Immunology_Janeway

Immunology_Janeway. Left panel: TLR-3, expressed by dendritic cells and macrophages, senses double-stranded viral RNA (dsRNA). TLR-3 signaling uses the adaptor protein TRIF, which recruits the E3 ligase TRAF3 to generate K63-linked polyubiquitin chains. This scaffold recruits NEMO and TANK (TRAF family member-associated NFκB activator), which associate with the serine– threonine kinases IKKε (IκB kinase ε) and TBK1 (TANK-binding kinase 1). TBK1 phosphorylates (red dot) the transcription factor IRF3, and IRF3 then enters the nucleus and induces expression of type I interferon genes. Right panel: TLR-7, expressed by plasmacytoid dendritic cells, detects single-stranded RNA (ssRNA) and signals through MyD88. Here, IRAK1 directly recruits and phosphorylates IRF7, which is also highly expressed in plasmacytoid dendritic cells. IRF7 then enters the nucleus to induce expression of type I interferons.


Immunology_Janeway

Immunology_Janeway. their carboxy termini. They then move to the nucleus as active transcription factors. Of the nine IRF family members, IRF3 and IRF7 are particularly important for TLR signaling and expression of antiviral type I interferons. For TLR-3, expressed by macrophages and conventional dendritic cells, the cytoplasmic TIR domain interacts with the adaptor protein TRIF. TRIF interacts with the E3 ubiquitin ligase TRAF3, which, like TRAF6, generates a polyubiquitin scaffold. In TLR-3 signaling, this scaffold recruits a multiprotein complex containing the kinases IKKε and TBK1, which phosphorylate IRF3 (Fig. 3.16). TLR-4 also triggers this pathway by binding TRIF, but the IRF3 response induced by TLR-4 is relatively weak compared with that induced by TLR-3, and its functional role in vivo remains elusive. In contrast to TLR-3, TLR-7, TLR-8, and TLR-9 signal uniquely through MyD88. For TLR-7 and TLR-9 signaling in plasmacytoid dendritic cells, the MyD88 TIR domain recruits the IRAK1/IRAK4 complex


Immunology_Janeway

Immunology_Janeway. In contrast to TLR-3, TLR-7, TLR-8, and TLR-9 signal uniquely through MyD88. For TLR-7 and TLR-9 signaling in plasmacytoid dendritic cells, the MyD88 TIR domain recruits the IRAK1/IRAK4 complex as described above. Here, the IRAK complex carries out a distinct function beyond recruiting TRAFs that generate a signaling scaffold. In these cells, IRAK1 can also physically associate with IRF7, which is highly expressed by plasmacytoid dendritic cells. This allows IRF7 to become phosphorylated by IRAK1, leading to induction of type I interferons (see Fig. 3.16). Not all IRF factors regulate type I interferon genes; IRF5, for example, plays a role in the induction of pro-inflammatory cytokines.


Immunology_Janeway

Immunology_Janeway. The collective ability of TLRs to activate both IRFs and NFκB means that they can stimulate either antiviral or antibacterial responses as needed. In human IRAK4 deficiency, for example, no extra susceptibility to viral infections has been noted. This would suggest that IRF activation is not impaired and the production of antiviral interferons is not affected. TLRs are expressed by different types of cells involved in innate immunity and by some stromal and epithelial cells, and the responses generated will differ in some respects depending on what type of cell is being activated. 3-8 The NOD-like receptors are intracellular sensors of bacterial infection and cellular damage.


Immunology_Janeway

Immunology_Janeway. The TLRs, being expressed on the cell’s plasma membrane or endocytic vesicles, are primarily sensors of extracellular microbial products. Since the discovery of Toll and the mammalian TLRs, additional families of innate sensors have been identified that detect microbial products in the cytoplasm. One large group of cytoplasmic innate sensors has a centrally located nucleotidebinding oligomerization domain (NOD), and other variable domains that detect microbial products or cellular damage or that activate signaling pathways; collectively, these are the NOD-like receptors (NLRs). Some NLRs activate NFκB to initiate the same inflammatory responses as the TLRs, while other NLRs trigger a distinct pathway that induces cell death and the production of pro-inflammatory cytokines. The NLRs are considered a very ancient family of innate immunity receptors because the resistance (R) proteins that are part of plant defenses against pathogens are NLR homologs.


Immunology_Janeway

Immunology_Janeway. Subfamilies of NLRs can be distinguished on the basis of the other protein domains they contain. The NOD subfamily has an amino-terminal caspase recruitment domain (CARD) (Fig. 3.17). CARD was initially recognized in a family of proteases called caspases (for cysteine-aspartic acid proteases), which are important in many intracellular pathways, including those leading to cell death by apoptosis. CARD is structurally related to the TIR death domain in MyD88 and can dimerize with CARD domains on other proteins to induce signaling (Fig. 3.18). NOD proteins recognize fragments of bacterial cell-wall peptidoglycans, although it is not known whether this occurs through direct binding or via accessory proteins. NOD1 senses γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of peptidoglycans of Gram-negative bacteria such as Salmonella and some Gram-positive bacteria such as Listeria, Pattern recognition by cells of the innate immune system.


Immunology_Janeway

Immunology_Janeway. Pattern recognition by cells of the innate immune system. LRR domain cytoplasm RIP2 RIP2 NF˜B I˜B Binding of bacterial ligands to NOD proteins induces recruitment of RIP2, which activates TAK1, leading to NF˜B activation NOD proteins reside in the cytoplasm in an inactive form CARD XIAP cIAP1/2 NOD CARD NOD intracellular bacteria, muramyl dipeptide or iE-DAP TAB1/2 NEMO polyubiquitin TAK1 IKK°IKK˛ Fig. 3.17 Intracellular NOD proteins sense the presence of bacteria by recognizing bacterial peptidoglycans and activate NFκB to induce the expression of pro-inflammatory genes. whereas NOD2 recognizes muramyl dipeptide (MDP), which is present in the peptidoglycans of most bacteria. NOD ligands may enter the cytoplasm as a result of intracellular infection, but may also be transported from materials captured by endocytosis, since mice lacking an oligopeptide transporter (SLC15A4) that is present in lysosomes have greatly reduced responses to NOD1 ligands.


Immunology_Janeway

Immunology_Janeway. When NOD1 or NOD2 recognizes its ligand, it recruits the CARD-containing serine–threonine kinase RIP2 (also known as RICK and RIPK2) (see Fig. 3.17). RIP2 associates with the E3 ligases cIAP1, cIAP2, and XIAP, whose activity generates a polyubiquitin scaffold as in TLR signaling. This scaffold recruits TAK1 and IKK and results in activation of NFκB as shown in Fig. 3.15. NFκB then induces the expression of genes for inflammatory cytokines and for enzymes involved in the production of nitric oxide (NO), which is toxic to bacteria and intracellular parasites. In keeping with their role as sensors of bacterial components, NOD proteins are expressed in cells that are routinely exposed to bacteria. These include epithelial cells forming the barrier that bacteria must cross to establish an infection in the body, and the macrophages and dendritic cells that ingest bacteria that have succeeded in entering the body. Macrophages and dendritic cells express TLRs as well as NOD1 and NOD2, and are


Immunology_Janeway

Immunology_Janeway. in the body, and the macrophages and dendritic cells that ingest bacteria that have succeeded in entering the body. Macrophages and dendritic cells express TLRs as well as NOD1 and NOD2, and are activated by both pathways. In epithelial cells, NOD1 is an important activator of responses against bacterial infections, and NOD1 may also function as a systemic activator of innate immunity. It seems that peptidoglycans from intestinal microbiota are transported via blood in amounts sufficient to


Immunology_Janeway

Immunology_Janeway. First panel: NOD proteins reside in an inactive state in the cytoplasm, where they serve as sensors for various bacterial components. Second panel: degradation of bacterial cell-wall peptidoglycans produces muramyl dipeptide, which is recognized by NOD2. NOD1 recognizes γ-glutamyl diaminopimelic acid (iE-DAP), a breakdown product of Gram-negative bacterial cell walls. The binding of these ligands to NOD1 or NOD2 induces aggregation, allowing CARD-dependent recruitment of the serine– threonine kinase RIP2, which associates with E3 ligases, including XIAP (X-linked inhibitor of apoptosis protein), cIAP1 (cellular inhibitor of apoptosis 1), and cIAP2. This recruited E3 ligase activity produces a polyubiquitin scaffold, as in TLR signaling, and the association of TAK1 and the IKK complex with this scaffold leads to the activation of NFκB, as shown in Fig. 3.15. In this pathway, RIP2 acts as a scaffold to recruit XIAP, and RIP2 kinase activity is not required for signaling.


Immunology_Janeway

Immunology_Janeway. Fig. 3.18 Protein-interaction domains contained in various immune signaling molecules. Signaling proteins contain protein-interaction domains that mediate the assembly of larger complexes. The table shows examples of proteins discussed in this chapter that contain the indicated domain. Proteins may have more than one domain, such as the adaptor protein MyD88, which can interact with TLRs via its TIR domain and with IRAK1/4 via its death domain (DD). MOVIE 3.6 Fig. 3.19 Cellular damage activates the NLRP3 inflammasome to produce pro-inflammatory cytokines.


Immunology_Janeway

Immunology_Janeway. The LRR domain of NLRP3 associates with chaperones (HSP90 and SGT1) that prevent NLRP3 activation. Damage to cells caused by bacterial pore-forming toxins or activation of the P2X7 receptor by extracellular ATP allows efflux of K+ ions from the cell; this may dissociate these chaperones from NLRP3 and induce multiple NLRP3 molecules to aggregate through interactions of their NOD domains (also called the NACHT domain). Reactive oxygen intermediates (ROS) and disruption of lysosomes also can activate NLRP3 (see text). The aggregated NLRP3 conformation brings multiple NLRP3 pyrin domains into close proximity, which then interact with the pyrin domains of the adaptor protein ASC (PYCARD). This conformation aggregates the ASC CARD domains, which in turn aggregate the CARD domains of pro-caspase 1. This aggregation of procaspase 1 induces proteolytic cleavage of itself to form the active caspase 1, which cleaves the immature forms of pro-inflammatory cytokines, releasing the mature


Immunology_Janeway

Immunology_Janeway. 1. This aggregation of procaspase 1 induces proteolytic cleavage of itself to form the active caspase 1, which cleaves the immature forms of pro-inflammatory cytokines, releasing the mature cytokines that are then secreted.


Immunology_Janeway

Immunology_Janeway. increase basal activation of neutrophils. A reduction in neutrophils activated in this way may explain why mice lacking NOD1 show increased susceptibility even to pathogens that lack NOD ligands, such as the parasite Trypanosoma cruzi.


