Regulation of Migration,Phagocytosis and Apoptosis of Human Neutrophils by Recombinant Human Intestinal Alkaline Phosphatase

Hui Mi-zhou ,Cong Zhen-yu ,Jia Xiao-xiao ,Guo Tian-tian ,Wu Shu-yin,and Shuang Bao*

1 School of Life Sciences,Northeast Agricultural University,Harbin 150030,China

2 School of Animal Technology Sciences,Qingdao Agricultural University,Qingdao 266109,China

3 School of Food Sciences,Northeast Agricultural University,Harbin 150030,China

Abstract:The purpose of this study was to explore the effects of recombinant human intestinal alkaline phosphatase (recIAP) on human neutrophils in vitro,and the migration,phagocytosis,apoptosis in presence and absence of LPS.In this study,freshly extracted human neutrophils were used to establish an inflammatory cell model,and the control group,recIAP group,LPS group and recIAP +LPS group were set up to stimulate the model.The migration of neutrophils was detected by agarose gel drop method.Fluorescent particles and fluorescent probes were added to different treatment groups,and the phagocytic rate of neutrophils and the release of reactive oxygen species (ROS) from neutrophils were detected by flow cytometry.The apoptosis rate of neutrophils was detected by flow cytometry according to Annexin V-FITC apoptosis detection kit.The results showed that regardless of the presence or absence of LPS,recIAP could inhibit the migration of neutrophils,phagocytosis and the release of ROS.In addition,recIAP could weaken the inhibitory effect of LPS on neutrophils apoptosis.

Key words:alkaline phosphatase,migration,phagocytosis,apoptosis,reactive oxygen species (ROS)

Introduction

Inflammation is an effective immune response against foreign invasion.When the body is injured,leucocytes in the blood exudate and migrate to the external vessel,causing inflammatory reactions by secreting a variety of inflammatory mediators.Endotoxin is the component of the outer wall of gram-negative bacteria,and lipopolysaccharide (LPS) is the main component of endotoxin.When inflammation occurs,LPS exerts its toxicity by binding to TLR4 receptors in the target cell membrane and activates inflammatory cascades.Leucocytes are the main inflammatory cells,including neutrophils.The accumulation and migration of inflammatory cells,incomplete phagocytosis and excessive production of reactive oxygen species (ROS)will aggravate the inflammatory response,causing extensive damage to the surrounding normal tissues,while the timely apoptosis of inflammatory cells will reduce the inflammatory response.So for the development of inflammatory diseases,neutrophils are not only the effector cells of inflammatory response,but also the effector medium of inflammatory conduction.By regulating neutrophils migration and accumulation,apoptosis,phagocytosis and ROS release,the inflammatory response of the body can be regulated,which plays an important role in the prevention and treatment of related inflammatory diseases.

Alkaline phosphatases (AP) are widely distributed in nature,including prokaryotes and higher eukaryotes.AP is a homodimeric enzyme.It is a glycosylated protein with a molecular weight of about 60 ku and has multiple disulfide bonds.Its structure is strong and difficult to degrade (Sharmaet al.,2014).AP is encoded by a multi-gene family and has a variety of tissue subtypes,such as tissue nonspecific alkaline phosphatase (TNAP),intestinal alkaline phosphatase(IAP) and placental alkaline phosphatase (PLAP).Three types of AP are connected to the cell membrane through phosphatidylinositol bonds,which are typical proteases connected to the cell membrane.Studies by Poelstraet al.(1997) have found that IAP can inactivate LPS,suggesting that IAP can inhibit LPS-induced inflammatory response and may have therapeutic effects on LPS-related diseases.Clinical studies of AM-Pharma have confirmed the effectiveness of recombinant human intestinal alkaline phosphatase(recIAP) as a new treatment for LPS-associated sepsis,acute kidney injury and colitis (Davidsonet al.,2019;Luká?et al.,2010).However,there are no researches on recIAP in inflammatory diseases under non-LPSdependent signal transduction pathway,which provides a theoretical basis for expanding the clinical application of recIAP and is of great significance.

