Increased TNF-alpha and decreased TGF-beta expression in peripheral blood leukocytes after acute myocardial infarction.

Inflammation contributes to the development of atherosclerosis and cardiovascular events. Counteracting pro- and anti-inflammatory responses of serum cytokines have been reported, but the relevance of TNF-alpha, TGF-beta and IL-6 gene expression in peripheral blood leukocytes and their contribution to systemic inflammation in atherosclerosis, especially after acute myocardial infarction (AMI), has not been investigated yet. Using quantitative RT-PCR, we determined temporal cytokine mRNA expression alterations in blood cells from patients with AMI (n = 51). Serum cytokine concentrations were analyzed in parallel using the ELISA technique. TNF-alpha mRNA expression rates and serum concentrations were significantly elevated in AMI patients compared to controls (n = 77), while mRNA expression and serum content of TGF-beta were decreased. Interestingly, we found no statistically significant correlation between transcript and protein levels, indicating that gene expression in leukocytes may be an independent sign for systemic inflammation. While IL-6 was significantly increased in serum from AMI patients with positive correlation to left ventricular dysfunction and negative correlation to ejection fraction, IL-6 mRNA levels did not differ between patients and controls. Gene expression alterations indicate a sophisticated regulation of counteracting TNF-alpha and TGF-beta cytokine expression in peripheral blood leukocytes after AMI with bias towards a pro-inflammatory situation.

Altered chemokine levels in individuals at risk of Type 1 diabetes mellitus.

AIMS: The hypothesis was tested in an exploratory study that individuals at high risk of developing Type 1 diabetes mellitus have altered systemic levels of cytokines and chemokines. SUBJECTS AND METHODS: Forty-two non-diabetic first-degree relatives of patients with Type 1 diabetes mellitus were recruited. Of these, 18 had multiple islet autoantibodies (islet cell antibody, glutamic acid decarboxylase antibody, IA-2 antibody). Follow-up for 9-11 years confirmed high vs. moderate diabetes risk in islet autoantibody-positive vs. -negative relatives. Cytokines and chemokines were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum concentrations of classic Th1-associated cytokines (IFN-gamma, IL-12, IL-18) or Th2/Treg-associated cytokines (IL-5, IL-10, IL-13) did not significantly differ in high vs. moderate diabetes risk group. However, of six chemokines analysed, levels of CCL3 and CCL4 were increased (P = 0.0442 and P = 0.0334) while CCL2 was decreased (P = 0.0318) in the multiple islet autoantibody-positive group. No significant differences were seen for CCL5, CCL11, CXCL10. There was a significant correlation between the two closely related chemokines CCL3 and CCL4 in individuals at risk (r = 0.84, P = 0.00005), but not in the autoantibody-negative group. CONCLUSION: Relatives at high risk of developing Type 1 diabetes mellitus have abnormal cellular immune regulation at the level of systemic chemokines. The up-regulation of CCL3 and CCL4 vs. down-regulation of CCL2 suggests opposed functions of these chemokines in the disease process. These findings need to be confirmed by independent studies.

Inflammation and type 2 diabetes: results from KORA Augsburg.

Type 2 diabetes is associated with a systemic low-grade inflammation. First data provided by cross-sectional studies from as early as the 1960s demonstrated elevated systemic levels of glycoproteins and acute-phase reactants and increased leukocyte counts in type 2 diabetes patients. Subsequently, prospective studies showed that elevated concentrations of several acute-phase proteins and cytokines are predictive of later type 2 diabetes. Immune gene variants in man and in animal models were found to affect insulin resistance and diabetes incidence. Antidiabetic treatment by medication, diet or physical activity results in a significant decrease of systemic immune mediator concentrations. Immunological analyses of the KORA Survey S4 (1999/2001) allowed us to show that levels of circulating acute-phase proteins like CRP and of IL-6 are highly correlated and associated not only with overt type 2 diabetes, but already with impaired glucose tolerance (IGT) pointing out a role of these mediators in the pathogenesis of type 2 diabetes. On the contrary, TNFalpha was neither coregulated with CRP nor associated with diabetes status. Our study therefore shows that type 2 diabetes is accompanied by a non-random and differential upregulation of components of the innate immunity and suggests that this inflammatory condition is involved in the aetiology of the disease. Future work will extend the range of analysed immune mediators to chemokines and will also investigate the association of immune markers with indices of obesity to elucidate the relevance of this traditional risk factor for low-grade inflammation.

Significant association of the interleukin-6 gene polymorphisms C-174G and A-598G with type 2 diabetes.

Elevated blood concentrations of IL-6 have been shown to predict type 2 diabetes. Because the impact of IL-6 gene polymorphisms on diabetes status, parameters of the metabolic syndrome, and low-grade systemic inflammation has not been analyzed in a population-based study, we investigated the association of the IL-6 single nucleotide polymorphisms C-174G and A-598G on these parameters in 704 elderly participants of the Kooperative Gesundheitsforschung im Raum Augsburg/Cooperative Research in the Region of Augsburg (KORA) Survey 2000. Both -174G and -598G alleles were significantly associated with type 2 diabetes (-174G: odds ratio = 1.51, 95% confidence interval = 1.11-2.07, P = 0.0096; -598G: odds ratio = 1.56, 95% confidence interval = 1.13-2.15, P = 0.0069) but not with impaired glucose tolerance. In subgroup analyses, the association reached statistical significance in men and in leaner subjects (body mass index

Identification of known and novel genes in activated monocytes from patients with rheumatoid arthritis.

