Patterns of C1-Inhibitor/Plasma Serine Protease Complexes in Healthy Humans and in Hereditary Angioedema Patients.

C1-inhibitor (C1-INH) is an important regulator of the complement, coagulation, fibrinolytic and contact systems. The quantity of protease/C1-INH complexes in the blood is proportional to the level of the in vivo activation of these four cascade-like plasma enzyme systems. Parallel determination of C1-INH-containing activation complexes could be important to understand the regulatory role of C1-INH in diseases such as hereditary angioedema (HAE) due to C1-INH deficiency (C1-INH-HAE). We developed in-house ELISAs to measure the concentration of complexes of C1-INH formed with active proteases: C1r, C1s, MASP-1, MASP-2, plasma kallikrein, factor XIIa, factor XIa, and thrombin, as well as to determine total and functionally active C1-INH. We measured the concentration of the complexes in EDTA plasma from 6 healthy controls, from 5 with type I and 5 with type II C1-INH-HAE patients during symptom-free periods and from five patients during HAE attacks. We also assessed the concentration of these complexes in blood samples taken from one C1-INH-HAE patient during the kinetic follow-up of a HAE attack. The overall pattern of complexed C1-INH was similar in controls and C1-INH-HAE patients. C1-INH formed the highest concentration complexes with C1r and C1s. We observed higher plasma kallikrein/C1-INH complex concentration in both type I and type II C1-INH-HAE, and higher concentration of MASP-1/C1-INH, and MASP-2/C1-INH complexes in type II C1-INH-HAE patients compared to healthy controls and type I patients. Interestingly, none of the C1-INH complex concentrations changed significantly during HAE attacks. During the kinetic follow-up of an HAE attack, the concentration of plasma kallikrein/C1-INH complex was elevated at the onset of the attack. In parallel, C1r, FXIIa and FXIa complexes of C1-INH also tended to be elevated, and the changes in the concentrations of the complexes followed rather rapid kinetics. Our results suggest that the complement classical pathway plays a critical role in the metabolism of C1-INH, however, in C1-INH-HAE, contact system activation is the most significant in this respect. Due to the fast changes in the concentration of complexes, high resolution kinetic follow-up studies are needed to clarify the precise molecular background of C1-INH-HAE pathogenesis.

MASP-1 Increases Endothelial Permeability.

Pathologically increased vascular permeability is an important dysfunction in the pathomechanism of life-threatening conditions, such as sepsis, ischemia/reperfusion, or hereditary angioedema (HAE), diseases accompanied by uncontrolled activation of the complement system. HAE for example is caused by the deficiency of C1-inhibitor (the main regulator of early complement activation), which leads to edematous attacks threatening with circulatory collapse. We have previously reported that endothelial cells become activated during HAE attacks. A natural target of C1-inhibitor is mannan-binding lectin-associated serine protease-1 (MASP-1), a multifunctional serine protease, which plays a key role in the activation of complement lectin pathway. We have previously shown that MASP-1 induces the pro-inflammatory activation of endothelial cells and in this study we investigated whether MASP-1 can directly affect endothelial permeability. All experiments were performed on human umbilical vein endothelial cells (HUVECs). Real-time micro electric sensing revealed that MASP-1 decreases the impedance of HUVEC monolayers and in a recently developed permeability test (XperT), MASP-1 dose-dependently increased endothelial paracellular transport. We show that protease activated receptor-1 mediated intracellular Ca2+-mobilization, Rho-kinase activation dependent myosin light chain (MLC) phosphorylation, cytoskeletal actin rearrangement, and disruption of interendothelial junctions are underlying this phenomenon. Furthermore, in a whole-transcriptome microarray analysis MASP-1 significantly changed the expression of 25 permeability-related genes in HUVECs-for example it up-regulated bradykinin B2 receptor expression. According to our results, MASP-1 has potent permeability increasing effects. During infections or injuries MASP-1 may help eliminate the microbes and/or tissue debris by enhancing the extravasation of soluble and cellular components of the immune system, however, it may also play a role in the pathomechanism of diseases, where edema formation and complement lectin pathway activation are simultaneously present. Our findings also raise the possibility that MASP-1 may be a promising target of anti-edema drug development.

