Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Oct 12.
Published in final edited form as: Circ Res. 2012 Aug 17;111(9):1190–1197. doi: 10.1161/CIRCRESAHA.112.277475

Neurohormonal Modulation of the Innate Immune System is Pro-inflammatory in the Pre-hypertensive Spontaneously Hypertensive Rat, a Genetic Model of Essential Hypertension

Sailesh C Harwani 1, Mark W Chapleau 1,2,3, Kevin L Legge 4, Zuhair K Ballas 1,3, Francois M Abboud 1,2
PMCID: PMC3477787  NIHMSID: NIHMS408863  PMID: 22904093

Abstract

Rationale

Inflammation and autonomic dysfunction contribute to the pathophysiology of hypertension. Cholinergic stimulation suppresses innate immune responses. Angiotensin II induces hypertension and is associated with pro-inflammatory immune responses.

Objective

Our goal was to define the innate immune response in a model of genetic hypertension and the influence of cholinergic stimulation and Ang II.

Methods and Results

Studies were conducted on 4–5 week old pre-hypertensive Spontaneously Hypertensive Rats (SHR) and age matched normotensive control, Wistar Kyoto (WKY) rats. Isolated splenocytes were pre-exposed to nicotine or Ang II prior to Toll-like Receptor (TLR) activation. Culture supernatants were tested for cytokines (TNFα, IL-10, and IL-6). TLR-mediated cytokine responses were most pronounced with TLR7/8 and TLR9 activation and similar between WKY and SHR. Nicotine and Ang II enhanced this TLR mediated IL-6 response in pre-hypertensive SHR splenocytes. In contrast, nicotine suppressed the TLR mediated IL-6 response in WKY, while Ang II had no effect. In vivo, nicotine enhanced plasma levels of TLR7/8 mediated IL-6 and IL-1β in pre-hypertensive SHR, but suppressed these responses in WKY. Flow cytometry revealed an increase in a CD161+ innate immune cell population, which was enhanced by nicotine in the pre-hypertensive SHR spleen, but not in WKY.

Conclusions

There is a pronounced anti-inflammatory nicotinic/cholinergic modulation of the innate immune system in WKY, which is reversed in pre-hypertensive SHR. The results support the novel concept that neurohormonal regulation of the innate immune system plays a role in the pathogenesis of genetic hypertension and provide putative molecular targets for treatment of hypertension.

Keywords: Hypertension, toll-like receptor, nicotine, angiotensin II, interleukin-6, innate immunity

INTRODUCTION

Inflammation, as measured by pro-inflammatory cytokines (IL-17, ICAM-1, IL-6, TNFα), is implicated in the development and/or maintenance of hypertension in patients and in experimental models of angiotensin-dependent hypertension [1, 2]. T-lymphocytes, members of the adaptive immune system, have been directly implicated in the development of hypertension [3, 4]. The Spontaneously Hypertensive Rat (SHR) is a well accepted genetic model of essential hypertension and is known to manifest dysregulation of the immune system [5–7]. Although dysregulated immune responses are implicated in the development of hypertension, the mechanisms remain unknown.

Autonomic dysfunction, characterized by increased sympathetic and decreased parasympathetic activity, is associated with increased mortality in cardiovascular disease [8] and correlates with the development of hypertension [9]. Notably, the SHR has also been shown to be a good model for autonomic dysfunction, with enhanced chemoreceptor and decreased baroreceptor activity [8, 10, 11]. Studies that show anti-inflammatory and pro-inflammatory effects of cholinergic and adrenergic stimulation, respectively [12–14], potentially help explain the mechanism by which autonomic dysfunction leads to hypertension and contributes to cardiovascular mortality. Tracey et al have shown that nicotine suppresses innate immune cytokine responses in macrophages in a murine model of sepsis [15, 16]. Furthermore, central delivery of angiotensin II (Ang II) activates peripheral sympathetic nerve activity, and enhances splenic cytokine gene expression, suggesting that Ang II may contribute to the development of hypertension by stimulating a neurally-mediated pro-inflammatory immune response in the spleen [17], in addition to its direct renal effects.

The SHR has been used as a genetic model for essential hypertension since it develops hypertension as it ages. Hypertension in SHR begins at about 6 weeks of age and at 4–5 weeks of age, the SHRs are in the pre-hypertensive period [18, 19]. Given the autonomic dysfunction and immunological abnormalities present in the SHR [5–8, 10], it serves as an ideal model for the investigation of the neurohormonal regulation of innate immune responses. Although the adaptive immune system has been implicated in hypertension [2, 3], the role of the innate immune system remains undefined. We hypothesized that there is a dysregulation of innate immune responses prior to the development of hypertension in the SHR. In the present work, we use Toll-like receptor (TLR) ligands to activate the innate immune response. These TLRs detect “pathogen associated molecular patterns” and “damage associated molecular patterns” and induce cytokine production and release that trigger the activation of the acquired/adaptive immune system [20]. The modulation of the cytokine response to TLR activation by nicotine and Ang II was contrasted in young Wistar Kyoto (WKY) normotensive rats and the young prehypertensive SHR. In the WKY, nicotine suppressed the cytokine response, but in the SHR, nicotine, as well as Ang II, significantly enhanced the cytokine response. We propose that this abnormal pro-inflammatory modulation of the innate immune system which precedes the onset of hypertension contributes significantly to its development in the SHR.

METHODS

Please see Online Supplement for detailed Materials and Methods.

Animals

Male Wistar Kyoto (WKY) and Spontaneous Hypertensive Rats (SHR) [Charles River Laboratories] were used at 4–5 weeks of age.

