Increased levels of interferon-gamma (IFN-gamma), IL-4 and transforming growth factor-beta (TGF-beta) mRNA expressing blood mononuclear cells in human HIV infection.

Evidence has been presented for the involvement of IFN-gamma, IL-4 and TGF-beta in AIDS. Measured plasma levels may, however, poorly reflect in vivo production, since cytokines act auto- and paracrinally and have very short half life in plasma. In situ hybridization with complementary DNA oligonucleotide probes was used to enumerate blood mononuclear cells expressing cytokine messenger RNA (mRNA). HIV-infected patients had elevated blood levels of cells expressing each of the cytokines, with predominance for cells expressing TGF-beta mRNA. All AIDS patients included had elevated numbers of IL-4 mRNA-expressing cells, and levels of cells expressing this cytokine correlated inversely with counts of CD4+ cells in blood, reflecting the involvement of Th2-like cells in later stages of HIV infection. The described approach should be useful in further studies of cytokines in HIV infection and other diseases.     

Tumor necrosis factor α-308 alleles in multiple sclerosis and optic neuritis

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is believed to play an important role in multiple sclerosis (MS) pathogenesis. A bi-allelic polymorphism in the TNF-α promoter region (TNFα-308), has been reported to influence levels of TNF-α production. In the present study, we investigated the TNFα-308 polymorphism in 93 patients with MS, 17 patients with optic neuritis (ON) and 95 healthy individuals using an allele-specific PCR technique. Allelic genotype was compared with TNF-α mRNA expression levels and HLA class II phenotypes. No significant difference regarding the TNFα-308 polymorphism was observed between MS patients and controls. Specifically, the less common allele, TNF2, which is associated with higher expression levels of TNF-α, was somewhat less frequent among MS patients. In fact, analysis of 19 patients homozygous for the MS associated HLA-DR-DQ haplotype HLA-Dw2 showed that this haplotype does not carry the TNF2 allele. In addition, in 47 patients, the TNF-α alleles did not correlate with expression levels measured as numbers of TNF-α expressing cells. Thus, we found no evidence for an important role of TNFα-308 polymorphism for genetic susceptibility to MS.     

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα‐chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll‐like receptor (TLR) ligand activation of TCR‐transgenic murine dNKT cells. IFN‐γ production by dNKT cells required dendritic cells (DC), cell‐to‐cell contact and presence of TLR ligands. TLR‐stimulated DC activated dNKT cells to secrete IFN‐γ in a CD1d‐, CD80/86‐ and type I IFN‐independent manner. In contrast, a requirement for IL‐12p40, and a TLR ligand‐selective dependence on IL‐18 or IL‐15 was observed. TLR ligand/DC stimulation provoked early secretion of pro‐inflammatory cytokines by both CD62L⁺ and CD62L^? dNKT cells. However, proliferation was limited. In contrast, TCR/co‐receptor‐mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L^? dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co‐receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.     

Interleukin-12 and Perforin mRNA Expression is Augmented in Blood Mononuclear Cells in Multiple Sclerosis

Cytokines are suggested to orchestrate an abnormal immune response in multiple sclerosis (MS). The regulatory cytokine interleukin (IL)-12 induces T-helper (Th) cell switch to the Th1 type and the production by cytotoxic T cells of perforin, a cell lysis-inducing factor. It has been suggested that Th1-like cytokines may promote the development of MS, and the production of perforin to induce oligodendrocyte damage. In-situ hybridization with radiolabelled synthetic oligonucleotide probes was used to detect and enumerate mononuclear cells (MNC) expressing IL-12 and perforin mRNA in blood and cerebrospinal fluid (CSF) from patients with MS and controls. Plasma and CSF levels of IL-12 (p70) were evaluated by ELISA. Higher numbers of IL-12 and perforin mRNA-expressing MNC were registered in blood in MS and also in controls with aseptic meningoencephalitis (AM) compared to healthy subjects. There were a few patients with other non-inflammatory neurological diseases who also had high levels of IL-12 or perforin mRNA expressing blood MNC. A parallel elevation was observed for IL-12 (p70) concentrations in plasma. In the MS patients' CSF, there was a further augmentation of IL-12 mRNA expressing MNC. To evaluate autoantigen-induced IL-12 and perforin mRNA expression, blood MNC were cultivated ± myelin basic protein (MBP), a candidate autoantigen in MS. Higher numbers of MBP-reactive IL-12 and perforin mRNA expressing blood MNC were detected in MS than controls. The augmentation of both IL-12 and perforin in MS might reflect ongoing inflammatory processes in MS and could represent targets for future treatments.     

