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Elevated neutrophil respiratory burst activity in essential hypertensive patients
Cellular Immunology 263 (2010) 230–234
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Elevated neutrophil respiratory burst activity in essential hypertensive patients Rajesh Ramasamy a,*, Maryam Maqbool a, Abdul Latiff Mohamed b, Rahim Md. Noah c a
Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia Faculty of Medicine, Cyberjaya University College of Medical Sciences, Malaysia c Medical Science Technology, Universiti Kuala Lumpur, Malaysia b
a r t i c l e
i n f o
Article history: Received 29 December 2009 Accepted 12 April 2010 Available online 18 April 2010 Keywords: Neutrophil Respiratory burst Reactive oxygen species
a b s t r a c t Neutrophils play a significant role in maintaining the integrity of innate immunity via their potent respiratory burst activity. However, the uncontrolled activation of respiratory burst in neutrophils also attributes to chronic diseases such as primary hypertension and atherosclerosis. In our study, we have investigated the activation of respiratory burst function of neutrophils harvested from essential hypertensive patients. In the presence of stimuli PMA and opsonized zymosan (OZ), hypertensive patients’ neutrophils secrete significantly higher amount of superoxide anions compared to normotensive control. Although the magnitude of activation varies between both groups, yet the kinetics of activation is similar. When normotensive control’s neutrophils were pre-treated with hypertensive serum, the cells failed to migrate toward fMLP which indicates the impairment of the migration property. In conclusion, the respiratory burst activity of neutrophils is affected by hypertension and their elevated superoxide anions production could be an aggravating factor in hypertension-related complication. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Neutrophils, also known as polymorphonuclear leukocytes (PMN) are the major cell type that constitutes the innate immune system. They comprise approximately 50–70% of leukocytes and are predominant in eliminating pathogens that induce acute inflammation [1–6]. Elimination of the pathogens by neutrophils involves a series of physiological sequence that comprise chemotaxis, phagocytosis and microbial killing. The success of pathogen elimination by neutrophils is depends on respiratory burst, which is a major process that mediates microbial killing through formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [3]. Phagocytosed microbes are internalized in phagosome, in which ROS are used as microbicides. The ROS production is initiated by NADPH phagosome oxidase (phox) enzyme complex which yields various oxygen reactive species such as hydrogen peroxide, superoxide anion, oxygen cations and other free radicals [3]. Whilst, RNS results from the catalyst of L-arginine by nitric oxide synthase (NOS) that produces nitric oxide reactive species. ROS and RNS are essential for neutrophil defense system and the failure of their production causes severe bacterial infection whilst the
* Corresponding author. Address: Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Fax: +603 8941 3802. E-mail address: [email protected] (R. Ramasamy). 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.04.004
overproduction triggers vascular damage in chronic diseases such as hypertension and arthrosclerosis [7]. An increasing body of evidence suggests that ROS play a crucial role in development of hypertension by creating a milieu of oxidative stress [8,9]. The skewed balance of free radical production such as superoxide anion and hydrogen peroxide coupled with diminished free radicals scavenger system has been demonstrated in human hypertension [8,10–12]. A significant elevation of oxidative stress embarks pathogenesis of coronary artery disease by altering the vasomotor tone thus exacerbating atherosclerosis and leads to development of hypertension [12–14]. Although vascular endothelial cells play major role in elevating systemic oxidative stress by their activities and cellular damage, blood immune cells also found to be a contributor to this phenomenon [15,16]. The elevated leukocytes count and enhanced PMN activation are correlated with the development and progress of hypertension and cardiovascular disease [15]. Spontaneous activation of neutrophils release proinflammatory factors and reactive oxygen species, which have negative effects on vascular tone and on their adhesion to the endothelium. Furthermore, the current study indicates that an excessive load of free radicals secreted by neutrophils at vascular sites advances the inflammation by limiting the bioavailability of nitric oxide (NO) secreted by endothelial cells [17]. The oxidative stress in hypertensive patients’ neutrophils is revealed by an increased NADPH oxidase production and lipid peroxidation and decreased cytosolic and mitochondrial superoxide dismutase concentrations [2,3,12,15,18]. Furthermore, the overexpression of
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adhesion molecules, such as beta2-integrin promotes PMN adhesion and leukocyte-endothelium interactions may contribute to the vascular damage and exacerbate the initiation or complication of arterial hypertension. Therefore the main objective of this study is to investigate the respiratory bust activity impairment of hypertensive patients’ neutrophils. Our study demonstrates that freshly isolated neutrophils from uncontrolled human subjects diagnosed with essential hypertension acquire the ability of producing a higher amount of ROS in response to phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) stimulation. Although many factors may have contributed to this dramatic impairment of neutrophils function, at least in our study we have shown that the humoral component of hypertensive circulation system may be involved in this impairment.
Briefly 1 106 of isolated neutrophils stimulated with either 0.1 lM of phorbol 12-myristate 13-acetate (PMA) (Sigma Chem Com) or 40 mg/ml opsonized zymosan (OZ). The signal derived from oxidative burst was amplified by adding of 0.2 lM 5-amino-2,3-dihydro-1,4-phythalazinedione (luminol) (Sigma Chem. Com). Neutrophils suspension in phenol free HBSS added with luminol and followed by addition of stimulator either PMA or OZ with quick vortex prior to oxidative burst measurement. Oxidative burst activity was characterized by measuring the amount ROS production at different time points at room temperature and the time difference between two readings was fixed at 10 s. The parameters such as the maximal amount ROS production and the time to be taken to reach maximal release of ROS were measured to determine the oxidative burst. The ROS production measured as relative micro voltage scale.
