This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bernard, C. Articles by Tedgui, A. Search for Related Content PubMed PubMed Citation Articles by Bernard, C. Articles by Tedgui, A.

(Hypertension. 1998;31:1350-1356.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Resistance to Endotoxin Shock in Spontaneously Hypertensive Rats

Catherine Bernard; Régine Merval; Bruno Esposito; ; Alain Tedgui

From INSERM U141 and IFR Circulation-Lariboisière, Hôpital Lariboisière, Paris, France.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Septic shock involves systemic vasodilation mediated by proinflammatory cytokines. In essential hypertension, vascular and immune dysfunctions are closely associated. The response of hypertensive animals compared with normotensive controls to endotoxin (lipopolysaccharide; LPS) challenge is not known. Age-matched (12 weeks) normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were exposed to intravenous injection of 10 mg/kg LPS. Survival rate at 24 hours was markedly higher in SHR than in WKY (12 of 15 and 3 of 15, respectively; P<0.01). Survival of LPS-injected SHR was not related to their hypertension because hydralazine-treated SHR with normalized pressure had similar survival rates, and WKY made hypertensive by clipping of one renal artery showed fatality similar to that of normotensive WKY. Continuous arterial pressure and sequential plasma levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF) were measured in LPS-treated SHR and WKY. Both the duration of the delayed hypotensive phase and the systemic release of IL-6 were much lower in SHR than WKY, whereas both acute hypotension and plasma TNF peak were equivalent. We further explored in vitro the inflammatory response and showed that LPS-activated whole blood from SHR produced less TNF and IL-6 than WKY LPS-activated whole blood. Our results indicate that SHR have a greater ability to resist endotoxic shock than WKY. This is not related to their hypertension but is associated with an attenuated inflammatory response to LPS.

Key Words: rats • hypertension, essential • endotoxins • shock • cytokines • nitric oxide

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
In essential hypertension, in animals as well as in humans, chronic vascular and immune dysfunctions are closely associated. The SHR is an animal model of hypertension that has been genetically selected from the Wistar strain and is now widely studied. Hypertension in SHR is established progressively during aging, and numerous vascular alterations have been reported including sympathetic hypertonia, endothelial dysfunction, arterial compliance decrease, medial hypertrophy, and microvascular rarefaction.^{1 2} On the other hand, SHR exhibit immune abnormalities with depressed T lymphocyte functions, decreased delayed-type hypersensitivity, and defective leukocyte–endothelial cell interactions.^{3 4} Moreover, a chronic inflammatory process with the presence of lymphocytes and macrophages develops early in the cardiovascular system of SHR.⁵ All these observations suggest that the cardiovascular and immune systems directly interact in SHR.

Septic shock involves an acute hemodynamic failure characterized by systemic vasodilation that is induced by bacterial products including LPS from Gram-negative bacteria. Host immune factors, namely proinflammatory cytokines such as TNF, mediate LPS actions, and their main target seems to be the cardiovascular system.^{6 7}

The survival response of SHR to endotoxic shock and its determinants is not known, although this acute inflammatory challenge interestingly targets both vascular and immune systems. We therefore examined the in vivo inflammatory responses induced by LPS in both SHR and normotensive WKY. Furthermore, we explored the effects of LPS in whole blood to evaluate the responses to LPS of the immune system in the 2 strains. We show here that SHR are greatly resistant to endotoxic shock and that this is associated with a lower production of the proinflammatory cytokines TNF and IL-6.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Animals
Twelve-week-old male SHR and age-matched male normotensive WKY were used in these studies. These animals were purchased from IFFA-CREDO (Lyon, France) as specific pathogen-free animals and housed under a 12-hour light/dark cycle for 3 to 5 days after delivery, with food and water given ad libitum. Twelve-week-old male Wistar rats were also used in this study.

Ten 12-week-old SHR were given a hypotensive drug, hydralazine, in their drinking water over an 8-day period before LPS injection. Ten 8-week-old WKY were made hypertensive by placing a constricting clip with an internal diameter of 0.2 mm on the right renal artery (2K-1C rats). After 4 weeks, these hypertensive animals were used to study the response to LPS.

Animals were weighed, and their systolic BP was measured using the tail-cuff method (blood pressure recorder 8005, W&W Electronic).

Animals were cared for in accordance with the European Community standards on the care and use of laboratory animals (No. 00577).

Mortality Studies
Escherichia coli endotoxin 026:B6 (Difco Laboratories, Detroit, Mich) was dissolved in sterile, pyrogen-free saline and sonicated before use. The same lot was used for all experiments. LPS (10 mg/kg) was rapidly injected within 1 minute into the dorsal penile vein in animals under light ether anesthesia.

The effect of intravenous LPS injection was evaluated in SHR, WKY, and Wistar rats, as well as in hydralazine-treated SHR and 2K-1C WKY. The behavior and survival of the animals was monitored over 24 hours. The survival rate of these rats was evaluated at 24 hours after LPS injection.

