Chapter II.2.3
Innate and Adaptive Immunity
The Immune Response to Foreign Materials
The goal of this chapter is to understand the general organization of the immune system (both specific and non-specific components), how the multiple different elements recognize apparent “invaders,” and what effector responses are generated to eliminate perceived threats. “Immunology,” as this subject is called, typically encompasses an entire course (with its own introductory text). Thus, an overview chapter can only paint with very broad brushstrokes what is a beautifully complex and intricate canvas of concepts regarding innate and adaptive immunity. The intent here is to provide a fundamental understanding of the body’s response to the insertion of a foreign device, so that the reader can anticipate the issues that arise at the biomaterials–tissue interface. More extensive discussion of any aspect of the immune system can be found in a number of excellent basic immunology texts (Coico et al., 2003; Abbas et al., 2007; Murphy et al., 2007), and from there to the primary literature (citations sprinkled throughout).
Overview
The immune system exists primarily to defend the host against infectious organisms. The general strategy begins with distinguishing “self” from “non-self.” Once the immune system determines that something does not belong, the responding population proliferates, recruits additional cells, and generates mediators to neutralize and destroy the invader. In most cases, the immune response is so exquisitely specific that “self” is not perceived as foreign, and so well-regulated that host tissues do not sustain any “innocent bystander” injury in the process.
Unfortunately, this is not always the case. Autoimmunity (inappropriate immune responses to self) can be the source of chronic, debilitating, and life-threatening disease. Moreover, infections can occasionally lead to substantially greater tissue injury, and even death, occurring as a direct consequence of host immune responses. Finally, one of the most powerful triggers of the immune system is tissue injury. This makes good functional sense, since evolutionarily most cases of injury are either caused by microbial agents, or tissues damaged by another cause often becoming secondarily infected. Thus, although the system evolved primarily to identify and eliminate infectious agents, noninfectious foreign materials (including biomaterials in medical devices or the process by which they are implanted) can also elicit immune responses. Occasionally, these can result – even if uninfected – in severe tissue damage.
Consequently, a more inclusive definition of immunity is the body’s reaction to any substance (microbes, proteins, polysaccharides, self, silastic implants, etc.) regardless of the pathologic consequences. In this chapter, we will initially focus on the physiologic pathways of immune activation in response to infectious agents, and describe the subsequent effector responses. We will then show how these same pathways can have pathologic outcomes, with special emphasis being given to the mechanisms underlying a response to a foreign biomaterial and/or implant.
An abbreviated glossary of terms is provided here to help the reader navigate the subsequent pages; other terms will be explained as they arise:
Adaptive immunity Immune responses conferred by T lymphocytes or antibodies. The complete repertoire of T cells and antibodies in any given host has the capacity to bind uniquely to a huge number of antigens or distinct molecular configurations (a feature called specificity). Subsequent exposure to the same molecular configuration leads to more robust, faster responses (a feature called memory).
Allogeneic Different than oneself, usually referring to the same molecule that has different configurations in different members of the same species (such as Major Histocompatibility Complex (MHC) molecules).
Allograft Transplanted tissue or organ from a different donor, usually of the same species. A heart transplant from one human to another is called an allograft.
Antigen A molecular configuration that is recognizable by the adaptive immune system. This molecular configuration can be a linear sequence of amino acids or a folded three-dimensional conformation; it can also be sugars, nucleotides or small molecules, or modified versions of any of these. Thus, a phosphorylated tyrosine in a short peptide can be antigenic. It is important to understand that antigens need not necessarily be foreign and can also be self-molecules. Moreover, just because a molecule is antigenic does not necessarily mean that it will elicit an immune response; issues related to the mode of antigen presentation, access, recognition, and tolerance induction all impact on host reactivity.
Autograft Transplanted tissue or organ from the same individual. Thus explanted skin that is expanded in culture ex vivo and returned to the same donor is an autograft.
Immunogenic Capable of eliciting a specific (adaptive) immune response.
Innate immunity Immune responses conferred by macrophages and neutrophils, as well as complement and other proteins that recognize a discrete set of fixed motifs. There is a limited repertoire of pathogen recognition receptors, and responding cells have the same response with each contact (i.e., there is no memory).
Isograft Transplanted tissue or organ from a different individual with identical genetic make-up. This is more typical in experimental situations involving inbred animal strains, but can occur in the setting of identical human twin donor and recipient pairs.
Syngeneic The same as oneself, usually referring to genetic make-up, and more specifically in transplantation, identity for Major Histocompatibility Complex (MHC) molecule expression.
Xenogeneic Differences across species. Thus, a porcine renal transplant into a baboon is a xenograft.
Innate and Adaptive Immunity
Defense against microbes is a two-stage process, beginning with a relatively non-specific innate response to “injury,” followed by a targeted adaptive response more uniquely focused on the specific causal agent (Figure II.2.3.1; Table II.2.3.1).
FIGURE II.2.3.1 Innate and adaptive immunity. Although with a limited ability to recognize invading organisms, the relatively primitive (evolutionarily-speaking) components of innate immunity provide a very effective first line of defense against the vast majority of microbial infections. Adaptive immunity, with exquisite specificity to any particular infectious agent, develops sometime later after innate components have responded. Notably, the elements of innate immunity not only respond first, but direct subsequent adaptive immunity; in turn, elements of the adaptive immune responses orchestrate a more efficient and vigorous response by the components of innate immunity. The specific kinetics of the responses shown are approximations, and will vary depending on the inciting agent.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
TABLE II.2.3.1 Components of Innate and Adaptive Immunity
| Innate | Adaptive | |
| Cellular and chemical barriers | Skin, mucosal epithelium, antimicrobial proteins (e.g., defensins, cathelicidins) | Lymphocytes in epithelia, secreted antibodies |
| Blood proteins | Complement, mannose-binding protein, C-reactive protein | Antibodies |
| Cells | Phagocytes (macrophages, neutrophils) natural killer cells | Lymphocytes |
Adapted from Abbas et al., 2007.
Components of Innate Immunity
Also called “natural” or “native” immunity, this is the initial, rapid (hours) response to infection or injury; it is mediated by both cellular and protein constituents (Figure II.2.3.2; Table II.2.3.2) (Medzhitov and Janeway, 2000; Medzhitov, 2007):
• physical and chemical barriers such as epithelia and anti-microbial proteins (e.g., defensins)
• phagocytic cells (neutrophils and macrophages) that ingest (phagocytize) and destroy microbes (see later)
• natural killer (NK) cells that kill virally-infected cells
• circulating proteins (complement, coagulation factors, C-reactive protein, etc.) that either directly insert pore-forming proteins in microbes that lead to cell death (e.g., complement, see below and also Chapter II.2.4), or that opsonize microbes (rendering them more “attractive” and readily phagocytized)
• cytokines: proteins secreted by cells of innate or adaptive immunity that regulate and coordinate the cellular response.
FIGURE II.2.3.2 Basic mechanisms of innate immunity. The principal cellular components of innate immunity in defense against microbial infection include phagocytes and natural killer (NK) cells; the principal protein constituents are cytokines and complement (a proteolytic cascade of related proteins, see also Chapter II.2.4). (A) Phagocytes (neutrophils and macrophages) will directly bind, ingest, and intracellularly degrade various microbes; in addition, macrophages can secrete cytokines to recruit and activate additional inflammatory cell types (e.g., neutrophils) to sites of infection, and will help drive the activation of the T lymphocytes of the adaptive immune response. Note that macrophages may also require the production of cytokines (e.g., interferon-γ) by other cell types in order to be most active. (B) Natural killer (NK) cells directly lyse virally-infected cells, as well as cells coated with bound antibody; in addition, they are a source of cytokines (e.g., interferon-γ) that can activate macrophages and T lymphocytes. (C) Cytokines are proteins produced by cells of innate and adaptive immunity that locally and systemically affect inflammation. In the figure, macrophages have been activated by lipopolysaccharide (LPS, a constituent of bacterial cell walls) to secrete tumor necrosis factor (TNF) and interleukin-12 (IL-12). TNF results (among other activities) in the production, recruitment, and activation of neutrophils. IL-12 drives NK cell activation, resulting in the production of interferon-γ that in turn activates macrophages. (D) Complement proteins form pores in the membranes of microbes to cause direct cell lysis; in addition, complement components will incite inflammatory cell recruitment, and augment phagocytosis (opsonize microbes).
