Quantitative image analysis in adipose tissue using an automated image analysis system: differential effects of peroxisome proliferator-activated receptor-alpha and -gamma agonist on white and brown adipose tissue morphology in AKR obese and db/db diabetic mice

Morphometric analysis of adipocytes is widely used to demonstrate the effects of antiobesity drugs or anti-diabetic drugs on adipose tissues. However, adipocyte morphometry has been quantitatively performed by manual object extraction using conventional image analysis systems. The authors have developed an automated quantitative image analysis method for adipose tissues using an innovative object-based quantitative image analysis system (eCognition). Using this system, it has been shown quantitatively that morphological features of adipose tissues of mice treated with peroxisome proliferator-activated receptor (PPAR) agonists differ dramatically depending on the type of PPAR agonist. Marked alteration of morphological characteristics of brown adipose tissue (BAT) treated with GI259578A, a PPAR-alpha agonist, was observed in AKR/J (AKR) obese mice. Furthermore, there was a 22.8% decrease in the mean size of adipocytes in white adipose tissue (WAT) compared with vehicle. In diabetic db/db mice, the PPAR-gamma agonist GW347845X decreased the mean size of adipocytes in WAT by 15.4% compared with vehicle. In contrast to changes in WAT, GW347845X increased the mean size of adipocytes in BAT greatly by 96.1% compared with vehicle. These findings suggest that GI259578A may activate fatty acid oxidation in BAT and that GW347845X may cause adipocyte differentiation in WAT and enhancement of lipid storage in BAT.     

Activation of peroxisome proliferator-activated receptor-gamma in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma

Peroxisome proliferator-activated receptors (PPARs) are activated by an array of polyunsaturated fatty acid derivatives, oxidized fatty acids, and phospholipids and are proposed to be important modulators of immune and inflammatory responses. Recently, we showed that activation of PPAR-gamma alters the maturation process of dendritic cells (DCs), the most potent antigen-presenting cells. In the present report, we investigated the possibility that, by targeting DCs, PPAR-gamma activation may be involved in the regulation of the pulmonary immune response to allergens. Using a model of sensitization, based on the intratracheal transfer of ovalbumin (OVA)-pulsed DCs, we show that rosiglitazone, a selective PPAR-gamma agonist, reduces the proliferation of Ag-specific T cells in the draining mediastinal lymph nodes but, surprisingly enough, dramatically increases the production of the immunoregulatory cytokine interleukin (IL)-10 by T cells, as compared to control mice sensitized with OVA-pulsed DCs. After aerosol challenge, the recruitment of eosinophils in the bronchoalveolar lavage fluids was strongly reduced compared to control mice. Finally, T cells from the mediastinal lymph nodes produced higher amounts of IL-10 and interferon-gamma. Inhibition of IL-10 activity with anti-IL-10R antibodies partly restored the inflammation. The specificity of the phenomenon was confirmed by treating OVA-pulsed DCs with ciglitazone, another PPAR-gamma agonist, and by using GW9662, a PPAR-gamma antagonist. Our data suggest that PPAR-gamma activation prevents induction of Th2-dependent eosinophilic airway inflammation and might contribute to immune homeostasis in the lung.     

Peroxisome proliferator-activated receptor gamma is expressed in airways and inhibits features of airway remodeling in a mouse asthma model

BACKGROUND: Allergic asthma is associated with persistent functional and structural changes in the airways and involves many different cell types. Peroxisome proliferator-activated receptor gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, is predominantly expressed in adipose tissue and plays a major role in regulating adipocyte differentiation and glucose metabolism. Recently, PPAR-gamma has been shown to play an important role in the control of inflammatory responses, including within the lung, acting on both immune and nonimmune cells. OBJECTIVE: Our aim was to assess the anti-inflammatory potential of a PPAR-gamma agonist locally delivered by means of nebulization. METHODS: We used a mouse model of asthma induced by sensitization and airway challenge with ovalbumin. Ciglitazone, a PPAR-gamma agonist, was administered by means of nebulization alone at the time of antigen challenge or by means of gavage and nebulization. Treatments with both ciglitazone and GW9662, a specific antagonist, were also performed to verify that ciglitazone's effects were mediated through PPAR-gamma activation. RESULTS: Our results show that PPAR-gamma is mainly expressed in airway epithelium on antigen sensitization. Treatment with ciglitazone reduced PPAR-gamma levels in the lung, whereas combined treatment with GW9662 abrogated this inhibition. Importantly, nebulization with ciglitazone decreased airway hyperresponsiveness, basement membrane thickness, mucus production, collagen deposition, and TGF-beta synthesis. A significant correlation was also found between airway hyperresponsiveness, basement membrane thickness, and TGF-beta levels. CONCLUSION: These results demonstrate that inhaled agonistic ligands of PPAR-gamma might have new therapeutic potential for airway asthmatic inflammation.     

