Hypertens Pregnancy, 2014; 33(2): 236–249 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2013.858744
ORIGINAL ARTICLE
An imbalance in the production of proinflammatory and anti-inflammatory cytokines is observed in whole blood cultures of preeclamptic women in comparison with healthy pregnant women Jorge Campos-Can ´ s Romo-Palafox,2 ˜as,1 Ine Mario Albani-Campanario,3 and Ce ´ sar Herna ´ ndez-Guerrero4 ´ ngeles Acoxpa, Colonia Ex-Hacienda Coapa, Me´xico D.F., Hospital A Estudiante de la Maestrı´a en Nutriologı´a Aplicada de la Universidad Iberoamericana Ciudad de Me´xico, Me´xico D.F., 3 RedCrea, Medicina Reproductiva, Lindavista, Me´xico D.F., and 4 Departamento de Salud, Universidad Iberoamericana Ciudad de Me´xico, Me´xico D.F. 1 2
Keywords Anti-inflammation, Cytokines, Hypertension, Preeclampsia, Proinflammation.
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Objective: To compare the production of pro- and anti-inflammatory cytokines by whole blood samples stimulated with lipopolysaccharide from normotensive and preeclamptic women. Methods: The synthesis of tumor necrosis factor-a, interleukyn-1b, interleukin6 and interleukin-10 were assayed by immunosorbent assays in 56 and 50 preeclamptic and normotensive women, respectively. Results: Preeclamptic women showed a higher synthesis (p50.05) of interleukin-1b and interleukin-6 versus normotensive group. Interleukin-10 synthesis from samples of normotensive women showed a higher production versus preeclamptic group (p50.05). Conclusion: Preeclamptic women showed an imbalance in the production of pro and anti-inflammatory cytokines in comparison with normotensive women.
INTRODUCTION Preeclampsia (PE) constitutes the most frequent complication during pregnancy, around 5–7% of pregnant women suffer this disease. PE is defined clinically as new onset in systolic blood pressure 140 mmHg or diastolic blood pressure 90 mmHg, plus the presence of proteinuria after 20th week of gestation. This phenomenon is responsible for 15% of maternal mortality and 15% of preterm birth mortality, being this medically indicated because of the pathology (1,2). Correspondence: Dr Ce´sar Herna´ndez Guerrero, Departamento de Salud, Universidad Iberoamericana Ciudad de Me´xico, Me´xico. Prolongacio´n Paseo de la Reforma 880, Lomas de Santa Fe, C.P. 01219, Me´xico, Distrito Federal. Tel: +52 555 950 40 00 Ext. 4976. E-mail: [email protected]
Proinflammatory cytokines in preeclampsia
Pregnant women who have this complication are susceptible to develop the potentially lethal affections like placenta abruption, cerebral hemorrhage, hepatic failure and acute renal failure. A specific multisystemic pregnancy syndrome characterized by a decrease in organic perfusion related to vasospasm and activation of the coagulation cascade is identified in women with the disease (3–5). The etiology of PE is unknown to date, however, the presence of an altered immunological response is an essential factor in its appearance. It has been described that women with PE present an ‘‘excessive inflammatory’’ response secondary to an inadequate placentation, and/or from an exaggerated maternal immunological response towards the proinflammatory branch (6,7). The proinflammatory maternal state promotes the appearance of oxidative stress and endothelial dysfunction; mechanisms that decrease the vascular relaxation. On the other hand, the proinflammatory cytokines activate the peripheral neutrophils which contribute to the endothelial damage throughout the liberation of elastase and lactoferrin (8). A higher concentration of soluble peripheral markers derived from the degranulation of activated neutrophils, as well as a higher activation of complement proteins have been identified in plasma of women with PE in comparison with normotensive (NT) (9–11). The activation of the complement amplifies the inflammatory process and promotes the chemotaxis and activation of monocytes and neutrophils, which are cells capable of increasing the endothelial damage (12,13). The maternal proinflammatory state observed in women with PE is explained as consequence of an inadequate placentation that reduces the uterine blood flow (placentary ischemia), which activates and promotes the endothelial dysfunction (14,15). Nevertheless, the inadequate vascular reorganization of the placenta because of a deficient trophoblast uterine invasion has been questioned, since a failure in the uterine vascular bed is associated with a restriction of the fetal intrauterine growth, phenomenon in which no signs of proteinuria or hypertension are present. It is clear that an altered immunological response contributes to the etiology of the PE, since diverse evidences in the literature have identified an increased concentration of proinflammatory cytokines in the plasma of women with PE, in comparison with NT women. However, the participation of the maternal compartment in the production of pro- and anti-inflammatory cytokines has not been completely understood (16–20). The present work has the objective to evaluate the relationship between the production of pro and anti-inflammatory cytokines by whole blood stimulated with lipopolysaccharides (LPS) from women with PE and NT.
