- Email: [email protected]
Phenotypic and metabolic characteristics of monocytes and granulocytes in preeclampsia
Phenotypic and metabolic characteristics of monocytes and granulocytes in preeclampsia Maria-Teresa Gervasi, MD,a Tinnakorn Chaiworapongsa, MD,a Percy Pacora, MD,a Nihal Naccasha, MD,b Bo Hyun Yoon, MD, PhD,c Eli Maymon, MD,a and Roberto Romero, MDa Bethesda, Md, Detroit, Mich, and Seoul, Korea OBJECTIVE: The maternal syndrome of preeclampsia has recently been attributed to a systemic intravascular inflammatory response and endothelial cell activation and dysfunction. This novel hypothesis has considerable clinical and biological implications. This study was designed to determine whether women with preeclampsia have evidence of intravascular inflammation by examination of the phenotypic and metabolic activity of granulocytes and monocytes. STUDY DESIGN: A cross-sectional study was performed that included patients with preeclampsia (n = 31) and normal pregnancies (n = 58) matched for gestational age at blood draw. Intravascular inflammation was studied with use of flow cytometry. Peripheral venous blood was assayed to determine granulocyte and monocyte phenotype with the use of monoclonal antibodies for selective cluster differentiation (CD) antigens. The panel of antibodies included CD11b, CD14, CD16, CD18, CD49d, CD62L, CD64, CD66b, and HLA-DR. The quantity of basal intracellular reactive oxygen species and oxidative burst was assessed. Results were reported as mean channel brightness or intensity of detected fluorescence. Analysis was conducted with nonparametric statistics. A P value < .01 was considered to be significant. RESULTS: Preeclampsia was associated with a significant increase in mean channel brightness for CD11b on granulocytes and monocytes but lower mean channel brightness for CD62L on granulocytes than those from women with normal pregnancy (P < .01 for each). Basal intracellular reactive oxygen species were increased in monocytes but not in granulocytes. The oxidative burst was higher in both cell types. CONCLUSION: Preeclampsia is associated with phenotypic and metabolic changes in granulocytes and monocytes. (Am J Obstet Gynecol 2001;185: 792-7.)
Key words: Preeclampsia, leukocyte phenotype, maternal intravascular inflammation
The importance of the innate component of the immune response has been increasingly recognized.1 Sacks et al2 proposed that normal human pregnancy is characterized by systemic activation of some of the cellular components of the innate immune system, specifically granulocytes and monocytes. Changes in the humoral components of the nonspecific immune response have also been reported. For example, the concentration of fibrinogen,3 certain clotting factors, and specific compo-
From the Perinatology Research Branch, National Institute of Child Health and Human Development,a the Department of Obstetrics and Gynecology, Wayne State University/Hutzel Hospital,b and the Department of Obstetrics and Gynecology, Seoul National University.c Presented at the Twenty-first Annual Meeting of the Society for MaternalFetal Medicine, Reno, Nev, February 5-10, 2001. Reprint requests: Roberto Romero, MD, Perinatology Research Branch, NICHD, Wayne State University/Hutzel Hospital, Department of Obstetrics and Gynecology, 4707 St Antoine Blvd, Detroit, MI 48201. E-mail: [email protected]. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/6/117311 doi:10.1067/mob.2001.117311
792
nents of the complement system4 are increased in the plasma of healthy pregnant women. Placental products (of particulate or soluble nature) have been implicated in the activation of the innate component and suppression of the specific component of the immune response during normal pregnancy,5 an adaptation thought to be important for viviparity.2 Moreover, an exaggerated activation of the innate immune response has been proposed to be the cause of the maternal syndrome of preeclampsia.6 This hypothesis proposes that placental debris deported into the maternal circulation results in excessive intravascular leukocyte activation which, in turn, leads to endothelial cell dysfunction and the clinical manifestations of preeclampsia.7, 8 Systemic inflammation is associated with phenotypic and metabolic changes in circulating leukocytes.9 Activated granulocytes and monocytes manufacture products from the reduction of oxygen that are known as intracellular reactive oxygen species (iROS) and whose detection has been used as an index of metabolic activity of the cells. There is controversy about whether or not the maternal syndrome of preeclampsia is caused by systemic maternal inflammation and even whether there is
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Table I. Leukocyte surface antigens analyzed and staining of leukocyte subpopulation with specific monoclonal antibodies with the use of whole-blood flow cytometry Percentage of positive-staining cells* Surface marker CD11b CD14 CD15 CD16 CD18 CD49d CD62L CD64 CD66b HLA-DR
Function
Monoclonal antibody subtype†
Integrin αM subunit, binds to ICAM-1 Receptor for lipopolysaccharide and its binding protein Fucosylated carbohydrate structure, recognizes endothelial selectins Low-affinity receptor for aggregated IgG Integrin β2 subunit, mediated firm adhesion of leukocytes to endothelium α4-Integrin, binds to VCAM-1 and fibronectin L-Selectin, mediates tethering and rolling of leukocytes High affinity receptor for IgG, mediates release of IL-1, IL-6, and tumor necrosis factor α Member of the CEA family, mediates cell-cell interaction Class II major histocompatibility complex antigen
Granulocytes
Monocytes
IgG1 IgG2a IgM
60.5 (21.3) 2.7 (1.2) 98.6 (1.3)
51.4 (28.0) 93.2 (4.3) 21.8 (17.1)
IgG1 IgG1
96.3 (2.6) 98.1 (3.4)
13.3 (5.4) 93.4 (7.1)
IgG1 IgG1 IgG1
4.5 (2.0) 78.0 (13.4) 5.9 (2.6)
29.0 (17.8) 53.5 (22.4) 77.8 (21.4)
IgG1 IgG1
70.6 (18.4) 3.2 (1.2)
3.5 (1.8) 68.5 (13.6)
ICAM-1, Intercellular adhesion molecule-1; IgG, immunoglobulin G; IgM, immunoglobulin M; VCAM-1, vascular cell adhesion molecule-1; IL-1, interleukin 1; IL-6, interleukin 6; CEA, carcinoembryonic antigen. *Antibodies supplied by Immunotech, Miami, Fla. †Granulocyte and monocyte subgroups distinguished by side-scatter and LDS-751 fluorescence characteristics. For each subpopulation, the percentage of positive cells is given as the mean and SD for healthy women who were not pregnant (n = 20).
