Role of fimbriae-mediated adherence for neutrophil migration across Escherichia coli-infected epithelial cell layers

This study examined the role of P and type 1 fimbriae for neutrophil migration across Escherichia coli-infected uroepithelial cell layers in vitro and for neutrophil recruitment to the urinary tract in vivo. Recombinant E. coli K-12 strains differing in P or type 1 fimbrial expression were used to infect confluent epithelial layers on the underside of transwell inserts. Neutrophils were added to the upper well, and their passage across the epithelial cell layers was quantified. Infection with the P- and type 1-fimbriated recombinant E. coli strains stimulated neutrophil migration to the same extent as a fully virulent clinical E. coli isolate, but the isogenic non-fimbriated vector control strains had no stimulatory effect. The enhancement of neutrophil migration was adhesion dependent; it was inhibited by soluble receptor analogues blocking the binding of P fimbriae to the globoseries of glycosphingolipids or of type 1 fimbriae to mannosylated glycoprotein receptors. P- and type 1-fimbriated E. coli triggered higher interleukin (IL) 8 secretion and expression of functional IL-8 receptors than non-fimbriated controls, and the increase in neutrophil migration across infected cell layers was inhibited by anti-IL-8 antibodies. In a mouse infection model, P- or type 1-fimbriated E. coli stimulated higher chemokine (MIP-2) and neutrophil responses than the non-fimbriated vector controls. The results demonstrated that transformation with the pap or fim DNA sequences is sufficient to convert an E. coli K-12 strain to a host response inducer, and that fimbriation enhances neutrophil recruitment in vitro and in vivo. Epithelial chemokine production provides a molecular link between the fimbriated bacteria that adhere to epithelial cells and tissue inflammation.     

Uropathogenic Escherichia coli as a model of host-parasite interaction

Resistance to mucosal infection varies greatly in the population, but the molecular basis of disease susceptibility is often unknown. Studies of host-pathogen infections are helpful to identify virulence factors, which characterise disease isolates, and successful defence strategies of hosts that resist infection. In the urinary tract infection (UTI) model, we have identified crucial steps in the pathogen-activated innate host response, and studied the genetic control of these activation steps. Furthermore, genetic variation in the innate host-response defence is investigated as a basis of disease susceptibility. The Toll-like receptor 4 (TLR4) controls initial mucosal response to uropathogenic Escherichia coli (UPEC). Bacterial TLR4 activation in epithelial cells leads to chemokine secretion and neutrophil recruitment and TLR4 mutant mice develop an asymptomatic carrier state. The chemokine receptor CXCR1 determines the efficiency of neutrophil migration and activation, and thus of bacterial clearance. CXCR1 mutant mice become bacteremic and develop renal scars and studies in UTI prone children have detected low CXCR1 expression, suggesting that CXCR1 is also essential for human disease susceptibility.     

Decreased CXCR1 and CXCR2 expression on neutrophils in anti-neutrophil cytoplasmic autoantibody-associated vasculitides potentially increases neutrophil adhesion and impairs migration

Introduction

In anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), persistent inflammation within the vessel wall suggests perturbed neutrophil trafficking leading to accumulation of activated neutrophils in the microvascular compartment. CXCR1 and CXCR2, being major chemokine receptors on neutrophils, are largely responsible for neutrophil recruitment. We speculate that down-regulated expression of CXCR1/2 retains neutrophils within the vessel wall and, consequently, leads to vessel damage.

Methods

Membrane expression of CXCR1/2 on neutrophils was assessed by flow cytometry. Serum levels of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), angiopoietin 1 and angiopoietin 2 from quiescent and active AAV patients and healthy controls (HC) were quantified by ELISA. Adhesion and transendothelial migration of isolated neutrophils were analyzed using adhesion assays and Transwell systems, respectively.

Results

Expression of CXCR1 and CXCR2 on neutrophils was significantly decreased in AAV patients compared to HC. Levels of IL-8, which, as TNFα, dose-dependently down-regulated CXCR1 and CXCR2 expression on neutrophils in vitro, were significantly increased in the serum of patients with active AAV and correlated negatively with CXCR1/CXCR2 expression on neutrophils, even in quiescent patients. Blocking CXCR1 and CXCR2 with repertaxin increased neutrophil adhesion and inhibited migration through a glomerular endothelial cell layer.

