Comparison of neutrophil elastase levels in obese and normal individuals

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INTRODUCTION
Obesity is often defined sim ply as a condition of abnormal or excessive fat accumulation in ad ipose tissue, to the ex tent th at h ealth m ay be im paired (Drewnowski A et al. 1989).According to World H ealth Organization (W HO), obesity is classified as chronic an d se vere disease in deve loped and developing countries, affecting both adults and children (Hoyt CL et al. 2014) Previous data showed that obesity has reached to an epidem ic proportions globally, w ith more than 1 billion adults overweight and at least 300 m illion of them clinically obese.Which is a m ajor contributor to the global burden of chronic disease and disability.Often coexisting in developing countries with under-nutrition, obes ity is a com plex condition, with serious social and psychologi cal dim ensions, affecting virtually all ages and socioeconom ic groups (Nathan C et al. 2006).Many recen t studies h ave suggested that obesity is associated with chronic inflammation in fat tissue.
Neutrophils being the most abundant leukocyt es in humans and the first cells to arrive on the site of inflammatory immune response.

Neutrophil Neutrophil Elastase PMA Obesity
Purpose: Neutrophils being the most abundant leukocy tes in humans and the first cells to arrive on the site of inflam matory immune response.These cells are classically characterized by their ability to act as phagocy tic cells, to release lytic enzy mes like neutrophil elastase from azurophil granules.Neutrophil elastase (NE) is known to prom ote inflam matory responses in several disease.The purpose of this study is to correlate neutrophil elastase expression level with obesity.
Method: Present study aim s to determ ine the differential expression and packaging (in azurophil granules of neutrophils) of human NE using Sodium Dodecy l Sulphate Poly Acry lamide Gel Electrophoresis (SDS-PAGE) in normal and obese individuals.
Results: It was observed that, the obese individuals tend to have an average elevated levels neutrophil elastase in blood neutrophils as confirmed by the bands observed in SDS-PAGE.The elevated levels of neutrophil elastase in obese individuals m ay be attributed to chronic inflammation in fat tissue.

Conclusion:
Obese individuals (Students of Hail University) have a slightly higher level of neutrophil elastase in blood leukocyte (neutrophils).
Upon stim ulation by pathogens or pharmacological agen ts such as phorbol myristate acetate (P MA), neutrophil elastase is excreted from the cell and exists either as free pro tein or associated with networks of extrace llular traps (Brinkmann V et al. 2004).Neutrophils are norm ally found in the blood stream.During the beginning (acute) phase of inflammation, particular ly as a resu lt of bacterial inf ection, envir onmental exposure, and some cancers (Nathan C et al. 2006;Levinsky R et al. 1980).Neutrophils are one of the firstresponders of infla mmatory cells to migrate towards the site of inflammation.They m igrate through the blood vessels, then through inter stitial tissue, f ollowing chemical signals su ch as Interleukin-8 (IL-8), C5a, and Leukotriene B4 in a process called chem otaxis.Neutrophils are rec ruited to the site of inju ry within minutes fol lowing traum a and are the hallmark of acute inf lammation (Takahashi H et al. 1988;Jacobs L et al. 2010).Together with other proteases released from activated neutrop hils, neutrophil elastase plays a critical ro le in degrading invading pathogens and thus provides the earliest line of defense in the immune sys tem (Boxer LA et al. 2010;Sato T et al. 2006).In addition to its expression in neutrophils, neutrophil elastase is also exp ressed in non -small cell lung cancer tum ors and cell lines ( Waugh DJJ and W ilson C 2008;De Larco JE et al. 2004).
Neutrophil elastase may also play a critical ro le in tum or invas ion and metastasis ( Yamashita Ji et al. 1997;Henriksen P A and Sallenave JM 2008), due to its ability to d egrade in soluble elastin and other extracellular matrix constituents.Mutations in ELA2, the gene encoding neutrophil elastase, are the major cause of the two m ain f orms of hereditary neutropenia (Cohen S and Burns RC 2002;Chua F and Laurent GJ 2006).NE has been found to play key role in insulin resistance in mice fed with high fat diet (Talukdar S et al. 2012).Neutrophil elastase has been found within athero sclerotic plaqu es contributing to m atrix degrada tion and weakening of the vessel walls (Henriksen PA and Sallenave JM et al. 2008).These studies identify a clear role of neutrophil elastase in fat metabolism and deposition.Present s tudy attem pts to determ ine a possible relation with NE levels and obesity.

MATERIAL AND METHOD Participants
Students (Num ber: 64) were enrolled in the study, divided into 4 groups (Table 1) Each Group included 16 students.Group 1: Non obese students and the other Group 2: Obese students.The mean age of the groups were 24±4.
All particip ants were s ubjected to anthropometric measurement and norm al body com position m easurement.Serum NE was measured by SDS-PAGE, while sample preparation and activation of Neutrophils was perform ed by Cayymann Assay kit for Neutrophil Ealsatse, Supplied by Caymann.