Immunology_Janeway

Immunology_Janeway. NOD2 seems to have a more specialized role, being strongly expressed in the Paneth cells of the gut, where it regulates the expression of potent antimicrobial peptides such as the αand β-defensins (see Chapter 2). Consistent with this, loss-of-function mutations in NOD2 in humans are associated with the inflammatory bowel condition known as Crohn’s disease (discussed in Chapter 15). Some patients with this condition carry mutations in the LRR domain of NOD2 that impair its ability to sense MDP and activate NFκB. This is thought to diminish the production of defensins and other antimicrobial peptides, thereby weakening the natural barrier function of the intestinal epithelium and leading to the inflammation characteristic of this disease. Gain-of-function mutations in human NOD2 are associated with the inflammatory disorders early-onset sarcoidosis and Blau syndrome, which are characterized by spontaneous inflammation in tissues such as the liver, or in the joints, eyes, and skin.


Immunology_Janeway

Immunology_Janeway. with the inflammatory disorders early-onset sarcoidosis and Blau syndrome, which are characterized by spontaneous inflammation in tissues such as the liver, or in the joints, eyes, and skin. Activating mutations in the NOD domain seem to promote the signaling cascade in the absence of ligand, leading to an inappropriate inflammatory response in the absence of pathogens. Besides NOD1 and NOD2, there are other members of the NOD family, such as the proteins NLRX1 and NLRC5, but their function is currently less well understood.


Immunology_Janeway

Immunology_Janeway. 3-9 NLRP proteins react to infection or cellular damage through an inflammasome to induce cell death and inflammation. Another subfamily of NLR proteins has a pyrin domain in place of the CARD domain at their amino termini, and is known as the NLRP family. Pyrin domains are structurally related to the CARD and TIR domains, and interact with other pyrin domains (Fig. 3.19). Humans have 14 NLR proteins containing pyrin domains. The best characterized is NLRP3 (also known as NALP3 or cryopyrin), although the molecular details of its activation are still under active investigation. NLRP3 resides in an inactive form in the cytoplasm, Potassium ef˜ux induces NLRP3 forms oligomers with dissociation of chaperones ASC causing proteolytic that keep NLRP3 in an cleavage of pro-caspase 1 inactive conformation Pattern recognition by cells of the innate immune system.


Immunology_Janeway

Immunology_Janeway. where its LRR domains are thought to bind the heat-shock chaperone protein HSP90 and the co-chaperone SGT1, which may hold NLRP3 in an inactive state (see Fig. 3.19). Several events seem to induce NLRP3 signaling: reduced intracellular potassium, the generation of reactive oxygen species (ROS), or the disruption of lysosomes by particulate or crystalline matter. The loss of intracellular potassium through efflux can occur during infection with, for example, intracellular bacteria such as Staphylococcus aureus that produce pore-forming toxins. Also, death of nearby cells can release ATP into the extracellular space; this ATP would activate the purinergic receptor P2X7, which itself is a potassium channel, and allow K+ ion efflux. In one model, it is the reduction of intracellular K+ concentration that triggers NLRP3 signaling by causing the dissociation of HSP90 and SGT1. A model proposed for ROS-induced NLRP3 activation involves the intermediate oxidization of sensor proteins


Immunology_Janeway

Immunology_Janeway. concentration that triggers NLRP3 signaling by causing the dissociation of HSP90 and SGT1. A model proposed for ROS-induced NLRP3 activation involves the intermediate oxidization of sensor proteins collectively called thioredoxin (TRX). Normally TRX proteins are bound to thioredoxin-interacting protein (TXNIP), but oxidation of TRX by ROS causes the dissociation of TXNIP from TRX. The free TXNIP may then displace HSP90 and SGT1 from NLRP3, again causing its activation. In both of these cases, NLRP3 activation involves aggregation of multiple monomers via their LLR and NOD domains to induce signaling. Finally, phagocytosis of particulate matter, such as the adjuvant alum, a crystalline salt of aluminum potassium sulfate, may lead to the rupture of lysosomes and release of the active protease cathepsin B, which can activate NLRP3 by an unknown mechanism.


Immunology_Janeway

Immunology_Janeway. Rather than activating NFκB as in NOD1 and NOD2 signaling, NLRP3 signaling leads to the generation of pro-inflammatory cytokines and to cell death through formation of a multiprotein complex known as the inflammasome (see Fig. 3.19). Activation of the inflammasome proceeds in several stages. The first is the aggregation of LRR domains of several NLRP3 molecules, or other NLRP molecules, by a specific trigger or recognition event. This aggregation induces the pyrin domains of NLRP3 to interact with pyrin domains of another protein named ASC (also called PYCARD). ASC is an adaptor protein composed of an amino-terminal pyrin domain and a carboxy-terminal CARD domain. Pyrin and CARD domains are each able to form polymeric filamentous structures (Fig. 3.20). The interaction of NLRP3 with ASC further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and CARD domains facing outward. These CARD domains then interact with CARD domains of the inactive


Immunology_Janeway

Immunology_Janeway. ASC further drives the formation of a polymeric ASC filament, with the pyrin domains in the center and CARD domains facing outward. These CARD domains then interact with CARD domains of the inactive protease pro-caspase 1, initiating


Immunology_Janeway

Immunology_Janeway. Fig. 3.20 The inflammasome is composed of several filamentous protein polymers created by aggregated CARD and pyrin domains. Top panel: an electron micrograph of structures formed by full-length ASC, the pyrin domain of AIM2, and the CARD domain of caspase 1. The central dark region represents anti-ASC staining with a gold-labeled (15 nm) antibody. The long outward filaments represent the polymer composed of the caspase 1 CARD domain. Bottom panel: Schematic interpretation of NLRP3 inflammasome assembly. In this model, CARD regions of ASC and caspase 1 aggregate into a filamentous structure. The adaptor ASC translates aggregation of NLRP3 into aggregation of pro-caspase 1. Electron micrograph courtesy of Hao Wu.


Immunology_Janeway

Immunology_Janeway. its CARD-dependent polymerization into discrete caspase 1 filaments. This aggregation seems to trigger the autocleavage of pro-caspase 1, which releases the active caspase 1 fragment from its autoinhibitory domains. Active caspase 1 then carries out the ATP-dependent proteolytic processing of pro-inflammatory cytokines, particularly IL-1β and IL-18, into their active forms (see Fig. 3.19). Caspase 1 activation also induces a form of cell death called pyroptosis (‘fiery death’) through an unknown mechanism that is associated with inflammation because of the release of these pro-inflammatory cytokines upon cell rupture.


Immunology_Janeway

Immunology_Janeway. For inflammasome activation to produce inflammatory cytokines, a priming step must first occur in which cells induce and translate the mRNAs that encode the pro-forms of IL-1β, IL-18, or other cytokines. This priming step can result from TLR signaling, which may help ensure that inflammasome activation proceeds primarily during infections. For example, the TLR-3 agonist poly I:C (see Section 3-5) can be used experimentally to prime cells for subsequent triggering of the inflammasome.


Immunology_Janeway

Immunology_Janeway. Several other NLR family members form inflammasomes with ASC and caspase 1 that activate these pro-inflammatory cytokines. NLRP1 is highly expressed in monocytes and dendritic cells and is activated directly by MDP, similar to NOD2, but can also be activated by other factors. For example, Bacillus anthracis expresses an endopeptidase, called anthrax lethal factor, which allows the pathogen to evade the immune system by killing macrophages. Lethal factor does this by cleaving NLRP1, activating an NLRP1 inflammasome and inducing pyroptosis in the infected macrophages. NLRC4 acts as an adaptor with two other NLR proteins, NAIP2 and NAIP5, that serve to detect various bacterial proteins that enter cells through specialized secretion systems used by pathogens to transport materials into or access nutrients from host cells. One such protein, PrgJ, from the pathogen Salmonella typhimurium, is a component of the type III secretion system (T3SS), a needle-like macromolecular complex. Upon


Immunology_Janeway

Immunology_Janeway. access nutrients from host cells. One such protein, PrgJ, from the pathogen Salmonella typhimurium, is a component of the type III secretion system (T3SS), a needle-like macromolecular complex. Upon infection of host cells by Salmonella, PrgJ enters the cytoplasm and is recognized by NLRC4 functioning together with NAIP2. Extracellular bacterial flagellin is recognized by TLR5, but flagellin may also enter host cells with PrgJ via the T3SS, and in this case can be recognized by NLRC4 in conjunction with NAIP5. Some NLR proteins may negatively regulate innate immunity, such as NLRP6, since mice lacking this protein exhibit increased resistance to certain pathogens. However, NLRP6 is highly expressed in intestinal epithelium, where it appears to play a positive role in promoting normal mucosal barrier function and is required for the normal secretion of mucus granules into the intestine by goblet cells. NLRP7, which is present in humans but not mice, recognizes microbial acylated


Immunology_Janeway

Immunology_Janeway. mucosal barrier function and is required for the normal secretion of mucus granules into the intestine by goblet cells. NLRP7, which is present in humans but not mice, recognizes microbial acylated lipopeptides and forms an inflammasome with ASC and caspase 1 to produce IL-1β and IL-18. Less is known about NLRP12, but like NLRP6, it initially was proposed to have an inhibitory function. Subsequent studies of mice lacking NLRP12 suggest it has a possible role in the detection of and response to certain bacterial species, including Yersinia pestis, the bacterium that causes bubonic plague, although the basis of this recognition is still unclear.


Immunology_Janeway

Immunology_Janeway. Inflammasome activation can also involve proteins of the PYHIN family, which contain an N-terminal pyrin domain but lack the LRR domains present in the NLR family. In place of an LRR domain, PYHIN proteins have a HIN (H inversion) domain, so named for the HIN DNA recombinase of Salmonella that mediates DNA inversion between flagellar H antigens. There are four PYHIN proteins in humans, and 13 in mice. In one of these, AIM2 (absent in melanoma 2), the HIN domain recognizes double-stranded DNA genomes and triggers caspase 1 activation through pyrin domain interactions with ASC. AIM2 is located in the cytoplasm and is important for responses in vitro to vaccinia virus, and its in vivo role has been demonstrated by the increased susceptibility of AIM2-deficient mice to infection by Francisella tularensis, the causative agent of tularemia. The related protein IFI16 (interferon inducible protein 16) contains two HIN domains; it is primarily located in the cell nucleus and recognizes viral


Immunology_Janeway

Immunology_Janeway. tularensis, the causative agent of tularemia. The related protein IFI16 (interferon inducible protein 16) contains two HIN domains; it is primarily located in the cell nucleus and recognizes viral double-stranded DNA, and will be described below in Section 3-11.