MATERIALS AND METHODS Patients The population consisted in 106 patients, from which 56 were diagnosed with PE (17 and 39 with mild and severe PE, respectively) and 50 were NT controls with normal blood pressure. Mild preeclamptic (PreMi) patients were clinically defined as such if they presented elevated blood pressure (4140 mmHg systolic or 490 mmHg diastolic after 20th week of gestation) and proteinuria
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(41þ dipstick or 40.3 g/24 h). Severe PE (PreSe) was defined as the presence of one or more of the following features: systolic blood pressure of 160 mmHg, diastolic pressure of 110 mmHg, proteinuria 45 g/24 h or 3þ by urine dipstick, neurological symptoms, elevated serum creatinine, thrombocytopenia or pulmonary edema. The control group solved their pregnancies at term (37–42 weeks of gestation), none of which were previously diagnosed with hypertension, diabetes or autoimmune disease. At the time of enrollment they appeared healthy by clinical and laboratory standards. Women with PE were matched for maternal and gestational age with control group women. All participants were previously informed of the characteristics and the scope of the study, and written informed consent was obtained from each participant. The project was examined and approved by the ethic and scientific committee of the Institute. Sample Collection and Peripheral Blood Stimulation About 5–10 mL of peripheral blood was drawn from subjects by venipuncture of humeral or radial vein. All the collector tubes contained sodium heparin with vacutainer system (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Once the samples were taken they were transported immediately to the laboratory for processing. The stimulation of peripheral whole blood was prepared under two conditions: The first step was to make a dilution 1:4 of peripheral blood with culture medium consisting of RPMI-1640 with L-glutamine, 25 mM HEPES without sodium (GIBCO GRL, Life Technologies, Breda, Netherlands) and supplemented with 10 U/mL of penicillin and streptomycin (GIBCO); namely unstimulated (UN) cells. The same blood dilution was made with RPMI-1640 with 6 mg/ml of LPS (E.coli O26:B6, Sigma, St. Louis, MO), which we added for the production of cytokines; namely stimulated (ST) cells. Once we had a homogeneous mixture of blood and medium, it was added to a 24-well plate with a flat bottom (Costar, Cambridge, Boston, MA). The plate was incubated for 24 h at 37 C of humidity at 5% of CO2. The period between the blood recollection and the initiation of the incubation was less than an hour. After 24 h of incubation, blood was withdrawn from the wells and centrifuged at 3000 rpm for 10 min at 4 C. The supernatant was kept in 1.5 mL eppendorf tubes at 70 C until cytokines were quantified. Cytokine Determination Determination of TNF-a, IL-1b, IL-4, IL-6, IL-10 and IFN-g was performed by enzyme linked-immunoassay (ELISA) using reagents of the PharMingen Company (PharMingen, San Diego, CA). A monoclonal antibody (Ab) specific for each cytokine was added to the binding solution (0.1 M Na2HPO4, pH was adjusted to 9.0 with 0.1 M de NaH2PO4). Plates were incubated at 4 C over night. The binding solution was removed 24 h later by washing each well three times with PBS/Tween solution. A biotinylated monoclonal anti TNF-a, IL-1b, IL-4, IL-6, IL-10 and IFN-g-specific antibody was added, incubated for 3 h at room temperature and excess biotinylated antibody removed by repeated
Proinflammatory cytokines in preeclampsia
washing with PBS/Tween. Samples were incubated all night. Then streptavidin-alkaline phosphatase with PBS-Tween was added, and plates were incubated for 2 h at room temperature. After the chromogenic substrate was added plates were read at 405 nm. Inter- and intra-assay variations were less than 10% and 5%, respectively, for all the cytokines assayed. STATISTICAL ANALYSIS The method of descriptive statistic was used to analyze the results of the variables obtained from the participants of the study. The difference in the cytokine productions by PE and control group was evaluated using Mann–Whitney rank sum test for two groups or analysis of variance (ANOVA) from Kruskal–Wallis, applying the Dunn test as pos hoc test. Statistical analyses were performed using the SigmaStat v.3.5 software (Systat Software Inc., San Jose, CA).
RESULTS Study Population The study population included 106 pregnant subjects hospitalized in the Instituto Nacional de Perinatologı´a (INPer) in Mexico City. The control group consisted in 50 pregnant women with normal blood pressure and 56 patients who were diagnosed with PE (17 with mild PE and 39 with severe PE). All participants were 18 years old or older, and denied previous history of hypertension, diabetes and autoimmune disease previous to the pregnancy. There were no statistical differences regardless the age and gestational age between women with PE and control group (Table 1). Data of concentration of protein in 24 h urine collection as well as diastolic and systolic blood pressure are shown in Table 1. Blood pressure readings were measured at the moment of the diagnosis, before any medication or intervention was made. At this moment the peripheral blood samples was taken.
Table 1. Characteristics of the participants included in the study. Control (n ¼ 50) Age (years) Gestational age (weeks) Protein (mg/mL) Diastolic BP (mmHg) Systolic BP (mmHg)
28 7.9 27 (18–44) 30 4.3 31 (22–36) – 73 6.6 75 (60–80) 114 4.2 115 (107–120)
PreAll (n ¼ 56) 28 4.9 28 (20–41) 32 3.6 32 (24–38) 1387 1159.1 858 (294–4300) 101 15.2 100 (100–120) 155 12.2 160 (130–186)
PreMi (n ¼ 17) 27 6.1 26 (20–41) 33 3.4
PreSe (n ¼ 39) 28 4.4 28 (21–38) 31 3.7
33 (27–38) 32 (24–38) 521 291.0 1765 1194.5 444 (300–1360) 1732 (294–4300) 93 4 104 16.9 90 (90–100) 105 (100–120) 143 6.6 161 9.6 140 (130–150) 160 (140–186)
Data show mean SD; median (minimum maximum). PreAll, PreMi plus PreSe; PreMi, mild preeclampsia; PreSe, severe preeclampsia; BP, blood pressure.
