Translational Inflammation

students.

Preface

Jeffrey K. Actor; Keri C. Smith

Understanding the role of inflammation as a function of clinical disease states is required for both design of treatment modalities and the development of therapeutics. A greater knowledge of inflammatory responses at all levels of disease is critical for health care workers, physicians, and students of all biological systems. It should be noted from the start that inflammation is critical for everyday health and homeostasis, without which the body cannot discern regular biological turnover and events from outside interference that causes harm.

There is a basic assumption that the overarching objective of the immune response is to protect an individual from pathogenic assault. When closely examined, the immune system is partitioned into segments that are involved in both health maintenance and protection. Inflammation is an essential component for these processes. We typically think of protection as a requirement for recognition of foreign nonhost molecules, with the end result of producing factors that are involved in effector processes. As a first step, inflammation is a by-product of those early induced events, primarily as an effective tool to trigger reactive paths that clear pathogens. However, inflammation is also critical for homeostasis, with common activities involved in control of deleterious responses. These pathways are critical for effective maintenance, such as normal cellular turnover during aging, healing of tissue damaged by trauma, and keeping the immune response in a ready state of surveillance for pathogenic components that may enter the body.

We have organized this book to allow the reader to gain a perspective on the integral role of the inflammatory response in medical applications, in a way that permits discussion of its control as a therapeutic mechanism to combat disease and manifestation of clinical disorders (Fig. 1). The text begins with an overview of the inflammatory process, with a higher level examination of processes involved in inflammatory activity. This will permit the reader a basic orientation to allow a more thorough understanding of subsequent chapters.

Fig. 1 Inflammation: overlapping information subsets discussed throughout this text. Our discussion begins with an overview of inflammation and a description of regulators of response. Subsequent chapters discuss the inflammatory response in medical applications, for both systemic and organ-specific pathologies and clinical disorders.

Chapters 2–4 focus on specific molecules, or sets of mediators, involved in the initiation and maintenance of inflammation. The application of these molecules to the resolution of inflammation and a return to homeostasis is also explored. From a clinical standpoint, the end result of the inflammatory process is manifested by damage to specific tissues or by dysregulation of an organ system. Therefore, molecules that have emerged as critical in regulation of inflammation are explored, with each explained as exerting control over distinct pathways.

The subsequent chapters are translational and represent an in-depth discussion of targeted applications which approach inflammation in a disease-specific fashion. Thus, Chapters 5–13 focus on the clinical consequences of unchecked inflammation and attempts at therapeutic modification of pathological processes. In this manner, we chose to highlight the role of inflammation in various disease states arising due to infection, organ dysfunction, autoimmunity, trauma, or other assault on human tissue caused by uncontrolled or aggressive inflammation.

The target audience for this assembled text ranges from graduate students in both medical and theoretical fields, to the resident needing a better understanding of the inflammatory response, to physicians in need of clarification of underlying mechanisms that cause pathological damage. We feel that these chapters and their concepts are ones that have value to multiple audiences. Each chapter contains an overview of the molecules involved in disease development and a link to clinical manifestation of related disorders.

Multiple events are involved in normal physiology of tissue maintenance. In reality there is a balance between factors necessary for response to damage and subsequent tissue repair. Indeed, we urge the reader to keep in mind that inflammation may be defined as a homeostatic fulcrum, integral in both sides of the equation to allow flexibility in function in normal homeostasis. Most often, inflammation is beneficial to the host, but the response may also cause tissue damage. Understanding how pathways control the balance of response is critical for therapeutic intervention and development of translational aspects to treat disease and disorders.

Chapter 1

Translational Inflammation

Jeffrey K. Actor; Keri C. Smith    Department of Pathology and Laboratory Medicine, McGovern Medical School, University of Texas Health Science Center, Houston, TX, United States

Abstract

Understanding the role of inflammation as a function of clinical disease is required for both the design of treatment modalities and the development of therapeutics. Inflammation is an essential component critical for everyday health and immune homeostasis, without which the body cannot discern regular biological turnover and events from outside harmful interference. This review examines the processes involved in the inflammatory response, with emphasis on defining the cellular players and chemical mediators of this process. We begin with a historical definition of inflammation and describe elements of inflammatory danger signals. The primary focus is to understand innate inflammation; however, consequences of hypersensitive inflammation are also discussed. This information provides an overview of the complex processes of inflammation that form the underlying basis for unique organ-specific pathologies.

