Calgranulins A (S100A8), B (S100A9) and C (S100A12) are members of the superfamily of EF-hand calcium binding proteins. The term calprotectin specifically refers to the Calgranulin A and B heterodimer that is formed by a non-covalent interaction. A distinguishing feature of calgranulins is their involvement in innate immunity and inflammation. As secreted proteins, calgranulins can directly inhibit microbial growth in the extracellular milieu, presumably through their ability to chelate zinc. Cells that actively express calgranulins are more resistant to bacterial adherence and invasion. Calgranulins also support optimal functioning of the cells that comprise the innate immune system. For example, granulocytes depend on the intracellular calgranulin A/B complex to adequately participate in cell adhesion, cytokinesis, cytokine secretion, and activation of the respiratory burst. Calgranulin A/B can down-regulate immune responses through inhibition of immunoglobulin production by lymphocytes and induction of myeloid derived suppressor cells. In addition, calgranulin induces apoptosis in cells that stimulate the inflammatory cascade. Calgranulin A expression may also be important in promoting the induction of a regulatory macrophage phenotype that down-regulates inflammation and facilitates a healing response.
Under normal conditions, calgranulins support an optimal immune response, leading to resolution of infection and inflammation. When normal expression and/or secretion of calgranulins are perturbed, inflammation is exacerbated with adverse implications for host tissues. As an example, dysregulation in the expression or secretion of calgranulins can have detrimental effects by promoting chronic active inflammation that leads to collateral tissue damage. Excessive amounts of calgranulin A/B in extracellular compartments of tissues correlates with a variety of inflammatory disorders. Expression and release of calgranulin A/B in the extracellular milieu can be triggered by IL-1α. Extracellular calgranulin A/B can then bind to and activate toll like receptor 4 (TLR 4), which in turn initiates further expression of pro-inflammatory mediators such as IL-1, IL-6, and IL-8. The interaction of these cytokines and chemokines with calgranulin A can create an autocrine / paracine mediated pro-inflammatory feedback loop that does not necessarily resolve infection. In the urinary tract, over expression of calgranulin A is not specific for infection. Over expression of calgranulins has been reported in a variety of inflammatory disorders of the urinary tract, including interstitial cystitis, interstitial nephritis, glomerulonephritis and neoplasia. This suggests that while calgranulin A may be a useful biomarker for chronic active inflammation, in general it may not be important in the defence against bacterial pathogens. Recent studies in rodent models of urinary tract infection (UTI) suggest that calgranulins are not required for innate defence against urogenital pathogens. Moreover, the exaggerated expression of these proteins during infection may be contributing to complicated urinary tract diseases such as struvite urolithiasis.
This review will focus on the role of calgranulin A and B in innate immune responses with particular attention to diseases of the urinary tract. The interaction of calgranulins with various host cell signal transduction pathways important in innate immunity and inflammation of the urinary tract will be reviewed. Further, potential consequences of aberrant signalling that may contribute to perturbations of regulation of calgranulins will also be discussed.
2. Classification and general function of calgranulins
Calgranulins are members of the superfamily of EF-hand calcium binding proteins that are involved in innate immune and pro-inflammatory processes. In the literature, these proteins have been given a variety of names including S100A8, myeloid related protein 8 (MRP), and L1 light chain for calgranulin A. Calgranulin B is also known as MRP-14 and S100A9. Calgranulin C may be referred to as S100A12 and EN-RAGE (Nacken et al., 2003). The name calprotectin specifically refers to the Calgranulin A and B heterodimer that is formed by a non-covalent interaction. Calgranulin C does not appear to form a heterodimer complex with either Calgranulin A or B (Foell et al., 2007).
