© 1995 American Heart Association, Inc.
Articles |
Circulating Leukocyte Counts, Activation, and Degranulation in Dahl Hypertensive Rats
From the Institute for Biomedical Engineering, University of California, San Diego, La Jolla.
Correspondence to Dr G.W. Schmid-Schönbein, Institute for Biomedical Engineering, University of California, San Diego, La Jolla, CA 92093-0412.
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Abstract |
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Abstract Previous evidence has shown that rats with spontaneous hypertension have on average about twice as many circulating leukocytes in comparison with their normotensive counterparts, the Wistar-Kyoto rats. Since such high levels of leukocytes may increase the risk for vascular complications for hypertensive animals, it is useful to ascertain whether a comparable derangement is present in other forms of hypertension. The present study deals with the properties of the circulating leukocytes in rats exhibiting another form of experimental hypertension; Dahl salt-sensitive (Dahl-S) hypertensive rats were compared with Dahl salt-resistant (Dahl-R) control rats. Measurements were performed to determine the following: circulating hematocrit levels, leukocyte counts, differential counts, number of activated leukocytes (by means of nitro blue tetrazolium [NBT] reduction), leukocyte adhesion in vitro and neutrophil CD-18 expression, alkaline phosphatase activity in individual neutrophils and in the plasma, and myeloperoxidase activity in neutrophils. The experimental cohort consisted of Dahl-S and Dahl-R rats maintained for a 6-week period on a 6% NaCl diet. The results show a highly significant elevation in the number of total leukocytes, neutrophil and monocyte counts, and NBT-positive neutrophils and monocytes in Dahl-S but not Dahl-R rats. There was a significant loss of alkaline phosphatase and myeloperoxidase activity in the neutrophils of the salt-treated Dahl-S rats but not in the neutrophils of the untreated Dahl-S or Dahl-R rats. No significant differences were found in neutrophil adhesion under in vitro test conditions between the two strains maintained on the salt diet. In salt-treated Dahl-S rats, there was a reduced expression of ß2 integrins (CD-18) in neutrophils, although a higher fraction of the circulating neutrophils exhibited above-threshold CD-18 expression. These results indicate an increased number of circulating neutrophils and monocytes in Dahl-S rats that are in an activated state and undergo spontaneous degranulation in the circulation without exhibiting elevated levels of membrane adhesive properties. Such circulating leukocyte kinetics may contribute to a range of vascular complications in the hypertensive rats.
Key Words: salt hypertension • adhesion • nitro blue tetrazolium reduction • alkaline phosphatase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals showing a sustained elevation of blood pressure in the central arteries have a prognosis for accelerated organ injury, such as stroke, heart disease, atherosclerosis, and other conditions.1 2 3 4 Although hypertension is a well-documented risk factor, the underlying cause for such complications in hypertensive animals is uncertain. During the last decade, an increasing body of evidence has accumulated that suggests that circulating leukocytes, as well as leukocytes sequestered in the microcirculation, may contribute to the pathophysiological complications in shock, ischemia, diabetes, and other conditions.5 6 7 8 9 10 11 12 13 14 The deterioration has been related to obstruction of blood vessels, production of oxygen free radicals,15 and release of proteolytic enzymes.16
To explore the potential role of leukocytes in hypertension, it is necessary that basic aspects of their behavior in the circulation be documented, but few data exist in this respect for any form of hypertension. The Okamoto spontaneously hypertensive rats have approximately twice the number of circulating leukocytes compared with control rats and continue to have skewed differential counts throughout their early and adult lifetime compared with normotensive Wistar-Kyoto rats. There is also evidence for a greater activation of circulating leukocytes in spontaneously hypertensive rats (SHR), as is manifested by elevated levels of oxygen free radical formation,17 but it is uncertain whether these leukocyte abnormalities are specific for the hypertensive state in the SHR or merely a feature characteristic of the SHR strain.
Accordingly, we present here the results of our study of leukocyte properties in the circulation of another hypertensive model, the Dahl salt-dependent hypertensive rat.18 Leukocyte counts, activation, and adhesion in vitro and neutrophil adhesion glycoprotein (CD-18) expression and degranulation are documented for Dahl salt-sensitive (Dahl-S) compared with salt-resistant (Dahl-R) rats.