Immunology_Janeway

Immunology_Janeway. A ‘non-canonical’ inflammasome (caspase 1-independent) pathway uses the protease caspase 11 to detect intracellular LPS. The discovery of this pathway was initially confused as being dependent on caspase 1 because of a specific genetic difference between experimental mouse strains. Caspase 11 is encoded by the murine Casp4 gene and is homologous to human caspases 4 and 5. The mice in which the caspase 1 gene (Casp1) was initially disrupted and studied were originally found to be resistant to lethal shock (see Section 3-20) induced by administration of LPS. This led researchers to conclude that caspase 1 acted in the inflammatory response to LPS. But researchers later discovered that this mouse strain also carried a natural mutation that inactivated the related Casp4 gene. Because the Casp1 and Casp4 genes reside within 2 kilobases of each other on mouse chromosome 9, they failed to segregate independently during subsequent experimental genetic backcrosses to other mouse strains. Thus,


Immunology_Janeway

Immunology_Janeway. and Casp4 genes reside within 2 kilobases of each other on mouse chromosome 9, they failed to segregate independently during subsequent experimental genetic backcrosses to other mouse strains. Thus, mice initially thought to lack only caspase 1 protein in fact lacked both caspase 1 and caspase 11. Later, mice lacking only caspase 1 were generated by expressing functional Casp4 as a transgene; these mice became susceptible to LPS-induced shock. Mice were also generated that lacked only caspase 11, and these were found to be resistant to LPS-induced shock. These results indicated that caspase 11, and not caspase 1 as originally thought, is responsible for LPS-induced shock. Caspase 11 is responsible for inducing pyroptosis, but not for processing of IL-1β or IL-18. It was suspected that TLR-4 was not the sensor for LPS that activated the non-canonical imflammasome, since mice lacking TLR-4 remain susceptible to LPS-induced shock. Recent evidence has suggested that caspase 11 itself is


Immunology_Janeway

Immunology_Janeway. was not the sensor for LPS that activated the non-canonical imflammasome, since mice lacking TLR-4 remain susceptible to LPS-induced shock. Recent evidence has suggested that caspase 11 itself is the intracellular LPS sensor, making it an example of a protein that is both a sensor and an effector molecule.


Immunology_Janeway

Immunology_Janeway. Inappropriate inflammasome activation has been associated with various diseases. Gout has been known for many years to cause inflammation in the cartilaginous tissues by the deposition of monosodium urate crystals, but how urate crystals caused inflammation was a mystery. Although the precise mechanism is still unclear, urate crystals are known to activate the NLRP3 inflammasome, which induces the inflammatory cytokines associated with the symptoms of gout. Mutations in the NOD domain of NLRP2 and NLRP3 can activate inflammasomes inappropriately, and they are the cause of some inherited autoinflammatory diseases, in which inflammation occurs in the absence of infection. Mutations in NLRP3 in humans are associated with hereditary periodic fever syndromes, such as familial cold inflammatory syndrome and Muckle–Wells syndrome (discussed in more detail in Chapter 13). Macrophages from patients with these conditions show spontaneous production of inflammatory cytokines such as IL-1β. We


Immunology_Janeway

Immunology_Janeway. syndrome and Muckle–Wells syndrome (discussed in more detail in Chapter 13). Macrophages from patients with these conditions show spontaneous production of inflammatory cytokines such as IL-1β. We will also discuss how pathogens can interfere with formation of the inflammasome in Chapter 13.


Immunology_Janeway

Immunology_Janeway. 3-10 The RIG-I-like receptors detect cytoplasmic viral RNAs and activate MAVS to induce type I interferon production and pro-inflammatory cytokines. TLR-3, TLR-7, and TLR-9 detect extracellular viral RNAs and DNAs that enter the cell from the endocytic pathway. By contrast, viral RNAs produced within a cell are sensed by a separate family of proteins called the RIG-I-like receptors (RLRs). These proteins serve as viral sensors by binding to viral RNAs using an RNA helicase-like domain in their carboxy terminal. The RLR helicase-like domain has a ‘DExH’ tetrapeptide amino acid motif and is a subgroup of DEAD-box family proteins. The RLR proteins also contain two amino-terminal CARD domains that interact with adaptor proteins and activate Fig. 3.21 RIG-I and other RLRs are cytoplasmic sensors of viral RNA.


Immunology_Janeway

Immunology_Janeway. First panel: before detecting viral RNA, RIG-I and MDA-5 are cytoplasmic and in inactive, auto-inhibited conformations. The adaptor protein MAVS is attached to the mitochondrial outer membrane. Second panel: detection of uncapped 5ʹ-triphosphate RNA by RIG-I, or viral dsRNA by MDA-5, changes the conformation of their CARD domains to become free to interact with the amino-terminal CARD domain of MAVS. This interaction involves the generation of K63-linked polyubiquitin from the E3 ligases TRIM25 or Riplet, although structural details are still unclear. Third panel: the aggregation induces a proline-rich region of MAVS to interact with TRAFs (see text) and leads to the generation of additional K63-linked polyubiquitin scaffold. As in TLR signaling, this scaffold recruits TBK1 and IKK complexes (see Figs. 3.15 and 3.16) to activate IRF and NFκB, producing type I interferons and pro-inflammatory cytokines, respectively.


Immunology_Janeway

Immunology_Janeway. signaling to produce type I interferons when viral RNAs are bound. The first of these sensors to be discovered was RIG-I (retinoic acid-inducible gene I). RIG-I is widely expressed across tissues and cell types and serves as an intracellular sensor for several kinds of infections. Mice deficient in RIG-I are highly susceptible to infection by several kinds of single-stranded RNA viruses, including paramyxoviruses, rhabdoviruses, orthomyxoviruses, and flaviviruses, but not picornaviruses.


Immunology_Janeway

Immunology_Janeway. RIG-I discriminates between host and viral RNA by sensing differences at the 5ʹ end of single-stranded RNA transcripts. Eukaryotic RNA is transcribed in the nucleus and contains a 5ʹ-triphosphate group on its initial nucleotide that undergoes subsequent enzymatic modification called capping by the addition of a 7-methylguanosine to the 5ʹ-triphosphate. Most RNA viruses, however, do not replicate in the nucleus, where capping normally occurs, and their RNA genomes do not undergo this modification. Biochemical studies have determined that RIG-I senses the unmodified 5ʹ-triphosphate end of the ssRNA viral genome. Flavivirus RNA transcripts have the unmodified 5ʹ-triphosphate, as do the transcripts of many other ssRNA viruses, and they are detected by RIG-I. In contrast, the picornaviruses, which include poliovirus and hepatitis A, replicate by a mechanism that involves the covalent attachment of a viral protein to the 5ʹ end of the viral RNA, so that the 5ʹ-triphosphate is absent, which


Immunology_Janeway

Immunology_Janeway. include poliovirus and hepatitis A, replicate by a mechanism that involves the covalent attachment of a viral protein to the 5ʹ end of the viral RNA, so that the 5ʹ-triphosphate is absent, which explains why RIG-I is not involved in sensing them.


Immunology_Janeway

Immunology_Janeway. MDA-5 (melanoma differentiation-associated 5), also called helicard, is similar in structure to RIG-I, but it senses dsRNA. In contrast to RIG-I-deficient mice, mice deficient in MDA-5 are susceptible to picornaviruses, indicating that these two sensors of viral RNAs have crucial but distinct roles in host defense. Inactivating mutations in alleles of human RIG-I or MDA-5 have been reported, but these mutations were not associated with immunodeficiency. The RLR family member LGP2 (encoded by DHX58) retains a helicase domain but lacks CARD domains. LGP2 appears to cooperate with RIG-I and MDA-5 in the recognition of viral RNA, since mice lacking LGP2 have impaired antiviral responses normally mediated by RIG-I or MDA-5. This cooperative viral recognition by LGP2 appears to depend on its helicase domain, since in mice, mutations that disrupt its ATPase activity result in impaired IFN-β production in response to various RNA viruses.


Immunology_Janeway

Immunology_Janeway. Sensing of viral RNAs activates signaling by RIG-I and MDA-5 that leads to type I interferon production appropriate for defense against viral infection (Fig. 3.21). Before infection by viruses, RIG-I and MDA-5 are in the cytoplasm in an autoinhibited configuration that is stabilized by interactions between the CARD and helicase domains. These interactions are disrupted upon infection when viral RNA associates with the helicase domains of RIG-I or MDA-5, freeing the two CARD domains for other interactions. The more amino-proximal portion of the two CARD domains can then recruit E3 ligases, including TRIM25 and Riplet (encoded by RNF153), which initiate K63-linked polyubiquitin scaffolds (see Section 3-7), either as free polyubiquitin chains or on linkages within the second CARD domain. Precise details are unclear, but this scaffold appears to help RIG-I and MDA-5 interact with a downstream adaptor protein called MAVS (mitochondrial antiviral signaling protein). MAVS is attached to the


Immunology_Janeway

Immunology_Janeway. details are unclear, but this scaffold appears to help RIG-I and MDA-5 interact with a downstream adaptor protein called MAVS (mitochondrial antiviral signaling protein). MAVS is attached to the outer mitochondrial membrane and contains a CARD domain that may bind RIG-I and MDA-5. This aggregation of CARD domains, as in the inflammasome, may initiate aggregation of MAVS. In this state, MAVS propagates signals by recruiting various TRAF family E3 ubiquitin ligases, including TRAF2, TRAF3, TRAF5, and TRAF6. The relative importance of each E3 ligase may differ between cell types, but their further production of K63linked polyubiquitin leads to activation of TBK1 and IRF3 and production of type I interferons, as described for TLR-3 signaling (see Fig. 3.16), and also to activation of NFκB. Some viruses have evolved countermeasures to thwart the protection conferred by RLRs. For example, even though the negative-sense RNA genome of influenza virus replicates in the nucleus, some viral RNA


Immunology_Janeway

Immunology_Janeway. viruses have evolved countermeasures to thwart the protection conferred by RLRs. For example, even though the negative-sense RNA genome of influenza virus replicates in the nucleus, some viral RNA transcripts produced during influenza infection are not capped but must be translated in the cytoplasm. The influenza A nonstructural protein 1(NS1) inhibits the activity of TRIM25, and thereby interrupts the antiviral actions that RIG-I might exert against infection.


Immunology_Janeway

Immunology_Janeway. 3-11 Cytosolic DNA sensors signal through STING to induce production of type I interferons. Innate sensors that recognize cytoplasmic RNA use specific modifications, such as the 5ʹ cap, to discriminate between host and viral origin. Host DNA is generally restricted to the nucleus, but viral, microbial, or protozoan DNA may become located in the cytoplasm during various stages of infection. Several innate sensors of cytoplasmic DNA have been identified that can lead to the production of type I interferon in response to infections. One component of the DNA-sensing pathway, STING (stimulator of interferon genes), was identified in a functional screen for proteins that can induce expression of type I interferons. STING (encoded by TMEM173) is anchored to the endoplasmic reticulum membrane by an amino-terminal tetraspan transmembrane domain; its carboxy-terminal domain extends into the cytoplasm and interacts to form an inactive STING homodimer.


Immunology_Janeway

Immunology_Janeway. STING is known to serve as a sensor of intracellular infection, based on its recognition of bacterial cyclic dinucleotides (CDNs), including cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP). These molecules are bacterial second messengers and are produced by enzymes present in most bacterial genomes. CDNs activate STING signaling by changing the conformation of the STING homodimer. This homodimer recruits and activates TBK1, which in turn phosphorylates and activates IRF3, leading to type I interferon production (Fig. 3.22), similar to signaling by TLR-3 and MAVS (see Figs. 3.16 and 3.21). TRIF (downstream of TLR3), MAVS, and STING each contain a similar amino acid sequence motif at their carboxy termini that becomes serine-phosphorylated when these molecules are activated. It appears that this motif, when phosphorylated, recruits both TBK1 and IRF3, allowing IRF3 to be efficiently phosphorylated and activated by TBK1.