(Figure reprinted with permission from Abbas et al. (2000), Cellular and Molecular Immunology, 4th ed.)
TABLE II.2.3.2 Components and Functions of Innate Immunity
Adapted from Abbas et al., 2000.
There is a bewildering plethora of cytokines with a wide array of activities influencing everything from endothelial cell function, to fibroblast synthetic activity, to lymphocyte differentiation (and more). Different cells will make different cohorts of cytokines, and any given cytokine can affect target cells differently. Multiple cytokines are in play in a particular location at any given time and can have synergistic or antagonistic effects on one another. Clearly, this is a fairly complex topic that warrants its own extensive treatment; for the purposes of this chapter, only selected cytokines will be discussed fleetingly, and the interested reader is encouraged to at least look at more comprehensive texts (Coico et al., 2003; Abbas et al., 2007; Murphy et al., 2007).
Innate immunity is an evolutionarily primitive system found even in invertebrates, and to some extent in plants (Litman et al., 2005); in most cases it can quite capably dispatch infections without the benefit of the lymphocytes or antibodies of adaptive immunity. Nevertheless, in cases where additional (and more specific) ammunition is required, the components of innate immunity are critical in mobilizing the subsequent lymphocyte and antibody effectors to clear invading microorganisms (see later).
Recognition in Innate Immunity
Innate immunity is triggered by molecular structures – called pathogen-associated molecular patterns (PAMPs) – that are common to groups of related microbes (Akira et al., 2006; Meylan et al., 2006; Palm and Medzhitov, 2009). These structures are characteristic of microbial pathogens and are not present on mammalian tissues; consequently, recognition via this pathway will distinguish self and non-self. Moreover, because the microbial products that are recognized are usually essential for the survival of the microorganism, they cannot be discarded or mutated. The pattern recognition receptors for pathogen-associated structures have a fairly limited diversity (numbering about 20 different types of molecules), and have no capacity to make fine distinctions between different substances; each cell of the innate system also expresses essentially the same cohort of receptors (Figure II.2.3.3). The components of innate immunity have no functional memory; they react in essentially the same way each time they encounter the same infectious agent, so that there is no mechanism to allow more rapid or specific responses upon a second encounter with the same agent.
FIGURE II.2.3.3 Pattern recognition receptors. Cells of innate immunity have a limited repertoire of receptors for foreign molecular structures; the same receptors are present in all cells, and they can be on the cell surface (e.g., the mannose receptor or scavenger receptor) or cytosolic (e.g., Nod-like receptors). Circulating molecules such as C-reactive protein and mannose-binding protein are also pattern recognition receptors; their binding to microbes can trigger subsequent complement activation or phagocytosis. Because the number of different receptors is relatively small, they are all encoded in the germline. In comparison, the recognition of antigens by the adaptive immune system is specific and unique for each potential antigen (see Figure II.2.3.10); to achieve the potential diversity of roughly 1010 different specificities requires somatic recombination of different gene segments.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
The PAMPs include:
• Double-stranded RNA found in cells containing replicating viruses. This induces cytokine production by infected cells leading to the destruction of the intracellular virus.
• Unmethylated CpG DNA sequences characteristic of bacterial infections. These induce autocrine macrophage activation and more effective intracellular killing of phagocytosed organisms.
• N-formylmethionine peptides from bacterial protein synthesis. Binding to receptors on neutrophils and macrophages causes chemotaxis (movement up a concentration gradient) and activation. Similar chemotaxis can be engendered by protein fragments released during complement activation, lipid mediators of inflammation, and chemokine proteins released by “stressed” cells.
• Mannose-rich oligosaccharides from bacterial or fungal cell walls. Engagement of receptors on macrophages induces phagocytosis; soluble mannose-binding protein in the plasma opsonizes or enhances phagocytosis of microbes bearing mannose.
• Bacterial or fungal wall oligosaccharides directly activate complement and induce either direct microbial lysis or microbial coating with complement that markedly enhances phagocytosis.
• Phosphorylcholine in bacterial cell walls binds to circulating C-reactive protein (CRP; also called pentraxin); CRP induces opsonization and also activates complement.
• Lipopolysaccharide (LPS) from bacterial cell walls binds to circulating LPS-binding protein which in turn binds to CD14 surface molecules on macrophages, activating it through an associated toll-like receptor (TLR). The macrophages respond by producing a host of cytokines including tumor necrosis factor (TNF) and interleukin-12 that recruit and activate neutrophils and NK cells, respectively. By similar pathways, LPS induces severe systemic responses that can culminate in septic shock (Hotchkiss and Karl, 2003).
Components of the innate system also recognize sites of injury, anticipating either that these may be primarily caused by infection or may be subject to subsequent infection. Heat shock proteins, altered membrane phospholipids, and urates released from the degradation of nucleic acids are among the danger signals released by necrotic tissues that can lead to macrophage activation (Kono and Rock, 2008). Moreover, components of the coagulation cascade, denatured connective tissue elements (such as might occur at sites of trauma), or denatured circulating proteins can also bind to macrophage cell surface receptors and induce activation. In particular, denatured proteins expressing previously cryptic RGD (arginine-glycine-aspartic acid) motifs that bind to cells via integrins can participate in the recruitment and activation of the cells of innate immunity. These become especially important in the context of the implantation of foreign bodies where otherwise minor trauma, and the presence of denatured proteins non-specifically adhering to “inert” surfaces, leads to macrophage activation (Tang and Eaton, 1999; Anderson et al., 2008).
Effector Mechanisms of Innate Immunity
As we will discuss in greater detail with the effector functions of adaptive immunity, the immune system needs to deal with pathogens lurking in two general environments – those that live in the extracellular space, and those that are intracellular. Extracellular pathogens are dispatched by a combination of direct killing (NK cells and complement) and ingestion (macrophages and neutrophils). Intracellular microbes (e.g., viruses) require either that the infected cell marshal its own defense mechanisms (such as interferon-α to reduce viral replication), or – failing that – be killed by NK cells (presumably taking down the virus at the same time), or even commit cellular suicide (apoptosis, see Chapter II.2.4).
Although the cells and proteins of innate immunity are primarily intended to clear infections, inappropriate, over-exuberant or poorly controlled effector activity can also result in host tissue injury. Once set into motion, activated proteases, macrophages and neutrophils, cytokines and other mediators will execute their functions somewhat blindly, and without regard to whether the “victim” is a microbe or self. This will continue until the initial stimulus abates or regulatory mechanisms supervene. Even business-as-usual for innate immunity can cause host cell death (i.e., the action of NK cells killing virally-infected cells), or loss of normal function (i.e., scarring of a tissue).
Complement
Complement is an important protein constituent of the recognition and effector arms of innate immunity (Roozendaal and Carroll, 2006), as well as an important mediator of adaptive immune function (Carroll, 2004) (see also Chapter II.2.4). It is comprised of a number of related proteins (called C1–C9, plus other co-factors B, D, and properdin) that form a self-amplifying proteolytic cascade – in other words, complement components earlier in the cascade enzymatically cleave and activate subsequent components. Late products in the cascade have the ability to form pores in cell membranes and cause cell lysis (a complex of C5b, C6, C7, C8, and C9 called the membrane attack complex or MAC), while many of the proteolytic fragments generated along the way (e.g., C3b, C5a, C5b) have additional useful activities (Figure II.2.3.4 and below) (Walport, 2001a,b). Complement in the circulation is synthesized by the liver; in tissues, macrophages constitute the major source. The complement cascade can be activated (albeit somewhat inefficiently) by binding directly to microbial surfaces or it can be activated by circulating proteins of the innate system (e.g., mannose binding protein) that bind to microbial PAMPs. However, it can be efficiently activated (103–104 better than direct activation) by antibody first binding to a particular antigen (discussed later).