2. Materials and methods
2.5. Chemotaxis assay
2.1. Subjects
The migration of neutrophils from normotensive controls in response to serum from hypertensive patients was measured by chemotaxis assays. One million neutrophils from healthy normotensive individuals were incubated with either in cell culture medium, serum from normotensive or hypertensive individual for 10 min at 37 °C. Incubation with media and normotensive serum serve as control. Treated neutrophils transferred into modified Boyden chamber whereby the neutrophils were physically separated from stimulus by 5 lm filter system. N-Formylmethionylleucyl-phenylalanine (fMLP) (Sigma Chem. Com) at 25 lM was used as chemo-attractant in this assay. After 1 h of incubation in 5% CO2 at 37 °C, the filter detached from modified Boyden chamber and stained with hematoxylin. The migration of neutrophils calculated by light microscope whereby the difference in distance from starting point of neutrophil migration to the highest distance traveled by neutrophil throughout the filter. A minimal number of three readings were taken at different area of filter to determine the highest migration distance.
The study group consisted of 30 primary hypertensive patients and healthy normotensive adults who gave informed consent for participation. Hypertensive patients were selected from pool of essential hypertensive patients with uncontrolled blood pressure exceeding 140/90 mmHg of systolic and diastolic pressure. The hypertensive patients were excluded if they present with chronic diseases and symptomatic complications. Normotensive control group were selected from healthy individuals. Both patients and controls were free from clinical evidence for acute or chronic infection and inflammation. The patient selections were made by consultant cardiologist at hypertension clinic of Hospital Universiti Kebangsaan Malaysia. This study was approved by University ethical review board and procedures followed by institutional guidelines. 2.2. Blood sampling Fifteen milliliter (15 ml) of peripheral venous blood sample from hypertensive patients and normotensive controls were collected by certified phlebotomist. Ten milliliter (10 ml) of sample deposited in anti-coagulated tube for neutrophil isolation and 5 ml of samples kept in plain tube to harvest the serum.
2.6. Statistical analysis The data were compared between hypertensive patients and normotensive controls. Data were presented as the mean ± standard deviation from three or more experiments. The statistical significance was determined by 2-tailed Student t-test.
2.3. Neutrophil isolation 3. Results The following methods were performed to optimize the neutrophil isolation. Ten milliliter (10 ml) of venous blood was collected and diluted in 1 Hank’s balanced salt solution (HBSS) (Flowlab) medium at 1:1 ratio. Ten milliliter (10 ml) diluted blood was then layered over 5 ml lympoprep (Nycomed) and centrifuged at 1500 rpm for 40 min at room temperature. Plasma and the mononuclear cell layer were discarded. The red cell pellet which contains the PMN and red blood cell (RBC) was suspended in 5 ml HBSS. Red cell suspension was layered on 3% Dextran and left at room temperature for 45–60 min to further sediment the RBC. Supernatant from dextran sedimentation was collected and the remnant RBC was lysed using hypotonic lysing procedure to obtain the pure PMN population. The morphological examination and cell count were performed to determine the cell count and purity of the PMN. 2.4. Assessment of neutrophil oxidative burst Oxidative burst by human neutrophils was measured by production of ROS using chemiluminescence meter (Bioorbit 1250).
3.1. Neutrophils from hypertensive patients are easily excitable and highly activated in response to stimulators The stimulators PMA and OZ activate the respiratory burst activity of neutrophils. However, the magnitude of activation which is a reflection of oxygen species production in neutrophils alters by intrinsic and extrinsic factors. In order to assess the impact of high blood pressure, neutrophils from hypertensive patients were stimulated and their ROS production was recorded from the time of administration of stimulators to the peak of respiratory burst activity. Regardless to the type of stimulations, neutrophils from hypertensive patients showed a higher secretion of ROS at all time points (Fig. 1A and B). At the initiation phase of respiratory burst activity (first 30 s), neutrophils from normotensive and hypertensive individuals responded similarly, however, once the respiratory burst machinery is accelerated and entered to the log phase of the kinetics, neutrophils from hypertensive patients display a significantly higher oxidative burst activity (Fig. 1A and B).
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Respiratory Burst (mV)
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time(s) Fig. 1. Temporal comparison of PMA and OZ activated neutrophils from hypertensive patients and normotensive controls. One million of neutrophils were activated either by PMA (A) or OZ (B) and their respiratory burst activity measured by superoxide production. Superoxide production determined at every 10 s beginning from the administration of stimulator to the time point where the superoxide production reached maximal level and started to decline. This experiment were repeated at least 10 times and the *significance was determined at p < 0.05.
