Barbara Frick

Crucial Role of Interferon-γ and Stimulated Macrophages in Cardiovascular Disease

Inflammation and immune activation are crucially involved in the pathogenesis of atherosclerosis and cardiovascular disease. Accordingly, markers of inflammation such as fibrinogen, ferritin, C-reactive protein or neopterin are found in patients with vascular diseases, correlating strongly with the extent of disease and predicting disease progression. Neopterin formation by human monocyte-derived macrophages and dendritic cells is induced by the pro-inflammatory cytokine interferon-γ, which is released by activated T-lymphocytes. Human macrophages are centrally involved in plaque formation, and interferon-γ and macrophages are also of importance in the development of oxidative stress for antimicrobial and antitumoural defence within the cell-mediated immune response. Interferon-γ also stimulates the enzyme indoleamine- 2,3-dioxygenase, which degrades tryptophan to kynurenine. Again, macrophages are the most important cell type executing this enzyme reaction, but also other cells like dendritic cells, endothelial cells or fibroblasts can contribute to the depletion of tryptophan. Likewise, enhanced tryptophan degradation was reported in patients with coronary heart disease and was found to correlate with enhanced neopterin formation. In chronic diseases such as in cardiovascular disease, biochemical reactions induced by interferon-γ may have detrimental consequences for host cells. In concert with other pro-inflammatory cytokines, interferon-γ is the most important trigger for the formation and release of reactive oxygen species (ROS). Chronic ROS-production leads to the depletion of antioxidants like vitamin C and E and glutathione, with a consequence that oxidative stress develope. Oxidative stress plays a major role in the atherogenesis and progression of cardiovascular disease, and it may also account for the irreversible oxidation of other oxidation-sensitive substances like B-vitamins (e.g. folic acid and B12). They are essential cofactors in homocysteine-methionine metabolism. Associations between moderate hyperhomocysteinaemia and cellular immune activation are found in several diseases including coronary heart disease, and data indicate that hyperhomocysteinaemia may develop as a consequence of immune activation. Homocysteine accumulation in the blood is established as an independent risk factor for cardiovascular disease. Homocysteine itself has the capacity to further enhance oxidative stress. Interferon-γ appears to be a central player in atherogenesis and in the development and progression of cardiovascular disease. Anti-inflammatory and immunosuppressive treatment (e.g. with non-steroidal anti-inflammatory drugs or statins) may among other consequences, also contribute to a slow-down of the adverse effects of interferon-γ.

Rapid measurement of total plasma homocysteine by HPLC

BACKGROUND Determination of plasma homocysteine has gained increasing interest during the past few years. Several HPLC methods for determination of homocysteine are available. Based on these methods, we developed a new HPLC assay for rapid and sensitive measurement of total plasma homocysteine. METHODS As a reducing reagent tris-(2-carboxylethyl)-phosphine is used, ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate serves as the derivatization agent. Separation is performed by reversed-phase HPLC using a precolumn and a 55-mm RP(18) cartridge; mobile phase: 0.1 mol/l KH(2)PO(4) with 5% methanol, adjusted to pH 2.7 with ortho-phosphoric acid, flow-rate 1.1 ml/min. RESULTS Homocysteine is clearly separated from other thiols, the retention time being 2.2 min, total analysis time is 6 min. Tests for linearity, recovery and precision are satisfactory, as well as the comparison with a commercial available assay method. Detection limit of the method is 0.5 micro mol/l, it could be further enhanced for measurements of even lower homocysteine concentrations in, e.g., cell culture supernatants. CONCLUSIONS The described method is well suited for analysis of thiols in blood specimens. It is more convenient and more rapid than methods described earlier.

Moderate Hyperhomocysteinemia and Immune Activation

Moderate hyperhomocysteinemia is associated with an increased risk of atherosclerosis, thrombosis and neurodegenerative diseases. Homocysteine accumulation in the blood can be due to many underlying causes, which may interact with each other, e.g. genetic disposition and B-vitamin status. The role of the sulfur-containing amino acid homocysteine in the pathogenesis of diseases remains unclear, even if many studies suggest a causal relationship between homocysteine-mediated processes like oxidative stress, NO-inactivation and endothelial deficiency and atherogenesis. Proposed mechanisms of action of homocysteine are discussed, and the question is addressed, whether effects that are attributed to homocysteine, are not rather the consequence of folate and vitamin B12-deficiency. Deficiency of these B-vitamins in parallel with moderate hyperhomocysteinemia is often found in patients with enhanced activation of the cellular immune system, like Alzheimers disease, rheumatoid arthritis and also vascular diseases. In patients with these diseases an association between homocysteine metabolism, oxidative stress and immune activation exists. On the one hand proliferation of immunocompetent cells having an enhanced demand for B-vitamins leads to the accumulation of homocysteine. On the other hand macrophages stimulated by TH1-type cytokine interferon-gamma form reactive oxygen species (ROS), which oxidize antioxidants, lipoproteins and oxidation-sensitive B-vitamins. Thereby Th1-type immune response could contribute importantly to the development of hyperhomocysteinemia, and may also be a major determinant of disease progression.

