INTRODUCTION

Polymorphonuclear leukocytes or granulocytes are hematopoietically derived phagocytes characterized by multilobed nuclei and the presence of multiple, distinct granules within their cytoplasm. Three different polymorphonuclear leukocytes are distinguished according to their granular staining properties: neutrophils (polymorphonuclear neutrophils or PMNs), basophils, and eosinophils. Neutrophil granules stain preferentially with neutral dyes, whereas basophil granules stain with basic dyes, and eosinophilic granules stain with acidic colorants such as eosin. These three types of leukocytes differ not only in their tinctorial properties, but also in their functions and roles during the inflammatory process. They constitute key effector cells in innate immunity, and the frontline of host defense in response to foreign antigens and microorganisms. In this chapter, we will focus on the biology and role of neutrophils. The other polymorphonuclear leukocytes are discussed elsewhere.

NEUTROPHIL HOMEOSTASIS: MYELOPOIESIS AND DESTRUCTION

Neutrophil myelopoiesis is a closely regulated pro cess that begins with the differentiation of pluripotent stem cells into primitive myeloid progenitors, which in turn differentiate into specific myeloid precursors. Contact with specific adhesion molecules, hematopoietic growth factors, and cytokines promotes the progression of myeloblasts along unique pathways to mature as neutrophils, eosinophils, and basophils, as well as monocytes. The sequence that leads to neutrophil formation begins with the neutrophilic promyelocyte and progresses through several maturation steps (neutrophilic myelocyte, metamyelocyte, band cell, and mature neutrophil) (Figure 4A.1).

RHEUMATOID ARTHRITIS

Rheumatoid arthritis (RA) is an extremely painful, debilitating, and destructive inflammatory disease of diarthrodial joints. It affects between 0.5% and 1% of the world's population, with women having a threefold prevalence. Using traditional therapy, the majority will develop moderate disability at 2 years, with 40% being unable to work at 5 years. The disease has a high cost in pain, disability, and deformity. Morbidity is high, with a reduced lifespan. The advent of new treatment paradigms has meant that disability has been reduced, orthopedic surgeries have a much reduced rheumatoid joint replacement case-load, and whilst still severe, the aim of therapy is to preserve normal lifestyle and work patterns.

This is achieved by the use of low-dose methotrexate, leflunomide, or sulphasalazine, followed by biologic therapies, mainly anti-tumor necrosis factor (anti-TNF-α), anti-B cell (anti-CD20), recombinant human IL-1 receptor antagonist (rhIL-1ra), or anti-interleukin-6 (anti-IL-6). There remains a severe problem that despite these regimes a significant proportion (up to 40% for anti-TNF-α) do not respond. In addition, these treatments are expensive in their own right, and heavy on clinical resources for administration and monitoring. There is thus a continuing requirement for the development of improved therapeutics through drug discovery and further development of current therapeutics and their targets. A detailed understanding of the pathogenic mechanisms of RA are also required to fulfill these aims. Animal models of rheumatic disease continue to play a significant role in this process.

INTRODUCTION

The rapid response of the innate immune system of humans depends in large part on the behavior of a subset of leukocytes known as polymorphonuclear leukocytes or neutrophils. Constituting the majority of white blood cells in the circulation, neutrophils provide continuous surveillance for microbial and noninfectious threats to the integrity of the host. Once recruited to an area of infection, neutrophils ingest microbes and thereby confine them in the phagosome, a membrane-bound compartment where an array of antibacterial factors can be delivered to kill and degrade the trapped microorganism. In addition to proteins that exhibit direct antimicrobial action, cytoplasmic granules in neutrophils contain enzymes that utilize microbial constituents as substrate, thereby compromising the structural integrity of the organism and indirectly contributing to microbial killing. Under normal circumstances, these complex and synergistic events occur in the presence of reactive oxygen species (ROS) generated in situ by activation of a multicomponent enzyme, the phagocyte NADPHdependent oxidase. Like the prefabricated granule constituents, ROS damage targets directly and act in concert with granule proteins to generate second derivative active agents. The concomitant release of granule proteins and activation of the NADPH oxidase thus provide phagocytic cells such as neutrophils with the capacity to respond rapidly to potentially life-threatening challenges and thus provide the initial wave of host defense.