TLR activation

Several Toll receptors recognize specific extra- or intracellular ligands and induce NFκβ activation in immune cells and the release of inflammatory cytokines. Responses to the TLR ligands activation were measured in supernatants of cultured splenocytes in vitro as well as in blood in vivo.

Splenocyte cultures

Splenocytes from 4–5 week old animals were stimulated in vitro with known TLR ligands in the presence or absence of pre-exposure to nicotine or Ang II. Culture supernatants were collected and IL-6, TNFα, and IL-10 were measured by ELISA.

In vivo studies

Subcutaneous (s.c.) osmotic minipumps were implanted and infused either saline or nicotine over 24 hours (15 mg/kg). At 20 hours, animals received an intraperitoneal (i.p.) injection of either saline or the TLR7/8 ligand (Clo97). Serum was collected from each animal and IL-6, IL-1β, TNFα, IL-10, and IL-17 were measured at 24 hours.

Flow cytometry

Splenocytes were stained with fluorochrome-conjugated antibodies to CD3, CD4, CD8, CD45R, CD11bc, CD161a, and MHCII, and analyzed by flow cytometry.

RESULTS

Cytokine release from splenocytes in response to TLR activation is pronounced with TLR7/8 and 9 and equivalent in SHR and WKY

We first asked whether the native innate immune response of the SHR differed from the normotensive WKY. Splenocytes were stimulated with three graded doses of the ligands for TLR2, TLR3, TLR4, TLR5, TLR7/8, and TLR9, as listed in the legend of Figure 1. Similar levels of TNFα (Figure 1A), IL-10 (Figure 1B), and IL-6 (Figure 1C) were induced in WKY and SHR splenocytes in response to TLR activation. The most robust responses were seen with TLR7/8 and TLR9 activation. Nicotine and Ang II alone did not induce cytokine secretion from either WKY or SHR splenocytes (Figure 1).

Figure 1. Dose response of isolated splenocytes from WKY (open bars) and SHR (red bars) to TLR ligands.

Figure 1

Open in a new tab

Three doses of each TLR ligand were tested to induce secretion of (A) TNF-α, (B) IL-10, (C) IL-6. Ligands for each TLR are listed above the TLR number. They are: Lipoteichoic Acid (LTA) for TLR2; Poly I:C (PIC) for TLR3; Lipopolysaccharide (LPS) for TLR4; Flagellin (Flag) for TLR5; Clo97 for TLR7; CpG2395 for TLR9. The effects of four doses of Ang II (= Angiotensin II) and Nic (= Nicotine) were also tested. The three doses of each ligand are indicated in micromoles/liter. Data represent means of three separate experiments. Error bars represent standard error of the mean. Responses to the intracellular receptors TLR7 (Clo97) and TLR9 (CpG) were significantly larger than those to ligands of the other TLRs.

Modulation of TLR-induced IL-6 secretion by nicotine and Ang II is markedly proinflammatory in SHR

Although nicotine and Ang II did not directly induce the secretion of cytokines, exposure of splenocytes to nicotine (10 micromolar) and Ang II (1 micromolar) for 2 hours prior to the addition of TLR ligands resulted in significant modulation of TLR-induced IL-6 secretion. In WKY splenocytes, nicotine pre-exposure suppressed TLR9-mediated IL-6 secretion (Figure 2A). In contrast, nicotine exposure of SHR splenocytes resulted in a dramatic increase in the release of IL-6 (Figure 2B) in response to TLR7/8 and TLR9. Ang II exposure of WKY splenocytes did not alter TLR mediated IL-6 release (Figure 2C), but led to a significant increase in TLR mediated IL-6 release in SHR splenocytes, similar to the effect of nicotine (Figure 2D).

Figure 2. Immunomodulation of IL-6 responses to TLR activation in WKY and SHR by prior exposure to nicotine or Ang II.

Figure 2

Open in a new tab

IL-6 concentrations in supernatants of cultured splenocytes following activation by various TLR ligands: TLR2 (LTA 100 µM), TLR3 (PIC 10 µM), TLR4 (LPS 10 µM), TLR5 (Flag 100 µM), TLR7 (Clo97 1 µM), and TLR9 (CpG 10 µM) are shown in WKY (Panels A and C in black and white) and in SHR (Panels B and D in red). The names of each TLR ligand are shown in Figure 1. Results were obtained after 48 hours in cultures. The effects of 10 µM nicotine (Panels A and B) and of 1 µM Ang II (Panels C and D) are contrasted in WKY and SHR. Bars represent the mean of three separate experiments. Error bars represent standard error of the mean. ***=p<0.001 and **=p<0.01 indicate significantly different responses to TLR alone (open bars) vs. TLR with exposure to either nicotine or Ang II (shaded bars). Of the six TLR ligands tested, nicotine and Ang II altered most significantly the responses to TLR7 and TLR9. Both nicotine and Ang II enhanced dramatically the IL-6 responses to TLR7 and 9 in SHR, whereas nicotine suppressed the response in WKY and Ang II did not alter it significantly. Responses of TNFα and IL-10 were not altered by nicotine or Ang II (data not shown).

Based on the robust responses to TLR7/8 and TLR9, we focused on these ligands in subsequent experiments (Figure 3). We continued to see uniformly suppressive effects of nicotine on TLR responses in WKY splenocytes (Figure 3A) and uniformly enhanced effects of both nicotine and Ang II in SHR splenocytes (Figure 3B, 3D). Ang II exposure did not appreciably alter the TLR responses in WKY splenocytes (Figure 3C).