Homocysteine accumulates in supernatants of stimulated human peripheral blood mononuclear cells

Moderate hyperhomocysteinaemia is associated with atherosclerosis, thrombosis and also with stroke and dementia. Elevated homocysteine concentrations are related to deficiency of folate and also vitamin‐B12, as these two vitamins are essential co‐factors in the remethylation of homocysteine to methionine. A causal role of homocysteine in the pathogenesis of vascular disease has been discussed over years. Immune activation appears to be involved strongly in atherogenesis as well as in other diseases found to be associated with moderate hyperhomocysteinaemia. To study a possible influence of immune stimulation on homocysteine metabolism, in vitro experiments were performed using peripheral blood mononuclear cells upon stimulation with mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen. In stimulated cells a dose‐dependent increase of homocysteine concentrations was found. When cells were kept in medium supplemented with methionine, homocysteine concentrations increased further, while supplementation with folate had only a slight effect. We conclude that in supernatants of stimulated peripheral blood mononuclear cells homocysteine is accumulating. T cell activation could be involved in the development of moderate hyperhomocysteinaemia.

Hyperhomocysteinemia and immune activation

Abstract Hyperhomocysteinemia is an established risk factor for atherosclerosis, thrombosis and other vascular diseases. Homocysteine auto-oxidation is considered to be crucially involved in the pathogenesis of these diseases. However, the question remains to be elucidated whether vitamin deficiency and homocysteine accumulation are causal for disease development or rather comprise a secondary phenomenon. Most diseases accompanied by hyperhomocysteinemia are also associated with ongoing activation of the immune system. In vitro experiments show homocysteine to accumulate in stimulated peripheral blood mononuclear cells. In patients with coronary heart disease, with rheumatoid arthritis and in patients with dementia, an association between cellular immune activation and homocysteine metabolism is found. Homocysteine concentrations not only correlate inversely with folate concentrations, they also show a positive relationship with concentrations of immune activation markers like neopterin. Moreover, in patients with various kinds of dementia, increased concentrations of serum peroxides, homocysteine and neopterin correlate with each other. Studies support a role of immune system activation in the development of hyperhomocysteinemia. Stimulation and proliferation of immune cells may lead to the production of reactive oxygen species that may oxidize antioxidants and oxidation-sensitive B-vitamins. An enhanced demand for antioxidants as well as folate and vitamin B12 may develop, together with hyperhomocysteinemia, despite sufficient dietary intake.

Moderate hyperhomocysteinaemia and immune activation in Parkinson's disease

Summary. Moderate hyperhomocysteinaemia has been linked to an increased risk for cardiovascular diseases. Increased homocysteine concentrations may follow folate depletion due to insufficient dietary intake of the vitamin, but there is also some indication that immune activation could play a role. In this preliminary study, homocysteine, folate, and vitamin B12 concentrations were measured in 19 patients with Parkinsons disease, 61–90 years of age, and compared to a healthy control group of similar age and to neopterin concentrations as an indicator of immune activation. A subgroup of patients presented with increased homocysteine and low folate concentrations. Homocysteine levels correlated inversely with vitamins folate and B12 and positively with neopterin concentrations. Disturbed homocysteine metabolism in Parkinsons disease may be associated with vitamin deficiency and with immune system activation which may underlie folate depletion.

Homocysteine but not neopterin declines in demented patients on B vitamins

Summary.Inflammation and immune system activation seem to play an important role in the development and progression of dementia. Hyperhomocysteinemia is common in various forms of dementia, and a significant relationship was found between concentrations of homocysteine and immune activation marker neopterin. B vitamin supplementation is able to slow-down homocysteine formation in patients.In an open-label study, effects of B vitamin supplementation (Beneuran compositum®) on concentrations of homocysteine and neopterin were investigated in 58 patients with Alzheimer’s disease (n = 30), vascular dementia (n = 12) and mild cognitive impairment (n = 16). In all groups of patients, a significant percentage of patients presented with homocysteine concentrations >15 µmol/L and with elevated concentrations of immune activation marker neopterin. Decline of homocysteine concentrations was observed after one month of B vitamin supplementation (all p < 0.01; paired Kruskal-Wallisn-test). By contrast, neopterin concentrations remained unchanged (all p > 0.05).B vitamin supplementation in patients with various forms of dementia did not influence neopterin concentrations, which indicates that the degree of immune activation and inflammation remained unchanged. The question remains, if lowering of homocysteine by folate supplementation alone could have any beneficial effect to modulate the course of dementia development and if longer period of supplementation would also ameliorate immune system activation status.

