Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli.

Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.

Interleukin 8 receptor deficiency confers susceptibility to acute experimental pyelonephritis and may have a human counterpart.

Neutrophils migrate to infected mucosal sites that they protect against invading pathogens. Their interaction with the epithelial barrier is controlled by CXC chemokines and by their receptors. This study examined the change in susceptibility to urinary tract infection (UTI) after deletion of the murine interleukin 8 receptor homologue (mIL-8Rh). Experimental UTIs in control mice stimulated an epithelial chemokine response and increased chemokine receptor expression. Neutrophils migrated through the tissues to the epithelial barrier that they crossed into the lumen, and the mice developed pyuria. In mIL-8Rh knockout (KO) mice, the chemokine response was intact, but the epithelial cells failed to express IL-8R, and neutrophils accumulated in the tissues. The KO mice were unable to clear bacteria from kidneys and bladders and developed bacteremia and symptoms of systemic disease, but control mice were fully resistant to infection. The experimental UTI model demonstrated that IL-8R-dependent mechanisms control the urinary tract defense, and that neutrophils are essential host effector cells. Patients prone to acute pyelonephritis also showed low CXC chemokine receptor 1 expression compared with age-matched controls, suggesting that chemokine receptor expression may also influence the susceptibility to UTIs in humans. The results provide a first molecular clue to disease susceptibility of patients prone to acute pyelonephritis.

Effects of epithelial and neutrophil CXCR2 on innate immunity and resistance to kidney infection.

The murine chemokine receptor 2 (mCXCR2) controls resistance to urinary tract infection. We have previously shown that mCXCR2 knockout mice develop severe acute pyelonephritis and renal tissue damage with sub-epithelial neutrophil entrapment. In this study we examined the relative importance of neutrophil- and epithelial-specific mCXCR2 expression for bacterial clearance in bone marrow chimeric mice infected with uropathogenic Escherichia coli. Mice expressing mCXCR2 on their neutrophils responded rapidly to experimental urinary tract infection, clearing the infection from the kidneys. Mice lacking epithelial mCXCR2, however, showed delayed exit of neutrophils from the tissues. Mice lacking neutrophil mCXCR2 and mice with no mCXCR2 had no neutrophil recruitment and bacterial clearance. Mice expressing mCXCR2 only in their epithelial cells had a transient epithelial chemokine response; however, neutrophil recruitment was inhibited and bacteria grew without constraint. Our study shows that the expression of mCXCR2 on hematopoietic cells was crucial for bacterial clearance, while its expression on non-bone marrow-derived cells influenced the neutrophil response. These results emphasize the importance of mCXCR2 for the innate defense against urinary tract infection.

TLR- and CXCR1-dependent innate immunity: insights into the genetics of urinary tract infections.

The susceptibility to urinary tract infection (UTI) is controlled by the innate immune response and Toll like receptors (TLRs) are the sentinels of this response. If productive, TLR4 signalling may initiate the symptomatic disease process. In the absence of TLR4 signalling the infected host instead develops an asymptomatic carrier state. The activation of mucosal TLR4 is also influenced by the properties of the infecting strain, and pathogens use their virulence factors to trigger 'pathogen-specific' TLR4 responses in the urinary tract but do not respond to the asymptomatic carrier strains in patients with asymptomatic bacteriuria (ABU). The TLR4 dependence has been demonstrated in mice and the relevance of low TLR4 function for protection for human disease was recently confirmed in children with asymptomatic bacteriuria, who expressed less TLR4 than age matched controls. Functional chemokines and functional chemokine receptors are crucial for neutrophil recruitment, and for the neutrophil dependent bacterial clearance. Interleukin (IL)-8 receptor deficient mice develop acute septic infections and chronic tissue damage, due to aberrant neutrophil function. This mechanism is relevant for human UTI as pyelonephritis prone children express low levels of the human CXCL8 (Il-8) receptor, CXC chemokine receptor 1 (CXCR1) and often have heterozygous CXCR1 polymorphisms. This review illustrates how intimately the innate response and the susceptibility to UTI are linked and sophisticated recognition mechanisms that rely on microbial virulence and on host TLR4 and CXCR1 signalling.

Interleukin-8 and the neutrophil response to mucosal gram-negative infection.