Materials and Methods

Cell and vector

Chinese hamster ovary cells CHO-S cells,pCAChimAP plasmid template and pMH3-pH20 expression vector plasmid were donated by Shaoxing Huihui Technology Company.

Reagents and instruments

Reagents: FBS (Tianhang Biotech Company,China);DMEM/F-12 culture medium (Gibco,USA);RPMI-1640 culture medium,RPMI-1640 dry powder,phorbo 12-myristate 13-acetate (PMA),latex beads,niacin modified polystyrene,fluorescent red,fluorescent probe dihydrorhodamine (DHR123) (Sigma,USA);PBS,penicillin-streptomycin (Hyclone,USA);agarose,LPS (Solarbio Biotech Company,China);sodium diphenyl phosphate colorimetric kit (Reagan Biotechnology Company,China);human peripheral blood neutrophils isolation kit (Haoyang Huake Biotechnology Company,China);Annexin V-FITC apoptosis kit (Biyuntian Biotechnology Company,China);SDS-PAGE gel preparation kit (Kangwei Century Biological Company,China);Diff-Quick-Stain kit (Solarbio Science &Technology Company,China).Instruments: CO2cell incubator (Thermo Fisher Scientific Company,USA);electronic balance (SQP,Sartorius Scientific Instruments Company,China);horizontal rotor centrifuge (CF0805,Shuoguang Optoelectronics Technology Company,China);inverted phase contrast microscope (Mingmei Scientific Company,China);cationic column spXK50,anion column QXK50 (GE Healthcare Systems,USA);hollow fiber column (Bogelong Biotechnology Company,China);high performance liquid chromatography analysis system (Waters Breeze HPLC,USA);flow cytometry (Becton Dickinson,USA).

Expression and production of recIAP

In this study,the cDNA of recIAP (Le and Millan,2002;Tinaet al.,2014) was inserted into the pMH3 vector (Rishton and Hui,2010) to construct the pMH3-recIAP expression vector.Then the pMH3-recIAP expression vector was transformed into CHO-S cell line,and cultured in DMEM/ F-12 medium supplemented with 10% FBS.The stable and high expression clones were screened by G418 pressure.The fermentation broth of recIAP was amplified and cultured in a mammalian cell bioreactor.The fermentation broth was centrifuged,and the heterogeneous proteins were removed by cationic column spXK50.The flow fluid containing recIAP fraction was collected,and the eluent containing recIAP was collected by anion column QXK50.All the solutions used in the purification process were removed LPS by hollow fiber column.

Determination of recIAP activity

The recIAP activity was determined by sodium diphenyl phosphate colorimetric method.Sodium diphenyl phosphate was hydrolyzed by alkaline phosphatase under alkaline conditions to produce free phenol and phosphoric acid.Under alkaline conditions,phenol was combined with amino antipyrine and oxidized to form red quinoid structures with different depths.The absorbance was measured at 510 nm,and the activity level of alkaline phosphatase was calculated by colorimetric analysis.The active unit of AP was defined as: at 37℃,100 mL of the sample to be tested reacted with disodium phenyl phosphate for 15 min to produce 1 mg phenol as a gold unit (U · L-1).

Isolation of neutrophils from human peripheral blood

With the approval of the Medical Ethics Committee of Changchun Jiahe Surgical Hospital and the consent of the volunteers themselves,venous blood was collected from five healthy volunteers aged 22±4 years.Human venous blood were collected on the same day of the experiment,and blood pressure was immediately processed.According to the sucrose density gradient centrifugation method,neutrophils in peripheral blood were separated using the human peripheral blood neutrophils separation kit.Human venous blood was collected at room temperature and placed in an anticoagulant tube.The 5 mL separation liquid was added to each sterile silicide centrifuge tube,and the blood was gently added to the separation liquid surface,and centrifuged at 1 800 r · min-1for 25 min.The neutrophil layer were transferred to a common sterile centrifuge tubes,respectively.The 10 mL red blood cell lysate was added to the centrifuge tube,and it was reversed up and down for 2 min.The red blood cells were fully lysed and centrifuged at 1 000 r · min-1for 10 min.After discarding the supernatant,if there was still obvious red precipitate,added 5 mL red blood cell lysate,upside down 2 min,again 1 000 r · min-1centrifugal 10 min.Blowed gently cell precipitation,added 10 mL cleaning solution,centrifuged at 1 000 r · min-1for 10 min,and washed twice.The 3 mL RPMI-1640 medium containing 10% FBS was resuspended for further use.The purity of isolated neutrophils was identified by Diff-Quick-Stain kit.