Immunology_Janeway

Immunology_Janeway. STING also plays a role in viral infections, since mice lacking STING are susceptible to infection by herpesvirus. But until recently, it was unclear whether STING activates the kinase TBK1 to phosphorylate IRF3, which enters the nucleus and induces expression of type I interferon genes First panel: cGAS resides in the cytoplasm and serves as a sensor of double-stranded DNA (dsDNA) from viruses. When cGAS binds dsDNA, its enzymatic activity is stimulated, leading to production of cyclic-GMP-AMP (cGAMP). Bacteria that infect cells produce second messengers such as cyclic dinucleotides, including cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP). Second panel: cGAMP and other bacterial dinucleotides can bind and activate the STING dimer present on the ER membrane. Third panel: in this state STING activates TBK1, although the details of this interaction are still unclear. Active TBK1 activates IRF3, as described in Fig. 3.16.


Immunology_Janeway

Immunology_Janeway. STING recognized viral DNA directly or acted only downstream of an unknown viral DNA sensor. It was found that the introduction of DNA into cells, even without live infection, generated another second messenger molecule that activated STING. This second messenger was identified as cyclic guano-sine monophosphate-adenosine monophosphate (cyclic GMP-AMP), or cGAMP. cGAMP, like bacterial CDNs, binds both subunits of the STING dimer and activates STING signaling. This result also suggested the presence of a DNA sensor acting upstream of STING. Purification of the enzyme that produces cGAMP in response to cytosolic DNA identified a previously unknown enzyme, which was named cGAS, for cyclic GAMP synthase. cGAS contains a protein motif present in the nucleotidyltransferase (NTase) family of enzymes, which includes adenylate cyclase (the enzyme that generates the second messenger molecule cyclic AMP) and various DNA polymerases. cGAS can bind directly to cytosolic DNA, and this stimulates


Immunology_Janeway

Immunology_Janeway. which includes adenylate cyclase (the enzyme that generates the second messenger molecule cyclic AMP) and various DNA polymerases. cGAS can bind directly to cytosolic DNA, and this stimulates its enzymatic activity to produce cGAMP from GTP and ATP in the cytoplasm, activating STING. Mice harboring an inactivated cGAS gene show increased susceptibility to herpesvirus infection, demonstrating its importance in immunity.


Immunology_Janeway

Immunology_Janeway. There are several other candidate DNA sensors, but less is known about the mechanism of their recognition and signaling, or their in vivo activity. IFI16 (IFN-γ-inducible protein 16) is a PYHIN family member related to AIM2, but appears to function in DNA sensing and acts through STING, TBK1, and IRF3 rather than activating an inflammasome pathway. DDX41 (DEAD box polypeptide 41) is an RLR related to RIG-I and is a member of the DEAD-box family, but appears to signal through STING rather than MAVS. MRE11A (meitotic recombination 11 homolog a) can sense cytosolic double-stranded DNA to activate the STING pathway, but its role in innate immunity is currently unknown. 3-12 Activation of innate sensors in macrophages and dendritic far-reaching effects on the immune response.


Immunology_Janeway

Immunology_Janeway. 3-12 Activation of innate sensors in macrophages and dendritic far-reaching effects on the immune response. Besides activating effector functions and cytokine production, another outcome of the activation of innate sensing pathways is the induction of co-stimulatory molecules on tissue dendritic cells and macrophages (see Section 1-15). We will describe these in more detail later in the book, but mention them now because they provide an important link between innate and adaptive immune responses. Two important co-stimulatory molecules are the cell-surface proteins B7.1 (CD80) and B7.2 (CD86), which are induced on macrophages and tissue dendritic cells by innate sensors such as TLRs in response to pathogen recognition (Fig. 3.23). B7.1 and B7.2 are recognized by specific co-stimulatory receptors expressed by cells of the adaptive immune response, particularly CD4 T cells, and their activation by B7 is an important step in activating adaptive immune responses.


Immunology_Janeway

Immunology_Janeway. Substances such as LPS that induce co-stimulatory activity have been used for years in mixtures that are co-injected with protein antigens to enhance their immunogenicity. These substances are known as adjuvants (see Appendix I, Section A-1), and it was found empirically that the best adjuvants contain microbial components that induce macrophages and tissue dendritic cells to express co-stimulatory molecules and cytokines. As we shall see in Chapters 9 and 11, the cytokines produced in response to infections influence the functional character of the adaptive immune response that develops. In this way the ability of the innate immune system to discriminate among different types of pathogens is used by the organism to ensure an appropriate module of adaptive immune response. 3-13 Toll signaling in Drosophila is downstream of a distinct set of pathogen-recognition molecules.


Immunology_Janeway

Immunology_Janeway. Before leaving innate sensing, we shall look briefly at how Toll, TLRs, and NODs are used in invertebrate innate immunity. Although Toll is central to defense against both bacterial and fungal pathogens in Drosophila, Toll itself is not a pattern recognition receptor, but is downstream of other proteins that detect pathogens (Fig. 3.24). In Drosophila, there are 13 genes encoding peptidoglycan-recognition proteins (PGRPs) that bind the peptidoglycan components of bacterial cell walls. Another family, the Gram-negative binding proteins (GNBPs), recognizes LPS and β-1,3-linked glucans. GNBPs recognize Gram-negative bacteria and, unexpectedly, fungi, rather than Gram-positive bacteria. The family members GNBP1 and PGRP-SA cooperate in the recognition of peptidoglycan from Gram-positive bacteria. They interact with a serine protease called Grass, which initiates a proteolytic cascade that terminates in the cleavage of the protein Spätzle. One of the cleaved fragments forms a homodimer


Immunology_Janeway

Immunology_Janeway. They interact with a serine protease called Grass, which initiates a proteolytic cascade that terminates in the cleavage of the protein Spätzle. One of the cleaved fragments forms a homodimer that binds to Toll and induces its dimerization, which in turn stimulates the antimicrobial response. A fungus-specific recognition protein, GNBP3, also activates the proteolytic cascade, causing cleavage of Spätzle and activation of Toll.


Immunology_Janeway

Immunology_Janeway. In Drosophila, fat-body cells and hemocytes are phagocytic cells that act as part of the fly’s immune system. When the Spätzle dimer binds to Toll, hemocytes synthesize and secrete antimicrobial peptides. The Toll signaling pathway in Drosophila activates a transcription factor called DIF, which is related to mammalian NFκB. DIF enters the nucleus and induces the transcription of genes for antimicrobial peptides such as drosomycin. Another Drosophila factor in the NFκB family, Relish, induces the production of antimicrobial peptides in response to the Imd (immunodeficiency) signaling pathway, which is triggered in Drosophila by particular PGRPs that recognize Gram-negative bacteria. Relish induces expression of the antimicrobial peptides diptericin, attacin, and cecropin, which are distinct from the peptides induced by Toll signaling. Thus, the Toll and Imd pathways activate effector mechanisms to eliminate infection by different kinds of pathogens. Four mammalian PGRP homologs have


Immunology_Janeway

Immunology_Janeway. from the peptides induced by Toll signaling. Thus, the Toll and Imd pathways activate effector mechanisms to eliminate infection by different kinds of pathogens. Four mammalian PGRP homologs have been identified, but act differently than in Drosophila. One, PGLYRP-2, is secreted and functions as an amidase to hydrolyze bacterial peptidoglycans. The others are present in neutrophil granules and exert a bacteriostatic action through interactions with bacterial cell-wall peptidoglycan.


Immunology_Janeway

Immunology_Janeway. Fig. 3.23 Bacterial LPS induces changes in dendritic cells, stimulating them to migrate and to initiate adaptive immunity by activating T cells. Top panel: immature dendritic cells in the skin are highly phagocytic and macropinocytic, but lack the ability to activate T lymphocytes. Dendritic cells residing in the skin ingest microbes and their products and degrade them. During a bacterial infection, the dendritic cells are activated by various innate sensors and the activation induces two types of changes. Second panel: the dendritic cells migrate out of the tissues and enter the lymphatic system and begin to mature. They lose the ability to ingest antigen but gain the ability to stimulate T cells. Third panel: in the regional lymph nodes, they become mature dendritic cells. They change the character of their cell-surface molecules, increasing the number of MHC molecules on their surface and the expression of the co-stimulatory molecules CD80 (B7.1) and CD86 (B7.2).


Immunology_Janeway

Immunology_Janeway. Fig. 3.24 Drosophila Toll is activated as a result of a proteolytic cascade initiated by pathogen recognition. The peptidoglycan-recognition protein PGRP-SA and the Gram-negative binding protein GNBP1 cooperate in the recognition of bacterial pathogens and activate the first protease in a protease cascade that leads to cleavage of the Drosophila protein Spätzle (first panel). Cleavage alters the conformation of Spätzle, enabling it to bind Toll and induce Toll dimerization (second panel). Toll’s cytoplasmic TIR domains recruit the adaptor protein dMyD88 (third panel), which initiates a signaling pathway very similar to that leading to the release of NFκB from its cytoplasmic inhibitor in mammals. The Drosophila version of NFκB is the transcription factor DIF, which then enters the nucleus and activates the transcription of genes encoding antimicrobial peptides. Fungal recognition also leads to cleavage of Spätzle and the production of antimicrobial peptides by this pathway, although


Immunology_Janeway

Immunology_Janeway. and activates the transcription of genes encoding antimicrobial peptides. Fungal recognition also leads to cleavage of Spätzle and the production of antimicrobial peptides by this pathway, although the recognition proteins for fungi are as yet unidentified.


Immunology_Janeway

Immunology_Janeway. 3-14 TLR and NOD genes have undergone extensive diversification in both invertebrates and some primitive chordates. There are only about a dozen mammalian TLR genes, but some organisms have diversified their repertoire of innate recognition receptors, especially those containing LRR domains, to a much greater degree. The sea urchin Strongylocentrotus purpuratus has an unprecedented 222 different TLR genes, more than 200 NOD-like receptor genes, and more than 200 scavenger receptor genes in its genome. The sea urchin also has an increased number of proteins that are likely to be involved in signaling from these receptors, there being, for example, four genes that are similar to the single mammalian MyD88 gene. However, there is no apparent increase in the number of downstream targets, such as the family of NFκB transcription factors, suggesting that the ultimate outcome of TLR signaling in the sea urchin may be very similar to that in other organisms.


