Cytokines affect nearly every biological process from embryonic development and disease pathogenesis, to changes in cognitive functions, non-specific responses to infection, specific responses to antigens, allograft rejection, and the degenerative processes of aging. Although cytokines are secreted by immune system cells to combat infection, the pro-inflammatory cytokines are targeted for therapeutic intervention because of their role in promoting or sustaining harmful inflammation.1 In fact, several intrinsic mechanisms also mitigate the detrimental effects of these mediators during cytokine-induced inflammation. First, in the case of interleukin-1 (IL-1), the IL-1 RII cell surface receptor serves as a decoy for the actual IL-1 receptor. IL-1 RII binds to IL-1 but does not transmit a signal, thus depriving the bona fide receptor of the opportunity to initiate an IL-1-dependent signal.2 Cytokine receptor extracellular domains (i.e. soluble IL-1 receptors) released during inflammation also act to neutralize IL-1 signaling. In addition, the IL-1 receptor antagonist (IL-1ra) can bind to the IL-1 receptor with greater affinity than either the IL-1 alpha or the IL-1 beta ligands3 but also does not elicit a signal. Since IL-1ra is present in high concentrations as an acute phase protein, the ratio of IL-1ra to IL-1 can impact disease severity. One final mechanism for limiting the intrinsic inflammatory response to IL-1 is the single immunoglobulin IL-1-related receptor (SIGIRR), which acts to reduce inflammatory responses in vitro4 and in vivo.5 The effects of IL-18, another member of the IL-1 family, can be reduced by a constitutively produced IL-18 binding protein (IL-18BP). IL-18BP binds to IL-18 with an affinity greater than either the cell-bound or the soluble forms of the IL-18 receptor and thus can prevent IL-18 signaling.6, 7
View Larger Image
|Figure 1: Inflammation is a complex biological response to pathogenic and harmful stimuli and involves both the local vasculature and the immune system. During an inflammatory response, eosinophils and neutrophils migrate from the bloodstream into tissues. Upon activation, they secrete pro-inflammatory cytokines and contribute to the recruitment of monocytes and T cells. A cascade of signals initiates and propagates the response, only later to be balanced by anti-inflammatory cytokines.|
Processing and Secretion of IL-1 beta. Tight regulation of processing and secretion provides another mechanism for suppressing the activity of IL-1 beta and IL-18. With few exceptions, members of the IL-1 family are first synthesized as inactive precursors that lack a classic signal peptide. Consistent with this pattern, the newest member of the IL-1 family, IL-33, is synthesized as an inactive precursor lacking a classic signal peptide. These cytokines cannot simply enter the classical secretory pathway and exit the cell as other secreted proteins do. Rather, they must first be converted from an inactive to an active form. For IL-1 beta, IL-18, and IL-33, an intracellular cysteine protease, caspase-1, is required for processing the inactive precursors into mature, active forms that can then be secreted from the cell. The activation of caspase-1 is itself tightly controlled and provides yet another unique mechanism to limit inflammation.
