Toll-Like Receptors

Work done by Janeway and Medzhitov revolutionized understanding of the critical role of the innate immune system as the first step in adaptive immunity.⁷ They identified the human counterpart of a protein found in fruit flies (Drosophila melanogaster) known as Toll. The Toll protein in fruit flies is responsible for infectious susceptibility to fungi. This protein in humans, human Toll-like receptor 4 (TLR4), recognizes LPS, and is located on antigen-presenting cells (APC) such as dendritic cells, macrophages, and monocytes. Through a complex signaling cascade TLR-4 results in activation of cytoplasmic nuclear factor-κB (NF-κB). NF-κB can then translocate into the nucleus and induce the transcriptional activation of a wide variety of inflammatory and immune responses.² These responses include the induction of cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, IL-12, and the induction of costimulatory molecules such as CD80 and CD86. The presentation of antigen by the major histocompatibility complex (MHC) II molecule on the APC is insufficient to induce the activation of the T-cell receptor, and thus the T cell. There must also be expression of CD80 or CD86 by the APC that is required for full T-cell receptor activation. Thus normally pathogen-specific T cells should only be activated and not self-antigen.

At present there are nine human TLR receptors.⁸ The TLRs recognize molecules common to pathogens ranging from protozoa to bacteria to fungi and to viruses. The TLRs that recognize bacterial cell wall components (TLR1, -2, -4, and -6) are located on the cell surface, forming homodimers and heterodimers. Once ligated, they then translocate to the endosome for signaling (see Chapter 103).⁹ TLR3, TLR7, and TLR9 reside in the endosome, recognize nucleic acids produced by viruses and bacteria, and thus are available for activity against intracellular viruses.¹⁰

Endogenous proteins such as heat shock proteins, surfactant protein A, high mobility group 1 (HMGB1), uric acid, and fibrinogen among many others may also function as ligands for TLRs and other PRRs. These endogenous proteins are termed damage-associated molecular patters (DAMPs).^11,12 The immune system has critical roles in normal physiologic processes, such as tissue remodeling after injury and development and scavenging of apoptotic cells; it is thought that the DAMPs participate in “alerting” the immune system. Mitochondria have evolved from an endosymbiont alpha-proteobacterium. They have their own DNA, enriched in hypomethylated CpG-containing sequences. Zhang et al.¹³ detected mitochondrial DNA in the blood of patients with systemic inflammatory response after major trauma at levels that would active TLR9 and phosphorylate signaling molecules downstream of TLR9. Accordingly, TLR9 could be signaled by mitochondrial DNA. Mitochondrial proteins from human tissues injected into animals activated formyl-peptide receptors on neutrophils, resulting in neutrophil activation and acute organ injury. As mitochondria are released from injured and dying cells (necrosis, not apoptosis), this may be the “signal” for systemic inflammation in these disease states.

There are several other classes of PRRs in addition to TLRs: RIG-I–like receptors (RLRs), Nod-like receptors (NLRs), and C-type lectin receptors (CLRs) (previously described). RLRs, an intracellular receptor, recognize single- and double-stranded viral RNA and transmit their signal through a common adaptor protein, interferon promoter stimulator-1 (IPS-1) and NF-κB, to induce type I interferon production and antiviral responses.¹⁰ Double-stranded RNA-dependent protein kinase (PKR) is a unique PRR in that it can turn off protein translation directly; its effect does not seem to be modulated by downstream effectors.¹⁴ Nod1 and Nod2 are NLRs that recognize PAMPs derived from the bacterial cell wall. Nod1 and Nod2 are localized to the cytoplasm and can elicit TLR-independent antibacterial responses. Other NLRs, such as NALP1, NALP3, IPAF, and NAIP5, are components of a molecular complex called the inflammasome. The inflammasome complex comprises one or some of the NLR proteins and caspase-1. Caspase is activated in this complex and cleaves critical inflammatory molecules such as pro-IL-1β and pro-IL-18 to produce mature proteins.¹⁰ The NALP3 inflammasome is activated by stimuli such as uric acid crystal, silica, and asbestos; thus NALP3 appears to be a receptor for danger-associated molecular patterns. Infections with the malaria parasite or the fungus Candida albicans were also reported to activate the NALP3 inflammasome.

