although the human T cell response to myelin basic protein (MBP) is well characterized in terms of antigen specificity, HLA restriction, and T cell-receptor (TCR) usage, little is known about the cytokine pattern of these autoreactive T cells. To gain such information, conditions for studying cytokine secretion by human autoreactive T cell clones (TCC) were established. The cytokine secretion profile of human autoreactive CD4+ TCC, specific for myelin basic protein peptide (83–89) [MBP(83–99)], a candidate autoantigen in MS, was investigated. Our results show that TCC cytokine production in long-term culture was stable. In addition, the correlation of various cytokines within specific TCC revealed differences compared to murine T cells. The comparison of 30 human MBP(83–99)-specific TCC demonstrated heterogeneity in cytokine secretion, with a continuum between Th1- and Th2-like cells rather than distinct Th1 or Th2 subsets. These data are important for further investigation of the potential role of cytokines in the inflammatory process of MS, and provide a powerful tool to investigate therapeutic interventions with respect to their influence on cytokine secretion of autoreactive T cells. © 1996 Wiley-Liss, Inc.     

Review: cytokines and the pathogenesis of multiple sclerosis

Perivascular accumulation of mononuclear cells (MNCs) in the central nervous system (CNS) and high levels of myelin autoantigen-reactive T cells in blood and further enriched in cerebrospinal fluid (CSF) are characteristic for multiple sclerosis (MS) and suggest a role for immunoregulatory cytokines in MS pathogenesis. The difficulties inherent to measurements of cytokine concentrations in body fluids have been partly overcome by adopting techniques allowing cytokine determinations on cellular level. MS is associated with the parallel up-regulation of proinflammatory [interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), lymphotoxin-α, and interleukin (IL)-12] and immune response-down-regulating [transforming growth factor-β (TGF-β), IL-10] cytokines systemically. A preferential up-modulation of TNF-α and lymphotoxin-α is observed in clinical exacerbations and of TGF-β and IL-10 in remissions. The B cell-stimulating IL-4 and IL-6 are also up-regulated in MS, as is the cytolysis-promoting perforin. Cytokine production is elevated to an even higher degree in the CSF than systemically, underlining the autonomy of the immune responses in the CSF. All cytokine abnormalities are demonstrable already in very early MS, manifested by acute unilateral optic neuritis associated with more than two MS-like lesions on brain magnetic resonance imaging and oligoclonal IgG bands in CSF. The cytokine abnormalities hitherto observed are not MS specific, because they can be found in other inflammatory CNS diseases, e.g., aseptic meningitis and even noninflammatory neurological diseases like stroke. The influence on cytokine profiles, e.g., suppressing proinflammatory cytokines and promoting TGF-β and IL-10, could be an important way to identify new and promising treatments of MS. © 1996 Wiley-Liss, Inc.     

Interferon γ, interleukin 4 and transforming growth factor β in experimental autoimmune encephalomyelitis in lewis rats: Dynamics of cellular mrna expression in the central nervous system and lymphoid cells

The potential role of certain important immunoregulatory and effector cytokines in autoimmune neuroinflammation have been studied. We have examined the expression of mRNA, with in situ hybridization, of interferon -γ (IFN-γ), interleukin 4 (IL-4) and transforming growth factor β (TGF-β) both in sections of spinal cords and the antigen-induced expression of these cytokines by lymphoid cells after stimulation with a dominant encephalitogenic peptide of MBP (MBP 63–88) during the course of actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. In spinal cords, the target organ in EAE, cells expressing mRNA for IFN-γ, first appeared at the onset of clinical signs, i.e., day 10 postimmunization (p.i.), peaked at the height of disease (day 13 p.i.), and then gradually decreased concomitant with recovery. Very few IL-4 mRNA-expressing cells appeared in the spinal cord with no clear relation to clinical signs or histopathology. In contrast, expression of mRNA for TGF-β did not increase until day 13 p.i., at height of the disease, shortly preceding recovery. These data are consistent with a disease upregulating role of IFN-γ, while TGF-β may act to limit central nervous system (CNS) inflammation. In lymphoid organs, primed MBP 63–88 reactive T cells showed an interesting time-dependent evolution of their cytokine production in vitro. Thus, early after immunization there was a conspicuous MBP 63–88-induced production of both IFN-γ and IL-4. Such cells may act in the initiation and promotion of the disease. Later, in the recovery phase, MBP 63–88 induced lymphoid cells to TGF-β production. Thus, an autoantigen-specific production of TGF-β occurred during EAE and hypothetically such a mechanism may serve to downregulate aggressive autoimmunity systemically. © 1995 Wiley-Liss, Inc.     

