Sára Tóth

Leptin-induced signal transduction pathways

Leptin is a multifunctional cytokine and hormone that primarily acts in the hypothalamus and plays a key role in the regulation of food intake and energy expenditure. In addition, it has direct effects on many cell types on the periphery. Leptin acts through its receptor, the product of the db gene, which has six isoforms. Only one of them (OB‐Rb) has full signalling capabilities and is able to activate the Jak/STAT pathway, the major pathway used by leptin to exert its effects. However, some signalling events can be initiated by the short isoforms. Besides Jak/STAT, other pathways, such as MAPK and the 5′‐AMP‐activated protein kinase (AMPK) pathway, are also involved in leptin signalling. Leptin also interacts with insulin signalling. In this paper, we give an overview of the signal transduction mechanisms that are related to the actions of leptin.

Antisense strategies: functions and applications in immunology.

The ability to manipulate gene expression by means of exogenously administered oligodeoxynucleotides complementary to specific sequences in the genome is clearly going to impact on many fields of biology and medicine including basic and clinical immunology. Also endogenously generated antisense RNA species are important in regulating gene expression. Antisense RNA has become a widely used tool for analysis of gene function and holds great promise for therapeutic use in the future. Thus, inhibition can take place on different levels (transcription, translation and aptamer binding). Avoiding unspecific reactions one has to use controls and well-designed oligonucleotides. Based on the studies described in this review, antisense oligonucleotides hold a great promise as a novel class of therapeutic agents in immunology as well as in oncology, neurology and viral infections.

Isomer-dependent daunomycin release and in vitro antitumour effect of cis-aconityl-daunomycin

Two isomers of cis-aconytil-daunomycin (cAD) were isolated after the reaction of daunomycin with cis-aconitic-anhydride. The structure of the isomers was identified by MS-spectroscopy and 1H and 13C NMR experiments. In contrast with the assumptions described earlier, our results show that the two isomers belong to the cis- and trans-isomers of the alpha-monoamide of cis-aconityl-daunomycin, respectively. We found that the pH dependent daunomycin release is different for the two isomers. Comparative analysis of the in vitro antitumour effect of the isomers on c26 colon carcinoma and on MDA-MB 435P human breast carcinoma cell lines showed that cAD-1 is more potent than cAD-2, but the extent of differences is tumour cell dependent. The results of this study might be appreciated in the light of the use of acid-labile spacer for the design and preparation of protein/peptide conjugates of drugs by indicating that isomers could possess markedly different biological activity.

Lymphocytes from rheumatoid arthritis patients have elevated levels of intracellular peroxiredoxin 2, and a greater frequency of cells with exofacial peroxiredoxin 2, compared with healthy human lymphocytes

Peroxiredoxin 2 has immune regulatory functions, but its expression in human peripheral blood lymphocytes and levels in extracellular fluid in healthy subjects and rheumatoid arthritis patients are poorly described. In the present study, the median intracellular peroxiredoxin 2 protein content of lymphocytes from rheumatoid arthritis patients was more than two-fold higher compared with healthy subjects’ lymphocytes. Intracellular peroxiredoxin 3 levels were similar in healthy and rheumatoid arthritis lymphocytes. Flow cytometry detected peroxiredoxin 2 on the surface of ca. 8% of T cells and ca. 56% of B cells (median % values) of all subjects analyzed. Exofacial thioredoxin-1 was also observed. In the total lymphocyte population from rheumatoid arthritis patients, few cells (median, 6%) displayed surface peroxiredoxin 2. In contrast, a significantly increased proportion of interleukin-17+ve lymphocytes were exofacially peroxiredoxin 2+ve (median, 39%). Prdx2 was also detected in human extracellular fluids. We suggest that crucial inflammatory cell subsets, i.e. interleukin-17+ve T cells, exhibit increased exofacial redox-regulating enzymes and that peroxiredoxin 2 may be involved in the persistence of pro-inflammatory cells in chronic inflammation.

Phenotypic Profiling of Engineered Mouse Melanomas with Manipulated Histamine Production Identifies Histamine H2 Receptor and Rho-C as Histamine-Regulated Melanoma Progression Markers

In the present study, the impact of acquired neoplastic L-histidine decarboxylase (HDC) expression, and its direct consequence, the release of histamine in the tumor environment, was assessed on melanoma tumor progression. B16-F10 mouse melanoma cells were manipulated via stable transfection, and nine novel transgenic variants were generated in triplicates, constitutively expressing the full-length sense mouse HDC mRNA, a mock control, and an antisense HDC RNA segment, respectively. Establishing both primary skin tumors and lung metastases in C57BL/6 mice, the nine variants with different histamine-releasing capacities were subjected to a comprehensive comparative progression profiling in vivo. Our analyses showed trends of markedly accelerated tumor growth (P < 0.001), and moderately increased metastatic colony-forming potential (P = 0.010) along with rising levels of local histamine production. Using RNase protection assay for screening of the melanoma progression profile, and Western blotting for subsequent result validation, we looked for molecular progression markers affected by melanoma histamine secretion. Investigation of 21 functionally clustered markers associated with tumor proliferation, angiogenesis, invasivity, metastasis formation, local or systemic immunomodulation, and histamine signaling revealed positive correlations between histamine production, tumor histamine H2 receptor and rho-C expression (P < 0.001, P = 0.002, respectively). These observations confirm the involvement of histamine in the molecular machinery of melanoma progression.