Tyrosine Kinase Inhibitors Ameliorate Autoimmune Encephalomyelitis in a Mouse Model of Multiple Sclerosis

Multiple sclerosis is an autoimmune disease of the central nervous system characterized by neuroinflammation and demyelination. Although considered a T cell-mediated disease, multiple sclerosis involves the activation of both adaptive and innate immune cells, as well as resident cells of the central nervous system, which synergize in inducing inflammation and thereby demyelination. Differentiation, survival, and inflammatory functions of innate immune cells and of astrocytes of the central nervous system are regulated by tyrosine kinases. Here, we show that imatinib, sorafenib, and GW2580—small molecule tyrosine kinase inhibitors—can each prevent the development of disease and treat established disease in a mouse model of multiple sclerosis. In vitro, imatinib and sorafenib inhibited astrocyte proliferation mediated by the tyrosine kinase platelet-derived growth factor receptor (PDGFR), whereas GW2580 and sorafenib inhibited macrophage tumor necrosis factor (TNF) production mediated by the tyrosine kinases c-Fms and PDGFR, respectively. In vivo, amelioration of disease by GW2580 was associated with a reduction in the proportion of macrophages and T cells in the CNS infiltrate, as well as a reduction in the levels of circulating TNF. Our findings suggest that GW2580 and the FDA-approved drugs imatinib and sorafenib have potential as novel therapeutics for the treatment of autoimmune demyelinating disease.     

Prolactin may be a promising therapeutic target for myasthenia gravis: hypothesis and importance

Myasthenia gravis (MG) is an autoimmune disease that affects the transmission signals from nerves to muscles. The basic pathology is the production of anti-acetylcholine receptor (AChR) antibodies (AChRAb) which is the consequence for the generation of autoreactive T lymphocytes responsing to AChR. However, the molecular mechanism of MG and the production of autoreactive T lymphocytes remain elusive. Recently beside its pivotal role in reproduction, the pituitary hormone prolactin (PRL) has been attributed to an immunomodulatory function. Furthermore it has been shown to be expressed in T cells and conversely it also affects the function of T cells, such as directly stimulating the proliferation and survival of T lymphocytes. In addition, elevated PRL levels frequently are described in autoimmune diseases, such as systemic lupus erythematosus (SLE) and multiple sclerosis (MS). So we hypothesized that the stimulating effect of PRL on T-cells function may be implied in the pathogenesis of MG and, perhaps, prolactin may be a promising therapeutic target for MG.     

The nuclear receptor PPAR gamma is expressed by mouse T lymphocytes and PPAR gamma agonists induce apoptosis

Peroxisome proliferator-activated receptor (PPAR)-gamma is a nuclear hormone receptor that serves as a trans factor to regulate lipid metabolism. Intense interest is focused on PPAR-gamma and its ligands owing to its putative role in adipocyte differentiation. Little is known, however, about the functions of PPAR-gamma in the immune system, especially in T lymphocytes. We demonstrate that both naive and activated ovalbumin-specific T cells from DO11.10-transgenic mice express PPAR-gamma mRNA and protein. In order to determine the function of PPAR-gamma, T cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin or antigen and antigen-presenting cells. Simultaneous exposure to PPAR-gamma ligands (e. g. 15-deoxy-Delta(12, 14)-prostaglandin J(2), troglitazone) showed drastic inhibition of proliferation and significant decreases in cell viability. The decrease in cell viability was due to apoptosis of the T lymphocytes, and occurred only when cells were treated with PPAR-gamma, and not PPAR-alpha agonists, revealing specificity of this response for PPAR-gamma. These observations suggest that PPAR-gamma agonists play an important role in regulating T cell-mediated immune responses by inducing apoptosis. T cell death via PPAR-gamma ligation may act as a potent anti-inflammatory signal in the immune system, and ligands could possibly be used to control disorders in which excessive inflammation occurs.     

Dopamine fails to regulate activation of peripheral blood lymphocytes from multiple sclerosis patients: effects of IFN-beta.