Conclusions

Expression of CXCR1 and CXCR2 is decreased in AAV, potentially induced by circulating proinflammatory cytokines such as IL-8. Down-regulation of these chemokine receptors could increase neutrophil adhesion and impair its migration through the glomerular endothelium, contributing to neutrophil accumulation and, in concert with ANCA, persistent inflammation within the vessel wall.     

Macrophage inflammatory protein-2 is required for neutrophil passage across the epithelial barrier of the infected urinary tract

IL-8 is a major human neutrophil chemoattractant at mucosal infection sites. This study examined the C-X-C chemokine response to mucosal infection, and, specifically, the role of macrophage inflammatory protein (MIP)-2, one of the mouse IL-8 equivalents, for neutrophil-epithelial interactions. Following intravesical Escherichia coli infection, several C-X-C chemokines were secreted into the urine, but only MIP-2 concentrations correlated to neutrophil numbers. Tissue quantitation demonstrated that kidney MIP-2 production was triggered by infection, and immunohistochemistry identified the kidney epithelium as a main source of MIP-2. Treatment with anti-MIP-2 Ab reduced the urine neutrophil numbers, but the mice had normal tissue neutrophil levels. By immunohistochemistry, the neutrophils were found in aggregates under the pelvic epithelium, but in control mice the neutrophils crossed the urothelium into the urine. The results demonstrate that different chemokines direct neutrophil migration from the bloodstream to the lamina propria and across the epithelium and that MIP-2 serves the latter function. These findings suggest that neutrophils cross epithelial cell barriers in a highly regulated manner in response to chemokines elaborated at this site. This is yet another mechanism that defines the mucosal compartment and differentiates the local from the systemic host response.     

Expression and functional analysis of chemokine receptors in human peripheral blood leukocyte populations

Emerging evidence indicates that chemokine receptor expression patterns are critical in determining the spectrum of action of the chemokines. We have analysed the expression patterns of 17 chemokine receptors and two orphan chemokine receptor-like genes in various freshly prepared human peripheral blood leucocyte populations, including neutrophils, lymphocytes, and na?ve and differentiated monocytes using real-time quantitative polymerase chain reaction (TaqMan). This is the first comprehensive study of chemokine receptor expression in such a wide variety of cell types. Human peripheral blood leukocyte populations were found to express a wide range of chemokine receptors that varies depending on cell type and differentiation state. Novel expression patterns of certain chemokine receptors were seen during our analysis. For example, the orphan chemokine receptor HCR was expressed at very high levels by both primary neutrophils and primary monocytes, and was further upregulated on neutrophil activation and during monocyte to macrophage differentiation. When neutrophil calcium transients were measured in response to a panel of 30 different chemokines the results clearly correlated with the chemokine receptor expression profile. For example strong calcium responses were seen in neutrophils following stimulation with the CXCR1 and CXCR2 ligands, interleukin (IL-)8, GCP-2 and Gro-beta. These data have implications for the study of the functional responses of leukocytes to external stimuli and will aid in our understanding of general leukocyte biology.     

CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity

Intestinal epithelial cell migration plays a key role in gastrointestinal mucosal barrier formation, enterocyte development, differentiation, turnover, wound healing, and adenocarcinoma metastasis. Chemokines, through engagement of their corresponding receptors, are potent mediators of directed cell migration and are critical in the establishment and regulation of innate and adaptive immune responses. The aim of this study was to define the role for the chemokine CXCL12 and its sole cognate receptor CXCR4 in regulating intestinal epithelial cell migration and to determine its impact on barrier integrity. CXCL12 stimulated the dose-dependent chemotactic migration of human T84 colonic epithelial cells. Epithelial cell migration was inhibited by CXCR4 neutralizing antibody, pertussis toxin, LY-294002, and PD-98059, thereby implicating Galpha(i), phosphatidylinositol 3-kinase (PI3-kinase), and the ERK1/2 MAP kinase pathways in CXCR4-specific signaling. CXCL12 was also shown to increase barrier integrity, as defined by transepithelial resistance and paracellular flux across differentiating T84 monolayers. To determine whether CXCL12 regulated epithelial restitution, we used the normal nontransformed intestinal epithelial cell-6 (IEC-6) wound healing model. By using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, we first showed expression of both CXCR4 and its ligand by IEC-6 cells. We then demonstrated that CXCL12 activated comparable signaling mechanisms to stimulate epithelial migration in the absence of proliferation in wounded IEC-6 monolayers. Taken together, these data indicate that CXCL12 signaling via CXCR4 directs intestinal epithelial cell migration, barrier maturation, and restitution, consistent with an important mechanistic role for these molecules in mucosal barrier integrity and innate host defense.     

CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1beta or TNF-alpha. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1alpha (SDF-1alpha) indicated that the CXCR4 receptors were functional. Incubation with SDF-1alpha resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene alpha. RPE cells also migrated in response to SDF-1alpha. As SDF-1alpha expression by RPE cells was detected constitutively, we postulate that SDF-1-CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.     

Molecular cloning and genomic structure of an interleukin-8 receptor-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Chemokines are small proteins (70-100 amino acids) which play an important role in recruitment and activation of leucocytes to migrate to the site of inflammation. Based on the position of the first two conserved cysteines, chemokines are classified into four subfamilies: C, CC, CXC and CX3C. To date, many members of CC and CXC have been found and studied extensively [1]. Chemokines exert effects on their target cell via chemokine receptors, which are G-protein coupled receptors containing seven transmembrane domains with an extracellular N-terminus and an intracellular C-terminus [2]. Interleukin 8 (IL-8) belongs to the CXC chemokine subfamily. It can activate and attract migratory neutrophils to an inflammation site. Two IL-8 receptors, CXCR1 and CXCR2, have been identified in mammals [3-6]; both of these receptors have high affinity for IL-8 and are expressed on the neutrophil. CXCR1 just binds IL-8; however, CXCR2 binds IL-8 and other structurally related chemokines such as growth-related oncogene (GRO) a, GRObeta, GROgamma, neutrophil-activating peptide-2 (NAP-2) and epithelial cell-derived neutrophil activating peptide-78 (ENA-78) [7, 8]. Several studies on fish chemokine receptors have been reported [9-11]. Thus far, however, IL-8 and CXCR1 and CXCR2 proteins from rainbow trout have not been reported: however, the sequence of a rainbow trout IL-8 has been noted (GenBank Accession No. AJ279069 [12]). Cloning of the IL-8 receptor is important to study the function of IL-8/CXCR1 and (CXCR2) in inflammation and signal transduction in fish. This paper reports the molecular cloning and genomic structure of an IL-8 receptor-like gene from four homozygous clones of rainbow trout: Oregon State University (OSU), Hot Creek (HC), Arlee (AR) and Swanson (SW).     

CXC chemokine receptor 2 but not C-C chemokine receptor 1 expression is essential for neutrophil recruitment to the cornea in helminth-mediated keratitis (river blindness)

Infiltration of neutrophils and eosinophils into the mammalian cornea can result in loss of corneal clarity and severe visual impairment. To identify mediators of granulocyte recruitment to the corneal stroma, we determined the relative contribution of chemokine receptors CXC chemokine receptor (CXCR)-2 (IL-8R homologue) and CCR1 using a murine model of ocular onchocerciasis (river blindness) in which neutrophils and eosinophils migrate from peripheral vessels to the central cornea. CXCR2(-/-) and CCR1(-/-) mice were immunized s.c. and injected into the corneal stroma with Ags from the parasitic helminth Onchocerca volvulus. We found that production of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1 alpha was localized to the corneal stroma, rather than to the epithelium, which was consistent with the location of neutrophils in the cornea. CCR1 deficiency did not inhibit neutrophil or eosinophil infiltration to the cornea or development of corneal opacification. In marked contrast, neutrophil recruitment to the corneas of CXCR2(-/-) mice was significantly impaired (p < 0.0001 compared with control, BALB/c mice) with only occasional neutrophils detected in the central cornea. Furthermore, CXCR2(-/-) mice developed only mild corneal opacification compared with BALB/c mice. These differences were not due to impaired KC and MIP-2 production in the corneal stroma of CXCR2(-/-) mice, which was similar to BALB/c mice. Furthermore, although MIP-1 alpha production was lower in CXCR2(-/-) mice than BALB/c mice, eosinophil recruitment to the cornea was not impaired. These observations demonstrate the critical role for CXCR2 expression in neutrophil infiltration to the cornea and may indicate a target for immune intervention in neutrophil-mediated corneal inflammation.     

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.     