Cytokines and the pathogenesis of myasthenia gravis

Myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG) are caused by autoantibodies against nicotinic acetylcholine receptor (AChR) in skeletal muscle. The production of anti-AChR antibodies is mediated by cytokines produced by CD4⁺ and CD8⁺ T helper (Th) cells. Emerging investigations of the roles of cytokines in MG and EAMG have revealed that the Th2 cell related cytokine interleukin 4 (IL-4), an efficient growth promoter for B-cell proliferation and differentiation, is important for anti-AChR antibody production. IL-6 and IL-10 have similar effects. The Th1 cytokine IFN-γ is important in inducing B-cell maturation and in helping anti-AChR antibody production and, thereby, for induction of clinical signs and symptoms. Results from studies of time kinetics of cytokines imply that IFN-γ is more agile at the onset of EAMG, probably being one of the initiating factors in the induction of the disease, and IL-4 may be mainly responsible for disease progression and persistance. Even though other Th1 cytokines like IL-2, tumor necrosis factor α (TNF-α), and TNF-β as well as the cytolytic compound perforin do not directly play a role in T-cell-mediated help for anti-AChR antibody production, they are actually involved in the development of both EAMG and MG, probably by acting in concert with other cytokines within the cytokine network. In contrast, transforming growth factor β (TGF-β) exerts immunosuppressive effects which include the down-regulation of both Th1 and Th2 cytokines in MG as well as EAMG. Suppressive effects are also exerted by interferon α (IFN-α). Based on elucidation of the role of cytokines in EAMG and MG, treatments that up-modulate TGF-β or IFN-α and/or suppress cytokines that help B-cell proliferation could be useful to improve the clinical outcome. © 1997 John Wiley & Sons, Inc. Muscle Nerve, 20, 543–551, 1997     

Interleukin-1β (IL-1β)-induced modulation of the hypothalamic IL-1β system,tumor necrosis factor-α, and transforming growth factor-β1 mRNAs in obese (fa/fa) and lean (Fa/Fa) Zucker rats: Implications to IL-1β feedback systems and cytokine–cytokine interactions

Interleukin-1β (IL-1β) induces anorexia, fever, sleep changes, and neuroendocrine alterations when administered into the brain. Here, we investigated the regulation of the IL-1β system (ligand, receptors, receptor accessory protein, and receptor antagonist), tumor necrosis factor-α (TNF-α), transforming growth factor (TGF)-β1, and TGF-α mRNAs in the hypothalamus of obese (fa/fa) and lean (Fa/Fa) Zucker rats in response to the intracerebroventricular microinfusion of IL-1β (8.0 ng/24 hr for 72 hr, a dose that yields estimated pathophysiological concentrations in the cerebrospinal fluid). IL-1β increased IL-1β, IL-1 receptor types I and II (IL-1RI and IL-1RII), IL-1 receptor accessory protein soluble form (IL-1R AcP II), IL-1 receptor antagonist (IL-1Ra), TNF-α, and TGF-β1 mRNAs in the hypothalamus from obese and lean rats. IL-1β–induced IL-1β system and ligand (IL-1β, TNF-α, and TGF-β1) mRNA profiles were highly intercorrelated in the same samples. Levels of membrane-bound IL-1R AcP and TGF-α mRNAs did not change. Heat-inactivated IL-1β had no effect. The data suggest 1) the operation of an IL-1β feedback system (IL-1β/IL-1RI/IL-1R Acp II/IL-1RII/IL-1Ra) and 2) potential cytokine–cytokine interactions with positive (IL-1β ←→ TNF-α) and negative (TGF-β1 → IL-1β/TNF-α) feedback. Dysregulation of the IL-1β feedback system and the TGF-β1/IL-1β-TNF-α balance may have implications for neurological disorders associated with high levels of IL-1β in the brain. J. Neurosci. Res. 49:541–550, 1997. © 1997 Wiley-Liss, Inc.     

