Approximately one-third of the Western population has hypertension, and this disease becomes more frequent with aging. This disease is also a major risk factor for cardiovascular disease, causing stroke, heart failure, renal failure, and cognitive decline. Despite the frequency of hypertension and its profound impact on human health, the precise cause of most cases of human hypertension remains essentially unknown. Rare monogenic causes of hypertension have been identified, but are extremely rare and are not thought to underlie most cases of hypertension. Dysregulation of central neural signaling, renal dysfunction, and alterations of vascular reactivity have all been implicated, but a concise understanding of how these become abnormal and how they interact to produce clinical hypertension has been elusive. Multiple genome-wide association studies (GWAS) have identified genetic loci associated with hypertension, but what might tie these together and actually instigates clinical disease is unknown. In the past several years, it has become apparent that hypertension is often accompanied by an inflammatory process in which immune cells infiltrate and alter function and structure of the kidney and the vasculature. As stressed in this chapter, an emerging paradigm is that this inflammatory reaction promotes not only blood pressure elevation, but also the end-organ damage associated with hypertension.
Inflammation is the biologic response to invading organisms, irritants or injury, and is essential to combat invading organisms, foreign bodies and neoplasia. Unfortunately, inflammation occasionally becomes excessive and persists beyond the initial insult, contributing to numerous chronic degenerative processes. Celsus described the cardinal signs of inflammation as dolor, rubor, calor and tumor (i.e., pain, heat, redness, and swelling). Notably, these are largely vascular phenomena mediated by vasodilatation, increased permeability and in some cases release of pain mediators such as endothelin-1 and substance P from vascular cells. A fundamental aspect of inflammation is infiltration of immune cells through the vessel wall into the interstitium of the affected tissue, governed by endothelial production of adhesion molecules that promote initially sticking of immune cells to the endothelial surface and chemokines that promote diapedesis of these cells through junctions between endothelial cells. This latter event involves molecular interactions between leukocytes and the endothelium and rearrangement and loosening of endothelial cell junctions. A truly miraculous aspect of this process is that there is a concomitant increase on the immune cells of ligands that recognize the adhesion molecules expressed by the activated endothelium, localizing the inflammatory process to sites of infection or injury.
General Considerations of the Immune System
The two major arms of the immune system are innate and adaptive immunity. Innate immunity includes chemical and humoral mediators such as nitric oxide, reactive oxygen species (ROS), complement, acute phase proteins, chemokines, and cytokines. Natural immunoglobulin M (IgM) and immunoglobulin G3 (IgG3) antibodies, produced largely by B1 cells, are present in infants and adults before exposure to an antigen and confer innate protection to viruses and bacteria but can also participate in autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus. Some natural antibodies target lectins, present on the surface of microbes and apoptotic cells. Cellular components of innate immunity include phagocytic cells, including granulocytes, monocytes and macrophages and natural killer (NK) cells. Other cells of the innate immune system include dendritic cells, newly identified innate γ/δ T cells and epithelial cells, which provide a barrier to invading organisms. These features of innate immunity have been reviewed in depth recently.
Several components of the innate immune system will be discussed in greater detail later, however special mention of monocytes and monocyte-derived cells is warranted. Approximately 5% to 10% of circulating leukocytes are monocytes. These survive in the circulation for approximately one to two days, and do not undergo further proliferation, but are capable of enormous phenotypic differentiation. A major impetus for this differentiation is transmigration of these cells through the endothelium which, as mentioned earlier, is triggered by various inflammatory stimuli. Upon entry of monocytes into the interstitial space, monocytes can undergo at least three fates ( Fig. 7.1 ). Most commonly recognized is their conversion to macrophages, which remain in the interstitium and can phagocytose injurious bodies and release potent mediators including reactive oxygen species (ROS), nitric oxide (NO), cytokines, and matrix metalloproteinases. It is now recognized that there is also a population of tissue resident macrophages, not derived from circulating monocytes, which participate in tissue healing. Unlike monocyte-derived macrophages, tissue resident macrophages undergo proliferation and self-renewal. A second fate of monocytes is to differentiate into dendritic cells, powerful antigen-presenting cells for T cell activation, which are discussed more fully later. A final fate of monocytes is to reemerge from the vessel wall without differentiation. It is now recognized that monocytes can enter tissues, acquire antigens and transport these to lymph nodes where they can activate T cells without becoming either dendritic cells or macrophages. These minimally differentiated monocytes are typified by their surface expression of major histocompatibility complex type II and the activation marker Ly-6C and an enhanced ability to activate T cells.
