Type 1 FimbriatedEscherichia coli–Mast Cell Interactions in Cystitis

The single most commonly expressed virulence factor on uropathogenic Escherichia coli is type 1 fimbriae. In addition to promoting avid bacterial adherence to the uroepithelium, it is increasingly becoming clear that these determinants of bacterial adherence also play a critical role in modulating inflammatory responses of various immune cells of the host. Type 1 fimbriae have been shown to avidly bind to neutrophils, macrophages, and lymphocytes, triggering the release of inflammatory mediators and in the case of lymphocytes, initiating cellular proliferation and secretion of antibodies [1]. An inflammatory cell found in close proximity to the bladder epithelium and which has the potential to play a key role in modulating inflammation in the urinary tract is the mast cell. Relatively little is known of the role of mast cells in urinary tract infections. In light of recent findings demonstrating that the adherence of type 1 fimbriated E. coli to the mouse bladder epithelium during infection denudes the bladder of its epithelial lining [2], mast cells are likely to be one of the first inflammatory cells to be encountered by infecting type 1 fimbriated bacteria Work from our laboratory has demonstrated that type 1 fimbriated E. coli bind mast cells, evoking vigorous exocytic and endocytic responses in these cells [3]. We have recently identified the specific receptor on mast cells that bind the type 1 fimbrial adhesin, FimH, and made some progress at elucidating the early signal transduction events initiated by this mast cell receptor. These studies are reviewed below in the context of urinary tract infections Type 1 fimbriae, a determinant of bacterial virulence in bladder infectionsAn overwhelming number of bladder infections are caused by the enteric bacteria E. coli [4]. The bladder is a relatively inhospitable site to bacterial colonization. Since a potential pathogen must withstand the dilution effects of the frequent exfoliation of epithelial cells as well as the flushing action associated with the voiding of urine, the adhesive property of the bacteria appears to be an essential determinant for colonizing the urinary tract. Over 80% of uropathogenic bacteria exhibit the capacity to adhere to uroepithelial cells, and the most commonly expressed organelle of adhesion on these bacteria is type 1 fimbriae, with P fimbriae being the second most common [4]. Indeed, almost all cystitis-causing strains of E. coli are known to express type 1 fimbriae [4]. Type 1 fimbriae are long, filamentous appendages that promote bacterial binding to epithelial cells by specifically recognizing mannosylated molecules on the cell surfaces. Each filament is a heteropolymer comprising a major subunit and several minor subunits, including FimH, the mannose-binding moiety, which is preferentially located at the fimbrial tips [1] While the adhesive property of bacteria is clearly important in establishing bacterial colonization of the uroepithelium, this property could also facilitate subsequent steps of the infectious process when the bacteria penetrate the epithelium and come in contact with immune cells of the host. However, the consequences of the interactions of type 1 fimbriated bacteria with immune cells of the host are diverse and not readily predictable. On one hand, activation of immune cells of the bladder by type 1 fimbriated bacteria could result in the early removal of the bacteria and, on the other hand, if the inflammatory response of these immune cells is inappropriate or unregulated, severe pathologic consequences to the host can result. During infection, both activities may be occurring, and which of these responses ultimately prevails depends on multiple factors, including the underlying immune status of the host and the presence of other virulence attributes in the infecting bacteria Activation of bladder mast cells by type 1 fimbriated E. coli.Mast cells are found in relatively large numbers in the mucosa, lamina propria, and muscle of the bladder. To assess whether these mast cells were activated by type 1 fimbriated bacteria, we instilled 1×108E. coli J96 suspended in 100 μL of PBS via a catheter into the bladders of anesthetized female BALB/c mice. E. coli J96 is a well-characterized type 1 fimbriated uropathogenic isolate that has been used in many in vitro and in vivo studies. To evaluate mast cell activation in vivo, we sought to measure histamine, a mediator of inflammation originating specifically from local mast cells. Since FimH is the critical determinant on type 1 fimbriae that is responsible for promoting bacterial binding to mucosae, we instilled into a second group of mice 1×108E. coli PilE, an isogenic FimH-minus derivative of E. coli J96 [5]. A