Cytokine mRNA profiles in mononuclear cells in acute aseptic meningoencephalitis.

Cytokines are important modulators of inflammation and immune responses. Using in situ hybridization with radiolabelled cDNA oligonucleotide probes, we studied the expression of mRNA encoding the cytokines gamma interferon (IFN-gamma), interleukin 4 (IL-4), IL-6, IL-10, transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), lymphotoxin, and perforin in mononuclear cells (MNC) from blood and cerebrospinal fluid (CSF) of patients with acute aseptic meningoencephalitis (AM) and from blood of healthy controls. Patients in the acute phase of AM had elevated numbers of IFN-gamma mRNA-expressing cells in the blood compared with that of controls and higher numbers of IFN-gamma mRNA-expressing cells in their CSF compared with that of convalescent-phase patients, which is in accordance with the antiviral effects of this cytokine. Upregulation of IL-4, IL-6, and IL-10 was found in convalescent-phase patients, which is consistent with the longstanding B-cell response found in AM. TGF-beta and perforin were upregulated in both stages of AM, while the numbers of blood and CSF MNC expressing cytokine mRNA of the TNF family (TNF-alpha and lymphotoxin) did not differ between patients with AM and controls. An even higher elevation in CSF was noticed for MNC expressing most of the cytokines, particularly IL-4 and TGF-beta, reflecting the autonomy of the immune response in the CSF. The definition of cytokine profiles in AM, a self-limiting and benign disease, provides a foundation for future comparisons with other infectious and inflammatory nervous system diseases.     

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     

Stromal cells and osteoclasts are responsible for exacerbated collagen‐induced arthritis in interferon‐β–deficient mice

Objective

Clinical trials using interferon‐β (IFNβ) in the treatment of rheumatoid arthritis have shown conflicting results. We undertook this study to understand the mechanisms of IFNβ in arthritis at a physiologic level.

Methods

Collagen‐induced arthritis (CIA) was induced in IFNβ‐deficient and control mice. The role of IFNβ was investigated in both the priming and effector phases of the disease. The effect of IFNβ deficiency on synovial cells, macrophages, and fibroblasts from preimmunized mice was analyzed by flow cytometry, immunohistochemistry, and enzyme‐linked immunosorbent assay. Differences in osteoclast maturation were determined in situ by histology of arthritic and naive paws and by in vitro maturation studies of naive bone marrow cells. The importance of IFNβ‐producing fibroblasts was determined by transfering fibroblasts into mice at the time of CIA immunization.

Results

Mice lacking IFNβ had a prolonged disease with a higher incidence compared with control mice. IFNβ deficiency was found to influence the effector phase, but not the priming phase, of arthritis. Compared with control mice, IFNβ‐deficient mice had greater infiltration of CD11b+ cells and greater production of tumor necrosis factor α in vivo, and their macrophages and fibroblasts were both more activated in vitro. Moreover, IFNβ‐deficient mice generated a greater number of osteoclasts in vitro, and mice immunized to induce arthritis, but not naive mice, had a greater number of osteoclasts in vivo compared with control mice. Importantly, IFNβ‐competent fibroblasts were able to ameliorate arthritis in IFNβ‐deficient recipients.

Conclusion

Our data indicate that IFNβ is involved in regulating the activation state of osteoclasts and stromal cells, including macrophages and fibroblasts, but that it has little effect on T cells.

     

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.     

Soluble CD30 levels in plasma and cerebrospinal fluid in multiple sclerosis, HIV infection and other nervous system diseases

The CD30 molecule, a member of tumor necrosis factor superfamily, has been suggested to be preferentially expressed and released in soluble form by activated T cells that produce T helper 2 type (Th2) cytokines. To evaluate whether determination of soluble CD30 (sCD30) levels could have a diagnostic value in diseases associated with Th1 and Th2 cytokine involvement, we investigated sCD30 in plasma and cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS), HIV infection and other nervous system diseases. There was no statistically significant difference for plasma sCD30 levels in these clinical groups. However, patients with HIV infection had higher levels of sCD30 in CSF than MS patients. The mean sCD30 values were 3 to 6 folds higher in plasma than in CSF in all patient groups. No relationships were found between sCD30 levels and different clinical variables of MS and HIV infection, except that higher plasma sCD30 levels in HIV-infected patients were found in those with higher CD4⁺ T cell counts (> 200 ± 10⁶) compared to the group with lower cell counts. The findings indicate that determinations of plasma and CSF sCD30 levels in MS and HIV infection have limited or no value as diagnostic or prognostic indicator.