Keywords

Inflammation; Myeloid cells; Lymphoid cells; Antibodies; Complement; PAMPs; DAMPs; Hypersensitivity

Introduction

Understanding the role of inflammation as a function of clinical disease states has become invaluable in both design of treatment modalities and the development of therapeutics. A greater knowledge of inflammatory responses at all levels of disease is critical for health care workers, physicians, and students of all biological systems. But it should be noted from the start that inflammation is critical for everyday health and homeostasis, without which the body cannot discern regular biological turnover and events from outside interference that causes harm. I.R. Cohen stated it most directly when he wrote The function of the immune system is to produce inflammation commensurate with the body’s need to keep itself fit for living [1].

There is a basic assumption that the objective of the immune response is to protect an individual from pathogenic assault. However, when closely examined, the immune system is partitioned into factors that are involved in both protection and in the maintenance of health. We typically think of protection as a response to recognition of nonhost molecules, with the end result of producing factors that are involved in effector processes. Inflammation is a by-product of those early induced events, where it functions primarily to trigger reactive paths that clear pathogens. Longer term, well-regulated inflammation is also critical for homeostasis. These pathways are critical for effector maintenance, such as normal cellular turnover during aging, healing of tissue due to trauma, and keeping the immune response in a ready state of surveillance for pathogenic components that may enter the body.

Inflammation: A Definition

It is critical to define inflammation to allow discussion of responses to be placed within a clinical or pathological setting. A simplistic working definition includes the response of living tissue to damage or assault of any kind. At its root, inflammation represents a response to insult to vascularized tissues, with a purpose to deliver defensive materials (blood cells and fluid) to a site of injury [2–5]. Main components of inflammation revolve around vascular reactions and cellular responses that activate chemical or cell-derived mediators. A strong feedback mechanism controls active participation of leukocytes and factors involved in maintaining normal healthy tissue status (Fig. 1.1). Upon injury, released factors in the form of chemical mediators diffuse from the region to function on vascular beds, triggering endothelial cells to alter surface conformation to manipulate delivery of circulating factors and cell populations into subvascular space. A process ensues whereby chemotactic factors allow activation in localized blood vessels (capillaries, venules, or arterioles) to permit diapedesis of specific leukocytes into the underlying parenchyma. Cells migrate toward inflammatory sites following a chemotactic gradient. In a similar manner, blood-borne components carried in exudate accumulate outside of vascular beds, resulting in the delivery of serum factors essential for a functional response. The optional endpoint of this feedback reaction is resolution of tissue damage and protection of the host from outside elements (pathogenic or physical). However, the inflammatory response may be caustic in and of itself; if it persists it may result in damage and extended pathology.

Fig. 1.1 Biological elements of inflammation. Multiple parameters drive the inflammatory response, leading to development of pathological consequences and clinical disease states.

Historical Definition of Acute Inflammation

As the subsequent chapters of this book will demonstrate, the clinical consequences of inflammation are varied and unique to each organ or tissue. Why do we then ascribe a single condition, inflammation to the processes that lead to this damage? The combination of signs and symptoms observed following injury or illness is visible and consistent. The word inflammation is derived from the Latin inflammare, which literally means to set on fire. Greek literature suggests that ancient healers had an understanding of inflammatory response. Indeed, the first written definition of the condition was provided in the 1st century by Cornelius Celsus who noted four cardinal signs: RUBOR ET TUMOR CUM CALORE ET DOLORE (redness and swelling with heat and pain) [6]. We now understand that redness occurs due to changes in localized blood flow (vasodilation); swelling occurs from influx of fluid and cells leaving blood vessels to enter tissue; heat is the result of increased blood arriving to areas of damage; and pain results from released molecular mediators and edema (fluid accumulation) which increases pressure on, and activates, local nerves surrounding the damaged site. The concept that this response was more than tissue damage in response to injury was introduced in the 3rd century by Galen, whose description of laudable pus suggested that inflammation was beneficial and necessary for wound healing. The advent of microscopy in the late 19th century allowed for the identification of the cellular components of inflammation, further underscoring the role of infiltrating immune cells in the response. At this time, Virchow added a fifth classical sign of an inflammatory response—FUNCIO LASEA (loss of function) [7]. It is this loss of tissue function that we still clinically grapple with today.

