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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 2  |  Page : 367-372

Evaluation of CD40 ligand in normal pregnancy and preeclampsia


1 Department of Clinical Pathology, Faulty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Obstetrics & Gynecology, Faulty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission12-Sep-2013
Date of Acceptance06-Jan-2014
Date of Web Publication18-Oct-2016

Correspondence Address:
Shereen M Abd-El Azeem Neweer
Department of Clinical Pathology, Faulty of Medicine, Menoufia University, Massjjed El-Houda St.,Sekat El-Omara, Sirs El-Layan, Menoufia Governorate, 33736
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.192423

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  Abstract 

Objective:
This study was carried out to assess the presence of an inflammatory response during preeclampsia by demonstrating the presence of the CD40 ligand in preeclamptic pregnancies compared with normal pregnancies.
Background:
Preeclampsia is a multisystem disorder generally appearing after the 20th week of gestation. Worldwide, the incidence of preeclampsia is seen in 2–10% of pregnancies. WHO estimates the incidence of preeclampsia to be seven times higher in developing countries (2.8% of live births) than in developed countries (0.4%). It is characterized by hypertension, proteinuria, vascular abnormalities, and often intrauterine growth retardation. The only effective treatment is delivery of the fetus and placenta.
Participants and methods:
Fifteen normal, apparently healthy nonpregnant women, 20 normal, apparently healthy pregnant women, and 68 pregnant women suffering from preeclampsia were included in this study. The serum concentration of sCD40 ligand was measured using ELISA. Statistical analysis was performed using Mann–Whitney U and ANOVA tests.
Results:
The serum concentration of soluble CD40L was significantly higher in women with preeclampsia than in normal pregnant women. In normal pregnancy the concentration of sCD40L was significantly lower than that in nonpregnant women. We conclude that the levels of inflammatory mediators are higher in women with preeclampsia than in normal pregnant women. In normal pregnancy the levels of these inflammatory mediators are lower than those seen in nonpregnant women.
Conclusion:
These results suggest that preeclampsia is associated with activation of the CD40–CD40L system. The activation of this system may contribute to the development or maintenance of the proinflammatory and prothrombic state found in preeclampsia.

Keywords: CD40 ligand, hypertension, inflammatory mediators, preeclampsia, pregnancy, proteinuria


How to cite this article:
Abd-Alhalim EF, Khodeer SA, El Damaty WG, Abd-El Azeem Neweer SM. Evaluation of CD40 ligand in normal pregnancy and preeclampsia. Menoufia Med J 2016;29:367-72

How to cite this URL:
Abd-Alhalim EF, Khodeer SA, El Damaty WG, Abd-El Azeem Neweer SM. Evaluation of CD40 ligand in normal pregnancy and preeclampsia. Menoufia Med J [serial online] 2016 [cited 2019 Nov 21];29:367-72. Available from: http://www.mmj.eg.net/text.asp?2016/29/2/367/192423


  Introduction Top


Preeclampsia is a major health concern, as it is a leading cause of fetal and maternal mortality and morbidity worldwide [1]. It is a multisystem disorder generally appearing after the 20th week of gestation [2]. It is characterized by hypertension, proteinuria, vascular abnormalities, and often intrauterine growth retardation [3].

In severe cases, preeclampsia is accompanied by the HELLP syndrome (Hemolysis, Elevated Liver enzymes and Low Platelets). Preeclampsia affects ∼7% of first pregnancies and is a leading cause of maternal death and a major contributor to maternal and perinatal morbidity. The only effective treatment is delivery of the fetus and placenta, often resulting in serious complications of prematurity for the neonate. The resulting preterm births and the associated increased infant morbidity and mortality are especially disheartening consequences of preeclampsia [4].

In fact, 15% of all preterm births are indicated early deliveries for preeclampsia. Preeclampsia also increases the risk for intrauterine growth restriction, resulting in low birth-weight babies at increased risk for long-term disabilities [4].

The underlying mechanisms responsible for the pathogenesis of preeclampsia remain poorly understood [4].

Although the etiology of preeclampsia remains unknown, endothelial cell dysfunction plays an important role in its pathogenesis [5].

Preeclampsia may be mediated by CD40/CD40L signal transduction [6]. In porcine coronary arteries, sCD40L significantly decreased endothelium-dependent vasorelaxation and endothelial nitric oxide synthase mRNA expression and increased O2[7].