In Vivo LPS Challenge
Levels of plasma TNF, IL-6, and NO2/NO3 as well as blood cell counts were determined up to 6 hours after LPS administration. This period of time was chosen to avoid selective bias from observation of only rats that would survive after 6 hours. Indeed, the earliest death was seen in the group of WKY at 6 hours after LPS injection. Different groups of rats were killed at different time points (1, 3, and 6 hours) after penile intravenous LPS administration, and their plasmas were harvested and frozen at -70°C for cytokine assays.

In a series of experiments, arterial BP was continuously recorded during endotoxin shock until 6 hours after LPS. WKY and SHR were always studied in parallel. The rats were anesthetized with pentobarbital, and sterile catheters were aseptically inserted into the left carotid artery and jugular vein for measurement of BP and for drug administration, respectively. The arterial catheter was connected to a Statham model P23ID pressure transducer (Gould). After a 1-hour equilibration period following the rapid awakening, conscious rats were injected with LPS.

Whole Blood Activation
Blood was collected from naive, ether-anesthetized 12-week-old SHR and WKY by vena cava puncture. Whole blood activation was studied using the method previously described for human blood.⁸ Briefly, heparinized (30 UI/mL) whole blood was diluted 10-fold in Dulbecco's modified Eagle's medium (DMEM) before incubation and exposed for 24 hours to increasing doses of LPS (from E. coli of the same serotype O26:B6 and lot as used in in vivo studies). The supernatant was then harvested by centrifugation and frozen at -70°C for cytokine assays. In parallel, blood cell counts were performed on each blood sample.

Cytokine Assays
TNF Assay
TNF activity was measured by a specific in vitro cell cytotoxicity assay using actinomycin-D–treated murine fibroblast L-M cells (American Type Culture Collection, Rockville, Md).⁹ L-M cells were plated onto 96-well microtiter plates at 7x10⁴ cells (150 µL) per well and incubated for 24 hours at 37°C in 5% CO₂. Medium (50 µL) containing 10 µg/mL actinomycin-D was then added to all wells, and the cells were incubated for 2 hours. Recombinant human TNF- (specific activity, 3x10⁶ U/mg) was diluted into medium standards. Standard or sample (200 µL) were pipetted in duplicate into the first column of wells and then serially diluted across the plate, and cells were incubated for 24 hours. Cytotoxicity was detected by a tetrazolium dye technique. The plates were read at 570 nm on a microtiter plate reader (model 650, Dynatech Laboratories) against n-propyl alcohol blanks. A standard curve relating cell cytotoxicity to doses of recombinant human TNF- was used to quantify TNF activity in the samples. The sensitivity of this bioassay had a range of 0.1 to 0.2 U/mL (300 to 600 pg/mL), and the coefficient of variation was <15%.

IL-6 Assay
IL-6 activity was measured by a specific cell proliferation bioassay using an IL-6–dependent B9 hybridoma cell line.¹⁰ The B9 cells were cultured in RPMI medium supplemented with human recombinant IL-6 (8 U/mL), 20 µmol/L 2-mercaptoethanol, 10% heat-inactivated fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were washed once in the above medium without added IL-6 before use for the IL-6 assay. Recombinant human IL-6 (specific activity, 10⁷ U/mg) was diluted into medium standards. Supernatants or plasma samples were diluted 1:100 with RPMI medium. Fresh medium (100 µL) was added to all wells, with the exception of the first column. IL-6 standard or diluted sample (200 µL) was pipetted into the first column of wells and then serially diluted across the plate. Cells were incubated for 72 hours. Cell proliferation was detected by a tetrazolium dye technique, as described above. A standard curve relating cell proliferation to doses of recombinant human IL-6 was used to quantify IL-6 activity in the samples. The sensitivity of this bioassay had a range of 0.1 to 0.2 U/mL (100 to 200 pg/mL), and the coefficient of variation was <15%.

Nitrite/Nitrate Assay
Total NO2/NO3 concentrations in plasma and whole blood samples, an index of NO production, were measured using the Griess reagent.¹¹ Nitrate was first reduced into nitrite by treating samples (100 µL of 1/8 diluted plasma sample) with nitrate reductase from Aspergillus (0.1 U) and 100 µmol/L NADPH for 45 minutes. NADPH, which can interfere with the Griess reaction, was then oxidized with methosulfate phenazine (1 mmol/L) and potassium ferrocyanide (1 mmol/L). After 30 minutes, 100 µL of Griess reagent was added, and the absorbance was read at 543 nm. Concentrations were determined from a linear standard curve at between 2 and 100 µmol/L sodium nitrite. The lower threshold of nitrite detection in this assay is 1 µmol/L.

Blood Cell Counts
Venous blood samples were processed for WBC, platelets, and hematocrit by routine methods (System 9000, automatic cell counter, Baker Instruments).