Figure 3. Immunomodulation of IL-6 and IL-1β responses to TLR7/8 and/or TLR9 by nicotine and Ang II in vitro and in vivo.

Figure 3

Open in a new tab
Individual responses of isolated splenocytes from 4–5 week old WKY (n=5) and SHR (n=5) rats are shown in upper panels 3A to D. In vivo changes in serum levels in response to subcutaneous (s.c.) infusions of nicotine or Ang II in WKY and SHR are in the lower panels 3E to 3H. Numbers above each bar graph represent the number of animals (n) in the particular group.
  • Splenocyte responses in vitro: Splenocytes were activated by ligands to TLR7/8 (Clo97, 1.0 µM) and to TLR9 (CpG, 10 µM). IL-6 was measured in culture supernatants. Panels A - D portray responses to activation of the TLRs with exposure to nicotine (10 µM) in WKY and SHR (Panels A and B respectively) and with exposure to Ang II (1 µM) in WKY and SHR (Panels C and D, respectively). Responses of WKY are in black and white (Panels A and C) and those of SHR are in red (Panels B and D). The suppressive effect of nicotine on responses to activation of both TLRs in WKY was significant (p=<0.01), and Ang II had generally small and inconsistent effects in WKY. In contrast, a significant augmentation of the responses to activation of both TLRs by nicotine and by Ang II was consistently observed in SHR (p=<0.01).
  • In vivo serum levels: The effects of s.c. infusions of nicotine (15 mg/kg) or Ang II (0.72 mg/kg) on IL-6 and IL-1β serum levels induced by the TLR7/8 ligand (Clo97, 1 mg/kg) i.p. are shown in Panels E through H. The infusions of saline, Nic, or Ang II were maintained for 24 hours. At 20 hours, animals received i.p. injection of Clo97 or saline. Sera were collected at 24 hours and assayed for IL6 or IL-1β. Nicotine (Panels E and F) and Ang II (Panels G and H) when given with i.p. saline increased IL-6 and IL-1β in WKY and to a lesser extent in SHR. In WKY, nicotine (s.c.) suppressed significantly IL-6 and IL-1β responses to i.p. Clo97 (Panel E). In contrast, in SHR, nicotine enhanced significantly both IL-6 and IL-1 β responses (Panel F). Ang II had no effect on the responses in WKY (Panel G), yet it significantly enhanced the IL-1β response in SHR (Panel H). * indicates significant differences between responses to Nic/TLR or Ang/TLR vs. saline/TLR in both WKY and SHR (p<0.01). The anti-inflammatory effects of nicotine in WKY and pro-inflammatory effects of nicotine and Ang II in SHR seen in vitro in splenocytes were thus confirmed in vivo.

In vivo effect of nicotine and Ang II on TLR7/8-mediated cytokine production: Inhibitory in WKY and proinflammatory in SHR

We asked whether the contrasting nicotine effects seen in vitro would be reproduced in vivo. We found that s.c. infusion of nicotine alone or Ang II alone demonstrated a trend of increased serum levels of IL-6 and IL-1β in both WKY (Fig. 3E,G) and SHR (Fig. 3F,H). We also found that the serum levels of both IL-6 and IL-1beta in response to TLR7/8 activation with Clo97 (i.p.) were markedly suppressed by the s.c. infusion of nicotine in WKY (Fig. 3E) and conversely significantly enhanced in SHR (Fig. 3F). Ang II did not alter the TLR responses of IL-6 and IL-1β in WKY (Fig. 3G), but in SHR the IL-1β response was significantly enhanced and the increase in IL-6 was not significant (Fig. 3H). Thus, the antiinflammatory modulation of TLR responses by nicotine seen in the WKY splenocytes and the proinflammatory enhancements by nicotine and Ang II seen in SHR splenocytes were reproduced in vivo.

Activated macrophages in the SHR are increased by nicotine

Based on the clearly opposite effects of nicotine on IL-6 secretion in WKY and SHR, both in vitro and in vivo, we asked whether there was a difference in the immune cell subpopulations in SHR vs. WKY, and used flow cytometry to answer this question.

Although, there was no difference in the distribution of CD3+/CD8+, CD3+CD4+, CD11bc+, or CD45R+ immune cells, we found a discordance in the composition of CD3-/CD8bright and CD3-/CD8dim cell populations with an increase in the latter in SHR compared to WKY (Fig. 4). Further analysis of the CD3−/ CD8dim cells was made by assessing CD161a, a marker of activated macrophages. We found a CD161a population that was more dominant in SHR (4.6%) compared to WKY (1.1%) and expanded in response to nicotine to 6.4% while the WKY population remained at 1.1%. Another marker of activated macrophages (CD3−/CD8−/CD161a+) was also increased in SHR and expanded further from 3.7% to 6.0% in response to nicotine (Fig.4). This population also remained low in WKY (1.5%), despite the presence of nicotine (Fig. 4). Hence, there appears to be a population of activated macrophages (CD161a+) present in the SHR, but not in the WKY, prior to the development of hypertension, that may account for the proinflammatory enhancement of the TLR-induced increase in IL-6 and IL-1β levels.

Figure 4. Effects of nicotine on CD8+CD161+ innate immune cells are discordantbBetween WKY and SHR.

Figure 4

Open in a new tab

Subpopulations of CD8 and CD161 are shown in WKY and SHR after 48 hours in culture in media alone (Panels A and B respectively) and in media plus nicotine (10 µmoles/L in Panels C and D respectively). After gating for CD3- cells, flow cytometry revealed an increase in the CD8dim/CD 161+ and CD8−/CD161+ populations in SHR (Panel B), compared to WKY (Panel A). Moreover, an inductive effect of nicotine on the CD161a+ immune cell populations is seen in SHR (Panel D) but not in WKY (Panel C). The markers indicate an increase in activated macrophages in SHR that is enhanced by nicotine in contrast to the suppressed values in WKY. This suggests that the proinflammatory effect of nicotine in SHR results from an increase in activated macrophages (CD161+) not seen in WKY. Results presented are representative of three individual experiments.