FIGURE II.2.3.4 The major functions of complement. (A) Complement fragment C3b on the surface of microbes (or other cells) promotes phagocytosis (also called opsonization). (B) C3a and C5a proteolytic fragments increase vascular permeability and cause vasodilation by releasing histamine from mast cells; C5a is also chemotactic and enhances leukocyte binding to endothelium (shown here is a neutrophil), while stimulating leukotriene synthesis and the production of reactive oxygen species. (C) The C5b–C9 complex forms a membrane attack complex (MAC) that punches holes in microbes (and other cells) leading to osmotic rupture.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
The complement cascade can induce microbial death and/or cellular injury by:
• direct cytolysis via C5b–9, the membrane-attack complex (MAC) punching holes in a cell’s plasma membrane
• opsonization (via the C3b fragment), enhancing phagocytosis by macrophages and neutrophils
• C3a and C5a (so-called anaphylotoxins) mediate increased vascular permeability and smooth muscle relaxation (vasodilation), mainly by inducing mast cells to degranulate, releasing histamine from mast cells
• C5a also activates the lipoxygenase pathway in arachidonic acid catabolism, resulting in increased leukotriene synthesis
• C5a mediates chemotaxis of polymorphonuclear cells (PMN; neutrophils) and monocytes.
Neutrophils and Macrophages: Cells of Innate Immunity
The primary function of all the various recruiting and activating factors is to attract phagocytes (literally, eating cells) into a site of infection to ingest and destroy microbes (Figure II.2.3.5). The primary responding cell in the earliest stages of injury or infection is the neutrophil, a short-lived (hours) phagocytic cell capable of ingesting and destroying microbes, as well as releasing a variety of potent proteases. Neutrophils are the characteristic cellular feature of acute inflammation; these cells are typically first on the scene following injury (within 6–12 hours), and this phase of the host response to damage lasts for roughly 24–48 hours. Thereafter, the host response enters a phase of chronic inflammation, characterized by macrophage infiltration. Macrophages generally constitute the second wave of inflammatory cells recruited to sites of injury; they are substantially longer-lived and can persist at sites of inflammation, making them the dominant effector cell type in late stage innate immunity.
FIGURE II.2.3.5 Phagocytosis and intracellular destruction of microorganisms. Surface receptors on phagocytes can either bind microbes directly, or may bind opsonized microbes (for example, Mac-1 integrin binds microorganisms after they have been coated with complement proteins). After binding to one (or more) of the variety of surface receptors, microbes are internalized into phagosomes, which subsequently fuse with intracellular lysosomes to form phagolysosomes. Following fusion, reactive oxygen species (ROS, e.g., superoxide and hydrogen peroxide) and nitric oxide (NO) are selectively generated within the phagolysosomes; these reactive oxygen species kill the microbes largely via free radical injury; the lysosomes also contain a wealth of catabolic enzymes that digest the microbes.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
Both neutrophils and macrophages are recruited to sites of injury by changes in adhesion molecule expression on endothelial cells in the vicinity, and by chemotactic signals (acting e.g., through G-protein coupled receptors on neutrophils) delivered by injured cells (i.e., chemokines), by complement components (generated during complement activation), and by microorganisms themselves. The successive waves of inflammatory cell recruitment (neutrophils followed by macrophages) are regulated by distinct subsets of adhesion molecules and chemokines with different specificities. The recruited phagocytic cells clear opsonized microorganisms, kill them with reactive oxygen intermediates (superoxide, oxyhalide molecules, nitric oxide, and hydrogen peroxide), and degrade them with proteases (Figure II.2.3.5) (Segal, 2005). However, release of such cytotoxic and degradative molecules into the neighboring environment will also cause local tissue injury. Severe local injury due to excessive neutrophil activation results in an abscess with total destruction of parenchyma and stroma. C3b opsonization can also lead indirectly to tissue injury; large, non-ingestible cells or tissue can result in “frustrated phagocytosis” by neutrophils or macrophages. The attempted intracellular lysis results instead in the extracellular release of proteases and toxic oxygen metabolites (neutrophils), and/or cytokines (macrophages).
In addition, activated macrophages (and neutrophils to a more limited extent) release a variety of cytokines and other factors that can have both local and systemic effects (Figure II.2.3.6):
• tumor necrosis factor (TNF) recruits and activates neutrophils
• interleukin-12 (IL-12) activates T cells and NK cells
• activates coagulation pathways (tissue factor elaboration)
• secretes angiogenic factors (new blood vessel formation)
• fibroblast activating factors (e.g., platelet-derived growth factor; PDGF) that induce fibroblast proliferation
• transforming growth factor-β (TGF-β) expression increases extracellular matrix (ECM) synthesis and down-regulates many aspects of inflammation
FIGURE II.2.3.6 Macrophage recruitment and local tissue effects after activation. Circulating monocytes are recruited to sites of tissue injury by changes in adhesion molecule expression on endothelial cells in the vicinity of the injury, as well as by chemotactic signals (chemokines) delivered by injured cells or neutrophils, by complement components (generated during complement activation), and by microorganisms themselves. Once monocytes emigrate from the vasculature, they become tissue macrophages. Macrophages are activated by interferon-γ (IFN-γ) from various sources – including activated NK cells or T cells – or by non-immunologic stimuli such as endotoxin. Activated macrophages aggressively phagocytize microorganisms and necrotic debris, and also secrete a number of eicosanoids (arachadonic acid or AA metabolites such as prostglandins and leukotrienes), reactive oxygen intermediates, and proteases that can sterilize the local environment, but will also incite tissue injury. Overall, the cytokine mediators produced by activated macrophages will tend to induce fibrosis (scar).
(Figure reprinted with permission from Kumar et al. (2007), Robbins Basic Pathology, 8th ed.)
Thus (as can be inferred from the preceding descriptions), in the setting of prolonged activation, innate immunity ultimately drives tissue fibrosis and scarring (Figure II.2.3.6) (Henry and Garner, 2003; Martin and Leibovich, 2005). This clearly has ramifications in the setting of inserted foreign bodies intended to persist for many years.
Adaptive Immunity
Adaptive immunity (also called “specific” or “acquired” immunity) is comprised of cellular (lymphocyte) and humoral circulating protein (antibody) mediators; temporally, these will follow innate immunity in recognizing and resolving infections.
Adaptive immunity is more evolutionarily advanced than its innate counterpart, and is first seen in phylogenic development with the jawed vertebrates. Lymphocytes have the cardinal features of: (1) exquisite specificity for distinct macromolecules; and (2) memory, the latter being the ability to respond more vigorously to subsequent exposures to the same microorganism. The substances that induce specific immune responses are called antigens. These can be proteins, carbohydrates, lipids or other small molecules; they can also be self or non-self, microbial or non-infectious. The cells and antibodies of adaptive immunity each have the theoretical capacity to recognize 109–1011 distinct antigenic determinants. Since each cell of the adaptive immune system can only recognize and respond to a single antigenic determinant, the impressive diversity of the system requires an equally large number of different cells. Given that the human body only has 1013–1014 cells in total, there obviously cannot be more than a few tens to hundreds of cells of any one specificity at baseline. This has important ramifications in eliminating an infection; the small numbers of cells that can initially recognize an invader will not be up to the task on their own. Rather, following recognition of a pathogen, the responding cell(s) must clonally expand, as well as recruit an additional army of additional effectors (see later).
The other important concept is that the elements of adaptive immunity (like innate immunity) need to be able to respond to two basic infectious challenges – pathogens that are extracellular and those that reside within cells. As we will see when we look at effector mechanisms, the humoral and cellular elements complement each other in this enterprise – and also take advantage of the innate immune system. Thus, not only does the initial innate response influence the strength and nature of adaptive immune responses (e.g., whether antibodies or cellular mediators are produced), but the same effectors of innate immunity are also conscripted by adaptive immunity to clear infectious agents.