Endotoxin Assay
A quantitative chromogenic limulus amoebocyte lysate assay (Biowhittaker, Bioproducts) was used according to the recommendations provided by the supplier.

Materials
LPS from E. coli 026:B6 serotype was from Difco, DMEM and Krebs-Ringer solution were from Gibco (Life Technologies SARL), and phenylephrine was from Sigma Chemical Co.

Statistics
Results are expressed as mean±SEM. The experimental designs allowed us to use a two-way repeated measures ANOVA to provide evidence of differences related to group (WKY versus SHR) and time. The ² test was used to compare LPS-induced lethality in the different groups.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Body Weight and Systolic BP
Body weights and systolic BP at the time of experiments in SHR, WKY, hydralazine-treated SHR, and 2K-1C WKY are given in Table 1. Body weights in SHR, WKY, and hydralazine-treated SHR were not significantly different but were lower in 2K-1C WKY (P<0.05). Systolic BP was significantly increased in SHR and 2K-1C WKY compared with WKY (P<0.01) and was almost normalized in hydralazine-treated SHR.

View this table: [in this window] [in a new window]   Table 1. Weight and Systolic BP in SHR, Hydralazine-Treated SHR, WKY, and 2K-1C Hypertensive WKY

Enhanced Survival to LPS Shock in SHR
Groups of 15 rats from each strain were given 10 mg/kg of LPS from E. coli by direct intravenous injection in the penile vein, and survival was recorded at 24 hours after LPS. The animals of each strain showed signs of sepsis such as apathy, piloerection, diarrhea, polypnea, and conjunctivitis, which were particularly prominent in WKY. SHR did not suffer from focalized neurological defects. Figure 1 shows the survival curves for groups of WKY and SHR after LPS injection. Of the SHR, 80% (12 of 15) were alive at 24 hours compared with only 20% of WKY (3 of 15) (²=10.8, P<0.001). The first deaths were observed at about 6 hours after LPS in WKY, and 50% of the WKY were dead at about 9 hours after LPS, indicating that the mechanisms leading to death operated soon after LPS administration in this endotoxic shock model.

View larger version (14K): [in this window] [in a new window]   Figure 1. Survival rates of SHR and WKY after intravenous injection of LPS (10 mg/kg). Survival for groups of 15 rats is expressed by Kaplan-Meier survival curves. **P<0.01.

Twelve-week-old male Wistar rats received LPS according to the same protocol and were observed until 24 hours after LPS administration. The survival rate of Wistar rats (2 of 10) was not different from that of WKY.

Resistance of SHR Is Not Related to Hypertensive State
In an attempt to evaluate the role of hypertension in the LPS resistance of SHR, we studied the survival rate to LPS shock in SHR treated with hydralazine to normalize the arterial BP and in WKY made hypertensive by clipping of one renal artery. Treatment of SHR with the antihypertensive drug did not modify the resistance state of these rats to LPS (survival rate, 6 of 10; ²=1.2, P>0.05). Similarly, the sensitivity of hypertensive 2K-1C WKY to LPS shock was not significantly different from that of WKY (survival rate, 0 of 10; ²=2.27, P>0.05).

Resistance State of SHR Is Also Observed in 5-Week-Old Prehypertensive Rats
Young male rats from the 2 strains were also studied at 5 weeks of age. Their systolic BP was 115±9 mm Hg in WKY and 128±10 mm Hg in SHR. Their survival rate after LPS administration was not significantly different compared with that of 12-week-old rats of their respective strains, with survival rates being 10 of 10 in 5-week-old SHR (²=1.62, P>0.05) and 3 of 10 in 5-week-old WKY (²=2.27, P>0.05). This result indicates that the sensitivity to LPS shock depends on rat strain and is unrelated to the arterial pressure.

Hemodynamic Profile
As shown in Figure 2, two hypotensive phases were observed. The first hypotensive phase occurred very rapidly after LPS intravenous injection, with a nadir obtained at 1 hour and the arterial pressure decreasing markedly to about 45% of the initial mean pressure levels. No difference was observed between SHR and WKY. This period was followed by a partial recovery until 3 hours after LPS. At that time, a secondary hypotensive phase was observed in WKY, while SHR tended to recover. The mean arterial pressures (expressed as a percentage of the initial values) were found to be significantly higher in SHR than in WKY at 4 hours and 6 hours after LPS (P<0.05).

View larger version (18K): [in this window] [in a new window]   Figure 2. Time course of mean arterial pressure expressed as a percentage of the value before the injection of LPS (T0) in SHR and WKY injected with LPS. n indicates number of animals. *P<0.05.

Effects of LPS on Blood Cells
Basal blood cell counts were measured by using a hemocytometer in heparinized blood samples obtained from WKY and SHR (Table 2). The total leukocyte counts as well as the number of lymphocytes and platelets were found to be significantly higher in SHR than in WKY, whereas the numbers of neutrophils and monocytes were not different.