INTRODUCTION

The kidney receives disproportionately high blood flow, Blood flows to each renal filtering unit, known as the nephron, through a specialized vascular bed called the glomerulus, which is by design uniquely leaky to solutes and plasma proteins (Figure 28B.1). Human glomeruli filter 144 liters of plasma filtrate a day from the bloodstream (Figure 28B.2). Disruption of the glomerulus interferes with blood flow to the downstream segments of the nephron due to the presence of a second capillary network deriving from the glomerular capillaries, frequently culminating in ischemia of the nephron, with consequent chronic inflammatory responses to ischemic injury of parenchymal cells. Furthermore, the unique regulation of vascular tone in the kidney paradoxically can worsen ischemic insults by triggering vasoconstriction. Physiologically, compromised blood flow in the medulla of the kidney is a prerequisite for effective reabsorption of ≈142 liters a day of filtered solute back into the blood compartment, but renders the high-energy-requiring components of the medulla especially susceptible to ischemic injury.

The glomerulus is particularly prone to inflammatory injury, perhaps due to the tendency of immune complexes and other abnormal proteins to become lodged in the specialized filtering basement membranes, which then trigger immune responses not seen in other regions of the vasculature. Rarely autoimmunity directed at unique proteins within the specialized glomerular filtering membranes also triggers stereotyped responses in the glomerulus, which we now recognize as immune cell and parenchymal cell responses to injury.

INTRODUCTION

Autacoids are locally acting substances that are rapidly biosynthesized in response to specific stimuli, act quickly, and are usually deactivated by metabolism. Eicosanoids are a chemically diverse family of arachidonic acid–derived autacoids that have critical roles in cardiovascular, inflammatory, and reproductive physiology. Pharmacologic interventions in eicosanoid pathways – including the nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, and leukotriene inhibitors – are useful in the clinical management of inflammation, pain, and fever. Given the many important bioactivities of lipid mediator eicosanoids, resolvins, and protectins, future research may lead to the development of new therapeutics for the treatment of inflammatory conditions, autoimmune diseases, asthma, glomerulonephritis, cancer, sleep disorders, and Alzheimer's disease.

BIOSYNTHESIS OF EICOSANOIDS

Eicosanoids are crucially involved in a number of metabolic pathways that have diverse roles in inflammation and cellular signaling. These pathways center on reactions involving the metabolism of arachidonic acid (Figure 12.1). The following considers the biochemical steps leading to arachidonic acid synthesis, then discusses the cyclooxygenase (COX), lipoxygenase (LOX), epoxygenase, and isoprostane pathways of arachidonic acid metabolism. Arachidonic acid (cis-, cis-, cis-, cis-5, 8, 11, 14-eicosatetraenoic acid), the common precursor to eicosanoids, must be synthesized from the essential fatty acid precursor linoleic acid (cis, cis-9, 12-octadecadienoic acid), which can be obtained only from dietary sources. In cells, arachidonic acid is esterified to the sn 2 position of the membrane phospholipids predominantly in phosphatidylcholine phosphatidylethanolamine, and phosphatidyl inositol.

CASE 1

Presentation and Initial Assessment

A 64-year-old male with a history of diabetes mellitus type II, hypertension, hyperlipidemia, and known mild renal insufficiency (chronic kidney disease, stage 2) presented with fevers and chills, and worsening of kidney function (baseline plasma creatinine 1.5, increased to 1.8 mg/dL). His standing medications were modified and renal function was noted to return to baseline. One month later at follow up he reported fever, his renal function had deteriorated further with plasma creatinine level of 3.0 mg/dL, and he was found to be anemic. He received a blood transfusion and a course of antibiotics for presumed upper respiratory tract bacterial infection but one month later returned with lethargy, oliguria, and further deterioration in renal function with creatinine 4.4 mg/dL. At this point, he was referred to a nephrologist for further assessment.

At that assessment by a nephrologist, his family history was notable for thromboembolic disease, but he denied current upper or lower respiratory tract symptoms, rashes, joint pains, areas of numbness, black or red bowel movements. His medications at this presentation included Hydralazine Metoprolol Felodipine, Insulin subcutaneously, Ezetimibe, Glimepiride, Zolpidem, multi-vitamin qd, calcium, vitamin E, fish oil, and acetaminophen.

Clinical examination: weight 200 lbs, temperature 96.7F, pulse 63 beats per minute, blood pressure 160/80 mmHg, respiratory rate 20 per minute, peripheral blood oxygen saturation 97% on room air. He was a middle-aged man, pleasant, in no distress. Pupils and eye movements and oropharyngeal exams were normal.