A Bird’s Eye View of the Inflammatory Process

Reaction to cellular injury can take multiple forms, due to initiating stimuli that either directly or indirectly manipulate responding cells in the area affected. The innate immune system is exquisitely sensitive to inflammatory triggering signals and has the ability to quickly ramp-up recognition upon real or perceived alterations in homeostasis [8]. It is now understood that infectious agents contain molecular motifs (e.g., danger signals) which trigger resting innate immune cells to initiate molecular activation of internal processes. However, it should be appreciated that physical agents, such as toxins and chemicals, can also activate similar responses. In fact, simple tearing of cellular membranes and subsequent release of phospholipids can result in vasoactive change (dilation or constriction) with the end result of altered permeability for blood-derived components to enter damaged areas. Often, these signals arise from localized cell death due to ischemia, yet they can also be elicited under the influence of blunt physical trauma.

The initiation through stimuli induces further cellular responses, generally characterized as those mediated by locally residing innate cells (macrophages, dendritic cells, or fibroblasts), and those recruited from vascular regions (neutrophils and specialized first responding lymphocytes such as natural killer cells). The cellular response is immediate, with explicit release of both preformed and induced substances from innate cell phenotypes. These effector factors (cytokines and chemokines) subsequently diffuse from the area of damage or danger to nearby blood vessels where they induce changes in endothelial cell membranes. A directed process of recruitment then occurs, based primarily on adhesion and migration properties of multiple circulating cell populations. Concurrently, fibrous matrix ladders assemble, providing a cellular roadway for trafficking through subcapillary beds.

Overt changes in vascular permeability result in the delivery of a fluid exudate, which allows nutrients and oxygen to flow into areas of need. With respect to microorganisms, the sheer influx of fluid (edema) creates a buffer by diluting toxins. This vasodilation also permits fluids (serum) containing circulating antibodies, complement and acute phase proteins to accumulate within damaged tissue. The antibodies that are present combine with toxins neutralizing destructive activities. Complement components, either by themselves or with the help of antibodies, directly lyse microorganisms and coat them (opsonization) with breakdown products of the complement enzymatic cascade. This greatly assists in directed uptake and phagocytosis of encountered pathogens.

While we tend to think of fluid and cellular flow into damaged tissue as a one-way process, it should be noted that it is in fact dynamic. Signals from reactive components leave damaged sites and enter the blood stream with the purpose of systemic activation. Distant organs are affected, including those responsible for thermoregulation (e.g., the hyptothalamic, pituitary, and adrenal axis), and those responsible for production of additional enzymes and acute reactants (such as the liver). Over time, activated innate immune cells make their way to draining lymphatics and to lymph nodes where they condition lymphocytes for secondary hypersensitivity reactions. This step is critical for the adaptive second phase of response where lymphocytes are actively recruited to the area of inflammation; these lymphocytes have the capability to respond with exquisite specificity to nonhost antigenic stimuli. It is this second wave of immune cells that are ultimately responsible for prolonged pathological consequences resulting from the initial stimuli. If the situation is limited in duration and scope, the acute reaction has minimal lasting effect. Resolution occurs with limited tissue reorganization. However, chronic persistence with repeated stimulation due to a contribution of innate and antigen-specific adaptive components can lead to progressive damage of lasting consequence and clinical scope.

Inflammation is thus a combination of desirable and nondesirable events, with these terms defined according to the underlying stimuli initiating reactivity. The clinical outcome is truly a balance between responses which benefit the health of the host, versus responses that lead to destructive pathology.