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Materials and Methods |
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Animals
Dahl-R and Dahl-S rats (
13 to 15 weeks old; their body
weights are listed in Table 1
) from Harlan Sprague-Dawley
(Indianapolis, Ind) were maintained on a diet of standard rat chow and
water ad libitum for an initial observation period of 1 week and then
given a salt-supplemented (6% NaCl–enriched, ICN Nutritional
Biochemicals) diet for a period of 6 weeks. This period was selected
because it leads in the Dahl-S strain to significantly higher blood
pressures at a time when no overt signs of vascular complications are
yet detectable. A femoral catheter was inserted under local anesthesia
(4% xylocaine SC, Antra Pharmaceuticals) for the purpose of blood
pressure measurements and blood sample withdrawal. Blood pressure was
recorded continuously via a transducer into a laboratory computer to
monitor systolic, diastolic, and mean blood pressure and heart rate.
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Blood Samples
Blood samples (1 mL with heparin as anticoagulant, 10 U/mL) were
taken from the femoral artery catheter after an initial stabilization
period of 30 to 45 minutes. Samples were drawn before general
anesthesia and euthanasia to circumvent the loss of circulating
leukocytes associated with the administration of the anesthetic
agents.17 Unless stated otherwise, all tests on the blood
cells were carried out without delay (within 2 to 3 minutes) to
avoid spontaneous activation of the leukocytes under in vitro
conditions.
Cell Counts
The following measurements were carried out: hematocrit, total
and differential leukocyte counts (with hemocytometer [Fisher
Scientific] and methyl blue–stained blood smear, respectively), and
nitro blue tetrazolium (NBT) reduction by granulocytes, as a measure
for spontaneous superoxide formation, according to a modified method of
Park et al19 and Barroso-Aranda and
Schmid-Schönbein.20 NBT reduction can be inhibited
by superoxide dismutase.21
Neutrophil Alkaline Phosphatase and Myeloperoxidase Activity
Measurement of alkaline phosphatase activity in rat neutrophils,
a membrane-associated enzyme, is based on a dye reduction reaction
(procedure No. 86-R, Sigma diagnostics) under standardized conditions.
Briefly, a smear of fresh whole blood was air-dried at room temperature
(20°C) for at least 1 hour under a light cover, fixed in citrate for
30 seconds, and rinsed for 45 seconds with distilled water. The
alkaline phosphatase dye mixture was gently poured onto the wet slide
and kept in the dark for 15 minutes (covered with an aluminum foil).
The slides were rinsed again with distilled water and dried at room
temperature. No counterstain was applied. The dye that accumulated in
the cytoplasm of individual neutrophils was recorded by television
microscopy with a x40 objective, a standardized substage optical
setting, and constant light intensity. Average light absorption by
individual granulocytes was determined with a video photometric window
technique (model 410, Instrumentation for Physiology and Medicine, Inc)
from the transmitted (IE) and incident (II)
average light intensity over the surface of a video window with and
without single neutrophils, respectively. From this measurement, the
absorption, A, due to the alkaline phosphatase–mediated neutrophil
staining was computed as follows:
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1.3% when measured
repeatedly on a single cell. The microscope substage condenser was
positioned as close as possible to the cell to minimize light
diffraction and optimize light absorption by the red alkaline
phosphatase reaction products. One hundred neutrophils were measured on
each glass slide. The histochemical procedures and cell preparation
were standardized, and reproducibility was tested by repeated light
absorption measurements on different slides from the same rat.
Reproducibility was found to be within 2% of the average light
absorption among 10 repeated measurements on the same cell. The level of myeloperoxidase activity in individual granulocytes after reaction with p-phenylenediamine, catechol, and H2O2 (according to procedure No. 390, Sigma diagnostics) was measured with the same optical technique as described above. This procedure requires counterstaining of the cells for optimal recognition and classification. Therefore, we measured the light absorption of neutrophils from the two strains after staining with the counterstain but without myeloperoxidase staining. These average values were identical, indicating that the counterstain had no detectable influence on the comparison between the four groups.
Plasma Phosphatase Activity
The plasma alkaline phosphatase activities were measured
quantitatively (in Sigma units per milliliter) with a colorimetric
determination (procedure No. 104, Sigma diagnostics; reissued February
1989) at 420 nm. Plasma was obtained after a 15-minute centrifugation
of fresh blood samples at 1200g at 6°C in polyethylene
sterile tubes.
Leukocyte Adhesion In Vitro
Cell adherence to nylon fibers in whole blood was determined
within 1 hour after phlebotomy according to a modified fiber filtration
method. Aliquots (0.8 mL) of heparinized whole blood at 37°C were
placed with a pipette onto nylon fiber columns (Leukopak, Travenol
Laboratories) and allowed to pass by gravity. The columns contained
40-mg nylon fibers packed into 1-mL tuberculin syringes from the 0- to
0.22-mL mark (13-mm length). Percent leukocyte adherence was computed
as follows: [1-(PMN count after filtration/PMN count before
filtration) x100, where PMN indicates polymorphonuclear leukocyte.