neutrophil activation in preeclampsia.10-13 The purpose of this study was to determine whether preeclampsia is associated with phenotypic and metabolic changes in monocytes and granulocytes. Patients and methods Study design. A cross-sectional study was designed to compare the phenotypic characteristics of peripheral blood granulocytes and monocytes obtained from healthy pregnant women and women with preeclampsia who were matched for gestational age. Patients with normal pregnancies met the following criteria: (1) no medical, obstetric, or surgical complications at the time of the study, (2) gestational age ranging from 20 to 41 weeks, and (3) delivery of a term infant, appropriate for gestational age, without complications. Preeclampsia was defined as hypertension (systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg on at least 2 occasions, 4 hours to 1 week apart) and proteinuria (≥300 mg in a 24-hour urine collection or 1 dipstick measurement ≥2+).14 However, proteinuria, concurrent with an increase in diastolic blood pressure of ≥15 mm Hg only, was not an inclusion criteria.15, 16 Eclampsia was diagnosed if convulsions developed. Venipuncture. Eligible patients were approached at the Detroit Medical Center of Wayne State University in Detroit, Mich. Each patient provided informed written consent, and venipuncture was performed. A sample of 2 mL of peripheral blood was obtained with a syringe, added to an anticoagulant solution (20 µg/mL of the protease in-
hibitor leupeptin), placed on ice, and transported to the laboratory. The blood was processed and analyzed within 60 minutes of phlebotomy. Flow cytometry studies. Evaluation of the granulocyte and monocyte surface markers was performed according to the methods described by McCarthy and Macey.17, 18 When the sample arrived at the laboratory, a vital nucleic acid dye (LDS-751; Molecular Probes, Eugene, Ore) was immediately added to the specimen (final concentration, 0.0001%). The objective of this step was to separate leukocytes from anucleated red blood cells. Monoclonal antibodies against the cluster differentiation (CD) markers and blood were mixed in precooled tubes. Our study included a panel of 12 tubes that contained optimal concentrations of negative isotype control antibodies (immunoglobulin G1 and immunoglobulin G2a), antibodies against CD11b, CD14, CD15, CD16, CD18, CD49d, CD62L, CD64, CD66b, and HLA-DR (Immunotech, Miami, Fla) that had been conjugated to the fluorescent dye fluorescein isothiocyanate. After a 10minute incubation with the reagents, samples were analyzed with a flow cytometer. Flow cytometric analysis was performed on a Coulter XL-MCL flow cytometer (Coulter Corp, Hialeah, Fla) (with an argon-ion 488-nm laser). Fluorescein isothiocyanate was detected at 525 nm, and LDS-751 was detected at 620 nm. Red blood cells, which do not label with LDS751, were excluded from the analysis. Granulocytes and monocytes were gated according to the characteristic staining pattern. For both cell types, the percentage of positive cells for the fluorescent antibody and the intensity of fluo-
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Table II. Demographic characteristics of study population
Age (y) Gestational age at blood draw (wk) Race Black White Hispanic Asian Other Gestational age at delivery (wk)
Healthy pregnant women (n = 58)
Women with preeclampsia (n = 31)
24.5 (17-36) 33 (24-40.4)
23 (14-40) 33.5 (25.4-40.1)
45 (77.6%) 8 (13.8%) 1 (1.7%) 1 (1.7%) 3 (5.2%) 39.1 (37-42)
26 (83.9%) 3 (9.7%) 1 (3.2%) — 1 (3.2%) 33.5 (25.5-40.1)
Statistical significance NS NS NS
P < .001*
Value expressed as median (range) or number (percent). *Statistically significant, P < .01; NS, not statistically significant.