What follows is a focus on the pathogen ligands of the PRRs, in particular TLR4. It had been recognized for a number of years that CD14, present on monocytes and blood-derived macrophages, and lipopolysaccharide binding protein (LBP), found in plasma, were critical for an LPS response. However, CD14 is a glycosylphosphatidylinositol-anchored protein lacking transmembrane and intracellular domains and would be unable to signal intracellular processes. CD14 is also present to a limited extent on neutrophils.¹⁵ In addition, a soluble form of CD14 could substitute for membrane-bound form of CD14 in LPS-mediated signaling. Thus it was clear that at least one other molecule present on the cell surface would be needed to elicit an LPS-dependent response. TLR4 knockout mice and human TLR4 mutations conferred such a role for this receptor for LPS-induced responses in humans and mice. TLR4 requires an additional molecule, MD-2, that is part of the complex on the cell surface. LPB is an acute phase reactant.^2,16 It catalyzes the transfer of LPS to CD14, which is then able to interact with TLR-4 and MD-2, resulting in intracellular signaling through the Toll/IL-1 receptor homologous region (TIR) adaptor molecules.⁹ Downstream signaling events result in release of transcriptional activating factors including NF-κB, that guide gene transcription of many genes, including TNF and interferon-β (IFN-β), a type I interferon. TNF is discussed at greater length later in this chapter; however, it is a central mediator in innate immune response pathway. IFN-β is critical in adaptive and antiviral immunity (see Chapter 103 for a description of other TLRs).

Endogenous Antimicrobials

The epithelial surface of the skin, gastrointestinal tract, and bronchial tree produce a number of antibacterial peptides.¹⁷ Leukocytes are also a rich source of these or similar proteins that can be secreted into the phagolysosome or alternatively secreted into biofilms protecting the mouth and gastrointestinal tract.^18,19 These antimicrobial proteins include the defensins, cathelicidin, lactoferrin, and bacterial permeability increasing factor (BPI).²⁰ Because the antimicrobial peptides are heavily positively charged (due to cationic amino acids), they specifically target bacterial cell membranes whose outermost leaflet of lipid bilayer is heavily populated with negatively charged phospholipid head groups.¹⁷ Thus they also act as pattern recognition receptors. It is still unclear exactly how these antimicrobial peptides actually kill microbes. Possibilities include the creation of physical holes that cause cell contents to “leak out,” disturbances of cell membrane function by “scrambling” the lipid bilayer, or fatal depolarization of the normally energized bacterial membrane.²¹

The α defensins are stored in the granules of neutrophils, monocytes, and macrophages, and are released extracellularly. The β defensins are produced by Paneth cells, reproductive tissues, epithelial cells, and keratinocytes; some are produced constitutively, some in response to LPS and proinflammatory products such as TNF and IL-1. Cathelicidin/LL37 is stored in neutrophil granules but is also produced by keratinocytes and epithelial cells in response to inflammatory stimuli. In addition to the antimicrobial effects of the defensins and cathelicidin, both are chemotactic for CD4 and CD8 T cells, phagocytes, and immature dendritic cells. They also enhance antigen-specific immunity and regulate complement activation.²⁰

Lactoferrin and BPI are found in secondary and primary granules of the neutrophils, respectively, but they are also secreted or presented on epithelial surfaces. Lactoferrin is found in milk, tears, saliva, and other secretions such as bile, pancreatic juice, and small intestinal secretions. Lactoferrin is structurally similar to transferrin, though its affinity for iron is about 300 times higher, allowing it to retain iron at low pH.²² Both lactoferrin and BPI have cationic-rich regions that are critical to binding and neutralizing LPS. Lactoferrin also has direct antimicrobial actions. Lactoferrin can potentiate the cytotoxic effects of monocytes and T cells.²² BPI was initially identified in neutrophil primary granules. Because of its high affinity for the lipid A region of LPS, it is particularly cytotoxic for gram-negative bacteria, and its antibacterial activities are synergistically enhanced by defensins and cathelicidin.²³ In addition, BPI can function as an opsonin, enhancing phagocytosis.

