The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae

Summary For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step start. The threshold amount of cell mass (reflected as a critical size) necessary for start is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at start, termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for start, causing the smaller cells, produced in the previous cell cycle, to be delayed at start while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and start, nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

The effect of central retinal lesions on optokinetic nystagmus in the monkey

Summary In alert monkeys (Macaca mulatta and fascicularis) the effect of central retinal lesions on fast optokinetic responses was investigated during high velocity optokinetic and visual-vestibular conflict stimulation. The fast component of the optokinetic response manifests itself as a rapid rise in the slow-phase eye velocity after light-on, during high velocity optokinetic stimulation; and a sudden drop in eye velocity after light-off. In contrast, the velocity storage component leads only to gradual changes in eye velocity during continuous optokinetic stimulation and after light-off (optokinetic after-nystagmus).Retinal lesions were placed by laser coagulation in and around the fovea. Responses of the normal and lesioned eye were compared. It was found that central lesions up to 12 deg (fovea diameter 6 deg) had only a negligible effect on fast optokinetic responses. With lesions of more than 25–30 deg diameter centered on the fovea definite fast responses could still be obtained, on average reduced to about 50% of the responses of the normal eye. Some monkeys showed initially no fast optokinetic responses and had, therefore, to be excluded from lesion experiments.The results demonstrate that fast optokinetic responses also can be obtained from extrafoveal areas, i.e. areas which are not generally involved in smooth pursuit eye movements. These results are discussed in relation to reports that the smooth pursuit eye movement system is also used to generate fast optokinetic responses.Supported by Swiss National Foundation for Scientific Research 3.343-2.78 and Deutsche Forschungsgemeinschaft, SFB 200 A2These experiments were performed at the Dept. of Neurology, University of Zürich. A preliminary report of this work was presented at the workshop on Physiological and pathological aspects of eye movements in Habay-la-Neuve (Belgium) and at the 8th Extraordinary Meeting of the Barany Society in Basel (Switzerland)     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Potential roles for tumour necrosis factorα during embryonic development

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNF) in development. It is proposed that TNF may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNF in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNF-mediated cytotoxicity; and a consideration of the role TNF may play in a free radical theory of development.     

Spontaneous Mucosal Lymphocyte Cytokine Profiles in Children with Autism and Gastrointestinal Symptoms: Mucosal Immune Activation and Reduced Counter Regulatory Interleukin-10

A lymphocytic enterocolitis has been reported in a cohort of children with autistic spectrum disorder (ASD) and gastrointestinal (GI) symptoms. This study tested the hypothesis that dysregulated intestinal mucosal immunity with enhanced pro-inflammatory cytokine production is present in these ASD children. Comparison was made with developmentally normal children with, and without, mucosal inflammation. Duodenal and colonic biopsies were obtained from 21 ASD children, and 65 developmentally normal paediatric controls, of which 38 had signs of histological inflammation. Detection of CD3⁺ lymphocyte staining for spontaneous intracellular TNF, IL-2, IL-4, IFN, and IL-10, was performed by multicolor flow cytometry. Duodenal and colonic mucosal CD3⁺ lymphocyte counts were elevated in ASD children compared with noninflamed controls (p<0.03). In the duodenum, the proportion of lamina propria (LP) and epithelial CD3⁺TNF⁺ cells in ASD children was significantly greater compared with noninflamed controls (p<0.002) but not coeliac disease controls. In addition, LP and epithelial CD3⁺IL-2⁺ and CD3⁺IFN⁺, and epithelial CD3⁺IL-4⁺ cells were more numerous in ASD children than in noninflamed controls (p<0.04). In contrast, CD3⁺IL-10⁺ cells were fewer in ASD children than in noninflamed controls (p<0.05). In the colon, LP CD3⁺TNF⁺ and CD3⁺IFN⁺ were more frequent in ASD children than in noninflamed controls (p<0.01). In contrast with Crohns disease and non-Crohns colitis, LP and epithelial CD3⁺IL-10⁺ cells were fewer in ASD children than in nondisease controls (p<0.01). There was a significantly greater proportion of CD3⁺TNF⁺ cells in colonic mucosa in those ASD children who had no dietary exclusion compared with those on a gluten and/or casein free diet (p<0.05). There is a consistent profile of CD3⁺ lymphocyte cytokines in the small and large intestinal mucosa of these ASD children, involving increased pro-inflammatory and decreased regulatory activities. The data provide further evidence of a diffuse mucosal immunopathology in some ASD children and the potential for benefit of dietary and immunomodulatory therapies.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Expression of transforming growth factor-alpha and its receptor during human liver development and maturation