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Cytokines Measurement In our study, it was not possible to determine the concentration of IL-4 and IFN-g neither in PE or control group samples. Both cytokines are synthesized mainly by T CD3þ/CD4þ helper lymphocytes, and the LPS stimuli has as a target monocyte and macrophage cells. The before mentioned cytokines were included in the panel of determinations with the intention to evaluate their basal synthesis, and/or the possible synergic effect of soluble or cellular factors present in the whole blood; however their synthesis was not detected. The synthesis of TNF-a only was detected when the samples were stimulated. There were no statistical differences in the concentrations of this cytokine between the control group and the PE groups when the samples were stimulated (Figure 1). IL-1b as well as TNF-a was not detected in samples without stimuli, however when comparing IL-1b production by stimulated samples, we found a statistical difference between the control group versus all the PE groups (Figure 1). The IL-6 synthesis by unstimulated samples from control group was higher in comparison (p50.05) with all the PE groups (Figure 2). However, when the samples were stimulated the results showed an inverted pattern between unstimulated and stimulated samples, since all PE groups showed a higher production of IL-6 (p50.05) respect to the control group (Figure 2). The IL-10 synthesis by unstimulated samples was very low in the PE groups in comparison with control group. In all the cases, there were statistically differences between control and PE groups (Figure 3). However, when the samples were stimulated (Figure 3), the synthesis of IL-10 in the PE groups was the same as the control group (p ¼ 0.774). On the other hand, when a comparison between the unstimulated and stimulated synthesis of the proinflammatory and anti-inflammatory cytokines was done, the IL-6 production by normotensive control samples showed an increment around 2.3 times, while preeclamptic women revealed an increment between 50 and 70 times (Figure 4). In all the cases, there were statistically differences (p50.001) between the unstimulated and stimulated conditions of culture. With respect to the anti-inflammatory cytokine IL-10, the control and the preeclamptic groups showed a statistically (p50.001) increment in the production of mentioned cytokine when the samples were stimulated (Figure 5). However, the increment in the production of IL-10 by the preeclamptic stimulated groups was not as dramatic as observed in IL-6, since the levels of IL-10 were the same between the experimental and control groups (Figure 3).
DISCUSSION The inflammatory response is a strongly associated factor with endothelial damage observed in the PE symptomatology. Inadequate placentation seen in women with PE is presumed to promote a placental ischemic process that conducts to the liberation of cytokines like IFN-g, TNF-a, IL-1b, IL-6, IL-8, which activate different cellular and humoral immunologic components that potentiate endothelial damage. However, it has been described that in the
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Figure 1. Synthesis of proinflammatory cytokines by culture whole blood samples. *p50.05 compared versus control group (Cont; n ¼ 50). PreAll (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (PreMi; n ¼ 17). Severe preeclampsia (PreSe; n ¼ 39). Media and standard deviation are shown in the bars.
inflammatory hyper-responsiveness detected in women with PE, immunologic maternal cells actively participate in this phenomenon, which seems the source of the elevated proinflammatory cytokine concentration identified in plasma (21). In our study, we stimulated whole blood of women with PE and NT with LPS, with the intention to observe the production of proinflammatory and anti-inflammatory cytokines at the nearest time of diagnostic of PE. Once the women were classified with the pathology and accepted to participate in the study, the samples of blood were taken and processed as fast as possible. We wanted to evaluate the production of both types of
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Figure 2. Synthesis of IL-6 by culture whole blood samples. *p50.05 compared versus control group (Cont; n ¼ 50). PreAll (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (PreMi; n ¼ 17). Severe preeclampsia (PreSe; n ¼ 39). Media and standard deviation are shown in the bars.
cytokines taking into account all possible soluble and cellular elements present in the blood compartment, as well as the ‘‘activation status’’ of peripheral immune cells prior to any treatment or maneuver implemented for women. The LPS applied as a stimulus is an endotoxin present in Gram negative bacteria, which consists in a phosphoglycolipid anchored to the bacteria membrane (lipid A), attached covalently to a hydrophilic heteropolysaccharide that confers the biologic activity to the molecule. The LPS binds to the CD (cluster differentiation)-14 molecule found in monocytes, macrophages and polymorphonuclear cells and/or TLR receptor (Toll-like receptors)-4, which
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conducts a complex cascade of intracellular activation that includes protein kinase (PKA and PKC), mitogen-activated proteinkinase (MAPK), tyrosine kinases, protein G and protein phosphatase and others, that when acting in a synergic and antagonist way results in the synthesis of inflammatory cytokines such as IFN-g, TNF-a, IL-1b, IL-6 and IL-8 (22,23). The results obtained were very interesting in the sense that in the basal form we did not identify the production of the cytokines TNF-a and IL-1b in the blood sample culture (BSC) in NT nor PE women. Nevertheless, we observe more synthesis of IL-6 in NT women in comparison with the values obtained from the PreAll, PreMi and PreSe groups (significantly different in all cases).
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Figure 4. Synthesis of IL-6 by unstimulated and stimulated culture whole blood samples. *p50.05 compared versus respective unstimulated group. Control (n ¼ 50). Preeclampsia all groups (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (n ¼ 17). Severe preeclampsia (n ¼ 39). Media and standard deviation are shown in the bars.
