Asthma is an inflammatory disorder of the airways resulting in their hyperreactivity. In the United States, approximately 25.7 million people have been diagnosed with asthma by a healthcare professional . Asthma initially starts out as an episodic disease in which repetitive environmental insults such as viral infection or allergen exposure result in a period of wheezing and airflow obstruction followed by resolution of the symptoms. After several bouts of wheezing, the threshold for airway reactivity is reduced and patients begin to experience mild symptoms between episodes . The persistence of symptoms and airway hyperreactivity between episodes suggest the existence of a memory-driven process (Fig. 1). Given that most asthma exhibits an immunologically based T-helper-2-type (Th2) inflammatory response with extensive upregulation of the interleukin (IL)-4 gene cluster, it has been difficult to discern whether this phenotype is mediated by an immunological or a tissue-based memory . Here, we present a hypothesis of disease-related memory and its relevance for the development and persistence of chronic asthma.
CONCEPT OF MEMORY IN BIOLOGICAL SYSTEMS
In physiological systems, memory is the retention and retrieval of an acquired or learned signal. Memory occurs through encoding of information, storage and then retrieval. Persistence of memory usually requires consolidation and reconsolidation (repetition of the encoding process). Retrieval of the memory may occur in various forms. In psychological terms, declarative (explicit) memory requires a conscious recall. On the contrary, procedural (implicit) memory does not require conscious recall but is based upon implicit learning through repetition (reviewed in ). Outside the central nervous system, the term memory is often applied to T and B cells, and more recently natural killer (NK) cells of the immune system. The T and B cell memory is induced and maintained through the expansion and persistence of antigen-specific clones. The immunological memory is akin to episodic/explicit memory, as it requires an act of ‘conscious recall’ through re-exposure to the inciting antigen [5,6].
NEUROLOGICAL SIGNALLING MECHANISMS OF MEMORY FORMATION
Neurological memory can be formed by a variety of short-term and long-term facilitation, augmentation and potentiation mechanisms. The order, frequency and duration of stimulation can alter the neuronal system such that the magnitude and frequency of the presynaptic input and the resulting postsynaptic output are no longer in a one-to-one correspondence [7,8]. This ability is termed synaptic plasticity. Synaptic plasticity alters the N-methyl-D-aspartate (NMDA) receptor, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and calcium that are directly required for synaptic signalling, as reviewed in . In addition, evidence demonstrates shifts in the signalling outputs including cyclic AMP (cAMP) (reviewed in [10,11]), mitogen-activated protein kinase (MAPK) amplification , and protein kinase C (PKC) and phosphatidylinositol-3-kinase (PI3K) [13▪]. Secondary activation of the systems produces neurotrophin-like neuropeptide in a PKC-dependent manner, thus increasing overall signalling during synaptic plasticity.
Coordinated activation of AMPA receptors in combination with the metabotropic glutamate receptors (mGluRs) or NMDA glutamate receptors results in sustained intracellular signalling. This coordinated receptor stimulation leads to activation of the Ca2+–PLC–PKC pathway, the PI3K pathway  and the ERK1/2 MAPK pathways . Many signalling pathways ultimately converge on ERK signalling, resulting in translational and posttranslational effects that maintain altered signalling patterns despite a lack of new activation triggers. Activation of cyclic AMP response element-binding protein downstream of cAMP and ERK1/2 can specifically increase immediate early genes including c-Fos during plasticity. Activation of immediate early genes can perpetuate intracellular signalling and prolong phenotypes following cessation of activation signals. c-Fos dimerizes with c-jun to form the activator protein 1 transcription factor, which is crucial for long-term memory and synaptic plasticity .
ROLE OF ENDOSOMAL RECYCLING OF RECEPTORS IN SUSTAINED SIGNALLING AND MEMORY GENERATION
Repeated stimulation of neurons leads to long-term potentiation, which manifests with prolonged and exaggerated outputs of action potentials. This eventually increases signalling output and expression of immediate early genes. Endosomal trafficking of certain receptors such as AMPA and NMDA receptors is critical, given that during long-term potentiation, AMPA receptors that become internalized will undergo preferential sorting via early endosomes and recycling endosomes to return to the plasma membrane. In contrast, long-term depression, which results in decreased excitatory signalling, will preferentially target AMPA receptors to the lysosomes (reviewed in [17,18]). These differentially regulated processes suggest that recycling of neurotransmitter receptors plays a seminal role in synaptic plasticity. Homer is a recently identified protein, whose deficiency impairs memory. Homer is an ERK1/2-inducible protein, and it functions to facilitate AMPA receptor recycling . Outside the nervous system, an interleukin 2 receptor α mutant with increased recycling causes sustained T cell signalling and proliferation . Similarly, reduced degradation and increased recycling of IL4RA in Dock2-/- mice lead to sustained signal transducers and activators of transcription 6 (STAT6) signalling and heightened Th2 response . The mechanisms surrounding receptor recycling are likely to be complex and involve many factors, but appear to be conserved between physiological systems.