Although the innate immune system is nonspecific and provides immediate protection, it does not have the ability to augment protection upon repeated antigenic challenges. In contrast, adaptive immunity provides powerful and specific defense against previously encountered antigens. Components of the adaptive immune response include T cells, responsible for cellular immunity, and B cells, which upon activation by T cells differentiate into either short or long-lived antibody producing plasma cells.
A critical step in initiation of the adaptive immune response is uptake and processing of antigens by antigen presenting cells (APCs). Although several cells can present antigens, the major APCs are macrophages, B cells, and dendritic cells (DCs). CD4 + T cells are generally activated by peptides that have been derived from extracellular antigens presented in the context of type II major histocompatibility complexes (MHCs), whereas CD8 + T cells are activated by intracellular antigens, such as invading viruses, that are presented by type I MHCs. APCs that have acquired antigen undergo maturation, increasing expression of costimulatory molecules and production of cytokines that can direct T cell polarization. The activated APC migrates to secondary lymphoid organs and seeks a T cell with a T cell receptor that recognizes and binds the MHC/peptide complex. The subsequent immunologic synapse formed by these two cells leads to T cell activation, involving proliferation, an increase in cytokine production and a change in surface receptors that arm the cell to leave the secondary lymphoid organ and migrate to inflamed peripheral tissues. B cells also phagocytose and present antigen, and their formation of an immunologic synapse with T helper cells promoting their proliferation and formation of germinal centers in lymph nodes and tertiary sites.
T cell subsets exhibit division of duty. CD4 + T cells are conditioned or polarized by the cytokines they encounter upon initial stimulation, leading to unique T helper phenotypes. T H 1 cells produce proinflammatory cytokines such as interferon (IFN)-γ, IL-2 and tumor necrosis factor (TNF)α. T H 2 cells produce IL-4, IL-5 and IL-13, cytokines involved in response to allergens and helminth infections. T H 17 cells produce the unique cytokine IL-17, and play critical roles in diseases such as psoriasis, experimental allergic encephalitis and inflammatory bowel disease. T regulatory cells represent another subset of T cells that suppress the immune response. Activated CD8 + T cells have cytolytic activity, manifested principally by their release of isoforms of granzyme and perforin, but they can also release cytokines, and in fact are major sources of IFN-γ. These features of adaptive immunity have been reviewed in depth elsewhere.
Historical Aspects Regarding Inflammation and Hypertension
It has been recognized that there is an inflammatory component in human hypertension for more than half of a century, and that immune cells contribute to blood pressure elevation in several experimental models. In 1953, Heptinstall reported that lymphocytic infiltrates were commonly observed in the kidneys of humans undergoing sympathectomy and or adrenalectomy for hypertension. In 1964 White and Olsen showed that cortisone and mercaptopurine could lower blood pressure in a rat model of hypertension caused by renal infarction. Subsequently Okuda and Grollman found that transfer of lymph node cells from these rats could passively raise blood pressure in otherwise normal recipients. In 1970, Olsen showed that chronic angiotensin II infusion in rats lead to a striking periarteriolar infiltrate of lymphocytic and monocytic cells. Soon thereafter, Dr. Olsen demonstrated a striking periarteriolar infiltration of immune cells in humans with hypertension, and pointed out that these appeared to be lymphocytes and monocytes. In 1976, Svendsen demonstrated that athymic nude mice exhibit blunted hypertension in response to deoxycorticosterone acetate (DOCA)-salt challenge but that this phenotype was normalized by grafting of thymus tissue into these animals. Subsequently, Olsen showed that transfer of splenocytes from rats with DOCA-salt hypertension conferred an increase in blood pressure in recipient rats. Antithymocyte serum was shown to reduce hypertension in spontaneously hypertensive rats. These studies among others strongly supported the idea that the immune system contributes to hypertension via mechanisms that were poorly understood at the time.