This major production of IL-6 from NT women in isolated way could be an atypical result, considering that the evidences indicate that such cytokine is identified in a higher concentration in plasma of PE women. However, when BSC was stimulated we observed a huge response in the production of IL-6, since the NT women generally duplicate the synthesis of that cytokine, whereas for PE women the value of the synthesis was quintupled in general. The IL-6 is produced by mononuclear phagocytes and T cells and is a powerful cytokine involved in the symptomatology of PE, due to enhances endothelial cell permeability likely by altering the ultrastructural distribution
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Figure 5. Synthesis of IL-10 by unstimulated and stimulated culture whole blood samples. *p50.05 compared versus respective unstimulated group. Control (n ¼ 50). Preeclampsia all groups (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (n ¼ 17). Severe preeclampsia (n ¼ 39). Media and standard deviation are shown in the bars.
of tight junctions. IL-6 contributes to the systemic endothelial activation and vascular damage, particularly in the kidney, a pivotal organ in the development of hypertension and proteinuria identified in this group of women (24). This cytokine appears to play a crucial factor in the generation of hypertension observed in these women, due to its capacity to improve the generation of autoantibodies that activate the angiotensin II type I receptor, and leads to activation of endothelin-1, oxidative stress, anti angiogenic factor, and increased sensitivity to angiotensina II (25). In fact, Gadonski have demonstrated in a model of pregnant rats that a chronic infusion of IL-6 increased
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arterial pressure and decreased renal plasma flow and glomerular filtration rate (26). Likewise, stimulated samples from women with PE not only showed a wider synthesis of IL-6 in comparison with NT women, also introduced a greater synthesis of IL-1b statistically different in the group and subgroups of PE women. IL-1b is produced by monocytes and macrophages after the stimulation with bacterial products and by TNF-a. IL-1b performs and enhances a lot of functions of TNF-a and both cytokines can alter the properties of vascular endothelial cells in women with PE through the activation of neutrophils, which contribute to the endothelial damage by means of the liberation of elastase and lactoferrin. Also, both cytokines induce the production of IL-6 in monocytes, macrophages and T cells by autocrine and paracrine ways; cytokine who contributes to the systemic endothelial activation and vascular damage act together with TNF-a and IL-1b (27,28). On the other hand, in the samples without stimuli it was possible to identify a lower synthesis of IL-10 (statistically different) in women with PE in comparison with women with NT, nevertheless when the samples were stimulated; the synthesis of IL-10 did not show differences between the control and the PE groups. We can point out that women with PE show a basal tendency to improve an immune response toward proinflammatory branch, being that the ratio of IL-6/IL-10 was statistically different versus control groups. Likewise, when the samples were stimulated that tendency was reinforced since the ratio of IL-1b/IL-10 and IL-6/IL-10 were higher and showed statistical difference versus control group (data not shown). The apparent loss of equilibrium between pro and anti-inflammatory response observed in PE women seems altered in two ways, due to in a basal condition those women showed a deficient capacity to synthesize IL-10, a potent anti-inflammatory cytokine synthesized mainly by Th2 lymphocytes, monocytes and B lymphocytes, which once it is bound to its receptor inhibits the synthesis of the proinflammatory cytokines TNF-a, IL-1b, lL-6, IL-8, IL-12, by monocytes, macrophages and Th1 lymphocytes. And the second way refers when the samples were stimulated and an increase capacity to produce IL-1b and lL-6 cytokines was identified, associated to a similar synthesis of IL-10 showed by the control group. We do not know the causes that PE maternal peripheral cells produce this specific cytokine imbalance, nevertheless it is clear that by performing the complete blood stimulation, all the soluble and cellular components capable of executing synergic functions and potentiate the immune response are found there. Moreover, although we have no experimental evidence, that higher capacity of immune cells to improve a proinflammatory response, could be as a consequence of a loss of the adequate maternal immune adaptation directed as an etiologic hypothesis of PE. Our data differ from those obtained by Beckmann (29) who by stimulating BSC obtained in NT women and with PE with LPS, identified after 24 h of incubation a higher production of TNF-a from samples with PE. Whereas Luppi (30) by stimulating mononuclear cells of NT women and with PE with LPS, established a lower percentage of IL-1b monocyte production in comparison with NT women using flow cytometer; being this an inverse
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pattern of synthesis of TNF-a and IL-b in non-stimulated cells from both the study groups. However, our findings reinforce the idea of a maternal proinflammatory hyper-response, combined to a lower synthesis capacity of the anti-inflammatory cytokines, which potentiates even more the inflammatory mechanism. The results present herein are in line with a previous report done by us (31), where a higher concentration of IFN-g was identified in peripheral plasma of women with PE in comparison with NT women, associated to a diminished concentration of IL-4, a typical anti-inflammatory cytokine. Nevertheless, we did not identify any differences at pro or anti-inflammatory cytokines between the coriodecidual and peripheral compartment of women with PE nor NT. Also, Vitoratos et al. (32) observed a higher concentration of IL-6 and TNF-a in plasma of women with PE in comparison with NT women at term, as well as similar values from both cytokines at the end of the pregnancy and after 12–14 weeks postpartum, showing that placental tissue seems not like the principal source of proinflammatory cytokines in women with PE. Data present here aid to increase the knowledge about the participation of the maternal compartment of the proinflammatory phenomenon in PE, with the intention to improve the strategies that decrease the maternal and fetal morbi-mortality that is detected in this pathology.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing the paper.
