(Circulation. 1995;91:1129-1134.)
© 1995 American Heart Association, Inc.
Articles |
From the Departments of Internal Medicine and Pharmacology, Center on Aging and Cardiovascular Center, University of Iowa College of Medicine, and Veterans Administration of Medical Center, Iowa City, Iowa.
Correspondence to Donald D. Heistad, MD, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242.
| Abstract |
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Methods and Results Human mononuclear and polymorphonuclear leukocytes were isolated from blood. To identify angiotensins in human leukocytes, we performed immunocytochemistry using both alkaline phosphatase and fluorescence methods. With light microscopy immunocytochemistry with akaline phosphatase, prominent staining of angiotensin II was observed in mononuclear leukocytes. Angiotensin I was also demonstrated in mononuclear leukocytes, but the signal was less pronounced than for angiotensin II. Polymorphonuclear leukocytes showed very little staining for angiotensin II. Fluorescence immunocytochemistry also demonstrated angiotensin II in mononuclear leukocytes. Angiotensins I and II in homogenate of leukocytes were quantified by radioimmunoassay. The concentration of angiotensins I and II in mononuclear leukocytes was 355±216 (mean±SEM) and 2331±106 fmol/mg protein, respectively, and the concentration in polymorphonuclear leukocytes was 36±10 and 336±120 fmol/mg protein.
Conclusions These findings suggest that human mononuclear leukocytes contain large amounts of angiotensin II and lesser amounts of angiotensin I. Human polymorphonuclear leukocytes contain small amounts of angiotensin I and II.
Key Words: leukocytes renin angiotensin radioimmunoassay
| Introduction |
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Activation of the renin-angiotensin system may contribute to the development of vascular lesions in hypertension and atherosclerosis.12 13 14 Monocytes have been observed to infiltrate the wall of blood vessels, and it has been proposed that they contribute to vascular lesions.15 16 Angiotensins have been identified in murine macrophages,17 18 and a preliminary report suggests that angiotensin II may be present in human polymorphonuclear leukocytes.19 The presence of angiotensins in monocytes and/or macrophages might have important implications for the development of vascular lesions.
The goals of the present study were to determine whether human leukocytes contain angiotensin, whether the peptide is present predominantly in polymorphonuclear leukocytes or monocytes, and whether angiotensin I or II is the predominant peptide in human leukocytes. To reach these goals, two different approaches were used. First, angiotensins I and II were identified in human mononuclear and polymorphonuclear leukocytes using two types of immunocytochemistry. Second, angiotensins I and II were measured by radioimmunoassay in homogenate from human mononuclear and polymorphonuclear leukocytes.
| Methods |
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Light Microscopy Immunocytochemistry
Light microscopy
immunocytochemistry was performed by an
alkaline phosphatase method. Cell smears were prepared on glass slides
and dried at room temperature. The slides were fixed in 3%
formaldehyde and 1% glutaraldehyde solution for 30 minutes. After
cells were permeabilized with 0.8% Triton X-100 solution and blocked,
the smears were incubated overnight with a rabbit antiserum to
angiotensin I or II diluted 1:200 at 4°C. A control study was
performed with healthy rabbit serum substituted for primary antibody
and with primary antibody preadsorbed with standard human angiotensin I
or II. The smears were incubated with biotin-conjugated anti-rabbit Ig
G and avidin-conjugated alkaline phosphatase. After they were washed,
the smears were incubated with naphthol-AS-BI-phosphate/fuchsin. The
slides were counterstained with hematoxylin and eosin solution.
Fluorescence Immunocytochemistry
Cell suspensions of both
mononuclear and
polymorphonuclear leukocytes were fixed in 3% buffered formalin
solution for 60 minutes. After cells were permeabilized with 0.8%
Triton X-100 solution and blocked, the fixed cells were incubated
overnight with a rabbit antiserum to angiotensin II diluted 1:200 at
4°C. A control study was performed with healthy rabbit serum
substituted for primary antibody and with primary antibody preadsorbed
with standard human angiotensin I or II. The cells were incubated with
FITC-conjugated goat antibody against rabbit Ig G. The cells were
mounted with aqueous mounting media.