Antimicrobial peptides have been used in a number of conditions and, though initially promising, their potential has not been completely realized. In the largest study to date, recombinant fragment of BPI, rBPI₂₁, was trialed in 400 children with severe meningococcal sepsis.²⁴ Although rBPI₂₁ had no effect on survival, fewer patients had multiple severe amputations, and by day 60 had a more functional outcome compared with those who did not receive rBPI₂₁²⁴ A mammalian cathelicidin, protegrin, had no effect on the development or reduction of mucositis among patients who received stomatotoxic chemotherapy.²⁵

C-Reactive Protein

CRP is phylogenetically ancient and is highly conserved in evolution. It is a member of the pentraxin family of calcium-dependent ligand-binding plasma proteins, the only other member of which is serum amyloid P (SAP). Although CRP is an acute-phase reactant in humans, SAP is not. In mice the major acute-phase reactant is SAP, with a minor fraction being CRP. In humans, plasma CRP is produced only by hepatocytes and is predominantly under the transcriptional control of IL-6. It is greatly elevated in infections, allergic complications of infection, inflammatory diseases, necrosis, trauma, and malignancy. It is modestly elevated or absent in systemic lupus erythematosus, scleroderma, dermatomyositis, ulcerative colitis, leukemia, and graft-versus-host disease. CRP binds to glycan and phospholipids of bacteria, fungi, and parasites. When aggregated or bound to macromolecular ligands, CRP is recognized by C1q and activates the classic complement pathway; thus it initiates direct microbial toxicity. Because it also recognizes phospholipids, in particular phosphocholine, it may be involved in handling apoptotic and necrotic cells.³

Complement System

The complement system is critically positioned to participate in both the innate and adaptive immune response.²⁶ However, it is also critical for the disposal of immune complexes from tissues and clearance of apoptotic cells.⁴ The three pathways in which complement may become activated are the classic, alternative, and mannose-binding lectin pathway. Although complement activation initiates differently in each, all three converge at the cleavage of C3 (Figure 91-1). The classic pathway is initiated by the binding of the C1 complex (consisting of C1q, C1r, and C1s) to antibodies bound to antigen on the cell wall. The mannose-binding lectin pathway is initiated by binding of the complex of mannose-binding lectin and the mannose-binding lectin-associate proteases 1 and 2 (MASP1 and MASP2) to arrays of mannose groups on the surface of the bacterial cell wall. Both MASP2 and C1q then function similarly in forming the C4bC2a complex, representing the convertase for C3. Bacterial products, LPS, yeast cell wall particles, and aggregated antibody, including immunoglobulin (Ig)A and IgE, are all capable of activating the alternative pathway. The alternative pathway is initiated via low-grade cleavage of C3 in plasma to C3b. C3b binds to the hydroxyl groups on cell surface carbohydrates. C3b binds factor B to form a C3bB complex, which is activated by factor D, forming C3bBb and stabilized with properdin. C3bBb then functions as an alternative convertase for C3, that cleaves many molecules of C3 to C3b. C3b binds to hydroxyl groups on the microorganism around the area of complement activation. C3a, an anaphylatoxin, is released by the C3 convertase. C3b can also bind to the C3 convertase to form C5 convertase. The C5 convertase releases the anaphylatoxin C5a and initiates the formation of the membrane attack complex (MAC), C5b6789.⁴ The membrane attack complex created inserts into the cell membrane, creates large pores, and leads to osmotic lysis of the target.

Figure 91–1 The three activation pathways of complement: the mannose-binding lectin, the classic, and alternative pathways. The three pathways converge at the point of cleavage of C3. The mannose-binding lectin (MBL) pathway is initiated by binding of the complex of the MBL and MBL-associated proteases 1 and 2 (MASP1, MASP2) to arrays of mannose on the bacterial cell wall. MASP2 then activates first C4, then C2 to form the C3 convertase, C4b2a. The classic pathway is initiated by the binding of the C1 complex (which consists of C1q, two molecules of C1r, and two molecules of C1s) to antibodies bound to antigen on the surface of the bacterial cell wall. C1s, similar to MASP2, then activates C4 followed by C2 to form C4b2a (the C3 convertase). The alternative pathway is initiated by covalent binding of a small amount of C3b to hydroxyl groups on the cell surface carbohydrates and is activated by low-grade C3 in plasma. This C3b binds factor B to form C3bB, which is then activated by factor D to then form C3bBb, the alternative pathways C3 convertase. Properdin stabilizes this activation step. The C3 convertase then cleaves many molecules of C3 to C3b and C3a (an anaphylatoxin). C3b binds covalently around the site of complement activation, some of which binds to the C4b and C3b of the classic and alternative pathway C3 convertase, respectively, to form the C5 convertase. The C5 convertase cleaves C5 to C5a (an anaphylatoxin) and C5b, which initiates the formation of the membrane attack complex (MAC).