MASP-1 induces a unique cytokine pattern in endothelial cells: a novel link between complement system and neutrophil granulocytes.

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms.

Dynamic ABCG2 expression in human embryonic stem cells provides the basis for stress response.

ABCG2 is a plasma membrane multidrug transporter with an established role in the cancer drug-resistance phenotype. This protein is expressed in a variety of tissues, including several types of stem cell. Although ABCG2 is not essential for life, knock-out mice were found to be hypersensitive to xenobiotics and had reduced levels of the side population of hematopoietic stem cells. Previously we have shown that ABCG2 is present in human embryonic stem cell (hESC) lines, with a heterogeneous expression pattern. In this study we examined this heterogeneity, and investigated whether it is related to stress responses in hESCs. We did not find any difference between expression of pluripotency markers in ABCG2-positive and negative hESCs; however, ABCG2-expressing cells had a higher growth rate after cell separation. We found that some harmful conditions (physical stress, drugs, and UV light exposure) are tolerated much better in the presence of ABCG2 protein. This property can be explained by the transporter function which eliminates potential toxic metabolites accumulated during stress conditions. In contrast, mild oxidative stress in hESCs caused rapid internalization of ABCG2, indicating that some environmental factors may induce removal of this transporter from the plasma membrane. On the basis of these results we suggest that a dynamic balance of ABCG2 expression at the population level has the advantage of enabling prompt response to changes in the cellular environment. Such actively maintained heterogeneity might be of evolutionary benefit in protecting special cell types, including pluripotent stem cells.

Doxorubicin selectively suppresses mRNA expression and production of endothelin-1 in endothelial cells.

Doxorubicin (DXR) is a widely used cytostatic agent, but its administration is limited by its cardiovascular side effects. The endothelium is one of the largest organs in the human body and due to its direct contact with blood; it is exposed to the toxic effects of DXR. The aim of this study was to investigate in endothelial cells the effects of DXR on the expression of genes involved in cardiovascular diseases. We used in vitro cultured human umbilical vein endothelial cells (HUVEC) as a model; gene expression was assessed by SuperArray and qPCR. Out of the 96 representative genes of cardiovascular importance, the expression of only the ET-1 gene changed significantly. ET-1 mRNA expression was 10.9% of the untreated control (p=0.0049). This result was confirmed by qPCR (2.41% of control, p=0.0022). DXR also suppressed ET-1 production at protein level (p=0.0116). Both the early decrease in endothelial ET-1 production in the presence of DXR and the high plasma level of DXR during chemotherapy may influence the toxic effects of the drug.

Proinflammatory activation pattern of human umbilical vein endothelial cells induced by IL-1ß, TNF-α, and LPS.

Endothelial cells play a critical role in inflammation by responding to several endogenous and exogenous proinflammatory stimuli. The three most studied factors that provide inflammatory signals to endothelial cells are lipopolysaccharide (LPS), tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß; however, their effects on endothelial cells were thoroughly compared at the level of gene expression only. Therefore, our aim was to assess the differences in the signaling pathways, adhesion molecules, and cytokines induced by proinflammatory factors in human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that signaling of LPS was less effective than that of IL-1ß, and was significantly slower than that ofTNF-α and IL-1ß, which can be partially explained by the special localization of Toll-like receptor 4 (TLR4). We showed that TLR4 is mainly localized in Golgi apparatus in HUVEC. The proinflammatory capacity of TNF-α was similar to that of IL-1ß in inducing NF-κB nuclear translocation, while IL-1ß was the strongest activator of MAPK pathways. Moreover, expression of E-selectin, IL-6, and IL-8 was induced most efficiently by IL-1ß, while LPS and TNF-α had less effect, whereas we did not find such a difference in ICAM-1 and MCP-1 expression. Due to the higher induction of E-selectin and IL-8, IL-1ß might have more important role in neutrophil recruitment than LPS and TNF-α. By above-mentioned parameters we identified a signaling and expression pattern for the three proinflammatory molecules. This pattern illustrates how complex a proinflammatory process can be, and may enable us to predict and compare the pathomechanism of various inflammatory diseases.

Plasma osteopontin concentrations in preeclampsia - is there an association with endothelial injury?