Components of Adaptive Immunity
The principal components of adaptive immunity are:
• B lymphocytes (also called B cells), responsible for making antibodies
• Antibodies: proteins secreted by B lymphocytes with specificity for a specific antigen
• T lymphocytes (also called T cells) are functionally divided into helper T cells (Th cells) that produce cytokines to orchestrate the activity of other cell types, and cytotoxic T cells (Tc cells) that kill selected target cells. Th cells express a cell surface protein designated CD4 (“CD” stands for “cluster of differentiation;” there are over 300 different CD molecules marking various cell types), and are therefore also generically referred to as CD4+ (“CD4-positive”) cells. Tc cells express CD8 and may be referred to as CD8+ cells. All T cells also express CD3 (actually a complex of γ, δ, and ε protein chains) that is involved in T cell activation.
• Cytokines: many of the cytokines produced by T cells are also secreted by cells of innate immunity; several, however, are relatively T-cell specific, and are involved in modulating immune responses or in effector cell differentiation. Although typically synthesized by Th cells, Tc cells and even B cells can be sources of these protein mediators.
Not all the possible adaptive responses are elicited concurrently in response to a particular pathogen. In some cases, it may be more advantageous to induce primarily a B cell antibody-mediated response; in other circumstances, a cytotoxic T cell response may be most warranted. Moreover, the adaptive immune response needs to be tightly regulated to prevent ongoing tissue injury, and therefore a negative-regulatory feedback must exist. The regulation of which of these potential outcomes occurs derives from the helper T cells, and more specifically the nature of the cytokines that they produce.
Antibodies
The generic antibody (also called an immunoglobulin or Ig) is a vaguely “Y”-shaped structure composed of four disulfide-linked proteins – two smaller (25 kD) light chains and two larger (50 kD) heavy chains (Figure II.2.3.7); for any given Ig the light chains on each arm of the “Y” are identical, as are the heavy chains. The N-termini of both chains have genetically (germline and somatic) variable domains (designated VL and VH in Figure II.2.3.7) that are the source of the tremendous antigen-binding diversity of the total antibody repertoire. The C-termini of each chain are genetically homogeneous for any given Ig, and are designated as constant domains (CL or CH). The two “arms” of the Ig are each composed of heavy and light chains, and are called the Fab or antigen-binding fragment of the molecule; the “body” of the Ig is composed of paired heavy chain constant regions, and is called the Fc portion of the molecule (for historical reasons, the “c” stands for “crystallizable” but “constant” is equally applicable).
FIGURE II.2.3.7 Generic antibody structure. Antibodies typically adopt a “Y”-shaped structure composed of two light chains and two heavy chains. The N-termini are the antigen binding fragments (Fab) and contain variable regions that allow the necessary diversity. The C-termini are constant for any particular immunoglobulin isotype (called Fc fragment). Antigen binding to the Fab area causes a conformational change in the Fc region that allows the antibody to activate complement, or to attach to Fc receptors.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
Once antigen is bound to the Fab portion of an Ig (more on this recognition step later), the protein undergoes a conformational change in the Fc region that allows the antibody to activate complement, or to attach to one of a variety of surface-bound Fc receptors on different cell types (e.g., macrophages, neutrophils, B cells, NK cells, etc.). Based on the structure of the heavy chain constant regions, antibodies occur as five major isotypes, IgA, IgD, IgE, IgG, and IgM; IgA has two subtypes and IgG has four. IgG is the most common of the antibody isotypes. All except IgD (which is only found in the membrane-bound form on naïve B cells) exist as circulating and B cell membrane-bound forms; the membrane-bound form of Ig is the antigen receptor for B cells and engagement drives B cell activation and differentiation. The different antibody isotypes are specialized to activate specific effector mechanisms (e.g., phagocytosis, complement activation or NK cell killing); we will also specifically revisit one (IgE) in detail later when we discuss allergy.
T Lymphocytes
As we will see, T cells can participate in the adaptive immune response and elimination of extracellular pathogens. However, they are also the main mechanism by which intracellular pathogens (e.g., viruses and certain bacteria) – not accessible to phagocytes and circulating antibodies – can be targeted (see later).
In broad strokes, the helper versus cytotoxic T cell dichotomy addresses the requirements of defending against extracellular (Th) and intracellular (Tc) pathogens; having said that, Th cells are also involved in immunity to intracellular microbes, and help drive the Tc response.
Th Cells
CD4+ T helper cells achieve their function by secreting specific cytokines to stimulate the proliferation and differentiation of other cells. Two basic subsets of Th cells are classically described, called Th1 and Th2; each produce a handful of specific cytokines that will induce a different general immunologic response (Moss et al., 2004; Pulendran, 2004). Once a Th1- or a Th2-specific reaction is established against a particular pathogen, the responding population tends to maintain that pattern of differentiation. This is accomplished because Th1 cell cytokines promotes further Th1 differentiation and inhibits Th2 cells, and vice versa.
Generally speaking, Th1 cells are induced by intracellular pathogens that infect or activate macrophages and NK cells. The Th1 cytokines include interferon-γ (IFN-γ, the signature cytokine of Th1 cells), lymphotoxin, and tumor necrosis factor (TNF); these promote phagocyte-mediated defenses, especially against intracellular microbes (Figure II.2.3.8).
FIGURE II.2.3.8 Th1 cell functions. Differentiation of CD4+ T cells into Th1 cells results in the production of interferon-γ (IFN-γ), lymphotoxin (LT), and tumor necrosis factor (TNF). IFN-γ drives macrophage activation increasing their capacity to kill intracellular microbes; IFN-γ also induces the differentiation of B cells to produce antibodies that are better at opsonization and complement activation (e.g., IgG). LT and TNF function to recruit and activate additional inflammatory cells at a site of injury; shown here is a neutrophil, but macrophages are also involved. The net result of the Th1 cytokines is the enhancement of phagocyte-mediated defenses against infection; this involves both extracellular pathogens and those that can persist within non-activated macrophages.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
Th2 cells are induced by extracellular pathogens (especially worms) and certain polymeric antigens (allergens). The cardinal cytokines of this subset include interleukin (IL)-4, -5, and -10; these promote IgE and mast cell/eosinophil activation (and inhibit macrophage activation); these pathways putatively evolved to deal with helminthic infections that are not effectively eradicated by macrophages and NK cells (Figure II.2.3.9); however, Th2 differentiation also underlies many allergic responses.
FIGURE II.2.3.9 Th2 cell functions. Differentiation of CD4+ T cells into Th2 cells results in the production of interleukin (IL)-4, IL-5, and Il-10. IL-4 drives the differentiation of B cells to produce antibody isotypes that either bind well to mast cell Fc receptors (IgE) or bind antigen but do not activate complement or induce opsonization (e.g., IgG4); IL-4 (along with IL-10) also modulates macrophage activation status, antagonizing the actions of IFN-γ. IL-5 is involved in the recruitment and activation of eosinophils that are potent mediators, particularly against helminths. The net effect is to augment responses that will be effective against large extracellular pathogens such as worms.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
More recently, a unique third Th subset has been described, generated in the context of TGF-β and IL-6 or IL-1 cytokine production; IFNγ and IL-4 actually inhibit the expansion of this subset. These are called Th17 cells because they characteristically produce IL-17, a cytokine that, in association with IL-22 and other mediators, induces neutrophil-rich inflammatory responses (Korn et al., 2007, 2009). Since Th17 cells can potentially significantly contribute to tissue damage in the setting of local inflammation (due to the neutrophil-rich infiltrates), and because they are lineage-related to anti-inflammatory regulatory T cells, the pathways controlling their differentiation and activation are a focus of intense research.