Table III. Clinical characteristics of patients with preeclampsia At blood draw (mean ± SD) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg) Highest value (mean ± SD) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg) Urine protein (+ dipstick) Median Range Aspartate aminotransferase (L) Median Range Platelet count (103 L) Median Range
153 ± 15 89 ± 9 110 ± 10 177 ± 20 107 ± 12 127 ± 14 3 2-4 40 14-305 177 52-308
rescence or mean channel brightness were recorded. The surface markers studied are described in Table I. The presence of iROS within granulocytes and monocytes were assessed by determination of the basal content, the production in response to a stimulant (ie, oxidative burst), and the stimulation index (ratio between oxidative burst and basal value of iROS). This was performed by use of the method described by Himmelfarb et al.19 In brief, 1 mL of peripheral blood, which was drawn with a syringe and into a tube that contained sodium heparin (10 IU/mL), was placed on ice and transported to the laboratory. The cells were incubated for 15 minutes at 37 °C with 2´,7´dichlorofluorescein diacetate, which diffuses across the cell membrane and is trapped within the cell by a deacetylation reaction. When the 2´,7´dichlorofluorescein diacetate is exposed to hydrogen peroxide, it is oxidized to the highly fluorescent 2´,7´dichlorofluorescein. The oxidative burst was studied by addition of 10 µL of N-formyl-methionyl-leucyl-phenylalanine (Sigma Chemical Co, St Louis, Mo), dissolved in ethanol to a tube containing 50 µL of blood and 2´,7´dichlorofluorescein diacetate. The contents of the tube were mixed gently and incubated for 30 minutes at 37°C. After this period of time, 5 µL of LDS-751 (final concentration, 0.0001%) in
methanol were added, mixed, and incubated for 1 minute at room temperature. The samples were then analyzed immediately on the flow cytometer. 2´,7´Dichlorofluorescein was detected at 525 nm, LDS-751 at 620 nm, and phycoerythrin at 575 nm. Fluorescein isothiocyanate mean channel density staining was calibrated before analysis with standard Brite Beads (Beckman Coulter, Miami, Fla). A discriminator was set to exclude red blood cells, which do not label with LDS-751. Granulocytes were gated with use of an LDS-751 versus side-scatter histogram, and the monocytes were gated with use of a CD14-phycoerythrin versus side-scatter histogram. The mean channel brightness was measured, and 10,000 events, excluding red blood cells, were collected for analysis. Statistical analysis. The Kolmogorov-Smirnov and Shapiro-Wilk tests were used to test for normality. MannWhitney U tests were used for analysis. A χ2 test was used for comparison of proportion (SPSS 10.0, SPSS Inc, Chicago, Ill). A P value <.01 was considered to be significant. Biological materials and some results of flow cytometric analysis of patients included in this study have been used for other studies of inflammation in pregnancy complications reported elsewhere.20-22 Results Patient characteristics. This study included 31 patients with preeclampsia and 58 healthy pregnant women who were matched for gestational age (within 2 weeks). Table II displays the clinical characteristics of the 2 groups. There were no differences in maternal age, parity, or ethnic group distribution between the 2 groups. Table III shows the clinical characteristics of the patients with preeclampsia. Eclampsia developed in 2 of the patients with preeclampsia. Flow cytometric analysis. Granulocytes from patients with preeclampsia had a significantly higher median mean channel brightness for CD11b but significantly lower mean channel brightness for CD62L than those from women with normal pregnancies (P < .001 for each). No significant differences between the 2 groups were found in mean channel brightness for CD14, CD15,
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Table IV. Mean channel brightness (median and range) of labeled antibody binding to peripheral blood granulocytes Healthy pregnant women (n = 58)
Women with preeclampsia (n = 31)
Surface marker
Median
Range
Median
Range
Statistical significance
CD11b CD14 CD15 † CD16 CD18 ‡ CD49d CD62L CD64 CD66b HLA-DR
4.13 1.28 87.45 36.90 8.74 0.99 6.36 1.96 4.15 0.41
2.3-12.6 0.7-1.9 2.8-112.5 0.9-65.2 5.6-14.7 0.7-1.3 4.0-9.3 1.2-5.7 2.4-9.2 0.3-0.6
6.63 1.18 85.15 31.40 8.31 0.96 4.37 2.02 4.44 0.42
2.6-12.6 0.7-2.8 63.6-114.9 0.7-68.3 5.9-16.3 0.7-1.3 2.1-10.2 1.0-3.2 2.8-6.7 0.3-0.6
P < .001* NS NS NS NS NS P < .001* NS NS NS
*Statistically significant, P < .01; NS, not statistically significant. †Preeclampsia, n = 22. ‡Preeclampsia, n = 26.
Table V. Mean channel brightness (median and range) of labeled antibody binding to peripheral blood monocytes Healthy pregnant women (n = 58)
Women with preeclampsia (n = 31)
Surface marker
Median
Range
Median
Range
CD11b CD14 CD15 † CD16 CD18 ‡ CD49d CD62L CD64 CD66b HLA-DR
6.05 45.20 2.22 0.83 16.70 3.50 6.15 13.65 0.75 9.19
3.4-13.5 33.8-58.9 1.3-4.1 0.6-1.7 12.3-26.1 2.2-5.3 3.9-10.7 8.8-23.6 0.5-1.2 4.1-23.5
7.70 43.50 2.18 0.96 15.70 2.88 5.73 12.7 0.77 8.83
3.8-15.5 13.3-62.4 1.0-10.2 0.6-2.9 9.5-24.3 1.7-7.2 2.5-10.0 3.3-19.7 0.6-4.6 4.1-23.8
Statistical significance P < .001* NS NS NS NS NS NS NS NS NS
*Statistically significant, P < .01; NS, not statistically significant. †Preeclampsia n = 22. ‡Preeclampsia n = 26.
Table VI. Mean channel brightness (median and range) of intracellular basal DCF intracellular DCF after stimulation with FMLP (oxidative burst) and stimulation index (ratio of iROS at stimulation over basal level) in peripheral blood Healthy pregnant women (n = 57)
Granulocytes Basal Oxidative burst Stimulation index Monocytes Basal Oxidative burst Stimulation index
Women with preeclampsia (n = 30)
Median
Range
Median
Range
Statistical significance
5.34 18.1 3.17
1.5-16.9 3.7-49.3 1.4-7.6
5.92 28.7 4.9
2.2-19.5 6.5-94.8 2.5-9.6
NS P < .001* P < .001*
4.88 5.43 1.21
2.4-9.9 2.5-14.7 0.7-2.6
6.20 9.38 1.46
1.9-13.0 2.0-24.2 0.9-2.6
P < .001* P < .001* P < .001*
DCF, Dichlorofluorescein diascetate; iROS, intracellular reactive oxygen species; FMLP, N-formyl-methionyl-leucyl-phenylalanine. *Statistically significant, P < .01; NS, not statistically significant.
