Received Date: February 20, 2017; Accepted Date: March 07, 2017; Published Date: March 16, 2017
Citation: Jana P, Khan M, De SK, Sinha AK, Guha S, et al. (2017) Estriol Inhibits TNF-Α and IL-6 Production, and Promotes Anti-Inflammatory Responses via Nitric Oxide Stimulation in Dermcidin (Isoform-2)-Stimulated Neutrophil. J Cell Sci Apo 1: 103.
Copyright: © 2017 Jana P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Increase in the level of cytokines like TNF-α and IL-6 causes the inflammatory surge in acute ischemic heart disease (AIHD). Occurrence of a high level dermcidin isoform-2 in AIHD demonstrates a possible regulation on cytokines expression. It was found that incubation of 120 nM of dermcidin isoform-2 (DCN-2) to the normal neutrophil solution for 2 h resulted in the increase of synthesis of TNF-α from 3.829 ± 1.53 pg/ml to 20.7 ± 6.9 pg/ml and IL-6 from 3.27 ± 1.52 pg/ml to 47.07 ± 3.4 pg/ml. Cytokines were determined in AIHD patient blood with TNF-α level 18.3-27.3 pg/ml, median value 21.863 pg/ml and IL-6 23.54-52.733 pg/ml, median value 42.163 pg/ml. Treatment with 0.6 nM estriol, a kind of female steroid hormone estrogen for 45 min decreased the elevated cytokine level in 120 nM DCN-2 treated normal neutrophils. The expression of DCN-2 induced TNF-α synthesis in neutrophils was further determined by Western blot technique with a thickened band intensity of TNF-α in DCN-2 induced neutrophil solution. The production of nitric oxide (NO) was also regulated with the effect of DCN-2 treatment from 1.61 nmol NO/ml to 0 nmol NO/ml. The subsequent reduction of TNF-α level due to 0.6 nM estriol treatment had shown the corresponding increase in NO level to 0.559 nmol/ml. It can be concluded that production of DCN-2 due to stress in AIHD after heart attack propagate the inflammatory response. Steroid molecule like estriol plays a protective role by reducing DCN-2 responses through the NO synthesis.
Acute ischemic heart disease; Inflammation; TNF-α; IL- 6; Dermcidin isoform-2
AIHD: Acute Ischemic Heart Disease; DCN- 2: Dermcidin Isoform-2; TNF-Α: Tumour Necrosis Factor-Α; IL- 6: Interleukin 6; LPS: Lipopolysaccharide; HBSS: Hank’s Balanced Salt Solution; NOS: Nitric Oxide Synthase; ELISA: Enzyme Linked Immunosorbent Assay; SDS-PAGE: Sodium Dodecyle Sulphate- Polyacralamide Gel Electrophoresis; ICAM: Intercellular Adhesion Molecule, Nsaids: Non-Steroidal Anti-Inflammatory Drugs, MDCs: Monocyte-Derived Cells; MMPs: Matrix Metalloproteinases; PGE2: Prostaglandin
Acute ischemic heart disease (AIHD) is a life threatening cardiac disorder that may terminate the life process all of a sudden. There have been several factors reported to be linked with the condition. Inflammatory process plays an important role for the development of AIHD. Both atherosclerosis and inflammation bring about narrowing of the coronary artery, resulting in diminishing blood supply, hence the nutrients and O2 to the heart muscle . Increase in the level of inflammatory components at the location of atherosclerotic lesion leads to the active inflammation . It is evident that neutrophils (most abundant 50-75% leukocytes) are major contributor among the inflammatory compound secreting cells . The inflammatory components play an important role in multi-step and diversified cascade of atherosclerosis. Eventually, it leads to the burst of the atherosclerotic plaque. Increase of inflammatory markers i.e. TNF-α  and IL-6  in the atherosclerotic lesion has been found to be elevated and so retain in the increased level in chronic situation as in the blood of AIHD patients .