Urinary tract infections activate a mucosal inflammatory response, which includes cytokine secretion and neutrophil influx. The mechanisms involved in the neutrophil influx have not been identified. Interleukin-8, a potent chemoattractant for neutrophils, is produced by urinary tract epithelial cell lines in vitro. This study analyzed the human IL-8 response to deliberate Escherichia coli infection of the urinary tract. Urine and serum samples were obtained before and after intravesical instillation of E. coli. Neutrophil numbers were determined on uncentrifuged urine, and IL-8 levels were measured by ELISA. A urinary IL-8 response was found in all patients after bacterial instillation, but no serum IL-8 was detected. There was a strong correlation between urinary IL-8 levels and urinary neutrophil numbers. The same E. coli strains used to colonize the patients stimulated IL-8 production in urinary tract epithelial cells. The level of IL-8 secreted by epithelial cell lines was influenced by the fimbrial properties of the E. coli. These results demonstrated that E. coli elicit a mucosal IL-8 response in humans, and suggested that IL-8 is involved in the onset of pyuria. Epithelial cells may be an important source of IL-8 during urinary tract infection.

Cytokine responses during mucosal infections: role in disease pathogenesis and host defence.

Mucosal pathogens use diverse and highly specific molecular mechanisms to activate mucosal inflammation. It may even be argued that their virulence depends on the inflammatory response that they induce. Some bacteria target epithelial cells and trigger them to produce inflammatory mediators but others cross the mucosa and activate macrophages or dendritic cells. Although systemic release of inflammatory mediators causes many symptoms and signs of infection, local chemokine production leads to the recruitment of inflammatory cells and lymphocytes that participate directly in the clearance of bacteria from mucosal sites. In this way, mucosal inflammation is a two-edged sword responsible for disease associated tissue destruction and crucial for the antimicrobial defence. Understanding of these pathways should create tools to enhance the defence and interfere with disease.

Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

Epithelial cytokine responses and mucosal cytokine networks.

Localized at the border between the external environment and the internal tissue, epithelial cells are exposed to stimulants from two directions. Microorganisms in the lumen can activate the transcription of cytokine mRNA and cytokine secretion, and cytokines in the mucosal environment can modify endogenous and microbially induced epithelial cytokine responses. Epithelial cells thus actively participate in mucosal immunity and inflammation.

Neutrophil recruitment, chemokine receptors, and resistance to mucosal infection.

Neutrophil migration to infected mucosal sites involves a series of complex interactions with molecules in the lamina propria and at the epithelial barrier. Much attention has focussed on the vascular compartment and endothelial cells, but less is known about the molecular determinants of neutrophil behavior in the periphery. We have studied urinary tract infections (UTIs) to determine the events that initiate neutrophil recruitment and interactions of the recruited neutrophils with the mucosal barrier. Bacteria activate a chemokine response in uroepithelial cells, and the chemokine repertoire depends on the bacterial virulence factors and on the specific signaling pathways that they activate. In addition, epithelial chemokine receptor expression is enhanced. Interleukin (IL)-8 and CXCR1 direct neutrophil migration across the epithelial barrier into the lumen. Indeed, mIL-8Rh knockout mice showed impaired transepithelial neutrophil migration, with tissue accumulation of neutrophils, and these mice developed renal scarring. They had a defective antibacterial defense and developed acute pyelonephritis with bacteremia. Low CXCR1 expression was also detected in children with acute pyelonephritis. These results demonstrate that chemokines and chemokine receptors are essential to orchestrate a functional antimicrobial defense of the urinary tract mucosa. Mutational inactivation of the IL-8R caused both acute disease and chronic tissue damage.

Haemophilus influenzae causing conjunctivitis in day-care children.

The role of Haemophilus influenzae in acute purulent conjunctivitis was studied during an outbreak among children in day care. Five day-care centers contributed 20 cases and 35 controls. All the children were subjected to culture of the nasopharynx and the eyes. H. influenzae was carried in the nasopharynx of 53% of the children (range between day care centers, 20 to 91%). Of the 20 children with acute conjunctivitis 8 had eye cultures positive for H. influenzae, 2 had Moraxella and the remaining were culture-negative. Ten colonies of H. influenzae were isolated from each positive culture and identified by capsular type, biotype and multi-locus enzyme electrophoresis. All but one of the isolates were nonencapsulated. They belonged to 4 biotypes and 8 electrophoretic types. The same strain was recovered from the eyes and nasopharynx of the symptomatic children, suggesting that the H. influenzae in the eyes originated from the nasopharynx. There was no evidence for spread of the same H. influenzae strains between day-care centers. Even within each center the Haemophilus strains recovered from the eyes varied among the symptomatic children. The in vitro capacity to attach to oropharyngeal epithelial cells was not increased among the H. influenzae recovered from the eyes. The results question if the majority of conjunctivitis cases were caused by H. influenzae and suggested that eyes were colonized with the nasopharyngeal carrier strain rather than infected by an isolate with special virulence for the eye.