Calgranulin C appears to be exclusively expressed in granulocytes (Vogl et al., 1999). However, calgranulins A and B are constitutively expressed in a variety of cells including granulocytes, monocytes, dendritic cells, epithelial cells, and keratinocytes (Foell et al., 2007; Frosch et al., 2004; Nacken et al., 2003). In healthy individuals, low concentrations of calprotectin can be detected in plasma, saliva, cerebrospinal fluid, urine and feces (Johne et al., 1997). Typically calgranulin gene expression is tightly regulated. However, during inflammation, expression is induced in cells such as macrophages and fibroblasts that do not normally produce calgranulins (Foell et al., 2007; Frosch et al.., 2004; Hsu et al., 2005; Nacken et al., 2003). Further, cells that constitutively express calgranulins are induced to secrete calprotectin into the extracellular milieu when stimulated (Kido et al., 2003). Therefore, calprotectin is considered a biomarker of inflammatory conditions such as rheumatoid arthritis, Crohn’s disease, and chorioamnionitis (Kostakis et al., 2010; Perere et al., 2010).
Calprotectin deficiency has never been reported in humans, suggesting that this protein is essential for life. The need for calgranulin A was confirmed with a null mutation in mice, which resulted in early resorption of mouse embryos (Passey et al., 1999a). Null mutation of the S100A9 gene (calgranulin B) was not lethal in the mouse (Hobbs et al., 2003). Interestingly, although the S100A9 -/- mouse expresses S100A8 (calgranulin A) mRNA, the protein is not detected in peripheral tissues.. Consequently, this mouse strain does not produce calprotectin, making it convenient for use in studying the role of calprotectin in various disease processes. Although the calgranulin genes are conserved among higher vertebrates, there are structural and functional differences among the various species (Nacken et al., 2003). For example, in rodents, calgranulin A is chemotactic for granulocytes but this is not the case in humans (Foell et al., 2007). Calgranulin C, which is not present in rodents, is the potent granulocyte chemoattractant in humans (Yang et al., 2001) and is highly expressed in activated tissue macrophages localized within sites of inflammation (Perera et al., 2010). Calgranulin C also serves as a ligand for RAGE (Perera et al., 2010). Although, rodents possess RAGE, there is no evidence that either calgranulin A, B, or calprotectin act as a ligand for this receptor. To date, there are no published studies demonstrating a role for calgranulin C in urinary tract infection. Therefore, this review will focus on calgranulins A and B.
2.1. The contribution of calgranulins to defense against infection
The innate immune system comprises the first attack against invading microorganisms. Both intracellular and extracellular calgranulins play an integral role in modulating leukocyte activation, trafficking, and amplification of immune responses during infection. Intracellular calgranulins regulate cell adhesion, migration, phagocytosis, and bacterial killing through direct interactions with the cell cytoskeleton and plasma membrane components. Calprotectin and calgranulin B facilitate phagocyte transmigration by coordinating microtubule dynamics (Vogl, et al., 2004). Specifically, the calprotectin complex induces polymerization of microtubules that can be disrupted by phosphorylation of calgranulin B by p38 mitogen-activated protein kinase. With disruption of the calprotectin complex the microtubule depolymerizes. A similar mechanism in squamous epithelial cells increases their resistance to intracellular invasion by mucosal pathogens such as
Active secretion of calgranulins from neutrophils can be induced through engagement of toll-like receptor/CD14 and nuclear factor (Kido et al., 2003) or through protein kinase C in monocytes (Nacken et al., 2003). Calgranulins can be found in mucosal body fluids that are normally colonized with bacteria, such as the oral cavity. Moreover, calprotectin concentrations increase in mucosal fluids in response to certain infections such as
Calprotectin microbicidal activity towards
Extracellular calgranulins also augment immune defense mechanisms in a cytokine like manner. Calgranulin B enhances neutrophil microbial killing by stimulating degranulation of matrix metalloproteinase through p38 mitogen activated protein kinase (Simard et al., 2010). Calprotectin stimulates production of interleukin-8 in airway epithelial cells (Ahmad et al, 2003). Calprotectin binding to microvascular endothelial cells promotes chemokine secretions, up regulation of cell adhesion molecules, and disruption of the endothelial barrier (Viemman et al., 2005, Eue et al., 2000), facilitating leukocyte migration into sites of infection. Calprotectin may also contribute to bacterial clearance from uroepithelium through its ability to enhance activation of toll-like receptor (TLR) 4 (Ehrchen, et al., 2009). Song et al. identified an additional alternative pathway in uroepithelium in which activation of TLR 4 by uropathogenic type 1 fimbriated