View this table: [in this window] [in a new window]   Table 2. Blood Cell Counts in WKY and SHR

The time-course analysis of leukocyte counts showed a peak depletion at 1 hour after LPS injection, with no difference between the 2 strains (Figure 3A). However, a progressive recovery up to 6 hours was observed only in SHR (Figure 3A). This was mainly due to the change in neutrophil counts, which showed a rapid recovery at 3 hours followed by significant neutrophilia at 6 hours in SHR while WKY remained profoundly neutropenic (Figure 3B). Mononuclear cell counts, while being greater in SHR than in WKY at the basal time, were depressed early at 1 hour after LPS and remained unchanged thereafter, but they were still significantly different between SHR and WKY at 1 and 3 hours after LPS (Figure 3C).

View larger version (15K): [in this window] [in a new window]   Figure 3. Time course of leukocyte (A), neutrophil (B), and mononuclear cell (C) counts after LPS injection in SHR and WKY. n indicates number of animals. **P<0.01, ***P<0.001.

Platelet analysis showed a progressive depletion appearing earlier in WKY than in SHR (Figure 4A). However, basal platelet counts were higher in SHR than WKY (P<0.001) and remained so throughout the experiment.

View larger version (15K): [in this window] [in a new window]   Figure 4. Time course of platelet count (A) and hematocrit (B) after LPS injection in SHR and WKY. n indicates number of animals. *P<0.05, **P<0.01, ***P<0.001.

An increase in blood hematocrit was seen in the 2 strains after LPS injection but was significantly greater in WKY, suggesting a greater water extravasation in these animals (Figure 4B).

Systemic Proinflammatory Cytokine and NO Release
TNF and IL-6 plasma levels were assayed in 10 rats from each strain injected in parallel with LPS and killed at different time points: 1, 3, and 6 hours after LPS. As shown in Figure 5A, plasma TNF peaks occurred at 1 hour and were not different between SHR (5046±1006 U/mL) and WKY (5469±1038 U/mL). Additional animals were killed at 30 and 90 minutes after LPS injection to ensure that the peak in TNF was indeed achieved by 1 hour. TNF plasma levels at 30 and 90 minutes were lower than those at 1 hour, but they were not significantly different between SHR and WKY. It is noteworthy that biological TNF activity persisted until 3 hours in WKY (185.4±37.2 U/mL), being significantly greater than that in SHR (36.9±9.9 U/mL, P<0.05).

View larger version (14K): [in this window] [in a new window]   Figure 5. Time course of TNF (A), IL-6 (B), and NO2/NO3 (C) plasma release after LPS injection in SHR and WKY. n indicates number of animals. *P<0.05, ***P<0.001.

Of particular interest are the IL-6 plasma kinetics observed in SHR versus WKY (Figure 5B). Basal values were not different between the 2 strains (814±131 U/mL in SHR and 863±143 U/mL in WKY). However, in SHR rats, IL-6 peaked at 3 hours after LPS (196.6±24.3x10³ U/mL) and decreased at 6 hours (70.3±24.4x10³ U/mL), whereas in WKY, IL-6 plasma levels were much higher at 3 hours (557.1±46.3x10³ U/mL, P<0.001) and continued to increase at 6 hours (797.1±142.1x10³ U/mL, P<0.001).

Basal NO2/NO3 plasma levels were not significantly different in SHR and WKY (16.4±2.8 µmol/L in SHR versus 11.8±2.3 µmol/L in WKY). NO2/NO3 plasma levels began to rise at 3 hours after LPS (100.5±10.7 µmol/L in SHR versus 100.8±11.2 µmol/L in WKY) and increased up to 6 hours (859.3±132.4 µmol/L in SHR versus 814.6 ±53.3 µmol/L in WKY), with no significant difference between SHR and WKY (Figure 5C).

LPS Clearance
LPS plasma levels, assayed at 1, 3, and 6 hours after LPS injection, decreased progressively with time in SHR and WKY, respectively: 0.66±0.19 versus 0.66±0.07 IU/mL at 1 hour; 0.45±0.09 versus 0.53±0.10 IU/mL at 3 hours; and 0.11±0.03 versus 0.19±0.07 IU/mL at 6 hours. The LPS plasma clearance was not significantly different in SHR and WKY.

Whole Blood Activation
Heparinized 10-fold diluted whole blood from SHR and WKY was exposed for 24 hours to increasing doses of LPS, and TNF and IL-6 biological activities were then assayed in the supernatants. Whole blood from SHR produced significantly less TNF and IL-6 than that from WKY (Figure 6A and 6B).

View larger version (26K): [in this window] [in a new window]   Figure 6. TNF (A), IL-6 (B), and NO2/NO3 (C) production in supernatants of 10-fold diluted whole blood from SHR and WKY in response to increasing doses of LPS after 24 hours of incubation. n indicates number of experiments. *P<0.05, **P<0.01, ***P<0.001.