Increase in cytokine level is stimulated by some endotoxins like lipopolysaccarides (LPS) and a few stress inducers. Dermcidin isoform-2 (DCN-2), a stress induced protein which is found to be over expressed in acute myocardial infarction (AMI) patients  has been reported to increase the level of TNF-α  and IL-6. But various agents including estrogens are known to inhibit the inflammatory response  that may play an important role in AIHD. In our previous work we have reported that estrogen is able to produce nitric oxide (NO) by stimulating the nitric oxide synthase (NOS)  and resulted in the inhibition of platelet aggregation  and fibrinolysis . We report herein, the presence of DCN-2 in AIHD patients results in the elevated synthesis of TNF-α and IL-6 in neutrophils. The incubation of estriol, a kind of estrogen with less estrogenic activity (in the aspect of stimulating female reproductive organs) can nullify the effect of DCN- 2 up to a level. We have also reported that the pre-existence of sufficient amount of estrogen can induce nitric oxide production  that acts as a good vaso-dilating agent.
In this background, we are intended to conduct our investigation. Here, we explore the induction of inflammatory responses and their markers TNF-α and IL-6 by the stress induced protein DCN- 2 in the neutrophil. We further investigated the role of estriol in the nullification of above mentioned inflammatory responses suggesting the anti-inflammatory therapeutic efficacy of this steroid against the present disease pathogenesis.
The protocol used in the study involved normal human subjects and was carried out in accordance with the Helsinki agreement which was approved by the Internal Review Board, Sinha Institute of Medical Science and Technology, Garia, Kolkata, West Bengal.
Each patient or next of kin of those patients with any type of cardiac disease (in severe cases where patient is unable to give any consent) had signed in an informed consent form before giving the blood (5.0 ml). Withdrawal of the blood was decided by an attending physician after confirming the occurrence of the disease.
Estriol (purity 98%), goat anti-rabbit immunoglobulin G-alkaline phosphatase and fibrinogen were obtained from Sigma-Aldrich. TNF-α, IL-6 and their primary and secondary antibody were obtained from AbCam. Polyclonal antibody against estriol was obtained from Thermo Scientific. Maxisorp plates for enzyme linked immunosorbent assay (ELISA) were obtained from Nunc, Roskilde, Denmark. All other chemicals used were of analytical grade.
Selection of the volunteers
The study was conducted by involving both male and female volunteers (n=40, M=20, F=20) between the ages of 20 to 40 years. They had no history of systemic hypertension or diabetes mellitus. They were also clarified for any life threatening infectious diseases if they had. The volunteers did not suffer from cardiovascular or cerebrovascular diseases and had not been hospitalized for any reason at least for 6 months before they participated in the study. Women volunteers had never received any contraceptive medications. No medications including acetyl salicylic acid (aspirin) was taken by the participants at least for 4 weeks before they donated blood.
Selection of the AIHD patients
Acute ischemic heart disease in all patients was characterized as chest pain over 120 minutes accompanied by ischemic electrocardiographic changes (ST-section changes and/or T-wave reversals). Infarction in cardiac muscle was diagnosed by elevation of troponin I or troponin T (0.8 ng/ml) or creatine kinase-myocardial band.
Preparation of neutrophil solution
Neutrophils were isolated from the citrated blood samples as described before . Cell counts were determined by optical microscopy. Isolated neutrophils suspended in Hank’s balanced salt solution (HBSS), pH 7.4 were incubated with different concentrations of dermcidin isoform-2 (DCN-2) for 2 h at 37°C under sterile conditions. When needed, the nucleic acids were isolated from these incubated samples for in vitro translation of TNF-α and IL-6 as described below.
Determination of NO synthesis in DCN-2 induced neutrophils
The media solution containing neutrophils were incubated with 10 μM of 1-arginine in the presence or absence of different concentrations of DCN-2 (90 min in 37°C). The mixtures were incubated in the presence or absence of pre-incubated estriol (0.6 nM for 45 min at 37°C) with triplicate experiments for each group. To determine the NO production in the control experiment, the vehicle (HBSS buffer without neutrophils) was treated similarly under the identical conditions.