Critical role of extracellular vesicles in modulating the cellular effects of cytokines

Under physiological and pathological conditions, extracellular vesicles (EVs) are present in the extracellular compartment simultaneously with soluble mediators. We hypothesized that cytokine effects may be modulated by EVs, the recently recognized conveyors of intercellular messages. In order to test this hypothesis, human monocyte cells were incubated with CCRF acute lymphoblastic leukemia cell line-derived EVs with or without the addition of recombinant human TNF, and global gene expression changes were analyzed. EVs alone regulated the expression of numerous genes related to inflammation and signaling. In combination, the effects of EVs and TNF were additive, antagonistic, or independent. The differential effects of EVs and TNF or their simultaneous presence were also validated by Taqman assays and ELISA, and by testing different populations of purified EVs. In the case of the paramount chemokine IL-8, we were able to demonstrate a synergistic upregulation by purified EVs and TNF. Our data suggest that neglecting the modulating role of EVs on the effects of soluble mediators may skew experimental results. On the other hand, considering the combined effects of cytokines and EVs may prove therapeutically useful by targeting both compartments at the same time.

Histamine Suppresses Fibulin-5 and Insulin-like Growth Factor-II Receptor Expression in Melanoma

We previously showed that transgenic enhancement of histamine production in B16-F10 melanomas strongly supports tumor growth in C57BL/6 mice. In the present study, gene expression profiles of transgenic mouse melanomas, secreting different amounts of histamine, were compared by whole genome microarrays. Array results were validated by real-time PCR, and genes showing histamine-affected behavior were further analyzed by immunohistochemistry. Regulation of histamine-coupled genes was investigated by checking the presence and functional integrity of all four known histamine receptors in experimental melanomas and by administering histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists to tumor-bearing mice. Finally, an attempt was made to integrate histamine-affected genes in known gene regulatory circuits by in silico pathway analysis. Our results show that histamine enhances melanoma growth via H1R rather than through H2R. We show that H1R activation suppresses RNA-level expression of the tumor suppressor insulin-like growth factor II receptor (IGF-IIR) and the antiangiogenic matrix protein fibulin-5 (FBLN5), decreases their intracellular protein levels, and also reduces their availability in the plasma membrane and extracellular matrix, respectively. Pathway analysis suggests that because plasma membrane-bound IGF-IIR is required to activate matrix-bound, latent transforming growth factor-beta1, a factor suggested to sustain FBLN5 expression, the data can be integrated in a known antineoplastic regulatory pathway that is suppressed by H1R. On the other hand, we show that engagement of H2R also reduces intracellular protein pools of IGF-IIR and FBLN5, but being a downstream acting posttranslational effect with minimal consequences on exported IGF-IIR and FBLN5 protein levels, H2R is rather irrelevant compared with H1R in melanoma.

Inverse regulation of interleukin-6 (IL-6) and IL-6 receptor in histamine deficient histidine decarboxylase-knock-out mice

Interleukin-6, a multifunctional cytokine upon binding to its receptor on hepatocytes regulates production of acute phase proteins involved in local and systemic inflammation. Gene expression and biosynthesis of IL-6 and its receptor (IL-6 R/gp130) is under complex regulation. Histamine, in addition to its principal role in immediate type hypersensitivity has been described to modulate IL-6 production and expression of IL-6 receptor. In this study, the IL-6 and IL-6 receptor expression was examined in histamine deficient histidine decarboxylase (HDC) knock-out mouse model. Our data suggest that in histamine deficient mice the inducibility of IL-6 is significantly reduced, whilst more IL-6 receptor/gp130 mRNA expresses in the liver than in wild type (HDC(+/+)) mice. These in vivo findings confirm earlier in vitro results and emphasize the efficacy of antihistamines in local IL-6 related processes.

Soluble interleukin-6 receptor (sIL-6R) makes IL-6R negative T cell line respond to IL-6; it inhibits TNF production.

The receptor for interleukin-6 (IL-6) consists of two subunits: a ligand specific IL-6Ralpha and gp130 that is responsible for signal-transduction. A soluble form of the ligand specific chain was described that when complexed to IL-6 is capable of binding to the membrane-bound gp130 subunit and thus can elicit signal-transduction. This soluble receptor can act on cells that express only the gp130 but not the ligand-specific subunit of the IL-6R. This phenomenon, called trans-signaling, introduced a novel aspect of cytokine action. In this study we examined the response of Jurkat cells, that are known not to express IL-6Ralpha, to IL-6, the soluble IL-6 receptor (sIL-6R) and a covalent complex of IL-6 and sIL-6R termed Hyper-IL-6. We studied the expression of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma). The complex of IL-6+sIL-6R and Hyper-IL-6 inhibited significantly the production of TNF in a gp130-dependent manner, whereas no differences in IFN-gamma expression were found. IL-6 and sIL-6R alone were not effective. Because we did not detect major differences in the TNF mRNA levels upon treatments, we conclude that the inhibition of TNF production should occur at the post-transcriptional level. These results provide another example of trans-signaling and underline the physiological importance of sIL-6R, and in the case of Hyper-IL-6 its possible therapeutic application can also be considered.

A novel galectin-1 and interleukin 2 receptor β haplotype is associated with autoimmune myasthenia gravis

Galectin-1 (LGALS1) and interleukin receptor 2β (IL2Rβ) are regulators of T-cell activation. Here we evaluated the association of regulatory region polymorphisms of the LGALS1 (rs4820293, rs4820294) and IL2Rβ (rs743777, rs228941) genes in 146 Caucasian myasthenia gravis patients compared to 291 ethnically matched controls. A significant difference was found in the distribution of the rs4820293/rs743777 polymorphism haplotypes (p<0.01). The rs4820293 polymorphism, previously not described to be associated with any disease, does not affect LGALS1 expression in peripheral mononuclear cells and skeletal muscle. Pathway analysis revealed interaction between LGALS1 and IL2Rβ suggesting a role of these proteins in this rare disease.