Microbiota Promotes Chronic Pulmonary Inflammation by Enhancing IL-17A and Autoantibodies

RATIONALE Changes in the pulmonary microbiota are associated with progressive respiratory diseases including chronic obstructive pulmonary disease. Whether there is a causal relationship between these changes and disease progression remains unknown. OBJECTIVE To investigate the link between an altered microbiota and disease, we utilized a model of chronic lung inflammation in specific pathogen free (SPF) mice and mice depleted of microbiota by antibiotic treatment or devoid of a microbiota (axenic). METHODS Mice were challenged with LPS/elastase intranasally over 4 weeks, resulting in a chronically inflamed and damaged lung. The ensuing cellular infiltration, histological damage and decline in lung function were quantified. MEASUREMENTS AND MAIN RESULTS Similar to human disease, the composition of the pulmonary microbiota was altered in disease animals. We found that the microbiota richness and diversity were decreased in LPS/Elastase-treated mice, with an increased representation of the genera Pseudomonas, Lactobacillus and a reduction in Prevotella. Moreover, the microbiota was implicated in disease development as mice depleted of microbiota exhibited an improvement in lung function, reduction in airway inflammation, decrease in lymphoid neogenesis and auto-reactive antibody responses. The absence of microbial cues also markedly decreased the production of IL-17A, whilst intranasal transfer of fluid enriched with the pulmonary microbiota isolated from diseased mice enhanced IL-17A production in the lungs of antibiotic treated or axenic recipients. Finally, in mice harboring a microbiota, neutralizing IL-17A dampened inflammation and restored lung function. CONCLUSIONS Collectively, our data indicate that host-microbial cross-talk promotes inflammation and could underlie the chronicity of inflammatory lung diseases.

The RNA binding proteins RBM38 and DND1 are repressed in AML and have a novel function in APL differentiation

The RNA binding proteins RBM binding motif protein 38 (RBM38) and DEAD END 1 (DND1) selectively stabilize mRNAs by attenuating RNAse activity or protecting them from micro(mi)RNA-mediated cleavage. Furthermore, both proteins can efficiently stabilize the mRNA of the cell cycle inhibitor p21(CIP1). Since acute myeloid leukemia (AML) differentiation requires cell cycle arrest and RBM38 as well as DND1 have antiproliferative functions, we hypothesized that decreased RBM38 and DND1 expression may contribute to the differentiation block seen in this disease. We first quantified RBM38 and DND1 mRNA expression in clinical AML patient samples and CD34(+) progenitor cells and mature granulocytes from healthy donors. We found significantly lower RBM38 and DND1 mRNA levels in AML blasts and CD34(+) progenitor cells as compared to mature neutrophils from healthy donors. Furthermore, the lowest expression of both RBM38 and DND1 mRNA correlated with t(8;21). In addition, neutrophil differentiation of CD34(+) cells in vitro with G-CSF (granulocyte colony stimulating factor) resulted in a significant increase of RBM38 and DND1 mRNA levels. Similarly, neutrophil differentiation of NB4 acute promyelocytic leukemia (APL) cells was associated with a significant induction of RBM38 and DND1 expression. To address the function of RBM38 and DND1 in neutrophil differentiation, we generated two independent NB4RBM38 as well as DND1 knockdown cell lines. Inhibition of both RBM38 and DND1 mRNA significantly attenuated NB4 differentiation and resulted in decreased p21(CIP1) mRNA expression. Our results clearly indicate that expression of the RNA binding proteins RBM38 and DND1 is repressed in primary AML patients, that neutrophil differentiation is dependent on increased expression of both proteins, and that these proteins have a critical role in regulating p21(CIP1) expression during APL differentiation.

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection

Background: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. Methods: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox−/− ) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. Results: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox−/− mice, indicating a larger inflammatory response in gp91phox−/− even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox−/− at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. Conclusion: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

Identification and characterization of antigens and potential virulence factors in Enterococcus

Enterococci are normal flora in the human intestinal tract, and also one of the leading causes of nosocomial infections, with most of the clinical isolates being Enterococcus faecalis and Enterococcus faecium. Despite extensive studies on the antibiotic resistance, the pathogenicity of enterococci is not well understood, especially for E. faecium. To identify potential virulence factors based on their antigenicity during infection, E. faecium genomic libraries were constructed and screened using sera from patients with E. faecium endocarditis. ^ As one of my projects, total polysaccharides were extracted from E. faecalis OG1RF and from two epa mutants constructed previously, TX5179 and TX5180, and western blots with patient sera showed that an immuno-reactive polysaccharide present in wild type OG1RF was not produced by either of the two epa mutants. The epa mutants were more sensitive to ethanol stress, neutrophil killing and neutrophil phagocytosis than the wild type OG1RF. ^ Expression of virulence factors is commonly regulated by two component systems. A BLAST search was performed to identify potential two component systems in the E. faecalis V583 genome database using PhoP/PhoS as query sequences, and 11 gene pairs were identified, seven of which were disrupted in E. faecalis OGIRF. ^ Finally, an in vitro translocation model was established for enterococci. E. faecalis strain OG1RF and E. faecium strain DO were shown to be able to translocate across a T84 monolayer, while E. coli strain DH5α and E. faecalis strain E1 could not. ^ In conclusion, several E. faecium antigens expressed in infection (whose antibodies present in sera from patients with E. faecium endocarditis) were identified, two of which, SagA and GlyA, were characterized and suggested to be involved in cell wall metabolism. E. faecalis epa gene cluster (involving in polysaccharide biosynthesis and known to be involved in virulence of E. faecalis in mice) was shown to be involved in hindering neutrophil killing. Several two-component systems were identified in E. faecalis and two of which, EtaRS and EtbRS, were involved in E. faecalis virulence in a mouse peritonitis model.^