Nitric oxide was assayed following the methemoglobin method, following a procedure described elsewhere, by use of a Beckman spectrophotometer (model DU6) . Assay validity was confirmed by the use of an independent chemiluminescence method .
Determination of the change in TNF-α and IL-6 synthesis in neutrophil solution
Briefly, neutrophil was incubated with an equal volume of phosphate buffer saline (PBS) in an assay plate overnight in 4°C. Nonspecific binding was blocked by 5% bovine serum albumin in the same buffer. The samples were then washed with PBS containing Tween-20, and incubated for 2 h with diluted primary antibody in PBS (1:200) obtained from AbCam. The samples were next washed with PBS-T20 and incubated with diluted goat anti-rabbit IgG-horse radish peroxidase (1:2000) in the same buffer for 1 h. After washing they were incubated with p-nitrophenyl phosphate (1 mg/ml) in carbonate buffer (pH 9.8) containing 10 mM MgCl2. The development of color was determined at 450 nm. The amount of TNF-α and IL-6 present in the sample was determined in an ELISA reader using respective monoclonal antibody according to the method described .
Level of TNF-α was checked in neutrophils from AIHD subjects as positive control reported to have high level of TNF-α. Normal neutrophil in the absence of DCN-2 was also subjected for the determination of TNF-α in control.
Western blot analysis of TNF-α in neutrophil solution after the treatment with DCN-2
The neutrophil solutions from different treatment groups were centrifuged and supernatant from each vial was collected separately. The supernatant was subjected for SDS-polyacralamide gel electrophoresis (SDS-PAGE). Protein bands were transferred in nitrocellulose membrane as described before using CAPS buffer. The level of TNF-α in nitrocellulose membrane was analysed by using goat anti-TNF-α antibody .
The results shown are mean ± standard deviation (SD) of at least 5 independent experiments using the blood samples from 5 different donors each in triplicate. MS excel was used to analyse these data.
The significance “p” of the results was determined by student’s t test, and p<0.001 was considered to be significant (n=15). The coefficient of correlation “r” was determined by Pearson test by using GraphPad Prism software.
Band intensity was determined by using ImageJ software.
Effect of DCN-2 in the inhibition of estriol induced NO production in normal neutrophils
Different concentrations of DCN-2 were incubated to the neutrophils for 120 min at 37°C to find out if the DCN-2 has any inhibitory property to estriol induced NO production. Neutrophils which were pre-treated with 0.6 nM estriol for 45 min at 37°C also subjected for NO assay with different concentrations of DCN-2. It was found that incubation of 120 nM DCN-2 to the neutrophils caused a distinctive inhibition of the NO production from 1.61 nmol NO/ ml to 0 nmol NO/ml. NO level was also decreased to 0.559 nmol/ ml in post treatment of 0.6 nM estriol after incubating with different concentration of DCN-2 (Figure 1). But the pre-treatment of 0.6 nM estriol showed the NO level 1.27 nmol/ml which did not affect too much due to incubation with DCN-2 (Figure 1).
Figure 1: DCN-2 induced inhibition of estriol mediated NO synthesis in neutrophils. Different concentrations of DCN-2 were incubated with neutrophil solution. Solid inverted triangles show synthesis of NO in normal neutrophils with 0.6 nM estriol but in the absence of DCN-2. Solid squares describe the change in the synthesis of NO in the absence of estriol but with different concentrations of DCN-2 (80-120 nM) for 120 min. Solid circles signify the NO synthesis with different concentrations of DCN-2 followed by 0.6 nM estriol incubation for 45 min. NO synthesis was also carried out with a preincubation of 0.6 nM estriol and different concentrations of DCN-2 for 120 min then which are denoted by solid triangle . Solid rhombus describe the synthesis of NO in the control sample were incubated with vehicle only.
