(Hypertension. 2000;36:813.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
Polymorphonuclear Integrins, Membrane Fluidity, and Cytosolic Ca2+ Content After Activation in Essential Hypertension
From Istituto di Clinica Medica e Malattie Cardiovascolari, Università degli Studi di Palermo, Italy.
Correspondence to Prof Gregorio Caimi, Via Leonardo da Vinci, 52, 90145 Palermo, Italy.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—The purpose of this research was to obtain further information about the role of polymorphonuclear leukocytes in essential hypertension. These cells could be involved in the pathogenesis of organ injury. Thirty subjects (14 men and 16 women) with essential hypertension were enrolled. In these subjects we determined, at baseline and after in vitro activation with 4-phorbol 12-myristate 13-acetate and N-formyl-methionyl-leucyl-phenylalanine, the polymorphonuclear leukocyte membrane fluidity, obtained by labeling the cells with 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, cytosolic Ca2+ concentration, obtained by marking the cells with Fura 2-AM, and integrin pattern (CD11a, CD11b, CD11c, and CD18), by using the indirect immunofluorescence with a flow cytometer. At baseline there was no difference in membrane fluidity between normal subjects and hypertensives, whereas hypertensives showed an increase in cytosolic Ca2+ content and an increase of the phenotypical expression of CD11a, CD11b, and CD18. In normal subjects and in hypertensives, after activation, no variation was found in membrane fluidity and cytosolic Ca2+ content. In normal subjects, after activation, we observed a significant increase of the expression of all adhesion molecules, whereas in hypertensives we found an increase of the expression of CD11b, CD11c, and CD18 but also a decrease of CD11a. The behavior of the polymorphonuclear leukocyte integrin profile may have several explanations, and in particular, the trend of CD11a after chemotactic activation may be related to its cleavage or to an altered integrin phosphorylation/dephosphorylation balance hypothetically present in this clinical condition.
Key Words: hypertension, essential • neutrophils • integrins • membranes • calcium
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In recent years, the knowledge about hypertensive disease was enriched by much evidence regarding the role of circulating leukocytes. In fact, from observations of an altered leukocyte count in experimental and clinical hypertension,1 2 3 4 5 some authors hypothesized that leukocytes could be activated and that this condition, strengthened by nitrobule tetrazolium (NBT) reduction test, could contribute to explaining organ injury.6 7 8 9 10 11
In our previous works,12 13 we evaluated, in essential hypertension, the leukocyte rheology, expressed as flow properties, polymorphonuclear leukocyte (PMN) membrane fluidity and PMN cytosolic Ca2+ content at baseline and after chemotactic activation.
Subsequently, our aim was to examine, in hypertensives, the PMN adhesion molecule profile. Our interest in this topic, for which there is little information in the literature, is derived from the fact that some experimental data underline how in this clinical condition an abnormality of the leukocyte adhesion is present.9 10 11 14 As we know, adhesion is particularly mediated by integrins; among these, ß2 integrins (CD11a, CD11b, CD11c, CD18) are very important.
Integrins15 16 are usually in a low-affinity state that makes them unable to interact with their corresponding ligands. Activation of integrins results in a conformational variation that modulates not only affinity but also avidity. Perhaps these variations are carried out by cytoskeletal readjustments involving talin. Calcium ions can have a stimulating or inhibiting action, based on the ligand-integrin interaction.
On the other hand, we must remember the role played in essential hypertension by free radicals. They make nitric oxide inactive, and the increase of nitric oxide inactivation or its reduced synthesis influences leukocyte adhesion.17 18 19 20 21
In this study, we evaluated in a group of hypertensives the PMN membrane fluidity, PMN cytosolic Ca2+ content, and PMN integrin pattern (CD11a, CD11b, CD11c, CD18) at baseline and after in vitro chemotactic activation.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Thirty subjects with essential hypertension (14 men and 16 women, mean age 48.2±11.3 years, range 26 to 69 years, 9 smokers and 21 nonsmokers) were enrolled. In this group, the mean systolic blood pressure was 160.3±10.8 mm Hg and the mean diastolic blood pressure was 95.6±7.6 mm Hg.