Study on migration of neutrophils

Neutrophils were resuspended in 1×RPMI-1640 medium,and the cell density was adjusted to 3×108cell · mL-1for further use.The 0.8% agarose sterilization solution was put in water at 37℃ for heat preservation.The equal amount of agarose solution was mixed with 2×RPMI-1640 medium containing 20% FBS and 1% streptomycin-penicillin.The equal amount of the above mixture was mixed with neutrophil suspension,respectively,and placed in a 37℃ water bath.Two pre-cooled 96-well plates were taken,and 2 μL of neutrophil agarose mixture was added to each well of the plate,respectively.The gel droplets with a diameter of about 2 mm were required to form at the center of the bottom of the hole.

The 96-well plate coated with rubber droplets was placed at 4℃ for 15 min.The control group,recIAP group,LPS group and recIAP+LPS group were set to treat neutrophils.RPMI-1640 medium containing 10% FBS,1.0 ng · mL-1LPS+10% FBS,1.0 ng · mL-1LPS+2.5 U · mL-1recIAP+10% FBS and 2.5 U · mL-1recIAP+10% FBS was prepared.After the agar drop in the well was solidified,100 μL of the above four groups of reagents was added to each well,with four parallels in each group.The 96-well plate was placed in a 37℃ incubator for 3 h and then removed.The migration distance was observed under an inverted microscope and photographed.Image J was used to calculate the cell migration area.

Study on phagocytosis of neutrophils

Neutrophils were resuspended with RPMI-1640 medium containing 10% FBS,and the cell density was adjusted to 4×106cell · mL-1and inoculated in 24-well plates.The control group,recIAP group,LPS group and recIAP+LPS group were set to treat neutrophils.RPMI-1640 medium containing 10% FBS,1.0 ng · mL-1LPS+10% FBS,1.0 ng · mL-1LPS+5.0 U · mL-1recIAP+10% FBS and 5.0 U · mL-1recIAP+10% FBS was prepared.The 100 μL of the above four reagents was added into each well to stimulate neutrophils,with four parallels in each group.Then 3.5 μL fluorescent particles with diameter of 2 μm were added to each well,and the density of fluorescent particles was adjusted to 2×107cell · mL-1.The best phagocytosis model of neutrophils and fluorescent particles was established and incubated at 37℃ in dark for 1 h.After the stimulation,quickly added precooled PBS to terminate the reaction and rinsed twice with PBS.The cells were resuspended with PBS and the phagocytic rate of neutrophils was detected by flow cytometry.

Study on apoptosis of neutrophils

Neutrophils were resuspended with RPMI-1640 medium containing 10% FBS,and the cell density was adjusted to 1×106cell · mL-1,which was inoculated on a 24-well plate.The control group,recIAP group,LPS group and recIAP+LPS group were set to treat neutrophils.RPMI-1640 medium containing 10%FBS,1.0 ng · mL-1LPS+10% FBS,1.0 ng · mL-1LPS+2.5 U · mL-1recIAP+10% FBS and 2.5 U · mL-1recIAP+10% FBS was prepared and added to neutrophils.After incubation at 37℃ in dark for 3 h,the cells were stained according to the Annexin V-FITC apoptosis detection kit.After staining,quickly added precooled PBS to terminate the reaction and rinsed twice with PBS.The cells were resuspended with PBS and the apoptosis rate of human neutrophils was detected by flow cytometry.