CXCR2 is critical to hyperoxia-induced lung injury

Hyperoxia-induced lung injury is characterized by infiltration of activated neutrophils in conjunction with endothelial and epithelial cell injury, followed by fibrogenesis. Specific mechanisms recruiting neutrophils to the lung during hyperoxia-induced lung injury have not been fully elucidated. Because CXCL1 and CXCL2/3, acting through CXCR2, are potent neutrophil chemoattractants, we investigated their role in mediating hyperoxia-induced lung injury. Under variable concentrations of oxygen, murine survival during hyperoxia-induced lung injury was dose dependent. Eighty percent oxygen was associated with 50% mortality at 6 days, while greater oxygen concentrations were more lethal. Using 80% oxygen, we found that lungs harvested at day 6 demonstrated markedly increased neutrophil sequestration and lung injury. Expression of CXCR2 ligands paralleled neutrophil recruitment to the lung and CXCR2 mRNA expression. Inhibition of CXC chemokine ligands/CXCR2 interaction using CXCR2(-/-) mice exposed to hyperoxia significantly reduced neutrophil sequestration and lung injury, and led to a significant survival advantage as compared with CXCR2(+/+) mice. These findings demonstrate that CXC chemokine ligand/CXCR2 biological axis is critical during the pathogenesis of hyperoxia-induced lung injury.     

Decreased neutrophil adhesion to human cytomegalovirus-infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas ligand independent of neutrophil apoptosis

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro alpha and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.     

Differential calcium signaling in dairy cows with specific CXCR1 genotypes potentially related to interleukin-8 receptor functionality

Neutrophil migration and activation are critical components of innate immunity and are mediated by a variety of inflammatory mediators, which include interleukin-8 (IL-8) and epithelial-derived neutrophil activating peptide-78 (ENA-78). Limited knowledge on the expression of receptors for these inflammatory mediators (CXCR1 and CXCR2) in bovine, in addition to the association of a polymorphism (G→C) in position +777 of the CXCR1 gene with impaired neutrophil function, prompted evaluation of CXCR1 and CXCR2 mRNA and protein expression, ligand binding affinity, and intracellular receptor signaling in neutrophils from cows with different CXCR1 genotypes. Initial observations revealed that overall IL-8 receptor numbers appeared to be lower in cows with a CC genotype compared to cows with a GG genotype. However, in the presence of SB225002, a CXCR2 inhibitor, CXCR1 affinity was about fivefold lower in cows with a CC genotype and may have resulted in an underestimation of receptor numbers in cows with this genotype. In addition, intracellular calcium ([Ca⁺⁺]_i) release was lower in cows with a CC genotype when cells were stimulated with IL-8 but not ENA-78. Furthermore, when neutrophils were stimulated with an optimal dose of IL-8 in the presence of SB225002, [Ca⁺⁺]_i release was lower in cows with a CC genotype, suggesting differential CXCR1 signaling among genotypes. These findings offer knowledge of the role that each of these receptors plays in the inflammatory response in the bovine and provide insight into the potential mechanisms that may be affected in neutrophils of cows with different CXCR1 genotypes.     

Constitutive expression of stromal derived factor-1 by mucosal epithelia and its role in HIV transmission and propagation

HIV particles that use the chemokine receptor CXCR4 as a coreceptor for entry into cells (X4-HIV) inefficiently transmit infection across mucosal surfaces [1], despite their presence in seminal fluid and mucosal secretions from infected individuals [2] [3] [4]. In addition, although intestinal lymphocytes are susceptible to infection with either X4-HIV particles or particles that use the chemokine receptor CCR5 for viral entry (R5-HIV) during ex vivo culture [5], only systemic inoculation of R5-chimeric simian-HIV (S-HIV) results in a rapid loss of CD4(+) intestinal lymphocytes in macaques [6]. The mechanisms underlying the inefficient capacity of X4-HIV to transmit infection across mucosal surfaces and to infect intestinal lymphocytes in vivo have remained elusive. The CCR5 ligands RANTES, MIP-1alpha and MIP-1beta suppress infection by R5-HIV-1 particles via induction of CCR5 internalization, and individuals whose peripheral blood lymphocytes produce high levels of these chemokines are relatively resistant to infection [7] [8] [9]. Here, we show that the CXCR4 ligand stromal derived factor-1 (SDF-1) is constitutively expressed by mucosal epithelial cells at sites of HIV transmission and propagation. Furthermore, CXCR4 is selectively downmodulated on intestinal lymphocytes within the setting of prominent SDF-1 expression. We postulate that mucosally derived SDF-1 continuously downmodulates CXCR4 on resident HIV target cells, thereby reducing the transmission and propagation of X4-HIV at mucosal sites. Moreover, such a mechanism could contribute to the delayed emergence of X4 isolates, which predominantly occurs during the later stages of the HIV infection.     

CXCR2 deficiency confers impaired neutrophil recruitment and increased susceptibility during Toxoplasma gondii infection.