Preparation of Cell Homogenate
Isolated cells from each donor
were suspended to a concentration
of 2.5x10-7 cells/mL in a phosphate-buffered saline
consisting of 131 mmol/L NaCl and 10 mmol/L
KH2PO4/K2HPO4
(pH 7.0). After the addition of protease inhibitors (15 µg/mL
leupeptin, 50 ng/mL pepstatin, and 5 µg/mL APMSF), 10 µmol/L
captopril, and 1 mmol/L EGTA, the cells were disrupted by sonification
with three 15-second bursts and immersed in an ice-water bath using the
microtip probe of a sonicator (Branson sonifier) set at low
power.22 More than 95% of the cells were disrupted by
this technique. Undisrupted cells, nuclei, and large debris were
removed by centrifugation at 1000g for 10 minutes. The
resultant supernatant was centrifuged at 100 000g for 30
minutes, and cytosolic fractions were obtained. Throughout all of these
procedures, extreme care was taken to maintain the sample at
<4°C.
Radioimmunoassay for Angiotensins I and II
The concentrations
of angiotensins I and II were measured
using radioimmunoassay (RIA) as described previously.23
Briefly, the cytosolic fraction from each donor was diluted with RIA
buffer (50 mmol/L Tris, pH 7.5) at three different concentrations, and
anti-angiotensin antibody and 125I-angiotensin were added
to each diluted fraction. After overnight incubation at 4°C,
polyethylene glycol (PEG 8000, 12.5%) was added to separate
antibody-bound angiotensin and centrifuged at 10 000g for
45 minutes. Radioactivity of the pellet was measured using a gamma
counter. Counts of the assay mixtures from three diluted fractions were
fitted to the standard curve, and the average of three points was
determined as a result from each donor. Protein concentration was
measured by the method of Lowry et al.24
Materials
Angiotensins I and II were obtained from Sigma
Chemical Co.
Anti-angiotensin I rabbit serum, anti-angiotensin II rabbit serum, and
healthy rabbit serum were obtained from Peninsula Laboratories Inc.
125I-Angiotensin I and 125I-angiotensin II were
obtained from New England Nuclear.
Statistical Analysis
The data are expressed as
mean±SEM. An ANOVA followed by
Schéffe's test was used for comparison of groups.
P<.05 was considered to be significant.
| Results |
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We also performed light microscopy immunocytochemistry with an
alkaline phosphatase method. As shown in Fig 2A
,
angiotensin II was distributed predominantly in large mononuclear
leukocytes. Immunostaining for angiotensin I was also observed in
mononuclear leukocytes, but the staining appeared to be less than
staining for angiotensin II (Fig 2B
). Control experiments in
which
healthy rabbit serum was substituted for primary antibody showed no
significant immunostaining (Fig 2C
). We performed another
control
experiment in which primary antibody was preadsorbed with standard
angiotensin II. This control experiment also showed no significant
immunostaining for angiotensin II (data not shown).
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Polymorphonuclear
leukocytes showed some staining for angiotensin
II, but the intensity appeared to be low (Fig 3A
)
without positive staining in control experiments (Fig 3B
).
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Angiotensins in Cell Homogenate
We measured concentrations of
angiotensins I and II in cell
homogenates from both human mononuclear and polymorphonuclear
leukocytes. The concentrations of angiotensins I and II in mononuclear
leukocytes were 355±216 (mean±SEM) and 2331±106 fmol/mg
protein,
respectively (P<.05) (Fig 4
). The
concentrations of angiotensins I and II in polymorphonuclear leukocytes
were 36±10 and 336±120 fmol/mg protein, respectively
(P<.05) (Fig 4
).