ROLE OF HYSTERESIS AND BISTABILITY OF SIGNALLING MOLECULES IN MEMORY FORMATION
Many biological processes manifest a behaviour known as hysteresis. Hysteresis in biology means that a cellular behaviour and a response are shaped not only by the current environment but also by the past history/experience. Biological processes manifesting hysteresis can toggle between two alternative stable steady states: bistability. A bistable system can exist in three states: an ‘on’ state, an ‘off’ state and an unstable intermediate state . Early examples of biological bistable systems include the lambda phage lysis-lysogeny switch and the hysteretic lac repressor system . Lisman in 1985  first suggested that a bistable system could serve as a self-sustaining biochemical memory. Bistability arises from a positive feedback loop or a mutually inhibitory, double-negative feedback loop .
Ferrell and associates have mathematically shown that when the strength of positive feedback exceeds a certain limit, the system shows hysteresis and becomes bistable . In confirmation, they have shown that progesterone-induced activation of MAPK and Cdc2 induces a self-sustained activation mechanism in frog oocytes, which is dependent upon a positive feedback loop . Disruption of the positive feedback loop at the level of c-Mos (Raf-1) abrogates the signalling memory. The concentration and activity of the kinases of the ERK module and their phosphatases play a pivotal role in bistability . ERK can exist in an ‘off’ state or an ‘on’ state on the basis of the concentrations of its immediate activator mitogen-activated protein kinase kinase (MEK) and its inactivating enzyme mitogen-activated protein kinase phosphatase 3. Within a narrow range of concentrations, an intermediate unstable state exists. Thus, the ERK pathway could function as a bistable module and the ‘on’ state of a bistable module could function as memory. In summary, there is strong theoretical, mathematical and experimental evidence that the ERK1/2 module can function as a system memory.
EVIDENCE FOR MEMORY-DRIVEN PROCESSES IN ASTHMA
In a similar fashion to synaptic plasticity in the central nervous system, a primed cell and tissue are able to respond faster and stronger because of prior experience. Tissue priming in asthma manifests as airway hyperreactivity, one of the most reliable features of asthma and detectable when the patient is asymptomatic. The mechanism of airway hyperreactivity is complex and likely involves memory-driven processes in the airway smooth muscle and epithelium. Priming of epithelium is also manifested through increased permeability and increased secretion of mucus, cytokines and other epithelial mediators.
EXTRACELLULAR SIGNAL REGULATED KINASE 1/2 MANIFESTS SUSTAINED ACTIVATION IN ASTHMA
Sustained ERK1/2 activation is critical for asthma. Pharmacological inhibition of ERK1/2 blocks airway inflammation, mucus production and airway hyperreactivity in a mouse model of asthma [26,29,30]. We have further shown that ERK1 knockout mice are unable to generate a Th2 immune response and features of asthma in the mouse model. Finally, we have demonstrated sustained expression of pERK1/2 in airway epithelium and smooth muscle biopsy samples from asthmatic patients. The expression level of the activated ERK1/2 correlated with the clinical severity of asthma and eosinophilic infiltrates. We have also reported increased expression of MEK and sustained levels of pERK1/2 in blood CD4 T cells from asthmatic patients. This increased pERK1/2 conferred T cells’ resistance against inhibition by glucocorticoids, transforming growth factor (TGF)-beta and IL-10 .
EXTRACELLULAR SIGNAL REGULATED KINASE 1/2 ACTIVATION IS SELF-SUSTAINED (MEMORY-DRIVEN) FOLLOWING REPETITIVE STIMULATION AND IS ASSOCIATED WITH EPITHELIAL CELL PRIMING
In epithelial cells, a rapid activation and a return to baseline of ERK1/2 result following a single stimulation with IL-4, IL-6, IL-13, eotaxin or epidermal growth factor (EGF). In contrast, repetitive stimulation with any of these cytokines results in sustained activation of ERK1/2 lasting more than 7 days after the last stimulation . This provides a strong evidence for ERK1/2 bistability and suggests that airway epithelial cells can maintain activated ERK1/2 through a memory-driven process. As the airways of a sensitized subject are repeatedly exposed to environmental triggers during the natural course of asthma, we speculate that this generates a cellular memory sustaining ERK1/2 activation at times when there are no apparent environmental triggers.