Effect of nicotine on myeloid dendritic cells in WKY

Preliminary findings reported in the Online Figures II-IV indicate suppression of an MHCII+ CD11bc+ myeloid dendritic cell population by nicotine exclusively in WKY but not in SHR, which may account for the anti-inflammatory effect of nicotine.

DISCUSSION

The major finding of this study is that the SHR exhibit a pro-inflammatory innate immune response (measured by IL-6 and IL-1β), prior to the development of hypertension, which suggests its contribution to the development of hypertension. It is most pronounced with activation of TLR7/8 and 9. Nicotine, a ligand for cholinergic receptors, is anti-inflammatory in the normotensive control WKY, but is paradoxically pro-inflammatory in the SHR. An accentuated pro-inflammatory response to Ang II is also seen in the SHR, but not in WKY. Changes in innate immune cell populations with exposure to nicotine correlate with the anti-inflammatory effects of nicotine in the WKY and pro-inflammatory effects in young SHR.

In the Discussion, we will address: 1) the contribution of the innate immune system in human hypertension and in SHR; 2) the functional relevance of immune responses to nicotine and angiotensin to the survival advantage of parasympathetic activation and angiotensin converting enzyme inhibition in cardiovascular disease; 3) the prominent effect of ligands of TLR 7/8 and 9 relative to other TLRs (which is presented in the Supplement); and 4) the changes in populations of CD161a+ activated macrophages and possibly myeloid dendritic cells that may account for the dramatically opposite effects of nicotine in WKY and SHR.

Inflammation and innate immunity in human hypertension and in SHR

Circulating inflammatory cytokines have been detected in the sera of hypertensive patients [1]; moreover, amounts of IL-6 and IL-1β in whole blood of patients with essential hypertension were exaggerated in response to TLR4 activation with lipopolysaccharide consistent with activation of monocytes [21]. Similarly the genetically hypertensive adult SHRs have elevated blood levels of innate immune cytokines (IL-6 and IL-1β) compared to WKY [22]. Moreover, the systemic inhibition of NFκβ in SHR decreases renal inflammation and results in significant reductions of systolic blood pressure.

Our results focused on the pre-hypertensive (normotensive) young SHR. Previous studies [18] and preliminary results from our laboratory [19] have shown that SHR are normotensive at 4–5 weeks of age. In the present study, we show that activation of isolated splenocytes with several TLR ligands induced increases in IL-6, TNFα and IL-10 that are comparable in the prehypertensive SHR to those of normotensive WKY. Also, in vivo TLR activation caused increases in IL-6 and IL-1β blood levels in both WKY and SHR. More importantly, however, an enhanced proinflammatory response to TLR activation similar to that seen in patients with essential hypertension was unmasked in our prehypertensive SHR by nicotine and Ang II in vitro and in vivo.

In contrast in the WKY rats, nicotine which had been reported to suppress inflammatory responses by activating nicotinic α7 cholinergic receptors (nAChR) [16], suppressed the increase of IL-6 in vitro and the increases in IL-6 and IL-1β in vivo. Ang II had no effect on the cytokine response to TLR activation in WKY.

The results indicate that there is an abnormal proinflammatory state of the innate immune system in the prehypertensive SHR, which is provoked by nicotine and by Ang II, when TLR 7/8 and TLR9 are activated, and may thereby contribute to the development of hypertension in this model. We also note an antiinflammatory effect of nicotine in the WKY which is probably mediated by α7 nicotinic cholinergic receptors.

Functional relevance of inflammatory responses to nicotine and Ang II to the survival advantage of parasympathetic stimulation and angiotensin converting enzyme inhibition in cardiovascular disease

Autonomic dysfunction with increased sympathetic nerve activity and decreased parasympathetic activity has been correlated with increased cardiovascular mortality [23] and the development of hypertension [24, 25]. The attempt to restore autonomic balance with direct carotid sinus and vagal stimulation has shown promise in the treatment of hypertension and heart failure [26]. Classically, autonomic dysfunction has referred to the imbalance of neural activity between the parasympathetic and the sympathetic nervous systems. Recently, the neurotransmitters of the autonomic nervous system have been shown to not only be produced by, but also exert effects on non-neuronal cells, expanding the conceptual importance of autonomic dysfunction [27].

There is direct sympathetic innervation of immunological organs [28]. Activation of adrenergic receptors has been shown to induce a pro-inflammatory immune response [29, 30]. In contrast, it has been shown that the vagus nerve stimulation exerts an anti-inflammatory effect in the gut in a murine model for sepsis [15]. Vagal nerve stimulation also exerts an immunomodulatory effect on a subset of splenic T-lymphocytes that produces and secretes acetylcholine in response to adrenergic input from the splenic nerve [28, 31, 32]. The results of the present study suggest novel mechanisms by which autonomic regulation of immune responses may increase cardiovascular mortality or be protective and beneficial.