2.2. Regulation of immunity by calgranulins
Successful defense against infection depends on a well controlled inflammatory response that is able to remove the pathogen without extensive collateral damage of the surrounding host tissues. During an effective immune response the signal transduction pathways trigger production and /or release of pro-inflammatory mediators that potentiate removal of the pathogen, and simultaneously or sequentially activate anti-inflammatory factors and protect surrounding tissues from proteases and reactive oxygen species. Calgranulins participate in several of these processes most of which have been identified in macrophages (Lim et al. 2009; Passey et al., 1999b; Xu et al., 2001). For example, bacterial lipopolysaccaride, IFN-γ, TNF-, and interleukin-10 all induce calgranulin gene expression in murine macrophages (Xu et al., 2001). Glucocorticoids also amplify calgranulin expression in macrophages that have been primed with lipopolysacarride (Hsu et al., 2005). Methylprednisolone treatment in rheumatoid arthritis patients significantly increases the number of calgranulin A and B expressing macrophages in synovium (Hsu et al., 2005).
Secreted calgranulins also exhibit anti-inflammatory properties in neutrophils. Sroussi et al., have shown that both calgranulin A and B can repel human neutrophils
Calgranulins that have undergone post-translational modifications can down-regulate inflammation or provide protection from granulocyte secreted products such as reactive oxygen species. Calgranulin A, in particular, is highly susceptible to oxidation by hydroxyapatite, hydrogen peroxide, and hypochlorite (Lim et al., 2009; Harrison et al, 1999). Calgranulins therefore act as sinks for reactive oxygen species, providing protection of the microenvironment from collateral damage. Both calgranulins A and B can be nitrosylated (addition of nitric oxide molecule) (Lim et al., 2008, 2009). Nitrosylated calgranulin A suppresses mast cell mediated activation, leukocyte adhesion, and extravasation into the microcirculation (Lim et al., 2008).
Calgranulins A and B modulate various aspects of adaptive immunity. Calgranulin A has been reported to inhibit immunoglobulin G production in lymphocytes (Brun et al., 1995). Calgranulin B may down regulate responsiveness to toll-like receptor mediated stimulation in dendritic cells (Averill et al., 2011). During murine myeloid differentiation, increased expression of calgranulin B in embryonic stem cells favors development into myeloid derived suppressor cells over dendritic cells (Lim et al. 2009). Myeloid derived suppressor cells promote immune tolerance during infection and inflammation by suppression of T cell activation and promotion of a T helper type 2 cytokine phenotype involving expression of interleukin-4 and interleukin-13 (Delano et al., 2007; Ezernitchi et al., 2006; Haile et al., 2008). Moreover, calprotectin secreted by myeloid derived suppressor cells prolongs their immunosuppressive effects through an autocrine positive feedback loop (Lim et al., 2009).
2.3. Pro-inflammatory effects of calgranulins
Increases in extracellular calgranulin concentrations are associated with several inflammatory and auto-immune disorders including cystic fibrosis, chronic bronchitis, sepsis, pyelonephritis, oral candidiasis, periodontitis,
One mechanism by which calprotectin amplifies inflammation is through direct activation of TLR 4. In the CD40L over-expressing mouse, calgranulin mediated activation of TLR 4 increases expression of interleukin-17, which activates autoreactive CD8+ T cells (Loser et al., 2010). In experimentally induced sepsis, calprotectin enhancement of TLR 4 activation induces exaggerated secretion of TNF- with endotoxic shock, which is not observed in S100A9 -/- mice. However, septic S100A9 -/- mice treated with recombinant calprotectin exhibit the same degree of endotoxic shock and mortality as their wild type counterparts. It is important to note that calprotectin mediated enhancement of TLR 4 pathway only occurs in systems that have already been immunologically activated. For example, in the case of the sepsis model, the initial activation of TLR 4 occurred by bacterial lipopolysaccaride. These results prompted us to re-evaluate our rat model of inflammation induced struvite urolithiasis secondary to experimental infection with
In an unpublished study of women with first time urinary tract infection, we identified the predominant pathogens present.