(Modified from Walport MJ: Complement. First of two parts, N Engl J Med 344:1058, 2001.)

Thus the complement system amplifies the initial response to the organism. In addition to lysing the target organism, opsonization with complement fragments C3b/C4b and C3bi (fragment C3b) occurs, resulting in phagocytosis by neutrophils, monocytes, and macrophages. Binding occurs through specific complement receptors, CR1 for C3b/C4b (CD35) and CR3 for C3bi (CD11b/CD18, also known as Mac-1 and α_Mβ2). The anaphylatoxins C3a, C4a, and C5a produced are low molecular weight, biologically active peptides defined by their actions on small blood vessels, smooth muscle, mast cells, and peripheral blood leukocytes. Blood vessels, smooth muscle cells, basophils, and mast cells respond to all three anaphylatoxins. Neutrophils, monocytes, and macrophages respond to C5a. The anaphylatoxins promote edema and increase vascular permeability through the release of histamine from mast cells and through the local production of vasodilatory prostaglandins such as prostaglandin E₂ (PGE₂) and edemogenic leukotrienes (LT) C₄, D₄, and E₄. C5a is a powerful activator of granulocyte function including chemotaxis, degranulation, and increased oxidative metabolism. C5a actions occur through its seven-transmembrane–spanning, G protein–coupled receptor, C5aR.²⁷ In animal models, C5a/C5aR is critical to the development of sepsis and multiple organ failure, and potentiates many early response cytokines and coagulation. C5aR is present not only on leukocytes, but also other tissue, including the brain, kidney, gastrointestinal tract.^27,28

Complement activation also occurs after oxidative stress such as occurs with ischemia/reperfusion. Complement activation is an early event after injury and the inhibition of complement activation or its components offer tissue protection after reperfusion.²⁹ Finally, complement proteins transduce various cell signals. Complement can activate B and T cells. It can regulate apoptosis of various cell types (see Chapter 100).^20,26

Immunoglobulin

The different immunoglobulins (Igs) secreted by B cells (IgG, IgA, IgM, IgD, and IgE) are known as isotopes. IgD plays little role, if any, in containment of microorganisms. Ig isotypes can be divided into subclasses, variants that show slight structural differences but are sufficiently alike structurally to be essentially identical to other members of the isotype class. There are four IgG subclasses: IgG1, IgG2, IgG3, and IgG4. As seen in Table 91-1, isotype subclasses have specialized roles in the immune response. For example, IgG2 has a major role in the formation of carbohydrate antibodies, but has poor complement fixation characteristics. The antibody system consists of Ig present in serum, protecting the blood and tissue spaces, and that present in the secretory system, lining the gastrointestinal and respiratory tracts and present in tears. The serum component is mostly IgG (85%), with lesser amounts of IgA and IgM. The secretory system consists mostly of secretory IgA (85%), which is structurally different than serum IgA, and lesser amounts of IgG and IgM. All Igs have a basic four-chain structure composed of an identical pair of heavy (H) and light (L) chains. The H-L pairs are held together by interchain disulfide bonds and noncovalent forces. The binding site for antigen is formed by one H and one L chain. IgM is a polymer of five four-chain units, and secretory IgA is a dimer. Polymeric forms (Figure 91-2) possess a J chain that is synthesized with the H and L chains and serves to stabilize the sulfhydryl groups during polymerization. At regular intervals along the peptide chain a disulfide bond forms an intrachain loop, known as the Ig domain. This motif is repeated among immunoglobulins, T-cell receptors, adhesion proteins, and histocompatibility antigens. Proteins with these Ig domains share close homology structurally and functionally and suggest a common evolutionary origin. They are termed members of the Ig superfamily (IgSF), and all are involved in cell interaction processes associated with recognition.