UNLABELLED: Abstract Background: It has been previously reported that plasma osteopontin (OPN) concentrations are increased in cardiovascular disorders. The goal of the present study was to determine plasma OPN concentrations in healthy pregnant women and preeclamptic patients, and to investigate their relationship to the clinical characteristics of the study subjects and to markers of inflammation [C-reactive protein (CRP)], endothelial activation [von Willebrand factor antigen (VWF:Ag)] or endothelial injury (fibronectin), oxidative stress [malondialdehyde (MDA)] and trophoblast debris (cell-free fetal DNA). METHODS: Forty-four patients with preeclampsia and 44 healthy pregnant women matched for age and gestational age were involved in this case-control study. Plasma OPN concentrations were measured with ELISA. Serum CRP concentrations were determined with an autoanalyzer using the manufacturer's reagents. Plasma VWF:Ag was quantified by ELISA, while plasma fibronectin concentrations were measured by nephelometry. Plasma MDA concentrations were estimated by the thiobarbituric acid-based colorimetric assay. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time PCR analysis of the sex-determining region Y (SRY) gene. For statistical analyses, non-parametric methods were applied. RESULTS: Serum levels of CRP, as well as plasma concentrations of VWF:Ag, fibronectin, MDA and cell-free fetal DNA were significantly higher in preeclamptic patients than in healthy pregnant women. There was no significant difference in plasma OPN concentrations between controls and the preeclamptic group. However, preeclamptic patients with plasma fibronectin concentrations in the upper quartile had significantly higher plasma OPN concentrations than those below the 75th percentile, as well as healthy pregnant women [median (interquartile range): 9.38 (8.10-11.99) vs. 7.54 (6.31-9.40) and 7.40 (6.51-8.80) ng/mL, respectively, p<0.05 for both]. Furthermore, in preeclamptic patients, plasma OPN concentrations showed a significant positive linear association with plasma fibronectin (Spearman R=0.38, standardized regression coefficient (beta)=0.41, p<0.05 for both). CONCLUSIONS: Plasma OPN concentrations are increased in preeclamptic patients with extensive endothelial injury. However, further studies are warranted to explore the relationship between OPN and endothelial damage. Clin Chem Lab Med 2010;48:181-7.

Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia.

BACKGROUND: The aim of our study was to examine whether increased circulating total cell-free DNA levels are related to the clinical characteristics and standard laboratory parameters of preeclamptic patients, to markers of inflammation, endothelial activation or injury, oxidative stress and to cell-free fetal DNA levels. METHODS: Circulating total cell-free DNA was measured by real-time quantitative PCR in plasma samples obtained from 67 preeclamptic and 70 normotensive pregnant women. Standard laboratory parameters, C-reactive protein, plasma von Willebrand factor antigen, plasma fibronectin, plasma malondialdehyde and cell-free fetal DNA levels were also determined. RESULTS AND CONCLUSION: Circulating total cell-free and fetal deoxyribonucleic acid levels were significantly elevated in pregnancies complicated by preeclampsia (median: 11.395 vs. 32.460 and 0.001 vs. 0.086 pg/microl; P < .001). The quantity of plasma total cell-free DNA did not correlate with most of the laboratory parameters, except for serum aspartate aminotransferase and alanine aminotransferase activities (correlation coefficient: 0.31; P = 0.012 and 0.46; P < .001). There was no correlation with clinical characteristics, including body mass index. The releases of both free fetal and total cell-free deoxyribonucleic acid were found to be affected in preeclampsia. Hepatocellular necrosis seems to be responsible--at least partly--for increased circulating total DNA levels in preeclampsia, as suggested by the significant correlation with liver enzyme activities.

Levels of von Willebrand factor antigen and von Willebrand factor cleaving protease (ADAMTS13) activity predict clinical events in chronic heart failure.