INTRODUCTION

Nonsteroidal anti-inflammatory drugs (NSAIDs), sometimes called the “ aspirin-like drugs,” are among the most widely used of all drugs. Aspirin itself was introduced by Bayer in 1898 as a replacement for salicylic acid, which had been available in synthetic form since the 1870s and as the active constituent of plant (e.g., willow bark) preparations for many centuries before that. Since the beginning of the 20th century, the number of NSAIDs has grown substantially: phenylbutazone was introduced in the 1940s, the fenamates in the 1950s, indomethacin in the 1960s, the proprionates in the 1970s, and the oxicams in the 1980s. The 1990s saw a radical new development – the introduction of “ coxibs ” and the new millennium has ushered in an era of reappraisal and reassessment of our understanding of the pharmacology of these drugs and their therapeutic and unwanted effects. There are now more than 50 different NSAIDs on the global market and some of the more prominent examples are listed in Figure 18.1. (Chemical structures of NSAIDs shown, grouped according to chemical structure.)

NSAIDs had been in clinical use for a long time prior to an understanding of their pharmacological mechanism of action. The real breakthrough in NSAID pharmacology was made in the early 1970s when John Vane identified that NSAIDs work by inhibition of prostaglandin (PG)-forming cyclo-oxygenase (COX) activity resulting in the reduction of prostanoid synthesis. The second breakthrough came about in the early 1990s with the discovery of COX-2 that eventually lead to the development of the COX-2-selective NSAIDs that lacked some of the major side effects of classical NSAIDs.

INTRODUCTION

The eye is one of the primary sense organs. This unique organ is composed of highly specialized tissues: an outgrowth of the central nervous system, the retina, on the posterior end and a highly modified transparent skin, the cornea, on the anterior end (Figure 31.1). In the simplest terms, the primary function of the eye is to convert light energy into nerve action potentials. This essential and evolutionarily conserved function directly depends on maintaining (1) the refractive properties of the cornea, lens, aqueous humor, and vitreous humor; (2) formation and reabsorption of fluids that keep intraocular pressure in the fluid-filled eyeball constant; and (3) the function of retinal neurons. The eye, as an organ that faces the external environment, provides a unique opportunity to directly access highly specialized and diverse tissues such as the immune-privileged avascular cornea and the conjunctiva, a mucosal tissue that directs host defense and immune response of the ocular surface. Moreover, since light has to pass through the eye, physiological processes and cells in the anterior (cornea), interior (lens, uvea), and posterior (neural and vascular retinal layers) tissues of the eye (Figure 31.1) can be studied by noninvasive procedures in animals and humans [1 – 7].

The visual axis is a delicate sensory organ that has to maintain an avascular and transparent state in order to preserve ocular function. Hence, in the eye, especially in the cornea and retina, the threat of inflammation is incompatible with good vision.

WHAT IS A STROMAL CELL?

The architecture of organs and tissues is closely adapted to their function to provide microenvironments in which specialized functions may be carried out efficiently. The nature and character of such microenvironments are primarily defined by the stromal cells that reside within the tissues. The most abundant cell types of the stroma are fibroblasts, which are responsible for the synthesis and remodeling of extracellular matrix (ECM) components. In addition, their ability to produce and respond to growth factors and cytokines allows reciprocal interactions with adjacent epithelial and endothelial structures and with infiltrating leukocytes. As a consequence, fibroblasts play a critical role during tissue development and homeostasis and have often been described as having a “landscaping” function. In this chapter, we concentrate on fibroblasts as the prototype stromal cell. However, the stroma also consists of blood and lymphatic vessels, and a wider definition of stromal cells might include endothelial cells, specialized cells such as pericytes (blood vessel supporting cells), and even tissue resident macrophages.

Stromal Cell Identity and Microenvironments

Tissue resident macrophages in the liver (Kupffer cells) and lung (alveolar macrophages) perform very different functions compared to macrophages in the brain (glial cells) or skin (Langerhans cells), yet they are all members of the monocyte/macrophage family. Until recently, fibroblasts had been thought of as ubiquitous, generic cells with a common phenotype even within different tissues.

INTRODUCTION

Inflammation has emerged as a key pathophysiological event in vascular diseases and the consequent cardiac and cerebral ischemic injury. There is ample evidence that inflammation is intimately involved in atherosclerosis. It mediates the initiation of atherosclerosis, promotes progression of the atherosclerotic lesions, and regulates atheromatous plaque stability [1, 2]. There is also good evidence that inflammation plays a crucial role in ischemia-reperfusion cardiac and cerebral injury [3, 4].