All the subjects included in this group were examined after a pharmacological washout of 3 weeks. None of the hypertensives was affected by impaired glucose tolerance or non–insulin-dependent diabetes mellitus. The mean fasting blood glucose level was 5.23±0.58 mmol/L, total serum cholesterol was 5.40±0.92 mmol/L, and serum triglycerides were 1.33±0.59 mmol/L.
Venous blood samples were drawn from patients in a fasting state and anticoagulated with EDTA-K3 (1.5 mg/mL). Unfractionated leukocyte suspension was prepared according to the method described by Mikita et al.22 In the final preparation, leukocytes were suspended in Dulbecco’s PBS containing EDTA-K3 (1 mg/mL). Leukocytes were separated into mononuclear and PMN cells,23 with a Ficoll-Hypaque medium of density 1.114 g/mL (Mono-Poly Resolving Medium, Flow Laboratories Ltd). The cells were resuspended in Dulbecco’s buffer containing EDTA-K3 (1 mg/mL).
We adopted this procedure, including the use of EDTA in the leukocyte suspensions, because in our experience and in agreement with other authors,24 it does not induce leukocyte activation. A possible interference of EDTA in the measurement of PMN cytosolic Ca2+ content should have, however, the same extent for PMN cells of normal and hypertensive subjects.
PMN Membrane Fluidity
PMNs were suspended in Dulbecco’s buffer at a concentration of
4x106 cells/mL and labeled with
1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene
(TMA-DPH Molecular Probes), previously dissolved in acetone. The
labeling was effected as follows: 4 minutes of preincubation at 4°C
followed by incubation for 20 minutes at 37°C, with a final probe
concentration of 2 µmol/L. Fluorescence measurement was
effected at 37°C, with the use of a spectrophotofluorimeter (model
LS5, Perkin-Elmer) equipped with polarization accessories. The
excitation wavelength was 360 nm and the emission wavelength was 430
nm. Examining the fluorescence intensity with the polarizers
oriented parallel and perpendicular to the plane of polarization, we
calculated the fluorescence polarization degree, reflecting PMN
membrane lipid fluidity.25 26 27
PMN Cytosolic Ca2+ Content
PMNs were suspended in Dulbecco’s buffer at a concentration of
2x106 cells/mL and marked with the
fluorescent probe Fura 2-AM (Molecular Probes), previously
dissolved in DMSO. The labeling was effected as follows: 4 minutes of
preincubation at 4°C followed by incubation for 30 minutes at 37°C,
with a final probe concentration of 1.0 µmol/L and a final DMSO
concentration of 0.5%. This procedure is sufficient to inhibit Fura
2-AM uptake in endocytic vesicles.28 Fluorescence
measurement was carried out with the Perkin-Elmer LS5
spectrophotofluorimeter. The excitation wavelength was 335 nm for the
Fura 2-Ca2+ complex and 385 nm for the unchelated
Fura 2; the emission wavelength was 505 nm. In accordance with Roe et
al28 and David-Dufilho et al,29 we considered
the ratio between the Fura 2–Ca2+ complex and
the fluorescence intensity of the unchelated Fura 2 (335 nm/385
nm).
PMN Integrin Pattern
For immunofluorescence analysis,
2x106 cells were suspended in PBS and incubated
for 20 minutes at room temperature with monoclonal antibody conjugated
with FITC. The monoclonal antibodies were directed against the
following antigens: CD18, CD11a, CD11b, and CD11c (Becton Dickinson).
Isotype-identical antibodies (IgG1, IgG2a, and IgG2b) FITC/phycoerytrin
(Becton Dickinson) served as controls. Cytofluorometric
analysis was performed with FACScan (Becton Dickinson) by
Cellquest software. Granulocytes, after PBS washing at 1800g at room
temperature for 5 minutes, were analyzed for mean of
fluorescence after forward scatter/side scatter dot-plot gate;
10 000 gated events were acquired for single fluorescence
analysis.
