Katharina Schroecksnadel

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.

Neopterin, a prognostic marker in human malignancies

Increased neopterin concentrations are established in patients with an activated cellular (= Th1-type) immune response which includes allograft rejection, viral infection and autoimmune disorders as well as various malignant tumors. In patients with several types of cancer, neopterin concentrations in body fluids like urine, serum/plasma or ascites parallel the course of the disease, and a higher neopterin concentration in patients is an independent predictor of a shorter survival period. Neopterin is released in large amounts from human monocyte-derived macrophages and dendritic cells preferentially following stimulation with the pro-inflammatory cytokine interferon-gamma, thus reflecting the immune activation status. Therefore, not only as a laboratory diagnostic tool, the measurement of neopterin concentrations allows studying the immunological network and its interaction with the pathogenesis of tumor development. It contributes to a better understanding how immune activation is involved in the development of tumor-induced immune escape and tumor antigen specific tolerance.

Antitumoral Activity of Interferon-γ Involved in Impaired Immune Function in Cancer Patients

Insufficient immunosurveillance is an important aspect in early tumorigenesis and in the pathogenesis of malignant disease. In the later course of cancer, the development of immunodeficiency is considered the major reason for disease progression and death. Within the anti-tumoral host defense reaction, Th1-type cytokine interferon-γ (IFN-γ) is of particular relevance. IFN-γ stimulates several anti-proliferative and thus tumoricidal biochemical pathways in macrophages and other cells and also in tumor cell lines. These include inducible nitric oxide synthase, indoleamine (2, 3)- dioxygenase, an enzyme degrading the essential amino acid tryptophan, and the production of reactive oxygen species and neopterin in human macrophages and dendritic cells. Although the anti-proliferative strategy of the immune system aims to inhibit the growth of malignant cells, it can also affect T-cell response and thus contribute to the development of immunodeficiency. Accelerated degradation of tryptophan and increased production of neopterin were found to parallel the course of malignant diseases. Moreover, a higher degree of these metabolic changes characterizes poor prognosis and is associated with the development of anemia, weight loss and depressive mood in patients. Available data suggest that immunodeficiency in cancer patients may develop as a long-term side-effect of the antiproliferative and pro-apoptotic mechanisms elicited within Th1-type immune response, and enhanced production of pro-inflammatory cytokine IFN-γ seems to be critically involved.

Inverse association between serum concentrations of neopterin and antioxidants in patients with and without angiographic coronary artery disease

Neopterin is released from human monocyte-derived macrophages upon stimulation with interferon-gamma and is a sensitive indicator for cellular immune activation. Furthermore, reactive oxygen species (ROS) are produced in case of immune activation and inflammation. In a cross-sectional approach, plasma concentrations of neopterin and of antioxidant compounds and vitamins were compared in 1463 patients investigated by coronary angiography, which were recruited within the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study. Serum neopterin concentrations were higher in patients with coronary artery disease (CAD; mean+/-S.D.: 8.7+/-7.3 nmol/L) compared to controls (7.4+/-5.0 nmol/L; Welchs t-test: p<0.001). Mean concentrations of ascorbic acid (p<0.0001), gamma-tocopherol (p<0.05), lycopene (p<0.001), lutein+zeaxanthin (p<0.05), alpha-carotene (p<0.05) and beta-carotene (p<0.05) were lower in CAD than in controls. Neopterin concentrations correlated with CAD-score (r(s)=0.156; p<0.0001) and inversely with antioxidants lycopene (r(s)=-0.277; p<0.0001) and lutein+zeaxanthin (r(s)=-0.175; p<0.0001) levels and with vitamins ascorbic acid (r(s)=-0.207; p<0.0001) and alpha-tocopherol (r(s)=-0.105; p<0.0001). The study demonstrates that higher neopterin production is associated with lower concentrations of antioxidant compounds in patients at risk for atherosclerosis. Results suggest that lower concentrations of antioxidant compounds may relate to higher grade of chronic immune activation in patients.

Tryptophan degradation increases with stage in patients with rheumatoid arthritis.

Immune system activation is known to be involved in the progression of rheumatoid arthritis (RA). The proinflammatory cytokine interferon-γ in various cells, including monocytes, induces the enzyme indoleamine (2,3)-dioxygenase (IDO), which converts tryptophan to kynurenine. In sera of 22 patients (17 women and 5 men) with RA stages 1 to 4 according to Steinbrocker, the concentrations of tryptophan and kynurenine were measured by high-pressure liquid chromatography. To estimate IDO activity, the kynurenine to tryptophan ratio (kyn/trp) was calculated. In parallel, concentrations of the macrophage activation marker neopterin were determined by enzyme-linked immunosorbent assay. Tryptophan concentrations were lower in patients with RA, and the decrease in serum tryptophan correlated with increase in stage (p<0.05). Kyn/trp correlated well with neopterin concentrations, which were elevated in most patients. Whereas higher C-reactive protein concentrations and erythrocyte sedimentation rates were observed in patients with greater disease activity, tryptophan and neopterin concentrations did not differ between patients with different subjective disease activity graded by the physician. Deficiency of the essential amino acid tryptophan in patients with RA most likely results from immune activation involved in the pathogenesis of the disease. It could also be relevant for the mood of patients, as tryptophan is the precursor of serotonin.