Inflammation is a complex process involving multiple cellular and molecular components. It is triggered by diverse proinflammatory mediators (PIM) which are generated directly and indirectly by microbial invasion, endotoxins, immune complexes, and cytokines. Vascular endothelium is subjected to pro inflammatory insults, as it is in constant contact with circulating blood and along with it many environmental stressful factors. Fortunately, endothelium is endowed with potent anti-inflammatory molecules that confer resistance to damage by transient proinflammatory attacks. Once the insulting factors dissipate, endothelial cells return to its basal state. The mechanisms by which endothelial cells resist insults are likely to be very complex. One model is stress-coupled induction of anti-inflammatory and cytoprotective genes [5]. This mechanism allows for timely defense against transient insults. However, when insults by PIMs become persistent, this protective property wears out resulting in endothelial cell damage and functional defects and eventually endothelial apoptosis and necrosis.

BIOLOGICAL FUNCTIONS OF ADHESION MOLECULES

Cell adhesion molecules are endowed with many biological functions, often crucial to life. For instance, in gut or lung epithelia they assure cell-to-cell contacts, thus making and maintaining barrier functions, while allowing passage of nutrients and other solutes. Besides maintaining body form and structure, cell adhesion molecules play important roles in tissue development as well as helping to determine its final organization. A common feature of adhesion molecules is their ability to sustain strong bonds between adjacent cells on either a permanent or intermittent basis.

Apart from these static functions of cell adhesion molecules, there are many mobile functions, whereby they are able to induce intermittent cell-to-cell bonds hence assuring particular cell movements either in the blood vessel (as during inflammation; topic to be investigated in this chapter) or within tissues and organs.

It is therefore evident that, contrary to previous simplistic visions and predictions, cells are never free floating but rather they move on surfaces such as the inflamed joint or gut subepithelial strata, penetrate into injured organs or circuit the body (think of T cells traveling the blood lymphatic circulation, and so forth) by activating processes all relating to cell adhesion molecule-mediated phenomena.

INTRODUCTION

Mast cells are tissue-dwelling hematopoietic effector cells that are endowed with a range of potent inflammatory effector molecules. They are implicated in both allergic and nonallergic diseases, as well as in innate and adaptive immunity to infectious agents based on animal studies. Mast cells constitutively reside in a perivascular distribution in connective tissues and at mucosal surfaces (Figure 5.1). They are especially abundant in tissues that form interfaces with the external environment (skin, conjunctivae, intestinal, and airway mucosa), suggesting a strategic placement so as to function in a first line of host defense. Mast cells are best known for their role as effectors of classic type 1 hypersensitivity (allergic) reactions. In such reactions, mast cell activation is initiated by the binding of multivalent allergen to membrane-bound IgE that is coupled with the tetrameric high-affinity Fc receptor for IgE (FcεRI) on mast cells. IgE-dependent activation of mast cells results in their release of preformed inflammatory mediators that are stored in their secretory granules, including histamine, neutral proteases, preformed cytokines, and proteoglycans (Figure 5.2). In addition, activated mast cells secrete newly synthesized lipid mediators that are the products of endogenous arachidonic acid metabolism, such as prostaglandin (PG)D2, leukotriene (LT)B4, and LTC4, the parent molecule of the cysteinyl leukotrienes (cys-LTs).

INTRODUCTION

Eosinophilic granulocytes, commonly referred to as eosinophils, or less commonly as acidophils, were first identified by Paul Ehrlich in 1879. He named these bilobed nucleated cells as eosinophils because of their intense staining with the acidic dye eosin. Ehrlich completed his original studies that defined the eosinophil's morphological features during a time when he was committed to a clinical practice as well as basic research, an early example of a physician scientist. Combining his clinical expertise with his laboratory discoveries, he proposed a variety of functions for eosinophils that included phagocytosis, granule secretion, and chemotaxis.

Eosinophils are normally present as a minority of the peripheral blood leukocyte pool and primarily reside within tissues containing mucosal surfaces such as the uterus and gastrointestinal tract. While their exact function is not certain, eosinophils are thought to possess both beneficial and deleterious properties as participants in both innate and adaptive host immune responses. As an arm of the innate defense system, eosinophils are strongly proposed to be responsible in part for combating parasitic infections, particularly with helminths. When recruited in excess, eosinophils are currently thought to participate in mediating the pathogenesis of allergic diseases such as asthma, atopic dermatitis, gastrointestinal diseases, and hypereosinophilic syndromes.