OBJECTIVE: To define gene activation patterns of monocytes (MO) in patients with rheumatoid arthritis (RA). METHODS: A complementary DNA (cDNA) library was constructed from first-leukapheresis MO obtained from an RA patient with active disease; 32P-labeled cDNA from first-leukapheresis MO (activated pool) and third-leukapheresis MO (nonactivated pool) were used as probes for differential hybridization. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assess gene activation in MO from an additional 26 RA patients and 6 normal controls. RESULTS: Subtraction of genes from first- and third-leukapheresis MO resulted in 482 differentially expressed clones. In first-leukapheresis MO, these clones included the following: 1) interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, tumor necrosis factor alpha, growth-related oncogene alpha (GROalpha)/melanoma growth-stimulatory activity, macrophage inflammatory protein 2/GRObeta, ferritin, alpha1-antitrypsin, lysozyme, transaldolase, Epstein-Barr virus-encoded RNA 1 (EBER-1)/EBER-2-associated-protein, thrombospondin 1, an angiotensin receptor II (ATRII) C-terminal homolog, and RNA polymerase II elongation factor (elongin); 2) two clones homologous to functionally undefined genes (BSK-67 and BSK-83); and 3) three unknown cDNA sequences (BSK-66, 80, 89). In third-leukapheresis MO, the clones included differentiation genes (HOX-B3, thymosin-beta4, PU.1, glucocerebrosidase, MEL-18, and glucose-6-phosphate dehydrogenase) and 3 unknown/functionally undefined sequences. Differential expression of most genes from the activated pool was confirmed in leukapheresis samples from 2 additional RA patients. In MO from RA patients, not only were IL-1beta and the ATRII homolog significantly overexpressed (maximum 36-fold), but also 4 of the unknown/functionally undefined genes (maximum 102-fold). Notably, messenger RNA levels of BSK-89 correlated positively with the erythrocyte sedimentation rate (ESR), whereas those of BSK-83 correlated negatively with the ESR and C-reactive protein level. CONCLUSION: The combined strategy of gene subtraction and semiquantitative RT-PCR may allow the definition of MO activation patterns during different disease phases (including therapy-induced remission) and the identification of novel MO genes with pathogenetic relevance in RA.

Preactivated peripheral blood monocytes in patients with essential hypertension.

The purpose of this study was to investigate the possible involvement of human peripheral blood monocytes in the pathology of hypertensive disease. We determined the in vitro secretion patterns of proinflammatory cytokines obtained from isolated peripheral monocytes from normal controls and from hypertensive patients either after in vitro stimulation with angiotensin II (Ang II) with or without preincubation with an Ang II type 1 receptor antagonist (losartan) or after stimulation with lipopolysaccharide. Blood samples were obtained from 22 patients with essential hypertension (before any drug administration or after interruption of antihypertensive therapy) and from 24 normotensive healthy individuals used as a control group. Peripheral blood monocytes were isolated by density gradient centrifugation and plastic adherence. The state of monocyte activity was determined by the capacity to secrete tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6, (IL-6) either spontaneously or after stimulation. Cytokine concentrations were determined in culture supernatants by specific ELISA. Proinflammatory cytokine levels were assessed by semiquantitative reverse transcribed polymerase chain reaction. After stimulation with Ang II, the IL-1beta secretion of peripheral blood monocytes was significantly increased in hypertensive patients versus healthy individuals (P<0.05). In contrast, in monocytes preincubated with losartan before exposure to Ang II, IL-1beta secretion was diminished in both groups to comparable levels. The secretion of IL-1beta and TNF-alpha was significantly increased in peripheral blood monocytes from hypertensive patients versus healthy individuals after stimulation with lipopolysaccharide (TNF-alpha, P<0.02; IL-1beta, P<0.05). Upregulation of IL-1beta and TNF-alpha secretion in peripheral blood monocytes from hypertensive patients was also seen at the RNA level. Our results indicate preactivated peripheral blood monocytes in hypertensive patients. Ang II may be directly involved in the process of monocyte activation.

Pinacidil relaxes porcine and human coronary arteries by activating ATP-dependent potassium channels in smooth muscle cells.

We investigated the effect of the potassium channel opener pinacidil on ATP-dependent K+ channels (KATP) in the relaxation of porcine and human coronary arteries by means of isometric contraction experiments in arterial rings. We also measured whole cell currents in freshly isolated porcine and human coronary artery vascular smooth muscle cells with patch clamp. We first characterized serotonin-induced precontractions in our vessels and proved that the contractions were mediated by Ca2+ influx through voltage-dependent Ca2+ channels. Similarly, we observed that serotonin-induced contractions were strongly enhanced by small K(+)-induced depolarizations. Pinacidil completely relaxed rings preconstricted with 5 microM serotonin and produced dose-dependent relaxations of 5 microM serotonin-preconstricted rings, with an IC50 of 1.26 microM. Similar results were observed (IC50 = 1.15 microM) when the endothelium was removed. The KATP blocker glibenclamide (3 microM), inhibited pinacidil-induced relaxations (5-10 microM) by approximately 25% although the KATP blocker tetrapentylammonium (10 microM), inhibited pinacidil-induced (5-10 microM) relaxations completely. Pinacidil 10 microM had only a minimal effect on rings precontracted with a 50 mM external K+ concentration (IC50 = 60 microM). Porcine and human arterial rings did not differ qualitatively in their responses. Moreover, in the patch clamp experiments pinacidil (1 microM and 20 microM) induced a large, nonrectifying, outward current in both human and porcine cells. The reversal potential was close to the K+ equilibrium potential, suggesting an induction of pinacidil-activated K+ current. The pinacidil-induced (1 microM) current was strongly inhibited by glibenclamide (3 microM). These data show that the relaxation of porcine and human coronary arteries by pinacidil is primarily induced by an opening of KATP in smooth muscle cells. Furthermore, the vasorelaxant effect of pinacidil is not endothelium dependent.