Aspirin Downregulates Homocysteine Formation in Stimulated Human Peripheral Blood Mononuclear Cells

Moderate hyperhomocysteinaemia is established as an independent risk factor for atherosclerosis, thrombosis, stroke and dementia. Hyperhomocysteinaemia is mostly caused by the deficiency of B‐vitamins folate and vitamin B12, which are essential cofactors in the remethylation of homocysteine to methionine. Interestingly, moderate hyperhomocysteinaemia is also often observed in chronic diseases, in which also elevated immune activation markers such as neopterin or sTNFR‐II are found. In order to simulate immune activation in vitro, human peripheral blood mononuclear cells (PBMC) were stimulated with mitogens. Stimulation significantly increased homocysteine production in comparison with unstimulated PBMC; in parallel also neopterin formation was induced. Homocysteine formation was due to cell proliferation, proliferating T lymphocytes, and also the myelomonocytic cell line U‐937 produced homocysteine. Treatment with the anti‐inflammatory drug aspirin dose‐dependently inhibited homocysteine production and also neopterin formation in human PBMC. Treatment with salicylic acid showed similar effects as aspirin; FACS analysis showed that both compounds inhibited cell proliferation by arresting cells in the G0/G1‐phase. In U‐937, both compounds also slightly induced apoptosis at 5 mm. Proliferation‐induced homocysteine formation and in parallel also monocyte activation can be suppressed effectively by aspirin and salicylic acid in vitro, suggesting that also in vivo aspirin may downregulate not only inflammation but also formation of homocysteine.

Association of hyperhomocysteinemia in Alzheimer disease with elevated neopterin levels.

In patients with dementias including Alzheimer disease (AD), elevated blood concentrations of homocysteine are common, often going along with low normal folate and vitamin B12. Immune activation leading to oxidative stress also seems to play an important role in the pathogenesis of AD. To find out a possible relationship between immune activation and the development of moderate hyperhomocysteinemia, we determined serum concentrations of homocysteine, folate, vitamin B12 and immune activation markers 75 kD soluble TNF receptor (sTNF-R75) and neopterin in 38 patients with clinically diagnosed AD. A subgroup of patients (45%) presented with increased homocysteine concentrations in comparison to reference ranges in healthy controls of similar age. Also, concentrations of immune activation markers were elevated in a significant proportion of patients. In 17 patients with moderate hyperhomocysteinemia, concentrations of neopterin were higher than in those with lower homocysteine (p < 0.001). Homocysteine correlated with folate (rs= −0.43; p < 0.01) and neopterin (rs= 0.506; p < 0.001). The data suggest that immune activation and concomitant production of reactive oxygen species in patients with AD could be involved in the development of hyperhomocysteinemia via an enhanced decomposition of folate.

Atorvastatin suppresses homocysteine formation in stimulated human peripheral blood mononuclear cells

Abstract Hyperhomocysteinemia is regarded as an independent risk factor for vascular diseases, and homocysteine is supposed to contribute to oxidative stress and endothelial damage. Statin therapy is an established intervention to reduce the risk of acute events in patients suffering from cardiovascular diseases. Apart from their lipid-lowering capacity, statins also exert anti-inflammatory and antioxidant effects. As cellular immune activation and oxidative stress play a major role in the pathogenesis of cardiovascular diseases, the anti-inflammatory capacity of statins could partly be responsible for the beneficial effects observed in patients. Earlier we reported that stimulated peripheral blood mononuclear cells (PBMCs) release homocysteine. Here we studied the influence of atorvastatin on homocysteine production in stimulated PBMCs and compared changes in cysteine concentrations and in neopterin production, which is a sensitive indicator of cellular immune activation. Stimulation of human PBMCs with the mitogens concanavalin A and phytohemagglutinin induced significant homocysteine and neopterin production compared to unstimulated cells, whereas cysteine concentrations remained unchanged. Treatment of PBMCs with increasing doses of atorvastatin (10–100μM) suppressed both biochemical pathways in a dose-dependent manner, and cell proliferation was inhibited in parallel. Again, cysteine levels were not influenced by any treatment. The down-regulating effect of atorvastatin on homocysteine formation in vitro indicates that statins may prevent homocysteine accumulation in the blood via immunosuppression.