Cholinergic anti-inflammatory immunomodulation –Role of α7 nAChR

Our data show that there is an anti-inflammatory modulation in the normotensive WKY by nicotine, which in preliminary results is blocked by α-bungarotoxin, a blocker of α7 nAChR (Online Figure I). The paradoxically enhanced pro-inflammatory modulation of the TLR response by nicotine in SHR was not altered by α-bungarotoxin and cannot be attributed to decreased expression of α7 nAChR in SHR. It has been shown that expression of α7 nAChR is similar in pre-hypertensive SHR and age matched WKY [33]. Interestingly, there are reports of decreased expression of α7 nAChR in the central nervous system of the stroke-prone SHR [34] and in older (20-week or more) hypertensive SHR [33]. Moreover, α7 nAChR agonists decrease the inflammatory end organ damage in the hypertensive SHR [33] and recent studies in α7 nAChR knockout mice show an enhanced inflammatory end-organ response in the two kidney/one clip model of hypertension [33]. There is also evidence that T-cells in the WKY and SHR produce acetylcholine (ACh), and express ACh receptors [35] and that circulating, thymic, and splenic levels of ACh in the very young SHR are elevated, but decreased with aging up to 20 weeks compared to the WKY age matched controls [35].

Nicotinic pro-inflammatory immunomodulation in SHR

Although α7 nAchRs are potent suppressors of the inflammatory response and can protect the end organs from hypertensive damage, many other nAChR subunits [36], and G protein-coupled receptors that may be involved in pro-inflammatory responses. In a recent study [37], it was reported that nicotine and Ang II activate respective G protein-coupled receptors on vascular muscle cells, increase intracellular reactive oxygen species and cytokines (IL-6, 1FN-gamma) that promote activation of AMP-Kα2 and its nuclear translocation with the induction of transcription factors such as matrix metalloproteinase 2.

Hence, there are clear indications of a cholinergic influence on the immune system and on end organs through α7 nAChR, ACh production, or activation of other nicotinic receptors. The interaction may be protective or paradoxically inflammatory. Our results identify a significant proinflammatory innate immune system abnormality that precedes the development of hypertension in SHR. Further characterization of the cellular and molecular determinants of that interaction needs to be examined.

Proinflammatory immunomodulation by Ang II in SHR

Ang II is clearly involved in the pathogenesis of hypertension through neural, renal, and vascular mechanisms. Additionally Ang II activates NFκβ causing the expression of proinflammatory cytokines in vitro and in vivo [38]. Inhibition of angiotensin converting enzyme reduces cardiac inflammatory markers in SHR by inactivating NFκβ [22]. AT1 receptor blockade reduces LPS-induced innate immune responses in rat spleen (38), reverses renal inflammation [39], and also reduces vascular and circulating inflammatory mediators in SHR [40]. Although we found that both nicotine and Ang II had no direct effect on cytokine release from splenocytes in culture, their systemic infusions did increase serum levels of IL-6 and IL-1beta in both WKY and SHR. This pro-inflammatory response may be secondary to an increase in sympathetic nerve activity through a central action of Ang II or ganglionic effect of nicotine.

Our results shed light on a novel mechanism by which Ang II may be pro-inflammatory in a model of genetic hypertension. It induces a marked enhancement of secretion of IL-6 and/or IL-1β from splenocytes of prehypertensive SHR in response to TLR activation. This pro-inflammatory response, similar to that elicited by nicotine, is immunomodulatory since Ang II alone did not induce secretion of detectable levels of cytokines. Interestingly, our preliminary results demonstrate that the AT1 receptor blocker losartan not only prevents the proinflammatory effect of Ang II in SHR, but also reduces IL-6 release to levels below those seen in the absence of Ang II (see Online Figure I). An inhibitory effect of losartan on inflammatory signaling mediated by PPAR gamma has been reported [41].

Prominent contribution of TLR in activating innate immunity in hypertension

A description of TLRs and their linkage to hypertension and to the innate immune system is discussed in the Supplement.

Changes in populations of innate immune cells with nicotine differ in WKY and SHR

Dendritic cells and macrophages are members of the innate immune system and serve as antigen presenting cells, thus activating T lymphocytes and playing a role in their differentiation. Natural Killer (NK) cells are also important members of the innate immune system. We show that there is a more prominent CD3−/CD161a+ cell population in the SHR splenocytes that appeared to be further induced by nicotine pre-exposure (Figure 4). We postulate that the expansion of this cell population may be mediated by IL-6 which is known to affect the differentiation of monocytes to macrophages [42]. CD161a is a marker for NK cells, dendritic cells, and activated macrophages [43, 44]. Thus, it appears that enhanced activation of macrophages, which is not seen in WKY, would explain the pro-inflammatory effect of nicotine. We are currently exploring the distribution of the various innate immune cells including dendritic cell subsets, NK cells and, possibly, NK T cells. Preliminary results included in the Supplement (Online Figures II-IV) suggest that the antiinflammatory effect of nicotine in WKY represents a suppression of a larger population of myeloid dendritic cells not seen in SHR.

CONCLUSIONS

The results of the current study support the novel concept of neuro-hormonal modulation of the innate immune system as a pathogenic mechanism in genetic hypertension prior to the onset of hypertension [45]. Further, the results begin to identify cellular targets of interest that may mediate these effects. We show evidence that the innate immune system is abnormally primed and sensitized in SHR to be highly proinflammatory in response to putative cholinergic and angiotensinergic stimuli.

Supplementary Material

01

Novelty and Significance.

What Is Known?

  • Inflammation characterized by the presence of activated immune cells and production of proinflammatory cytokines has been linked to hypertension. Recent studies have implicated T-lymphocytes in angiotensin II-induced hypertension.

  • Activation of nicotinic cholinergic receptors inhibits innate inflammatory immune responses while binding of angiotensin II (Ang II) to angiotensin type 1 (AT1) receptors is proinflammatory.

What New Information Does This Article Provide?