Furthermore, increased soluble CD40L and upregulated CD40/CD40L expression were detected in the whole-blood platelets and monocytes of women with preeclampsia [8].

CD40, a transmembrane glycoprotein, and its ligand, CD40L, a member of the TNF superfamily of molecules, are widely distributed in human endothelial cells, smooth muscle cells, lymphocytes, and activated platelets. CD40/CD40L plays an important role in antigen presentation and autoimmune disease. CD40/CD40L signaling induces the expression of adhesion molecules and metalloproteinases. In addition, CD40–CD40L interactions transduce immune and inflammatory signals, promoting the generation of Th1 and Th2 cytokines and stimulating blood cells to produce interleukin 1b (IL-1b) precursors and IL-b-converting enzymes [9].

Increased IL-1 activity prolongs the inflammatory responses, resulting in endothelial cell damage [9].

Furthermore, the relationship between CD40/CD40L expression and oxidative endothelial cell damage in human umbilical vascular endothelial cells is confirmed [6]. High concentrations of sCD40L have been demonstrated in women with preeclampsia [0].

The aim of this work is to assess the presence of an inflamatory response during preeclampsia by demonstrating the presence of CD40 ligand in preeclamptic pregnancies compared with normal pregnancies.


  Participants and Methods Top


This study included 103 women; 15 apparently healthy nonpregnant women (normal nonpregnant women) whose ages ranged from 25 to 33 years, 20 apparently healthy normal pregnant women at gestational age greater than 20 weeks whose ages ranged from 25 to 30 years, and 68 pregnant women suffering from preeclampsia at gestational age greater than 20 weeks whose ages ranged from 25 to 33 years.

All women included in this study were subjected to the following: thorough history taking with stress on the history of headache, blurring of vision, chronic diseases, and past history of preeclampsia; complete clinical examination with stress on general examination including measurement of blood pressure; and laboratory examination including liver function tests for aspartate aminotransferase (AST), alanine amino transferase (ALT), serum albumin, prothrombin time and concentration, kidney function tests for urea, creatinine, and urine protein, CBC, and CD154 antigen (CD40L) using ELISA (Quantikine, Human soluble CD40 Ligand Immunoassay; R&D system, USA). This assay uses the quantitative sandwich enzyme immunoassay technique. A polyclonal antibody specific for CD40 ligand is precoated onto a microplate. Standards and samples are pipetted into the wells and any CD40 ligand present is bound by the immobilized antibody.

After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for CD40 ligand is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of CD40 ligand bound in the initial step.

The color development is stopped and the intensity of the color is measured.

Statistical analysis

Two types of statistical analyses were performed: descriptive statistics such as percentage, mean (X), and SD, and analytic statistics such as the χ2-test, Student's t-test, Mann–Whitney U-test, ANOVA test, and Pearson Correlation test. Receiver operator characteristic (ROC) curve was also used in this study.


  Results Top


Statistical analysis was performed using the Mann–Whitney U and ANOVA tests, which revealed no significant statistical differences between the studied groups with respect to age (P > 0.05). There were highly significant statistical differences between group III and group II with respect to systolic and diastolic blood pressure (P<0.001), with higher levels in group III compared with group II. There were nonsignificant statistical differences between group III and group II with respect to AST and ALT (P > 0.05). Meanwhile, the mean values of serum albumin and prothrombin concentration showed highly significant statistical differences between group III and group II, with lower levels in group III compared with group II (P<0.001). There were highly significant statistical differences between group III and group II with respect to urea and protein in 24 h urine with higher levels in group III compared with group II (P<0.001), and significant statistical differences between group III and group II with respect to creatinine, with higher levels in group III compared with group II (P<0.05). There were significant statistical differences between group III and group II with respect to platelets, with lower levels in group III compared with group II (P<0.05). According to the mean values of soluble CD40L in serum, there were significantly higher levels in group III (1.64 ± 1.51 ng/ml) compared with group II (0.33 ± 0.11 ng/ml) (P<0.001). The mean values of soluble CD40 ligand in serum were also significantly lower in group III (1.64 ± 1.51 ng/ml) compared with group I (2.36 ± 0.78 ng/ml) (P<0.001). The mean values of soluble CD40 ligand in serum were significantly lower in group II (0.33 ± 0.11 ng/ml) compared with group I (2.36 ± 0.78 ng/ml) (P<0.001). The ROC curve for CD40 ligand in serum revealed that the best cutoff level of CD40 ligand in serum was 0.57, with a sensitivity of 100%, specificity of 95.0%, positive predictive value of 98.6%, negative predictive value of 100%, and diagnostic accuracy of 98.9%; the area under the curve was 0.998.