Recent Evidence Implicating Immune Cells in Hypertension
Advancements in the field of immunology have markedly enhanced our ability to understand the role of immune cells in hypertension. As an example, the development of mice lacking recombination activating gene-1 allowed Guzik et al to show that T cells are essential for the development of hypertension. These mice lack both T and B lymphocytes, and were found to exhibit blunted hypertensive responses to either chronic Ang II infusion or DOCA-salt challenge. Reconstituting T cells completely restored hypertension in these mice. Studies of Dahl salt sensitive rats have confirmed a role of T cells in this salt sensitive model of hypertension. Mechanistic studies in mice lacking T cell costimulatory proteins or various cytokines including IL-17A, IFN-γ and IL-6 have illustrated an important role for these mediators in hypertension. An evolving notion is that CD8 + T cells seem to play an important role, via mechanisms that are incompletely understood. Mice specifically lacking CD8 + T cells are more protected against hypertension than mice lacking CD4 + T cells. Moreover, circulating CD8 + T cells in humans with hypertension display a senescent phenotype and evidence of activation. These cells produce large amounts of IFN-γ, which has been implicated in producing renal injury and promoting local angiotensinogen production in the kidney.
B lymphocytes have also been implicated in hypertension. Chen et al found that Ang II-induced hypertension is associated with a striking increase in serum IgG and in the aortic adventitia and that the hypertensive response to Ang II is attenuated in mice lacking B cells. The precise roles of B cells, their production of antibody and their ability to present antigen to T cells in hypertension remain unclear. In preeclampsia, agonistic antibodies against the angiotensin II type 1 receptor play an important role in blood pressure elevation.
There is also substantial evidence supporting a role of innate immune cells in hypertension. Wenzel et al showed that hypertension increases the accumulation of monocyte/macrophages in the artery wall and deletion of monocyte/macrophages completely prevents Ang II-induced hypertension in mice. The authors provided evidence that these cells contribute to the production of vascular reactive oxygen species and vascular dysfunction. There is also increasing evidence that dendritic cells, and in particular inflammatory dendritic cells derived from monocytes, play a crucial role in T cell activation and contribute to hypertension. Natural killer (NK) cells, which are important sources of IFN-γ, infiltrate the arterial wall in hypertension and seem to contribute to vascular dysfunction and formation of reactive oxygen species.
Recently, myeloid-derived suppressor cells (MDSCs) have been found to have a protective effect in hypertension. These are immature cells that can suppress T cells responses and reduce inflammation, most notably in the setting of neoplasia. MDSCs increase in experimental models of hypertension and depletion of these cells exacerbates blood pressure elevation and kidney injury, whereas adoptive transfer of these cells blunts hypertension.
The previous discussion illustrates that almost all components of the immune system contribute to hypertension. This is typical of many inflammatory conditions, and reflects the interdependence of innate, adaptive, cellular and humoral immunity.
Basic Mechanisms by Which Inflammation Contributes to Hypertension
Before discussing how immune cells contribute to hypertension, it is useful to consider currently accepted mechanisms of hypertension to begin to understand how inflammatory cells might affect these processes. As discussed in the introduction, there is substantial debate as to the etiology of most cases of adult hypertension. Indeed, the origins of hypertension are probably diverse. Nonetheless, perturbations of renal function, vascular function, and central neural control are supported by extensive investigation. There is compelling evidence that some degree of renal dysfunction must be present to sustain hypertension. This is based on the concept of the pressure natriuresis curve in which an elevation of blood pressure leads to a brisk diuresis, restoring blood pressure to its initial set point. In contrast, a lowering of blood pressure leads to a reduction in urine output, leading to volume and sodium retention until blood pressure rises to the set point. Guyton pointed out that all forms of hypertension are therefore associated with a resetting of this set point to a higher level, such that the kidneys no longer respond with diuresis at this higher level of pressure. Although overt renal failure is often associated with hypertension, alterations of the pressure natriuresis curve can involve subtle alterations of renal function not manifested by reduced glomerular filtration rate or elevations of blood urea nitrogen or serum creatinine. In fact, monogenic causes of hypertension, including Liddle syndrome and pseudohyperaldosteronism type II, involve enhanced sodium resorption in the distal nephron with otherwise normal renal function. Autocrine and paracrine factors including Ang II, nitric oxide, reactive oxygen species, endothelin-1 and prostaglandins influence renal sodium transport and their actions on the nephron have been implicated in hypertension. Many extrarenal stimuli, including catecholamines, aldosterone, vasopressin and as discussed later, inflammatory cytokines, can affect the pressure natriuresis curve without causing overt changes in renal function parameters. As discussed later in this chapter, several immune cell released cytokines affect tubular and vascular function and seem to promote sodium and volume retention in hypertension ( Fig. 7.2 ).