third group mice (controls) was instilled with 100 μL of PBS. After 1 h of incubation, the mice from all 3 groups were sacrificed, the urine from each mouse was collected, and the histamine content in the urine was measured. Bladder mast cells were activated by E. coli J96 and responded with the release of appreciable amounts of histamine (298±55 ng/mL). This contrasts with the significantly milder response (92±22 ng/mL) (P<.05) to PBS. Furthermore, the mast cell histamine response to FimH-minus E. coli (94±18 ng/mL) was comparable to that of PBS, indicating the pivotal role of FimH in facilitating activation of bladder mast cells To confirm that the effect of FimH was attributable to its capacity to promote bacterial binding to the bladder mucosae, we compared the number of adherent bacteria in the bladder after 1 h of incubation for each group of mice. As predicted, the number of type 1 fimbriated E. coli associated with bladder mucosae (5±1×103/bladder) was at least 5-fold higher than the number of FimH-deficient E. coli PilE (1±0.2×103). Thus, the magnitude of the histamine response of bladder mast cells correlated with the number of adherent bacteria on the bladder mucosae. Furthermore, electron microscopic analysis of infected bladder tissue revealed the presence of bacteria within mast cells, suggesting that bladder mast cell activation was, at least in part, through direct contact with type 1 fimbriated bacteria (unpublished data) We have not determined the in vivo significance of the mast cell response to bacteria in the bladder. We have shown previously that the in vivo histamine response of mast cells to type 1 fimbriated E. coli is accompanied by the release of a variety of other proinflammatory mediators, including tumor necrosis factor (TNF)-α and leukotriene B4 [6]. These mediators, along with histamine, can play a critical part in mediating the inflammatory response at the site of bacterial infection. Indeed, it is conceivable that the bladder mast cell response to bacteria could play a critical role in the clearance of the bacteria. On the other hand, this response could also contribute to the pathophysiology of bladder infection. The known function of mast cell products may hypothetically explain the signs and symptoms of cystitis, which typically include marked frequency, nocturia, urgency, and pain on filling of the bladder and at the end of micturition Several mast cell products induce fibrosis, which could prevent distention of the bladder, resulting in urgency and frequency [6, 7]. Pain is induced by histamine and bradykinin and also by prostaglandins that act on nerve endings and lower the pain threshold [7]. Histamine also causes detrusor irritability and bladder contraction. TNF-α and leukotriene B4, which initiate influx of various inflammatory cells, are likely responsible for pyuria [6]. While it may be difficult to predict whether mast cell responses to bacterial type 1 fimbriae in the bladder benefit or harm the host, it is apparent that mast cells can be pivotal in determining both the duration and pathophysiology of infection Identification of the mast cell receptor for E. coli type 1 fimbriaeBecause of its relevance in identifying the molecular events leading to mast cell responses to enterobacteria, we sought to identify the mast cell receptor for FimH, the type 1 fimbrial adhesin. We chose to isolate the FimH receptor from the rat mast cell line, RBL-2H3, because, unlike primary cultures of mast cells or freshly isolated mouse peritoneal mast cells, this cell line could be readily cultured, generating large numbers of cells. Using a Con A affinity column, we isolated several mannosylated and potential FimH receptor molecules and ultimately identified a 45-kDa protein as our putative receptor moiety [8]. Microsequencing of the amino terminus of this moiety revealed it to be rat CD48. This molecule is a glycosylphosphatidylinositol (GPI)-linked moiety that has been reported to be present primarily on cells of hematopoietic lineage. Since GPI-linked moieties are cleaved off the surface of cells with phospholipase C (PLC), we incubated bone marrow–derived mouse mast cells with increasing amounts of PLC prior to exposure to FimH-expressing E. coli. PLC pretreatment of mast cells inhibited bacterial binding in a dose-dependent fashion [8]. More direct evidence implicating CD48 as the putative E. coli FimH receptor on rodent mast cells comes from the observation that pretreatment of these mast cells with antibodies to CD48 inhibited the adherence of FimH-expressing E. coli in a dose-dependent fashion, while antibodies to CD117 (c-kit), a well-known mast cell membrane marker, did not [8] We also wanted to know whether expression of CD48 on cells that do not normally express this molecule could promote