Effect of DCN-2 in the synthesis of TNF-α and IL-6 in normal neutrophils
Incubation of different concentrations of DCN-2 to the neutrophils have shown to elevate TNF-α and IL-6 level in the solution. It has been found that TNF-α level has been increased from 3.829 ± 1.53 pg/ml to 20.7 ± 6.9 pg/ml due to incubation of 130 nM DCN-2 to the neutrophils solution for 120 min at 37°C (Figure 2a). The incubation of 0.6 nM estriol before the treatment with different concentrations of DCN-2 resulted to produce a decreased level of TNF-α with 11.97 ± 0.92 pg/ml due to incubation in the same environment (Figure 2a).
Figure 2: (a) DCN-2 induced synthesis of TNF-α. Incubation of DCN-2 to the neutrophils was followed by the determination of TNF-α. Control group (C) shows the synthesis of TNF-α in the absence of DCN-2. Group E describes the synthesis of TNF-α in the presence of different concentration of DCN-2. In the next group (Group F) the neutrophils were pre-incubated with 0.6 nM estriol and different concentrations of DCN-2 then. (b) Expression of DCN-2 induced TNF-α in neutrophil represented by western blot technique. Lane 1 describes TNFα expression in neutrophil from AMI patients. Lane 2 shows TNFα level in dermcidin induced normal neutrophils. Lane 3 describes TNFα level in dermcidin induced normal neutrophils treated with estrogen. Lane 4 describes TNFα level in normal neutrophils. (c) Measurement of the intensity of band from western blotted film.
Expression of TNF-α was also determined by Western blot technique and the bands obtained on the photographic film have described different level of intensity (Figure 2b). It was found that the band intensity for the TNF-α protein in AIHD patients was highest as 1234.44 Units (Figure 2c). Whereas in neutrophil obtained from age and sex matched normal control was found to be 375.74 Units which increases to 762.72 Units as 120 nM of DCN-2 was incubated to normal neutrophil solution for 2 h at 37°C (Figure 2c). A 45 min treatment of 0.6 nM estriol after 120 nM DCN-2 treated neutrophil solution the expression of TNF-α was inhibited and the band intensity was 463.28 Units (Figure 2c).
Another cytokine IL-6 also found to be increased due to incubating with different concentrations of DCN-2 as described before. The level of IL-6 has been changed from 3.27 ± 1.52 pg/ml to 47.07 ± 3.4 pg/ml due to incubation of 130 NM of DCN-2 to the neutrophils for 120 min at 37°C (Figure 3). The inhibitory effect of 0.6 nM of estriol for 45 min pre-incubation has shown a decreased level of IL-6 to 15.34 ± 2.91 pg/ ml from 43.218 ± 4.2 pg/ml (Figure 3). But gradual increase of DCN-2 to the neutrophil solution was found to nullify the inhibitory effect of 0.6 nM estriol on IL-6 level. And, at the concentration of 130 nM DCN- 2 the cytokine IL-6 level was 47.07 ± 3.4 pg/ml whereas 0.6 nM estriol was able to restore the level to 39.03 ± 1.82 pg/ml.
Figure 3: Synthesis of IL-6 in neutrophils by DCN-2. The level of cytokine IL-6 was determined by ELISA. Here, Group D denotes the synthesis of IL-6 in control. Group-C describes the synthesis of IL-6 in 120 min incubated neutrophils. In group F, the synthesis of IL-6 was characterised after incubating 0.6 nM estriol followed by different concentrations of DCN-2.