Blood pressure is the product of cardiac output and systemic vascular resistance, and thus an increase in blood volume and cardiac output would be expected to increase blood pressure. Vascular resistance, and particularly renal vascular resistance, is elevated in many cases of human essential hypertension, suggesting a vascular etiology. Indeed hypertension is associated with several perturbations of resistance vessel function and structure ( Fig. 7.3 ). Vasodilatation, particularly that mediated by endothelial nitric oxide production, is often compromised in hypertension and vascular remodeling, involving an increase in medial thickness and a decrease in lumen diameter, is common. These effects are in part mediated by cytokine stimulation of reactive oxygen species. Vascular fibrosis occurs at both the level of the resistance circulation and as discussed later, in larger vessels. Vascular rarefaction, or disappearance of capillaries and small resistance vessels, is also a common consequence of hypertension. These processes disable proper autoregulation and result in increased systemic vascular resistance.
An emerging vascular mechanism of hypertension relates to stiffening of the central large arteries, and particularly the aorta. Although large vessels have not been considered important in regulating systemic vascular resistance, it has become clear that aortic stiffening is a common harbinger of hypertension. Central arteries expand during systole, accommodating a portion of the ejected blood, and recoil in diastole, propelling blood to the distal tissues. In this way healthy arteries maintain diastolic perfusion. Aortic stiffening is clinically detected as an increase in pulse wave velocity and becomes abnormal in a variety of conditions, including aging, diabetes, obesity, tobacco abuse and hypertension. In large population studies, aortic stiffening precedes the development of hypertension by several years. The precise mechanisms linking aortic stiffening to gradual onset of hypertension remain undefined, but likely involve alterations of pulse wave contour reaching peripheral tissues like the kidney, the microcirculation and the brain, ultimately leading to damage of these tissues. Indeed, aortic stiffening portends conditions such as renal failure, heart failure, atherosclerosis, stroke and dementia.
In addition to renal and vascular causes of hypertension, there is convincing evidence that perturbations of the central nervous system contribute to hypertension. The lamina terminalis of the forebrain is composed of the subfornical organ (SFO), the median preoptic area (MPO) and the organum vasculosum of the lateral terminalis (OVLT). The SFO and OVLT possess poorly developed blood-brain barriers, and are sensitive to circulating mediators such as angiotensin II and salt, which increase neuronal firing in these structures and have input into the hypothalamus, and in particular the paraventricular nucleus. The paraventricular nucleus (PVN) in turn provides input to the rostroventral lateral medulla of the brainstem. This latter structure integrates baroreflex input with signals arising from the higher centers to regulate blood pressure. Hypertension is associated with abnormal neuronal firing, increased angiotensin II signaling and oxidative signaling in all of these structures. Importantly, lesions within these structures and local blockade of the renin angiotensin system have profound effects on blood pressure. As an example, lesions of the anteroventral third ventricle (AV3V) region, which disrupt fibers from the SFO to the OVLT, prevent most forms of experimental hypertension. Likewise, injection of angiotensin receptor blocking agents into the rostral ventrolateral medulla (RVLM) prevents hypertension. An emerging concept is that of the neuroimmune axis, in which sympathetic outflow modulates immune cell activation ( Fig. 7.4 ), whereas afferent signals from the periphery inhibit further sympathetic outflow. There is evidence that this inhibitory loop is disrupted in hypertension.
