Orphan Nuclear Receptor NR2F6 Suppresses T Follicular Helper Cell Accumulation through Regulation of IL-21.

CD4 T follicular helper (Tfh) cells are specialized in helping B cells during the germinal center (GC) reaction and ultimately promote long-term humoral immunity. Here we report that loss of the nuclear orphan receptor NR2F6 causes enhanced survival and accumulation of Tfh cells, GC B cells, and plasma cells (PCs) following T cell-dependent immunization. Nr2f6-deficient CD4 T cell dysfunction is the primary cause of cell accumulation. Cytokine expression in Nr2f6-deficient Tfh cells is dysregulated, and Il21 expression is enhanced. Mechanistically, NR2F6 binds directly to the interleukin 21 (IL-21) promoter and a conserved noncoding sequence (CNS) near the Il21 gene in resting CD4+ T cells. During Tfh cell differentiation, this direct NR2F6 DNA interaction is abolished. Enhanced Tfh cell accumulation in Nr2f6-deficient mice can be reverted by blocking IL-21R signaling. Thus, NR2F6 is a critical negative regulator of IL-21 cytokine production in Tfh cells and prevents excessive Tfh cell accumulation.

Increased serum cadmium and strontium levels in young smokers: effects on arterial endothelial cell gene transcription.

OBJECTIVE: Metal constituents of tobacco have long been suspected to contribute to cardiovascular diseases. In this study, we determined the serum concentrations of aluminum, cadmium (Cd), cobalt, copper, iron, manganese, nickel, lead, strontium (Sr), and zinc of young nonsmokers, passive smokers, and smokers. METHODS AND RESULTS: Cd and Sr were found to be significantly increased in smokers compared with nonsmokers. The effects of these metals on primary arterial endothelial cells were then assessed using microarray technology and real-time polymerase chain reaction (RT-PCR). The data showed that Sr does not interfere with endothelial cell transcription. In contrast, the effects of Cd in amounts delivered to the human body by smoking were dramatic. CONCLUSIONS: Arterial endothelial cells responded to Cd exposure by massively upregulating metal and oxidant defense genes (metallothioneins) and by downregulating a number of transcription factors. In addition, the mRNA of the intermediate filament protein vimentin, crucial for the maintenance of cellular shape, was reduced. Surprisingly, a number of pro-inflammatory genes were downregulated in response to Cd. The present data suggest that by delivering Cd to the human body, smoking deregulates transcription, exerts stress, and damages the structure of the vascular endothelium; furthermore, in contrast to the effects of cigarette smoke as a whole, Cd seems to possess anti-inflammatory properties.

Gene expression profiling of human endothelial cells exposed to 50-Hz magnetic fields fails to produce regulated candidate genes.

To address the question of a possible effect of magnetic fields (MF) at 50 Hz on living systems, gene expression analyses were performed on human primary vascular endothelial cells exposed to MF of various intensities compared to control cells. Exposure protocols included continuous exposure at a single intensity (10 and 700 microT), intermittent exposure at a single intensity (700 microT), and continuous exposure to a variable-intensity field (10-30 microT). The transcriptional response of the cells was investigated using oligonucleotide microarrays containing up to 30 000 unique features. Although in individual experiments genes were identified where the expression appeared to be affected by exposure to MF, none of these genes were regulated in the same manner in subsequent repetition experiments. This is the first report of a transcriptome-wide analysis of the effects of MF exposure on human cells. The lack of a reproducible effect of MF on the expression of any genes in our investigation adds further weight to the evidence that 50-Hz MF are not capable of interacting with biological systems and thus do not represent an endothelial stress factor.

Cigarette smoke metal-catalyzed protein oxidation leads to vascular endothelial cell contraction by depolymerization of microtubules.

Smoking is a significant risk factor for development of atherosclerosis. However, the pathophysiology of smoking-mediated vessel wall damage is not understood. With tools ranging from analytical chemistry to cell biology, we show that cigarette smoke contains metals that catalyze the direct oxidation of cellular proteins by smoke oxidants. Oxidation of cellular proteins causes a loss of microtubule function, culminating in microtubule depolymerization and proteasome-dependent degradation of alpha-tubulin. As a consequence of the microtubule collapse, cytoskeletal structures as well as intermediate filaments break down, leading finally to a contraction of vascular endothelial cells. We observed a smoke extract-induced, calpain-dependent degradation of the intracellular form of platelet-endothelial cell adhesion molecule 1/CD31, as well as a release of P-selectin/CD62P, IL-6, and IL-8 from endothelial cells into the supernatant. Increased levels of soluble CD62P and IL-6 are well known to be associated with smoking in humans. Increased permeability of the vascular endothelium is a crucial event in atherogenesis. This work highlights the compounds and mechanisms by which cigarette smoke induces leakiness of the vascular endothelium.