Immunology_Janeway

Immunology_Janeway. Sea urchin TLR genes fall into two broad categories. One is a small set of 11 divergent genes. The other is a large family of 211 genes, which show a high degree of sequence variation within particular LRR regions; this, together with the large number of pseudogenes in this family, indicates rapid evolutionary turnover, suggesting rapidly changing receptor specificities, in contrast with the few stable mammalian TLRs. Although the pathogen specificity of sea urchin TLRs is unknown, the hypervariability in the LRR domains could be used to generate a highly diversified pathogen-recognition system based on Toll-like receptors. A similar expansion of innate receptors has occurred in some chordates, the phylum to which vertebrates belong. Amphioxus (the lancelet) is a nonvertebrate chordate lacking an adaptive immune system. The amphioxus genome contains 71 TLRs, more than 100 NOD-like receptors, and more than 200 scavenger receptors. As we will see in Chapter 5, a primitive vertebrate


Immunology_Janeway

Immunology_Janeway. lacking an adaptive immune system. The amphioxus genome contains 71 TLRs, more than 100 NOD-like receptors, and more than 200 scavenger receptors. As we will see in Chapter 5, a primitive vertebrate lineage—the jawless fish, which lack immunoglobulinand T-cell-based adaptive immunity—uses somatic gene rearrangement of LRR-containing proteins to provide a version of adaptive immunity (see Section 5-18).


Immunology_Janeway

Summary.

Immunology_Janeway. Summary.


Immunology_Janeway

Immunology_Janeway. Innate immune cells express several receptor systems that recognize microbes and induce rapid defenses as well as delayed cellular responses. Several scavenger and lectin-like receptors on neutrophils, macrophages, and dendritic cells help rapidly eliminate microbes through phagocytosis. G-protein-coupled receptors for C5a (which can be produced by activation of the complement system’s innate pathogen-recognition ability) and for the bacterial peptide fMLF synergize with phagocytic receptors in activating the NADPH oxidase in phagosomes to generate antimicrobial reactive oxygen intermediates. Toll-like receptors (TLRs) on the cell surface and in the membranes of endosomes detect microbes outside the cell and activate several host defense signaling pathways. The NFκB and IRF pathways downstream of these receptors induce pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and antiviral cytokines including type I interferons. Other receptor families detect microbial infection


Immunology_Janeway

Immunology_Janeway. of these receptors induce pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and antiviral cytokines including type I interferons. Other receptor families detect microbial infection in the cytosol. NOD proteins detect bacterial products within the cytosol and activate NFκB and the production of pro-inflammatory cytokines. The related NLR family of proteins detects signs of cellular stress or damage, as well as certain microbial components. NLRs signal through the inflammasome, which generates pro-inflammatory cytokines and induces pyroptosis, a form of cell death. RIG-I and MDA-5 detect viral infection by sensing the presence of viral RNAs and activate the MAVS pathway, while sensors of cytosolic DNA, such as cGAS, activate the STING pathway; both of these pathways induce type I interferons. The signaling pathways activated by all of these primary sensors of pathogens induce a variety of genes, including those for cytokines, chemokines, and co-stimulatory molecules that


Immunology_Janeway

Immunology_Janeway. I interferons. The signaling pathways activated by all of these primary sensors of pathogens induce a variety of genes, including those for cytokines, chemokines, and co-stimulatory molecules that have essential roles in immediate defense and in directing the course of the adaptive immune response later in infection.


Immunology_Janeway

Induced innate responses to infection.

Immunology_Janeway. Induced innate responses to infection.


Immunology_Janeway

Immunology_Janeway. We will now examine the responses of innate immunity induced as an immediate consequence of pathogen recognition by the sensors described in the last section. We will focus on the major phagocytes—neutrophils, macrophages, and dendritic cells—and the cytokines they produce that induce and maintain inflammation. First, we will introduce the families of cytokines and chemokines that coordinate many cellular responses, such as the recruitment of neutrophils and other immune cells to sites of infection. We will discuss the various adhesion molecules that are induced on immune cells circulating in the blood and on endothelial cells of blood vessels to coordinate movement of cells out of the blood and into infected tissues. We will consider in some detail how macrophage-derived chemokines and cytokines promote the continued destruction of infecting microbes. This is achieved both by stimulating the production and recruitment of fresh phagocytes and by inducing another phase of the innate


Immunology_Janeway

Immunology_Janeway. cytokines promote the continued destruction of infecting microbes. This is achieved both by stimulating the production and recruitment of fresh phagocytes and by inducing another phase of the innate immune response—the acute-phase response—in which the liver produces proteins that act as opsonizing molecules, helping to augment the actions of complement. We will also look at the mechanism of action of antiviral interferons, the type I interferons, and finally examine the growing class of innate lymphoid cells, or ILCs, which include the NK cells long known to contribute to innate immune defense against viruses and other intracellular pathogens. ILCs exert a diverse array of effector function that contribute to a rapid innate immune response to infection. They respond to early cytokine signals provided by innate sensor cells, and amplify the response by producing various types of effector cytokines. If an infection is not cleared by the induced innate response, an adaptive response


Immunology_Janeway

Immunology_Janeway. signals provided by innate sensor cells, and amplify the response by producing various types of effector cytokines. If an infection is not cleared by the induced innate response, an adaptive response will ensue that uses many of the same effector mechanisms used by the innate immune system but targets them with much greater precision. The effector mechanisms described here therefore serve as a primer for the focus on adaptive immunity in the later parts of this book.


Immunology_Janeway

Immunology_Janeway. 3-15 Cytokines and their receptors fall into distinct families of structurally related proteins. Cytokines are small proteins (about 25 kDa) that are released by various cells in the body, usually in response to an activating stimulus, and that induce responses through binding to specific receptors. Cytokines can act in an autocrine manner, affecting the behavior of the cell that releases the cytokine, or in a paracrine manner, affecting adjacent cells. Some cytokines are even stable enough to act in an endocrine manner, affecting distant cells, although this


Immunology_Janeway

Immunology_Janeway. Fig. 3.25 Cytokine receptors belong to families of receptor proteins, each with a distinctive structure. Many cytokines signal through receptors of the hematopoietin receptor superfamily, named after its first member, the erythropoietin receptor. The hematopoietin receptor superfamily includes homodimeric and heterodimeric receptors, which are subdivided into families on the basis of protein sequence and structure. Examples of these are given in the first three rows. Heterodimeric class I cytokine receptors have an α chain that often defines the ligand specificity of the receptor; they may share with other receptors a common β or γ chain that confers the intracellular signaling function. Heterodimeric class II cytokine receptors have no common chain and include receptors for interferons or interferon-like cytokines. All the cytokine receptors signal through the JAK-STAT pathway. The IL-1 receptor family have extracellular immunoglobulin domains and signal as dimers through TIR domains


Immunology_Janeway

Immunology_Janeway. interferon-like cytokines. All the cytokine receptors signal through the JAK-STAT pathway. The IL-1 receptor family have extracellular immunoglobulin domains and signal as dimers through TIR domains in their cytoplasmic tails and through MyD88. Other superfamilies of cytokine receptors are the tumor necrosis factor receptor (TNFR) family and the chemokine receptor family, the latter belonging to the very large family of G-protein-coupled receptors. The ligands of the TNFR family act as trimers and may be associated with the cell membrane rather than being secreted.


Immunology_Janeway

Immunology_Janeway. depends on their ability to enter the circulation and on their half-life in the blood. In an attempt to develop a standardized nomenclature for molecules secreted by, and acting on, leukocytes, many cytokines are called by the name interleukin (IL) followed by a number (for example, IL-1 or IL-2). However, not all cytokines are included in this system; thus students of immunology are still faced with a somewhat confusing and difficult task. The cytokines are listed alphabetically, together with their receptors, in Appendix III.


Immunology_Janeway

Immunology_Janeway. Cytokines can be grouped by structure into families—the IL-1 family, the hematopoietin superfamily, the interferons (described in Section 3-7), and the TNF family—and their receptors can likewise be grouped (Fig. 3.25). The IL-1 family contains 11 members, notably IL-1α, IL-1β, and IL-18. Most members of this family are produced as inactive proproteins that are cleaved (removing an amino-terminal peptide) to produce the mature cytokine. The exception to this rule is IL-1α, for which both the proprotein and its cleaved forms are biologically active. As discussed earlier, mature IL-1β and IL-18 are produced by macrophages through the action of caspase 1 in response to TLR signaling and inflammasome activation. The IL-1-family receptors have TIR domains in their cytoplasmic tails and signal by the NFκB pathway described earlier for TLRs. The IL-1 receptor functions in concert with a second transmembrane protein, the IL-1 receptor accessory protein (IL1RAP), that is required for IL-1


Immunology_Janeway

Immunology_Janeway. by the NFκB pathway described earlier for TLRs. The IL-1 receptor functions in concert with a second transmembrane protein, the IL-1 receptor accessory protein (IL1RAP), that is required for IL-1 signal transduction.


Immunology_Janeway

Immunology_Janeway. The hematopoietin superfamily of cytokines is quite large and includes non-immune-system growth and differentiation factors such as erythropoietin (which stimulates red blood cell development) and growth hormone, as well as interleukins with roles in innate and adaptive immunity. IL-6 is a member of this superfamily, as is the cytokine GM-CSF, which stimulates the production of new monocytes and granulocytes in the bone marrow. Many of the soluble cytokines made by activated T cells are members of the hematopoietin family. The receptors for the hematopoietin cytokines are tyrosine kinase-associated receptors that form dimers when their cytokine ligand binds. Dimerization initiates intracellular signaling from the tyrosine kinases associated with the cytoplasmic domains of the receptor. Some types of cytokine receptors are composed of two identical subunits, but others have two different subunits. An important feature of cytokine signaling is the large variety of different receptor


Immunology_Janeway

Immunology_Janeway. Some types of cytokine receptors are composed of two identical subunits, but others have two different subunits. An important feature of cytokine signaling is the large variety of different receptor subunit combinations that occur.


Immunology_Janeway

Immunology_Janeway. Tumor necrosis factor (TNF) receptors I and II, CD40, Fas (Apo1, CD95), CD30, CD27, nerve growth factor receptor CCR1–10, CXCR1–5, XCR1, CX3CR1 Homodimeric receptors Receptors for erythropoietin and growth hormone Heterodimeric receptors (no common chain) IL-1 family receptors Receptors for IL-13, IFN-˜, IFN-°, IFN-˛, IL-10 Heterodimeric receptors with a common chain Receptors for IL-3, IL-5, GM-CSF share a common chain, CD131 or °c (common °chain) °c Receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 share a common chain, CD132 or ˛c (common ˛chain). IL-2 receptor also has a third chain, a high-affnity subunit IL-2R˜(CD25) ˛c TNF receptor family Chemokine receptor family


Immunology_Janeway

Immunology_Janeway. These cytokines and their receptors can also be further divided into subfamilies characterized by functional similarities and genetic linkage. For instance, IL-3, IL-4, IL-5, IL-13, and GM-CSF are related structurally, their genes are closely linked in the genome, and they are often produced together by the same kinds of cells. In addition, they bind to closely related receptors, which belong to the family of class I cytokine receptors. The IL-3, IL-5, and GM-CSF receptors form a subgroup that shares a common β chain. Another subgroup of class I cytokine receptors is defined by the use of the common γ chain (γc) of the IL-2 receptor. This chain is shared by receptors for the cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, and is encoded by a gene located on the X chromosome. Mutations that inactivate γc cause an X-linked severe combined immunodeficiency (X-linked SCID) due to inactivation of the signaling pathways for several cytokines—IL-7, IL-15, and IL-2—that are required for


Immunology_Janeway

Immunology_Janeway. that inactivate γc cause an X-linked severe combined immunodeficiency (X-linked SCID) due to inactivation of the signaling pathways for several cytokines—IL-7, IL-15, and IL-2—that are required for normal lymphocyte development (see Section 13-3). More distantly related, the receptor for IFN-γ is a member of a small family of heterodimeric cytokine receptors with some similarities to the hematopoietin receptor family. These so-called class II cytokine receptors (also known as interferon receptors) include the receptors for IFN-α and IFN-β, and the IL-10 receptor. The hematopoietin and interferon receptors all signal through the JAK–STAT pathway described below and activate different combinations of STATs with different effects.