3.2. Neutrophils from hypertensive patients reach the maximal respiratory burst activity at higher magnitudes To determine the peak of respiratory burst activity, the maximal secretion of ROS was extrapolated. Both PMA and OZ significantly increase the maximal superoxide production of hypertensive patients’ neutrophils compare to normotensive controls (Fig. 2B). The mean maximal respiratory burst induced by PMA in hypertensive patients’ neutrophils reached at 277 ± 125 mV compared to normotensive controls at 192 ± 41 mV (Fig. 2B). Meanwhile OZ stimulation yields maximal superoxide production at 360 ± 144 and 185 ± 44 mV for hypertensive patients and normotensive control neutrophils, respectively (Fig. 2B). Although both stimulators were profoundly increase the magnitudes of respiratory burst in hypertensive neutrophils, however, the impact of OZ was much robust when compared to PMA stimulation. The mean time taken to reach the maximal respiratory burst in response to PMA and OZ is varies in which PMA stimulation reach the peak of the kinetics much faster than OZ. In the presence of PMA, neutrophils from hypertensive patients and normotensive control reach the maximum respiratory burst at mean 70 ± 12 s and 68 ± 15 s (Fig. 2A). OZ stimulation induces the hypertensive patients and normotensive control’s neutrophils to reach the maximal respiratory burst at 99 ± 22 s and 87 ± 12 s, respectively (Fig. 2A). 3.3. Serum from hypertensive patients inhibit the migration of normotensive control’s neutrophils To evaluate whether the serum from hypertensive patients affects the migration property of normotensive control’s neutrophils,
the cellular migration of neutrophils towards fMLP was conducted after the pre-treatment with hypertensive patient’s serum. Neutrophils pre-treated with culture medium, normal human serum (NHS) and hypertensive serum (PS) render differences in as 20 ± 5, 17 ± 3 and 11 ± 2 lm migrations toward the chemo-attractant (Fig. 3). 4. Discussion The cellular component of our immune system is an important tool in maintaining the integrity of vascular system. A close interaction between immune cells and endothelial cells via adhesion molecules, secretion of cytokines and cognate antigenic synapses contribute to chronic vascular diseases due to this broken integrity. This is best exemplified in essential hypertension whereby the vascular and migrated neutrophils acquired an impaired activation of respiratory burst which may exacerbate the pathogenesis of hypertension. In our study, we have investigated the role of neutrophils as our subject since it utilizes the respiratory burst function as a major tool for their immune responses. It has been proved by others that, hypertension enhances the respiratory burst activity of neutrophils in response to various stimuli [8,9,19]. In line with this, our report suggests that, in the presence of PMA and OZ, neutrophils from hypertensive patients release a significantly higher ROS compared to normotensive controls (Fig. 1A and B). Neutrophils responded well with OZ stimulation whereby the magnitude of OZ stimulation is much higher than PMA stimulation. Although the difference in magnitude of respiratory burst activation varies, PMA stimulation has certainly exhibited a significant difference at earlier time points (Fig. 1A and B).
R. Ramasamy et al. / Cellular Immunology 263 (2010) 230–234
A
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Fig. 2. Higher activation of respiratory burst activity and the time taken to reach the maximal respiratory burst in neutrophils from hypertensive patients in response to stimulators. The maximal time taken to reach the maximal superoxide production (A) and the maximal amount of secreted superoxide species (B) by 1 106 neutrophils were determined in response to PMA and OZ. This experiment were repeated at least 10 times and the *significance was determined at p < 0.05.
30
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25 20 15
*
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NHS-Normal Healthy Serum, PS- Patient’s Serum Fig. 3. Serum from hypertensive patients inhibits the chemotaxis or migratory property of normotensive control neutrophils. The effect of serum from hypertensive patients were assessed by incubating with 1 106 normotensive neutrophils. The chemotaxis of neutrophils determined by modified Boyden chamber whereby the neutrophils and fMLP were physically separated by filter system. The migration of neutrophils calculated by the distance traveled within the filter towards the fMLP. This experiment were repeated at least five times and the *significance was determined at p < 0.05.
This discrepancy may be due to involvement of different signaling mechanisms in activating respiratory burst machinery [20]. In accordance with this, studies on human neutrophils have shown that PMA stimulates the cell through the protein kinase C signaling pathway (PKC) directly [2] meanwhile OZ mimics a resemblance of pathogen-mediated phagocytosis. Based on kinetics of the stimulation, PMA stimulation through PKC signaling pathway favors a rapid transmission of signals that induces synthesis of ROS. This notion has been further supported by the fact that, the length of time taken to reach the maximal respiratory burst in response to