MHC-restriction,cytokine profile,and immunoregulatory effects of human T cells specific for TCR Vβ CDR2 peptides: Comparison with myelin basic protein-specific T cells

HLA-DR2+ patients with multiple sclerosis (MS) that respond to vaccination with TCR Vβ5.2-38-58 peptides have increased frequencies of TCR peptide-specific T cells, reduced frequencies of myelin basic protein (MBP)-specific T cells, and a better clinical course than non-responders. To evaluate possible network regulation of MBP responses by TCR peptide-specific T cells, we compared properties of both cell types. Both MBP- and TCR peptide-specific T cell clones were CD4⁺ and predominantly HLA-DR restricted. HLA-DR2, which is in linkage disequilibrium in MS patients, preferentially restricted TCR peptide-specific clones as well as MBP-specific responses in HLA-DR2 and DR2,3+ donors. Within the DR2 haplotype, however, both DRβ1*1501 and DRβ5*0101 alleles could restrict T cell responses to Vβ CDR2 peptides, whereas responses to MBP were restricted only by DRβ5*0101. TCR peptide-specific clones expressed message for Th2 cytokines, including IL-4, IL-5, IL-6, IL-10, and TGF-β, whereas MBP-specific T cell clones expressed the Th1 cytokines IFN-γ and IL-2. Consistent with the Th2-like cytokine profile, TCR peptide-specific T cell clones expressed higher levels of CD30 than MBP-specific T cells. Culture supernatants from TCR peptide-specific T cell clones, but not from MBP- or Herpes simplex virus-specific T cells, inhibited both proliferation responses and cytokine message production of MBP-specific T cells. These results demonstrate distinct properties of MBP and TCR peptide-specific T cells, and indicate that both target and bystander Th1 cells can be inhibited by Th2 cytokines secreted by activated TCR peptide-specific T cells. These data support the rationale for TCR peptide vaccination to regulate pathogenic responses mediated by oligoclonal T cells in human autoimmune diseases. © 1996 Wiley-Liss, Inc.     

Organ-specific autoantigens induce transforming growth factor-β mRNA expression in mononuclear cells in multiple sclerosis and myasthenia gravis

Multiple sclerosis (MS) is characterized by patchy accumulations of inflammatory cells combined with demyelination. There are mononuclear cells in blood and cerebrospinal fluid of patients with MS that produce interferon-γ and interleukin-4 in response to myelin basic protein (MBP) and proteolipid protein (PLP). Here we describe autoantigen-induced production of transforming growth factor-β (TGF-β). This multifunctional cytokine has inhibitory effects on the growth, differentiation, and effector functions of activated T cells. Blood and cerebrospinal fluid cells were exposed in short-term cultures to MBP and PLP and, after hybridization with complementary DNA oligonucleotide probes, they were evaluated for TGF-β mRNA expression. Patients with MS had higher numbers of MBP- and PLP-responsive TGF-β mRNA expressing cells in blood compared with control patients with other neurological diseases or myasthenia gravis and a five- and threefold further increment in their cerebrospinal fluid. In blood of patients with myasthenia gravis, where the acetylcholine receptor (AChR) is a target for autoaggressive immunity, there were increased levels of AChR-responsive TGF-β mRNA expressing cells. Thymectomized myasthenia gravis patients showed higher levels of TGF-β mRNA expressing cells compared with patients not thymectomized. Numbers of cells responding to AChR in MS and MBP in myasthenia gravis did not differ from numbers found in absence of antigen. Patients with other neurological diseases showed infrequent and low responses to MBP, PLP, and AChR. Diseases with presumed autoimmune pathogenesis are associated with organ-specific autoantigen-induced TGF-β production, which is increased after thymectomy.     

