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(Circulation Research. 1995;76:276-283.)
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Articles

Circulating Leukocyte Counts, Activation, and Degranulation in Dahl Hypertensive Rats

K. Shen, F. A. DeLano, B. W. Zweifach, G. W. Schmid-Schönbein

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.


*    Abstract
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 *Abstract
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 down arrowMaterials and Methods
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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


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Animals
Dahl-R and Dahl-S rats ({approx}13 to 15 weeks old; their body weights are listed in Table 1Down) 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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  		Table 1. Central Hemodynamic Parameters in Dahl Hypertensive Rats After 6- to 10-Week Salt Supplement

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 {approx}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 {approx}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:

The microscope light intensities and analog voltage output from the video window were adjusted to yield a measurement accuracy of {approx}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.


*    Results
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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 1Up). 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 {approx}28% (Fig 1Down). Before salt treatment, the Dahl-S rats already had a small but significant elevation of circulating leukocyte count (Fig 1Down, P=.034). The number of circulating leukocytes in salt-treated Dahl-S rats is on average {approx}38% higher than in the untreated Dahl-S rats (Fig 1Down). 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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  		Figure 1. Bar graphs showing hematocrit and total circulating leukocyte counts in Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after being placed on a 6% NaCl diet. Values are mean±SD (n=15 and 15 for Dahl-R rats and n=21 and 18 for Dahl-S rats without and with salt diet, respectively; *P<.04).

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 2Down).



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  		Figure 2. Bar graphs showing mean±SD values for circulating neutrophil, monocyte, and lymphocyte counts in Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet. The number of rats is the same as listed in the Fig 1Up legend (*P<.003 for neutrophils, *P<.008 for monocytes).

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 3Down and 4Down, 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 3Down and 4Down, 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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  		Figure 3. Bar graphs showing mean±SD values for nitro blue tetrazolium (NBT)-positive neutrophils in Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet. The top panel shows the percentage of NBT-positive neutrophils (counted among 100 cells) in unseparated whole blood; the bottom panel shows the circulating counts per blood volume. The number of rats is the same as listed in the Fig 1Up legend (*P<.015).



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  		Figure 4. Bar graphs showing mean±SD values for nitro blue tetrazolium (NBT)-positive monocytes in Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet. The top panel shows the fraction of NBT-positive monocytes in unseparated whole blood; the bottom panel shows the circulating counts per blood volume. The number of rats is the same as listed in the Fig 1Up legend (*P<.01).

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 5Down, top panel) after salt treatment, whereas in the paired blood samples the plasma alkaline phosphatase levels were significantly higher (Fig 5Down, 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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  		Figure 5. Bar graphs showing light absorption in alkaline phosphatase–stained neutrophils and plasma levels of alkaline phosphatase in the same samples of Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet. The reduced absorption in the Dahl-S neutrophils and elevated plasma levels after the salt diet suggests spontaneous degranulation by these hypertensive animals. Values are mean±SD (top panel, n=9 and 10 for Dahl-R rats and n=10 and 11 for Dahl-S rats without and with salt diet, respectively; bottom panel, n=7 and 6 for Dahl-R rats and n=8 and 7 for Dahl-S rats without and with salt diet, respectively; *P=.000 for alkaline phosphatase levels in the neutrophils; *P=.0012 for plasma levels).

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 5Up), which in light of the 112% higher neutrophil count in the salt-treated Dahl-S rats (Fig 3Up) 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 5Up).

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 6Down). The reduction of myeloperoxidase activity in the circulating neutrophils amounts on average to {approx}18% in the Dahl-S rat after salt exposure.



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  		Figure 6. Bar graph showing light absorption in myeloperoxidase-stained neutrophils of Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet. The reduced absorption in the Dahl-S neutrophils suggests spontaneous degranulation by these hypertensive animals. Values are mean±SD (n=6 and 5 for Dahl-R rats and n=11 and 7 for Dahl-S rats without and with salt diet, respectively; *P<.05).

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 7Down). 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 8Down, top). Since the Dahl-S strain after salt addition has significantly higher neutrophil counts (Fig 3Up, bottom), the absolute number of CD-18–positive neutrophils is significantly elevated in the Dahl-S rat after salt supplementation (Fig 8Down, middle panel). The average level of CD-18 expression on the neutrophil membrane is, however, low in the hypertensive Dahl-S rat (Fig 8Down, bottom panel), in line with the lack of a shift in adhesion under in vitro conditions (Fig 7Down).



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  		Figure 7. Bar graphs showing mean±SD values for in vitro neutrophil (top panel) and monocyte (bottom panel) adhesion of fresh heparinized unseparated cells to nylon fibers in Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet.



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  		Figure 8. Bar graphs showing mean±SD values for neutrophil membrane CD-18 glycoprotein expression in same samples of Dahl salt-resistant (Dahl-R) and salt-sensitive (Dahl-S) rats before and after a 6-week period of 6% NaCl–enhanced diet. Top, Percentage of CD-18–positive neutrophils. Middle, Number of CD-18–positive neutrophils per blood volume (percent CD-18–positive neutrophilsxneutrophil cell count). Bottom, Mean fluorescent density of CD-18–positive neutrophils for each group of rats computed from the individual average fluorescence intensities in each rat. The number of rats is n=5 and 6 for Dahl-R rats and n=7 and 11 for Dahl-S rats without and with salt diet, respectively (*P<.05).


*    Discussion
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 up arrowMaterials and Methods
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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 2Down), 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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  		Table 2. Spleen Weight in the Dahl Hypertensive Rat After a 6-Week Salt Supplement

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 8Up, 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 8Up, 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.


*    Acknowledgments
 
This study was supported by US Public Health Service grant HL-10881.

Received July 28, 1994; accepted October 5, 1994.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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