There is a spatial difference in ERK1/2 subcellular localization between acute/single and repetitive stimulation. Nuclear translocation of pERK1/2 dominates following an acute stimulation. In contrast, pERK1/2 primarily localizes to Rab4+ recycling endosomes and Rab5+ early endosomes with modest translocation to the nucleus in the repeated stimulation model. The nuclear localized pERK1/2 drives transcription of many genes and induces an acute state of cellular activation. In contrast, the endosomally localized pERK1/2 behaves more like a latent activator of the cell, which is akin to memory storage (Fig. 2). The endosomally localized pERK1/2 primes epithelial cells for heightened production of cytokines and chemokines and is associated with epithelial resistance against growth factor withdrawal induced apoptosis .
We have shown in vivo that repetitive intranasal exposure to multiple allergens (dustmite, ragweed, asperguillus) results in sustained ERK1/2 activation in the airway tissue that persists greater than 12 weeks after the last allergen exposure. This sustained ERK1/2 activation is associated with the development of persistent asthma (heightened airway hyperreactivity, sustained inflammation, mucus production and airway remodelling) despite the cessation of allergen exposure 3 months earlier . In accordance with prior in-vitro data, a significant portion of the pERK1/2 was localized to the endosomal compartment in airway epithelial cells. We have reported a similar endosomal localization of pERK1/2 in airway epithelial cells from human asthma. The results suggest that the sustained ERK1/2 activation in asthma is, at least, in part memory-driven.
MECHANISM OF MEMORY-DRIVEN ACTIVATION OF EXTRACELLULAR SIGNAL REGULATED KINASE 1/2
Several potential mechanisms have been proposed to explain the sustained pERK signalling following repeated stimulation. In cultured cells, EGF stimulation activates ERK1/2 prior to Cbl-dependent ubiquitination and downregulation of epidermal growth factor receptor (EGFR). However, when sprouty 2 (Spry 2) is overexpressed, there is direct binding and sequestration of Cbl by Spry 2 . As a result, EGFR is rescued from degradation and recycled via endosomes back to the cell surface producing sustained activation of EGFR and ERK1/2 signalling. Indeed, we have demonstrated that spry 2 is increasingly induced at a high level during repetitive stimulation of epithelial cells in a Fyn kinase-dependent manner. Spry 2 plays an essential role in sustained ERK1/2 activation, as its knockdown in epithelial cells and its biallelic deletion in fibroblasts result in inability to sustain ERK1/2 activation in this model. Our results indicate the development of multiple positive feedback and double-negative feedback loops, which lead to ERK1/2 bistability in our model (Fig. 3).
In addition to spry 2, other molecules that have been reported to promote sustained ERK1/2 signalling include B-Raf , Rap1  and beta-arrestin . The involvement of beta-arrestin is of particular interest, as mice with beta-arrestin deficiency are unable to manifest features of asthma. Subcellular localization of ERK1/2 is also regulated by phosphatidylinositol-4-phosphate 5-kinase and related FYVE finger-containing proteins 1 and neurofibromin 1 . An imbalance between ERK1/2 and its phosphatases in favour of ERK1/2 also promotes sustained ERK1/2 signalling [38▪].
OTHER EXAMPLES OF MEMORY-DRIVEN SIGNALLING PROCESSES IN ASTHMA
A classic example of memory-driven signalling processes is the auto-induction of GATA-binding protein 3 (GATA3) during a Th2 immune response. Following an initial induction of GATA3 by IL4-driven STAT6, the induced GATA3 binds to its own promoter and drives its transcription . This positive feedback loop establishes a bistable system, which functions as a memory for sustained GATA3 induction.
ROLE OF AUTOCRINE AND PARACRINE SECRETION IN GENERATION OF CELLULAR MEMORY
Positive feedback loops can be established not only through intracellular signalling networks but also through autocrine and paracrine secretory processes. Eosinophils partially regulate their own survival and activation through autocrine secretion of pro-survival IL-5 and pro-apoptotic TGF-beta . A similar promotion of survival and priming occurs in basophils through autocrine production of IL-3 . IL-17 induces sustained mucus production by autocrine secretion of epithelial IL-6 . It is unclear whether endosomal recycling of receptors and ligands is involved in the foregoing processes.