Study on release of ROS from neutrophils

Neutrophils were resuspended in RPMI-1640 medium containing 10% FBS,and the cell density was adjusted to 4.5×106cells/well.The cells were seeded in 24-well plates,and the final concentration of 5 μmol · L-1fluorescent probe dihydrorhodamine (DHR123) was added to each well.The cells were incubated at 37℃in dark for 15 min.The control group,recIAP group,LPS group,recIAP+LPS group,PMA group and recIAP+PMA group were set.RPMI-1640 medium containing 10% FBS,2.0 μg·mL-1LPS+10% FBS,2.0 μg · mL-1LPS+25 U · mL-1recIAP+10% FBS,50 nmol · L-1PMA+10% FBS and 50 nmol · L-1PMA+25 U · mL-1recIAP+10% FBS was prepared and added into neutrophils.After incubation at 37℃ in dark for 1 h,the precooled PBS was quickly added to terminate the reaction and rinsed twice with PBS.Cells were resuspended with PBS,and 10 000 cells were collected from each sample.The release of ROS in neutrophils was detected by flow cytometry,and the amount of ROS released by neutrophils was calculated according to the following formula.

Neutrophil activation rate (%)=ROS cells produced/total cell×100%

The amount of ROS produced by neutrophils=the activation rate of neutrophils×MFI (average fluorescence intensity)

Statistical analysis

Image J software was used to calculate the moving area,and Graph prism 8.2.1 was used to analyze the data.The data were expressed as mean ± standard deviation,and the results were compared by group or pairedttest,withP＜0.05 as the standard of statistical significance.Five samples were collected in each experiment,and each sample was repeated at least three times.

Results

Purification and activity detection of recIAP target protein

The cell culture medium was collected and centrifuged and filtered,and the 30 ku membrane package was concentrated by about six folds.The protein was purified by sp XK50 ion exchange column.The flowthrough fluid was collected and subjected to pyrogenfree treatment by QXK50 ion exchange column.The SDS-PAGE results of the purified target protein were 60 ku,as shown in Fig.1.The purity of protein detected by HPLC was 95.51%,as shown in Fig.2.The activity was determined as 137.28 U · mL-1according to the alkaline phosphatase activity detection kit(phenyl disodium phosphate colorimetric method).

Fig.1 SDS-PAGE detection of recIAP

Fig.2 HPLC detection of recIAP

Purity of neutrophils separation

The purity of neutrophils isolated from human peripheral blood was detected by Div rapid staining.The coated slides were prepared by adding 20 μL leucocytes suspension to the clean slides.The coated slides were placed on the dyeing rack,and 100 μL Diff-Quick-Fixative solution was added dropwise to make it quickly cover the entire suspension area.The coated slides were fixed for 20 s,washed with distilled water,and dried slightly.Added 100 μL Diff-Quick I solution to cover the dyeing area for 20 s.Washed the dyeing solution from one end of the slide with distilled water and dried it slightly.Added 100 μL Diff-Quick II solution to cover the dyeing area,and stood for 20 s.Washed the dyeing solution from one end of the slide with distilled water,and dried it.The red blood cells were light red,the granules in the leucocytes cytoplasm were clear,and the nucleus was purple red.The cells with multiple nuclei in leucocytes were neutrophils.The purity of freshly extracted neutrophils was calculated to be more than 90% from three random fields,as shown in Fig.3.

Fig.3 Purity of isolated neutrophils

Inhibition of neutrophils migration by recIAP

The effect of recIAP on the migration of neutrophils in the presence and absence of LPS was detected by agarose gel.The migration areas of neutrophils in each group are shown in Fig.4A.The area calculated by Image J software and statistical analysis,the results are shown in Fig.4B.Compared with the control group,LPS(1.0 ng · mL-1) promoted the migration of neutrophils.Compared with the control group,the cell migration area of recIAP group (2.5 U · mL-1) was significantly decreased,indicating that recIAP could inhibit the migration of neutrophils without LPS.Compared with LPS group,the cell migration area of recIAP+LPS group was significantly reduced,indicating that recIAP could inhibit the promotion of LPS on neutrophils migration.In conclusion,recIAP inhibited neutrophils migration regardless of whether LPS existed or not.