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ORIGINAL ARTICLE
An imbalance in the production of proinflammatory and anti-inflammatory cytokines is observed in whole blood cultures of preeclamptic women in comparison with healthy pregnant women Jorge Campos-Can ´ s Romo-Palafox,2 ˜as,1 Ine Mario Albani-Campanario,3 and Ce ´ sar Herna ´ ndez-Guerrero4 ´ ngeles Acoxpa, Colonia Ex-Hacienda Coapa, Me´xico D.F., Hospital A Estudiante de la Maestrı´a en Nutriologı´a Aplicada de la Universidad Iberoamericana Ciudad de Me´xico, Me´xico D.F., 3 RedCrea, Medicina Reproductiva, Lindavista, Me´xico D.F., and 4 Departamento de Salud, Universidad Iberoamericana Ciudad de Me´xico, Me´xico D.F. 1 2
Keywords Anti-inflammation, Cytokines, Hypertension, Preeclampsia, Proinflammation.
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Objective: To compare the production of pro- and anti-inflammatory cytokines by whole blood samples stimulated with lipopolysaccharide from normotensive and preeclamptic women. Methods: The synthesis of tumor necrosis factor-a, interleukyn-1b, interleukin6 and interleukin-10 were assayed by immunosorbent assays in 56 and 50 preeclamptic and normotensive women, respectively. Results: Preeclamptic women showed a higher synthesis (p50.05) of interleukin-1b and interleukin-6 versus normotensive group. Interleukin-10 synthesis from samples of normotensive women showed a higher production versus preeclamptic group (p50.05). Conclusion: Preeclamptic women showed an imbalance in the production of pro and anti-inflammatory cytokines in comparison with normotensive women.
INTRODUCTION Preeclampsia (PE) constitutes the most frequent complication during pregnancy, around 5–7% of pregnant women suffer this disease. PE is defined clinically as new onset in systolic blood pressure 140 mmHg or diastolic blood pressure 90 mmHg, plus the presence of proteinuria after 20th week of gestation. This phenomenon is responsible for 15% of maternal mortality and 15% of preterm birth mortality, being this medically indicated because of the pathology (1,2). Correspondence: Dr Ce´sar Herna´ndez Guerrero, Departamento de Salud, Universidad Iberoamericana Ciudad de Me´xico, Me´xico. Prolongacio´n Paseo de la Reforma 880, Lomas de Santa Fe, C.P. 01219, Me´xico, Distrito Federal. Tel: +52 555 950 40 00 Ext. 4976. E-mail: [email protected]
Proinflammatory cytokines in preeclampsia
Pregnant women who have this complication are susceptible to develop the potentially lethal affections like placenta abruption, cerebral hemorrhage, hepatic failure and acute renal failure. A specific multisystemic pregnancy syndrome characterized by a decrease in organic perfusion related to vasospasm and activation of the coagulation cascade is identified in women with the disease (3–5). The etiology of PE is unknown to date, however, the presence of an altered immunological response is an essential factor in its appearance. It has been described that women with PE present an ‘‘excessive inflammatory’’ response secondary to an inadequate placentation, and/or from an exaggerated maternal immunological response towards the proinflammatory branch (6,7). The proinflammatory maternal state promotes the appearance of oxidative stress and endothelial dysfunction; mechanisms that decrease the vascular relaxation. On the other hand, the proinflammatory cytokines activate the peripheral neutrophils which contribute to the endothelial damage throughout the liberation of elastase and lactoferrin (8). A higher concentration of soluble peripheral markers derived from the degranulation of activated neutrophils, as well as a higher activation of complement proteins have been identified in plasma of women with PE in comparison with normotensive (NT) (9–11). The activation of the complement amplifies the inflammatory process and promotes the chemotaxis and activation of monocytes and neutrophils, which are cells capable of increasing the endothelial damage (12,13). The maternal proinflammatory state observed in women with PE is explained as consequence of an inadequate placentation that reduces the uterine blood flow (placentary ischemia), which activates and promotes the endothelial dysfunction (14,15). Nevertheless, the inadequate vascular reorganization of the placenta because of a deficient trophoblast uterine invasion has been questioned, since a failure in the uterine vascular bed is associated with a restriction of the fetal intrauterine growth, phenomenon in which no signs of proteinuria or hypertension are present. It is clear that an altered immunological response contributes to the etiology of the PE, since diverse evidences in the literature have identified an increased concentration of proinflammatory cytokines in the plasma of women with PE, in comparison with NT women. However, the participation of the maternal compartment in the production of pro- and anti-inflammatory cytokines has not been completely understood (16–20). The present work has the objective to evaluate the relationship between the production of pro and anti-inflammatory cytokines by whole blood stimulated with lipopolysaccharides (LPS) from women with PE and NT.