PMN Activation
The PMN integrin pattern was also evaluated, following the
method described above, after activation with chemotactic agents. After
separation, a part of the PMN cells were subdivided into several
fractions, each of which had a concentration of
5x106 cells/mL. Each fraction was treated with 2
activating agents: 4-phorbol 12-myristate 13-acetate (PMA,
Sigma Chemical) and N-formyl-methionyl-leucyl-phenylalanine
(fMLP, Sigma Chemical). The activation was carried out in vitro, in
accordance with the methods described by Yasui et
al30 and Masuda et al,31 modified in
line with the techniques used by us for the evaluation of the PMN
parameters, as follows: the fractions of PMN suspension
were treated, in separate experiments, with 4.5 µmol/L of PMA or
with 10 µmol/L of fMLP and incubated for 5 minutes at 37°C;
additional PMN suspensions, submitted to the same treatment, were
incubated for 15 minutes at 37°C. At the end of incubation, the
activation was stopped by plunging the tubes into melting ice for a few
minutes and, soon after, the PMN suspensions were centrifuged
at 200g for 10 minutes at 20°C and resuspended in 1 mL of
Dulbecco’s buffer containing EDTA-K3 (1 mg/mL).
The PMN parameters were tested in a control group of 24 normal subjects (16 men and 8 women, mean age 34.7±8.0 years, range 25 to 52 years, 6 smokers and 18 nonsmokers). The mean fasting blood glucose level was 5.11±0.39 mmol/L, total serum cholesterol was 4.98±0.77 mmol/L, and serum triglycerides were 1.12±0.54 mmol/L. The mean systolic blood pressure was 121.9±9.5 mm Hg and the mean diastolic blood pressure was 71.8±5.6 mm Hg. The study was approved by the ethics committee, and each subject gave informed consent.
Statistical Analysis
The results are expressed as mean±SD. The mean difference
between control and hypertensive subjects was evaluated according to
the Student’s t test for unpaired data. The null hypothesis
was rejected for a probability value <0.05. The difference between the
means of PMN parameters at baseline and after activation
was investigated after the repeated-measures, 1-way ANOVA.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
At baseline, there was no significant difference in PMN membrane fluidity between normal subjects and hypertensives, whereas hypertensives showed an increase in PMN cytosolic Ca2+ concentration (Table 1). In hypertensives it was evident, in comparison with normal subjects, a significant increase of the phenotypical expression of CD11a, CD11b, and CD18. No difference was observed regarding the expression of CD11c (Table 1).
|
In normal subjects and in hypertensives after PMN activation with PMA (Table 1) and fMLP (Table 2), no variation in PMN membrane fluidity and cytosolic Ca2+ concentration was found. In normal subjects, after activation with both agents, we noted a constant and significant increase of the expression of all adhesion molecules. In hypertensives, after activation, we observed an increase of the expression of CD11b, CD11c, and CD18 but also a significant decrease of the expression of CD11a; this decrease of CD11a was more evident after activation with PMA. The PMN parameters were not different between smoking and nonsmoking essential hypertensives, nor between smoking and nonsmoking control subjects.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In hypertensives, the baseline behavior of PMN membrane fluidity and cytosolic Ca2+ concentration is similar to that previously described.14 PMN membrane fluidity does not differentiate normal subjects from hypertensives, whereas in hypertensives, an increase in PMN cytosolic Ca2+ content is present.
In hypertensives, instead, it is evident that the expression of PMN adhesion molecules CD11a, CD11b, and CD18 is upregulated if compared with normal subjects. It is possible that the upregulation of the integrins is consistent with a picture of degranulation of PMN. Such degranulation was observed previously in the spontaneously hypertensive rats, possibly because of prolonged circulation time in light of a P-selectin deficiency, that is, in hypertension, older PMN may not be properly removed from the circulation because of an adhesion deficiency.1 11 32 We know that the granulocyte activation is accompanied by an increase of the cytosolic Ca2+ content,33 34 35 36 and in our research this finding is constantly present.