  • The innate immune system is abnormally pro-inflammatory in a genetic model of hypertension prior to the onset of hypertension.

  • An antiinflammatory effect of nicotine on splenocytes isolated from control Wistar Kyoto (WKY) rats (inhibition of IL-6 release) is reversed to a proinflammatory increase of IL-6 release from splenocytes of young, pre-hypertensive spontaneously hypertensive rats (SHR).

  • Ang II evokes a proinflammatory response in pre-hypertensive SHR, but not in WKY.

  • In vivo changes in serum levels of IL-6 and IL-1β matched the in vitro responses when nicotine and Ang II were infused subcutaneously in WKY and SHR.

  • Alpha-7 nicotinic receptors and AT1 Ang II receptors mediate the anti-and pro-inflammatory responses, respectively.

  • Proinflammatory responses in SHR are associated with a unique innate immune cell population (activated macrophages) that proliferates in response to nicotine in SHR.

Little is known regarding regulation of the innate immune system in hypertension. This study shows that the innate immune system is abnormally primed and sensitized in SHR to be highly proinflammatory, in response to both nicotine and angiotensin II, even before blood pressure increases. These results support the concept of neurohumoral modulation of the innate immune system as a pathogenic mechanism in the development of hypertension and could help in identifying new molecular targets for the treatment of hypertension.

ACKNOWLEDGMENTS

We would like to acknowledge Justin Fishbaugh and the Flow Cytometry Facility staff for use of the equipment and assistance in flow cytometry. We also thank Carol Whiteis for assistance in experiments. We would also like to acknowledge the supportive efforts of Dr. Fayaz Sutterwala.

SOURCES OF FUNDING

This study was funded by NIH grant HL-14388 and NIH T32 HL07121-36.

Non-standard Abbreviations

PPARγ

peroxisome proliferator-activated receptor gamma

s.c.

subcutaneous

IL-6

Interleukin-6

IL-1β

Interleukin-1 beta

SHR

spontaneously hypertensive rats

WKY

Wistar-Kyoto rats

Ang II

angiotensin II

Ach

acetylcholine

α7 n AChR

alpha-7 nicotinic acetylcholine receptor

TLR

Toll-like Receptor

TNFα

tumor necrosis factor-alpha

AT1

angiotensin type 1

NFκB

nuclear factor kappa-B

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DISCLOSURES

There are no financial disclosures.