  Discussion Top


The present study showed that there were nonsignificant statistical differences among all studied groups with respect to mean age (P > 0.05). These results agreed with those of Lukanov et al. [1], Molvarec et al. [2], and Moslmizade et al. [3].

The current study showed that there were highly significant statistical differences with respect to systolic and diastolic blood pressure between group III and group II (P<0.001) with higher levels in group III compared with group II. The study also showed that there were highly significant statistical differences with respect to systolic and diastolic blood pressure between group III and group I (P<0.001), with higher levels in group III compared with group I (P<0.001).

These results agreed with those obtained by Levine et al. [4], and were in accordance with the results obtained by Lukanov et al. [1], who reported that there were highly significant statistical differences in the mean value of blood pressure among women with preeclampsia and normal, healthy pregnant women as blood pressure was highly elevated during preeclampsia (P<0.001).

With respect to liver function tests, there were nonsignificant statistical differences between group III and group II regarding ALT and AST (P > 0.05). Meanwhile, the mean values of serum albumin and prothrombin concentration showed highly significant statistical differences between group III and group II, with lower levels in group III compared with group II (P<0.001). In addition, there were nonsignificant statistical differences between group III and group I in terms of AST and ALT (P > 0.05). Meanwhile, the mean values of serum albumin and prothrombin concentration showed highly significant statistical differences between group III and group I, with lower levels in group III compared with group I (P<0.001).

These results agreed with those obtained by Makris et al. [5]. They were also nearly similar to those reported by Szarka et al. [6], who reported that there were nonsignificant statistical differences in the mean values of AST and ALT of liver enzymes, being normal among women with preeclampsia and healthy women with normal pregnancy. These results also agreed with those of Benoit and Rey [7], who found that women with severe preeclampsia had a significantly lower albumin concentration compared with women with mild preeclampsia (P<0.05).

The current study showed highly significant statistical differences between group III and group II regarding urea, creatinine, and protein in 24 h urine (P<0.001), with higher levels in group III compared with group II (P<0.001).

These results were in contrast with those obtained by Makris et al. [5], who reported that there were nonsignificant statistical differences in the mean value of urea and creatinine, being normal among women with preeclampsia and among healthy pregnant women with normal pregnancy especially after 20 weeks of pregnancy.

With regard to protein in 24 h urine, the results of the current study agreed with the results obtained by Laivuori [8], who reported that there were highly significant statistical differences in the mean value of total protein in urine in 24 h urine between women with preeclampsia and healthy pregnant women with normal pregnancy.

The obtained results showed that there were significant statistical differences between group III and group II regarding platelet count (P<0.05), with lower levels in group III (162.79 ± 34.55) compared with group II (180.0 ± 16.22). Our results also showed highly significant statistical differences between group III and group I regarding platelet count, with lower levels in group III (162.79 ± 34.55) compared with group I (211.33 ± 25.59) (P<0.001). The obtained results in this study also showed that there were significant statistical differences between group II and group I regarding platelet count, with lower levels in group II (180.0 ± 16.22) compared with group I (211.33 ± 25.59) (P<0.05). These results agreed with those of Mohapatra et al. [9], who found that the mean value of platelet count was significantly lower in patients with preeclampsia compared with the control group (P<0.01). Similar results were obtained by Lukanov et al. [1].

In the present study the mean values of sCD40L in serum were significantly higher in group III (1.64 ± 1.51 ng/ml) than in group II (0.33 ± 0.11 ng/ml) (P<0.001); however, the mean values of sCD40L were significantly lower in group III (1.64 ± 1.51 ng/ml) than in group I (2.36 ± 0.78 ng/ml) (P<0.001). Further, the mean values of soluble CD40 ligand in serum were significantly lower in group II (0.33 ± 0.11 ng/ml) than in group I (2.36 ± 0.78 ng/ml) (P<0.001).