binding of FimH-expressing E. coli. We stably transfected CHO cells with the full-length cDNA encoding CD48. We then examined the transfectants for their capacity to bind FimH-expressing E. coli. The association of bacteria with these CD48 expressing cells was at least 4-fold higher than the number associated with CHO cells transfected with control cDNA [8]. Thus, CD48 molecules on transfected CHO cells are functional as FimH receptors. Taken together, the data indicate that the FimH receptor on mast cells is a GPI-linked moiety, CD48 We have already shown that CD48-specific antibody inhibits FimH-mediated bacterial binding and subsequent uptake by mast cells [8]. We have also reported previously that the early mast cell TNF-α response to bacteria plays a critical role in triggering the innate immune response [3]. We sought to demonstrate the role of CD48 in eliciting the mast cell TNF-α response to FimH-expressing bacteria after (1) exposing mast cell surface CD48 to CD48-specific antibody and (2) removing CD48 from mast cell surface with PLC. Antibody to CD48 but not antibody to CD117 blocked mast cell TNF-α release in a dose-dependent manner [8]. Further confirmation of the critical role of CD48 in the mast cell TNF-α response comes from the finding that pretreatment of mouse bone marrow–derived mast cells with increasing concentrations of PLC significantly reduced the mast cells’ capacity to release TNF-α following exposure to FimH-expressing E. coli [8]. Taken together, these observations provide definitive evidence that the mast cell TNF-α response to FimH-expressing bacteria is mediated by CD48 molecules present on the mast cell surface. Although there are likely to be other mannosylated moieties on the mast cell membrane capable of binding FimH-expressing E. coli these studies show that CD48 is the biologically relevant receptor Bacterial signaling of mast cells via caveolaeCD48 is the first physiologically relevant receptor identified on mast cells for an infectious agent. CD48 has been referred to as BCM1 in mice, OX 45 in rats and Blast-1 in humans. The expression of CD48 is restricted to cells of hematopoietic lineage, particularly lymphocytes, monocytes, and mast cells. CD48 was discovered as a cell surface molecule expressed by human B lymphocytes in response to Epstein-Barr virus infections, but its physiologic role in the body is still unclear. The involvement of CD48 in bacterial recognition and in triggering TNF-α release in inflammatory cells represents a novel function for this molecule. Rodent CD48 is glycosylated, and the primary structure reveals several potential sites of glycosylation. The importance of the sugar moiety on CD48, and specifically its mannose residues, is indicated by the observation that binding of FimH-expressing E. coli to mast cells is mannose sensitive CD48 joins a growing class of GPI-anchored cell surface molecules that serve as receptors for microbes and their toxins. Some notable examples of these receptors include CD14 for lipopolysaccharide, Thy-1 for aerolysin, and CD55 for echovirus and Group B coxsackieviruses [9, 10]. Engagement of these receptors by microbes or their products has been shown to trigger cellular responses [9]. How CD48 and other GPI-anchored receptors, which are only linked to the exoplasmic leaflet of the lipid bilayer of the plasma membrane, actually transduce intracellular signals is an important question. Many GPI-anchored moieties are typically found in special microdomains or “glycolipid-enriched rafts” called caveolae in the plasma membranes of cells [11]. Although initially thought to exist only as distinct cave-like structures on the cell surface, it is now clear that caveolae are pleomorphic membrane structures characterized biochemically by their buoyant density, resistance to nonionic detergents, and enrichment in cholesterol, glycolipids, such as the glycosphingolipid GM1, and a specific protein, caveolin [11] These caveolae are rich in signaling molecules, such as the heterotrimeric GTP binding proteins, which can potentially mediate signal transduction from GPI-anchored proteins. G proteins are involved in many signal transduction pathways, including stimulation of adenylate cyclase, regulation of Ca++ channels, stimulation of phospholipase A2, and stimulation of phosphoinositol 3-kinase or PLC [11]. A recent immunochemical study has shown that CD48 in lymphocytes is physically associated with GTP binding proteins [12]. Thus, engagement of CD48 could potentially trigger a mast cell response via a signaling pathway involving GTP binding proteins Thus, we sought to investigate whether mouse bone marrow–derived mast cells possessed caveolae and if CD48, the FimH receptor, was associated with caveolae. Using standard biochemical approaches, we demonstrated