Utilization of learned skills to gain evolutionary advantage is critical in human development. The learning of these skills and their application are driven by memory. This memory allows adaptation to the environment. Many human diseases including asthma and allergic diseases are elicited by environmental factors in genetically susceptible individuals. We believe that the disease-inducing environmental factors generate a tissue memory upon multiple iterations. This tissue memory develops independent of the immunological memory. The immunological memory facilitates a robust response but requires the presence of the inciting antigen. In contrast, the tissue memory can sustain the pathological process in the absence of the environmental trigger. In the natural course of a disease such as asthma, we speculate that these two mechanisms complement each other. Allergens (and other nonallergenic asthma triggers) elicit asthma through direct activation of a sensitized immune system in a periodic manner. When allergens are absent in the environment, the tissue memory maintains the pathologic processes, albeit at a low intensity, until the next exposure to the allergen. Repetitive allergen exposure not only reinvigorates the disease process but also reinforces the disease memory and the cycles continue (Fig. 1).
System bistability provides a mathematical explanation for a tissue-based memory. In order to take advantage of the mathematical understanding of system bistability and memory, we will have to generate quantitative data about the involved biochemical reactions (substrates, products, dissociation constants of the reactions). Only then will we be able to understand and interfere with the disease memory and curtail the persistence of the disease.
This work is supported by NIH funding AI091614, HL 36577 and HHSN272200700048C.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 123).
1. Akinbami LJ, Moorman JE, Bailey C, et al
. Trends in asthma prevalence, healthcare use, and mortality in the United States, 2001–2010. NCHS Data Brief 2012; 94:1–8.
2. Weinberger M. Clinical patterns and natural history of asthma. J Pediatr 2003; 142:S15–S19.discussion S19–S20.
3. Kabesch M, Tzotcheva I, Carr D, et al. A complete screening of the IL4 gene: novel polymorphisms and their association with asthma and IgE in childhood. J Allergy Clin Immunol 2003; 112:893–898.
4. Dienes Z, Perner J. A theory of implicit and explicit knowledge. Behav Brain Sci 1999; 22:735–755.discussion 755–808.
5. Lanzavecchia A, Sallusto F. Understanding the generation and function of memory T cell subsets. Curr Opin Immunol 2005; 17:326–332.
6. Crotty S, Ahmed R. Immunological memory in humans. Semin Immunol 2004; 16:197–203.
7. Bliss TV, Lomo T. Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol 1973; 232:331–356.
8. Malenka RC, Nicoll RA. Long-term potentiation: a decade of progress? Science 1999; 285:1870–1874.
9. Kerchner GA, Nicoll RA. Silent synapses and the emergence of a postsynaptic mechanism for LTP. Nat Rev Neurosci 2008; 9:813–825.
10. Wang H, Zhang M. The role of Ca(2)(+)-stimulated adenylyl cyclases in bidirectional synaptic plasticity and brain function. Rev Neurosci 2012; 23:67–78.
11. Ferguson GD, Storm DR. Why calcium-stimulated adenylyl cyclases? Physiology (Bethesda) 2004; 19:271–276.
12. Kelleher RJ 3rd, Govindarajan A, Jung HY, et al. Translational control by MAPK signaling in long-term synaptic plasticity and memory. Cell 2004; 116:467–479.
13▪. Hu JY, Baussi O, Levine A, et al. Persistent long-term synaptic plasticity requires activation of a new signaling pathway by additional stimuli. J Neurosci 2011; 31:8841–8850.
The activation of persistent long-term synaptic plasticity requires additional stimulation and the activity of a secondary signalling pathway, PKC in addition to the primary PI3K activity.
14. Pezet S, Marchand F, D’Mello R, et al. Phosphatidylinositol 3-kinase is a key mediator of central sensitization in painful inflammatory conditions. J Neurosci 2008; 28:4261–4270.
15. Karim F, Wang CC, Gereau RWt. Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice. J Neurosci 2001; 21:3771–3779.
16. Alberini CM. Transcription factors in long-term memory and synaptic plasticity. Physiol Rev 2009; 89:121–145.
17. Gomes AR, Correia SS, Carvalho AL, Duarte CB. Regulation of AMPA receptor activity, synaptic targeting and recycling: role in synaptic plasticity. Neurochem Res 2003; 28:1459–1473.
18. van der Sluijs P, Hoogenraad CC. New insights in endosomal dynamics and AMPA receptor trafficking. Semin Cell Dev Biol 2011; 22:499–505.
19. Petrini EM, Lu J, Cognet L, et al. Endocytic trafficking and recycling maintain a pool of mobile surface AMPA receptors required for synaptic potentiation. Neuron 2009; 63:92–105.