The neurotransmitter dopamine counteracts T cell functions through its specific receptor subtype D5R but favors T cell proliferation and adhesion when acting on D3R. We found diminished mRNA and protein levels of D5R, but not of D3R, in peripheral blood mononuclear cells (PBMCs) from untreated multiple sclerosis (MS) patients. Dopamine reduced T cell proliferation, secretion of interferon-gamma (IFN-gamma), and production of matrix metalloproteinase-9 (MMP-9) mRNA in PBMCs from controls but not from MS patients. By contrast, reduced levels of D3R and renewed dopamine-associated regulatory functions were found in PBMCs from IFN-beta treated MS patients. Failure of the dopaminergic system of lymphocytes may lessen the threshold of T cell activation and sustain the pathogenic cascade of MS.     

Production of a monoclonal antibody to autoantigenic components of human glomerular basement membrane

Histamine (10(-3)-10(-8) M) inhibits PHA-induced proliferation of human peripheral blood lymphocytes (HPBL). Inhibition is detected at low concentrations of PHA but is rarely observed at high PHA concentrations. The histamine type II (H2) receptor agonists dimaprit, impromidine and 4-methylhistamine (4MH) inhibit HPBL proliferation and the H2 antagonist, cimetidine, reverses histamine-induced suppression of HPBL proliferation. Lymphocyte proliferation is also inhibited by the H1 receptor agonists, 2-pyridylethylamine and 2-thiazoylylethylamine, but only at high concentrations (10(-3) and 10(-4) M). The H1 agonist 2-methylhistamine, suppresses PHA-induced proliferation of HPBL in analogous fashion to histamine. This effect is reversed by cimetidine but not by diphenhydramine suggesting that an H2 receptor interaction is involved.     

Prostaglandin D(2), its metabolite 15-d-PGJ(2), and peroxisome proliferator activated receptor-gamma agonists induce apoptosis in transformed, but not normal, human T lineage cells

Prostaglandin D(2) (PGD(2)) is abundantly produced by mast cells, platelets, and alveolar macrophages and has been proposed as a key immunoregulatory lipid mediator. 15-Deoxy-Delta(12,14)-PGJ(2) (15-d-PGJ(2)), a key PGD(2) metabolite, is under intense study as a potential anti-inflammatory mediator. Little is known about PGD(2) or the role of 15-d-PGJ(2), if any, in regulating the activities of human T lineage cells. In this report we demonstrate that both PGD(2) and 15-d-PGJ(2) have potent antiproliferative effects, and in fact kill human T lymphocyte lines derived from malignant cells by an apoptotic mechanism. Interestingly, normal human T cells were not similarly affected. Although the T lymphocyte lines express mRNA for the PGD(2) receptor (DP-R), a potent DP receptor agonist, BW245C, did not inhibit the proliferation or viability of the cells, suggesting an alternative mechanism of action. PGD(2) and 15-d-PGJ(2) can bind to the peroxisome proliferator activated receptor-gamma (PPAR-gamma) which is implicated in lipid metabolism and apoptosis. Exposure to synthetic PPAR-gamma ligands (e.g. ciglitazone, troglitazone) mimicked the inhibitory responses of PGD(2) and 15-d-PGJ(2), and induced apoptosis in the transformed T cells consistent with a PPAR-gamma-dependent mechanism. These observations suggest that PPAR-gamma ligands (which may include PGD2) provide strong apoptotic signals to transformed, but not normal T lymphocytes. Thus, the efficacy of utilizing PPAR-gamma and its ligands as therapeutics for human T cell cancers needs to be further evaluated.     

Generation of functionally active suppressor cells by haemorrhage and haemorrhagic serum.

Mitogen (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMC) was reduced by more than 70% 2 h after the haemorrhage of 30% of blood volume. Experiments using isolated macrophages and lymphocytes showed that post-haemorrhage macrophages were functionally normal and that lymphocytes were responsible for the observed haemorrhage-induced depression of proliferative response. Surface marker determinations showed that at least some, if not all, of the haemorrhage-induced suppressor cells are of the OX8+ phenotype. Exposure of PBMCs to serum from bled animals also brought about activation of OX8+ suppressor T cells. These results suggest that the depressed proliferative response of PBMCs induced by haemorrhage or by exposing the cells to haemorrhagic serum (serum from bled animals) is due to the activation of OX8+ suppressor T cells.     