Neutrophil Membrane Adhesion Glycoprotein Expression
The expression of ß2 integrin on the neutrophil
membrane was determined with a fluorescence-activated cell sorter
(FACS) facility using a monoclonal antibody against rat CD-18 (WT.3,
Serotec) and a secondary antibody (goat anti-mouse IgG, Jackson
ImmunoResearch Laboratories Inc) conjugated with fluorescein
isothiocyanate.22 Heparinized whole blood (50 µL) was
incubated with 50 µL CD-18 (1 mg/mL) and 50 µL secondary antibody
at room temperature in the dark for 15 minutes. The erythrocytes were
lysed by adding 2.0 mL of FACS lysing solution (Becton Dickinson) for
10 minutes in the dark at room temperature. The cells were centrifuged
at 300g for 5 minutes and washed in 1 mL of
phosphate-buffered saline containing 0.1% azide and centrifuged again
at 200g for 5 minutes. Thereafter, the supernatant was
removed, and the cells were gently fixed in 0.5% formaldehyde and
analyzed with a tabletop flow cytometer (FACScan). Neutrophils were
determined from the forward-side scatter diagrams. The percent
CD-18–positive cells (with fluorescence intensity above background
without primary antibody, WT.3) and their cell counts were determined,
and the mean intensity of at least 5000 neutrophils was computed from
the fluorescence intensity histograms.
Statistics
The blood samples were processed in pairs of Dahl-R and Dahl-S
rats by the same investigator. The majority of blood samples were
analyzed as blinds to avoid bias in the measurements. Comparisons
between the two animal strains based on mean values (Student's
t test) were carried out by using a MINITAB
statistical software program. Probabilities of P<.05 were
considered statistically significant.
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Results |
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Blood Pressure
After 6 weeks on a 6% NaCl–enriched diet, the average central pressures and diastolic and systolic pressures were elevated in Dahl-S but not Dahl-R rats (Table 1
). The basal heart rates
remained unchanged.
Circulating Cell Counts
After salt treatment, the average hematocrit in the Dahl-R rats
remained unchanged, whereas in the Dahl-S rats it was decreased by
28% (Fig 1). Before salt treatment, the Dahl-S rats
already had a small but significant elevation of circulating leukocyte
count (Fig 1, P=.034). The number of circulating leukocytes
in salt-treated Dahl-S rats is on average 38% higher than in the
untreated Dahl-S rats (Fig 1). A comparable trend was also seen in the
Dahl-R rats, but it did not reach a significant value
(P=.226).
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The number of neutrophils and monocytes in Dahl-S rats was on average
more than twice as high in the salt-treated compared with the untreated
rats. In contrast, the salt diet had no statistically significant
effect on the neutrophil and monocyte counts in the Dahl-R strain. No
significant differences in lymphocyte counts could be detected,
although there was a tendency in salt-treated rats toward higher values
(Fig 2).
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Activation in Form of NBT Reduction
The percentage of circulating NBT-positive neutrophils and
monocytes was similar in the two strains on a normal diet, although
there was a significant increase in the number of neutrophils in the
Dahl-S rats that were activated after the salt diet (Figs 3 and 4, top
panels). Because of the shift in both the
leukocyte and differential counts, the average number of spontaneously
NBT-positive neutrophils or monocytes (in cells per cubic millimeter)
in fresh unseparated blood was higher in Dahl-S rats after the salt
treatment (Figs 3 and 4, bottom panels). Consequently, the Dahl-S
strain after salt supplement had on average about a 400% increase in
NBT-positive neutrophils and about a 350% increase in NBT-positive
monocytes in the circulation. In contrast, no comparable trend toward
higher activation was found after the salt diet in the Dahl-R strain.
Visual inspection of the typical size of NBT crystals in the leukocytes
suggested no noticeable difference among the four groups.
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Spontaneous Neutrophil Degranulation
In preparations stained for alkaline phosphatase, the light
absorption in neutrophils is on average significantly lower in the
Dahl-S strain (Fig 5, top panel) after salt treatment,
whereas in the paired blood samples the plasma alkaline phosphatase
levels were significantly higher (Fig 5, bottom panel). There were no
significant shifts in the Dahl-R strain after the salt supplement. This
observation suggests that circulating neutrophils tend to undergo
spontaneous degranulation after salt treatment in the Dahl-S but not
the Dahl-R strain.