MATERIALS AND METHODS Patients The population consisted in 106 patients, from which 56 were diagnosed with PE (17 and 39 with mild and severe PE, respectively) and 50 were NT controls with normal blood pressure. Mild preeclamptic (PreMi) patients were clinically defined as such if they presented elevated blood pressure (4140 mmHg systolic or 490 mmHg diastolic after 20th week of gestation) and proteinuria
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(41þ dipstick or 40.3 g/24 h). Severe PE (PreSe) was defined as the presence of one or more of the following features: systolic blood pressure of 160 mmHg, diastolic pressure of 110 mmHg, proteinuria 45 g/24 h or 3þ by urine dipstick, neurological symptoms, elevated serum creatinine, thrombocytopenia or pulmonary edema. The control group solved their pregnancies at term (37–42 weeks of gestation), none of which were previously diagnosed with hypertension, diabetes or autoimmune disease. At the time of enrollment they appeared healthy by clinical and laboratory standards. Women with PE were matched for maternal and gestational age with control group women. All participants were previously informed of the characteristics and the scope of the study, and written informed consent was obtained from each participant. The project was examined and approved by the ethic and scientific committee of the Institute. Sample Collection and Peripheral Blood Stimulation About 5–10 mL of peripheral blood was drawn from subjects by venipuncture of humeral or radial vein. All the collector tubes contained sodium heparin with vacutainer system (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ). Once the samples were taken they were transported immediately to the laboratory for processing. The stimulation of peripheral whole blood was prepared under two conditions: The first step was to make a dilution 1:4 of peripheral blood with culture medium consisting of RPMI-1640 with L-glutamine, 25 mM HEPES without sodium (GIBCO GRL, Life Technologies, Breda, Netherlands) and supplemented with 10 U/mL of penicillin and streptomycin (GIBCO); namely unstimulated (UN) cells. The same blood dilution was made with RPMI-1640 with 6 mg/ml of LPS (E.coli O26:B6, Sigma, St. Louis, MO), which we added for the production of cytokines; namely stimulated (ST) cells. Once we had a homogeneous mixture of blood and medium, it was added to a 24-well plate with a flat bottom (Costar, Cambridge, Boston, MA). The plate was incubated for 24 h at 37 C of humidity at 5% of CO2. The period between the blood recollection and the initiation of the incubation was less than an hour. After 24 h of incubation, blood was withdrawn from the wells and centrifuged at 3000 rpm for 10 min at 4 C. The supernatant was kept in 1.5 mL eppendorf tubes at 70 C until cytokines were quantified. Cytokine Determination Determination of TNF-a, IL-1b, IL-4, IL-6, IL-10 and IFN-g was performed by enzyme linked-immunoassay (ELISA) using reagents of the PharMingen Company (PharMingen, San Diego, CA). A monoclonal antibody (Ab) specific for each cytokine was added to the binding solution (0.1 M Na2HPO4, pH was adjusted to 9.0 with 0.1 M de NaH2PO4). Plates were incubated at 4 C over night. The binding solution was removed 24 h later by washing each well three times with PBS/Tween solution. A biotinylated monoclonal anti TNF-a, IL-1b, IL-4, IL-6, IL-10 and IFN-g-specific antibody was added, incubated for 3 h at room temperature and excess biotinylated antibody removed by repeated
Proinflammatory cytokines in preeclampsia
washing with PBS/Tween. Samples were incubated all night. Then streptavidin-alkaline phosphatase with PBS-Tween was added, and plates were incubated for 2 h at room temperature. After the chromogenic substrate was added plates were read at 405 nm. Inter- and intra-assay variations were less than 10% and 5%, respectively, for all the cytokines assayed. STATISTICAL ANALYSIS The method of descriptive statistic was used to analyze the results of the variables obtained from the participants of the study. The difference in the cytokine productions by PE and control group was evaluated using Mann–Whitney rank sum test for two groups or analysis of variance (ANOVA) from Kruskal–Wallis, applying the Dunn test as pos hoc test. Statistical analyses were performed using the SigmaStat v.3.5 software (Systat Software Inc., San Jose, CA).
RESULTS Study Population The study population included 106 pregnant subjects hospitalized in the Instituto Nacional de Perinatologı´a (INPer) in Mexico City. The control group consisted in 50 pregnant women with normal blood pressure and 56 patients who were diagnosed with PE (17 with mild PE and 39 with severe PE). All participants were 18 years old or older, and denied previous history of hypertension, diabetes and autoimmune disease previous to the pregnancy. There were no statistical differences regardless the age and gestational age between women with PE and control group (Table 1). Data of concentration of protein in 24 h urine collection as well as diastolic and systolic blood pressure are shown in Table 1. Blood pressure readings were measured at the moment of the diagnosis, before any medication or intervention was made. At this moment the peripheral blood samples was taken.
Table 1. Characteristics of the participants included in the study. Control (n ¼ 50) Age (years) Gestational age (weeks) Protein (mg/mL) Diastolic BP (mmHg) Systolic BP (mmHg)
28 7.9 27 (18–44) 30 4.3 31 (22–36) – 73 6.6 75 (60–80) 114 4.2 115 (107–120)
PreAll (n ¼ 56) 28 4.9 28 (20–41) 32 3.6 32 (24–38) 1387 1159.1 858 (294–4300) 101 15.2 100 (100–120) 155 12.2 160 (130–186)
PreMi (n ¼ 17) 27 6.1 26 (20–41) 33 3.4
PreSe (n ¼ 39) 28 4.4 28 (21–38) 31 3.7
33 (27–38) 32 (24–38) 521 291.0 1765 1194.5 444 (300–1360) 1732 (294–4300) 93 4 104 16.9 90 (90–100) 105 (100–120) 143 6.6 161 9.6 140 (130–150) 160 (140–186)
Data show mean SD; median (minimum maximum). PreAll, PreMi plus PreSe; PreMi, mild preeclampsia; PreSe, severe preeclampsia; BP, blood pressure.