We investigated the expression of transforming growth factor-alpha (TGF-) and its receptor during human liver development and maturation, using immunohistochemistry. In the fetal liver, strong immunoreactivity for TGF- and its receptor was noted in intrahepatic bile duct cells of various developmental stages; moderate immunoreactivity for TGF- and mild immunoreactivity for TGF- receptor were found in immature hepatocytes. In the postnatal liver, reactivity for TGF- in hepatocytes decreased gradually and was negative or only weakly positive in the adult liver, while reactivity for TGF- receptor in hepatocytes increased gradually and was strongly positive in the adult liver. In contrast, immunoreactivity of TGF- and its receptor in intrahepatic bile duct cells persisted in the postnatal liver and was positive in the adult liver. These data suggest that the system of TGF- and its receptor has an important role in the proliferation and differentiation of intrahepatic biliary cells and hepatocytes in the fetal liver. The decreasing expression of TGF- in hepatocytes in the postnatal liver may indicate that proliferative activity of hepatocytes gradually decreases with liver maturation. The presence of TGF- and its receptor in intrahepatic bile ducts in the postnatal liver suggests that the system of TGF- and TGF- receptor is operative postnatally.     

Molecular Mechanisms of Changes in TNF-α Production by Blood Mononuclears in Chronic Viral Hepatitis C

Chronic viral hepatitis C is associated with decreased production of TNF- by the peripheral blood mononuclear leukocytes irrespective of virus genotype and degree of the morphological activity of the process in the liver. This process positively correlates with the increase in the content of TNF- soluble receptor (molecular weight 55 kDa), which can play a role in the mechanisms of immunopathogenesis of long persistence of hepatitis C virus in the body.__________Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 139, No. 2, pp. 191–195, February, 2005     

Increased Production of a Th2 Cytokine Profile by Activated Whole Blood Cells from Rheumatoid Arthritis Patients

T cells produce regulatory cytokines which control inflammation. In rheumatoid arthritis (RA), a Th1 cytokine profile has been described in the synovium. In order to assess the Th1/Th2 cytokine balance in blood, a one step culture-immunoassay procedure was used to measure the ex vivo production of IFN and IL-4 by whole blood cells from 26 RA patients and 25 controls. For comparison, the same cytokines were measured by ELISA in supernatants of activated whole blood cells. The direct whole blood assay was 10-fold more sensitive than standard ELISA to measure IL-4 levels. IL-4 production was higher in RA patients than in controls, whereas that of IFN was lower. Accordingly, the IL-4/IFN ratio, which reflects the Th2/Th1 cytokine balance in blood, was higher in RA patients (P < 0.0001). The present findings indicate a Th2-over-Th1 cytokine balance profile in RA blood. These results are in contrast with the Th1-over-Th2 pattern previously found in the joint, indicating that the two compartments appear to be differently regulated.     

alpha1-acid glycoprotein possesses in vitro pro- and antiinflammatory activities

We studied the effects of ₁-acid glycoprotein on tumor necrosis factor- (TNF-) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to ₁-acid glycoprotein: first, stimulation of TNF- and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-/IL-10 ratio remained unchanged after addition of native ₁-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of ₁-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.     

Defective NF-κB Signaling in Dedifferentiated Hepatoma Cells

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1 pathway. We examined induction of NF-B, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-B induction. IB- levels, but not NF-B subunit p65 or IB- levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-B was found to be independent of degradation of IB- or IB-. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.     

Expression of matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) induced by tumour necrosis factor α correlates with metastatic ability in a human osteosarcoma cell line

We have examined the correlation between matrix metalloproteinase (MMP) expression and metastatic properties of a low metastatic osteosarcoma cell line, osteosarcoma takase (OST), under stimulation by tumour necrosis factor (TNF). In vivo, OST cells exhibited significantly increased colonization in the lungs of nude mice in a dose-dependent manner when they were treated by TNF prior to injection. In vitro, TNF enhanced tumour cell invasion through the reconstituted basement membrane in a transwell chamber up to 2.5-fold. Gelatin zymography and sandwich enzyme immunoassays demonstrated marked production of MMP-9 [92-kDa gelatinase/type IV collagenase (gelatinase B)] but not MMP-2 [72-kDa gelatinase/type IV collagenase (gelatinase A)], MMP-3 (stromelysin-1) or MMP-7 (matrilysin). Motility of the tumour cells and adhesion to cultured endothelial cells were slightly increased by the TNF treatment up to 1.6-fold and 1.4-fold, respectively, while the growth rate was decreased. These results suggest that upregulation of MMP-9 together with enhanced motility and endothelial adhesion contribute to the increased metastatic ability of OST cells induced by TNF treatment.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.