Sample Preparation
15 mL of whole blood sam ples were co llected from each ind ividual in EDTA blood collecti on tubes.This whole blood is then transferred to 50 m l conical tube (using st erilized dispo sable syringe).Blood collection tubes were then rinsed with 15 m L of filtered Assay Buffer (provided with th e kit) and added to the 50 mL conical tubes containing whole blood (total di luted blood volum e 30 m L). 10 m l of Cell-Based Assay Neutrophil Isolation Histopaque ® (provided with the kit) was then pipetted to fresh 50 ml conical tubes.30 m l of the diluted blood was then slowly added on the top of Cell-Based Assay Neutrophil Isolation H istopaque ® in each 50 mL conical tube.
Thereafter the samples were centrifuged at 500 x g for 20-30 m inutes at 18-26°C followed by carefully aspirating the yellowish and clear top layers and leaving the reddish pellets containing neutrophils and red blood cells in the tube.30 ml of Red Blood Cell (RBC) Lysis Buffer (provided with the kit) is then pipetted in to conical tubes containing the pellet of neutrophils and red blood cells.This m ixture is then vortexed and rocked over a rocker for 10-15 m inutes to lyse the red blood cells.Thereafter Centrifuging at 1,200 x rpm for 10 m inutes to pe llet the neutro phils followed by carefully aspirating the reddish supernatant.To this 5 ml of Roswell Park Memorial Institute medium (RPMI) Solutions containing 1% B ovine Serum Album in (BSA) is added and mixed well.Again centrifuge the tubes at 1,200 x rpm for five m inutes to pellet the neutrophils.Addition of RPMI and the centrifugation process were done twice to get more neutrophils.Thereafter the cells were resuspended in 20 m l RPMI containing 1% BSA and well m ixed to ensure sufficient separation of th e cells.PMA (provided with th e kit) was then added at a f inal dilution of 1:30,000 into each cu lture m edium.PMA at these concentrations causes a significant release of Neutrophil Elastase.Now these tubes are then centr ifuged at 1200x rpm for ten m inutes at 20 o C. The supernatant now contains neutrophil elastase.Now the samples are o rganised for SDS PAGE as per Table 1.SDS-PAGE was performed on Biorad Tetracel.Lane 1 was loaded with biorad precision plus protein standard.Lane 2 was loaded with NE standard (caym an).Lane 3 and 4 were loaded with Group 1 and Group 2 samples respectively.L ane 5 and 6 were loaded with Group 3 and Group 4 samples respectiv ely.SDS-PAGE was performed a s per standard protocols of Biorad (not all gel im ages can be shown due to journals page limit).

RESULTS AND DISCUSSION
Neutrophil elastase is an im portant protease en zyme that when expressed apparently can cause em physema changes; this involves the breakdown of the connective tissues.So the natural proteinase elastase is rele ased f rom polymorph nuclear (PMN) leukocytes in various physiological and pathological conditions.Many researchers have found that increase in the neutrophils elastase could be an indicator to abnormal affects.Figure 1 shows a comparasion of NE from 4 different group s as classified on the basis of their B ody Mass Index (Table 1).L ane 3 and 4 belongs to obese individuals and lane 5 & 6 belongs to normal individuals.It can be observed that bands in all the lanes 3,4,5,6 corrosponds to the standard NE in the lane 2.  This conform s, that the protein in the bands 3, 4, 5 and 6 is NE.PMA induces th e releas e of NE from Neutrophils without the lysis of neutrophils.This reduces the load of other proteins in the sample.It can be observed, that the bands in lane 3 & 4 are sharper and well defined as com pare to the bands in lane 5 & 6 (Figure 1).The sharpness and the inte nsity of the bands is proportional to the concentration of NE in the sam ple loaded in their resp ective wells.Hence it can be concluded that the samples loaded in the lane 3 & 4 h ave a higher concentration of NE as com pared to lane 5 & 6.
The sharpness of th e bands in lane 3 and 4 explains the elevated levels of NE in the blood neutrophils of the O bese individuals.However this is only an indication of such behaviour and f urther confirmation can be done in future studies involving W estern B lot and ELISA based analysis.

CONCLUSION
Serum neutrophil elastase concentration were found to be elev ated in obese stu dents com parative to n ormal students.T his m ay be attributed to chronic fatty tissue inflammation, insulin resistance and som etimes undetected prehypertension in obese individuals.Many researches showed the relatio nship of NE with incurab le diseases.There is a wide scope of research on the implications and the regulation of NE in obese individuals.

Table 1 :
Group characterization Group