Immunology_Janeway

Immunology_Janeway. The TNF family, of which TNF-α is the prototype, contains more than 17 cytokines with important functions in adaptive and innate immunity. Unlike most of the other immunologically important cytokines, many members of the TNF family are transmembrane proteins, a characteristic that gives them distinct properties and limits their range of action. Some, however, can also be released from the membrane in some circumstances. They are usually found as homotrimers of a membrane-bound subunit, although some heterotrimers consisting of different subunits also occur. TNF-α (sometimes called simply TNF) is initially expressed as a trimeric membrane-bound cytokine but can be released from the membrane. The effects of TNF-α are mediated by either of two TNF receptors. TNF receptor I (TNFR-I) is expressed on a wide range of cells, including endothelial cells and macrophages, whereas TNFR-II is expressed largely by lymphocytes. The receptors for cytokines of the TNF family are structurally unrelated


Immunology_Janeway

Immunology_Janeway. on a wide range of cells, including endothelial cells and macrophages, whereas TNFR-II is expressed largely by lymphocytes. The receptors for cytokines of the TNF family are structurally unrelated to the receptors described above and also have to cluster to become activated. Since TNF-family cytokines are produced as trimers, the binding of these cytokines induces the clustering of three identical receptor subunits. The signaling pathway activated by these receptors is described in Chapter 7, where we see that signaling uses members of the TRAF family to activate the so-called non-canonical NFκB pathway.


Immunology_Janeway

Immunology_Janeway. Members of the chemokine receptor family are listed in Appendix IV, along with the chemokines they recognize. These receptors have a 7-transmembrane structure and signal by interacting with G-proteins as described in Section 3-2. 3-16 Cytokine receptors of the hematopoietin family are associated with the JAK family of tyrosine kinases, which activate STAT transcription factors. The signaling chains of the hematopoietin family of cytokine receptors are noncovalently associated with protein tyrosine kinases of the Janus kinase (JAK) family—so called because they have two tandem kinase-like domains and thus resemble the two-headed mythical Roman god Janus. There are four members of the JAK family: Jak1, Jak2, Jak3, and Tyk2. As mice deficient for individual JAK family members show different phenotypes, each kinase must Chapter 3: The Induced Responses of Innate Immunity MOVIE 3.7 Fig. 3.26 Many cytokine receptors signal using a rapid pathway called the JAK–STAT pathway.


Immunology_Janeway

Immunology_Janeway. First panel: many cytokines act via receptors that are associated with cytoplasmic Janus kinases (JAKs). The receptor consists of at least two chains, each associated with a specific JAK. Second panel: binding of ligand brings the two chains together, allowing the JAKs to phosphorylate and activate each other, and then to phosphorylate (red dots) specific tyrosines in the receptor tails. The STAT (signal transducer and activator of transcription) family of proteins have an N-terminal domain that homodimerizes STATs in the cytosol before activation, and an SH2 domain that binds to the tyrosinephosphorylated receptor tails. Third panel: upon binding, the STAT homodimers are phosphorylated by JAKs. Fourth panel: after phosphorylation, STAT proteins reconfigure into a dimer that is stabilized by SH2 domain binding to phosphotyrosine residues on the other STAT. They then translocate to the nucleus, where they bind to and activate the transcription of a variety of genes important for


Immunology_Janeway

Immunology_Janeway. by SH2 domain binding to phosphotyrosine residues on the other STAT. They then translocate to the nucleus, where they bind to and activate the transcription of a variety of genes important for adaptive immunity.


Immunology_Janeway

Immunology_Janeway. have a distinct function. For example, Jak3 is used by γc for signaling by several of the cytokines described above. Mutations that inactivate Jak3 cause a form of SCID that is not X-linked. The dimerization or clustering of receptor signaling chains brings the JAKs into close proximity, causing phosphorylation of each JAK on a tyrosine residue that stimulates its kinase activity. The activated JAKs then phosphorylate their associated receptors on specific tyrosine residues. This phosphotyrosine, and the specific amino acid sequence surrounding it, creates a binding site that is recognized by SH2 domains found in other proteins, in particular members of a family of transcription factors known as signal transducers and activators of transcription (STATs) (Fig. 3.26).


Immunology_Janeway

Immunology_Janeway. There are seven STATs (1–4, 5a, 5b, and 6), which reside in the cytoplasm in an inactive form until activated by cytokine receptors. Before activation, most STATs form homodimers, due to a specific homotypic interaction between domains present at the amino termini of the individual STAT proteins. The receptor specificity of each STAT is determined by the recognition of the distinctive phosphotyrosine sequence on each activated receptor by the different SH2 domains within the various STAT proteins. Recruitment of a STAT to the activated receptor brings the STAT close to an activated JAK, which can then phosphorylate a conserved tyrosine residue in the carboxy terminus of the particular STAT. This leads to a rearrangement, in which the phosphotyrosine of each STAT protein binds to the SH2 domain of the other STAT, forming a configuration that can bind DNA with high affinity. Activated STATs predominantly form homodimers, with a cytokine typically activating one type of STAT. For


Immunology_Janeway

Immunology_Janeway. SH2 domain of the other STAT, forming a configuration that can bind DNA with high affinity. Activated STATs predominantly form homodimers, with a cytokine typically activating one type of STAT. For example, IFN-γ activates STAT1 and generates STAT1 homodimers, whereas IL-4 activates STAT6, generating STAT6 homodimers. Other cytokine receptors can activate several STATs, and some STAT heterodimers can be formed. The phosphorylated STAT dimer enters the nucleus, where it acts as a transcription factor to initiate the expression of selected genes that can regulate growth and differentiation of particular subsets of lymphocytes.


Immunology_Janeway

Immunology_Janeway. Since signaling by these receptors depends on tyrosine phosphorylation, dephosphorylation of the receptor complex by tyrosine phosphatases is one way that cells can terminate signaling. A variety of tyrosine phosphatases have been implicated in the dephosphorylation of cytokine receptors, JAKs, and STATs. These include the nonreceptor tyrosine phosphatases SHP-1 and SHP-2 (encoded by PTPN6 and PTPN11), and the transmembrane receptor tyrosine phosphatase CD45, which is expressed as multiple isoforms on many hematopoietic cells. Cytokine signaling can also be terminated by negative feedback involving specific inhibitors that are induced by cytokine activation. The suppressor of cytokine signaling (SOCS) proteins are a class of inhibitors that terminate the signaling of many cytokine and hormone receptors. SOCS proteins contain an SH2 domain that can recruit them to the phosphorylated JAK kinase or receptor, and they can inhibit JAK kinases directly, compete for the receptor, and direct


Immunology_Janeway

Immunology_Janeway. receptors. SOCS proteins contain an SH2 domain that can recruit them to the phosphorylated JAK kinase or receptor, and they can inhibit JAK kinases directly, compete for the receptor, and direct the ubiquitination and subsequent degradation of JAKs and STATs. SOCS proteins are induced by STAT activation, and thus inhibit receptor signaling after the cytokine has had its effect. Their importance can be seen in SOCS1-deficient mice, which develop a multiorgan inflammatory infiltrate caused by increased signaling from interferon receptors, γc-containing receptors, and TLRs. Another class of inhibitory proteins consists of the protein inhibitors of activated STAT (PIAS) proteins, which also seem to be involved in promoting the degradation of receptors and pathway components.


Immunology_Janeway

Immunology_Janeway. Phosphorylated STATs form dimers that translocate into the nucleus to initiate gene transcription Transcription factors (STATs) bind to the phosphorylated receptors, and are in turn phosphorylated by the activated JAKs JAK kinase active STAT dimer 3-17 Chemokines released by macrophages and dendritic cells recruit effector cells to sites of infection.


Immunology_Janeway

Immunology_Janeway. All the cytokines produced by macrophages in innate immune responses have important local and systemic effects that contribute to both innate and adaptive immunity, and these are summarized in Fig. 3.27. The recognition of different classes of pathogens by phagocytes and dendritic cells may involve signaling through different receptors, such as the various TLRs, and can result in some variation in the cytokines expressed by stimulated macrophages and dendritic cells. This is one way in which appropriate immune responses can be selectively activated, as the released cytokines orchestrate the next phase of host defense. In response to activation by PRRs, macrophages and dendritic


Immunology_Janeway

Immunology_Janeway. Fig. 3.27 Important cytokines and chemokines secreted by dendritic cells and macrophages in response to bacterial products include IL-1β, IL-6, CXCL8, IL-12, and TNF-α. TNF-α is an inducer of a local inflammatory response that helps to contain infections. It also has systemic effects, many of which are harmful (discussed in Section 3-20). The


Immunology_Janeway

Immunology_Janeway. Local effects Systemic effects IL-12IL-1˜Fever Production of IL-6 Fever Mobilization of metabolites Shock Fever Induces acute-phase protein production Activated macrophages secrete a range of cytokines CXCL8TNF-°IL-6 Activates vascular endothelium Activates lymphocytes Local tissue destruction Increases access of effector cells Lymphocyte activation Increased antibody production Chemotactic factor recruits neutrophils, basophils, and T cells to site of infection Activates NK cells Induces the differentiation of CD4 T cells into TH1 cells Activates vascular endothelium and increases vascular permeability, which leads to increased entry of IgG, complement, and cells to tissues and increased ˜uid drainage to lymph nodes chemokine CXCL8 is also involved in the local inflammatory response, helping to attract neutrophils to the site of infection. IL-1β, IL-6, and TNF-α have a crucial role in inducing the acute-phase response in the liver and inducing fever, which favors effective host


Immunology_Janeway

Immunology_Janeway. helping to attract neutrophils to the site of infection. IL-1β, IL-6, and TNF-α have a crucial role in inducing the acute-phase response in the liver and inducing fever, which favors effective host defense in various ways. IL-12 activates natural killer (NK) cells and favors the differentiation of CD4 T cells into the TH1 subset in adaptive immunity.