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Cytokines Measurement In our study, it was not possible to determine the concentration of IL-4 and IFN-g neither in PE or control group samples. Both cytokines are synthesized mainly by T CD3þ/CD4þ helper lymphocytes, and the LPS stimuli has as a target monocyte and macrophage cells. The before mentioned cytokines were included in the panel of determinations with the intention to evaluate their basal synthesis, and/or the possible synergic effect of soluble or cellular factors present in the whole blood; however their synthesis was not detected. The synthesis of TNF-a only was detected when the samples were stimulated. There were no statistical differences in the concentrations of this cytokine between the control group and the PE groups when the samples were stimulated (Figure 1). IL-1b as well as TNF-a was not detected in samples without stimuli, however when comparing IL-1b production by stimulated samples, we found a statistical difference between the control group versus all the PE groups (Figure 1). The IL-6 synthesis by unstimulated samples from control group was higher in comparison (p50.05) with all the PE groups (Figure 2). However, when the samples were stimulated the results showed an inverted pattern between unstimulated and stimulated samples, since all PE groups showed a higher production of IL-6 (p50.05) respect to the control group (Figure 2). The IL-10 synthesis by unstimulated samples was very low in the PE groups in comparison with control group. In all the cases, there were statistically differences between control and PE groups (Figure 3). However, when the samples were stimulated (Figure 3), the synthesis of IL-10 in the PE groups was the same as the control group (p ¼ 0.774). On the other hand, when a comparison between the unstimulated and stimulated synthesis of the proinflammatory and anti-inflammatory cytokines was done, the IL-6 production by normotensive control samples showed an increment around 2.3 times, while preeclamptic women revealed an increment between 50 and 70 times (Figure 4). In all the cases, there were statistically differences (p50.001) between the unstimulated and stimulated conditions of culture. With respect to the anti-inflammatory cytokine IL-10, the control and the preeclamptic groups showed a statistically (p50.001) increment in the production of mentioned cytokine when the samples were stimulated (Figure 5). However, the increment in the production of IL-10 by the preeclamptic stimulated groups was not as dramatic as observed in IL-6, since the levels of IL-10 were the same between the experimental and control groups (Figure 3).
DISCUSSION The inflammatory response is a strongly associated factor with endothelial damage observed in the PE symptomatology. Inadequate placentation seen in women with PE is presumed to promote a placental ischemic process that conducts to the liberation of cytokines like IFN-g, TNF-a, IL-1b, IL-6, IL-8, which activate different cellular and humoral immunologic components that potentiate endothelial damage. However, it has been described that in the
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Figure 1. Synthesis of proinflammatory cytokines by culture whole blood samples. *p50.05 compared versus control group (Cont; n ¼ 50). PreAll (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (PreMi; n ¼ 17). Severe preeclampsia (PreSe; n ¼ 39). Media and standard deviation are shown in the bars.
inflammatory hyper-responsiveness detected in women with PE, immunologic maternal cells actively participate in this phenomenon, which seems the source of the elevated proinflammatory cytokine concentration identified in plasma (21). In our study, we stimulated whole blood of women with PE and NT with LPS, with the intention to observe the production of proinflammatory and anti-inflammatory cytokines at the nearest time of diagnostic of PE. Once the women were classified with the pathology and accepted to participate in the study, the samples of blood were taken and processed as fast as possible. We wanted to evaluate the production of both types of
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Figure 2. Synthesis of IL-6 by culture whole blood samples. *p50.05 compared versus control group (Cont; n ¼ 50). PreAll (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (PreMi; n ¼ 17). Severe preeclampsia (PreSe; n ¼ 39). Media and standard deviation are shown in the bars.
cytokines taking into account all possible soluble and cellular elements present in the blood compartment, as well as the ‘‘activation status’’ of peripheral immune cells prior to any treatment or maneuver implemented for women. The LPS applied as a stimulus is an endotoxin present in Gram negative bacteria, which consists in a phosphoglycolipid anchored to the bacteria membrane (lipid A), attached covalently to a hydrophilic heteropolysaccharide that confers the biologic activity to the molecule. The LPS binds to the CD (cluster differentiation)-14 molecule found in monocytes, macrophages and polymorphonuclear cells and/or TLR receptor (Toll-like receptors)-4, which
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conducts a complex cascade of intracellular activation that includes protein kinase (PKA and PKC), mitogen-activated proteinkinase (MAPK), tyrosine kinases, protein G and protein phosphatase and others, that when acting in a synergic and antagonist way results in the synthesis of inflammatory cytokines such as IFN-g, TNF-a, IL-1b, IL-6 and IL-8 (22,23). The results obtained were very interesting in the sense that in the basal form we did not identify the production of the cytokines TNF-a and IL-1b in the blood sample culture (BSC) in NT nor PE women. Nevertheless, we observe more synthesis of IL-6 in NT women in comparison with the values obtained from the PreAll, PreMi and PreSe groups (significantly different in all cases).
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Figure 4. Synthesis of IL-6 by unstimulated and stimulated culture whole blood samples. *p50.05 compared versus respective unstimulated group. Control (n ¼ 50). Preeclampsia all groups (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (n ¼ 17). Severe preeclampsia (n ¼ 39). Media and standard deviation are shown in the bars.