Total NO2/NO3 production was amplified in WKY whole blood but was not significantly different from SHR whole blood production (Figure 6C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We show here for the first time that SHR are markedly resistant to endotoxic shock compared with their normotensive controls, WKY. The resistance of SHR to in vivo LPS challenge was not due to their high BP. Indeed, on one hand, SHR previously rendered normotensive by treatment with an arteriolar vasodilator (hydralazine) remained equally resistant to endotoxic shock as untreated SHR. On the other hand, WKY rendered hypertensive by a unilateral clip on the renal artery were highly sensitive to endotoxic shock. Inasmuch as hypertension in SHR is progressively established during aging, beginning at about 6 weeks with a plateau at 10 weeks of age, we were interested in following the survival rate of young rats from the 2 strains after LPS challenge, especially that of prehypertensive young SHR. Interestingly, the same strain-dependent difference in LPS sensitivity was obtained in 5-week-old rats as in 12-week-old rats.

The relative resistance of SHR to endotoxic shock therefore depends most likely on their genetic background, which differs from that of WKY in a polyallelic way^{12 13 14} and seems to affect the immune system in addition to the cardiovascular system.

The early hypotension that follows LPS injection was of similar intensity in SHR and WKY, with the mean arterial pressure being 50% of the respective basal value, which indicates that the fatal evolution of LPS shock in rats did not depend on the early hemodynamic response to LPS but rather on the delayed one. The time course of this secondary hypotensive phase was different between the 2 strains. SHR clearly showed a partial recovery when WKY did not.

Many authors have reported immune dysfunctions in SHR associated with T lymphocyte and humoral defects, which may be genetically determined and progress in parallel with hypertension.^{3 4 15 16} In the present work, some features of the immune response to LPS were found markedly different in SHR compared with WKY.

The time course of blood cell counts during endotoxic shock differed dramatically between the 2 strains. The basal values of leukocyte and neutrophil counts observed in our rats were similar to those reported in previous studies, being 50% greater in SHR than in WKY,¹⁷ whereas monocyte counts were not different. WBC counts showed a similar peak of depletion in both SHR and WKY at 1 hour after LPS administration. It is noteworthy that this early phase was similar in the 2 strains, as were the early hypotension and the early systemic TNF release, outlining the crucial role of secondary events. A prompt recovery in WBC counts was found in SHR, while WKY remained profoundly leukopenic. This differential time course of WBC count was essentially due to neutrophil counts. In agreement with these results, an impairment of selectin-mediated leukocyte adhesion in SHR in both basal and inflammatory conditions has been shown¹⁸ and has been suggested to result in relative leukocytosis in SHR. This defect of leukocyte adhesion to the endothelium could represent a crucial protective factor for SHR in endotoxemia, since neutrophil influx in various tissues is known to be a determinant of organ failure. Neutrophilia followed early neutropenia in SHR, as previously reported in nonlethal endotoxic shock.¹⁹

TNF plasma peaks were similar in the 2 strains, and only the residual plasma levels at 3 hours after LPS were significantly lower in SHR. This finding is in contrast to studies by Sirén et al²⁰ and Hallenbeck et al,²¹ who reported an enhanced release of TNF in both serum and cerebrospinal fluid after intravenous injection of nonlethal doses of LPS in SHR compared with WKY. Being aware of the biological variability described for WKY among different commercial suppliers,^{13 22 23} we repeated the LPS lethality protocol in SHR and WKY purchased from 2 other animal centers, Charles Rivers (using congenic reproduction) and CERJ, and reproduced our results. It is noteworthy that our animals were pathogen-free as confirmed by histological, microbial, and serum tests made in randomized samples from each group. In addition, the lethality protocol was repeated at different periods of the year, and similar results were found.

The lack of direct relationships between plasma TNF levels and LPS-induced mortality in the present work is in agreement with studies by Feuerstein et al¹⁹ in conscious rats and more recently by Basu et al²⁴ in a model of granulocyte-macrophage colony-stimulating factor-deficient mice. Even though TNF has been shown to be directly involved in LPS toxicity, plasma TNF levels may not necessarily reflect the actual tissue production and may represent only a small fraction thereof. The possibility that SHR produce less TNF at the organ level is in fact suggested by our in vitro results in the whole blood model showing that diluted whole blood from SHR produced significantly less TNF than that from WKY. The differences in cytokine production from whole blood were not accounted for by a difference in monocyte counts.