REFERENCES

  • 1.Chae CU, Lee RT, Rifai N, Ridker PM. Blood pressure and inflammation in apparently healthy men. Hypertension. 2001;38:399–403. doi: 10.1161/01.hyp.38.3.399. [DOI] [PubMed] [Google Scholar]
  • 2.Madhur MS, Lob HE, McCann LA, Iwakura Y, Blinder Y, Guzik TJ, Harrison DG. Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction. Hypertension. 2010;55:500–507. doi: 10.1161/HYPERTENSIONAHA.109.145094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Guzik TJ, Hoch NE, Brown KA, McCann LA, Rahman A, Dikalov S, Goronzy J, Weyand C, Harrison DG. Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction. J Exp Med. 2007;204:2449–2460. doi: 10.1084/jem.20070657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Svendsen UG. Evidence for an initial, thymus independent and a chronic, thymus dependent phase of DOCA and salt hypertension in mice. Acta Pathol Microbiol Scand A. 1976;84:523–528. doi: 10.1111/j.1699-0463.1976.tb00150.x. [DOI] [PubMed] [Google Scholar]
  • 5.Ba D, Takeichi N, Kodama T, Kobayashi H. Restoration of T cell depression and suppression of blood pressure in spontaneously hypertensive rats (SHR) by thymus grafts or thymus extracts. J Immunol. 1982;128:1211–1216. [PubMed] [Google Scholar]
  • 6.Pascual VH, Oparil S, Eldridge JH, Jin H, Bost KL, Pascual DW. Spontaneously hypertensive rat: lymphoid depression is age dependent and mediated via a mononuclear cell subpopulation. Am J Physiol. 1992;262:R1–R7. doi: 10.1152/ajpregu.1992.262.1.R1. [DOI] [PubMed] [Google Scholar]
  • 7.Takeichi N, Suzuki K, Okayasu T, Kobayashi H. Immunological depression in spontaneously hypertensive rats. Clin Exp Immunol. 1980;40:120–126. [PMC free article] [PubMed] [Google Scholar]
  • 8.Abboud FM. The Walter B. Cannon Memorial Award Lecture, 2009. Physiology in perspective: The wisdom of the body. In search of autonomic balance: the good, the bad, the ugly. Am J Physiol Regul Integr Comp Physiol. 2010;298:R1449–R1467. doi: 10.1152/ajpregu.00130.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Guyenet PG. The sympathetic control of blood pressure. Nat Rev Neurosci. 2006;7:335–346. doi: 10.1038/nrn1902. [DOI] [PubMed] [Google Scholar]
  • 10.Abboud FM. The sympathetic system in hypertension. State-of-the-art review. Hypertension. 1982;4:208–225. [PubMed] [Google Scholar]
  • 11.Tan ZY, Lu Y, Whiteis CA, Simms AE, Paton JF, Chapleau MW, Abboud FM. Chemoreceptor hypersensitivity, sympathetic excitation, and overexpression of ASIC and TASK channels before the onset of hypertension in SHR. Circ Res. 2010;106:536–545. doi: 10.1161/CIRCRESAHA.109.206946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Schauenstein K, Felsner P, Rinner I, Liebmann PM, Stevenson JR, Westermann J, Haas HS, Cohen RL, Chambers DA. In vivo immunomodulation by peripheral adrenergic and cholinergic agonists/antagonists in rat and mouse models. Ann N Y Acad Sci. 2000;917:618–627. doi: 10.1111/j.1749-6632.2000.tb05427.x. [DOI] [PubMed] [Google Scholar]
  • 13.Skok M, Grailhe R, Changeux JP. Nicotinic receptors regulate B lymphocyte activation and immune response. Eur J Pharmacol. 2005;517:246–251. doi: 10.1016/j.ejphar.2005.05.011. [DOI] [PubMed] [Google Scholar]
  • 14.Zimring JC, Kapp LM, Yamada M, Wess J, Kapp JA. Regulation of CD8(+) cytolytic T lymphocyte differentiation by a cholinergic pathway. Journal of Neuroimmunology. 2005;164:66–75. doi: 10.1016/j.jneuroim.2005.03.018. [DOI] [PubMed] [Google Scholar]
  • 15.Borovikova LV, Ivanova S, Zhang M, Yang H, Botchkina GI, Watkins LR, Wang H, Abumrad N, Eaton JW, Tracey KJ. Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin. Nature. 2000;405:458–462. doi: 10.1038/35013070. [DOI] [PubMed] [Google Scholar]
  • 16.Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Yang H, Ulloa L, Al-Abed Y, Czura CJ, Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 2003;421:384–388. doi: 10.1038/nature01339. [DOI] [PubMed] [Google Scholar]
  • 17.Ganta CK, Lu N, Helwig BG, Blecha F, Ganta RR, Zheng L, Ross CR, Musch TI, Fels RJ, Kenney MJ. Central angiotensin II-enhanced splenic cytokine gene expression is mediated by the sympathetic nervous system. Am J Physiol Heart Circ Physiol. 2005;289:H1683–H1691. doi: 10.1152/ajpheart.00125.2005. [DOI] [PubMed] [Google Scholar]
  • 18.Andresen MC, Kuraoka S, Brown AM. Baroreceptor function and changes in strain sensitivity in normotensive and spontaneously hypertensive rats. Circ Res. 1980;47:821–828. doi: 10.1161/01.res.47.6.821. [DOI] [PubMed] [Google Scholar]
  • 19.Whiteis CA, Post CE, Morgan DA, Rahmouni K, Chapleau MW, Abboud FM. Peripheral chemoreceptors contribute significantly to hypertension in Spontaneously Hypertensive Rats (SHR) (Abstract) FASEB J. 2012;26:15. [Google Scholar]
  • 20.Takeda K, Akira S. Toll-like receptors in innate immunity. Int Immunol. 2005;17:1–14. doi: 10.1093/intimm/dxh186. [DOI] [PubMed] [Google Scholar]
  • 21.Peeters AC, Netea MG, Janssen MC, Kullberg BJ, Van der Meer JW, Thien T. Pro-inflammatory cytokines in patients with essential hypertension. Eur J Clin Invest. 2001;31:31–36. doi: 10.1046/j.1365-2362.2001.00743.x. [DOI] [PubMed] [Google Scholar]
  • 22.Miguel-Carrasco JL, Zambrano S, Blanca AJ, Mate A, Vazquez CM. Captopril reduces cardiac inflammatory markers in spontaneously hypertensive rats by inactivation of NF-kB. J Inflamm (Lond) 2010;7:21. doi: 10.1186/1476-9255-7-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med. 1984;311:819–823. doi: 10.1056/NEJM198409273111303. [DOI] [PubMed] [Google Scholar]
  • 24.Donohue SJ, Stitzel RE, Head RJ. Time course of changes in the norepinephrine content of tissues from spontaneously hypertensive and Wistar Kyoto rats. J Pharmacol Exp Ther. 1988;245:24–31. [PubMed] [Google Scholar]
  • 25.Grassi G, Quarti-Trevano F, Dell'oro R, Mancia G. Essential hypertension and the sympathetic nervous system. Neurol Sci. 2008;29(Suppl 1):S33–S36. doi: 10.1007/s10072-008-0882-9. [DOI] [PubMed] [Google Scholar]