20. Fallon EM, Liparoto SF, Lee KJ, et al. Increased endosomal sorting of ligand to recycling enhances potency of an interleukin-2 analog. J Biol Chem 2000; 275:6790–6797.
21. Tanaka Y, Hamano S, Gotoh K, et al. T helper type 2 differentiation and intracellular trafficking of the interleukin 4 receptor-alpha subunit controlled by the Rac activator Dock2. Nat Immunol 2007; 8:1067–1075.
22. Hervagault JF, Canu S. Bistability and irreversible transitions in a simple substrate cycle. J Theor Biol 1987; 127:439–449.
23. Yildirim N, Santillan M, Horike D, Mackey MC. Dynamics and bistability in a reduced model of the lac operon. Chaos 2004; 14:279–292.
24. Lisman JE. A mechanism for memory storage insensitive to molecular turnover: a bistable autophosphorylating kinase. Proc Natl Acad Sci U S A 1985; 82:3055–3057.
25. Ferrell JE, Xiong W. Bistability in cell signaling: how to make continuous processes discontinuous, and reversible processes irreversible. Chaos 2001; 11:227–236.
26. Chialda L, Zhang M, Brune K, Pahl A. Inhibitors of mitogen-activated protein kinases differentially regulate costimulated T cell cytokine production and mouse airway eosinophilia. Respir Res 2005; 6:36.
27. Xiong W, Ferrell JE Jr. A positive-feedback-based bistable ’memory module’ that governs a cell fate decision. Nature 2003; 426:460–465.
28. Markevich NI, Hoek JB, Kholodenko BN. Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades. J Cell Biol 2004; 164:353–359.
29. Inoue H, Kato R, Fukuyama S, et al. Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness. J Exp Med 2005; 201:73–82.
30. Duan W, Chan JH, Wong CH, et al. Anti-inflammatory effects of mitogen-activated protein kinase kinase inhibitor U0126 in an asthma mouse model. J Immunol 2004; 172:7053–7059.
31. Liang Q, Guo L, Gogate S, et al. IL-2 and IL-4 stimulate MEK1 expression and contribute to T cell resistance against suppression by TGF-beta and IL-10 in asthma. J Immunol 2010; 185:5704–5713.
32. Liu W, Tundwal K, Liang Q, et al. Establishment of extracellular signal-regulated kinase 1/2 bistability and sustained activation through Sprouty 2 and its relevance for epithelial function. Mol Cell Biol 2010; 30:1783–1799.
33. Goplen N, Karim MZ, Liang Q, et al. Combined sensitization of mice to extracts of dust mite, ragweed, and Aspergillus species breaks through tolerance and establishes chronic features of asthma. J Allergy Clin Immunol 2009; 123:925–932.e911.
34. Haglund K, Schmidt MH, Wong ES, et al. Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation. EMBO Rep 2005; 6:635–641.
35. Andreadi C, Noble C, Patel B, et al. Regulation of MEK/ERK pathway output by subcellular localization of B-Raf. Biochem Soc Trans 2012; 40:67–72.
36. von Kriegsheim A, Baiocchi D, Birtwistle M, et al. Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 2009; 11:1458–1464.
37. Emery AC, Pshenichkin S, Takoudjou GR, et al. The protective signaling of metabotropic glutamate receptor 1 Is mediated by sustained, beta-arrestin-1-dependent ERK phosphorylation. J Biol Chem 2010; 285:26041–26048.
38▪. Geetha N, Mihaly J, Stockenhuber A, et al. Signal integration and coincidence detection in the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) cascade: concomitant activation of receptor tyrosine kinases and of LRP-1 leads to sustained ERK phosphorylation via down-regulation of dual specificity phosphatases (DUSP1 and -6). J Biol Chem 2011; 286:25663–25674.
Simultaneous occupancy of tyrosine kinase receptors and low-density lipoprotein receptors produces sustained ERK activity.
39. Ouyang W, Lohning M, Gao Z, et al. Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment. Immunity 2000; 12:27–37.
40. Goplen N, Gorska MM, Stafford SJ, et al. A phosphosite screen identifies autocrine TGF-beta-driven activation of protein kinase R as a survival-limiting factor for eosinophils. J Immunol 2008; 180:4256–4264.
41. Schroeder JT, Chichester KL, Bieneman AP. Human basophils secrete IL-3: evidence of autocrine priming for phenotypic and functional responses in allergic disease. J Immunol 2009; 182:2432–2438.
42. Chen Y, Thai P, Zhao YH, et al. Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop. J Biol Chem 2003; 278:17036–17043.
© 2013 Lippincott Williams & Wilkins, Inc.