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If one assumes a linear relation between enzyme activity, stain
intensity, and light absorption, we can estimate from the single-cell
absorption data the anticipated increase of plasma enzyme activity in
the salt-treated Dahl-S strain. The reduction of the average neutrophil
alkaline phosphatase activity in salt-treated Dahl-S rats is equivalent
to 32% of the average value in control Dahl-S rats (Fig 5), which in
light of the 112% higher neutrophil count in the salt-treated Dahl-S
rats (Fig 3) amounts to an average loss of enzyme activity per unit
volume of blood in the salt-treated Dahl-S rats equal to 71% of the
activity in control Dahl-S rats. This value is similar to the actual
measured increase of average plasma alkaline phosphatase activity in
the salt-treated Dahl-S rats of 81% compared with the activity in
untreated Dahl-S rats (Fig 5).
The trend for degranulation after salt supplementation was also
observed in fresh neutrophils stained for myeloperoxidase in secondary
granules of Dahl-S rats but not Dahl-R rats (Fig 6). The
reduction of myeloperoxidase activity in the circulating neutrophils
amounts on average to 18% in the Dahl-S rat after salt exposure.
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Leukocyte Adhesion
There were no significant differences in the level of
adhesion to the packed nylon fibers between Dahl rats with or without
the salt diet in either strain of hypertensive rats (Fig 7).
This form of adhesion can be almost completely blocked with WT 3
antibody against CD-18 membrane integrin (data not shown). The
percentage of circulating neutrophils expressing CD-18 tended to be
elevated on average in both strains after salt supplementation, but the
Dahl-S strain had significantly higher values (Fig 8, top).
Since the Dahl-S strain after salt addition has significantly higher
neutrophil counts (Fig 3, bottom), the absolute number of
CD-18–positive neutrophils is significantly elevated in the Dahl-S rat
after salt supplementation (Fig 8, middle panel). The average level of
CD-18 expression on the neutrophil membrane is, however, low in the
hypertensive Dahl-S rat (Fig 8, bottom panel), in line with the lack of
a shift in adhesion under in vitro conditions (Fig 7).
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Discussion |
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One of the clinically important features of the hypertensive syndrome is the susceptibility of such individuals to an array of pathophysiological complications involving the central and small blood vessels. In the majority of studies, emphasis was placed on factors associated with the actual elevation of the central blood pressure, such as the peripheral resistance, the arteriolar smooth muscle properties and their regulation, and the performance of the heart.23 Few of the current hypotheses, however, have provided a link between the high blood pressure per se and the complications that accompany the hypertensive state. The data in the literature provide no substantive indication of the mechanisms that predispose hypertensive strains to organ injury.
The Dahl hypertensive model exhibits a range of central vascular and
microvascular abnormalities associated with an elevated peripheral
resistance and a notable elevation of the arteriolar
tone.24 25 There is less evidence of structural
modification of the microvascular network in Dahl hypertensive
rats26 compared with SHR, which exhibit distinctive
structural modifications in the microvascular
network.27 28 Such animals develop a range of vascular
complications,1 including cataracts29 and
higher mortality,30 the severity of which depends on the
amount of salt in the diet, not unlike control rats.31 32
We observed in the present study that the Dahl-S strain also
develops a severe enlargement of the spleen (Table 2),
which was not encountered in the Dahl-R cohort. This may indicate an
abnormal turnover of circulating cells, as reflected by the reduction
of the hematocrit in the Dahl-S strain on salt supplementation.
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It is instructive to compare leukocyte properties in the Dahl hypertensive rats with those recorded in SHR. Although there are no differences in the hematocrit between the two control strains (ie, Dahl-R and Wistar-Kyoto rats), the Dahl-R strain exhibits an elevated circulating leukocyte count. The observations on leukocytes in the salt-treated Dahl-S strain closely mirror those recorded in the spontaneously hypertensive strain. Both hypertensive strains have elevated numbers of neutrophils, NBT-positive neutrophils, monocytes, and NBT-positive monocytes, and both exhibit spontaneous neutrophil degranulation.17 33
The present results present a conceptual framework for a better understanding of the vascular and organ complications in salt-dependent hypertensive rats stemming from the abnormal leukocyte kinetics in the circulation. High circulating leukocyte levels, especially in the case of the neutrophils and monocytes, represent a potential risk factor for a number of cardiovascular complications, including myocardial infarction and stroke.34 35 High circulating leukocyte counts will contribute to an increase in microvascular hemodynamic resistance,36 37 38 39 especially since the cells may exhibit pseudopod projections. Their interaction with the endothelium may have a direct influence on the modulation of the tone of the arterioles40 and may modify the production of humoral factors that influence vasoconstriction and blood pressure elevation.41 42 43 Recent evidence suggests that leukocytes contribute significantly to stroke,6 12 14 44 45 46 47 48 one of the main complications in hypertension. Monocytes and neutrophils also play a key role in atherosclerosis49 and in microvascular lesion formation.13
The increased tendency for circulating neutrophils to undergo
spontaneous degranulation can have considerable pathophysiological
consequences, since the degranulation phenomenon is associated with the
release of other proteolytic enzymes contained in
neutrophils.50 When such enzymes are disseminated by way
of the free bloodstream, their site of action is broadened and is no
longer limited to an intracellular phagocytic vacuole, as in the case
of ingestion processes. Cathepsin G or elastase, two enzymes of the
neutrophil primary granules, is known to activate prorenin to renin,
which in turn leads eventually to angiotensin II
production.41 Other reactions may be triggered by the
release of proteases after spontaneous degranulation. Since the ß2
integrin (CD-18) is stored in the cell granules,51 the
observed degranulation is consistent with upregulation of the protein
expression among the cell population (Fig 8, middle panel), whereas the
level of membrane CD-18 expression on individual cells is depressed
either because of either internalization or shedding of the
glycoprotein.