Role of estriol to reduce the level of TNF-α and IL-6 in neutrophils from AIHD
Neutrophils from AIHD (n=9, M=7, F=2) patients were collected and was assayed for both TNF-α and IL-6 level. It is noticed that TNF-α level was 18.3-27.3 pg/ml, median value 21.863 pg/ml (Figure 4) and IL-6 was 23.54-52.733 pg/ml, median value 42.163 pg/ml (Figure 5) in CAD patients. Age and sex matched controls have 0.29-1.54 pg/ ml, median value 0.98 pg/ml TNF-α level (Figure 4) and IL-6 level was 1.56-5.923 pg/ml, median value 4.433 pg/ml (Figure 5).
Figure 5: Role of estriol on IL-6 level in CAD patients. IL-6 was determined in same CAD patient neutrophils by ELISA method. IL-6 level in patient group were described as solid circle and has the median value with 42.163 pg/ ml. The solid squares show IL-6 level in CAD neutrophils after 0.6 nM estriol treatment (median value 4.433 pg/ml). Age and sex matched controls were determined for IL-6 level, described in the solid triangles with median value 4.76 pg/ml.
Incubation with 0.6 nM estriol for 45 min at 37°C showed level of both cytokines TNF-α andIL-6. A high level of TNF-α and IL-6 in CAD patient have been found to be declined to 4.9-9.13 pg/ml, median value 7.43 pg/ml (Figure 4) and 7.91-21.873 pg/ml, median value 16.733 pg/ ml (Figure 5) respectively for TNF-α and IL-6 after treating with estriol as described above (Figure 6).
The present results demonstrate that stress induced protein DCN- 2, plays a critical role to propagate the risk in AMI. It also induces the cytokine level particularly of TNF-α and IL-6 which causes an inflammatory response after atherosclerotic plaque rupture. It has been demonstrated by other researchers that incubation of TNF-α to cell suspension in vitro inspire membrane bound cell adhesion proteins to be activated  and causes aggregation between cells. In case of platelet aggregation which occurs as the post-atherosclerotic plaque rupture phenomenon, the platelets got activated cytokines and those helps to form inter-platelet fibrin mass from activated fibrinogen . Though aggregation of platelets acts as a lifesaving process (reference  for comprehensive literatures) but occurrence of this phenomenon at the wrong place causes the loss of life. As other investigators reported before that DCN-2 plays a role on platelet aggregation  like other platelet aggregating agents i.e. ADP, l-epinephrine, thrombin and collagen, our results describe that the promotion of DCN-2 induced platelet aggregation and its enhanced severity occurs through the elevation of cytokine level like TNF-α and IL-6. In our study estriol has been shown to induce strong anti-inflammatory responses by inhibiting TNF-α and IL-6 production in neutrophil. Since the contributing volume of this leukocyte in blood is large so its effect could have been immense. And DCN-2 has been shown to induce inflammatory events. It has been reported before that the platelet membrane have a sufficient number of TNF receptor (TNF-R) and help to activate platelets . In other way, the IL-6 is a multifunctional cytokine and plays its role on haematopoietic and immune-regulation. It has been reported that IL-6 is able to activate platelet through various modes either from thrombin regulated IL-6 receptor alpha (IL-6R-alpha) activation  or through the activation of GPIIb/IIIa followed by increase in the expression of p-selectin  an intercellular adhesion molecule (ICAM). This elevation of cytokines level causes a worst prognostic outcome leading to even death in an AIHD patient .
Different non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the expression of inflammatory cytokines . Aspirin is a good example as the inhibitor of inflammatory molecules and is the member of NSAIDs . With the inhibition of inflammatory molecules aspirin repress platelet aggregation by activating cyclooxygenase . At the same time, it induces NOS activity and production of NO causing a significant degree of vaso-dilation .
It is described before that steroid molecule plays an important role in the inhibition of cytokines expression  both through genomic and non-genomic pathway  and are being popularly used as the last possible drug in severe inflammatory response . Particularly estrogens, female steroid hormone have a protective role on cardiovascular disease during child bearing ages [29,30]. It has also been found that estriol like aspirin induces NO synthesis and inhibits DCN-2 induced cytokine synthesis. This phenomenon infers that synthesis of NO could inhibit the expression of inflammatory cytokines.