Leukocytes may be activated by interaction with platelets, by means of functional cooperation between P-sel and Mac-1 (CD11b/CD18), a ß2-integrin whose activity is especially regulated by tyrosine-kinases; the inhibition of these enzymes causes blocking of platelet-leukocyte interactions.37
In normal subjects and hypertensives, after PMN activation with PMA (not receptor-mediated) and fMLP (receptor-mediated), no significant variation was observed in PMN membrane fluidity and cytosolic Ca2+ content, and these results confirm our previous data.13 14
It is difficult to explain the trend of the PMN integrin pattern during activation in normal subjects and in hypertensives. In our group of normal subjects, an increase of all PMN adhesive molecules is evident, whereas in hypertensives we observed a significant increase of CD11b, CD11c, and CD18 and a significant decrease of CD11a.
The fall in CD11a expression, after chemotactic activation (especially with PMA), might be due to the different mechanisms that regulate the adhesion molecules profile. As we know, CD11a is constitutively expressed on PMN surface, whereas other adhesive molecules (CD11b and CD11c) can be taken out from intracellular storage pools situated in 3 mobilizable organelles (specific granules, gelatinase granules, and secretory vesicles).38 39
We must remember the role played by endocytosis mechanisms in regulating leukocyte adhesiveness. A mechanism of internalization, in fact, has been demonstrated, especially after activation with PMA, for CD18 and CD11b but not for CD11a.40 This finding could suggest the hypothesis that the fall in CD11a, after activation, might be related to its cleavage; alternatively, it could be lost in the medium.
It must be also pointed out that granulocyte activation is accompanied by the integrin phosphorylation39 that affects only the plasma membrane–associated molecules and not the presynthesized intracytoplasmic reserves; an altered integrin phosphorylation/dephosphorylation balance, related to the PMN spontaneous activation, might explain the decrease of the phenotypical expression of CD11a that cannot be taken back into storage pool.
The limits of our results may be due to possible selection bias and to the intrinsic limits of the laboratory methods. Our group of patients with mild essential hypertension might be not representative of the whole hypertensive population. Moreover, data obtained from a cross-sectional in vitro study are difficult to generalize to other patient groups. However, the rather high level of statistical significance observed in our study induces us to attribute to it a specific pathophysiological interest.
Received March 3, 2000; first decision March 22, 2000; accepted May 10, 2000.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Schmid-Schönbein GW, Seiffge D, DeLano FA, Shen K, Zweifach BW. Leukocyte counts and activation in spontaneously hypertensive and normotensive rats. Hypertension. 1991;17:323–330.
2.
Shen K, DeLano FA, Zweifach BW, Schmid-Schönbein
GW. Circulating leukocyte counts, activation, and degranulation in Dahl
hypertensive rats. Circ Res. 1995;76:276–283.
3. Friedman GD, Selby JV, Quesenberry CP. The leukocyte count: a predictor of hypertension. J Clin Epidemiol. 1990;43:907–911.[Medline] [Order article via Infotrieve]
4. Lawrence JD. White blood cell count and hypertension. J Clin Epidemiol. 1991;44:961. Comment/letter.[Medline] [Order article via Infotrieve]
5. Gillum RF, Mussolino ME. White blood cell count and hypertension incidence: the NANHES I epidemiologic follow-up study. J Clin Epidemiol. 1994;47:911–919.[Medline] [Order article via Infotrieve]
6. Anachenko VG, Vakolyuk RM, Kunetsov SV, Malashenkova IK, Streistova TV, Strizhova NV. The NBT test in hypertensive patients. Sov Med. 1986;10:3–5.
7. Anachenko VG, Kuzetsov SV, Vakolyuk RM, Vakolyuk VS, Strizhova NV, Malashenkova IK, Kim IS. Neutrophil and lymphocyte activity in essential hypertension. Sov Med. 1988;7:32–35.
8.
Mügge A, Lopez JAG. Do leukocytes have a role in
hypertension? Hypertension.. 1991;17:331–333.
9.
Arndt H, Smith CW, Granger DN.