Ureaplasmal infections are asymptomatic as long as their colonization is restricted to the urogenital mucosa. However, if these bacteria invade the deeper stromal layers, they induce an exaggerated host immune response that does not eliminate infection, but rather causes tremendous tissue damage at sites of colonization. In our rodent model of infection, invasion of bacteria into the submucosa triggered a smoldering inflammation within the submucosa that persisted. By two weeks post-inoculation, animals with ongoing submucosal infection developed struvite uroliths and an exaggerated pro-inflammatory cytokine profile in their urine. Since
In our rodent model of
We also evaluated the distribution of calprotectin within the bladder tissues of animals infected with
Danger-associated pattern molecules (DAMP) are endogenous ligands of toll like receptors. These molecules are often by-products of inflammation such as reactive oxygen species (Frantz et al., 2001) or tissue degradation products from the extracellular matrix (Okamura et al., 2001). Calprotectin is now considered a DAMP because of its ability to activate TLR4 (Ehrchen et al., 2009). Since we observed a correlation between uroepithelial calprotectin expression and pro-inflammatory cytokines in the urine of struvite positive animals, we measured the amount and distribution of TLR 4 in the bladder tissues of rats infected for 72 hours and 2 weeks post-inoculation.There were no appreciable differences in the amount of TLR 4 detected in bladder tissues from sham inoculated controls, or
2.4. Regulation of calgranulin gene expression in different cell populations
In addition to participating in immune defence, calgranulins are involved in embryogenesis, growth, and differentiation. Therefore, it is not surprising that the expression of these proteins is tightly regulated in a cell and tissue specific manner, which can change in response to environmental cues. For example, most mucosal epithelial cells have a basal level of calgranulin expression that changes in response to the appropriate stimulus (Ross & Hertzberg, 2001, Hsu et al., 2005, 2009). The appropriate stimulus for one tissue type is not necessarily the same for another. For example, calgranulin expression in oral squamous epithelial cells increases in response to interleukin-1α, but bacterial lipopolysaccaride does not induce an effect (Ross & Hertzberg, 2001). This may pertain to the fact that the oral cavity is heavily colonized with bacteria. On the other hand, bronchiolar epithelium increases its calgranulin expression in response to lipopolysaccaride mediated activation of TLR4 (Henke et al., 2006). Uroepithelium has a low basal expression of calgranulins A and B, which is significantly up regulated in cancer and inflammation (Yao et al., 2007; Tyagi et al., 2008). It is not known what specific cytokines or factors induce up regulation of calgranulin gene expression in these cells, but the studies performed by our laboratory and Dressing et al., imply that uroepithelium would respond to both interleukin-1α and toll like receptor activation.
Varieties of cell types that reside within bladder submucosa either repress or express calgranulins in response to changes within the tissue microenvironment. Cells pertinent to urinary tract infection are fibroblasts, dendritic cells, macrophages, and neutrophils (Cresswell, et al., 2001; Shafik, et al., 2004; Christmas & Botazzo, 1992; Shimizu et al., 2011). Fibroblasts do not participate in antimicrobial processes during urinary tract infection, but their response to mediators released by uroepithelial cells exposed to
Dendritic cells are important in immune surveillance and can express calgranulins in varying intensity depending on their level of maturation (Kumar et al., 2003; Koga et al., 2008). Immature dendritic cells express higher amounts of calgranulins A and B than mature cells (Koga et al., 2008). Interleukin-10 promotes induction of tolerogenic dendritic cells, which then exhibit increased expression of calgranulins (Kumar et al., 2003). Further, calgranulin B may down regulate cytokine release in dendritic cells after toll-like receptor stimulation (Averill et al., 2011).