EOSINOPHIL MORPHOLOGY

Eosinophils are approximately 12–17 μm in diameter, and represent 1%–6% of the total blood leukocyte population (Figure 7.1).

INTRODUCTION

The inflammatory response consists of an innate system of cellular and humoral responses following injury (such as after heat or cold exposure, ischemia/reperfusion, blunt trauma, etc.), in which the body attempts to restore the tissue to its preinjury state. In the acute inflammatory response, there is a complex orchestration of events involving leakage of water, salt, and proteins from the vascular compartment; activation of endothelial cells; adhesive interactions between leukocytes and the vascular endothelium; recruitment of leukocytes; activation of tissue macrophages; activation of platelets and their aggregation; activation of the complement; clotting and fibrinolytic systems; and release of proteases and oxidants from phagocytic cells, all of which may assist in coping with the state of injury. Whether due to physical or chemical causes, infectious organisms, or any number of other reasons that damage tissues, the earliest in vivo hallmark of the acute inflammatory response is the adhesion of neutrophils (polymorphonuclear leukocytes, PMNs) to the vascular endothelium (“margination”) (Figure 1.1). The chronic inflammatory response is defined according to the nature of the inflammatory cells appearing in tissues. The definition of chronic inflammation is not related to the duration of the inflammatory response. Reversal or resolution of the inflammatory response implies that leukocytes will be removed either via lymphatics or by apoptosis (programmed cell suicide) and that the ongoing acute inflammatory response is terminated.

RHEUMATOID ARTHRITIS

Rheumatoid Arthritis: Clinical Case

A 60-year-old man presented with a 4-week history of a gradually worsening ankle pain and swelling. He gave no history of morning stiffness, or symptoms in any other joints. There was no preceding history of infection (including of the gastrointestinal or genitourinary tract) and no history of previous inflammatory disease (including the skin and eye). There was a family history of rheumatoid arthritis with his daughter having been diagnosed with this condition at the age of 30. He smoked 10 cigarettes per day and drank 6 units of alcohol per week. He had been treated with diclofenac by his primary care physician but had derived little benefit from this.

On examination, he had tenderness with clinically apparent synovial swelling at the left ankle (Figure 22.1A). The remainder of the physical examination was normal.

The differential diagnosis of an inflammatory monoarthritis includes septic arthritis and crystal arthritis and to exclude these diagnoses the patient underwent an ultrasound guided joint aspiration (Figure 22.2). No organisms were identified on synovial fluid microscopy or culture and no crystals were seen on polarized light microscopy. Further investigations revealed the following: ESR 16 mm/h, CRP 17 mg/L (normal < 5 mg/L), rheumatoid factor 72 IU/mL (positive > 20 IU/ mL), anti-CCP antibody 81 U/mL (positive > 10 U/mL), chest radiograph normal.

INTRODUCTION

Asthma is a disease of chronic airway inflammation. This condition is prevalent worldwide and accounts for significant morbidity, excess mortality, and substantial health care expenditures [1, 2]. Asthma is clinically defined by three characteristics, namely reversible airflow obstruction, airway hyperresponsiveness, and airway inflammation [1]. There is no cure for asthma, but many therapies have been developed to lessen the burden of the disease. In light of the need for additional therapeutics to treat and ultimately cure asthma, several animal experimental models have been developed to perform preclinical investigation of asthma pathogenesis and novel therapeutics. Simply stated, asthma is only a human disease. None of the current animal models entirely recapitulates asthma [3], but they have proven very useful in the investigation of asthma traits. In this chapter, the most common animal models of asthma and their features will be described with particular attention to the airway inflammatory responses.

ASTHMA PATHOBIOLOGY

Asthma has a complex pathogenesis and can be considered a clinical syndrome of intermittent dyspnea, wheezing, chest tightness, and/or cough. In most subjects, airway inflammation is present [1]. The inflammatory cell infiltrate is enriched with eosinophils, T lymphocytes and, in some cases, neutrophils, especially in the setting of asthma exacerbations. This complex, chronic airway inflammation is likely initiated and driven by signals from sentinel cells in the airway, including airway epithelia and dendritic cells, responding to provocative stimuli.