CD16, CD18, CD49, CD64, CD66, or HLA-DR (Table IV). Monocytes of patients with preeclampsia had a significantly higher median mean channel brightness for CD11b (P < .001) but only a trend toward a lower mean channel brightness for CD49 (P = .018) than those of
healthy pregnant women (Table V). In the patients with preeclampsia, basal iROS values were significantly increased in monocytes but not in granulocytes. The response to N-formyl-methionyl-leucylphenylalanine was significantly higher in both
796 Gervasi et al
granulocytes and monocytes of women with preeclampsia than that of healthy pregnant women (Table VI). There was no difference in surface marker expression, baseline oxygen radical, or oxidative burst in response to N-formyl-methionyl-leucyl-phenylalanine among women with preeclampsia subdivided according to gestational age (20 to 30 weeks, 30 to 36 weeks, and >36 weeks). Comment Preeclampsia was associated with up-regulation of CD11b on both granulocytes and monocytes and downregulation of CD62L on granulocytes but not on monocytes. CD62L mediates low-affinity binding of leukocytes to endothelium, which allows rolling, the first step in the process of leukocyte adhesion. CD62L is shed during this process. CD11b mediates firm adhesion of leukocytes to endothelium by binding to intracellular adhesion molecule-1, the second discrete step of leukocyte adhesion. These changes are consistent with an alteration of the leukocyte phenotype in preeclampsia. Further evidence of this is that the values for baseline iROS in monocytes and the oxidative burst in granulocytes and monocytes were elevated in preeclampsia. The baseline iROS indicates that there is an enhanced intracellular metabolic activity in monocytes, whereas the increased oxidative burst suggests that priming of granulocytes and monocytes had occurred. The phenotypic changes observed in preeclampsia differ from those observed in normal pregnancy in which granulocytes overexpressed CD14 and CD64 and downregulated CD16 and HLA-DR and monocytes up-regulated not only CD11b but also CD14, CD18, CD62L, and CD64 and down-regulated HLA-DR. Moreover, they were also different from those we observed in patients with acute infection during pregnancy,20 preterm labor,21 and preterm premature rupture of membranes.22 Collectively, these findings suggest that there are qualitative differences in the leukocyte phenotype of preeclampsia and in that of other complications of pregnancy. The mechanism responsible for the phenotypic alterations of neutrophils and monocytes in preeclampsia is unknown. A role for cytokines such as granulocyte colony-stimulating factor,23 placental hormones,2 and deported trophoblast5 has been proposed. Further studies will be required to determine the precise nature of the factor responsible for the changes observed in preeclampsia. It is noteworthy that there was some overlap in mean channel brightness between normal pregnancy and preeclampsia, even for markers that were found to be statistically significant. The clinical significance of the changes in neutrophil and monocyte phenotype and metabolic activity in preeclampsia remains to be determined. It is unknown whether these changes precede the clinical onset of the disease. We were not able to show a relationship between leukocyte phenotype and metabolic activity and the clini-
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cal severity of the disease (blood pressure, lowest platelet count, highest serum glutamic-oxaloacetic transaminase, or severity of proteinuria). The observation that in vivo systemic inflammation is present in mothers with small for gestational age fetuses,24 preterm labor,21 preterm premature rupture of membranes,22 as well as patients with acute infection20 who do not have the clinical syndrome of preeclampsia, suggests that leukocyte activation is not specific to preeclampsia.25 The authors wish to acknowlege the contributions of Dr Marion Macey from the Royal London Hospital, London, UK, for her advice and suggestions in the design of this study; Dr Joseph Kaplan from Wayne State University; Ms Nancy Fine and Mr Steve Buck of the Children’s Hospital of Michigan Flow Cytometry Unit; Mr Michael Kruger, Biostatistician at C. S. Mott Center, Wayne State University; and the nursing staff of the Detroit Medical Center: Ms Sandy Field, Ms Vicki Ineson, Ms Penny Clinansmith, Ms Mabubah Mahoudieh, Ms Cindy Urbanik, Ms Jean Thayer-Seitz, Ms Audrey Miliken, Ms Leandra Ga-Pinlac, Ms Lorraine Lajeunesse, Ms Karen Scharret, and Ms Kathy Firchau. We also recognize the contributions of Ms Lisa Palmer for expert and timely data entry and verification of accuracy. REFERENCES
1. Janeway CA, Goodnow CC, Medzhitov R. Danger —pathogen on the premises! Immunological tolerance. Curr Biol 1996;6:519-22. 2. Sacks G, Sargent I, Redman C. An innate view of human pregnancy. Immunol Today 1999;20:114-8. 3. Stirling Y, Woolf L, North WR, Seghatchian MJ, Meade TW. Haemostasis in normal pregnancy. Thromb Haemost 1984;52:176-82. 4. Hopkinson NC, Powell RJ. Classical complement activation induced by pregnancy: implications for management of connective tissue diseases. J Clin Pathol 1992;45:66-7. 5. Johansen M, Redman CW, Wilkins T, Sargent IL. Trophoblast deportation in human pregnancy —its relevance for pre-eclampsia. Placenta 1999;20:531-9. 6. Redman CW, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499-506. 7. Redman CW, Sargent IL. Placental debris, oxidative stress and pre-eclampsia. Placenta 2000;21:597-602. 8. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, MacLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200-4. 