Figure 91–2 Basic immunoglobulin (Ig) structure. Sites of cleavage for papain or pepsin are shown with resulting fragments. The intrachain disulfide bond forms the region of the Ig domain. Insets show polymeric immunoglobulins, secretory IgA (left) and IgM (right). J chain is common to all polymeric forms; SC is the secretory component. V_L, Variable domain of light chain; C_L, constant domain of light chain; V_H, variable domain of heavy chain; C_H, constant domains of heavy chain.

The Ig molecule is divided into regions, the amino acid sequence of which is similar, such as regions needed for complement fixation or attachment to receptors on leukocytes. However, other regions are highly variable; those that bind to antigen have the highest divergency and are collectively known as the hypervariable region. Thus each Ig chain can be divided into constant and variable regions. The H chain has three constant regions and one variable region; the L chain has one variable region and one constant region. The hypervariable region of the L and H chain is tightly apposed and forms the combining site for antigen (see Figure 91-2). The digestion of Ig with papain and pepsin generates fragments with varying biologic capability. The Fc region of the Ig molecule accounts for its isotypic biologic capability. This is the region critical for complement fixation and recognition by Fc receptors on the leukocytes. The Fab region provides for specific unique antigen-antibody interactions. The F(ab′)2 fragment is formed with pepsin cleavage; the affinity for antigen is twice as great as Fab alone.

Contact Activation System

There are four major plasma protein systems that contribute to the host’s defense and participate in the development of inflammatory tissue injury: the complement system (previously discussed), the contact activation system (also known as Hageman factor or intrinsic coagulation system), the extrinsic coagulation cascade, and the fibrinolytic system (see Chapter 80). The contact activation system is critical to host defense and control of local blood flow at sites of injury. Hageman factor (Factor XII) is activated spontaneously (XIIa) on contact with negatively charged surfaces, such as lipid A of LPS and vascular basement membranes (Figure 91-3). High-molecular-weight kininogen (HMWK), prekallikrein, and factor XI circulate in the plasma as complexes. Factor XIIa will activate factor XI and cleave prekallikrein to kallikrein. Kallikrein will then cleave HMWK to bradykinin. The kallikrein-kinin system also encompasses the tissue (or glandular) kallikrein-kinin system (see Figure 91-3). Tissue kallikrein is immunologically distinct from plasma kallikrein and present throughout the body as an inactive “pro-” substance. In the presence of intracellular enzymes, plasmin, or plasma kallikrein, tissue kallikrein is produced, secreted, and active in the tissue where it is made. Tissue kallikrein can then cleave HMWK to bradykinin directly, or it can cleave low-molecular-weight kininogen (LMWK) or tissue kininogen (T-kininogen) to kallidin that is then directly converted to bradykinin. In the plasma, 80% of kininogen is low molecular weight.

Figure 91–3 Contact activation system and prostanoid production. Bradykinin can be produced by either the plasma kallikrein (top left) or tissue (glandular) kallikrein system (bottom left). Once formed, bradykinin can activate phospholipase A₂, and thus drive prostanoid production. In addition, it can potentiate the affects of the prostaglandins and vice versa. Bradykinin is metabolized into inactive peptides via several steps, the last of which is enzymatically driven by angiotensin-converting enzyme (ACE). HMW, High molecular weight; LMW, low molecular weight; PAF, platelet activating factor; COX-1, cyclooxgenase-1; COX-2, cyclooxygenase-2; 5LOX, 5-lipoxygenase; LT, leukotriene; TXA₂, thromboxane A₂; PG, prostaglandin.

(Modified from Ueno A, Oh-ishi S: Roles for the kallikrein-kinin system in inflammatory exudation and pain: lessons from studies on kininogen-deficient rats, J Pharmacol Sci 93:1, 2003.)

Bradykinin is an exceedingly potent vasoactive peptide.³¹ It can cause venous dilation, increased vascular permeability, hypotension, bronchoconstriction, and activation of phospholipase A₂. Phospholipase A₂ releases arachidonic acid from cell membrane and initiates the production of both proinflammatory and antiinflammatory phospholipids-derived products (see subsequent section and Figure 91-3). Bradykinin is metabolized by angiotensin-converting enzyme (ACE) to inactive peptides. Bradykinin, along with prostanoids, stimulates the pain response through polymodal receptors and C-fibers (capsaicin sensitive).³²