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PMA is much shorter when compared to OZ stimulation (Fig. 2A). Regardless to the type of stimuli, both PMA and OZ equally raised the production of ROS at all time points. Nevertheless, one study which was conducted using human un-fractionated leukocytes documented that, normotensive controls, borderline and essential hypertensive patients did not show a significant difference in respiratory burst in whole leukocytes population [19]. This contradictory result may originate from utilization of various subpopulations of leukocytes in their samples. Respiratory burst activity is considerably higher in neutrophils compared to lymphocytes. Our unpublished data show that lymphocytes produce very low amounts of superoxide anions which make us difficult to compare normotensive controls with hypertensive patients’ lymphocytes (data not shown). However, using lymphoblast model of human lymphocytes, Pettit et al. have shown that immortalized lymphocytes from hypertensive patients secretes higher amount of ROS due to over-activation of NADPH oxidase [21]. They have speculated that genetic predisposition of an increased expression of p22phox in hypertensive patients may account for this impairment. The recent therapeutical focus for hypertension is taken a reasonable consideration on the pathological role of neutrophils function in order to provide a better control mechanism by harnessing other auxiliary systems such as immune cells. An animal study had shown that fMLP-stimulated rabbit neutrophils, treated with the beta-adrenoreceptor blocker labetalol, demonstrate a dose-dependent antioxidant activity on neutrophils oxidative burst [22]. Moreover, this work suggested the mechanism by which labetalol acts in the treatment of hypertension may occur from an interaction in the signaling pathway of PKC activation. The antioxidant properties demonstrated in this mechanism contribute to the drug’s antihypertensive action and thus, may reduce the risk of injuries inflicted by reactive oxygen species involved in the pathogenesis of hypertension. Although there is unambiguous evidence of an elevated neutrophil respiratory burst in hypertension, yet whether this mutilation is an intrinsic defect of neutrophils or the sheer stress and hypertension associated deregulations force the neutrophil to be more vulnerable is still unknown. One clinical study showed that, occurrence of genetic polymorphisms in oxidative and anti-oxidative enzymes might alter the outcome of cellular susceptibility of neutrophils towards respiratory burst. Wyche et al. had demonstrated that C242T CYBA polymorphism of the NADPH oxidase is strongly associated with reduced respiratory burst in human neutrophils [23]. However, in our study, we have examined the effect of serum from hypertensive patients on migration property of neutrophils from normotensive controls. Hypertensive serum treated with normotensive controls’ neutrophils showed a decline in migration toward fMLP stimulus. The inhibition of chemotaxis may be due to existence of blocking factors in hypertensive serum that disrupt the optimal communication with chemotactic factors or downregulation of chemotaxis or chemokine receptors due to pre-treatment with hypertensive serum. However, our limited experiments did not identify the route of this migratory inhibition or the measurement of migration by hypertensive neutrophil in response to stimuli. Nonetheless one should bear in mind that, neutrophil migration is a crucial step in acute inflammation and failure of performing this task will risk the individual with a potential infection. This might complicate the outcome of hypertension with a susceptibility to bacterial infection. In summary our study has investigated the activation property of neutrophils from essential hypertension patients using a chemiluminescence method. PMA and OZ stimulators were used in study as PMA induces a quick eruption of respiratory burst through mitogen-mediated PKC signaling pathway while OZ serves similarly as a bacterial-mediated immune response. In both systems, neutrophils from hypertensive patients showed an elevated level of respi-
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ratory burst; however, the kinetics of activation does not differ between normotensive controls and hypertensive patients’ neutrophils. The migratory impairment of normotensive neutrophils that pre-treated with hypertensive serum may be due to occurrence of inhibitory humoral factors. However, harnessing the effect of hypertension on neutrophils will be a supplementary therapy for hypertensive patients to maintain the oxidative/anti-oxidative balance and integrity of immune system. Acknowledgment This project is partially supported by Research University Grant Scheme (RUGS), Universiti Putra Malaysia Project No: 04-01-090781RU. References [1] S. Greenberg, S. Grinstein, Phagocytosis and innate immunity, Curr. Opin. Immunol. 14 (2002) 136–145. [2] F.R. Sheppard, M.R. Kelher, E.E. Moore, N.J.D. McLaughlin, A. Banerjee, C.C. Silliman, Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation, J. Leukoc. Biol. 78 (2005) 1025–1042. [3] M.B. Hampton, A.J. Kettle, C.C. Winterbourn, Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing, Blood 92 (1998) 3007–3017. [4] L. Raffaghello, G. Bianchi, M. Bertolotto, F. Montecucco, A. Busca, F. Dallegri, L. Ottonello, V. Pistoia, Human mesenchymal stem cells inhibit neutrophil apoptosis: a model for neutrophil preservation in the bone marrow niche, Stem Cells 26 (2008) 151–162. [5] C. Kantari, M. Pederzoli-Ribeil, V. Witko-Sarsat, The role of neutrophils and monocytes in innate immunity, Contrib. Microbiol. 15 (2008) 118–146. [6] A.S. Cowburn, A.M. Condliffe, N. Farahi, C. Summers, E.R. Chilvers, Advances in neutrophil biology: clinical implications, Chest 134 (2008) 606–612. [7] C. Elbim, G. Lizard, Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations, Cytometry A 75 (2009) 475–481. [8] S. Sagar, I.J. Kallo, N. Kaul, N.K. Ganguly, B.K. Sharma, Oxygen free radicals in essential hypertension, Mol. Cell. Biochem. 111 (1992) 103–108.