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| Discussion |
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Presence of Angiotensin II in Monocytes
We used two different
immunocytochemical techniques to
demonstrate angiotensin II in human monocytes. Fluorescence
immunocytochemical findings indicate that angiotensin II may be
present primarily in mononuclear leukocytes. An alkaline
phosphatase method showed strong signals for angiotensin II in
mononuclear leukocytes. With this method, the size and shape of the
nucleus are well defined. Large cells with kidney-shaped nuclei
demonstrated marked staining, which suggests that monocytes contain
angiotensin II. The cytoplasm of each monocyte showed granular
staining, suggesting that some granules may contain angiotensin II.
Immunocytochemistry with electron microscopy will be required to define
the subcellular localization of this peptide.
Cell homogenate from human mononuclear leukocytes contains a considerable amount of angiotensin II. Relative to mononuclear leukocytes, human polymorphonuclear leukocytes do not contain much angiotensin. A preliminary report19 suggested that cell homogenates from human leukocytes have more angiotensin I than angiotensin II. The present findings suggest that angiotensin II is the predominant peptide in human mononuclear and polymorphonuclear leukocytes. Although we used a cocktail of three protease inhibitors and 1 mmol/L EGTA to prevent conversion of angiotensinogen and angiotensin I to angiotensin II during preparation of homogenate, we cannot exclude the possibility that activated proteases from leukocytes cleaved angiotensin I to form angiotensin II.25 Our immunocytochemical findings, however, support strongly the findings with RIA. Thus, our findings suggest that the predominant peptide in human mononuclear leukocytes is angiotensin II. Because the antibody against angiotensin II used in the present study cross-reacts completely with angiotensin III, we cannot exclude the possibility that angiotensin III may be present in human mononuclear leukocytes.
Gomez et al7 demonstrated mRNA of angiotensinogen in rat mononuclear leukocytes by using a Northern blot analysis. They have also shown that angiotensinogen, but not angiotensin I or angiotensin II, is released from rat mononuclear leukocytes in a constitutive manner. Thus, mononuclear leukocytes appear to be capable of delivering angiotensinogen to tissues. In the present study, we have shown that human monocytes contain angiotensin II. It is not known whether angiotensin II in monocytes is converted from angiotensinogen intracellularly or is taken up from blood. Monocytes contain cathepsin G, which can cleave angiotensinogen to produce angiotensin II directly.8 9 10 In addition, monocytes and macrophages have receptors for angiotensin II.26 27 When angiotensin II binds to its receptors on monocytes, endocytosis occurs and angiotensin II is taken up by monocytes.26 Thus, both synthesis and uptake of angiotensin II are possible in monocytes.
Study Implications
Angiotensin II has mitogenic effects on
vascular
muscle28 29 and a chemotactic effect on monocytes and
macrophages.30 31 32 33 The
finding that angiotensin-converting
enzyme inhibitors attenuate atherosclerosis in some
hypercholesterolemic animal models12 13 suggests a
role
for the renin-angiotensin system in atherogenesis. Thus, we speculate
that angiotensin II that is derived from monocytes may play an
important role in atherogenesis.34 35
Activation of the renin-angiotensin system may contribute to the development of vascular lesions in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP).14 Inhibition of the renin-angiotensin system by low concentrations of angiotensin-converting enzyme inhibitors, which does not decrease blood pressure, reduces arterial lesions in salt-loaded SHRSP36 37 and in malignant hypertension in SHRSP.38 Monocytes have been observed to infiltrate the subendothelial space of blood vessels, and they may contribute to endothelial dysfunction during hypertension.15 16 Macrophages in granulomas also contain and release angiotensins.17 18 We speculate that release of angiotensin II from monocytes and/or macrophages may contribute to the arterial lesions during chronic hypertension.
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| Acknowledgments |
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Received June 30, 1994; accepted September 20, 1994.
| References |
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