Inhibitory effects of fluvastatin on cytokine and chemokine production by peripheral blood mononuclear cells in patients with allergic asthma

BACKGROUND: Statins have anti-inflammatory effects on immune cells. OBJECTIVE: To investigate the immunomodulatory effects of fluvastatin on peripheral blood mononuclear cells (PBMCs) after allergen-specific and non-allergen-specific stimulation in patients with asthma and in healthy subjects. METHODS: PBMCs from seven patients with asthma who showed elevated immunoglobulin (Ig)E to house dust mite were isolated and stimulated with Dermatofagoides farinae, purified protein derivative, and phytohaemagglutinin (PHA) in the presence or absence of fluvastatin. PBMCs from seven healthy subjects were stimulated with PHA. The effects of fluvastatin on cell proliferation and production of cytokines (interferon [IFN]-gamma and interleukin [IL]-5) and chemokines (chemokine CXC motif, ligand [CXCL10], and CC chemokine ligand [CCL17]) were measured. Migration of T helper (Th)1 and Th2 cell lines was also investigated. The expression of CXCR3 and CCR4 was analysed with flow cytometry. Steroid-insensitive PBMCs induced by preculture with IL-2 and IL-4 were also evaluated. Some experiments were performed in the presence of mevalonic acid. RESULTS: Fluvastatin inhibited the proliferation of PBMCs and decreased the production of IL-5, IFN-gamma, CCL17, and CXCL10 after allergen-specific and non-allergen-specific stimulation; all these effects, except for decreased CXCL10 production, were partially reversed by mevalonic acid. Culture supernatants obtained in the presence of fluvastatin prevented the migration of Th1 and Th2 cell lines in a dose-dependent manner. In addition, CCR4 and CXCR3 expression on CD4(+) T cells was not affected by the presence of fluvastatin. Fluvastatin inhibited the proliferative response of steroid-insensitive PBMCs to phytohaemagglutinin. CONCLUSION: Fluvastatin has inhibitory effects on cytokine and chemokine production, and thus might be used as a potential therapeutic agent in severe asthma.     

T lymphocyte proliferation is suppressed by the opioid growth factor ([Met(5)]-enkephalin)-opioid growth factor receptor axis: implication for the treatment of autoimmune diseases

Opioid peptides function as immunomodulatory molecules. Reports have linked the opioid growth factor (OGF), [Met⁵]-enkephalin, and its receptor OGFr to autoimmune diseases. OGF repressed the incidence and magnitude of myelin oligodendrocyte-induced experimental autoimmune encephalomyelitis in mice. Given the extensive connection between the immune system and autoimmune diseases, the present study was conducted to examine the relationship of the OGF-OGFr axis and T lymphocyte proliferation. Splenic-derived mouse lymphocytes were stimulated with phytohemagglutin (PHA). All non-stimulated and PHA-stimulated T lymphocytes had immunoreactivity for OGF-like enkephalin and OGFr. OGF markedly suppressed T lymphocyte number in a dose-dependent manner. However, PHA-stimulated T lymphocytes were not altered in cell number by a variety of natural and synthetic opioid-related compounds, some specific for μ, δ, and κ opioid receptors. Persistent blockade of opioid receptors with the general opioid antagonist naltrexone (NTX), as well as antibody neutralization of OGF-like peptides, had no effect on cell number. Non-stimulated T lymphocytes exhibited no change in cell number when subjected to OGF or NTX. Treatment of T lymphocytes with siRNAs for μ, δ, or κ opioid receptors did not affect cell number, and the addition of OGF to these siRNA-exposed cultures depressed the population of cells. T lymphocytes treated with OGFr siRNA also had a comparable number of cells to control cultures, but the addition of OGF did not alter cell number. DNA synthesis in PHA-stimulated T lymphocytes exposed to OGF was markedly decreased from PHA-stimulated cultures receiving vehicle, but the number of cells undergoing apoptosis or necrosis in these cultures was similar to control levels. T lymphocytes subjected to siRNA for p16 and/or p21 had a comparable number of cells compared to controls, and treatment with OGF did not depress cell number in preparations transfected with both p16 and p21 siRNA. These data reveal that the OGF-OGFr axis is present in T lymphocytes and is capable of suppressing cell proliferation. However, T lymphocytes are not dependent on the regulation of cell proliferation by this system. The results showing that the OGF-OGFr axis is an immunosuppressant, offers explanation for reports that autoimmune diseases can be modulated by this system.     

Lack of IFN-gamma production in response to antigenic stimulation in human IFN-tau-treated lymphocytes.