From the present results it has been found that the pre-treatment of estriol to the normal neutrophil solution affects more on the DCN- 2 induced cytokine synthesis while the addition of estriol after DCN- 2 treatment has shown a diminished effect. This demonstrates that a preliminary upper level of estrogen like molecules in the system may perform a protective role on stress and cytokine induced platelet aggregation but the pre-occurrence of higher level DCN-2 could repress the protective role of estrogen. It was found that estriol has the ability to partially decrease the level of the cytokine as TNF-α and IL-6. But, IL-6 level was not diminished even after estriol treatment, when neutrophil solution was incubated with 120 nM DCN-2. This signifies a serious prognosis of outcome in higher level of stress-induced protein like DCN-2.
Stress induction in the mouse model of chronic ischemia suggests that tissues experiencing hypoxic condition are characterized with higher population of monocyte-derived cells (MDCs). These tissues are also recruited with elevated number of neutrophil in response to local inflammatory stimuli . Macrophage invasion and foam cell formation are the characteristic of the local areas of ischemic cells/tissues developed in an atherosclerotic plaque-rupture area . Importance of neutrophil (most abundant leukocyte) as an imflammatory marker (neutrophil-lymphocyte ratio) is recognized in previous a study , which is in line with agreement of our present studies. Neutrophilmediated inflammatory responses in vivo have been reported to be regulated by the autophagic phenomenon and degranulation of the neutrophil . Not only as noticed in the elevation of TNF-α and IL-6 in the current investigation, other cytokine like interleukin- 1β (IL-1β) also contribute the pathogenesis of atherosclerosis. Cholesterol which plays an important role in atherogenesis also activates neutrophil in crystal form by upregulating the synthesis of this cytokine . Other than inducer sources from neutrophil, several matrix metalloproteinases (MMPs) are reported to be produced from macrophages which contribute to plaque rupture, atherothrombosis and myocardial infarction. The contributing inflammatory mediators such as TNFα, IL-1 and toll-like receptor-ligands may also be produced dependently or independently to prostaglandin (PGE2) stimulation which activates MMP production also . With regard to our current study, it may be suggested that important inflammatory marker like TNF-α production may take place in diversified ways.
The onset of inflammatory response is associated with neutrophil recruitment into infected or injured tissues. Adhesion of circulating neutrophils to the endothelium represents a crucial step in their recruitment into the inflamed tissues. The hypothesis that the upregulation of the anti-adhesive proteins may represent an antiinflammatory mechanism that contributes to the resolution of inflammation . In the current study estrogen strongly performed as an anti-inflammatory role. A probable mechanism of the estrogen induced anti-adhesive protein expression via NO mediated pathway may occur in the system.
Not only during haematopoetic cells recruitment in cardiacendothelium injury, triggering of secondary complications such as inflammation may also occur in Spinal cord injury (SCI). Here, TNF-α and iNOS genes expression increased significantly after SCI. The results showed that injection of estrogen hormone reduced the TNF-α . Lack of estrogen is a cause of cardiovascular disease in men and postmenopausal women. One recent report suggests that estrogen receptor (ER) activation counteracts endothelial dysfunction induced by TNF-α  pointing out the positive outcome of estrogen as noticed in our study.
In conclusion, our present result suggests the inhibitory role of DCN-2 on the estriol induction of NO. But when the DCN-2 level is low estriol dominate in the maintenance of higher NO level. The antiinflammatory effects of estriol are highly distinct even at a high level of DCN-2 in case TNF-α. Very high level of DCN-2 may nullify the effects of anti-inflammatory effects of estriol, at least in neutrophil. But up to the 100-110 nM of DCN-2, estriol very efficiently support the NO production and anti-inflammatory responses.
Authors sincerely acknowledge University Grants Commission, New Delhi for supporting fellowship (JRF & SRF) to M. Khan.