  • 26.De Ferrari GM, Crijns HJ, Borggrefe M, Milasinovic G, Smid J, Zabel M, Gavazzi A, Sanzo A, Dennert R, Kuschyk J, Raspopovic S, Klein H, Swedberg K, Schwartz PJ. Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure. Eur Heart J. 2011;32:847–855. doi: 10.1093/eurheartj/ehq391. [DOI] [PubMed] [Google Scholar]
  • 27.Rosas-Ballina M, Olofsson PS, Ochani M, Valdes-Ferrer SI, Levine YA, Reardon C, Tusche MW, Pavlov VA, Andersson U, Chavan S, Mak TW, Tracey KJ. Acetylcholine-synthesizing T cells relay neural signals in a vagus nerve circuit. Science. 2011;334:98–101. doi: 10.1126/science.1209985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Nance DM, Sanders VM. Autonomic innervation and regulation of the immune system (1987–2007) Brain Behav Immun. 2007;21:736–745. doi: 10.1016/j.bbi.2007.03.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Grisanti LA, Evanson J, Marchus E, Jorissen H, Woster AP, DeKrey W, Sauter ER, Combs CK, Porter JE. Pro-inflammatory responses in human monocytes are beta1-adrenergic receptor subtype dependent. Mol Immunol. 2010;47:1244–1254. doi: 10.1016/j.molimm.2009.12.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Grisanti LA, Woster AP, Dahlman J, Sauter ER, Combs CK, Porter JE. alpha1-adrenergic receptors positively regulate Toll-like receptor cytokine production from human monocytes and macrophages. J Pharmacol Exp Ther. 2011;338:648–657. doi: 10.1124/jpet.110.178012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Antonica A, Ayroldi E, Magni F, Paolocci N. Lymphocyte traffic changes induced by monolateral vagal denervation in mouse thymus and peripheral lymphoid organs. J Neuroimmunol. 1996;64:115–122. doi: 10.1016/0165-5728(95)00157-3. [DOI] [PubMed] [Google Scholar]
  • 32.Rosas-Ballina M, Goldstein RS, Gallowitsch-Puerta M, Yang L, Valdes-Ferrer SI, Patel NB, Chavan S, Al-Abed Y, Yang H, Tracey KJ. The selective alpha7 agonist GTS-21 attenuates cytokine production in human whole blood and human monocytes activated by ligands for TLR2, TLR3, TLR4, TLR9, and RAGE. Mol Med. 2009;15:195–202. doi: 10.2119/molmed.2009.00039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Li DJ, Evans RG, Yang ZW, Song SW, Wang P, Ma XJ, Liu C, Xi T, Su DF, Shen FM. Dysfunction of the cholinergic anti-inflammatory pathway mediates organ damage in hypertension. Hypertension. 2011;57:298–307. doi: 10.1161/HYPERTENSIONAHA.110.160077. [DOI] [PubMed] [Google Scholar]
  • 34.Ferrari R, Frasoldati A, Leo G, Torri C, Zini I, Agnati LF, Zoli M. Changes in nicotinic acetylcholine receptor subunit mRNAs and nicotinic binding in spontaneously hypertensive stroke prone rats. Neurosci Lett. 1999;277:169–172. doi: 10.1016/s0304-3940(99)00879-4. [DOI] [PubMed] [Google Scholar]
  • 35.Fujimoto K, Matsui M, Fujii T, Kawashima K. Decreased acetylcholine content and choline acetyltransferase mRNA expression in circulating mononuclear leukocytes and lymphoid organs of the spontaneously hypertensive rat. Life Sci. 2001;69:1629–1638. doi: 10.1016/s0024-3205(01)01237-1. [DOI] [PubMed] [Google Scholar]
  • 36.Graham A, Court JA, Martin-Ruiz CM, Jaros E, Perry R, Volsen SG, Bose S, Evans N, Ince P, Kuryatov A, Lindstrom J, Gotti C, Perry EK. Immunohistochemical localisation of nicotinic acetylcholine receptor subunits in human cerebellum. Neuroscience. 2002;113:493–507. doi: 10.1016/s0306-4522(02)00223-3. [DOI] [PubMed] [Google Scholar]
  • 37.Wang S, Zhang C, Zhang M, Liang B, Zhu H, Lee J, Viollet B, Xia L, Zhang Y, Zou MH. Activation of AMP-activated protein kinase alpha2 by nicotine instigates formation of abdominal aortic aneurysms in mice in vivo. Nat Med. 2012 doi: 10.1038/nm.2711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Sanchez-Lemus E, Benicky J, Pavel J, Larrayoz IM, Zhou J, Baliova M, Nishioku T, Saavedra JM. Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen. Am J Physiol Regul Integr Comp Physiol. 2009;296:R1376–R1384. doi: 10.1152/ajpregu.90962.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Chen S, Ge Y, Si J, Rifai A, Dworkin LD, Gong R. Candesartan suppresses chronic renal inflammation by a novel antioxidant action independent of AT1R blockade. Kidney Int. 2008;74:1128–1138. doi: 10.1038/ki.2008.380. [DOI] [PubMed] [Google Scholar]
  • 40.Sanz-Rosa D, Oubina MP, Cediel E, de Las Heras N, Vegazo O, Jimenez J, Lahera V, Cachofeiro V. Effect of AT1 receptor antagonism on vascular and circulating inflammatory mediators in SHR: role of NF-kappaB/IkappaB system. Am J Physiol Heart Circ Physiol. 2005;288:H111–H115. doi: 10.1152/ajpheart.01061.2003. [DOI] [PubMed] [Google Scholar]
  • 41.An J, Nakajima T, Kuba K, Kimura A. Losartan inhibits LPS-induced inflammatory signaling through a PPARgamma-dependent mechanism in human THP-1 macrophages. Hypertens Res. 2010;33:831–835. doi: 10.1038/hr.2010.79. [DOI] [PubMed] [Google Scholar]
  • 42.Chomarat P, Banchereau J, Davoust J, Palucka AK. IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol. 2000;1:510–514. doi: 10.1038/82763. [DOI] [PubMed] [Google Scholar]
  • 43.Pozo D, Vales-Gomez M, Mavaddat N, Williamson SC, Chisholm SE, Reyburn H. CD161 (human NKR-P1A) signaling in NK cells involves the activation of acid sphingomyelinase. J Immunol. 2006;176:2397–2406. doi: 10.4049/jimmunol.176.4.2397. [DOI] [PubMed] [Google Scholar]
  • 44.Steiniger B, Stehling O, Scriba A, Grau V. Monocytes in the rat: phenotype and function during acute allograft rejection. Immunol Rev. 2001;184:38–44. doi: 10.1034/j.1600-065x.2001.1840104.x. [DOI] [PubMed] [Google Scholar]
  • 45.Abboud FM, Harwani SC, Chapleau MW. Autonomic neural regulation of the immune system: implications for hypertension and cardiovascular disease. Hypertension. 2012;59:755–762. doi: 10.1161/HYPERTENSIONAHA.111.186833. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS408863-supplement-01.pdf (675.9KB, pdf)

RESOURCES