Caspase-1 and Inflammation. Caspase-1, formerly called IL-1 beta converting enzyme (ICE), is a member of a family of nine cysteine proteases that specifically recognizes an aspartic acid residue in the P1 position of their substrates. Most members of this family of proteases are involved in mediating programmed cell death by promoting the cleavage of critical intracellular proteins upon apoptopic activation. Caspase-1, however, seems to be uniquely involved in participating in the inflammatory response by cleaving the precursors of IL-1 beta, IL-18, and IL-33. Indeed, the rate-limiting step in inflammation due to IL-1 beta or IL-18 is the activation of caspase-1. Inactive pro-caspase-1 is converted to an active enzyme via dimerization, followed by an autocatalytic reaction that generates an active molecule composed of two large and two small subunits. The autocatalysis of pro-caspase-1 to active caspase-1 is tightly controlled by the caspase-1 inflammasome.8
A great deal of investigation has focused on caspase-1 activation and the release of active IL-1 beta, whereas far less is known about the role of caspase-1 in the release of IL-18 or IL-33. The discovery of caspase-1 and its importance in IL-1 beta secretion was recognized 15 years ago when researchers demonstrated that caspase-1 inhibitors could cause a reduction in the secretion of active IL-1 beta from cultured monocytes.9, 10 Since that time, studies on caspase-1-deficient mice have confirmed the importance of this enzyme in the control of inflammation.11, 12 For example, it was found that mice deficient in caspase-1 do not develop inflammatory bowel disease.13 Currently, however, it is unclear whether reduced inflammation in caspase-1-deficient mice is due to a reduction in the processing and release of IL-18 or IL-1 beta.14
View Larger Image
|Figure 2: NALP3 and NALP1 Inflammasomes. (A) NALP3 is a protein with four
domains: PYR (Pyrin), NACHT (domain found in NAIP, CIITA, HET-E, and TP-1), NAD (NALP-associated Domain), and LRR (Leucine Rich Repeats). NALP3 associates with pro-caspase-1 via its CARD (Caspase-1 Recruitment Domain). Since NALP3 lacks its own CARD, the CARD on the adaptor protein ASC (Apoptosis-associated Speck-like protein containing CARD) provides the linkage. The pyrin domain of ASC associates with the pyrin on NALP3. Mutations in the NACHT domain of NALP3 are associated with three autoinflammatory diseases (FCAS, MWS, NOMID; see Table 1).26, 27, 28 Other proteins in the NALP3 complex, such as Cardinal (CARD Inhibitor of NFkB-activating Ligands), participate in the oligomerization of the inflammasome and activation of caspase-1. (B) The CARD on the carboxyl end of pro-caspase-1 associates with the carboxyl end CARD on NALP1. The presence of ASC appears to enhance the function of the NALP1 inflammasome. NALP1 also participates in the activation of caspase-5. The CARD and the pyrin domains of ASC link pro-caspase-5 to the NALP1 protein.
The Discovery of Cryopyrin (NALP3). In 2001, a critical discovery related to the processing of pro-caspase-1 was made by researchers studying patients that are exquisitely sensitive to cold. Upon exposure to cold temperatures, these patients exhibit urticarial rash (hives) and develop systemic symptoms of fatigue and joint aches. In addition, these patients develop fevers and show elevated white blood cell counts. In fact, their response to cold temperatures mimics symptoms of an acute infection. Clinicians knew this disease as Familial Cold Auto-inflammatory Syndrome (FCAS), but its cause was unknown. Genetic analysis of affected patients from an extended family indicated that the cold-sensitive trait had an autosomal dominant pattern of inheritance and patients all had mutations in one particular gene. The gene was named "Cold-induced Auto-inflammatory Syndrome-1" or CIAS-1, and the intracellular protein encoded by the gene was called "cryopyrin" since the patients developed fever following exposure to cold.15 An alternative name for cryopyrin is NALP3. Since that initial discovery, subsequent studies have indicated that NALP3 associates with intracellular proteins such as pro-caspase-1 via protein-protein interactions.16, 17 As discussed below, this association is important for the processing and activation of pro-caspase-1.