The Role of Immunity and Inflammation as Mediators of Hypertension
It has been difficult to understand how the kidney, vasculature, and brain interact to regulate blood pressure. An emerging concept is that immune cells are activated and infiltrate these various organs, and may serve to transmit and intensify alterations of function of the brain, vasculature, and kidney. It should be stressed that inflammation is generally not thought to cause hypertension on its own, but to intensify dysfunction of the kidney, vasculature, and central nervous system and exacerbate hypertension. Thus, the transformation of mild or prehypertension to clinical hypertension might mark activation of the immune system and inflammation in various end organs. Several general events mediated by inflammatory cells contribute to this process as discussed later.
Cytokine Release
A predominant role of almost all immune cells is to release various cytokines. These are extremely powerful and act locally to affect adjacent cells, including vascular and renal tubular cells. Several T cell, macrophage and dendritic cell-derived cytokines have been implicated in hypertension, including interleukin IL-6, IL-17A, IFN-γ and TNFα. As an example, hypertension in mice lacking IL-17A is blunted and these mice do not exhibit alterations of endothelium-dependent vasodilatation or increases in vascular superoxide production in response to Ang II infusion. Direct application of this cytokine to vascular segments inhibits endothelial NO production and infusion of IL-17A elevates blood pressure via mechanisms involving activation of Rho kinase. IFN-γ has likewise been implicated in causing tissue damage and dysfunction in hypertension. Ang II-induced hypertension increases T cell production of IFN-γ in rats and mice, and this cytokine has been implicated in cardiac and renal injury. Mice lacking the transcription factor T box expressed in T cells (Tbet), which is essential for IFN-γ production, are protected against endothelial dysfunction and have reduced vascular expression of several NADPH oxidase subunits. Likewise, mice lacking IL-6 are markedly protected against Ang II-induced hypertension via mechanisms involving the Janus kinase 2/signal transduction and activator of transcription 3 (JAK2/STAT3) pathway.
Although substantial research has focused on the vascular actions of these cytokines, there is also substantial evidence that these mediators alter renal function in ways that could promote hypertension. Interleukin 6 produced by innate cells plays a critical role in skewing T cells to produce IL-17, but also promotes production of angiotensinogen by renal tubular cells, promoting intrarenal production of Ang II. IFN-γ has also been shown to induce angiotensinogen production in these cells. Another major effect of cytokines is to enhance renal tubular sodium retention. We have recently shown that the changes in renal tubular sodium transporter expression and activation caused by Ang II are blunted in mice lacking either IFN-γ or IL-17A. These changes are paralleled by a reduction in the antidiuretic and antinatriuretic effects of Ang II. Thus, immune cells that infiltrate the kidney in hypertension likely produce these cytokines, which in turn affect local Ang II production and enhance sodium retention.
There is increasing evidence that inflammation in the central nervous system contributes to hypertension. Shi et al demonstrated that microglial cells within the paraventricular nucleus (PVN) of the hypothalamus of rats with Ang II-induced hypertension produce increased amounts of IL-1β, IL-6 and TNFα compared to sham-infused rats. Intracerebroventricular (ICV) infusion of the antibiotic minocycline, which suppresses microglial activation, decreased production of these cytokines, lowered systemic norepinephrine levels and reduced both blood pressure and left ventricular hypertrophy in these animals. The authors further demonstrated that adenoviral expression of the antiinflammatory cytokine IL-10 in the paraventricular nucleus also lowered blood pressure and reduced left ventricular hypertrophy whereas ICV infusion of IL-1β induced hypertension. In a related study, these investigators showed that transplant of bone marrow from normal Wistar Kyoto rats to spontaneously hypertensive rats reduced activation of microglial cells and blood pressure in the recipient mice. Pallow et al showed that Ang II-induced hypertension is associated with an increase in infiltration of T cells around the subfornical organ of the brain, and that this is sex-dependent Deletion of an antioxidant enzyme in the paraventricular organs of the brain, which increased sympathetic outflow, raised both blood pressure and activation of T cells. In contrast, deletion of a component of NADPH oxidase, which decreases sympathetic outflow, reduced T cell activation. Recently, we showed that renal denervation markedly reduced activation of dendritic cells and T cells in the kidney and provided evidence that the kidney might be a major site of immune activation. Mice exposed to an emotional stress paradigm develop hypertension and T cell activation whereas mice lacking T cells are protected against stress-induced hypertension.