Plasma IL-6 levels in response to LPS were markedly lower in SHR than in WKY in terms of both net production and plasma kinetics. Plasma IL-6 levels in WKY continuously increased up to 6 hours after LPS injection, while plasma IL-6 levels in SHR returned to baseline values at that time. A correlation between IL-6 and mortality rate has been already observed in human septic shock and in murine models of endotoxemia.²⁵ Kelly and Cross²⁶ showed that the relative disappearance of IL-6 after 10 hours in nonlethally injected mice corresponded with their symptomatic recovery, whereas IL-6 continued to circulate up to the time of death in lethally injected mice. The low IL-6 systemic production in SHR was associated with a low IL-6 production by SHR whole blood activated in vitro by increasing doses of LPS. This latter result is in agreement with the findings of Nakamura et al,²⁷ who reported lower IL-6 expression in renal tissue of SHR compared with WKY or Wistar rats.

Elevation of plasma NO2/NO3 occurs especially in the rodent model after LPS exposure and results from the high activity of the inducible NO synthase (iNOS) isoform in rats as well as in mice. In vivo plasma NO2/NO3 levels after LPS injection were not significantly different between SHR and WKY either before or after LPS injection. Furthermore, NO production from whole blood was not different between SHR and WKY. Some authors found different abilities to produce NO in vitro in response to inflammatory stimuli between SHR and WKY, which vary, however, according to the cellular system studied. Splenic macrophages from SHR produce exaggerated NO in response to Con A,²⁸ whereas vascular smooth muscle cells from SHR release less NO in response to IL-1ß.²⁹ It is noteworthy that the locus for iNOS does not cosegregate with BP in SHR, in contrast to Dahl sodium-sensitive hypertensive rats.³⁰ Thus, the expression of the iNOS pathway does not seem to be the determinant of mortality in this endotoxic model, in agreement with previous mouse studies, especially those in iNOS knockout mice.³¹

We assayed in vivo LPS plasma kinetics in both SHR and WKY and found no difference between the 2 strains. This result indicates that differences in in vivo LPS clearance are unlikely to account for the differential responses of the two strains. In fact, the observation of a differential LPS-activated state of whole blood from the 2 strains indicates that either a cellular and/or a soluble circulating factor is involved. Indeed, different anti-inflammatory pathways may be involved in the relative resistance of SHR to LPS shock compared with WKY; these pathways are under current investigation, in particular, the anti-inflammatory cytokine and hormonal pathways. Genetic variations in the stress response have profound effects on the immune system and may be implicated in LPS response variability.³² Stress hormones have been shown to drive a shift in the Th1/Th2 balance toward Th2,^{33 34} and this neuroendocrine regulation of the immune profile may operate in the SHR strain, as already suggested by some authors.^{35 36} In support of this hypothesis, it has been shown in studies on mucosal immune responses that SHR may preferentially develop a Th2 profile as opposed to WKY, which may favor a Th1 profile.^{37 38 39} In addition, numerous studies have reported that cellular immunity, including delayed hypersensitivity, is depressed in SHR.^{4 16} Interestingly, a Th2 environment has been shown to protect mice from endotoxic shock.⁴⁰

In conclusion, LPS-induced inflammatory response is genetically determined in rat strains, with SHR being relatively resistant to endotoxic shock compared with WKY. In this context, the proper immunoinflammatory response of SHR seems to be determinant, whereas the hypertensive state per se is not. Because immune factors are linked to hypertension in humans, the inflammatory response to LPS needs to be explored in hypertensive patients compared with normotensive ones.

	Selected Abbreviations and Acronyms

 

BP = blood pressure IL = interleukin 2K-1C = two-kidney, one clip LPS = lipopolysaccharide NO2/NO3 = nitrite/nitrate NO = nitric oxide SHR = spontaneously hypertensive rats TNF = tumor necrosis factor WBC = white blood cells WKY = Wistar-Kyoto rats

	Acknowledgments

 
This work was supported by grants from CNAMTS/INSERM via 4API12. We thank Michel Lupart from the Servier laboratory, Jean-Baptiste Michel from INSERM U 367, and Michel Wassef from the cytopathology laboratory of Hôpital Lariboisière for technical assistance. We thank Stéphanie Lehoux for editorial assistance.

	Footnotes

 
Reprint requests to Catherine Bernard, INSERM U141, 41 Blvd de La Chapelle, 75475 Paris, Cedex 10, France.

Received August 18, 1997; first decision September 23, 1997; accepted January 30, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Chobanian AV. Corcoran Lecture: adaptive and maladaptive responses of the arterial wall to hypertension. Hypertension. 1990;1989:15:666–674.