9. Barclay AN, Brown MH, Law SK, McKnight AJ, Tomlinson MG, van der Merwe PA. The leucocyte antigen facts book. 2nd ed. San Diego: Academic Press; 1997. 10. Haller H, Ziegler EM, Homuth V, Drab M, Eichhorn J, Nagy Z, et al. Endothelial adhesive molecules and leukocyte integrins in preeclamptic patients. Hypertension 1997;29:291-9. 11. Barden A. Graham D, Beilin LJ, Ritchie J, Baker R, Walters BN, et al. Neutrophil CD11b expression and neutrophil activation in preeclampsia. Clin Sci 1997;92:37-44. 12. Sacks GP, Studena K, Sargent IL, Redman CW. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leucocytes akin to those of sepsis. Am J Obstet Gynecol 1998;179:80-6. 13. Crocker IP, Wellings RP, Fletcher J, Baker PN. Neutrophil function in women with pre-eclampsia. Br J Obstet Gynaecol 1999;106:822-8. 14. Sibai BM, Ewell M, Levine RJ, Klebanoff MA, Esterlitz J, Catalano PM, et al. Risk factors associated with preeclampsia in healthy
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nulliparous women. The Calcium for Preeclampsia Prevention (CPEP) Study Group. Am J Obstet Gynecol 1997;177:1003-10. Levine RJ, Ewell MG, Hauth JC, Curet LB, Catalano PM, Morris CD, et al. Should the definition of preeclampsia include a rise in diastolic blood pressure of ≥15 mm Hg to a level <90 mm Hg in association with proteinuria? Am J Obstet Gynecol 2000;183:787-92. Martin JN, Rinehart BK, May WL, Magann EF, Terrone DA, Blake PG. The spectrum of severe preeclampsia: comparative analysis by HELLP (hemolysis, elevated liver enzyme levels, and low platelet count) syndrome classification. Am J Obstet Gynecol 1999;180:1373-84. McCarthy DA, Macey MG. A simple flow cytometric procedure for the determination of surface antigens on unfixed leucocytes in whole blood. J Immunol Methods 1993;163:155-63. McCarthy DA, Macey MG. Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood. Cytometry 1996;23:196-204. Himmelfarb J, Hakim RM, Holbrook DG, Leeber DA, Ault KA. Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry. Cytometry 1992;13:83-9.
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20. Naccasha N, Gervasi MT, Chaiworapongsa T, Berman S, Yoon BH, Maymon E, et al. Is normal pregnancy characterized by a state of leukocyte activation akin to sepsis? [abstract]. Am J Obstet Gynecol 2001;184:S15. 21. Gervasi MT, Chaiworapongsa T, Naccasha N, Bianco K, Yoon BH, Maymon E, et al. Evidence for a systemic maternal inflammatory response in preterm parturition [abstract]. Am J Obstet Gynecol 2001;184:S46. 22. Gervasi MT, Chaiworapongsa T, Naccasha N, Pacora P, Berman S, Maymon E, et al. Evidence of subclinical systemic maternal inflammation in preterm premature rupture of membranes [abstract]. Am J Obstet Gynecol 2001;184:S46. 23. Crocker IP, Baker PN, Fletcher J. Peripheral blood leukocytes antigen expression in pregnancy and preeclampsia. Am J Obstet Gynecol 1999;180:1310-1. 24. Sabatier F, Bretelle F, d’Ercole C, Boubli L, Sampol J, DigantGeorge F. Neutrophil activation in preeclampsia and isolated intrauterine growth restriction. Am J Obstet Gynecol 2000; 183:1558-63. 25. Dekker GA, Sibai BM. Etiology and pathogenesis of preeclampsia: current concepts. Am J Obstet Gynecol 1998;179:1359-75.
Key words: Preeclampsia, leukocyte phenotype, maternal intravascular inflammation
The importance of the innate component of the immune response has been increasingly recognized.1 Sacks et al2 proposed that normal human pregnancy is characterized by systemic activation of some of the cellular components of the innate immune system, specifically granulocytes and monocytes. Changes in the humoral components of the nonspecific immune response have also been reported. For example, the concentration of fibrinogen,3 certain clotting factors, and specific compo-
From the Perinatology Research Branch, National Institute of Child Health and Human Development,a the Department of Obstetrics and Gynecology, Wayne State University/Hutzel Hospital,b and the Department of Obstetrics and Gynecology, Seoul National University.c Presented at the Twenty-first Annual Meeting of the Society for MaternalFetal Medicine, Reno, Nev, February 5-10, 2001. Reprint requests: Roberto Romero, MD, Perinatology Research Branch, NICHD, Wayne State University/Hutzel Hospital, Department of Obstetrics and Gynecology, 4707 St Antoine Blvd, Detroit, MI 48201. E-mail: [email protected]. Copyright © 2001 by Mosby, Inc. 0002-9378/2001 $35.00 + 0 6/6/117311 doi:10.1067/mob.2001.117311
792
nents of the complement system4 are increased in the plasma of healthy pregnant women. Placental products (of particulate or soluble nature) have been implicated in the activation of the innate component and suppression of the specific component of the immune response during normal pregnancy,5 an adaptation thought to be important for viviparity.2 Moreover, an exaggerated activation of the innate immune response has been proposed to be the cause of the maternal syndrome of preeclampsia.6 This hypothesis proposes that placental debris deported into the maternal circulation results in excessive intravascular leukocyte activation which, in turn, leads to endothelial cell dysfunction and the clinical manifestations of preeclampsia.7, 8 Systemic inflammation is associated with phenotypic and metabolic changes in circulating leukocytes.9 Activated granulocytes and monocytes manufacture products from the reduction of oxygen that are known as intracellular reactive oxygen species (iROS) and whose detection has been used as an index of metabolic activity of the cells. There is controversy about whether or not the maternal syndrome of preeclampsia is caused by systemic maternal inflammation and even whether there is