Escherichia coli in patients with renal scarring: genotype and phenotype of Gal alpha 1-4Gal beta-, Forssman- and mannose-specific adhesins.

The frequency of Escherichia coli with Gal alpha 1-4Gal beta-specific adhesins is reduced among children who develop renal scars. The adhesion-negative phenotype may be due to the absence of the pap DNA sequences which encode these adhesins or to a phase variation event induced by in vitro culture. In the present study the frequency of pap and pil homologous DNA was determined by dot blot analysis with probes specific for the respective sequence using E. coli strains from children with recurrent pyelonephritis with and without renal scarring. The frequency of pap was 79% in the strains isolated from the nonscarring group compared with 39% in the strains from the scarring group (P less than 0.001). The Gal alpha 1-4Gal beta phenotype was expressed by 89% of the pap-positive strains from the nonscarring group compared with 71% in the scarring group (P less than 0.05). In addition 13 of 77 of the pap-positive E. coli strains agglutinated sheep erythrocytes but not the Gal alpha 1-4Gal beta latex beads; a reaction attributed to reactivity with the Forssman glycolipid. DNA sequences homologous with pil were found in 95% of all strains and there was no significant difference between the nonscarring and the scarring groups. The low frequency of Gal alpha 1-4Gal beta specific strains in the scarring group was therefore due to the absence of pap-homologous DNA sequences and to a reduced rate of phenotypic expression among pap-positive scarring strains. There was no support for a relationship between type 1 fimbriae and renal scarring.

Bacterial adherence as a virulence factor in urinary tract infection.

Escherichia coli (E. coli) causes greater than 90% of urinary tract infections, UTI, in childhood. The capacity to adhere to urinary tract epithelial cells characterizes E. coli strains that cause acute pyelonephritis. Adherence of uropathogenic E. coli is the result of a specific interaction between bacterial adhesins and glycolipid receptors on the host cells, especially the globoseries of glycolipids which share the Galactose alpha 1-greater than 4Galactose beta disaccharide (Gal alpha 1-greater than 4Gal beta). In childhood UTI, Gal alpha 1-greater than 4Gal beta-binding bacteria caused significantly higher body temperature, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and pyuria, and lower renal concentrating capacity, than E. coli lacking this specificity. The Gal alpha 1-greater than 4Gal beta-binding bacteria thus appeared to be more potent inducers of inflammation than other strains. Since inflammation may lead to tissue damage we examined the relationship of infection with Gal alpha 1-greater than 4Gal beta-positive bacteria to renal scarring. The frequency of renal scarring was 5% in boys with Gal alpha 1-greater than 4Gal beta-positive and 40% in boys with Gal alpha 1-greater than 4Gal beta-negative E. coli. Bacterial binding to Gal alpha 1-greater than 4Gal beta can be detected with a commercially available test reagent. This reagent can thus be used as an effective predictor of risk for renal scarring. Interleukin-6 (IL-6) is a pyrogen and inducer of the acute phase reactants. It was shown to be produced locally in the urinary tract, in response to UTI, and to spread systemically. Mucosal challenge with dead bacteria was sufficient to induce the IL-6 response. Circulating IL-6, and/or IL-1 and tumor necrosis factor could explain the fever, as well as increased ESR and CRP found in association with acute symptomatic UTI.

Nasopharyngeal colonization in Costa Rican children during the first year of life.