This major production of IL-6 from NT women in isolated way could be an atypical result, considering that the evidences indicate that such cytokine is identified in a higher concentration in plasma of PE women. However, when BSC was stimulated we observed a huge response in the production of IL-6, since the NT women generally duplicate the synthesis of that cytokine, whereas for PE women the value of the synthesis was quintupled in general. The IL-6 is produced by mononuclear phagocytes and T cells and is a powerful cytokine involved in the symptomatology of PE, due to enhances endothelial cell permeability likely by altering the ultrastructural distribution
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Figure 5. Synthesis of IL-10 by unstimulated and stimulated culture whole blood samples. *p50.05 compared versus respective unstimulated group. Control (n ¼ 50). Preeclampsia all groups (preeclampsia mild plus severe; n ¼ 56). Mild preeclampsia (n ¼ 17). Severe preeclampsia (n ¼ 39). Media and standard deviation are shown in the bars.
of tight junctions. IL-6 contributes to the systemic endothelial activation and vascular damage, particularly in the kidney, a pivotal organ in the development of hypertension and proteinuria identified in this group of women (24). This cytokine appears to play a crucial factor in the generation of hypertension observed in these women, due to its capacity to improve the generation of autoantibodies that activate the angiotensin II type I receptor, and leads to activation of endothelin-1, oxidative stress, anti angiogenic factor, and increased sensitivity to angiotensina II (25). In fact, Gadonski have demonstrated in a model of pregnant rats that a chronic infusion of IL-6 increased
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arterial pressure and decreased renal plasma flow and glomerular filtration rate (26). Likewise, stimulated samples from women with PE not only showed a wider synthesis of IL-6 in comparison with NT women, also introduced a greater synthesis of IL-1b statistically different in the group and subgroups of PE women. IL-1b is produced by monocytes and macrophages after the stimulation with bacterial products and by TNF-a. IL-1b performs and enhances a lot of functions of TNF-a and both cytokines can alter the properties of vascular endothelial cells in women with PE through the activation of neutrophils, which contribute to the endothelial damage by means of the liberation of elastase and lactoferrin. Also, both cytokines induce the production of IL-6 in monocytes, macrophages and T cells by autocrine and paracrine ways; cytokine who contributes to the systemic endothelial activation and vascular damage act together with TNF-a and IL-1b (27,28). On the other hand, in the samples without stimuli it was possible to identify a lower synthesis of IL-10 (statistically different) in women with PE in comparison with women with NT, nevertheless when the samples were stimulated; the synthesis of IL-10 did not show differences between the control and the PE groups. We can point out that women with PE show a basal tendency to improve an immune response toward proinflammatory branch, being that the ratio of IL-6/IL-10 was statistically different versus control groups. Likewise, when the samples were stimulated that tendency was reinforced since the ratio of IL-1b/IL-10 and IL-6/IL-10 were higher and showed statistical difference versus control group (data not shown). The apparent loss of equilibrium between pro and anti-inflammatory response observed in PE women seems altered in two ways, due to in a basal condition those women showed a deficient capacity to synthesize IL-10, a potent anti-inflammatory cytokine synthesized mainly by Th2 lymphocytes, monocytes and B lymphocytes, which once it is bound to its receptor inhibits the synthesis of the proinflammatory cytokines TNF-a, IL-1b, lL-6, IL-8, IL-12, by monocytes, macrophages and Th1 lymphocytes. And the second way refers when the samples were stimulated and an increase capacity to produce IL-1b and lL-6 cytokines was identified, associated to a similar synthesis of IL-10 showed by the control group. We do not know the causes that PE maternal peripheral cells produce this specific cytokine imbalance, nevertheless it is clear that by performing the complete blood stimulation, all the soluble and cellular components capable of executing synergic functions and potentiate the immune response are found there. Moreover, although we have no experimental evidence, that higher capacity of immune cells to improve a proinflammatory response, could be as a consequence of a loss of the adequate maternal immune adaptation directed as an etiologic hypothesis of PE. Our data differ from those obtained by Beckmann (29) who by stimulating BSC obtained in NT women and with PE with LPS, identified after 24 h of incubation a higher production of TNF-a from samples with PE. Whereas Luppi (30) by stimulating mononuclear cells of NT women and with PE with LPS, established a lower percentage of IL-1b monocyte production in comparison with NT women using flow cytometer; being this an inverse
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pattern of synthesis of TNF-a and IL-b in non-stimulated cells from both the study groups. However, our findings reinforce the idea of a maternal proinflammatory hyper-response, combined to a lower synthesis capacity of the anti-inflammatory cytokines, which potentiates even more the inflammatory mechanism. The results present herein are in line with a previous report done by us (31), where a higher concentration of IFN-g was identified in peripheral plasma of women with PE in comparison with NT women, associated to a diminished concentration of IL-4, a typical anti-inflammatory cytokine. Nevertheless, we did not identify any differences at pro or anti-inflammatory cytokines between the coriodecidual and peripheral compartment of women with PE nor NT. Also, Vitoratos et al. (32) observed a higher concentration of IL-6 and TNF-a in plasma of women with PE in comparison with NT women at term, as well as similar values from both cytokines at the end of the pregnancy and after 12–14 weeks postpartum, showing that placental tissue seems not like the principal source of proinflammatory cytokines in women with PE. Data present here aid to increase the knowledge about the participation of the maternal compartment of the proinflammatory phenomenon in PE, with the intention to improve the strategies that decrease the maternal and fetal morbi-mortality that is detected in this pathology.
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing the paper.
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