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Table I. Leukocyte surface antigens analyzed and staining of leukocyte subpopulation with specific monoclonal antibodies with the use of whole-blood flow cytometry Percentage of positive-staining cells* Surface marker CD11b CD14 CD15 CD16 CD18 CD49d CD62L CD64 CD66b HLA-DR
Function
Monoclonal antibody subtype†
Integrin αM subunit, binds to ICAM-1 Receptor for lipopolysaccharide and its binding protein Fucosylated carbohydrate structure, recognizes endothelial selectins Low-affinity receptor for aggregated IgG Integrin β2 subunit, mediated firm adhesion of leukocytes to endothelium α4-Integrin, binds to VCAM-1 and fibronectin L-Selectin, mediates tethering and rolling of leukocytes High affinity receptor for IgG, mediates release of IL-1, IL-6, and tumor necrosis factor α Member of the CEA family, mediates cell-cell interaction Class II major histocompatibility complex antigen
Granulocytes
Monocytes
IgG1 IgG2a IgM
60.5 (21.3) 2.7 (1.2) 98.6 (1.3)
51.4 (28.0) 93.2 (4.3) 21.8 (17.1)
IgG1 IgG1
96.3 (2.6) 98.1 (3.4)
13.3 (5.4) 93.4 (7.1)
IgG1 IgG1 IgG1
4.5 (2.0) 78.0 (13.4) 5.9 (2.6)
29.0 (17.8) 53.5 (22.4) 77.8 (21.4)
IgG1 IgG1
70.6 (18.4) 3.2 (1.2)
3.5 (1.8) 68.5 (13.6)
ICAM-1, Intercellular adhesion molecule-1; IgG, immunoglobulin G; IgM, immunoglobulin M; VCAM-1, vascular cell adhesion molecule-1; IL-1, interleukin 1; IL-6, interleukin 6; CEA, carcinoembryonic antigen. *Antibodies supplied by Immunotech, Miami, Fla. †Granulocyte and monocyte subgroups distinguished by side-scatter and LDS-751 fluorescence characteristics. For each subpopulation, the percentage of positive cells is given as the mean and SD for healthy women who were not pregnant (n = 20).
neutrophil activation in preeclampsia.10-13 The purpose of this study was to determine whether preeclampsia is associated with phenotypic and metabolic changes in monocytes and granulocytes. Patients and methods Study design. A cross-sectional study was designed to compare the phenotypic characteristics of peripheral blood granulocytes and monocytes obtained from healthy pregnant women and women with preeclampsia who were matched for gestational age. Patients with normal pregnancies met the following criteria: (1) no medical, obstetric, or surgical complications at the time of the study, (2) gestational age ranging from 20 to 41 weeks, and (3) delivery of a term infant, appropriate for gestational age, without complications. Preeclampsia was defined as hypertension (systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg on at least 2 occasions, 4 hours to 1 week apart) and proteinuria (≥300 mg in a 24-hour urine collection or 1 dipstick measurement ≥2+).14 However, proteinuria, concurrent with an increase in diastolic blood pressure of ≥15 mm Hg only, was not an inclusion criteria.15, 16 Eclampsia was diagnosed if convulsions developed. Venipuncture. Eligible patients were approached at the Detroit Medical Center of Wayne State University in Detroit, Mich. Each patient provided informed written consent, and venipuncture was performed. A sample of 2 mL of peripheral blood was obtained with a syringe, added to an anticoagulant solution (20 µg/mL of the protease in-
hibitor leupeptin), placed on ice, and transported to the laboratory. The blood was processed and analyzed within 60 minutes of phlebotomy. Flow cytometry studies. Evaluation of the granulocyte and monocyte surface markers was performed according to the methods described by McCarthy and Macey.17, 18 When the sample arrived at the laboratory, a vital nucleic acid dye (LDS-751; Molecular Probes, Eugene, Ore) was immediately added to the specimen (final concentration, 0.0001%). The objective of this step was to separate leukocytes from anucleated red blood cells. Monoclonal antibodies against the cluster differentiation (CD) markers and blood were mixed in precooled tubes. Our study included a panel of 12 tubes that contained optimal concentrations of negative isotype control antibodies (immunoglobulin G1 and immunoglobulin G2a), antibodies against CD11b, CD14, CD15, CD16, CD18, CD49d, CD62L, CD64, CD66b, and HLA-DR (Immunotech, Miami, Fla) that had been conjugated to the fluorescent dye fluorescein isothiocyanate. After a 10minute incubation with the reagents, samples were analyzed with a flow cytometer. Flow cytometric analysis was performed on a Coulter XL-MCL flow cytometer (Coulter Corp, Hialeah, Fla) (with an argon-ion 488-nm laser). Fluorescein isothiocyanate was detected at 525 nm, and LDS-751 was detected at 620 nm. Red blood cells, which do not label with LDS751, were excluded from the analysis. Granulocytes and monocytes were gated according to the characteristic staining pattern. For both cell types, the percentage of positive cells for the fluorescent antibody and the intensity of fluo-
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Table II. Demographic characteristics of study population
Age (y) Gestational age at blood draw (wk) Race Black White Hispanic Asian Other Gestational age at delivery (wk)
Healthy pregnant women (n = 58)
Women with preeclampsia (n = 31)
24.5 (17-36) 33 (24-40.4)
23 (14-40) 33.5 (25.4-40.1)
45 (77.6%) 8 (13.8%) 1 (1.7%) 1 (1.7%) 3 (5.2%) 39.1 (37-42)
26 (83.9%) 3 (9.7%) 1 (3.2%) — 1 (3.2%) 33.5 (25.5-40.1)
Statistical significance NS NS NS
P < .001*
Value expressed as median (range) or number (percent). *Statistically significant, P < .01; NS, not statistically significant.