BACKGROUND: The establishment of the nasopharyngeal flora was followed in Costa Rican children from birth to 1 year of age. METHODS: Nasopharyngeal cultures were obtained at 1 (n = 413), 3 (n = 393), 6 (n = 376) and 12 months (n = 356) of age from children representative of the population in the Puriscal district. Weekly cultures were obtained from a subcohort of these children (n = 101). Mother-infant diads (n = 95) and preschool children (n = 208) attending day-care centers were also studied. RESULTS: The estimated proportion of colonized children in the population differed markedly depending on the frequency of culture. Quarterly cultures showed a slow increase in carrier rates from 3.9% for Haemophilus influenzae, 3.1% for Streptococcus pneumoniae and 6.5% for Moraxella catarrhalis at 1 month of age to 10.1% carrying H. influenzae and 19.4% carrying S. pneumoniae by the end of the first year. By quarterly culture the proportion of children colonized at least once was 36% for S. pneumoniae, 26% for H. influenzae and 28% for M. catarrhalis. In contrast weekly sampling showed that 95 to 100% of the children were colonized at least once during the first year of life with H. influenzae, S. pneumoniae or M. catarrhalis. Nasopharyngeal carriage of H. influenzae, S. pneumoniae and M. catarrhalis was low in the mothers, and very few mother-infant pairs carried identical bacteria at the same time. In contrast carrier rates were high in the siblings attending day care (H. influenzae 27.9%, S. pneumoniae 39.4%, both organisms 26.6%). Infants with siblings had significantly higher bacterial carriage at all ages than infants without siblings. CONCLUSIONS: Quarterly nasopharyngeal cultures showed that Costa Rican infants acquire their nasopharyngeal flora at a rate comparable with that for infants in developed countries and that siblings are an important source of the bacteria. Weekly samplings showed that virtually all children were colonized at least once during the first year of life.

Interleukin 6 response to urinary tract infection in childhood.

This study analyzed the interleukin 6 (IL-6) response in 114 children with suspected urinary tract infection (UTI). Urine and serum samples were obtained at the time of enrollment. There were 90 children with UTI, 41 with and 49 without a temperature > or = 38.5 degrees C. The remaining 24 children did not have bacteriuria; 11 were febrile and 13 were not. The urinary IL-6 concentrations were higher in the children with UTI (mean, 129 units/ml) than in the children without bacteriuria (mean, 7 units/ml, P < 0.01). In contrast the serum IL-6 did not differ between children with or without UTI or between children with or without a temperature > or = 38.5 degrees C. The urinary IL-6 response was higher in children who were infected with P fimbriated Escherichia coli than in other children with UTI (P < 0.05). There was a correlation of urinary IL-6 with the degree of proteinuria, hematuria and urinary leukocyte counts (P < 0.001, P < 0.05, P < 0.05, respectively) but not with serum IL-6, CRP or temperature, and of serum IL-6 to C-reactive protein (P = 0.053) and renal concentrating capacity (P < 0.05). The results demonstrate that infections of the urinary tract activate an IL-6 response in children and that the magnitude of the IL-6 response is influenced by the properties of the infecting strain.

Antibodies to pneumococcal polysaccharides in human milk: lack of relationship to colonization and acute otitis media.

BACKGROUND: This study analyzed antibodies to pneumococcal polysaccharides in human milk and their effect on nasopharyngeal colonization and acute otitis media in breast-fed infants. METHODS: A total of 503 milk samples were collected from 310 mothers. Nasopharyngeal cultures were obtained from their children at 2, 6 and 10 months postpartum, and the capsular groups/types of the Streptococcus pneumoniae isolates were determined. RESULTS: Types 6A, 6B, 19A, 19F and 23F accounted for 54% of the pneumococcal isolates, but type 3 isolates were uncommon. Milk samples were analyzed for antibody activity to the common capsular polysaccharide types 6A, 19F and 23F; to the type 3 polysaccharide; to C-polysaccharide; and to phosphorylcholine (PC), a major component of the pneumococcal cell wall polysaccharide (CWPS). Anti-capsular antibody activity was low or absent in > 90% of the milk samples. In contrast anti-PC antibody activity was detected in 88% and anti-CWPS in 84% of the samples. The frequency of acute otitis media did not vary with the milk anti-capsular, anti-PC or anti-CWPS antibody activity. CONCLUSIONS: There was no reduction in nasopharyngeal carriage of S. pneumoniae among children fed milk with anti-capsular or anti-PC antibody activity, but carriage was increased in those children who received milk with anti-CWPS antibody activity. A protective role of antipolysaccharide or anti-CWPS antibodies in milk was not detected under the study conditions.

Bacterial adherence and mucosal cytokine production.