The adhesion molecule and cytokine profile of multiple sclerosis lesions

The expression of the adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and their respective receptors on leukocytes, very late activation antigen-4 (VLA-4) and lymphocyte function–associated antigen-1 (LFA-1), together with a selection of proinflammatory and immunomodulatory cytokines (interleukin [IL]-1, IL-2, IL-4, IL-10, tumor necrosis factor-α [TNF-α], transforming growth factor-β [TGF-β], and interferon-γ [IFN-γ]) was examined by immunocytochemistry in multiple sclerosis (MS) lesions of different ages and compared with central nervous system (CNS) tissue from other neurological diseases, both inflammatory and noninflammatory, and normal CNS tissue. These molecules play key roles in lymphocytic infiltration and interactions during tissue inflammation and are in large part normally not expressed by CNS cells. High levels of expression of all the molecules tested were found in MS, particularly in chronic active lesions. Positivity for all molecules was also seen in other neurological diseases, even in noninflammatory conditions. There was some suggestion that the VCAM-1/VLA-4 adhesion pathway was expressed at higher levels in chronic MS lesions, while ICAM-1/LFA-1 was used more uniformly in lesions of all ages. Of the cytokines examined, there was increased expression of TNF-α and IL-4 in MS; this was found to be statistically significant when compared with noninflammatory neurological diseases. The expression of most adhesion molecules and some cytokines was negligible in normal CNS tissue although low-level reactivity for ICAM-1 TGF-β, IL-4, TNF-α, and IL-10 was detected, perhaps indicative of immunoregulatory mechanisms. Microglial cells and astrocytes were the major CNS cell types expressing cytokines. The results indicate a potential in the CNS for widespread induced expression of molecules involved in the inflammatory cascade. No adhesion or cytokine molecule or pattern of expression unusual for MS was apparent.     

Transforming growth factor–beta inhibition of cytokine-induced vascular cell adhesion molecule-1 expression in human astrocytes

Leukocyte transmigration across the blood-brain barrier (BBB) is a cardinal feature of central nervous system (CNS) inflammation. Astrocytes form an integral part, both structurally and functionally, of the BBB. Vascular cell adhesion molecule-1 (VCAM-1), a member of the immunoglobulin gene superfamily, is involved in extravasation into inflamed tissues and activation of T-lymphocytes. In this study, we investigated the role of TGF-β, an immunosuppressive cytokine, in regulating cytokine-induced VCAM-1 expression in astrocytes. Human astroglioma cell lines and primary human fetal astrocytes were examined for VCAM-1 gene expression after treatment with proinflammatory cytokines (TNF-α, IL-1β, IFN-γ) in the absence or presence of TGF-β. Astroglioma cell lines as well as primary human fetal astrocytes expressed low levels of VCAM-1 constitutively, and the proinflammatory cytokines induced marked increases in VCAM-1 expression, particularly TNF-α and IL-1β. The inclusion of TGF-β1 or TGF-β2 with the proinflammatory cytokines inhibited VCAM-1 gene expression to varying degrees (33–93%) in all the astroglioma cell lines and primary fetal cells. These results indicate that TGF-β is an important regulator of cytokine induced VCAM-1 expression on astrocytes and may prove useful clinically in controlling CNS inflammation. GLIA 22:171–179, 1998. © 1998 Wiley-Liss, Inc.     

Influence of IFN-β1b (Betaferon) on cytokine mRNA profiles in blood mononuclear cells and plasma levels of soluble VCAM-1 in multiple sclerosis

Inflammatory cell infiltration within the central nervous system (CNS) and upregulation of both pro- and anti-inflammatory cytokines are characteristic for multiple sclerosis (MS). Treatment with interferon-β1b (IFN-β1b) reduces the number and severity of MS relapses. To examine whether treatment with IFN-β1b affects levels of cytokine mRNA expressing blood mononuclear cells (MNC) we employed in-sit hybridization with synthetic oligonucleotide probes to detect and enumerate IFN-γ, TNF-α, IL-10, TGF-β and perforin mRNA expressing cells in MS patients before treatment with IFN-β1b and during tretmetn for 3–6 weeks and for 3–6 monts. Numbers of blood MNC spontaneously expressing TNF-α and IL-10 mRNA were lower after 3–6 months of treatment, while numbers of IFN-γ, TGF-β and perforin mRNA expressing MNC were not affected by treatment. IFN-β1b had no influence on levels of MBP-reactive IFN-γ, TNF-α, TGF-β, IL-10 or perforin mRNA expressing blood MNC determined after 3–6 weeks 3–6 months of treatment. Parallel measurements of plasma concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) revealed elevated levels after 3–6 weeks of treatment and these levels remained higher after 3–6 months of treatment. The results suggest that IFN-β1b treatment upregulates plasma levels of sVCAM-1, but has little effects on numbers of blood MNC expressing mRNA of the pro- and anti-inflammatory cytokines under study.     