the presence of caveolar fractions in these mast cells and also the presence of CD48 within these fractions [13]. Confocal microscopy of mouse mast cells revealed that CD48 and caveolin, a specific marker of caveolae, were co-associated on the mast cell surface. The involvement of mast cell caveolae in signal transduction could be deduced by the fact that caveolae materials were recruited to sites of bacterial attachment from other cellular sites [13]. Using specific probes for various caveolar markers, we observed that recruited caveolar material completely encased the adherent bacteria as they were internalized. Thus, caveolae appeared to mediate the entry of type 1 fimbriated E. coli into mast cells. Support for this notion comes from the finding that specific disrupters of caveolae (methyl-β-cyclodextrin) or usurpers of caveolae (B subunit of cholera toxin) blocked uptake of type 1 fimbriated E. coli but not uptake of inert beads or antibody-opsonized FimH-minus E. coli by mast cells [13] To more definitively prove the contribution of cellular caveolae in mast cell uptake of bacteria, we demonstrated biochemically that cellular caveolae of mast cells was diverted to intracellular bacteria–containing chambers following bacterial infection of mast cells [13]. A diagrammatic representation of the uptake of type 1 fimbriated E. coli by caveolae of mast cells is shown in figure 1. Since intracellular compartments composed of caveolar material do not fuse with endosomes, it is not surprising that E. coli contained in caveolar chambers avoid the intrinsic bactericidal activity of mast cells and remain viable (unpublished data). Plasmalemmal caveolae have been implicated in the import of macromolecules and in signal transduction mediated by hormones and growth factors [11]. Our findings demonstrate that these highly specialized cellular entities can be co-opted by type 1 fimbriated bacteria to gain entry into mast cells as well as other immune cells [14] Model depicting bacteria-induced mobilization of caveolae in bone marrow–derived mast cells. A Specific coupling of a FimH-expressing bacterium to CD48 localized within plasmalemmal caveolae. This triggers movement of vesicular caveolae from an intracellular pool to the site of bacterial attachment. The molecular composition of plasmalemmal caveolae is shown in greater detail in the box. B The recruited caveolae form plasmalemmal extensions around the adherent bacterium. C The plasmalemmal extensions eventually form a distinct intracellular caveolar compartment Whereas the contribution of type 1 fimbriae to bacterial colonization of the bladder mucosae has been definitively demonstrated, their contribution to subsequent stages of the infectious process is unclear. In particular, the nature and outcome of their interactions with the host’s immune cells in the bladder is unknown. We have investigated the interactions of type 1 fimbriated E. coli with mast cells, which are one of the prominent inflammatory cells of the bladder. We have found that instillation of type 1 fimbriated E. coli into the bladder lumen results in the activation of bladder mast cells. Although activation of bladder mast cells can occur without direct contact with bacteria, our data suggest that mast activation is through direct contact with invading bacteria Studies directed at elucidating the molecular aspects of FimH-mediated stimulation of mast cells revealed that CD48, a GPI-anchored cell surface moiety, is the specific FimH receptor on the mouse mast cell. CD48 is specifically found in the plasma membrane of mast cells in cholesterol and glycolipid-rich microdomains called caveolae. Plasmalemmal caveolae are rich in signaling molecules and are conduits for transmembrane signals triggered by many hormones and growth factors. Upon binding of type 1 fimbriated bacteria to mast cell CD48, the mast cell discharges its mediators and concurrently internalizes adherent bacteria within compartments encased by membranes composed of caveolar material. These bacteria-containing caveolar chambers do not fuse with lysosomes of the mast cell, and, thus, the intracellular bacteria are maintained in a viable state Despite our understanding of the early signaling events in the mast cell following binding of type 1 fimbriated E. coli many questions remain unanswered. For example, what ultimately happens to these intracellular bacteria contained in caveolae and what is the relevance of this phenomenon during bladder infection? Examining these questions and elucidating the cellular and molecular processes involved will not only unveil an intriguing cycle of pathogenesis by an opportunistic pathogen but also will provide valuable clues on the contribution of mast cells to the host’s inflammatory response to bacterial infection