Interferon-tau (IFN-tau) is a type I IFN responsible for maternal recognition of the fetus in ruminants. In addition to its physiologic role, IFN-tau also inhibits HIV replication in human lymphocytes and macrophages and displays immunomodulatory effects but lacks the toxicity associated with other type I IFNs. Human IFN-alpha promotes a Th1 response, whereas IFN-tau has anti-inflammatory properties, inducing the production of Th2 cytokines in murine models of experimental autoimmune encephalitis (EAE) or fetal loss. We compared the effects of ovine IFN-tau (OvIFN-tau) and human IFN-alpha (HuIFN-alpha) on cytokine mRNA and protein production in human peripheral blood mononuclear cells (PBMCs) activated with a recall antigen, such as purified protein derivative (PPD) of tuberculin or with a proinflammatory stimulus, such as lipopolysaccharide (LPS). In both cases, IFN-alpha increased IFN-gamma production, whereas IFN-tau did not and thereby promoted Th2 cytokine production. This original property renders IFN-tau a potential candidate for therapeutic applications in immune disorders, such as multiple sclerosis (MS), but its therapeutic use in the treatment of HIV infection should be considered with caution.     

Evaluation of a rat model of experimental autoimmune encephalomyelitis with human MBP as antigen

Experimental autoimmune encephalomyelitis(EAE) is a good model for human multiple sclerosis(MS)research.However,there are some defects in the traditional models.Here,we improved the model by using thehuman myelin basic protein(MBP) as antigen.EAE was induced by immunization of female Wistar rats withhuman MBP.Compared with the traditional models,the new model was evaluated by clinical signs topathological changes.The immune state of the model was assessed by the lymphocyte infiltrative response andlevels of TNF-α,IFN-γ,IL-10.It was found that most of rats exhibited tail tone loss and hind-limb paralysis,also there were demyelination,infiltrative lymphocyte foci,“neuronophagia”in the cortex of cerebra and thewhite matter of spinal cords.PBMCs and spleen lymphocytes were strongly responsive to the stimulation ofMBP and PHA.The levels of TNF-α and IFN-γ were altered with the severity of EAE.In the remitting phase,IL-10 was increased significantly.This study demonstrates that the animal model of EAE induced by humanMBP bears resemblance to the features of human multiple sclerosis and promises to be a better model than everbefore for the study of MS.Cellular & Molecular Immunology.2004;1(5):387-391.     

Short‐chain fatty acids: Bacterial messengers modulating the immunometabolism of T cells

Short‐chain fatty acids (SCFAs) are mainly generated by bacterial fermentation of non‐digestible carbohydrates such as dietary fiber. In the last decade, new investigations have revealed that SCFAs have a very specific function and serve as active microbial metabolites, which are able to modulate the function of immune cells in the intestine and other tissues. Recent studies have highlighted the immunomodulatory potential of SCFAs in several autoimmune and inflammatory disorders such as multiple sclerosis, colitis, type 1 diabetes and rheumatoid arthritis. While the SCFA‐mediated activation of GPR41/GPR43 signalling pathways and their inhibitory activity on histone deacetylases have been extensively investigated, the impact of SCFAs on the T cell metabolism is poorly understood. SCFAs induce metabolic alterations in T cells by enhancing the activity of the mTOR complex and by regulating their glucose metabolism. Once taken up into T lymphocytes, SCFA‐derived acetyl groups contribute to the cellular acetyl‐CoA pool, which influences the histone acetylation and cytokine gene expression. This article reviews how SCFAs modulate the metabolic status of T cells, thereby impacting on epigenetic modifications and T cell function. We will also discuss how the recent findings from SCFA biology might be utilized for potential immune therapies of various autoimmune diseases.     

MHC-restricted autoantigen-reactive T cell clones in multiple sclerosis

Multiple sclerosis is a demyelinating disease of the central nervous system with genetic, viral and autoimmune characteristics. Myelin basic protein (MBP) is a suspected target autoantigen since it induces experimental autoimmune encephalomyelitis, an animal model closely resembling multiple sclerosis. The disease is mediated by Class II restricted, MBP-reactive T cells possessing the T helper/inducer phenotype. In the present study, we have isolated MBP-reactive T cell clones from the peripheral blood of a chronic progressive multiple sclerosis patient. The clones displayed blastogenic memory responses when rechallenged with the autoantigen and irradiated autologous lymphocytes. MBP recognition by the autoantigen-reactive T lymphocytes was restricted by major histocompatibility complex Class II antigens. Both CD4+8- and CD4-8+ MBP-reactive T cell clones were obtained.