Table III. Clinical characteristics of patients with preeclampsia At blood draw (mean ± SD) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg) Highest value (mean ± SD) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Mean arterial pressure (mm Hg) Urine protein (+ dipstick) Median Range Aspartate aminotransferase (L) Median Range Platelet count (103 L) Median Range
153 ± 15 89 ± 9 110 ± 10 177 ± 20 107 ± 12 127 ± 14 3 2-4 40 14-305 177 52-308
rescence or mean channel brightness were recorded. The surface markers studied are described in Table I. The presence of iROS within granulocytes and monocytes were assessed by determination of the basal content, the production in response to a stimulant (ie, oxidative burst), and the stimulation index (ratio between oxidative burst and basal value of iROS). This was performed by use of the method described by Himmelfarb et al.19 In brief, 1 mL of peripheral blood, which was drawn with a syringe and into a tube that contained sodium heparin (10 IU/mL), was placed on ice and transported to the laboratory. The cells were incubated for 15 minutes at 37 °C with 2´,7´dichlorofluorescein diacetate, which diffuses across the cell membrane and is trapped within the cell by a deacetylation reaction. When the 2´,7´dichlorofluorescein diacetate is exposed to hydrogen peroxide, it is oxidized to the highly fluorescent 2´,7´dichlorofluorescein. The oxidative burst was studied by addition of 10 µL of N-formyl-methionyl-leucyl-phenylalanine (Sigma Chemical Co, St Louis, Mo), dissolved in ethanol to a tube containing 50 µL of blood and 2´,7´dichlorofluorescein diacetate. The contents of the tube were mixed gently and incubated for 30 minutes at 37°C. After this period of time, 5 µL of LDS-751 (final concentration, 0.0001%) in
methanol were added, mixed, and incubated for 1 minute at room temperature. The samples were then analyzed immediately on the flow cytometer. 2´,7´Dichlorofluorescein was detected at 525 nm, LDS-751 at 620 nm, and phycoerythrin at 575 nm. Fluorescein isothiocyanate mean channel density staining was calibrated before analysis with standard Brite Beads (Beckman Coulter, Miami, Fla). A discriminator was set to exclude red blood cells, which do not label with LDS-751. Granulocytes were gated with use of an LDS-751 versus side-scatter histogram, and the monocytes were gated with use of a CD14-phycoerythrin versus side-scatter histogram. The mean channel brightness was measured, and 10,000 events, excluding red blood cells, were collected for analysis. Statistical analysis. The Kolmogorov-Smirnov and Shapiro-Wilk tests were used to test for normality. MannWhitney U tests were used for analysis. A χ2 test was used for comparison of proportion (SPSS 10.0, SPSS Inc, Chicago, Ill). A P value <.01 was considered to be significant. Biological materials and some results of flow cytometric analysis of patients included in this study have been used for other studies of inflammation in pregnancy complications reported elsewhere.20-22 Results Patient characteristics. This study included 31 patients with preeclampsia and 58 healthy pregnant women who were matched for gestational age (within 2 weeks). Table II displays the clinical characteristics of the 2 groups. There were no differences in maternal age, parity, or ethnic group distribution between the 2 groups. Table III shows the clinical characteristics of the patients with preeclampsia. Eclampsia developed in 2 of the patients with preeclampsia. Flow cytometric analysis. Granulocytes from patients with preeclampsia had a significantly higher median mean channel brightness for CD11b but significantly lower mean channel brightness for CD62L than those from women with normal pregnancies (P < .001 for each). No significant differences between the 2 groups were found in mean channel brightness for CD14, CD15,
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Table IV. Mean channel brightness (median and range) of labeled antibody binding to peripheral blood granulocytes Healthy pregnant women (n = 58)
Women with preeclampsia (n = 31)
Surface marker
Median
Range
Median
Range
Statistical significance
CD11b CD14 CD15 † CD16 CD18 ‡ CD49d CD62L CD64 CD66b HLA-DR
4.13 1.28 87.45 36.90 8.74 0.99 6.36 1.96 4.15 0.41
2.3-12.6 0.7-1.9 2.8-112.5 0.9-65.2 5.6-14.7 0.7-1.3 4.0-9.3 1.2-5.7 2.4-9.2 0.3-0.6
6.63 1.18 85.15 31.40 8.31 0.96 4.37 2.02 4.44 0.42
2.6-12.6 0.7-2.8 63.6-114.9 0.7-68.3 5.9-16.3 0.7-1.3 2.1-10.2 1.0-3.2 2.8-6.7 0.3-0.6
P < .001* NS NS NS NS NS P < .001* NS NS NS
*Statistically significant, P < .01; NS, not statistically significant. †Preeclampsia, n = 22. ‡Preeclampsia, n = 26.
Table V. Mean channel brightness (median and range) of labeled antibody binding to peripheral blood monocytes Healthy pregnant women (n = 58)
Women with preeclampsia (n = 31)
Surface marker
Median
Range
Median
Range
CD11b CD14 CD15 † CD16 CD18 ‡ CD49d CD62L CD64 CD66b HLA-DR
6.05 45.20 2.22 0.83 16.70 3.50 6.15 13.65 0.75 9.19
3.4-13.5 33.8-58.9 1.3-4.1 0.6-1.7 12.3-26.1 2.2-5.3 3.9-10.7 8.8-23.6 0.5-1.2 4.1-23.5
7.70 43.50 2.18 0.96 15.70 2.88 5.73 12.7 0.77 8.83
3.8-15.5 13.3-62.4 1.0-10.2 0.6-2.9 9.5-24.3 1.7-7.2 2.5-10.0 3.3-19.7 0.6-4.6 4.1-23.8
Statistical significance P < .001* NS NS NS NS NS NS NS NS NS
*Statistically significant, P < .01; NS, not statistically significant. †Preeclampsia n = 22. ‡Preeclampsia n = 26.