Inflammatory Danger Signals

PAMPs and DAMPs

Encounter with external stimuli can initiate the inflammatory response (Table 1.1), with the end result often dictating downstream (adaptive) immunity [9]. Response to invading bacteria or viruses has evolved to combat their attempts at colonization. Basic phagocytic innate cells, macrophages and dendritic cells in particular, are hardwired to respond to motifs, or danger signals, present on and within microorganisms [10]. These signals are common pathogenic patterns (pathogen-associated molecular patterns; PAMPs) specific for noneukaryotic cells, usually linked to pathogen survival. Examples of PAMPs include peptidoglycan and lipomannans, lipopolysaccharide and lipoteichoic acid, bacterial DNA, double-stranded RNA, and flagellin. Recognition occurs via unique surface molecules expressed by phagocytes (e.g., mannose receptors, scavenger receptors, Toll-like receptors (TLRs), or C-type lectin receptors (CLRs)) which trigger subsequent endocytosis.

Table 1.1

Binding of the specific motifs to intracellular NOD-like receptors (NLRs) allows translation of encounter of the danger signal to initiate a protective proinflammatory response via the assembly of an inflammasome [11]. These are multicomponent molecular structures that link the bound NLR to the downstream activation of caspase-1 ultimately culminating in the production of small chemical mediators (cytokines, chemokines) that work in concert to allow cells to communicate with each other and facilitate cross-talk. These molecules also lay the groundwork for destruction of the unwanted pathogen. Because of their varied nature, these mediators are quite pleiotropic and exert actions on multiple different cell types, often with overlapping and redundant functionality.

Realize that in addition to pathogens, danger signals presented by physical agents may also induce inflammation. Damage-associated molecular patterns (DAMPs) represent a second class of inflammatory inducing signals, usually host-derived biomolecules that initiate and perpetuate a noninfectious inflammatory response [12]. In many cases, these alarmins are released by stressed tissue undergoing necrosis [13]. A class of molecules that should not be overlooked are those represented by foreign objects that trigger this type of stress inflammation. Irritants and corrosive chemicals (acids, alkalis, oxidizing agents) can cause cellular damage. This can also be seen in response to trauma, ultraviolet light, radiation, and even frostbite. In all of these examples, inflammation is elicited; the underlying mechanisms are initiated via damage to cellular membranes and/or apoptotic-induced events. Released membrane phospholipids combine with natural lipases to produce arachidonic acid, which then triggers prostaglandin and leukotriene production which in turn mediates vasoactive properties, endothelial cell permeability, and chemotaxis of polymorphonuclear cells. In fact, any cause of gross tissue necrosis, be it lack of oxygen or nutrients, inadequate blood flow due to infarction, or physical dysplasia, will initiate similar inflammatory events.

By definition, a toxin initiates a destructive process which often triggers inflammation. Bacterial toxins are biological virulence factors that prepare the host for colonization. A classic example is the lipopolysaccharide (LPS), a powerful endotoxin representing a portion of the outer membrane of Gram-negative bacteria. In contrast to endotoxins, bacterial exotoxins are soluble mediators released during bacterial cell lysis or destruction. A specific class of exotoxins, called enterotoxins, are especially toxic to the intestinal tract causing vomiting and diarrhea. Well-defined toxins (neurotoxins, leukocidins, or hemolysins) are classified in terms of the specific target cell or site affected.

The Cellular Players in the Inflammatory Response

To understand the development and control of the inflammatory response, it is imperative to understand the roles that each phenotypic cell plays in the process. A global chart for these cells is given in Table 1.2. Each individual cell type is further described to allow a context for activity in the overall process. These cells include the myeloid and lymphoid phenotypes, as well as fibroblasts, endothelial cells, and platelets.