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.

Indoleamine-2, 3-Dioxygenase and Other Interferon-γ-Mediated Pathways in Patients with Human Immunodeficiency Virus Infection

Human immunodeficiency virus type 1 (HIV) infection is characterized by progressive immunodeficiency despite of an overwhelming cellular immune activation. Patients show highly elevated serum/plasma concentrations of the proinflammatory cytokine interferon-γ (IFN-γ ), which induces human monocytes to form neopterin, to produce reactive oxygen species (ROS) and in parallel, to degrade tryptophan. Enhanced tryptophan degradation by the enzyme indoleamine-2, 3-dioxygenase (IDO) contributes importantly to disease progression and “complications” of HIV infection: By a subsequent impairment of protein metabolism and serotonin formation, the development of neuropsychiatric disorders and weight loss in HIV infected patients can be enforced. Furthermore, increased IDO-activation efficiently suppresses the growth and proliferation of pathogens as well as host T-cells. IDO and other IFN-γ-mediated pathways are strongly induced in patients with HIV infection and are also linked with disease progression: Neopterin formation by GTPcyclohydrolase I sensitively reflects the stage of the disease, and determination of the pteridine in body fluids is useful to monitor the efficacy of antiretroviral therapy. Neopterin is an independent prognostic factor for the outcome of disease, and well suited to estimate the degree of immune activation in vivo and the responsiveness of immunocompetent cells to stimulation in vitro. ROS formation may contribute to the development of oxidative stress in HIV infection, resulting in depletion of antioxidants. The cause-effective role of an overwhelming Th1-type immune response together with the activation of IDO and other IFN-γ-mediated biochemical pathways for the course of HIV infection, the development of immunodeficiency, anemia and weight loss in HIV patients is discussed.

Aspirin down-regulates tryptophan degradation in stimulated human peripheral blood mononuclear cells in vitro

Acetylsalicylic acid (aspirin) is one of the most widely used drugs worldwide, due mainly to its broad therapeutic spectrum with anti‐inflammatory, antipyretic, antithrombotic and analgesic effects. However, the exact mechanisms by which aspirin influences inflammation, pain and immune system activation are only partly understood. Within activation of the cellular immune system, Th1‐type cytokine interferon (IFN)‐γ induces enzyme indoleamine‐2,3‐dioxygenase (IDO) which converts tryptophan to kynurenine. In parallel, IFN‐γ induces enzyme GTP‐cyclohydrolase I, which gives rise to neopterin production by activated human macrophages. Similarly, tryptophan degradation and neopterin formation increase during several disease states involving Th1‐type immune activation. Using stimulated human peripheral blood mononuclear cells (PBMC), the effect of aspirin on tryptophan degradation and neopterin production was investigated. Stimulation of PBMC with mitogens concanavalin A, phytohaemagglutinin and pokeweed mitogen induced significant tryptophan catabolism as was reflected by a decline in tryptophan levels and a parallel increase in kynurenine concentrations compared with unstimulated cells. In parallel, neopterin production was enhanced. Treatment of stimulated PBMC with increasing doses of 1–5 mM aspirin significantly decreased stimulation‐induced tryptophan degradation and neopterin production as well. All the effects of aspirin were dose‐dependent. The parallel influence of aspirin on both biochemical pathways implies that there was no direct inhibitory effect of aspirin on IDO; rather, it inhibits production of IFN‐γ in mitogen‐treated PBMC. The influence of aspirin on biochemical pathways induced by IFN‐γ may represent an important part of its broad pharmacological effect.

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.

Quality of life and immune activation in patients with HIV-infection

Depression and impaired quality of life (QoL) are frequently observed in patients suffering from HIV-infection. As an enhanced degradation of the serotonin precursor tryptophan is well documented in HIV-infected patients, disturbances in tryptophan metabolism may be causally linked to HIV-related depression. In this study, the relationship between QoL, depression, various laboratory parameters and tryptophan metabolism was investigated. To estimate QoL and mood, 152 HIV-infected patients (classified according to CDC-criteria) were requested to complete the following psychological questionnaires: BDI and MQoL-HIV. Disease progression was monitored by determination of viral load (VL), CD4(+) cell counts, haemoglobin and urinary/plasma neopterin, tryptophan and kynurenine concentrations. Increasing VL, decreasing CD4(+) cell counts, and enhanced tryptophan degradation reflected disease progression. Forty-one patients presented with mild, 22 with moderate and 14 with severe depression. BDI and MQoL scores were associated strongly with each other (rs=-0.838; p<0.001). Patients without depression had significantly lower plasma neopterin concentrations, higher CD4(+) cell counts and haemoglobin concentrations and better QoL scores (all p<0.01) than depressive patients. Furthermore, they showed lower rates of tryptophan degradation (p<0.05). Significant associations were observed between tryptophan degradation and immune activation. Haemoglobin and viral load were predictive for impaired QoL, while high urinary neopterin concentrations and low haemoglobin were the best predictors for depression. In HIV-infected patients, depressive mood and impaired QoL appear to be related to clinical parameters like immune activation, haemoglobin values and viral load.