Oxidative Injury
Many of the effects of these cytokines and immune cells are related to oxidative stress and injury. Phagocytic cells like monocyte/macrophages use the NADPH oxidase to generate large amounts of ROS upon activation and are likely sources of ROS when they accumulate in blood vessels or kidney. Mice lacking various components of this enzyme complex are partially protected against various forms of experimental hypertension. Moreover, cytokines like IFN-γ, TNFα, IL-6 and IL-17 enhance expression of NADPH oxidase subunits in various cells increasing vascular superoxide production. In vessels, ROS inactivate nitric oxide, promote vasoconstriction, enhance vascular smooth muscle hypertrophy and growth, and activate matrix metalloproteinases, which facilitate vascular remodeling. ROS also promote apoptosis, which contributes to vascular rarefaction. In the kidney, in addition to promoting renal vasoconstriction, ROS have been shown to activate sodium transport within the proximal tubule, the medullary thick ascending limb and in the cortical collecting duct. There is also evidence to support a role of ROS in modulating podocyte injury. These effects of ROS in hypertension have previously been reviewed in depth.
Matrix Reorganization
Vascular hypertrophy, luminal narrowing and rarefaction are commonplace in hypertension, and predispose to the increases in systemic vascular resistance encountered in this disease. For cells to proliferate, change size or migrate, there must be degradation of the extracellular matrix that encases cells. Matrix metalloproteinases (MMPs) are major mediators of this process. Although almost all cells can produce these potent enzymes, cells of the innate immune system, including macrophages, monocytes, neutrophils, and mast cells are major sources. These pluripotent enzymes not only degrade matrix, but can activate other enzymes, liberate cell bound mediators, promote tissue calcification and enhance apoptosis, necrosis and cell senescence. Short peptides released by MMPs have been referred to as matrikines that activate other immune cells. An important consequence of MMP activation is the conversion of latent transforming growth factor β (TGFβ) to active TGFβ, which via its signaling through Smad pathways, plays multiple roles in matrix deposition. The Smads act as transcription factors that promote collagen synthesis and also signal formation of myofibroblasts, major sources of extracellular matrix. TGFβ is a product of antigen presenting cells, including macrophages and dendritic cells, and is also an important modulator of T regulatory cell formation. IL-17A, released by activated CD4 + T cells stimulates fibroblasts to produce several isoforms of collagen and fibronectin via p38MAP kinase. An important renal consequence of matrix deposition is nephrosclerosis, a hallmark of hypertensive renal disease, a major cause of renal failure.
It has become increasingly apparent that specialized monocyte-like cells, often termed fibrocytes, play a major role in tissue fibrosis. These are bone marrow-derived cells that are recruited to sites of inflammation in a fashion similar to other monocytes, and are characterized by robust production of collagen and fibronectin. We recently found that about 60% of the collagen forming cells in the aorta are bone marrow derived in experimental hypertension. Interestingly, resident fibroblasts seem to represent less than 20% of the collagen-forming cells of the aorta, emphasizing the complexity of this process and the importance of immune cells in tissue fibrosis.
Enhanced Chemotaxis
A recurring observation in hypertension is that products of immune cells in the kidney and blood vessels enhance accumulation of other immune cells. As an example, when hypertension is induced in mice lacking IL-17A, there is a marked decrease in the presence of T cells and monocyte/macrophages in blood vessels. Likewise, the vascular accumulation of all leukocytes caused by chronic Ang II infusion is reduced in mice lacking lymphocytes. This is likely because cytokines such as IL-17A and TNFα promote production of chemokines that attract other immune cells. In addition, these cytokines can promote endothelial activation and expression of adhesion molecules that enhance attraction of other immune cells. This interaction emphasizes the feed forward nature of the inflammatory response that can occur in hypertension and illustrates the importance of local interactions between the innate and adaptive immune cells.
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