Cytokine production in the central nervous system of Lewis rats with experimental autoimmune encephalomyelitis: dynamics of mRNA expression for interleukin-10, interleukin-12, cytolysin,tumor necrosis factor α and tumor necrosis factor β

The kinetics of mRNA expression in the central nervous system (CNS) for a series of putatively disease-promoting and disease-limiting cytokines during the course of experimental autoimmune encephalomyelitis (EAE) in Lewis rats were studied. Cytokine mRNA-expressing cells were detected in cryosections of spinal cords using in situ hybridization technique with synthetic oligonucleotide probes. Three stages of cytokine mRNA expression could be distinguished: (i) interleukin (IL)-12, tumor necrosis factor (TNF)-β (=lymphotoxin-α) and cytolysin appeared early and before onset of clinical signs of EAE; (ii) TNF-α peaked at height of clinical signs of EAE; (iii) IL-10 appeared increasingly at and after clinical recovery. The early expression of IL-12 prior to the expression of interferon-γ (IFN-γ) mRNA shown previously is consistent with a role of IL-12 in promoting proliferation and activation of T helper 1 (Th1) type cells producing IFN-γ. The TNF-β mRNA expression prior to onset of clinical signs favours a role for this cytokine in disease initiation. A pathogenic effector role of TNF-α was suggested from these observations that TNF-α mRNA expression roughly paralleled the clinical signs of EAE. This may be the case also for cytolysin. IL-10-expressing cells gradually increased to high levels in the recovery phase of EAE, consistent with a function in down-regulating the CNS inflammation. From these data we conclude that there is an ordered appearance of putative disease-promoting and -limiting cytokines in the CNS during acute monophasic EAE.     

丙戊酸对实验性自身免疫性神经炎大鼠保护作用的机制

目的观察丙戊酸(VAP)对实验性自身免疫性神经炎(EAN)大鼠的保护作用及其机制。方法实验大鼠随机分为VAP高剂量组、VAP低剂量组、EAN模型组、正常组,应用P2 57-81多肽与完全弗氏佐剂的混合液诱导EAN模型。VAP于免疫当天至第15d每天腹腔内注射。观察各组大鼠发病情况和坐骨神经组织病理学变化,检测外周血中Th17细胞和Foxp3+Treg细胞含量,检测淋巴结中TNF-α、IFN-γ、IL-17、TGF-βmRNA表达。结果 VAP高剂量组的最初发病时间迟于EAN组(P<0.05),其高峰期临床评分显著低于EAN组(P<0.05),坐骨神经炎性细胞浸润较EAN组明显减少;VAP高剂量组和低剂量组外周血中Th17细胞比例较EAN组显著减少(P<0.05),Foxp3+Treg细胞比例较EAN组显著增加(P<0.05),淋巴结中促炎细胞因子TNF-α、IFN-γ及IL-17mRNA表达与EAN组比较明显下降(P<0.05),VAP高剂量组抑炎细胞因子TGF-βmRNA表达与EAN组比较明显升高(P<0.05)。结论 VAP对EAN有治疗作用,这种作用可能与其能够增加Foxp3+Treg细胞和抑炎细胞因子TGF-β含量、减少TH17细胞含量和促炎细胞因子的表达有关。     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

Interleukin 1-β- and tumor necrosis factor-α-mediated regulation of ICAM-1 gene expression in astrocytes requires protein kinase C activity

Several adhesion molecules including intracellular adhesion molecule-1 (ICAM-1) are expressed by astrocytes, the predominant glial cell of the central nervous system (CNS). Such molecules are important in the trafficking of leukocytes to sites of inflammation, and in lymphocyte activation. ICAM-1 is constitutively expressed by neonatal rat astrocytes, and expression is enhanced by the proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), with IL-1β and TNF-α being the strongest inducers. In this study, we have examined the nature of the second messengers involved in ICAM-1 gene expression induced by the cytokines IL-1β and TNF-α. Our results indicate that stimuli related to protein kinase C (PKC) such as the phorbol ester phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 increase ICAM-1 mRNA expression, whereas cyclic nucleotide analogs and PKA agonists have no effect. Pharmacologic inhibitors of PKC such as H7, H8, and calphostin C inhibit ICAM-1 gene expression inducible by IL-1β and TNF-α. Prolonged treatment of astrocytes with PMA results in a time-dependent downregulating of the PKC isoforms α, δ, and ?, and a concomitant diminution of ICAM-1 mRNA expression induced by IL-1β, TNF-α, and PMA itself at specific time points post-PMA treatment. These data, collectively, demonstrate a role for various PKC isoforms in IL-1β and TNF-α enhancement of ICAM-1 gene expression in rat astrocytes. © 1995 Wiley-Liss, Inc.     