Immunology_Janeway

MOVIE 3.8

Immunology_Janeway. MOVIE 3.8


Immunology_Janeway

Immunology_Janeway. Fig. 3.28 Properties of selected human chemokines. Chemokines fall mainly into two related but distinct groups: the CC chemokines, which have two adjacent cysteine residues near the amino terminus; and the CXC chemokines, in which the equivalent cysteine residues are separated by a single amino acid. In humans, the genes for CC chemokines are mostly clustered in one region of chromosome 4. Genes for CXC chemokine genes are found mainly in a cluster on chromosome 17. The two groups of chemokines act on different sets of receptors, all of which are G-proteincoupled receptors. CC chemokines bind to receptors designated CCR1–10. CXC chemokines bind to receptors designated CXCR1–7. Different receptors are expressed on different cell types, and so a particular chemokine can be used to attract a particular cell type. In general, CXC chemokines with a Glu-Leu-Arg tripeptide motif immediately before the first cysteine promote the migration of neutrophils. CXCL8 is an example of this type. Most


Immunology_Janeway

Immunology_Janeway. particular cell type. In general, CXC chemokines with a Glu-Leu-Arg tripeptide motif immediately before the first cysteine promote the migration of neutrophils. CXCL8 is an example of this type. Most of the other CXC chemokines, including those that interact with receptors CXCR3, 4, and 5, lack this motif. Fractalkine is unusual in several respects: it has three amino acid residues between the two cysteines, and it exists in two forms, one that is tethered to the membrane of the endothelial and epithelial cells that express it, where it serves as an adhesion protein, and a soluble form that is released from the cell surface and acts as a chemoattractant for a wide range of cell types. A more comprehensive list of chemokines and their receptors is given in Appendix IV.


Immunology_Janeway

Immunology_Janeway. cells secrete a diverse group of cytokines that includes IL-1β, IL-6, IL-12, TNF-α, and the chemokine CXCL8 (formerly known as IL-8).


Immunology_Janeway

Immunology_Janeway. Among the cytokines released by tissues in the earliest phases of infection are members of a family of chemoattractant cytokines known as chemokines. These small proteins induce directed chemotaxis in nearby responsive cells, resulting in the movement of the cells toward the source of the chemokine. Because chemokines were first detected in functional assays, they were initially given a variety of names, which are listed along with their standardized nomenclature in Appendix IV. All the chemokines are related in amino acid sequence, and their receptors are G-protein-coupled receptors (see Section 3-2). The signaling pathway stimulated by chemokines causes changes in cell adhesiveness and changes in the cell’s cytoskeleton that lead to directed migration. Chemokines can be produced and released by many different types of cells, not only those of the immune system. In the immune system they function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils, and other


Immunology_Janeway

Immunology_Janeway. by many different types of cells, not only those of the immune system. In the immune system they function mainly as chemoattractants for leukocytes, recruiting monocytes, neutrophils, and other effector cells of innate immunity from the blood into sites of infection. They also guide lymphocytes in adaptive immunity, as we will learn in Chapters 9–11. Some chemokines also function in lymphocyte development and migration and in angiogenesis (the growth of new blood vessels). There are more than 50 known chemokines, and this striking multiplicity may reflect their importance in delivering cells to their correct locations, which seems to be their main function in the case of lymphocytes. Some of the chemokines that are produced by or that affect human innate immune cells are listed in Fig. 3.28 along with their properties.


Immunology_Janeway

Immunology_Janeway. Chemokines fall mainly into two related but distinct groups. CC chemokines have two adjacent cysteine residues near the amino terminus, whereas in CXC chemokines the corresponding two cysteine residues are separated by a single amino acid. The CC chemokines promote the migration of monocytes, lymphocytes, and other cell types. One example relevant to innate immunity is CCL2, which attracts monocytes through the receptor CCR2B, inducing their migration from the bloodstream to become tissue macrophages. In contrast, neutrophil migration is promoted by CXC chemokines. CXCL8, acting through CXCR2, mobilizes neutrophils from bone marrow and induces them to leave the blood and migrate into the surrounding tissues. CCL2 and CXCL8 therefore have similar but complementary functions in the innate immune response, attracting monocytes and neutrophils respectively.


Immunology_Janeway

Immunology_Janeway. The role of chemokines in cell recruitment is twofold. First, they act on the leukocyte as it rolls along endothelial cells at sites of inflammation, converting this rolling into stable binding by triggering a change of conformation in the adhesion molecules known as leukocyte integrins. These conformational changes enable integrins to bind strongly to their ligands on the endothelial cells, which allows the leukocyte to cross the blood vessel walls by squeezing between the endothelial cells. Second, the chemokine directs the migration of the leukocyte along a gradient of chemokine molecules bound to the extracellular matrix and the surfaces of endothelial cells. This gradient increases in concentration toward the site of infection.


Immunology_Janeway

Immunology_Janeway. Chemokines are produced by a wide variety of cell types in response to bacterial products, viruses, and agents that cause physical damage, such as silica, alum, or the urate crystals that occur in gout. Complement fragments such as C3a and C5a, and fMLF bacterial peptides, also act as chemoattractants for neutrophils. Thus, infection or physical damage to tissues induces the production of chemokine gradients that can direct phagocytes to the sites where they are needed. Neutrophils arrive rapidly in large numbers at a site of infection. The recruitment of monocytes occurs simultaneously, but they accumulate more slowly at the site of infection, perhaps because they are less abundant in the circulation. The complement fragment C5a and the chemokines CXCL8 and CCL2 activate their respective target cells, so that not only are neutrophils and monocytes brought to potential sites of infection but, in the process, they are armed to deal with the pathogens they encounter there. In


Immunology_Janeway

Immunology_Janeway. respective target cells, so that not only are neutrophils and monocytes brought to potential sites of infection but, in the process, they are armed to deal with the pathogens they encounter there. In particular, the signaling induced by C5a or CXCL8 in neutrophils serves to augment the respiratory burst that generates oxygen radicals and nitric oxide and to induce the neutrophils to release their stored antimicrobial granule contents (see Section 3-2).


Immunology_Janeway

Immunology_Janeway. Chemokines do not act alone in cell recruitment. They require the action of vasoactive mediators that bring leukocytes close to the blood vessel wall (see Section 3-3) and cytokines such as TNF-α to induce the necessary adhesion molecules on endothelial cells. We will return to the chemokines in later chapters, where they are discussed in the context of the adaptive immune response. Now, however, we turn to the molecules that enable leukocytes to adhere to the endothelium, and we shall then describe step by step the extravasation process by which monocytes and neutrophils enter infected sites. response. The recruitment of activated phagocytes to sites of infection is one of the most important functions of innate immunity. Recruitment occurs as part of the inflammatory response and is mediated by cell-adhesion molecules that are induced on the surface of the endothelial cells of local blood vessels. Here we MOVIE 3.9


Immunology_Janeway

Immunology_Janeway. MOVIE 3.9 Chapter 3: The Induced Responses of Innate Immunity consider those functions that participate in the recruitment of inflammatory MOVIE 3.10 cells in the hours to days after the establishment of an infection.


Immunology_Janeway

Immunology_Janeway. As with the complement components, a significant barrier to understanding the functions of cell-adhesion molecules is their nomenclature. Most adhesion molecules, especially those on leukocytes, which are relatively easy to analyze functionally, were originally named after the effects of specific monoclonal antibodies directed against them. Their names therefore bear no relation to their structural class. For instance, the leukocyte functional antigens LFA-1, LFA-2, and LFA-3 are actually members of two different protein families. In Fig. 3.29, the adhesion molecules relevant to innate immunity are grouped according to their molecular structure, which is shown in schematic form alongside their different names, sites of expression, and ligands. Three structural families of adhesion molecules are important for leukocyte recruitment. The selectins are membrane glycoproteins with a distal lectin-like domain that binds specific carbohydrate groups. Members of this family are induced on


Immunology_Janeway

Immunology_Janeway. are important for leukocyte recruitment. The selectins are membrane glycoproteins with a distal lectin-like domain that binds specific carbohydrate groups. Members of this family are induced on activated endothelium and initiate endothelium–leukocyte interactions by binding to fucosylated oligosaccharide ligands on passing leukocytes (see Fig. 3.29).


Immunology_Janeway

Immunology_Janeway. The next step in leukocyte recruitment depends on tighter adhesion, which is due to the binding of intercellular adhesion molecules (ICAMs) on the endothelium to heterodimeric proteins of the integrin family on leukocytes. ICAMs are single-pass membrane proteins that belong to the large superfamily of immunoglobulin-like proteins, which contain protein domains similar to those of immunoglobulins. The extracellular regions of ICAMs are composed of several immunoglobulin-like domains. An integrin molecule is composed


Immunology_Janeway

Immunology_Janeway. Fig. 3.29 Adhesion molecules involved in leukocyte interactions. Several structural families of adhesion molecules have a role in leukocyte migration, homing, and cell–cell interactions: the selectins, the integrins, and proteins of the immunoglobulin superfamily. The figure shows schematic representations of an example from each family, a list of other family members that participate in leukocyte interactions, their cellular distribution, and their ligand in adhesive interactions. The family members shown here are limited to those that participate in inflammation and other innate immune mechanisms. The same molecules and others participate in adaptive immunity and will be considered in Chapters 9 and 11. The nomenclature of the different molecules in these families is confusing because it often reflects the way in which the molecules were first identified rather than their related structural characteristics. Alternative names for each of the adhesion molecules are given in


Immunology_Janeway

Immunology_Janeway. because it often reflects the way in which the molecules were first identified rather than their related structural characteristics. Alternative names for each of the adhesion molecules are given in parentheses. Sulfated sialyl-LewisX, which is recognized by Pand E-selectin, is an oligosaccharide present on the cell-surface glycoproteins of circulating leukocytes.


Immunology_Janeway

Immunology_Janeway. ˜°LFA-1 Integrins Bind to cell-adhesion molecules and extracellular matrix. Strong adhesion Monocytes, macrophages Fibronectin ˜5:°1 (VLA-5, CD49d:CD29) Monocytes, T cells, macrophages, neutrophils, dendritic cells, NK cells Neutrophils, monocytes, macrophages, NK cells Dendritic cells, macrophages, neutrophils, NK cells ICAM-1, ICAM-2 ICAM-1, iC3b, ÿbrinogen iC3b ˜L:°2 (LFA-1, CD11a:CD18) ˜M:°2 (CR3, Mac-1, CD11b:CD18) ˜X:°2 (CR4, p150.95, CD11c:CD18) of two transmembrane protein chains, α and β, of which there are numerous different types. Subsets of integrins have a common β chain partnered with different α chains. The leukocyte integrins important for extravasation are LFA-1 (αL:β2, also known as CD11a:CD18) and CR3 (αM:β2, complement receptor type 3, also known as CD11b:CD18 or Mac-1). We described CR3 in Section 2-13 as a receptor for iC3b, but it also binds other ligands. Both LFA-1 and CR3 bind to ICAM-1 and to ICAM-2 (Fig. 3.30). Even in the absence of infection, circulating


Immunology_Janeway

Immunology_Janeway. We described CR3 in Section 2-13 as a receptor for iC3b, but it also binds other ligands. Both LFA-1 and CR3 bind to ICAM-1 and to ICAM-2 (Fig. 3.30). Even in the absence of infection, circulating monocytes are continuously leaving the blood and entering certain tissues, such as the intestine, where they become resident macrophages. To navigate out of the blood vessel, they may adhere to ICAM-2, which is expressed at low levels by unactivated endothelium. CR3 also binds to fibrinogen and factor X, both substrates of the coagulation cascade.