Neutrophil migration to the site of infection is a critical early step in host immunity to microbial pathogens, in which chemokines and their receptors play an important role. In this work, mice deficient in expression of the chemokine receptor CXCR2 were infected with Toxoplasma gondii and the outcome was monitored. Gene-deleted animals displayed completely defective neutrophil recruitment, which was apparent at 4 h and sustained for at least 36 h. Kit(W)/Kit(W-v) animals also displayed defective polymorphonuclear leukocyte migration, suggesting mast cells as one source of chemokines driving the response. Tachyzoite infection and replication were accelerated in CXCR2(-/-) animals, resulting in establishment of higher cyst numbers in the brain relative to wild-type controls. Furthermore, serum and spleen cell IFN-gamma levels in infected, gene-deleted mice were reduced 60-75% relative to infected normal animals, and spleen cell TNF-alpha was likewise reduced by approximately 50%. These results highlight an important role for CXCR2 in neutrophil migration, which may be important for early control of infection and induction of immunity during Toxoplasma infection.     

Staphylococcus aureus Staphopain A inhibits CXCR2-dependent neutrophil activation and chemotaxis

The CXC chemokine receptor 2 (CXCR2) on neutrophils, which recognizes chemokines produced at the site of infection, plays an important role in antimicrobial host defenses such as neutrophil activation and chemotaxis. Staphylococcus aureus is a successful human pathogen secreting a number of proteolytic enzymes, but their influence on the host immune system is not well understood. Here, we identify the cysteine protease Staphopain A as a chemokine receptor blocker. Neutrophils treated with Staphopain A are unresponsive to activation by all unique CXCR2 chemokines due to cleavage of the N-terminal domain, which can be neutralized by specific protease inhibitors. Moreover, Staphopain A inhibits neutrophil migration towards CXCR2 chemokines. By comparing a methicillin-resistant S. aureus (MRSA) strain with an isogenic Staphopain A mutant, we demonstrate that Staphopain A is the only secreted protease with activity towards CXCR2. Although the inability to cleave murine CXCR2 limits in-vivo studies, our data indicate that Staphopain A is an important immunomodulatory protein that blocks neutrophil recruitment by specific cleavage of the N-terminal domain of human CXCR2.     

P-fimbriae trigger mucosal responses to Escherichia coli in the human urinary tract

Uropathogenic Escherichia coli elicit a host response that determines the severity of urinary tract infection (UTI). Specific adherence mechanisms allow the bacteria to initiate this process by targeting epithelial cells in the urinary tract mucosa. Epidemiological studies show a strong association of P-fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo . The present study examined the ability of P-fimbriae to induce inflammation in the human urinary tract. Patients were subjected to intravesical inoculation with a non-fimbriated E. coli strain or transformants of this strain expressing P-fimbriae. The inflammatory response was analysed as a function of P-fimbrial expression. The P-fimbriated transformants invariably caused higher interleukin (IL)-8, IL-6 and neutrophil responses in the urinary tract than the ABU strain. Furthermore, loss of P-fimbrial expression in vivo was accompanied by a return to background levels of neutrophils, IL-6 and IL-8 in individual patients. The results demonstrate that the pap sequences confer on a non-fimbriated, avirulent strain the ability to induce a host response in the human urinary tract. P-fimbriae thus fulfil the 'molecular Koch–Henle postulates' linking a single virulence factor to host response induction.     

Milk fat globule-epidermal growth factor-factor 8 attenuates neutrophil infiltration in acute lung injury via modulation of CXCR2

Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule epidermal growth factor-factor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and Mfge8(-/-) mice were intratracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology, and the neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, whereas caspase-3 and myeloperoxidase activities were assessed spectrophotometrically. CXCR2 and G protein-coupled receptor kinase 2 expressions in neutrophils were measured by flow cytometry. Following LPS challenge, Mfge8(-/-) mice exhibited extensive lung damage due to exaggerated infiltration of neutrophils and production of TNF-α, MIP-2, and myeloperoxidase. An increased number of apoptotic cells was trapped into the lungs of Mfge8(-/-) mice compared with WT mice, which may be due to insufficient phagocytosis of apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2-mediated chemotaxis revealed higher migration of neutrophils of Mfge8(-/-) mice than those of WT mice via increased surface exposures to CXCR2. Administration of recombinant murine MFG-E8 reduces neutrophil migration through upregulation of GRK2 and downregulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-α(v) integrin Abs. These studies clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic potential for ALI.