Tc Cells
CD8+ cytotoxic T lymphocytes (also called CTL) are effector cells that function to eliminate intracellular infections. The strategy is similar to NK cells, in that infected cells are induced to undergo apoptotic cell death. However, there is greater specificity of these cells versus NK cells, and CD8+ T cells also have the attribute of memory – subsequent exposure to the same antigen results in a faster, more robust secondary response.
Recognition in Adaptive Immunity
B cells, antibodies, and T cells can potentially recognize 1011 different antigenic specificities; this is achieved by somatic mutation and rearrangement of limited numbers of germline sequences, but the genetic details are beyond the scope of this discussion. However, what these different components of adaptive immunity recognize is important. Thus, B cell receptor and secretory Ig binds to intact antigens, although the antigen need not necessarily be in its native conformation (Figure II.2.3.10). In comparison, T cells cannot recognize antigens until they have been degraded into smaller fragments and associated with self histocompatibility molecules on antigen presenting cells (Figure II.2.3.11). The following sections will elaborate on these basic tenets.
FIGURE II.2.3.10 Antigen–antibody binding. (A) Antibodies can bind to three-dimensional conformations of proteins with determinants (or epitopes) comprised of multiple amino acids (or other small molecules) from different loops; denaturation of the protein will result in loss of antibody binding to this particular kind of antigen. (B) Antibodies can bind to primary sequences of amino acids (or other small molecules) that can be present in the native molecule or be occult – that is, hidden within the folded tertiary structure. Denaturation of the protein will not affect antibody binding to this antigenic structure, and indeed, may expose previous inaccessible determinants. (C) Antibodies can bind to determinants that are only exposed or created by proteolytic degradation.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
FIGURE II.2.3.11 T cells cannot recognize unprocessed antigen. Intact protein antigens will not bind the T cell receptor (TCR). Instead, the TCR can only bind to smaller (10–15 amino acids in length) polypeptides, and then only when complexed to self major histocompatibility (MHC) molecules. Thus, exogenous antigen must be taken up by antigen presenting cells (APC), delivered to intracellular endosomes for proteolytic processing, and the resulting fragments joined to newly synthesized MHC before being displayed on the cell surface.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
B Cell and Antibody Recognition
As shown in Figure II.2.3.10, antibodies can bind to conformations that depend on some combination of primary, secondary, and tertiary structure. Antigens can be linear sequences of amino acids or sugars, or can be composed of elements that are juxtaposed by virtue of three-dimensional folding; the same molecule can have multiple different potential antigenic epitopes (also called determinants). The nature of the epitopes is significant because in native, intact molecules, certain determinants may be internally folded and inaccessible to antibody. However, denaturation or proteolytic cleavage (e.g., at sites of tissue injury) can conceivably expose previously occult epitopes, and allow antibody binding and subsequent effector activity.
T Cell Recognition
T cells have surface receptors that cannot “see” intact antigens, but rather only recognize proteolytically digested antigen fragments (“processed antigen”) presented on the surface of certain host cells in association with major histocompatibility complex molecules (Figure II.2.3.11). For Th cells, these accessory or antigen-presenting cells (APC) include macrophages, one of the major cell types of the innate immune response; in this manner, the innate system can influence adaptive immune responses.
Histocompatibility molecules are grouped together on chromosomes into clusters generically called Major Histocompatibility Complexes or MHC. Proteins of this complex are denoted as “histocompatibility” molecules because they were first recognized as the major determining element in tissue (“histo”) compatibility in organ transplantation. When inbred strains of animals shared the same MHC determinants, tissue grafts could be transplanted with relative impunity; if the donor and host were MHC-disparate, grafts were said to be histo-incompatible, and the organs ultimately failed by a process called rejection. This is due to the host “interpreting” foreign MHC as a potential pathogen, and then engaging the full fury of the various immune effectors. Tissue incompatibility clearly has ramifications in the assembly and implantation of devices prepared from non-autologous (autologous = self) viable human cells; in most cases, these cells will express host-incompatible MHC and trigger specific immune responses (see discussion at the end of this chapter).
In humans, this MHC cluster occurs on chromosome 6, and the molecules are called Human Leukocyte Antigens or HLA. There are two general categories of MHC molecules, called class I and class II. In humans, MHC class I (MHC I) molecules are called HLA-A, -B, and -C; MHC class II (MHC II) molecules are called HLA-DP, -DQ, and -DR. In the general population, each type of HLA molecule (e.g., HLA-A, HLA-B, etc.) has hundreds of possible different alleles. Thus, outside of haplo-identical twins, it is virtually impossible to have a perfect match among all the possible class I and class II MHC combinations.
The MHC class I molecules present peptide fragments derived from intracellular antigens to CD8+ Tc cells; in this manner, intracellular infections can be detected and the infected cell can be killed. In comparison, MHC class II molecules present peptide fragments from extracellular antigens to CD4+ Th cells. In this pathway, the APC is not killed; rather, extracellular infections are detected by the Th cells that subsequently release cytokines to recruit additional effectors (Figure II.2.3.12) (Klein and Sato, 2000a,b).
FIGURE II.2.3.12 Presentation of extracellular versus intracellular antigens to cytotoxic versus helper T cells. (A) Extracellular antigens (e.g., from extracellular bacteria) are ingested and degraded by macrophages or other antigen presenting cells (APC, including B cells as shown, and dendritic cells), and are then presented in association with MHC II surface molecules to CD4+ helper T cells. Helper T cells activated in this manner lead to macrophage and/or B cell activation that will generate effector functions to more proficiently eliminate the extracellular microbe. (B) Intracellular antigens (e.g., from intracellular viruses) are degraded and presented in association with MHC I surface molecules to CD8+ cytotoxic T cells. Killer T cells activated in this manner then lyse (kill) the cell that originally harbored the intracellular pathogen.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
T cell recognition of antigen fragments bound to MHC molecules results in T cell activation. This recognition step is accomplished by T cell receptors (TCR) on the surface of T lymphocytes interacting with the CD3 complex and additional molecules to transduce a signal to the nucleus, resulting in selected gene transcription (Kuhns et al., 2006). Complete activation of T cells also requires additional interplay between other molecules (called co-stimulator molecules) on the surface of T cells and antigen presenting cells of the innate immune system. Incomplete activation of T cells (i.e., without the co-stimulators) may result in anergy (no response) to the antigen (Peter and Warnatz, 2005) (Figure II.2.3.13).
FIGURE II.2.3.13 Role of co-stimulation in T cell activation. (A) Antigen presenting cells (APC) that are not activated will express few or no co-stimulator molecules. In that setting, even though the APC display processed antigen in the appropriate MHC context, the T cells will fail to respond. Indeed, such co-stimulator-poor APC presentation may result in a long-term anergy (inability to respond) to particular antigens. (B) Microbes and cytokines produced during innate immune responses activate APC to make co-stimulator molecules (such as B7, shown here) that will result in “complete” activation of the T cells. Activated APC also produce additional cytokines such as interleukin-12 (IL-12) that also participate in stimulating T cell activation and differentiation.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
Effector Pathways in Adaptive Immunity
Depending on the nature of the inciting pathogen and the preceding innate response, different elements of adaptive immunity will be engaged; as a result, different effector functions will also obviously be involved.
Figure II.2.3.14 depicts schematically how antibodies can be marshaled to defend against pathogens. Binding to microbial surface receptor molecules can prevent access to target cells; similarly, binding to circulating toxins can serve to block their activity. Antibody opsonization will increase the phagocytosis (and intracellular destruction) of pathogens, as well as facilitate NK cell killing; both of these happen through the interaction of bound Ig and cells bearing specific Fc receptors. Finally, antibody binding also efficiently activates complement with subsequent MAC formation, additional opsonization, and the augmented recruitment and activation of innate inflammatory cells. The downside to these activities occurs when antibody is bound – either specifically or non-specifically – to self tissues: the full array of antibody-mediated effector functions are then brought to bear and can cause substantial injury (see later).