Leukocyte-endothelial cell adhesion in spontaneously
hypertensive and normotensive rats. Hypertension. 1993;21:667–673.
10.
Suzuki H, Schmid-Schönbein GW, Suematsu M, DeLano
FA, Forrest MJ, Miyasaka M, Zweifach BW. Impaired
leukocyte-endothelial cell interaction in spontaneously
hypertensive rats. Hypertension. 1994;24:719–727.
11. Suematsu M, Suzuki H, Tamatani T, Iigou Y, DeLano FA, Miyasaka M, Forrest MJ, Kannagi R, Zweifach BW, Ishimura Y, Schmid-Schönbein GW. Impaired of selectin-mediated leukocyte adhesion to venular endothelium in spontaneously hypertensive rats. J Clin Invest. 1995;96:2009–2016.
12. Caimi G. Erythrocyte, platelet and polymorphonuclear leukocyte membrane dynamic properties in essential hypertension. Clin Hemorheol Microcirc. 1997;17:199–208.[Medline] [Order article via Infotrieve]
13. Caimi G, Lo Presti R, Canino B, Montana M, Ferrara L, Oddo G, Ventimiglia G, Cerasola G. Essential hypertension: leukocyte rheology and polymorphonuclear cytosolic Ca2+ content at baseline and after activation. Clin Hemorheol Microcirc. 1998;19:281–289.[Medline] [Order article via Infotrieve]
14.
Mervaala EMA, Müller DN, Park J-K, Schmidt F,
Löhn M, Breu V, Dragun D, Ganten D, Haller H, Luft FC. Monocyte
infiltration and adhesion molecules in a rat model of high human renin
hypertension. Hypertension. 1999;33:389–395.
15. Longhurst CM, Jennings LK. Integrin-mediated signal transduction. Cell Mol Life Sci. 1998;54:514–526.[Medline] [Order article via Infotrieve]
16. Gahmberg CG, Valmu L, Fagerholm S, Kotovuori P, Ihanus E, Tian L, Pessa-Morikawa T. Leukocyte integrins and inflammation. Cell Mol Life Sci. 1998;54:549–555.[Medline] [Order article via Infotrieve]
17. Seifert R, Hilgenstock G, Fassbender M, Distler A. Regulation of the superoxide-forming NADPH oxidase of human neutrophils is not altered in essential hypertension. J Hypertens. 1991;9:147–153.[Medline] [Order article via Infotrieve]
18. Sagar S, Kallo IJ, Kaul N, Ganguly NK, Sharma BK. Oxygen free radicals in essential hypertension. Mol Cell Biochem. 1992;111:103–108.[Medline] [Order article via Infotrieve]
19. Kopprasch S, Graessler J, Seibt R, Naumann H-J, Scheuch K, Henßge, Schwanebeck U. Leukocyte responsiveness to substances that activate the respiratory burst is not altered in borderline and essential hypertension. J Hum Hypertens. 1996;10:69–76.[Medline] [Order article via Infotrieve]
20. Lacy F, O’Connor DT, Schmid-Schönbein GW. Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J Hypertens. 1998;16:291–303.[Medline] [Order article via Infotrieve]
21.
Suzuki H, DeLano FA, Parks DA, Jamshidi N, Granger DN,
Ishii H, Suematsu M, Zweifach BW, Schmid- Schönbein GW. Xanthine
oxidase activity associated with arterial blood pressure in
spontaneously hypertensive rats. Proc Natl Acad Sci U S A. 1998;95:4754–4759.
22. Mikita J, Nash G, Dormandy J. A simple method of preparing white blood cells for filterability testing. Clin Hemorheol. 1986;6:635–639.
23. Lennie SE, Lowe GDO, Barbenel JC, Forbes CD, Foulds WS. Filterability of white blood cell subpopulations, separated by an improved method. Clin Hemorheol. 1987;7:811–816.