Table 1.2

Myeloid Cells (Monocyte/Macrophages and Polymorphonuclear Cells)

Monocytes/Macrophages

Monocytes and macrophages are directly involved in phagocytosis and intracellular killing of microorganisms [14]. Macrophages are differentiated monocytes, which are one of the principal cells found to reside for long periods in the reticuloendothelial system. These monocytes/macrophages are highly adherent, motile, and phagocytic; they marshal and regulate other cells of the immune system, such as T lymphocytes; they also serve as antigen-processing/presenting cells. Upon encountering different stimuli, monocytes differentiate into highly microbicidal (M1) or into immunosuppressive macrophage (M2) phenotypes [15]. Macrophages are immediate responders during inflammation, changing phenotype in concert with immune evolution in response to threats and needs for homeostatic and surveillance tasks, as well as for tissue regeneration and repair [16]. They bear functional pattern recognition receptors on their surface, as well as complement and antibody receptors, which make them key regulators for recognition of inflammatory stimuli. Upon activation, they secrete chemokines and cytokines that both regulate and attract other immune cells, as well as having strong impact on local vascular endothelium [15]. Indeed, release of these inflammatory mediators strongly impacts the direction of subsequent immune function, both in a localized manner as well as systemically to affect tissues throughout the body. It should be stressed that while macrophages are critical in initial inflammatory responses, they also contribute to chronic responses. Finally, macrophages also regulate healing properties such as renewed vascularization of tissue and wound closure [17].

It should be noted that the monocytic macrophages are called macrophages when they are in the connective tissue, spleen, and lymph nodes. However, many submucosal macrophages have specific nomenclature, dependent upon tissue localization. For example, they are called Kupffer cells in the liver, Dust cells in the lung, Microglia in the central nervous system, osteoclasts in the bone, and Langerhans cells when found in the skin.

Dendritic Cells

Dendritic cells (DCs) provide a link between innate and adaptive immunity by interacting with T cells in a manner to deliver strong signals for development of functional and memory responses. They represent an intriguing and diverse population [18, 19] whose phenotypic control is under precise regulation [20]. They direct local responses during infection [14] and inflammation, as well as participate in the generation of matured responses posttrafficking to lymph node tissues [21, 22]. Immature DCs are called to the site of inflammation via chemokines, in a complex signaling path which is dependent upon both physical location and necessity for specific triggering by other immune cells responding to the insult or assault [23]. Dendritic cells recognize foreign agents and pathogens through a series of pattern recognition receptors (nonspecific) and are similar to macrophages in that they are able to present antigen to lymphocytes to direct maturation and immune functionality. They play critical roles within the skin (where they are referred to as Langerhans cells) and have special properties to assist in wound healing and repair [24].

Neutrophils

Neutrophils are the most highly abundant, motile, phagocytic leukocytes which are one of the first cells recruited to acute inflammatory sites. They are extremely important in development of acute and chronic inflammation [25, 26]. They ingest, kill, and digest pathogens, with their functions dependent upon special proteins, such as adherence molecules, or via biochemical pathways (respiratory burst). Neutrophils contain primary lysosomes which contain myeloperoxidase and acid hydrolases; they also contain secondary granules containing inflammatory mediators, such as lactoferrin, which can be released upon activation. Neutrophils express high levels of antibody receptors and complement receptors which allow increased phagocytosis of invading organisms. As a mechanism to help control the spread of intracellular pathogens, neutrophils are capable of deploying extracellular traps (NETs) which consist of decondensed nucleosomes, neutrophil elastase, and myeloperoxidase [27]. Critical to the inflammatory process, neutrophils are recruited via specific chemokines (interleukin-8; CXCL8), through a dynamic process where they extravasate from endothelial-lined vessels to enter tissue. Activation leads to respiratory burst producing reactive oxygen and nitrogen intermediates, and subsequently release primary and secondary granules containing proteases, phospholipases, elastases, and collagenases.

Eosinophils

Eosinophils defend against larger parasites and participate in hypersensitive reactions via cytotoxic events [28]. Their cytotoxicity is mediated by large cytoplasmic granules, which contain eosinophilic basic and cationic proteins. These granules are somewhat unique and contain a dense filamentous core of major basic protein, forming structures known as crystalloids. The matrix of the granules contains lysosomal enzymes, and in particular has a high content of peroxidase, arylsulfatase, acid phosphatase, RNase, and cathepsin. While the functionality of eosinophils is classic ascribed to parasitic infections, they also play a major role as responders in allergic reactivity to a variety of stimuli including pollen, and in some drug hypersensitivity reactions. These cells are known to phagocytose antigen-antibody complexes, which can also trigger responsiveness. They also have effects on leukotrienes, one major mediator of the inflammatory