FIGURE II.2.3.14 Effector functions of antibodies. From top to bottom, antibodies can: bind pathogens (or their toxins) and neutralize them (i.e., prevent their attachment or entrance into host cells); bind to microbes and induce opsonization (ingestion by Fc-receptor-bearing phagocytes); bind to microbes and direct natural killer (NK) cell cytolysis; and activate complement to cause direct lysis, opsonization, or additional inflammatory cell recruitment.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
In comparison, Figure II.2.3.15 shows the general effector mechanism of cell-mediated immunity. Recognition of infected cells by CD8+ CTL leads to their cytolysis. Antigen recognition by CD4+ Th cells leads to cytokine production that recruits and activates additional effector cells to more efficiently carry out their microbicidal activities. The down-side to CTL is that inappropriate targeting of host cells (autoimmunity) will cause their destruction; even the appropriate targeting of infected cells can wreak substantial havoc if the infection is widespread. If Th activity is not well-regulated or if Th cells begin recognizing self-antigens, then exuberant innate immunity activation will also cause significant tissue damage and scarring (see also below).
FIGURE II.2.3.15 Effector functions of T lymphocytes. (A) When CD4+ T cells encounter antigenic fragments presented appropriately by antigen presenting cells (APC) they respond by the production of cytokines that will drive subsequent effector cell recruitment and activation (see also Figures II.2.3.8 and II.2.3.9); shown here is the response of a Th1 cell producing cytokines that increase macrophage activation and inflammatory cell recruitment. CD8+ T cells can also be a source of cytokines that affect the local inflammatory response. (B) Recognition of processed antigen on target cells by CD8+ cytotoxic T lymphocytes (CTL) typically results in the directed CTL killing of the target.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
Overview: Immunity to Pathogens
Recall that following pathogen recognition – and depending on the location of the invader – effector functions need to be appropriately coordinated to neutralize and/or destroy either intracellular or extracellular microbes.
Extracellular pathogens: Innate immunity deals with an extracellular challenge largely through the activation of complement and phagocytosis, as well as by being effective at recruiting additional cells that can do more of the same. Antibodies are the major components of adaptive immunity that contribute to the battle against extracellular pathogens; they do so by binding and neutralizing microbes or their toxins, and by highly effectively inducing complement activation and phagocytosis. Th cells can also participate by secreting cytokines that recruit neutrophils, and by recruiting and activating macrophages, augmenting their phagocytic capacity to kill.
Intracellular pathogens: Intracellular pathogens trigger the involvement of innate immunity through interferon-α and -β production, by the activation of macrophages to more effectively kill ingested pathogens, and by the activation of NK cells to kill infected cells. On the adaptive immune side, CTL kill virally infected cells, and Th cells mediate highly effective macrophage activation. As it turns out, the most important immune response to intracellular bacteria – as well as large extracellular pathogens such as fungi – involves T cell-driven activation of macrophages to more avidly ingest microbes, and produce more reactive oxygen species (ROS) and nitric oxide. If these activities fail to completely eliminate the inciting pathogens, ongoing recruitment and activation of macrophages leads to the formation of a granuloma, a collection of activated macrophages that effectively form a vigilant ring of inflammation to wall off the invader (Figure II.2.3.16). A potentially negative outcome of persistent granulomatous inflammation is tissue injury due to compression atrophy by the expanding sphere of activated macrophages, and by the surrounding fibrosis these activated cells will engender.
FIGURE II.2.3.16 Granulomatous inflammation. Schematic illustration of the events that lead to granuloma formation in response to large or persistent antigens. Antigen-presenting cells (APC) of the innate immune system process antigen and subsequently present it to CD4+ helper Th1 cells; the APC also provide interleukin-12 (IL-12) and other cytokines to drive T cell activation. Activated T cells, in turn, elaborate cytokines such as tumor necrosis factor (TNF) that will recruit inflammatory cells, and interferon-γ (IFN-γ) that will induce the activation of the recruited cells, in particular macrophages (large, activated macrophages are also called “epithelioid cells”). These cytokines can also induce macrophage fusion to generate multinucleated “giant cells.” If the antigen is not effectively eliminated, the constant cycle of T cell and macrophage activation leads to the accumulation of an aggregate of activated cells. Activated macrophages will also elaborate mediators that result in tissue injury, as well as cytokines resulting in fibroblast activation and tissue fibrosis (see also Figure II.2.3.6). The end-result is loss of tissue function and scar formation. In the case of “inert” foreign bodies, absorption of host proteins onto the foreign body surface with subsequent denaturation and modification can lead to direct macrophage activation via the receptors involved in innate immunity.
(Figure reprinted with permission from Kumar et al. (2005), Robbins and Cotran Pathologic Basis of Disease, 7th ed.)
Pathology Associated with Immune Responses
The chapter began with the assertion that innate and adaptive immune systems exist primarily to defend the human body against infection. Unfortunately, immune activation leads not only to the activation of host defenses and production of protective Ig and T cells, but also occasionally to the development of responses that may potentially damage host tissues.
Both innate and adaptive immune responses are implicated in causing disease states. As described above, in the setting of prolonged activation, macrophages will ultimately mediate tissue fibrosis and scarring. Indeed, the response to foreign materials – causing much of the local pathology associated with implants – is attributable to such persistent macrophage activation. Moreover, certain bacterial toxins (e.g., LPS) non-specifically stimulate macrophages (as well as other cell types), and result in systemic pathology from excessive cytokine elaboration.
By having increased specificity, adaptive immunity might be expected to lead overall to less secondary damage. Normally, an exquisite system of checks and balances optimizes the antigen-specific eradication of infecting organisms with only trivial innocent bystander injury. However, certain types of infection (e.g., virus) may require destroying host tissues to eliminate the disease (see Figure II.2.3.15B). Still other types of infections (e.g., tuberculosis) may only be controlled by a granulomatous response that walls off the offending agent with activated macrophages and scar, often at the expense of adjacent normal parenchyma (similar to foreign body responses).
Even when the host response to an infectious agent is a specific antibody, the antibody may occasionally cross-react with self-antigens (e.g., anti-cardiac antibodies following certain streptococcal infections, causing rheumatic heart disease). Immune complexes composed of specific antibody and circulating antigens can also precipitate at inappropriate sites (see below) and cause injury by activation of the complement cascade or by facilitating binding of neutrophils and macrophages (e.g., in a disease such as post-streptococcal glomerulonephritis). If the antibody made in response to a particular antigen is IgE, any subsequent response to that antigen will be immediate hypersensitivity (allergy), potentially culminating in anaphylaxis. Finally, not all antigens that attract the attention of lymphocytes are exogenous and infectious. The immune system occasionally (but fortunately, rarely), loses tolerance for endogenous self-antigens, which results in autoimmune disease.
All of these forms of immune-mediated injury are collectively denoted as hypersensitivity reactions. As discussed below and in Chapter II.2.4, they are traditionally subdivided into four types; three are variations on antibody (Ig)-mediated injury, while the fourth is T cell-mediated:
Pathogenesis of Antibody-Mediated Disease
Antibodies involved in immune-mediated diseases can bind to antigenic determinants that are intrinsic to a particular tissue or cell, or which are exogenous and have been passively adsorbed (e.g., certain antibiotics or foreign proteins). Regardless of what they recognize or how they got there, antibodies bound to the surfaces of cells or to extracellular matrix cause injury by certain basic mechanisms.