[9] S. Pontremoli, F. Salamino, B. Sparatore, R. De Tullio, M. Patrone, A. Tizianello, E. Melloni, Enhanced activation of the respiratory burst oxidase in neutrophils from hypertensive patients, Biochem. Biophys. Res. Commun. 158 (1989) 966– 972. [10] K.K. Griendling, D. Sorescu, M. Ushio-Fukai, NAD(P)H oxidase: role in cardiovascular biology and disease, Circ. Res. 86 (2000) 494–501. [11] P. Puddu, G.M. Puddu, E. Cravero, M. Rosati, A. Muscari, The molecular sources of reactive oxygen species in hypertension, Blood Press. 17 (2008) 70–77. [12] C. Delles, W.H. Miller, A.F. Dominiczak, Targeting reactive oxygen species in hypertension, Antioxid. Redox Signal. 10 (2008) 1061–1077. [13] J. Torok, Participation of nitric oxide in different models of experimental hypertension, Physiol. Res. 57 (2008) 813–825. [14] E. Grossman, Does increased oxidative stress cause hypertension? Diabetes Care 31 (Suppl 2) (2008) S185–S189. [15] E. Hopps, R. Lo Presti, G. Caimi, Pathophysiology of polymorphonuclear leukocyte in arterial hypertension, Clin. Hemorheol. Microcirc. 41 (2009) 209– 218. [16] E. Schulz, T. Jansen, P. Wenzel, A. Daiber, T. Munzel, Nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension, Antioxid. Redox Signal. 10 (2008) 1115–1126. [17] M. Chatterjee, R. Saluja, S. Kanneganti, S. Chinta, M. Dikshit, Biochemical and molecular evaluation of neutrophil NOS in spontaneously hypertensive rats, Cell. Mol. Biol. (Noisy-le-grand) 53 (2007) 84–93. [18] M. Sedeek, R.L. Hebert, C.R. Kennedy, K.D. Burns, R.M. Touyz, Molecular mechanisms of hypertension: role of Nox family NADPH oxidases, Curr. Opin. Nephrol. Hypertens. 18 (2009) 122–127. [19] S. Kopprasch, J. Graessler, R. Seibt, H.J. Naumann, K. Scheuch, R. Henssge, U. Schwanebeck, Leukocyte responsiveness to substances that activate the respiratory burst is not altered in borderline and essential hypertension, J. Hum. Hypertens. 10 (1996) 69–76. [20] J.-P. Tung, J. Fraser, P. Wood, Y.L. Fung, Respiratory burst function of ovine neutrophils, BMC Immunol. 10 (2009) 25. [21] A.I. Pettit, R.K.M. Wong, V. Lee, S. Jennings, P.A. Quinn, L.L. Ng, Increased free radical production in hypertension due to increased expression of the NADPH oxidase subunit p22phox in lymphoblast cell lines, J. Hypertens. 20 (2002) 677–683. [22] F. Kouoh, B. Gressier, T. Dine, M. Luyckx, C. Brunet, L. Ballester, J.C. Cazin, In vitro and ex vivo antioxidant activities of labetalol on rabbit neutrophil respiratory burst, Adv. Ther. 21 (2004) 178–185. [23] K.E. Wyche, S.S. Wang, K.K. Griendling, S.I. Dikalov, H. Austin, S. Rao, B. Fink, D.G. Harrison, A.M. Zafari, C242T CYBA polymorphism of the NADPH oxidase is associated with reduced respiratory burst in human neutrophils, Hypertension 43 (2004) 1246–1251.
Contents lists available at ScienceDirect
Cellular Immunology journal homepage: www.elsevier.com/locate/ycimm
Elevated neutrophil respiratory burst activity in essential hypertensive patients Rajesh Ramasamy a,*, Maryam Maqbool a, Abdul Latiff Mohamed b, Rahim Md. Noah c a
Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Malaysia Faculty of Medicine, Cyberjaya University College of Medical Sciences, Malaysia c Medical Science Technology, Universiti Kuala Lumpur, Malaysia b
a r t i c l e
i n f o
Article history: Received 29 December 2009 Accepted 12 April 2010 Available online 18 April 2010 Keywords: Neutrophil Respiratory burst Reactive oxygen species
a b s t r a c t Neutrophils play a significant role in maintaining the integrity of innate immunity via their potent respiratory burst activity. However, the uncontrolled activation of respiratory burst in neutrophils also attributes to chronic diseases such as primary hypertension and atherosclerosis. In our study, we have investigated the activation of respiratory burst function of neutrophils harvested from essential hypertensive patients. In the presence of stimuli PMA and opsonized zymosan (OZ), hypertensive patients’ neutrophils secrete significantly higher amount of superoxide anions compared to normotensive control. Although the magnitude of activation varies between both groups, yet the kinetics of activation is similar. When normotensive control’s neutrophils were pre-treated with hypertensive serum, the cells failed to migrate toward fMLP which indicates the impairment of the migration property. In conclusion, the respiratory burst activity of neutrophils is affected by hypertension and their elevated superoxide anions production could be an aggravating factor in hypertension-related complication. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Neutrophils, also known as polymorphonuclear leukocytes (PMN) are the major cell type that constitutes the innate immune system. They comprise approximately 50–70% of leukocytes and are predominant in eliminating pathogens that induce acute inflammation [1–6]. Elimination of the pathogens by neutrophils involves a series of physiological sequence that comprise chemotaxis, phagocytosis and microbial killing. The success of pathogen elimination by neutrophils is depends on respiratory burst, which is a major process that mediates microbial killing through formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [3]. Phagocytosed microbes are internalized in phagosome, in which ROS are used as microbicides. The ROS production is initiated by NADPH phagosome oxidase (phox) enzyme complex which yields various oxygen reactive species such as hydrogen peroxide, superoxide anion, oxygen cations and other free radicals [3]. Whilst, RNS results from the catalyst of L-arginine by nitric oxide synthase (NOS) that produces nitric oxide reactive species. ROS and RNS are essential for neutrophil defense system and the failure of their production causes severe bacterial infection whilst the
* Corresponding author. Address: Immunology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Fax: +603 8941 3802. E-mail address: [email protected] (R. Ramasamy). 0008-8749/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cellimm.2010.04.004