24. Nash GB, Jones JG, Mikita J, Christopher B, Dormandy JA. Effects of preparative procedures and of cell activation on flow of white cells through micropore filters. Br J Hematol. 1988;70:171–176.[Medline] [Order article via Infotrieve]
25. Shinitzky M, Barenholtz Y. Fluidity parameters of lipids regions determined by fluorescence polarization. Biochim Biophys Acta. 1978;515:367–394.[Medline] [Order article via Infotrieve]
26. Donner M, Stoltz JF. Comparative study on fluorescent probes distributed in human erythrocyte and platelet. Biorheology. 1985;22:385–397.[Medline] [Order article via Infotrieve]
27. Donner M, Muller S, Stoltz JF. Molecular rheology of white blood cells. Rev Port Hemorreol. 1989;3:235–247.
28. Roe MW, Lemasters JJ, Herman B. Assessment of Fura 2 for measurements of cytosolic free calcium. Cell Calcium. 1990;11:63–73.[Medline] [Order article via Infotrieve]
29. David-Dufilho M, Montenay-Garestier T, Devynck MA. Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2. Cell Calcium. 1988;9:167–179.[Medline] [Order article via Infotrieve]
30. Yasui K, Masuda M, Matsuoka T, Yamazaki M, Komiyama A, Akabane T, Hasui M, Kobayashi Y, Murata K. Abnormal membrane fluidity as a cause of impaired dynamics of chemoattractant receptors on neonatal polymorphonuclear leukocytes: lack of modulation of the receptors by a membrane fluidizer. Pediatr Res. 1988;24:442–446.[Medline] [Order article via Infotrieve]
31. Masuda M, Komiyama Y, Murakami T, Murata K, Hasui M, Hirabayashi Y, Kobayashi Y. Difference in changes of membrane fluidity of polymorphonuclear leukocytes stimulated with phorbol myristate acetate and formyl-methionyl-leucyl-phenilalanine: role of excited oxygen species. J Leukoc Biol. 1990;47:105–110.[Abstract]
32. Suzuki H, Zweifach BW, Forrest MJ, Schmid-Schonbein GW. Modification of leukocyte adhesion in spontaneously hypertensive rats by adrenal corticosteroids. J Leukoc Biol. 1995;57:20–26.[Abstract]
33.
Pozzan T, Lew DP, Wollheim CB, Tsien RY. Is cytosolic
ionized calcium regulating neutrophil activation? Science.. 1983;221:1413–1415.
34. White JR, Naccache PH, Molski TPP, Borgeat P, Sha’afi RL. Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor. Biochem Biophys Res Commun. 1983;113:44–50.[Medline] [Order article via Infotrieve]
35.
Korchak HM, Vienne K, Rutherford LE, Wilkenfeld C,
Finkelstein MC, Weissmann G. Stimulus response coupling in the human
neutrophil. J Biol Chem. 1984;259:4076–4082.
36. Marks PW, Maxfield FR. Local and global changes in cytosolic free calcium in neutrophils during chemotaxis and phagocytosis. Cell Calcium. 1990;11:181–190.[Medline] [Order article via Infotrieve]
37.
Evangelista V, Manarini S, Rotondo S, Martelli N,
Polischuk R, McGregor JL, De Gaetano G, Cerletti C.
Platelet/Polymorphonuclear leukocyte interaction in dynamic
conditions: evidence of adhesion cascade and cross talk between
P-selectin and the ß2 integrin CD11b/CD18. Blood. 1996;88:4183–4194.
38.
Carlos TM, Harlan JM.
Leukocyte-endothelial adhesion molecules.
Blood. 1994;84:2068–2101.
39. Buyon JP, Philips MR, Merrill JT, Slade SG, Leszczynska-Piziak J, Abramson SB. Differential phosphorylation of the ß2 integrin CD11b/CD18 in the plasma and specific granule membranes of neutrophils. J Leukoc Biol. 1997;61:313–321.[Abstract]
40. Chambers JD, Simon SI, Berger EM, Sklar LA, Arfon KE. Endocytosis of ß2 integrins by stimulated human neutrophils analyzed by flow cytometry. J Leukoc Biol. 1993;53:462–469.[Abstract]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||