Fig.4 Effect of recIAP on migration of neutrophils

Inhibition of neutrophils phagocytosis by recIAP

The phagocytosis rate of neutrophils to fluorescent microspheres was measured by flow cytometry,as shown in Fig.5A.Statistical results are shown in Fig.5B.Compared with the control group,the phagocytosis rate of neutrophils in LPS group(1 ng · mL-1) was significantly increased.Compared with the control group,the phagocytosis rate of recIAP group (5 U · mL-1) was significantly decreased,indicating that recIAP could inhibit the phagocytosis of human neutrophils without LPS.Compared with LPS group,the phagocytosis rate of recIAP+LPS group was significantly decreased,indicating that recIAP could inhibit the promotion of LPS on neutrophils phagocytosis.In conclusion,recIAP inhibited neutrophils phagocytosis regardless of whether LPS existed or not.

Fig.5 Effect of recIAP on neutrophil phagocytosis of fluorescent granules by flow cytometry

Regulation of neutrophils apoptosis by recIAP

The effect of the four treatment groups on neutrophil apoptosis was detected by flow cytometry,and the results are shown in Fig.6A.The results after statistical analysis are shown in Fig.6B.Compared with the control group,the apoptosis rate of LPS group (1 ng · mL-1) was significantly reduced.Compared with LPS group,the apoptosis rate in recIAP+LPS group was significantly increased,indicating that recIAP could reduce the inhibitory effect of LPS on apoptosis of human neutrophils and achieve the purpose of reducing inflammatory response.The above results showed that although recIAP inhibited neutrophils apoptosis in absence of LPS,recIAP could weaken the inhibitory effect of LPS on human neutrophil apoptosis in presence of LPS.

Fig.6 Flow cytometry analysis of effect of recIAP on neutrophils apoptosis

Inhibition of neutrophil ROS release by recIAP

The effects of recIAP on the release of ROS from neutrophils in presence and absence of LPS were studied.The control group,PMA group,recIAP+PMA group,LPS group and recIAP+LPS group were set for the experiment.PMA group was the positive control group.The effects of five treatment groups on the release of ROS from neutrophils were detected by flow cytometry in Fig.7A.Statistical results are shown in Fig.7B.Compared with the control group,2 μg · mL-1LPS significantly promoted the release of neutrophil ROS.At the same time,the ROS released by neutrophils in the 50 nmol · L-1PMA group of the positive control group was also significantly increased.Compared with LPS group,the ROS released by cells in recIAP+LPS group (25 U · mL-1+2 μg · mL-1) was significantly decreased,indicating that recIAP could weaken the promoting effect of LPS on the release of ROS from neutrophils.Compared with PMA group,the release of ROS in recIAP+PMA group (25 U · mL-1+50 nmol · L-1) was significantly decreased,indicating that recIAP could inhibit the release of ROS in human neutrophils without LPS.

Fig.7 Effect of recIAP on ROS release from neutrophils

Disscusion

Various diseases caused by inflammation seriously threatened human health (Frascaet al.,2017;Monteiro and Azevedo,2010;Hansen,2018).Many studies had shown that leucocytes played an important role in the development of human inflammatory diseases (Suzuki,2017;Powell and Huttenlocher,2016;Jasperet al.,2019;Kovtunet al.,2018;Williams and Chambers,2016;Wrightet al.,2010;Suzuki,2018).Neutrophils had chemotaxis,phagocytosis and bactericidal effects.Due to the strong destructiveness of inflammatory cells,the body must strictly control the number of neutrophils produced every day.