In principle, two possible mechanisms lead to elevated leukocyte
counts: enhancement of the output of the hemopoietic pool or a
diminished removal of leukocytes from the circulation. We could not
find evidence from the blood smears that premature leukocytes may be
present in the circulation of either animal group. However, it
should be noted that 18-hydroxycorticosterone secretion in adrenal
venous blood is about two times higher in Dahl-S rats than in Dahl-R
rats52 because of a mutation in the cDNA sequence of
P450(11b) from the Dahl-R adrenal gland and suppression of its
steroidogenic activity under sodium loading.53 Expression
of steroids under salt supplementation in Dahl-S rats may serve to
downregulate various membrane-related functions in leukocytes and
endothelial cells.54 Thus, circulating leukocytes may be
kept from regular attachment to the endothelium by virtue of a
reduction of the membrane adhesion protein expression (Fig 8, bottom),
mediated by way of a corticosteroid mechanism, without abrogating
oxygen radical formation or spontaneous degranulation. This may
represent an early form of cytotoxicity that is expressed by
freely suspended circulating cells. There is also recent evidence to
suggest that neutrophils of the SHR strain are deficient in the
membrane adhesion glycoprotein CD-18 complex.55 In the
SHR, there is evidence for downregulation of P-selectin expression on
endothelial cells56 ; P-selectin is an adhesion molecule
required for rolling of leukocytes on the endothelium.57
L-Arginine treatment, designed to enhance the release of an endothelium-derived relaxing factor (EDRF; eg, nitric oxide), can prevent the development of hypertension in Dahl-S rats.58 Such evidence supports the notion that either an inherent endothelium deficit in EDRF release caused by the salt diet or a breakdown by superoxide, possibly in part derived from circulating and nonadhesive activated leukocytes,59 may lead to a deficiency in EDRF production. Since both Dahl-S and SHR exhibit elevated levels of neutrophils and monocytes and their activation, whereas the effect of L-arginine is quite different in the two strains, the abnormal leukocyte findings may develop as a secondary response to the elevation of arterial blood pressure. This observation is in line with the proposal that the elevation in blood pressure per se is not the sole factor that leads to the complications in hypertension but requires the involvement of a cofactor. The leukocytes may be one such cofactor.
Depression of EDRF release in normotensive animals enhances the attachment of leukocytes to the endothelium.60 The suppression of EDRF is accompanied by hydroperoxide formation in endothelial cells, degranulation of mast cells, and early expression of P-selectin, precipitating the attachment of leukocytes to the endothelium.61 Although these results suggest that EDRF plays an important anti-inflammatory role, the observed suppression of EDRF release in the Dahl hypertensive model appears to be accompanied by inhibition of the inflammatory signal in the form of suppression of leukocyte–endothelial membrane attachment.
Although it has been known for several years that the leukocyte cytoplasmic Na+ content is elevated62 because of lowered Na+,K+-ATPase activity,63 further information regarding properties of leukocytes in human hypertension is still limited. Enhanced levels of superoxide by circulating neutrophils, together with the loss of immune competent cells, has been reported.64 65 Neutrophils in hypertensive patients exhibit decreased activity of cytosolic and mitochondrial superoxide dismutase and enhanced lipid peroxidation.66 A leukocyte count in the upper quartile of a normotensive population has been identified as a risk factor for developing hypertension. Observations along these lines need to be expanded in light of the current observations involving hypertensive animal models.
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Acknowledgments |
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This study was supported by US Public Health Service grant HL-10881.
Received July 28, 1994; accepted October 5, 1994.
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