2. Folkow B. Physiological aspects of primary hypertension. Physiol Rev. 1982;62:347–504.[Free Full Text]

3. Dzielak DJ. AIDS, lupus, rheumatoid arthritis—hypertension? Hypertension. 1990;15:95–96.[Free Full Text]

4. Dzielak DJ. The immune system and hypertension. Hypertension. 1992;19:I-36–I-44.

5. Hinglais N, Heudes D, Nicoletti A, Mandet C, Laurent M, Bariéty J, Michel JB. Co-localization of myocardial fibrosis and inflammatory cells. Lab Invest. 1994;70:286–294.[Medline] [Order article via Infotrieve]

6. Zimmerman JJ, Ringer TV. Inflammatory host responses in sepsis. Crit Care Clin. 1992;8:163–189.[Medline] [Order article via Infotrieve]

7. Parrillo JE. Pathogenetic mechanisms of septic shock. N Engl J Med. 1993;328:1471–1477.[Free Full Text]

8. Desch CE, Kovach N, Present W, Broyles C, Harlan JM. Production of human tumor necrosis factor from whole blood ex vivo. Lymphokine Res. 1989;8:141–144.[Medline] [Order article via Infotrieve]

9. Meager A, Leung H, Wolley J. Assays for tumor necrosis factor and related cytokines. J Immunol Methods. 1989;116:1–8.[Medline] [Order article via Infotrieve]

10. Aarden LA, De Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growth by human monocytes. Eur J Immunol. 1987;17:1411–1416.[Medline] [Order article via Infotrieve]

11. Green LC, Wagner DA, Glogowski J, Skipper PL, Wisnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and (¹⁵N) in biological fluids. Anal Biochem. 1982;126:131–138.[Medline] [Order article via Infotrieve]

12. Lindpaintner K, Kreutz R, Ganten D. Genetic variation in hypertensive and `control' strains. Hypertension. 1992;19:428–430.[Free Full Text]

13. Johnson ML, Ely DL, Turner ME. Genetic divergence between the Wistar-Kyoto rat and the spontaneously hypertensive rat. Hypertension. 1992;19:425–427.[Abstract/Free Full Text]

14. Bottger A, Lith H, Kren V, Krenova D, Bila V, Vorlicek J, Zidek V, Musilova A, Zdobinska M, Wang JM, Zutphen BFM, Kurtz TW, Pravenec M. Quantitative trait loci influencing cholesterol and phospholipid phenotypes map to chromosomes that contain genes regulating blood pressure in the spontaneously hypertensive rat. J Clin Invest. 1996;98:856–862.[Medline] [Order article via Infotrieve]

15. Khraibi AA. Association between disturbances in the immune system and hypertension. Am J Hypertens. 1991;4:635–641.[Medline] [Order article via Infotrieve]

16. Fu ML. Do immune system changes have a role in hypertension? J Hypertens. 1995;13:1259–1265.[Medline] [Order article via Infotrieve]

17. Schmidt-Schönbein GW, Seiffge D, DeLano FA, Shen K, Zweichach BW. Leukocyte counts and activation in spontaneously hypertensive and normotensive rats. Hypertension. 1991;17:323–330.[Abstract/Free Full Text]

18. Suzuki H, Schmidt-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.[Abstract/Free Full Text]

19. Feuerstein G, Hallenbeck JM, Vanatta B, Rabinovici R, Perera PY, Vogel SN. Effects of gram-negative endotoxin on levels of serum corticosterone, TNF, circulating blood cells, and the survival of rats. Circ Shock. 1990;30:265–278.[Medline] [Order article via Infotrieve]

20. Sirén AL, Heldman E, Doron D, Lysko PG, Yue TL, Liu Y, Feuerstein G, Hallenbeck JM. Release of proinflammatory and prothrombotic mediators in the brain and peripheral circulation in spontaneously hypertensive and normotensive Wistar-Kyoto rats. Stroke. 1992;23:1643–1651.[Abstract/Free Full Text]

21. Hallenbeck JM, Dutka AJ, Vogel SN, Heldman E, Doron DA, Feuerstein G. Lipopolysaccharide-induced production of tumor necrosis factor activity in rats with and without risk factors for stroke. Brain Res. 1991;541:115–120.[Medline] [Order article via Infotrieve]

22. Kurtz TW, Morris RC Jr. Biological variability in Wistar-Kyoto rats: implications for research with the spontaneous hypertensive rat. Hypertension. 1987;10:127–131.[Abstract/Free Full Text]

23. Rapp JP. Use and misuse of control strains for genetically hypertensive rats. Hypertension. 1987;10:7–10.[Free Full Text]

24. Basu S, Dunn AR, Marino MW, Savoia H, Hodgson G, Lieschke GJ, Cebon J. Increased tolerance to endotoxin by granulocyte-macrophage-colony-stimulating factor-deficient mice. J Immunol. 1997;159:1412–1417.[Abstract]

25. Yoshimoto T, Nakanishi K, Hirose S, Hiroishi K, Okamura H, Takemoto Y, Kanamaru A, Hada T, Tamura T, Kakishita E, Higashino K. High serum IL-6 level reflects susceptible status of the host to endotoxin and IL-1/tumor necrosis factor. J Immunol. 1992;148:3593–3603.

26. Kelly NM, Cross AS. Interleukin-6 is a better marker of lethality than tumor necrosis factor in endotoxin treated mice. FEMS Microbiol Immunol. 1992;89:317–322.