overproduction triggers vascular damage in chronic diseases such as hypertension and arthrosclerosis [7]. An increasing body of evidence suggests that ROS play a crucial role in development of hypertension by creating a milieu of oxidative stress [8,9]. The skewed balance of free radical production such as superoxide anion and hydrogen peroxide coupled with diminished free radicals scavenger system has been demonstrated in human hypertension [8,10–12]. A significant elevation of oxidative stress embarks pathogenesis of coronary artery disease by altering the vasomotor tone thus exacerbating atherosclerosis and leads to development of hypertension [12–14]. Although vascular endothelial cells play major role in elevating systemic oxidative stress by their activities and cellular damage, blood immune cells also found to be a contributor to this phenomenon [15,16]. The elevated leukocytes count and enhanced PMN activation are correlated with the development and progress of hypertension and cardiovascular disease [15]. Spontaneous activation of neutrophils release proinflammatory factors and reactive oxygen species, which have negative effects on vascular tone and on their adhesion to the endothelium. Furthermore, the current study indicates that an excessive load of free radicals secreted by neutrophils at vascular sites advances the inflammation by limiting the bioavailability of nitric oxide (NO) secreted by endothelial cells [17]. The oxidative stress in hypertensive patients’ neutrophils is revealed by an increased NADPH oxidase production and lipid peroxidation and decreased cytosolic and mitochondrial superoxide dismutase concentrations [2,3,12,15,18]. Furthermore, the overexpression of
R. Ramasamy et al. / Cellular Immunology 263 (2010) 230–234
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adhesion molecules, such as beta2-integrin promotes PMN adhesion and leukocyte-endothelium interactions may contribute to the vascular damage and exacerbate the initiation or complication of arterial hypertension. Therefore the main objective of this study is to investigate the respiratory bust activity impairment of hypertensive patients’ neutrophils. Our study demonstrates that freshly isolated neutrophils from uncontrolled human subjects diagnosed with essential hypertension acquire the ability of producing a higher amount of ROS in response to phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) stimulation. Although many factors may have contributed to this dramatic impairment of neutrophils function, at least in our study we have shown that the humoral component of hypertensive circulation system may be involved in this impairment.
Briefly 1 106 of isolated neutrophils stimulated with either 0.1 lM of phorbol 12-myristate 13-acetate (PMA) (Sigma Chem Com) or 40 mg/ml opsonized zymosan (OZ). The signal derived from oxidative burst was amplified by adding of 0.2 lM 5-amino-2,3-dihydro-1,4-phythalazinedione (luminol) (Sigma Chem. Com). Neutrophils suspension in phenol free HBSS added with luminol and followed by addition of stimulator either PMA or OZ with quick vortex prior to oxidative burst measurement. Oxidative burst activity was characterized by measuring the amount ROS production at different time points at room temperature and the time difference between two readings was fixed at 10 s. The parameters such as the maximal amount ROS production and the time to be taken to reach maximal release of ROS were measured to determine the oxidative burst. The ROS production measured as relative micro voltage scale.
2. Materials and methods
2.5. Chemotaxis assay
2.1. Subjects
The migration of neutrophils from normotensive controls in response to serum from hypertensive patients was measured by chemotaxis assays. One million neutrophils from healthy normotensive individuals were incubated with either in cell culture medium, serum from normotensive or hypertensive individual for 10 min at 37 °C. Incubation with media and normotensive serum serve as control. Treated neutrophils transferred into modified Boyden chamber whereby the neutrophils were physically separated from stimulus by 5 lm filter system. N-Formylmethionylleucyl-phenylalanine (fMLP) (Sigma Chem. Com) at 25 lM was used as chemo-attractant in this assay. After 1 h of incubation in 5% CO2 at 37 °C, the filter detached from modified Boyden chamber and stained with hematoxylin. The migration of neutrophils calculated by light microscope whereby the difference in distance from starting point of neutrophil migration to the highest distance traveled by neutrophil throughout the filter. A minimal number of three readings were taken at different area of filter to determine the highest migration distance.
The study group consisted of 30 primary hypertensive patients and healthy normotensive adults who gave informed consent for participation. Hypertensive patients were selected from pool of essential hypertensive patients with uncontrolled blood pressure exceeding 140/90 mmHg of systolic and diastolic pressure. The hypertensive patients were excluded if they present with chronic diseases and symptomatic complications. Normotensive control group were selected from healthy individuals. Both patients and controls were free from clinical evidence for acute or chronic infection and inflammation. The patient selections were made by consultant cardiologist at hypertension clinic of Hospital Universiti Kebangsaan Malaysia. This study was approved by University ethical review board and procedures followed by institutional guidelines. 2.2. Blood sampling Fifteen milliliter (15 ml) of peripheral venous blood sample from hypertensive patients and normotensive controls were collected by certified phlebotomist. Ten milliliter (10 ml) of sample deposited in anti-coagulated tube for neutrophil isolation and 5 ml of samples kept in plain tube to harvest the serum.
2.6. Statistical analysis The data were compared between hypertensive patients and normotensive controls. Data were presented as the mean ± standard deviation from three or more experiments. The statistical significance was determined by 2-tailed Student t-test.
2.3. Neutrophil isolation 3. Results The following methods were performed to optimize the neutrophil isolation. Ten milliliter (10 ml) of venous blood was collected and diluted in 1 Hank’s balanced salt solution (HBSS) (Flowlab) medium at 1:1 ratio. Ten milliliter (10 ml) diluted blood was then layered over 5 ml lympoprep (Nycomed) and centrifuged at 1500 rpm for 40 min at room temperature. Plasma and the mononuclear cell layer were discarded. The red cell pellet which contains the PMN and red blood cell (RBC) was suspended in 5 ml HBSS. Red cell suspension was layered on 3% Dextran and left at room temperature for 45–60 min to further sediment the RBC. Supernatant from dextran sedimentation was collected and the remnant RBC was lysed using hypotonic lysing procedure to obtain the pure PMN population. The morphological examination and cell count were performed to determine the cell count and purity of the PMN. 2.4. Assessment of neutrophil oxidative burst Oxidative burst by human neutrophils was measured by production of ROS using chemiluminescence meter (Bioorbit 1250).