Additive effects of copolymer-1 and interferon β-1b on the immune response to myelin basic protein

Copolymer-1 (Cop-1) inhibits the T cell response to myelin basic protein (MBP), suppresses experimental autoimmune encephalomyelitis in many animal species, and was recently shown to be effective in the treatment of multiple sclerosis (MS). Interferon beta-1b (IFN-β), an immune modulator with no antigenic specificity, is already approved for treatment of relapsing-remitting MS. We investigated the combined effect of these two agents on the cellular immune response to MBP. Antigen-specific Th1-like cell lines were generated from two healthy individuals with different MHC phenotypes. Cop-1 inhibited the proliferation of all MBP-specific lines but had no suppressive effect on tuberculin (PPD) or tetanus toxoid (TT)-specific T cell lines from either donor, while IFN-β non-specifically reduced proliferation of all T cell lines. When combined in vitro, Cop-1 and IFN-β had additive suppressive effects on proliferation of MBP-specific T cell lines, with 70–100% inhibition depending on the concentration of antigen. Synthesis of the pro-inflammatory cytokines interleukin-2 and IFN-γ by MBP-specific lines was also inhibited additively (up to 100%). When antigen-presenting cells (APC) were pretreated with Cop-1, IFN-β or both, T cell proliferation was inhibited in the same additive pattern, even though the inhibitors were not present in culture, indicating that they acted primarily through modulation of APC function. Additive effects were not found with PPD- or TT-specific cell lines. Pretreatment of APC with IFN-β resulted in dose-dependent reduction in HLA-DR and HLA-DQ expression, which paralleled inhibition of T cell proliferation. Pretreatment of APC with Cop-1 had no effect on MHC class II expression. Reductions in proliferation and MHC class II expression were not the result of toxicity, as viability of T cell lines and APC was not altered by exposure to Cop-1 or IFN-β. These results suggest that inhibition of proliferation and pro-inflammatory cytokine production by MBP-specific T cell lines may be mediated through complementary effects on antigen presentation, with IFN-β down-regulating the expression of MHC class II molecules on APC, and Cop-1 competing with MBP for antigen binding sites on the remaining MHC molecules.     

Interleukin-1α (IL-1α), IL-1β, IL-1 receptor type I, IL-1 receptor antagonist, and TGF-β1 mRNAs in pediatric astrocytomas, ependymomas, and primitive neuroectodermal tumors

Interleukin-1α (IL-1α), IL-1β, interleukin-1 receptor type I (IL-1RI, signaling receptor), and IL-1 receptor antagonist (IL-1Ra, endogenous inhibitor) are pivotal components of the IL-1 system. IL-1 and other cytokines induced by IL-1, such as TGF-β1, may participate in the growth of various tumor cells. In children, primary nervous system tumors represent the most common solid malignancy. We investigated the levels of IL-1α, IL-1β, IL-1RI, IL-1Ra, and TGF-β1 mRNAs in pediatric astrocytomas (n=19), ependymomas (n=13), and primitive neuroectodermal tumors (n=22) using sensitive and specific RNase protection assays. The data show a significant distinct cytokine mRNA profile among brain tumor types. Pilocytic, nonpilocytic, and anaplastic astrocytomas have significant increased levels of IL-1β, IL-1RI, and TGF-β1 mRNAs, but low levels of IL-1Ra mRNA; this may have implications for an IL-1β feedback system and IL-1β?TGF-β1 interactions in astrocytomas. Ependymomas show increased levels of IL-1α and IL-1β mRNAs associated with low levels of IL-1Ra mRNA; primitive neuroectodermal tumors do not exhibit increased levels of any cytokine component examined. The data also suggest that a dysregulation of the balance between stimulatory and inhibitory cytokines may be involved in the growth and development of brain tumors via autocrine/paracrine mechanisms.