The NALP3 Inflammasome. The inflammasome is comprised of protein components that assemble into a complex mediated by weak protein-protein interactions.18 Although the NALP3 inflammasome can be pre-activated under some conditions, most studies report that activation of NALP3 requires a sudden fall in the levels of intracellular potassium. This efflux of potassium from the cell can be triggered by extracellular ATP via the P2X7 purinergic channel or by other natural by-products from dead cells. This potassium efflux, or other microbial triggers, activates oligomerization of the NALP3 inflammasome. Pro-caspase-1 associates with the inflammasome through its CARD domain. This domain associates with the CARD domain on the ASC adaptor protein, which then interacts with NALP3 through its pyrin (PYR) domain (Figure 2). The association of pro-caspase-1 with the NALP3 triggers an autocatalytic event that activates pro-caspase-1 and leads to the processing of the IL-1 beta precursor. Processed, active IL-1 beta exits the cell either directly from the cytosol or through the specialized secretory lysosomes (Figure 3).19
In freshly obtained human blood monocytes, the inflammasome appears to be constitutively activated. This level of activation is such that stimulation by endotoxin, other Toll-like Receptor (TLR) ligands, or cytokines (including IL-1 itself) induces processing of the IL-1 beta precursor followed by secretion of the active cytokine. The release of processed IL-1 beta occurs steadily over hours. In contrast, the inflammasome is not constitutively active in macrophages, macrophage cell lines, or dendritic cells derived from circulating monocytes. In these cells, both a stimulant for the synthesis of the IL-1 beta precursor plus the specific efflux of potassium is required for the activation of the inflammasome. In patients with auto-inflammatory diseases, the inflammasome is already activated and does not require a decrease in intracellular potassium levels.20
In addition to auto-inflammatory diseases, the processing of the IL-1 beta and IL-18 precursors by the NALP3 inflammasome plays a role in contact hypersensitivity. In the skin, exposure to chemical contact sensitizers causes an immune response called contact hypersensitivity or eczema. Keratinocytes contain components of the NALP3 inflammasome, and contact sensitizers induce caspase-1-dependent IL-1 beta and IL-18 processing and secretion. In fact, ASC- and NALP3-deficient mice exhibit impaired contact dermatitis, suggesting that contact hypersensitivity is caused, in part, by activation of the NALP3 inflammasome.21
The NALP1 Inflammasome. Patients with vitiligo, an autoimmune disease that attacks the melanocytes in the skin, may also have a second or even a third autoimmune disease such as thyroiditis, rheumatoid arthritis, diabetes, or inflammatory bowel disease (Crohn's Disease). A genetic analysis of families of patients with vitiligo and a second autoimmune disease identified mutations in another NALP family gene, NALP1.22 Although NALP1 and NALP3 both participate in the conversion of pro-caspase-1 to an active enzyme, NALP1 can directly associate with pro-caspase-1 via its CARD domain (Figure 2). The oligomerization of the NALP3 inflammasome requires the ASC adaptor protein to link NALP3 with pro-caspase-1, while ASC augments but is not required for NALP1 complex formation.
Processing of caspases by NALP1 appears to play a role in a specific pro-inflammatory cell death process called pyroptosis, which unlike apoptosis requires caspase-1. For example, in macrophages infected with Salmonella, activation of caspase-1 via NALP1 results in the production of IL-1 beta and IL-18 followed by a rapid release of intracellular contents through pores in the cell membrane.23 Likewise, macrophage cell death by anthrax toxin occurs via pyroptosis and requires NALP1 activation of caspase-1. In addition to activating caspase-1, activation of caspase-5 by NALP1 may also be required for pyroptosis (Figure 2).