IgE-Mediated (Immediate) Hypersensitivity (Kay, 2001a,b)
Mast cells and basophils express surface Fc-receptors that can bind the Fc constant region of immunoglobulin E (IgE). When circulating IgE binds to the Fc-receptors on these cells and is then cross-linked by specific allergen (typically a polyvalent antigen), this induces mast cell or basophil degranulation with release of preformed mediators, as well as synthesis of other potent effectors (Figure II.2.3.17):
• pre-formed mediators: amines such as histamine and serotonin (cause vasodilation and increased vascular permeability)
• arachidonate, lipid, and cytokine mediators synthesized de novo:
– prostaglandins (e.g., PGD2) that can affect vessel and airway contraction, and vascular permeability
– leukotrienes (e.g., LTC4, LTD4, and LTE4) that are exceptional vasoconstrictors and bronchoconstrictors
– platelet activating factor (PAF), a rapidly catabolized phospholipid derivative that increases vascular permeability and diminishes vascular smooth muscle tone; it also causes bronchoconstriction
– cytokines, in particular TNF (recruits sequential waves of neutrophils and monocytes), and IL-4 (interleukin 4, induces local epithelial and macrophage expression of chemokines like eotaxin, and also increases endothelial adhesion molecule expression: the combined effect will be to recruit eosinophils).
FIGURE II.2.3.17 Events in immediate-type hypersensitivity (allergy). Immediate hypersensitivity is initiated following contact with a specific allergen (typically a polyvalent antigen). For unclear reasons, allergens induce in a susceptible host a predominant Th2 response that ultimately promotes an IgE antibody response. IgE binds to mast cells in tissues (and basophils in the circulation, not shown) via specific IgE Fc receptors. Subsequent encounter with the relevant allergen results in IgE-Fc receptor cross-linking that activates the mast cells and basophils. Once activated, the cells release granules containing pre-formed mediators (e.g., histamine) causing the characteristic immediate response (vasodilation and increased vascular permeability, with bronchoconstriction). Over the next few hours (up to 24 hours), these activated cells will also synthesize and release additional mediators (prostaglandins, leukotrienes, platelet activating factor, and cytokines; see text).
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
In most vascular beds, the overall result is vasodilation and increased vascular permeability, with a variable infiltrate classically predominated by eosinophils. Eosinophils are an inflammatory cell type classically associated with parasitic infections (especially worms), as well as with allergies; they contain specific granules with potent cytotoxic activity for a variety of cell types. In the respiratory tree, the net result of an allergic stimulus is increased mucus secretion and bronchoconstriction.
The nature of the symptoms in any particular instance will depend on the portal of antigen entry, e.g., cutaneous (hives and rash, although these can also occur with inhaled or ingested allergen), inhaled (wheezing, airway congestion), ingested (diarrhea, cramping) or systemic (hypotension). The associated diseases range from the merely annoying (seasonal rhinitis or “hay fever”) to debilitating (asthma) to life-threatening (anaphylaxis). In the last case, systemic vasodilation leads to hypotension (low blood pressure), increased vascular permeability leads to increased tissue edema – most problematic around the larynx – and bronchoconstriction narrows the airways and impedes airflow; the combination can culminate in fatal anaphylactic shock.
Antibody Bound to Cell Surfaces or Fixed Tissue Antigens
Antibodies bound to either intrinsic or extrinsic tissue antigens can induce tissue injury by promoting complement activation, inducing opsonization, or by interacting with important cell surface molecules (Figure II.2.3.18):
• Complement: Recall that complement may induce injury either by direct cytolysis via the C5b–9 membrane-attack complex (MAC) punching holes in a cell’s plasma membrane, or by opsonization (via the C3b fragment), enhancing phagocytosis by macrophages and neutrophils. In addition to direct cell killing, local activation of the complement cascade will result in the generation of complement fragments such as C3a and C5a that alter vascular tone and permeability (Figure II.2.3.4 and Chapter II.2.4).
• Opsonization: Antibody on the surface of cells will promote phagocytosis by cells bearing Fc receptors.
– On circulating blood cells, bound complement may directly mediate cell lysis; in addition, bound antibody and opsonizing complement fragments induce efficient uptake and destruction by cells of the splenic and hepatic mononuclear phagocyte system.
– Antibody binding in conjunction with C3b opsonization can also lead indirectly to tissue injury. Large, non-phagocytosable cells or tissue may promote “frustrated phagocytosis” by neutrophils or macrophages; the attempted intracellular lysis results instead in the extracellular release of proteases and toxic oxygen metabolites (Figures II.2.3.18B and II.2.3.19A).
FIGURE II.2.3.19 Antibody-mediated pathology. (A) Direct binding of antibodies to tissue antigens will cause tissue injury by recruiting inflammatory cells and activating complement. (B) Circulating antigen–antibody complexes (also called immune complexes) can deposit in vessels and tissues also leading to inflammatory cell recruitment and complement activation. The classic vascular lesion in the setting of immune complex deposition is called vasculitis; such vessel inflammation will typically lead to thrombosis and tissue ischemia.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
• Antibody binding to cell receptors: Binding of antibodies to certain receptors can induce pathology even without causing tissue injury. For example, in the case of Grave’s disease, antibodies bind to the thyroid stimulating hormone (TSH) receptor on thyroid epithelial cells and mimic authentic TSH ligand interaction; the result is autonomous stimulation of the gland with hyperthyroidism. Alternatively, antibodies that bind to the acetylcholine receptor at the nerve–muscle synapse can block binding of acetylcholine and result in the weakness seen in the disease myasthenia gravis (Figure II.2.3.18C).
FIGURE II.2.3.18 Effector mechanisms in antibody-mediated disease. (A) Antibodies, with or without complement activation, will opsonize cells leading to phagocytosis and destruction. (B) Antibodies, and secondarily-generated complement fragments bound to large non-phagocytosable cells or tissues will recruit inflammatory cells such as neutrophils and macrophages. If these inflammatory cells cannot completely ingest the target, frustrated phagocytosis will result in the release of lysosomal contents and reactive oxygen intermediates into the tissues with subsequent cellular and matrix damage. (C) Antibodies can also elicit pathology without causing tissue damage. In the panel on the left, antibodies to the thyroid stimulating hormone (TSH) receptor can mimic authentic TSH to cause hyperstimulation of the thyroid (Grave’s disease). In the panel on the right, antibodies to the acetylcholine (ACh) receptor at the neuromuscular junction can block normal ACh stimulation of muscle contraction leading to weakness (myasthenia gravis).
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
Immune Complex (IC)-Mediated Injury
In many circumstances, circulating antigen and antibody combine to form insoluble aggregates called immune complexes (IC). These are usually efficiently cleared by macrophages in the spleen and liver, but can occasionally deposit in certain vascular beds. Once IC are deposited, the mechanism of injury is basically the same, regardless of where or for what reason IC have accumulated; the major sources of pathology are complement activation (see above) and interactions with cells bearing Fc receptors (e.g., neutrophils and macrophages) (Figure II.2.3.19B).
Pathogenesis of T Cell-Mediated Disease
T cell-mediated responses are of two general types (Figure II.2.3.20):
FIGURE II.2.3.20 Mechanisms of T cell-mediated disease. (A) In delayed-type hypersensitivity responses, T cells (typically CD4+ helper T cells) respond to tissue or cellular antigens by secreting cytokines that stimulate inflammation, and ultimately promote tissue injury (APC, antigen-presenting cell). (B) In some diseases, CD8+ cytotoxic T cells directly kill tissue cells.
(Figure reprinted with permission from Abbas et al. (2007), Cellular and Molecular Immunology, 6th ed.)
T cell-mediated cytolysis: In CTL-mediated reactions, CD8+ T lymphocytes recognize specific antigen in association with class I MHC and induce direct cytolysis. CTL-mediated cytolysis is highly specific, without significant “innocent bystander” injury.
Delayed-type hypersensitivity (DTH): In the case of cell-mediated immunity, CD4+ helper T cells recognize specific antigen in the context of class II MHC, and respond by producing a variety of soluble antigen-nonspecific cytokines. These soluble mediators induce further T lymphocyte recruitment and proliferation, and attract and activate antigen non-specific macrophages and other inflammatory cells; at the site of a CD4+ T cell-mediated response, the vast majority (greater than 90%) of newly recruited cells is not specific for the original inciting antigen. Although tightly regulated, the relatively non-specific effector components of cell-mediated immunity (cytokines and activated macrophages) are largely responsible for the injury seen in delayed-type hypersensitivity (DTH).