3.1. Neutrophils from hypertensive patients are easily excitable and highly activated in response to stimulators The stimulators PMA and OZ activate the respiratory burst activity of neutrophils. However, the magnitude of activation which is a reflection of oxygen species production in neutrophils alters by intrinsic and extrinsic factors. In order to assess the impact of high blood pressure, neutrophils from hypertensive patients were stimulated and their ROS production was recorded from the time of administration of stimulators to the peak of respiratory burst activity. Regardless to the type of stimulations, neutrophils from hypertensive patients showed a higher secretion of ROS at all time points (Fig. 1A and B). At the initiation phase of respiratory burst activity (first 30 s), neutrophils from normotensive and hypertensive individuals responded similarly, however, once the respiratory burst machinery is accelerated and entered to the log phase of the kinetics, neutrophils from hypertensive patients display a significantly higher oxidative burst activity (Fig. 1A and B).
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time(s) Fig. 1. Temporal comparison of PMA and OZ activated neutrophils from hypertensive patients and normotensive controls. One million of neutrophils were activated either by PMA (A) or OZ (B) and their respiratory burst activity measured by superoxide production. Superoxide production determined at every 10 s beginning from the administration of stimulator to the time point where the superoxide production reached maximal level and started to decline. This experiment were repeated at least 10 times and the *significance was determined at p < 0.05.
3.2. Neutrophils from hypertensive patients reach the maximal respiratory burst activity at higher magnitudes To determine the peak of respiratory burst activity, the maximal secretion of ROS was extrapolated. Both PMA and OZ significantly increase the maximal superoxide production of hypertensive patients’ neutrophils compare to normotensive controls (Fig. 2B). The mean maximal respiratory burst induced by PMA in hypertensive patients’ neutrophils reached at 277 ± 125 mV compared to normotensive controls at 192 ± 41 mV (Fig. 2B). Meanwhile OZ stimulation yields maximal superoxide production at 360 ± 144 and 185 ± 44 mV for hypertensive patients and normotensive control neutrophils, respectively (Fig. 2B). Although both stimulators were profoundly increase the magnitudes of respiratory burst in hypertensive neutrophils, however, the impact of OZ was much robust when compared to PMA stimulation. The mean time taken to reach the maximal respiratory burst in response to PMA and OZ is varies in which PMA stimulation reach the peak of the kinetics much faster than OZ. In the presence of PMA, neutrophils from hypertensive patients and normotensive control reach the maximum respiratory burst at mean 70 ± 12 s and 68 ± 15 s (Fig. 2A). OZ stimulation induces the hypertensive patients and normotensive control’s neutrophils to reach the maximal respiratory burst at 99 ± 22 s and 87 ± 12 s, respectively (Fig. 2A). 3.3. Serum from hypertensive patients inhibit the migration of normotensive control’s neutrophils To evaluate whether the serum from hypertensive patients affects the migration property of normotensive control’s neutrophils,
the cellular migration of neutrophils towards fMLP was conducted after the pre-treatment with hypertensive patient’s serum. Neutrophils pre-treated with culture medium, normal human serum (NHS) and hypertensive serum (PS) render differences in as 20 ± 5, 17 ± 3 and 11 ± 2 lm migrations toward the chemo-attractant (Fig. 3). 4. Discussion The cellular component of our immune system is an important tool in maintaining the integrity of vascular system. A close interaction between immune cells and endothelial cells via adhesion molecules, secretion of cytokines and cognate antigenic synapses contribute to chronic vascular diseases due to this broken integrity. This is best exemplified in essential hypertension whereby the vascular and migrated neutrophils acquired an impaired activation of respiratory burst which may exacerbate the pathogenesis of hypertension. In our study, we have investigated the role of neutrophils as our subject since it utilizes the respiratory burst function as a major tool for their immune responses. It has been proved by others that, hypertension enhances the respiratory burst activity of neutrophils in response to various stimuli [8,9,19]. In line with this, our report suggests that, in the presence of PMA and OZ, neutrophils from hypertensive patients release a significantly higher ROS compared to normotensive controls (Fig. 1A and B). Neutrophils responded well with OZ stimulation whereby the magnitude of OZ stimulation is much higher than PMA stimulation. Although the difference in magnitude of respiratory burst activation varies, PMA stimulation has certainly exhibited a significant difference at earlier time points (Fig. 1A and B).