Table VI. Mean channel brightness (median and range) of intracellular basal DCF intracellular DCF after stimulation with FMLP (oxidative burst) and stimulation index (ratio of iROS at stimulation over basal level) in peripheral blood Healthy pregnant women (n = 57)
Granulocytes Basal Oxidative burst Stimulation index Monocytes Basal Oxidative burst Stimulation index
Women with preeclampsia (n = 30)
Median
Range
Median
Range
Statistical significance
5.34 18.1 3.17
1.5-16.9 3.7-49.3 1.4-7.6
5.92 28.7 4.9
2.2-19.5 6.5-94.8 2.5-9.6
NS P < .001* P < .001*
4.88 5.43 1.21
2.4-9.9 2.5-14.7 0.7-2.6
6.20 9.38 1.46
1.9-13.0 2.0-24.2 0.9-2.6
P < .001* P < .001* P < .001*
DCF, Dichlorofluorescein diascetate; iROS, intracellular reactive oxygen species; FMLP, N-formyl-methionyl-leucyl-phenylalanine. *Statistically significant, P < .01; NS, not statistically significant.
CD16, CD18, CD49, CD64, CD66, or HLA-DR (Table IV). Monocytes of patients with preeclampsia had a significantly higher median mean channel brightness for CD11b (P < .001) but only a trend toward a lower mean channel brightness for CD49 (P = .018) than those of
healthy pregnant women (Table V). In the patients with preeclampsia, basal iROS values were significantly increased in monocytes but not in granulocytes. The response to N-formyl-methionyl-leucylphenylalanine was significantly higher in both
796 Gervasi et al
granulocytes and monocytes of women with preeclampsia than that of healthy pregnant women (Table VI). There was no difference in surface marker expression, baseline oxygen radical, or oxidative burst in response to N-formyl-methionyl-leucyl-phenylalanine among women with preeclampsia subdivided according to gestational age (20 to 30 weeks, 30 to 36 weeks, and >36 weeks). Comment Preeclampsia was associated with up-regulation of CD11b on both granulocytes and monocytes and downregulation of CD62L on granulocytes but not on monocytes. CD62L mediates low-affinity binding of leukocytes to endothelium, which allows rolling, the first step in the process of leukocyte adhesion. CD62L is shed during this process. CD11b mediates firm adhesion of leukocytes to endothelium by binding to intracellular adhesion molecule-1, the second discrete step of leukocyte adhesion. These changes are consistent with an alteration of the leukocyte phenotype in preeclampsia. Further evidence of this is that the values for baseline iROS in monocytes and the oxidative burst in granulocytes and monocytes were elevated in preeclampsia. The baseline iROS indicates that there is an enhanced intracellular metabolic activity in monocytes, whereas the increased oxidative burst suggests that priming of granulocytes and monocytes had occurred. The phenotypic changes observed in preeclampsia differ from those observed in normal pregnancy in which granulocytes overexpressed CD14 and CD64 and downregulated CD16 and HLA-DR and monocytes up-regulated not only CD11b but also CD14, CD18, CD62L, and CD64 and down-regulated HLA-DR. Moreover, they were also different from those we observed in patients with acute infection during pregnancy,20 preterm labor,21 and preterm premature rupture of membranes.22 Collectively, these findings suggest that there are qualitative differences in the leukocyte phenotype of preeclampsia and in that of other complications of pregnancy. The mechanism responsible for the phenotypic alterations of neutrophils and monocytes in preeclampsia is unknown. A role for cytokines such as granulocyte colony-stimulating factor,23 placental hormones,2 and deported trophoblast5 has been proposed. Further studies will be required to determine the precise nature of the factor responsible for the changes observed in preeclampsia. It is noteworthy that there was some overlap in mean channel brightness between normal pregnancy and preeclampsia, even for markers that were found to be statistically significant. The clinical significance of the changes in neutrophil and monocyte phenotype and metabolic activity in preeclampsia remains to be determined. It is unknown whether these changes precede the clinical onset of the disease. We were not able to show a relationship between leukocyte phenotype and metabolic activity and the clini-
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cal severity of the disease (blood pressure, lowest platelet count, highest serum glutamic-oxaloacetic transaminase, or severity of proteinuria). The observation that in vivo systemic inflammation is present in mothers with small for gestational age fetuses,24 preterm labor,21 preterm premature rupture of membranes,22 as well as patients with acute infection20 who do not have the clinical syndrome of preeclampsia, suggests that leukocyte activation is not specific to preeclampsia.25 The authors wish to acknowlege the contributions of Dr Marion Macey from the Royal London Hospital, London, UK, for her advice and suggestions in the design of this study; Dr Joseph Kaplan from Wayne State University; Ms Nancy Fine and Mr Steve Buck of the Children’s Hospital of Michigan Flow Cytometry Unit; Mr Michael Kruger, Biostatistician at C. S. Mott Center, Wayne State University; and the nursing staff of the Detroit Medical Center: Ms Sandy Field, Ms Vicki Ineson, Ms Penny Clinansmith, Ms Mabubah Mahoudieh, Ms Cindy Urbanik, Ms Jean Thayer-Seitz, Ms Audrey Miliken, Ms Leandra Ga-Pinlac, Ms Lorraine Lajeunesse, Ms Karen Scharret, and Ms Kathy Firchau. We also recognize the contributions of Ms Lisa Palmer for expert and timely data entry and verification of accuracy. REFERENCES
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