Treatment of Auto-inflammatory Diseases. Several chronic and often debilitating inflammatory diseases are characterized as auto-inflammatory. These disorders are accompanied by fever, painful joints and muscles, fatigue associated with high white blood cell counts (mostly neutrophils), elevated acute phase proteins, anemia, and high platelet counts (thrombocytosis). More importantly, a disease classified as being auto-inflammatory will show a rapid and sustained reduction in clinical, hematological, and biochemical parameters after employing one of several treatment options that specifically reduces IL-1 activity, particularly IL-1 beta.24 This reduction in IL-1 activity can be accomplished with IL-1ra, neutralizing anti-IL-1 beta monoclonal antibodies, IL-1 Trap, or orally active caspase-1 inhibitors. In contrast, reducing TNF-alpha activity has little or no effect indicating that auto-inflammatory diseases are essentially IL-1 beta-mediated. These disorders represent a failure of the caspase-1 inflammasome to control the processing of the IL-1 beta precursor. Blocking IL-1 beta in auto-inflammatory diseases often results in a decrease in the activity of the inflammasome. This suggests that IL-1 beta itself is driving its own synthesis, as well as the activation of the inflammasome (Figure 3).20 Table 1 lists several diseases that can be classified as auto-inflammatory. Some hereditary auto-inflammatory diseases are also referred to as Familial Periodic Fever Syndromes.25 The symptoms of auto-inflammatory syndromes and diseases constitute a portfolio of destructive inflammation events.127, 28
View Larger Image
|Figure 3: Activation of Caspase-1 by NALP3 and Secretion of IL-1 beta in a Tissue Macrophage. Binding of IL-1 beta to the IL-1 receptor (step 1) results in transcription (Step 2) and translation of the IL-1 beta precursor (Step 3). The IL-1 beta precursor remains diffusely in the cytosol as an inactive molecule. Upon activation of the P2X7 receptor by extracellular ATP (Step 4), there is a rapid efflux of potassium from the cell (Step 5a) resulting in a fall in intracellular potassium levels (Step 5b). The decrease in intracellular potassium triggers the assembly of the components of the caspase-1 inflammasome (Step 6) and its association with pro-caspase-1. The processing of pro-caspase-1 results in the formation of the active caspase-1 heterodimer and the cleavage of the IL-1 beta precursor. The enzymatic processing of the IL-1 beta precursor by caspase-1 may take place in the cytosol (Step 7a), in the secretory lysosome (Step 7b), or in both. An influx of calcium into the cell (Step 8) provides a mechanism by which mature IL-1 beta is released from the cell19, 40 (Step 9). In freshly obtained blood monocytes, the inflammasome and caspase-1 are sufficiently activated such that triggering of TLR or IL-1 receptors is the sole stimulus required for synthesis, processing, and secretion of mature IL-1 beta.|
Patients with mutations in NALP3 exhibit the most severe manifestations of an overactive inflammasome. Although IL-1 beta is clearly causing the symptoms of the disease, levels of IL-1 beta in the circulation are not significantly elevated. Instead, serum IL-6 levels are high.26 Significantly, IL-6 levels decrease with a reduction in IL-1 beta activity. Regardless of mutations in NALP3, however, an increase in the secretion of IL-1 beta from cultured blood monocytes can be demonstrated in nearly all auto-inflammatory diseases. For example, Systemic Onset Juvenile Idiopathic Arthritis is a debilitating childhood disease with recurrent fevers and arthritis. Blood monocytes from these patients secrete more IL-1 beta compared to monocytes from healthy children. While no specific genetic basis seems to account for this disease, treatment with IL-1 beta blockers is highly effective; thus standard corticosteroid doses can be lowered, allowing these children to grow normally. Similarly, blood monocytes from patients with Neonatal Onset Multi-system Inflammatory Disease also secrete more IL-1 beta compared to monocytes from healthy controls.20, 27, 29
Recent studies in patients with type 2 diabetes reveal that the administration of IL-1ra, Anakinra, protects insulin-secreting beta cells in the pancreatic islet.30 These clinical findings confirm studies published over 20 years ago that indicate IL-1 beta is toxic for the beta cell and participates in the loss of beta cell mass in diabetes. The source of IL-1 beta in type 2 diabetic patients is likely to be the insulin-producing beta cell itself, which releases IL-1 beta in response to high glucose exposure.31 In general, successful treatments for auto-inflammatory diseases are directed against IL-1 beta. The actual "triggering" mechanism for the recurrent bouts of systemic inflammation appears to be IL-1 beta itself, as IL-1 beta exhibits positive feedback (Figure 3).