27. Nakamura A, Kohsaka T, Johns EJ. Differential regulation of interleukin-6 production in the kidney by the renal sympathetic nerves in normal and spontaneously hypertensive rats. J Hypertens. 1993;11:491–497.[Medline] [Order article via Infotrieve]

28. Pascual DW, Pascual VH, Bost KL, McGhee JR, Oparil S. Nitric oxide mediates immune dysfunction in the spontaneously hypertensive rat. Hypertension. 1993;21:185–194.[Abstract/Free Full Text]

29. Singh A, Svetenk P, Larivière R, Thibault G, Schiffrin EL. Inducible nitric oxide synthase in vascular smooth muscle cells from prehypertensive spontaneously hypertensive rats. Am J Hypertens. 1996;9:867–877.[Medline] [Order article via Infotrieve]

30. Deng AY, Rapp JP. Locus for the inducible, but not a constitutive, nitric oxide synthase cosegregates with blood pressure in the Dahl salt-sensitive rat. J Clin Invest. 1995;95:2170–2177.

31. Laubach VE, Shesely EG, Smithies O, Sherman PA. Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc Natl Acad Sci U S A. 1995;92:10688–10692.[Abstract/Free Full Text]

32. Mason D. Genetic variation in the stress response: susceptibility to experimental allergic encephalomyelitis and implications for human inflammatory disease. Immunol Today. 1991;12:57–60.[Medline] [Order article via Infotrieve]

33. Rook GAW, Hernandez-Pando R, Lightman SL. Hormones, peripherally activated prohormones and regulation of the Th1/Th2 balance. Immunol Today. 1994;15:301–303.[Medline] [Order article via Infotrieve]

34. Van der Poll T, Coyle SM, Barbosa K, Braxton CC, Lowry SF. Epinephrine inhibits tumor necrosis factor- and potentiates interleukin 10 production during human endotoxemia. J Clin Invest. 1996;97:713–719.[Medline] [Order article via Infotrieve]

35. Pascual DW, Kiyono H, McGhee JR. The enteric nervous and immune systems: interactions for mucosal immunity and inflammation. Immunomethods. 1994;5:56–72.[Medline] [Order article via Infotrieve]

36. Nakamura A, Kohsaka T, Johns EJ. Neuroregulation of interleukin-6 gene expression in the spontaneously hypertensive rat kidney. J Hypertens. 1996;14:839–845.[Medline] [Order article via Infotrieve]

37. Chen CM, Schachter D. Elevation of plasma immunoglobulin A in the spontaneous hypertensive rat. Hypertension. 1993;21:731–738.[Abstract/Free Full Text]

38. Plissonnier D, Levy BI, Salzmann JL, Nochy D, Watelet J, Michel JB. Allograft-induced arterial wall injury and response in normotensive and spontaneously hypertensive rats. Arterioscler Thromb. 1991;11:1690–1699.[Abstract/Free Full Text]

39. Coste M, Wakefield T, McGhee JR, Kiyono H, Pascual DW. Hypertensive rats manifest high IgA levels and elevated responses to oral cholera toxin. FASEB J. 1994;8(pt 1):A515. Abstract.

40. Fowler DH, Kurasawa K, Husebekk A, Cohen PA, Gress RE. Cells of Th2 cytokine phenotype prevent LPS-induced lethality during murine graft-versus-host reaction. J Immunol. 1994;152:1004–1013.[Abstract]

This article has been cited by other articles:

				C. C. McGown and Z. L. S. Brookes Beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide Br. J. Anaesth., February 1, 2007; 98(2): 163 - 175. [Abstract] [Full Text] [PDF]

				R. Hernanz, A. M. Briones, M. J. Alonso, E. Vila, and M. Salaices Hypertension alters role of iNOS, COX-2, and oxidative stress in bradykinin relaxation impairment after LPS in rat cerebral arteries Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H225 - H234. [Abstract] [Full Text] [PDF]

				J. Y.H. Chan, L.-L. Wang, K. L.H. Wu, and S. H.H. Chan Reduced Functional Expression and Molecular Synthesis of Inducible Nitric Oxide Synthase in Rostral Ventrolateral Medulla of Spontaneously Hypertensive Rats Circulation, October 2, 2001; 104(14): 1676 - 1681. [Abstract] [Full Text] [PDF]

				A. Tsujikawa, J. Kiryu, K. Yamashiro, A. Nonaka, K. Nishijima, Y. Honda, and Y. Ogura Interactions Between Blood Cells and Retinal Endothelium in Endotoxic Sepsis Hypertension, August 1, 2000; 36(2): 250 - 258. [Abstract] [Full Text] [PDF]

				P. Andrew, Y. Deng, and S. Kaufman Fluid extravasation from spleen reduces blood volume in endotoxemia Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2000; 278(1): R60 - R65. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bernard, C. Articles by Tedgui, A. Search for Related Content PubMed PubMed Citation Articles by Bernard, C. Articles by Tedgui, A.