Macrophages represent differentiated monocytes that have migrated into tissues in response to pro-inflammatory signals or part of a homeostatic process. When differentiation is complete these cells cease to express calgranulins unless they become activated (Nacken et al., 2003). It is important to note that not all activated macrophages express calgranulins. Presently, three macrophage phenotypes with distinctly different functions have been identified (Benoit et al., 2008; Mosser & Edwards, 2008). The phenotype is determined by the type of stimulus the cell receives during the process of activation. For example, macrophages exposed to interferon-γ in combination with TNF-α, and induction of TLR transforms them into the classically activated or microbicidal macrophage (Benoit et al., 2008; Mosser & Edwards, 2008). Classically activated macrophages are known for their enhanced microbial killing through induction of nitric oxide synthase, secretion of pro-inflammatory cytokines (TNF, interleukin-1β, interleukin-6, and interleukin-12) and secretion of chemokines (CCL2, CCL5, and CXCL8) (Benoit et al., 2008). Resident macrophages that are exposed to interleukin-4 and/or interleukin-13 transform into a wound healing phenotype. Instead of participating in pro-inflammatory events, these cells primarily secrete extracellular matrix proteins and contribute to tissue remodelling. The third activated phenotype is referred to as the regulatory macrophage because these cells express high amounts of co-regulatory molecules that bind to T cell receptors. In addition, regulatory macrophages secrete high amounts of interleukin-10, and these cells are primarily involved in down-regulation of the inflammatory response (Mosser & Edwards, 2008). Of the three phenotypes, only regulatory macrophages express calgranulins, in particular calgranulin A (Benoit et al., 2008; Hsu et al., 2009). Therefore, calgranulin A may be a useful molecular marker for the
A common feature of regulatory macrophages is that they develop from simultaneous exposure to two stimuli (Mosser & Edwards, 2008). The first signal has little to no stimulatory function. Weak agonists known to induce a regulatory phenotype include immunoglobulin complexes, prostaglandins, apoptotic cells, glucocorticoids, interleukin-10, or G-protein coupled receptor ligands. The second signal involves TLR activation. It has been postulated that calgranulin expression by regulatory macrophages contributes to their immunosuppressive function (Hsu et al., 2009). Support for this argument is that regulatory macrophages exclusively express calgranulin A, which can reduce the damaging effects of inflammation by acting as a scavenger for reactive oxygen species. Furthermore, secreted calgranulin A also repels or inhibits recruitment of neutrophils (Hsu et al., 2009; Sroussi et al., 2010; Xu et al., 2001). Although regulatory macrophages may be highly beneficial during sterile inflammation, their presence at sites of infection may be potentially detrimental. Regulatory macrophages can be exploited by bacterial, parasitic, and viral pathogens. For example, during invasion of macrophages,
The biological function of calgranulins is complex, diverse and paradoxical. Therefore, extrapolations from studies outside of the context of urinary tract infection must be made with caution. To date, there are only two published reports that refer to calgranulins during urinary tract infection. Based on these reports, we can only conclude that the antimicrobial properties of calgranulins are minimally effective in the urinary tract. More than likely, the pro-inflammatory properties of calgranulins contribute to complicated inflammatory diseases such as struvite urolithiasis, pyelonephritis, and possibly interstitial cystitis. The positive correlation between increased calgranulin expression and inflammation in tissues, support the argument that these proteins may serve as biomarkers for chronic complicated urinary tract infections. However, additional studies are required in order to determine which clinical settings would benefit from monitoring calgranulin concentrations during urinary tract infections. Of more immediate benefit may be the use of calgranulins as molecular markers in basic science research. For example, monitoring these proteins during maturation and activation of different cell types that comprise innate defense may provide new insights into mechanisms of immune dysregulation induced by various microbial pathogens of the urinary tract.
Portions of this work were supported by award number K08DK075651 from the National Institute of Diabetes And Digestive And Kidney Diseases (NIDDK), and award number RO1 AI45875 from the National Institute of Allergy and Infectious Disease (NIAID). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDDK, NIAID or the National Institutes of Health.