Because of the destructive nature of inflammatory cells,the body must exercise very strict control over the number of leucocytes produced each day.Inflammatory cells acted like a double-edged sword,both as a killer against pathogens and as a serious damage to the host itself,and its migration from the inflammatory site over-accumulation and uncontrolled activation might lead to damage to normal tissue structure and expansion of inflammatory responses.Phagocytosis was critical in the body's resistance to microbial pathogens.When inflammatory cells spotted the chemical signals released by invading pathogens,they chased pathogens and bound to some antigen epitopes on the pathogen through their surface receptors,but if they couldn't be killed,they would not be completely swallowed.Incomplete phagocytosis could exacerbate inflammatory responses.Neutrophils were the main inflammatory cells in the body.Under physiological conditions,the survival cycle of neutrophils was very short and was eliminated by apoptosis and necrosis.Neutrophils necrosis intensified inflammatory response and damaged normal tissue cells.Neutrophils apoptosis maintained cell membrane integrity,intracellular pro-inflammatory cytokines didn't leak,inflammatory response would not expand.Therefore,timely apoptosis of neutrophils could effectively avoid the damage of surrounding tissue cells,and then controlled the inflammatory response.ROS was an important signaling factor to regulate inflammatory response,and excessive accumulation of ROS in the body could aggravate inflammatory response.Therefore,the discussion of the antiinflammatory effect of recIAP was very important to study its effects on neutrophils migration,neutrophils phagocytosis,neutrophils apoptosis and ROS release,which was of great significance to the treatment of inflammatory diseases and provided new fields and evidence for the anti-inflammatory effect of recIAP.

IAP was a nonspecific dephosphase that effectively removed phosphorus from various organophosphate compounds.Phosphorus migration from LPS was one of the physiological functions of IAP,and studies by Poelstraet al.(1997) have found that IAP could inactivate LPS,suggesting that IAP could inhibit inflammatory reactions caused by LPS and might have therapeutic effects on LPS-related diseases.In this study,an LPS-induced and non-induced inflammation model was established,and the results showed that recIAP inhibited the migration of neutrophils in both LPS-induced and non-induced cases,and recIAP inhibited the phagocytosis of neutrophils.In addition,recIAP could reduce the inhibition of LPS on neutrophil apoptosis,and recIAP could inhibit the release of neutrophil ROS,thus reducing the inflammatory response.Therefore,it was assumed that under the condition of the existence of LPS,recIAP regulated the functions of migration of neutrophils,phagocytosis and apoptosis of neutrophils,and the output of ROS through the mechanism of inactivated LPS.Qin (2020) found that recIAP inhibited the secretion of leucocytes TNF-αand IL-6,whether LPS existed or not.Other studies had shown that recIAP,as an exonynucleotide enzyme,dephosphoricated ATP,ADP and AMP in the form of dose dependence and pH dependence,resulting in the final adenosine product (Davidsonet al.,2016;Sowaet al.,2009).Extracellular adenosine was a powerful immune regulator that accumulated in an inflammatory state.Nucleotides such as ATP and ADP were released from damaged and necrotic cells and hydrolyzed into adenosine phosphate and adenosine under the synergy of exnucleotides CD39 and CD73(Deaglioet al.,2007;Menget al.,2019;Regateiroet al.,2011;Robertset al.,2014;Szaboet al.,2012).Huiet al.(2020) found that adenosine inhibited leucocyte secretion of TNF-αin non-dependent LPS signal transduction pathways.The above results suggested that there was a cross between recIAP and the exinite nucleoside enzymes CD39 and CD73,and that adenosine might be connected to the exonuclebent enzymes CD39 and CD73,and recIAP as an antiinflammatory medium (Antonioliet al.,2013;Bonoet al.,2015;Lennonet al.,1998;Schneideret al.,2019).Therefore,it was assumed that under the condition that LPS didn't exist,recIAP might regulate the migration of neutrophils,the phagocytosis and apoptosis of neutrophils,and the role of ROS production through the mechanism of adenosine products from dephosphate ATP,ADP and AMP,and it needed to be explored later.

Conclusions

In this study,LPS-induced and non-induced inflammatory models were established.The results showed that recIAP could inhibit the migration of neutrophils under LPS-induced and non-induced conditions,and recIAP could also inhibit the phagocytosis of neutrophils and the apoptosis of neutrophils.In addition,recIAP could inhibit the release of ROS on neutrophils,so as to reduce the inflammatory response.This study by using freshly extracted human leukocytes from human volunteers more closely represented anex vivowhole blood study or a human study.Taken together,the results indicated that recIAP was a promising medicine candidate for inflammatory diseases that were prominently mediated by human neutrophils.Finally,this study also established a cell-based assay for measuring recIAP activity on neutrophils migration,neutrophils phagocytosis,neutrophils apoptosis and the release of ROS in neutrophils.