In comparison to CTL, cytokine-mediated immunity may ultimately develop an antigen non-specific component; that is, after the initial antigen-specific T cell response, the recruited antigen non-specific T cells and macrophages can cause significant bystander injury. Macrophages, in particular, are an important component of the recruited inflammatory cells in DTH, and mediate much of the subsequent immune effector responses. By virtue of the release of reactive oxygen intermediates, prostaglandins, lysosomal enzymes, and cytokines such as TNF (which, in turn, have potent effects, e.g., on the synthetic function of fibroblasts, lymphocytes, and endothelium), activated macrophages can potentially wreak significant havoc.
Recall also that granulomas (nodules of granulomatous inflammation) are the characteristic response of the immune system to large, persistent, and/or non-degradable antigens. Besides antigenic stimulation, direct macrophage activation can also occur by binding to denatured or modified host proteins that have adsorbed on the surfaces of the foreign materials via the receptors used for innate immunity (Tang and Eaton, 1999; Anderson et al., 2008). Thus, foreign objects (such as implanted devices) can frequently elicit a granulomatous response that becomes a significant impediment to local tissue integrity and normal function. With persistent stimulation, for example, chronic macrophage activation leads to cytokines that culminate in a surrounding fibrosis. Injury associated with granulomas is also due to displacement, compression, and necrosis of adjacent healthy tissue. Granulomas associated with a variety of “autoimmune disorders” such as temporal arteritis, Crohn’s disease, and Wegener’s granulomatosis presumably reflect diseases with persistent antigen stimulation or a heightened DTH response to specific self-antigens.
Immune Responses to Transplanted Tissues, Biomaterials, and Synthetic Substances
When foreign cells or organs are transplanted into a new host, the histocompatibility proteins on the cell surfaces of the graft are recognized by the components of adaptive immunity as being non-self. Note that, except for minor genetic polymorphisms, most of the structural proteins and other molecular components in a graft are nearly identical to those that the host will also express (e.g., the contractile proteins in heart muscle, the collagenous extracellular matrix, the usual housekeeping proteins, etc.). The MHC molecules, however, are distinctly different between most humans (except identical twins) and will elicit helper and cytotoxic T cell activation, as well as B cell antibody production. Clearly, once these pathways have been activated, the usual physiologic effector mechanisms (direct cell killing, complement activation, phagocytosis, cytokine elaboration, etc.) will be brought to bear on the graft, and can effect its destruction. Besides direct killing of parenchymal cells (e.g., myocytes in a heart, hepatocytes in the liver, tubular epithelial cells in the kidney), the adaptive immune system and its various effectors also attack the graft vasculature, and can cause graft failure by vessel thrombosis and ischemia. Even in the absence of infarction or direct killing, cytokines and other inflammatory mediators such as prostaglandins and reactive oxygen species can cause significant graft dysfunction (i.e., a loss of myocardial contractility) that may be as clinically deleterious as frank injury.
Again, although components of innate immunity are recruited and activated in the process of graft damage, the initial recognition step and the driving force for transplant rejection is via the cells of adaptive immunity (you are also referred to the basic immunology texts for excellent overviews of the rejection phenomenon: Coico et al., 2003; Abbas et al., 2007; Murphy et al., 2007). To prevent or reverse such rejection requires a substantial armamentarium of immunosuppressive agents (e.g., cytotoxic drugs or agents such as cyclosporine), which put the recipient at risk of infections and malignancies.
The point is emphasized here because in the immunologic sense, synthetic devices are not rejected; moreover, if non-cellular biomaterials are derived from the same species (or sufficiently-related species so that there are no major antigenic differences in, e.g., collagen proteins), such engineered devices are also not rejected. Such devices do not elicit specific (adaptive) immune responses, and therefore will not have antibodies or lymphocytes that recognize the materials and cannot therefore drive the overall response. As a corollary statement, simply finding inflammatory cells (and even T cells and antibodies) does not in any way prove that the response is “rejection;” such elements will accrue at any site of injury in a non-specific way. For example, recall that some 90% of T cells in a DTH response are not antigen-specific, but are non-specifically recruited to the site of injury.
This is much more than a semantic point, in that synthetic or biomaterial device functions or longevity are not likely to benefit from specific immunosuppression. Of course, if a device incorporates viable cells in its manufacture (e.g., endothelial cells lining a vascular conduit), those cells will express MHC proteins, and will elicit adaptive immune responses that materially contribute to device failure. In that instance, it will either be necessary to engineer such devices using cells derived from the individual who will eventually receive the implant (making them an autograft), or to rely on long-term immunosuppression as is routinely required for organ transplants.
It should also be emphasized that although synthetic and biomaterials are not rejected in the immunologic sense, components of the immune system (particularly innate immunity) can contribute to device dysfunction and failure. In particular, and as described above, non-specific activation of macrophages and complement will lead to local tissue damage via proteolysis, accumulation of other inflammatory cells, and/or cytokine elaboration; in most cases, with an ongoing, persistent innate response to a device that cannot be eliminated, fibrous scar tissue will also occur in and around the implant (Anderson et al., 2008).
Thus, synthetic materials and biomaterials can have failure modes that are attributable to activation of the immune system (particularly innate immunity). An “inert” silastic-clad breast prosthesis, for example, can accumulate dense scar tissue around it (secondary to persistent macrophage activation) that is not aesthetically ideal. Similarly a titanium hip prosthesis can induce ongoing macrophage activation that in the bone will lead to cytokine production that ultimately drives bone resorption and prosthesis loosening. In some individuals, idiosyncratic IL-4 or IL-10 cytokine responses to stent grafts may underlie local aneurysm formation and “stent creep” associated with intravascular stenting (Levisay et al., 2008). Heavy metal hypersensitivities (e.g., to nickel) occur because the metal complexes with host proteins, creating a modified version of “self;” when these modified proteins are then processed and presented as part of normal immune surveillance, Th-mediated DTH responses can occur with associated inflammation and swelling. Notably, heavy metal hypersensitivities are localized to the immediate vicinity of the implant. While administration of steroids in any of these settings may have some beneficial effect by limiting macrophage activation, they may also induce complications since steroids (among other side-effects) also inhibit healing and increase susceptibility to infections.
Finally, there is a body of controversial work suggesting that denatured host proteins non-specifically adhering on device surfaces or complexing with fragmented and circulating implant materials can potentially break host self-tolerance for these molecules. In this hypothetical model, novel previously occult antigenic determinants become exposed (see Figure II.2.3.10). These can then elicit antibodies that can bind to damaged (or even normal) versions of the same or cross-reactive proteins, and cause local tissue damage or subsequent immune complex formation and deposition. Although the experiments that purport to support this are problematic, and the evidence for such a pathologic pathway is weak, such models are frequently invoked to explain new onset autoimmune disease following device implantation (or following infection, vaccine, or other external trigger) (Molina and Shoenfeld, 2005; Wolfram et al., 2008).
In deciding whether a particular response to biomaterials is driven by immune mechanisms, it may be well to consider applying an immunologic variant of Koch’s Postulates. In the late 19th century, a microbiologist named Robert Koch proposed a set of objective criteria to be used in demonstrating that a particular organism was responsible for a particular disease (actually used the first time in proving the pathogenic basis of anthrax!). Koch’s Postulates basically state that the presumed microbe should be routinely recovered from the pathologic lesions of the human host; should be culturable in a pure form and cause the same disease when injected into a new host; and should be recoverable again in a pure form from the secondary host.
Thus, Koch’s Immunologic Postulates would provide a more rigorous basis for implicating host immunity in biomaterial failures or subsequent host pathology:
• antigen-specific elements (antibodies or cells) should be directly associated with the pathology of interest
• antibodies or cells from affected hosts should cause the same pathology by transfer into an appropriate secondary host
• adoptively transferred antibodies or cells should be recoverable (or at least demonstrable) in the pathology of the secondary host.
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