Mellembakken et al. [0] suggest that enhanced platelet activation during preeclampsia might result in increased release of inflammatory mediators, possibly contributing to inflammation, leukocyte activation, and endothelial dysfunction. These results agreed with those of Oron et al. [0], who reported that the mean levels of sCD40L were significantly different among the three groups (P = 0.026). The highest levels (23 767 ± 15 637 pg/ml) were detected in nonpregnant women and preeclamptic women (21 025 ± 45 386 pg/ml); the lowest level (8292 ± 5926 pg/ml) was found in normal pregnant women. The higher levels of sCD40L in women with preeclampsia compared with those in normotensive pregnant controls may indicate an exaggerated activation of platelets and endothelial cells in the disorder. Laskwowska et al. [1] found increased serum concentrations of sCD40L in maternal and umbilical cord blood in pregnancies complicated by preeclampsia in comparison with normal pregnant women.

Further, a study by Alacacioglu et al. [2] on the plasma level of sCD40L during preeclamptic and normal pregnancies to emphasize inflammatory response in preeclampsia found that the mean sCD40L levels were 1.08 ± 0.43 ng/ml in patients with preeclampsia and 0.76 ± 0.24 ng/ml in healthy pregnant women, which was statistically significant (P = 0.01).

Moreover, the results agreed with those of Darmochwal-Kolarz et al. [3], who studied the concentration of the inflammatory markers CD40L and sCD40L in normal pregnancy and preeclampsia. They observed significantly higher serum concentrations of sCD40L in patients with preeclampsia compared with the level in normal pregnant women in the third trimester. They found significantly lower concentrations of sCD40L in the first, second, and third trimesters of normal pregnancy compared with healthy nonpregnant women. Lower concentrations of sCD40L may suggest decreased CD40–CD40L interactions of CD4+ T lymphocytes with antigen-presenting cells and decreased activation of innate immunity during normal pregnancy. This might be one of the mechanisms protecting the developing fetus against the activation of the maternal immune system.

Also, a study by Lukanov et al. [1] reported that there was a significantly higher expression of CD40 and CD40L on platelets and CD40 on monocytes in women with preeclampsia compared with normotensive pregnant women (P<0.001 for all comparisons). The serum concentration of sCD40L was significantly higher in women with preeclampsia compared with normotensive pregnant women (P = 0.012). Preeclampsia is associated with activation of the CD40–CD40L system. The activation of this system may contribute to the production and maintenance of proinflammatory and prothrombotic responses, increased cytokine production, and endothelial cell dysfunction found in preeclampsia.

A study by Lukanov et al. [8] reported that preeclamptic women showed a significant increase in CD40L and CD40 in monocytes when compared with normotensive pregnant women and nonpregnant women (all P<0.001). There was a significant increase in platelet-monocyte aggregates in preeclamptic women (P<0.001) and normotensive pregnant women (P = 0.003) compared with nonpregnant women. Also, a study by Azzam et al. [4] reported that the platelet surface expression of CD40L was significantly higher in women with mild and severe preeclampsia and HELLP compared with that in normal pregnant women (P = 0.001; P ≤ 0.001; P = 0.003, respectively).

On the basis of the ROC curve for sCD40L to differentiate between group III and group II, the best cutoff level of CD40 ligand in serum was 0.57, with a sensitivity of 100%, specificity of 95.0%, positive predictive value of 98.6%, negative predictive value of 100%, and diagnostic accuracy of 98.9%; the area under the curve was 0.998.


  Conclusion Top


The higher levels of soluble CD40 ligand concentrations in serum in women with preeclampsia compared with those in normotensive pregnant women may indicate an exaggerated activation of platelets that help in increased release of inflammatory mediators and endothelial cells in the disorder.

In conclusion, the present study has shown that preeclampsia is associated with activation of the CD40/CD40L system. These results have implications for understanding the platelet function in preeclampsia. Moreover, specific therapeutic interventions can suppress CD40/CD40L-mediated prothrombotic effects. The increased activation of the CD40/CD40L system is particularly involved in the physiological processes of thrombosis and inflammation, and we propose that the activation of this system might contribute to the development or maintenance of the proinflammatory and prothrombotic state, to increased cytokine production, and to endothelial cell dysfunction found in preeclampsia ([Table 1],[Table 2],[Table 3],[Table 4],[Table 5]).
Table 1: Statistical comparison of the three studied groups regarding age

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Table 2: Statistical comparison between GIII and GII regarding clinical parameters

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Table 3: Statistical comparison between GIII and GII regarding liver functions

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Table 4: Statistical comparison between GIII and GII regarding kidney functions

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Table 5: Statistical comparison between GIII and GII regarding platelets and CD40 ligand in serum

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Conflicts of interest

There are no conflicts of interest.[26]

 
  References Top

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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