Cardiovascular risk factors and atherosclerosis in young males: ARMY study (Atherosclerosis Risk-Factors in Male Youngsters).

BACKGROUND: Necropsy studies suggest that atherosclerosis begins in childhood, but in vivo confirmation of this concept is sparse and limited to selected population samples. Furthermore, new risk concepts of atherosclerosis focusing on inflammation, infections, and immunity have not yet been evaluated in this age group. METHODS AND RESULTS: This study was conducted in a sample of 141 17- to 18-year-old white males homogenous in age and sex. In addition to classic risk factors, C-reactive protein and the humoral and cellular immune reactivity to heat-shock proteins (HSPs) were assessed. Intima-media thickness (IMT) was quantified at 4 vessel segments of the carotid and femoral arteries. High IMT was considered to be present if the IMT of at least 1 vessel segment exceeded the 90th percentile. In a multivariate logistic regression analysis, cigarette smoking, high diastolic blood pressure, prominent immune reactivity to human and/or mycobacterial HSP60s, alcohol consumption (inverse), and low HDL cholesterol levels were all associated with high IMT. The prevalence of high IMT substantially increased from 0 to 60% when the number of risk conditions in a single individual increased from 0 to 4 (P<0.001 for linear trend). CONCLUSIONS: Our study supports the concept that atherosclerosis begins in the first decades of life and suggests a role of the immune system, especially immunoreactivity against HSP60s, in atherosclerosis of young individuals.

Disruption of vascular endothelial homeostasis by tobacco smoke: impact on atherosclerosis.

The World Health Organization (WHO) predicts that by 2020 tobacco will become the largest single health problem worldwide and will cause an estimated 8.4 million deaths annually (http://www5.who.int/tobacco/). Although the impact of smoking on human health is well defined from the medical point of view, surprisingly little is known about the mechanisms by which tobacco smoke mediates its disastrous effects. Here, we demonstrate that tobacco smoke dramatically changes vascular endothelial cell and tissue morphology, leading to a loss of endothelial barrier function within minutes. Long-term exposure of endothelial cells to tobacco smoke extracts induces necrosis that may trigger a pro-inflammatory status of the vessel wall. Pre-incubation of the extracts without cells for 6 h at 37 degrees C led to a complete loss of activity. Further, the endothelium could be rescued by changing to fresh medium even at times when the extracts had lost their activity. Finally, we show that N-acetyl cysteine and statins inhibit the adverse tobacco smoke effects.

Cigarette smoke is an endothelial stressor and leads to cell cycle arrest.

The molecular mechanisms underlying the atherogenic activity of cigarette smoke have yet to be fully elucidated. In the present study, genome-wide microarray analysis was performed on endothelial cells exposed to an aqueous cigarette smoke extract (CSE) for 3, 7, and 24 h, to obtain a better insight into how smoking may lead to endothelial damage. Microarray analysis showed the transcriptional response to CSE was dominated by heat shock, stress responsive, and inflammatory genes, along with genes encoding for anti-oxidant and metal detoxification proteins. The heat shock response was shown to be a result of short lived reactive species of CSE, with the abrogation of the effect by the addition of old CSE, the anti-oxidant N-acetyl cysteine, or the removal of metals from CSE implying that reactive oxygen species are the main culprit. This was further supported by a strong decline in the level of intracellular protein oxidation levels seen under these conditions compared to freshly prepared CSE. Mitochondrial integrity was also found to be significantly compromised after CSE treatment, resulting in a threefold increase in depolarised mitochondria after 6 h. Finally, cell cycle analysis showed the induction of G1 cell cycle arrest. An increased stress and inflammation response indicates that endothelial damage from smoking could contribute to immune cell infiltration, while decreased growth rates reduce endothelial layer repair, promoting atherogenesis.

Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy.

The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59+/-2 pN, which correlated nicely to the 51+/-1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.