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This discrepancy may be due to involvement of different signaling mechanisms in activating respiratory burst machinery [20]. In accordance with this, studies on human neutrophils have shown that PMA stimulates the cell through the protein kinase C signaling pathway (PKC) directly [2] meanwhile OZ mimics a resemblance of pathogen-mediated phagocytosis. Based on kinetics of the stimulation, PMA stimulation through PKC signaling pathway favors a rapid transmission of signals that induces synthesis of ROS. This notion has been further supported by the fact that, the length of time taken to reach the maximal respiratory burst in response to
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PMA is much shorter when compared to OZ stimulation (Fig. 2A). Regardless to the type of stimuli, both PMA and OZ equally raised the production of ROS at all time points. Nevertheless, one study which was conducted using human un-fractionated leukocytes documented that, normotensive controls, borderline and essential hypertensive patients did not show a significant difference in respiratory burst in whole leukocytes population [19]. This contradictory result may originate from utilization of various subpopulations of leukocytes in their samples. Respiratory burst activity is considerably higher in neutrophils compared to lymphocytes. Our unpublished data show that lymphocytes produce very low amounts of superoxide anions which make us difficult to compare normotensive controls with hypertensive patients’ lymphocytes (data not shown). However, using lymphoblast model of human lymphocytes, Pettit et al. have shown that immortalized lymphocytes from hypertensive patients secretes higher amount of ROS due to over-activation of NADPH oxidase [21]. They have speculated that genetic predisposition of an increased expression of p22phox in hypertensive patients may account for this impairment. The recent therapeutical focus for hypertension is taken a reasonable consideration on the pathological role of neutrophils function in order to provide a better control mechanism by harnessing other auxiliary systems such as immune cells. An animal study had shown that fMLP-stimulated rabbit neutrophils, treated with the beta-adrenoreceptor blocker labetalol, demonstrate a dose-dependent antioxidant activity on neutrophils oxidative burst [22]. Moreover, this work suggested the mechanism by which labetalol acts in the treatment of hypertension may occur from an interaction in the signaling pathway of PKC activation. The antioxidant properties demonstrated in this mechanism contribute to the drug’s antihypertensive action and thus, may reduce the risk of injuries inflicted by reactive oxygen species involved in the pathogenesis of hypertension. Although there is unambiguous evidence of an elevated neutrophil respiratory burst in hypertension, yet whether this mutilation is an intrinsic defect of neutrophils or the sheer stress and hypertension associated deregulations force the neutrophil to be more vulnerable is still unknown. One clinical study showed that, occurrence of genetic polymorphisms in oxidative and anti-oxidative enzymes might alter the outcome of cellular susceptibility of neutrophils towards respiratory burst. Wyche et al. had demonstrated that C242T CYBA polymorphism of the NADPH oxidase is strongly associated with reduced respiratory burst in human neutrophils [23]. However, in our study, we have examined the effect of serum from hypertensive patients on migration property of neutrophils from normotensive controls. Hypertensive serum treated with normotensive controls’ neutrophils showed a decline in migration toward fMLP stimulus. The inhibition of chemotaxis may be due to existence of blocking factors in hypertensive serum that disrupt the optimal communication with chemotactic factors or downregulation of chemotaxis or chemokine receptors due to pre-treatment with hypertensive serum. However, our limited experiments did not identify the route of this migratory inhibition or the measurement of migration by hypertensive neutrophil in response to stimuli. Nonetheless one should bear in mind that, neutrophil migration is a crucial step in acute inflammation and failure of performing this task will risk the individual with a potential infection. This might complicate the outcome of hypertension with a susceptibility to bacterial infection. In summary our study has investigated the activation property of neutrophils from essential hypertension patients using a chemiluminescence method. PMA and OZ stimulators were used in study as PMA induces a quick eruption of respiratory burst through mitogen-mediated PKC signaling pathway while OZ serves similarly as a bacterial-mediated immune response. In both systems, neutrophils from hypertensive patients showed an elevated level of respi-
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ratory burst; however, the kinetics of activation does not differ between normotensive controls and hypertensive patients’ neutrophils. The migratory impairment of normotensive neutrophils that pre-treated with hypertensive serum may be due to occurrence of inhibitory humoral factors. However, harnessing the effect of hypertension on neutrophils will be a supplementary therapy for hypertensive patients to maintain the oxidative/anti-oxidative balance and integrity of immune system. Acknowledgment This project is partially supported by Research University Grant Scheme (RUGS), Universiti Putra Malaysia Project No: 04-01-090781RU. References [1] S. Greenberg, S. Grinstein, Phagocytosis and innate immunity, Curr. Opin. Immunol. 14 (2002) 136–145. [2] F.R. Sheppard, M.R. Kelher, E.E. Moore, N.J.D. McLaughlin, A. Banerjee, C.C. Silliman, Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation, J. Leukoc. Biol. 78 (2005) 1025–1042. [3] M.B. Hampton, A.J. Kettle, C.C. Winterbourn, Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing, Blood 92 (1998) 3007–3017. [4] L. Raffaghello, G. Bianchi, M. Bertolotto, F. Montecucco, A. Busca, F. Dallegri, L. Ottonello, V. Pistoia, Human mesenchymal stem cells inhibit neutrophil apoptosis: a model for neutrophil preservation in the bone marrow niche, Stem Cells 26 (2008) 151–162. [5] C. Kantari, M. Pederzoli-Ribeil, V. Witko-Sarsat, The role of neutrophils and monocytes in innate immunity, Contrib. Microbiol. 15 (2008) 118–146. [6] A.S. Cowburn, A.M. Condliffe, N. Farahi, C. Summers, E.R. Chilvers, Advances in neutrophil biology: clinical implications, Chest 134 (2008) 606–612. [7] C. Elbim, G. Lizard, Flow cytometric investigation of neutrophil oxidative burst and apoptosis in physiological and pathological situations, Cytometry A 75 (2009) 475–481. [8] S. Sagar, I.J. Kallo, N. Kaul, N.K. Ganguly, B.K. Sharma, Oxygen free radicals in essential hypertension, Mol. Cell. Biochem. 111 (1992) 103–108.
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