Decreased activity of ADAMTS13, the von Willebrand factor (VWF) cleaving protease, was recently reported in cardiovascular diseases and in hepatic failure. Chronic heart failure (CHF) is characterised by abnormalities of left ventricular function accompanied by the failure of the liver and dysregulation of endothelial activation. Therefore, the aim of our study was to measure ADAMTS13 activity in CHF, and determine the prognostic value of VWF and ADAMTS13 on major clinical events in CHF. ADAMTS13 activity (measured by FRETS-VWF73 substrate) was decreased in CHF (n = 152, left ventricular ejection fraction <45%), and it correlated negatively with B-type natriuretic peptide (BNP) NYHA (New York Heart Association) classes, markers of synthetic capacity of the liver and endothelial dysfunction (all p < 0.005). Both, high VWF:Ag levels (hazard ratio [HR] 1.52, 95% confidence interval [CI] 1.189-1.943), and low ADAMTS13/VWF:Ag ratios (HR 0.70, 95% CI 0.58-0.84) independently and significantly predicted short-term (1 year follow-up) clinical adverse events in heart failure (HF). Decreased activity of ADAMTS13 with concomitant high VWF:Ag levels is a significant independent predictor of clinical events in CHF. The levels of the two molecules may integrate the impaired synthetic capacity of the liver and the disturbed endothelial regulation and can therefore be a useful tool to predict clinical events in CHF.

Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function.

Activation of the complement system can induce and enhance inflammatory reaction. Mannose-binding lectin-associated serine protease-1 (MASP-1) is an abundant protease of the complement lectin pathway; however, its physiological function is unclear. In this study, we demonstrate for the first time that MASP-1 is able to activate Ca(2+) signaling, NF-kappaB, and p38 MAPK pathways in cultured HUVECs. Activation was initiated by MASP-1 only; the related protease, MASP-2, had no such effect. The phenomenon was dependent on the proteolytic activity of MASP-1, suggesting modulation of endothelial cell function through a protease-activated receptor (PAR). Using synthetic peptide substrates representing the protease-sensitive regions of PARs, we were able to demonstrate that PAR4 is a target of MASP-1. The presence of functionally active PAR4 in HUVECs was demonstrated using PAR4 agonist peptide and mRNA quantification. Finally, we showed that the amount of membrane-bound intact PAR4 decreases after MASP-1 treatment. All of these results provide a novel link between the regulation of endothelial cell function and complement system activation, and they suggest that MASP-1-induced PAR4 activation could contribute to the development of the inflammatory reaction.

Protein kinase inhibitor-induced endothelial cell cytotoxicity and its prediction based on calculated molecular descriptors.

Protein kinase inhibitors (PKIs) as potent signal transduction therapeutic compounds represent a very rapidly expanding group of anticancer drugs. These agents may be toxic for endothelial cells, however, very few experimental data exist on the cytotoxicity of PKIs. The aim of this study was to set up an appropriate test system for endothelial cells and to assess the structure-related cytotoxic effects of a selected library of PKIs. The inhibitor library contains several lead molecules with different basic structures and a set of modified derivatives of the lead compounds. The toxicity of PKIs did not correlate directly with the structural features of the molecules. However, we successfully built up a model based on 15 calculated molecular descriptors, which is capable of predicting cytotoxicity with acceptable probability. Our results show that the cytotoxic effects of PKIs should be taken into account for optimal drug development to overcome endothelial cell-related side effects.

Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia.

It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer's kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.

Increased plasma von Willebrand factor antigen levels but normal von Willebrand factor cleaving protease (ADAMTS13) activity in preeclampsia.

The activity of ADAMTS13, the von Willebrand factor (VWF) cleaving protease is low in several conditions, including HELLP (haemolysis, elevated liver enzymes, and low platelet count) syndrome. As HELLP syndrome develops in most cases on the basis of preeclampsia, our aim was to determine whether plasma ADAMTS13 activity is decreased in preeclampsia. Sixty-seven preeclamptic patients, 70 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Plasma ADAMTS13 activity was determined with the FRETS-VWF73 assay, while VWF antigen (VWF:Ag) levels with an enzyme-linked immunosorbent assay. The multimeric pattern of VWF was analyzed by SDS-agarose gel electrophoresis. There was no significant difference in plasma ADAMTS13 activity between the preeclamptic and the healthy pregnant and non-pregnant groups (median [25-75 percentile]: 98.8 [76.5-112.8] %, 96.3 [85.6-116.2] % and 91.6 [78.5-104.4] %, respectively; p > 0.05). However, plasma VWF:Ag levels were significantly higher in preeclamptic patients than in healthy pregnant and non-pregnant women (187.1 [145.6-243.1] % versus 129.3 [105.1-182.8] % and 70.0 [60.2-87.3] %, respectively; p < 0.001). The multimeric pattern of VWF was normal in each group. Primiparas had lower plasma ADAMTS13 activity than multi-paras (92.6 [75.8-110.6] % versus 104.2 [92.1-120.8] %; p = 0.011). No other relationship was found between clinical characteristics, laboratory parameters and plasma ADAMTS13 activity in either study group. In conclusion, plasma ADAMTS13 activity is normal in preeclampsia despite the increased VWF:Ag levels. However, further studies are needed to determine whether a decrease in plasma ADAMTS13 activity could predispose preeclamptic patients to develop HELLP syndrome.

Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia.

It has been previously reported that serum levels of 70-kDa heat-shock protein (Hsp70) are elevated in preeclampsia. The aim of the present study was to examine whether increased serum Hsp70 levels are related to clinical characteristics and standard laboratory parameters of preeclamptic patients, as well as to markers of inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) or endothelial injury (fibronectin), trophoblast debris (cell-free fetal DNA) and oxidative stress (malondialdehyde). Sixty-seven preeclamptic patients and 70 normotensive, healthy pregnant women were involved in this case-control study. Serum Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Standard laboratory parameters (clinical chemistry) and C-reactive protein (CRP) levels were determined by an autoanalyzer using the manufacturer's kits. Plasma von Willebrand factor antigen (VWF:Ag) levels were quantified by ELISA, and plasma fibronectin concentration by nephelometry. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time polymerase chain reaction analysis of the sex-determining region Y gene. Plasma malondialdehyde levels were measured by the thiobarbituric acid-based colorimetric assay. Serum Hsp70 levels were increased in preeclampsia. Furthermore, serum levels of blood urea nitrogen, creatinine, bilirubin and CRP, serum alanine aminotransferase and lactate dehydrogenase (LDH) activities, as well as plasma levels of VWF:Ag, fibronectin, cell-free fetal DNA and malondialdehyde were also significantly higher in preeclamptic patients than in normotensive, healthy pregnant women. In preeclamptic patients, serum Hsp70 levels showed significant correlations with serum CRP levels (Spearman R = 0.32, p = 0.010), serum aspartate aminotransferase (R = 0.32, p = 0.008) and LDH activities (R = 0.50, p < 0.001), as well as with plasma malondialdehyde levels (R = 0.25, p = 0.043). However, there was no other relationship between serum Hsp70 levels and clinical characteristics (age, parity, body mass index, blood pressure, gestational age, fetal birth weight) and laboratory parameters of preeclamptic patients, including markers of endothelial activation or injury and trophoblast debris. In conclusion, increased serum Hsp70 levels seem to reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Nevertheless, further studies are required to determine whether circulating Hsp70 plays a causative role in the pathogenesis of the disease.

Interleukin-6 -174 promoter polymorphism does not influence IL-6 production after LPS and IL-1 beta stimulation in human umbilical cord vein endothelial cells.

The IL-6 is a typical pleiotropic cytokine, which regulates T cell response, B cell differentiation and immunoglobulin production. Endothelial cells can produce large amounts of IL-6. SNP at position -174 (G/C) in the IL-6 promoter region was found to be associated with a series of complex diseases. In this study we analyzed whether IL-6 -174 G/C polymorphism has any effect on IL-6 production of in vitro cultured HUVECs. Thirty-three fresh umbilical cords were recruited from healthy pregnancies. The endothelial cells isolated from human umbilical cords were genotyped for IL-6 -174 SNP. C allele frequency was 0.379. The IL-6 production of each primary HUVEC line was measured after IL-1beta or LPS treatment by ELISA. Serial dilutions of the stimulating agents were applied and maximum amount of produced IL-6 (R(max)) and stimulator concentrations at half-maximal IL-6 response (MR(50)) were calculated for each of the cell lines. IL-6 production was not associated with IL-6 -174 SNP genotypes or with presence of C allele. Our results showed that IL-6 production of HUVEC after proinflammatory stimulation was not influenced by IL-6 -174 SNP. Further functional studies are required to compare differences and similarities in IL-6 -174 SNP dependent expression of IL-6 among various cell types.