1. Uropathogenic E. coli adhere to mucosal sites. 2. In the urinary tract, adherence is followed by inflammation, including a mucosal cytokine response. 3. Bacteria activate epithelial cells to secrete IL-6 and IL-8. IL-6 may cause the fever and acute phase response that accompany systemic urinary tract infections. IL-8 may function as a neutrophil chemoattractant. 4. E. coli up-regulate adhesion molecule expression on epithelial cell lines and neutrophil migration through epithelial cell monolayers. This process is inhibited by antibodies to CD18 and ICAM-1. 5. Cytokines released by nonepithelial cells (T cells and monocytes) modify the epithelial cell cytokine response to bacteria.

Bacterial adherence and mucosal cytokine responses. Receptors and transmembrane signaling.

By attaching to cells or secreted mucosal components, microbes are thought to avoid elimination by the flow of secretions that constantly wash mucosal surfaces. The attached state enhances their ability to trap nutrients and allows the bacteria to multiply more efficiently than do unattached bacterial cells. Attachment is therefore regarded as an end result in itself, and emphasis has been placed on the role of adherence for colonization of mucosal surfaces. Specific adherence was shown to be essential for the tissue tropism that is to guide microbes to their respective sites of colonization/infection. Attachment is not only a mechanism of tissue targeting but also a first step in the pathogenesis of many infections. The attaching bacteria engage in a "cross-talk" with the host cells through the mutual exchange of signals and responses. Enteropathogenic E. coli induce attaching and effacing lesions (Finley et al., this issue). Shigella and Listeria sp. invade the cells and cause actin polymerization (Sansonetti et al., this issue). This review describes the ability of bacteria to trigger mucosal inflammation through activation of cells in the mucosal lining. The results suggest that receptors for bacterial adhesins bind their ligands with a high degree of specificity and that ligand-receptor interactions trigger transmembrane signaling events that cause cell activation. Receptors for microbial ligands thus appear to fulfill also the same criteria as those used to define receptors for other classes of ligands such as hormones, growth factors, and cytokines.

Bacterial virulence in urinary tract infection.

Urinary tract infections (UTI) are caused by a variety of gram-negative bacteria that ascend into the urinary tract and establish bacteriuria often at levels greater than or equal to 10(5) bacteria/ mL of urine. Escherichia coli dominate as the causative agent in all patient groups, with Staphylococcus saprophyticus as the second most common, accounting for about 10% to 30% of the infections in young adult women depending on the season. This article covers the pathogenesis and inflammatory response of UTI and the virulence factors of uropathogenic E. coli.

The host response to urinary tract infection.

The authors use the UTI model to identify basic mechanisms of disease pathogenesis, host response induction, and defense. Their studies hold the promise to provide a molecular and genetic explanation for susceptibility to UTI, and to offer more precise tools for diagnosis and therapy of these infections. There are few infections where the host response is understood in such detail and where pathologic host responses can be linked to distinct disease states. The susceptibility to UTI varies greatly in the population. The studies suggest that distinct molecular defects can cause the clinical entity of acute pyelonephritis with renal scarring, and suggest that the susceptibility to UTI in certain patient groups may have a genetic basis. In addition, the distinct signal transduction pathways explain the development of symptoms, and propose that defects in those signaling mechanisms may occur in patients with ABU. In the future, it may be useful to include these host response parameters in the diagnostic arsenal, to help in early detection of patients susceptible to recurrent UTI and renal scarring. These patients may then be offered therapies that strengthen their defense, and be offered close surveillance for recurrences and other complications.

Interaction of P-fimbriated Escherichia coli with human meconium.

The ability of Escherichia coli with different receptor specificities to interact with meconium was studied. E. coli strains expressing P-fimbriae, specific for Gal alpha 1-4Gal beta-containing receptors, were agglutinated by meconium at high titres. This reaction was inhibited by globotetraosylceramide. The attachment of P-fimbriated E. coli to human colonic epithelial cells of the HT-29 cell line was inhibited by meconium. Some type 1 fimbriated strains were agglutinated by meconium, but the agglutination was rarely blocked by methyl alpha-D-mannoside. The attachment by type 1 fimbriated strains to HT-29 cells was reduced by meconium only in some cases. These results suggest that meconium interacts with the P-fimbriae of E. coli, in a way that may influence bacterial colonization of the neonatal intestine.