Immunology_Janeway

Immunology_Janeway. Strong adhesion between leukocytes and endothelial cells is promoted by the induction of ICAM-1 on inflamed endothelium together with a conformational change in LFA-1 and CR3 that occurs on the leukocyte. Integrins can switch between an ‘active’ state, in which they bind strongly to their ligands, and an ‘inactive’ state, in which binding is easily broken. This enables cells to make and break integrin-mediated adhesions in response to signals received by the cell either through the integrin itself or through other receptors. In the activated state, an integrin molecule is linked via the intracellular protein talin to the actin cytoskeleton. In the case of migrating leukocytes, chemokines binding to their receptors on the leukocyte generate intracellular signals that cause talin to bind to the cytoplasmic tails of the β chains of LFA-1 and CD3, forcing the integrin extracellular regions to assume an active binding conformation. The importance of leukocyte integrin function in


Immunology_Janeway

Immunology_Janeway. bind to the cytoplasmic tails of the β chains of LFA-1 and CD3, forcing the integrin extracellular regions to assume an active binding conformation. The importance of leukocyte integrin function in inflammatory cell recruitment is illustrated by leukocyte adhesion deficiencies, which can be caused by defects in the integrins themselves or in the proteins required for modulating adhesion. People with these diseases suffer from recurrent bacterial infections and impaired healing of wounds.


Immunology_Janeway

Immunology_Janeway. Endothelial activation is driven by macrophage-produced cytokines, particularly TNF-α, which induce the rapid externalization of granules called Weibel–Palade bodies in the endothelial cells. These granules contain preformed P-selectin, which appears on the surfaces of local endothelial cells just minutes after macrophages have responded to the presence of microbes by producing TNF-α. Shortly after P-selectin gets to the cell surface, mRNA encoding E-selectin is synthesized, and within 2 hours the endothelial cells are expressing mainly E-selectin. Both P-selectin and E-selectin interact with sulfated sialyl-LewisX, a sulfated form of a carbohydrate structure that is also an important blood group antigen. Sulfated sialyl-LewisX is present on the surface of neutrophils, and its interactions with P-selectin and E-selectin are important for neutrophil rolling on the endothelium. Mutations in enzymes involved in its synthesis, such as fucosyltransferase, cause defective sialyl-LewisX


Immunology_Janeway

Immunology_Janeway. with P-selectin and E-selectin are important for neutrophil rolling on the endothelium. Mutations in enzymes involved in its synthesis, such as fucosyltransferase, cause defective sialyl-LewisX expression that results in an immunodeficiency, leukocyte adhesion deficiency type 2.


Immunology_Janeway

Immunology_Janeway. Integrins are also convenient cell-surface markers for distinguishing different cell types. Dendritic cells, macrophages, and monocytes express different integrin α chains and thus display distinct β2 integrins on their surface. The predominant leukocyte integrin on conventional dendritic cells is αX:β2, also known as CD11c:CD18 or complement receptor 4 (CR4) (see Fig. 3.29). This integrin is a receptor for the complement C3 cleavage product iC3b, fibrinogen, and ICAM-1. In contrast to conventional dendritic cells, most monocytes and macrophages express low levels of CD11c, and predominantly express the integrin αM:β2 (CD11b:CD18; CR3). However, patterns of integrin expression can vary, with some tissue macrophages, such as those in the lung, expressing high levels of CD11c:CD18. In the mouse, the two major branches of conventional dendritic cells can be distinguished by expression of CD11b:CD18: one branch characterized by high expression of CD11b:CD18,


Immunology_Janeway

Immunology_Janeway. Fig. 3.30 Phagocyte adhesion to vascular endothelium is mediated by integrins. When vascular endothelium is activated by inflammatory mediators it expresses two adhesion molecules, namely ICAM-1 and ICAM-2. These are ligands for integrins expressed by phagocytes— αM:β2 (also called CR3, Mac-1, or CD11b:CD18) and αL:β2 (also called LFA-1 or CD11a:CD18) MOVIE 3.11 MOVIE 3.12 MOVIE 3.13 and a second branch that lacks CD11b:CD18. Plasmacytoid dendritic cells (pDCs) express lower levels of CD11c, but can be distinguished from conventional dendritic cells using other markers; human pDCs express the C-type lectin BDCA-2 (blood dendritic cell antigen 2), and mouse pDCs express BST2 (bone marrow stromal antigen), neither of which is expressed by conventional dendritic cells. 3-19 Neutrophils make up the first wave of cells that cross the blood vessel wall to enter an inflamed tissue.


Immunology_Janeway

Immunology_Janeway. 3-19 Neutrophils make up the first wave of cells that cross the blood vessel wall to enter an inflamed tissue. The migration of leukocytes out of blood vessels, the process known as extravasation, occurs in response to signals generated at sites of infection. Under normal conditions, leukocytes travel in the center of small blood vessels, where blood flow is fastest. Within sites of inflammation, the vessels are dilated and the consequent slower blood flow allows leukocytes to interact in large numbers with the vascular endothelium. During an inflammatory response, the induction of adhesion molecules on the endothelial cells of blood vessels within the infected tissue, as well as induced changes in the adhesion molecules expressed on leukocytes, recruits large numbers of circulating leukocytes to the site of infection. We will describe this process with regard to monocytes and neutrophils (Fig. 3.31).


Immunology_Janeway

Immunology_Janeway. Extravasation proceeds in four stages. In the first, induction of selectins induces leukocyte rolling along the endothelium. P-selectin appears on endothelial cell surfaces within a few minutes of exposure to leukotriene B4, C5a, or histamine, which is released from mast cells in response to C5a. P-selectin can also be induced by TNF-α or LPS, and both of these induce synthesis of E-selectin, which appears on the endothelial cell surface a few hours later. When the sulfated sialyl-LewisX on monocytes and neutrophils contacts these exposed P-and E-selectins, these cells adhere reversibly to the vessel wall and begin to ‘roll’ along endothelium (see Fig. 3.31, top panel), permitting stronger interactions of the next step in leukocyte migration. Neutrophils are particularly efficient at rolling along endothelium even under flow rates that prevent rolling by other cells. Such ‘shear-resistant rolling’ by neutrophils uses long extensions of plasma membrane, termed slings, that bind the


Immunology_Janeway

Immunology_Janeway. along endothelium even under flow rates that prevent rolling by other cells. Such ‘shear-resistant rolling’ by neutrophils uses long extensions of plasma membrane, termed slings, that bind the endothelium and wrap around the cell as it rolls, serving to tether the cell firmly to the endothelium and to promote rapid entry to sites of infection.


Immunology_Janeway

Immunology_Janeway. The second step depends on interactions between the leukocyte integrins LFA-1 and CR3 with adhesion molecules such as ICAM-1 (which can be induced on endothelial cells by TNF-α) and ICAM-2 on endothelium (see Fig. 3.31, bottom panel). LFA-1 and CR3 normally bind their ligands only weakly, but CXCL8 (or other chemokines), bound to proteoglycans on the surface of endothelial cells, binds to specific chemokine receptors on the leukocyte and signals the cell to trigger a conformational change in LFA-1 and CR3 on the rolling leukocyte; this greatly increases the adhesive properties of the leukocyte, as discussed in Section 3-18. The cell then attaches firmly to the endothelium, and its rolling is arrested.


Immunology_Janeway

Immunology_Janeway. In the third step the leukocyte extravasates, or crosses the endothelial wall. This step also involves LFA-1 and CR3, as well as a further adhesive interaction involving an immunoglobulin-related molecule called PECAM or CD31, which is expressed both on the leukocyte and at the intercellular junctions of endothelial cells. These interactions enable the phagocyte to squeeze between the endothelial cells. It then penetrates the basement membrane with the aid of enzymes that break down the extracellular matrix proteins of the basement membrane. The movement through the basement membrane is known as diapedesis, and it enables phagocytes to enter the subendothelial tissues. The fourth and final step in extravasation is the migration of leukocytes through the tissues under the influence of chemokines. Chemokines such as Selectin-mediated adhesion to leukocyte sialyl-Lewisx is weak, and allows leukocytes to roll along the vascular endothelial surface


Immunology_Janeway

Immunology_Janeway. CXCL8 and CCL2 (see Section 3-17) are produced at the site of infection and bind to proteoglycans in the extracellular matrix and on endothelial cell surfaces. In this way, a matrix-associated concentration gradient of chemokines is formed on a solid surface along which the leukocyte can migrate to the focus of infection (see Fig. 3.31). CXCL8 is released by the macrophages that first encounter pathogens; it recruits neutrophils, which enter the infected tissue in large numbers in the early part of the induced response. Their influx usually peaks within the first 6 hours of an inflammatory response. Monocytes are recruited through the action of CCL2, and accumulate more slowly than neutrophils. Once in the inflamed tissue, neutrophils are able to eliminate many pathogens by phagocytosis. In an innate immune response, neutrophils use their complement receptors and the direct pattern recognition receptors discussed earlier in this chapter (see Section 3-1) to recognize and phagocytose


Immunology_Janeway

Immunology_Janeway. In an innate immune response, neutrophils use their complement receptors and the direct pattern recognition receptors discussed earlier in this chapter (see Section 3-1) to recognize and phagocytose pathogens or pathogen components directly or after opsonization with complement (see Section 2-13). In addition, as we will see in Chapter 10, neutrophils act as phagocytic effectors in humoral adaptive immunity, taking up antibody-coated microbes by means of specific receptors.


Immunology_Janeway

Immunology_Janeway. The importance of neutrophils in immune defense is dramatically illustrated by diseases or medical treatments that severely reduce neutrophil numbers. Patients suffering this affliction are said to have neutropenia, and they are highly susceptible to deadly infection with a wide range of pathogens and commensal organisms. Restoring neutrophil levels in such patients by transfusion Induced innate responses to infection. 117


Immunology_Janeway

Immunology_Janeway. Fig. 3.31 Neutrophils leave the blood and migrate to sites of infection in a multi-step process involving adhesive interactions that are regulated by macrophage-derived cytokines and chemokines. Top panel: the first step involves the reversible binding of a neutrophil to vascular endothelium through interactions between selectins induced on the endothelium and their carbohydrate ligands on the neutrophil, shown here for E-selectin and its ligand, the sialyl-LewisX moiety (s-Lex). This interaction cannot anchor the cells against the shearing force of the flow of blood, and thus they roll along the endothelium, continually making and breaking contact. Bottom panel: the binding does, however, eventually trigger stronger interactions, which result only when binding of a chemokine such as CXCL8 to its specific receptor on the neutrophil triggers the activation of the integrins LFA-1 and CR3 (Mac-1; not shown). Inflammatory cytokines such as TNF-α are also necessary to induce the expression