Not all active IL-1 beta is due to cleavage by intracellular caspase-1. Extracellularly, the IL-1 beta precursor can be cleaved by proteinase-3, a serine proteinase prominent in neutrophils.32 Since this type of enzymatic processing also yields active IL-1 beta, these patients respond to IL-1 beta blocking therapies as well. For example, diseases such as urate crystal arthritis (gout), in which neutrophils play a dominant role, can be treated with IL-1 receptor antagonist.33
Familial Mediterranean Fever. Familial Mediterranean Fever (FMF) is a classic auto-inflammatory disease marked by recurrent attacks of fever, leukocytosis, and pain in the serosal membranes of the lung and peritoneal wall. This disease is inherited, and although all patients have nearly identical clinical symptoms, approximately 50% do not carry the autosomal recessive gene.34 Anakinra reduces the signs and symptoms of FMF attacks.35 The dysregulation of caspase-1 activation and increased secretion of active IL-1 beta from blood monocytes accounts for the systemic and local disease, but the mechanism for caspase-1 activation in FMF is different from that of the NALP1 inflammasome.36 The mutations that result in FMF are found in a protein called pyrin. Whereas the N-terminus of pyrin interacts with the ASC adaptor protein, the disease-causing mutations in pyrin are found in the C-terminal domain. Pyrin normally suppresses IL-1 beta production, but when mutated or knocked down by siRNA, it does not.35 These studies suggest that in cases of FMF, the increase in IL-1 beta secretion is independent of the function of the ASC adaptor protein and dependent on the pyrin protein. Other studies have shown that the C-terminus of pyrin actually binds to caspase-1.37 This research demonstrates that the knock-down of pyrin by siRNA increases caspase-1 activation, whereas over-expression of the C-terminus of pyrin reduces caspase-1 activation.37 The results of these studies indicate that inhibition of the interaction between pyrin and caspase-1 leads to an increase in caspase-1 activity and a subsequent increase in IL-1 beta secretion. This diminished control of the processing and secretion of IL-1 beta may be responsible for the symptoms associated with FMF.
Hyper IgD Syndrome. Patients with Hyper IgD Syndrome share several clinical and biochemical characteristics with patients having FMF such as attacks of fever accompanied with abdominal pain. In addition, patients with Hyper IgD Syndrome also have markedly elevated serum IgD levels. Similar to other auto-inflammatory syndromes, the blood monocytes from these patients secrete more IL-1 beta than cells from unaffected individuals.38 The basis for increased IL-1 beta secretion in this autosomal recessive disease are mutations in the gene encoding mevalonate kinase and not in NALP1 or NALP3.39 It is unclear how mutations in mevalonate kinase result in increased production of IL-1 beta. Nevertheless, treatment with IL-1 receptor blockade reduces the frequency and severity of the attacks. In addition, because mevalonate kinase participates in cholesterol synthesis, treatment of these patients with inhibitors of HMG-CoA reductase such as simvastatin also reduces disease activity.
|Table 1 Auto-inflammatory Diseases|
|Familial Mediterranean Fever (FMF)|
|Familial Cold Auto-inflammatory Syndrome (FCAS)|
|Muckle-Wells Syndrome (MWS)|
|Neonatal Onset Multi-System Inflammatory Disease (NOMID)|
|Hyper IgD Syndrome|
|Adult Onset Still's Disease|
|Systemic Onset Juvenile Idiopathic Arthritis|
|TNF Receptor-Associated Periodic Syndrome|
|Macrophage Activation Syndrome|
|Normocomplementemic Urticarial Vasculitis|
|Pericarditis in Adult Still's Disease|
|Urate Crystal Arthritis (Gout)|
|Type 2 Diabetes|
Conclusions. Auto-inflammatory diseases are best defined as IL-1 beta-dependent because affected patients respond dramatically to treatments that inhibit IL-1 beta activity, but do not respond to those treatments that target other pro-inflammatory molecules.25 The common characteristic associated with each of the auto-inflammatory syndromes and diseases listed in Table 1 is increased levels of IL-1 beta secretion. This increase occurs as a result of defects in the regulatory pathways that control the processing of the IL-1 beta precursor into an active cytokine. Whether constitutively active or triggered by a fall in intracellular potassium, the cleavage of the IL-1 beta precursor is in most cases dependent upon caspase-1 and the caspase-1 inflammasome. Although mutations in the caspase-1 inflammasome result in greater disease severity, some auto-inflammatory diseases are caspase-1-independent since extracellular enzymes can also cleave the IL-1 beta precursor. Nevertheless, similarities in the clinical diseases are due to a common cause, the increased production of IL-1 beta. The auto-inflammatory diseases discussed here reveal the biological potency of IL-1 beta in humans. Interestingly, however, while the increase in IL-1 beta secretion from blood monocytes of affected patients cultured in vitro is significant, it is relatively small compared to that of unaffected individuals. This being the case, the diagnosis of an auto-inflammatory disease is not made by measuring circulating levels of IL-1 beta itself, but rather by clinical characteristics and a sustained reduction in disease severity upon treatments targeting IL-1 beta activity.