Molecular mechanisms of signal transducer and activator of transcription (STAT) protein function in hematopoiesis

Hematopoietic growth factors play important roles in regulating blood cell growth and development in vivo. In this work, we investigated the signaling mechanisms of two growth factors with clinical significance, erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF). Epo is essential for the survival, proliferation and differentiation of red blood cell progenitors, while G-CSF plays an important role in controlling mature neutrophil production. To identify which amino acid(s) and/or motif in EpoR is responsible for cell survival, wild type or mutant EpoR isoforms were transfected into the growth factor-dependent 32D cell line. Proliferation and apoptosis assays demonstrated that an EpoR isoform that lacks intracellular tyrosine residues and is truncated after 321 amino acids in the cytoplasmic tail (EpoR 1-321) mediates Epo-dependent cell survival. Furthermore, in absence of fetal calf serum (FCS), Epo signaling through wild type or mutant receptors supported anti-apoptosis, but not proliferation during 72 hours in response to Epo. To investigate the signaling pathway by which EpoR regulates cell survival, a dominant negative Stat5b (dnStat5b) isoform was generated and coexpressed with EpoR in stable cell lines. Expression of dnStat5b causes a significant induction of apoptosis in the presence of Epo in cells expressing EpoR 1-321, indicating that Stat5 is essential for survival signaling through tyrosine independent sequences in the EpoR. In a second project to investigate G-CSF signaling, we studied mechanisms by which G-CSF regulates the expression of PU.1, an important transcription factor in myeloid and B cell development. We demonstrated, by immunoblot and real time RT-PCR, that PU.1 is induced by G-CSF ex vivo as well as in vivo. To test whether G-CSF signaling through Stat3 is required for PU.1 regulation, the upstream region of the PU.1 gene was analyzed for potential Stat3 binding motifs. Four potential sites were identified; chromatin immunoprecipitations demonstrated that G-CSF activated Stat3 binds to 3 of the 4 binding motifs. In addition, PU.1 induction by G-CSF was completely abrogated in bone marrow from hematopoietic conditional Stat3 knockout mice. These results indicate an important role for Stat3 in G-CSF-dependent PU.1 gene regulation. Collectively, our works demonstrate that Stat protein play important and diverse roles in hematopoietic growth factor signaling. ^

Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

Effects of Estrogen on Muscle Damage in Response to an Acute Resistance Exercise Protocol

Creatine Kinase (CK) is used as a measure of exercise-induced muscle membrane damage. During acute eccentric (muscle lengthening) exercise, muscle sarcolemma, sarcoplasmic reticulum, and Z-lines are damaged, thus causing muscle proteins and enzymes to leak into the interstitial fluid. Strenuous eccentric exercise produces an elevation of oxygen free radicals, which further increases muscle damage. Muscle soreness and fatigue can be attributed to this membrane damage. Estradiol, however, may preserve membrane stability post-exercise (Brancaccio, Maffulli, & Limongelli, 2007; Carter, Dobridge, & Hackney, 2001; Tiidus, 2001). Because estradiol has a similar structure to Vitamin E, which is known to have antioxidant properties, and both are known to affect membrane structure, researchers have proposed that estrogen acts as an antioxidant to provide a protective effect on the post-exercise muscle of women (Sandoval & Matt, 2002). As a result, it has been postulated that muscles in women incur less damage in response to an acute strenuous exercise as compared to men. PURPOSE: To determine if circulating estrogen concentrations are related to muscle damage, as measured by creatine kinase activity and to determine gender differences in creatine kinase as a marker of muscle damage in response to an acute heavy resistance exercise protocol. METHODS: 7 healthy, resistance-trained, eumenhorrheic women (23±3 y, 169±9.1 cm, 66.4±10.5 kg) and 8 healthy, resistance-trained men (25±5 y, 178±6.7 cm, 82.3±9.33 kg) volunteered to participate in the study. Subjects performed an Acute Resistance Exercise Test (ARET) consisting of 6 sets of 5 repetitions Smith machine squats at 90% of their previously determined 1-RM. Blood samples were taken pre-, mid-, post-, 1 hour post-, 6 hours post-, and 24 hours post-exercise. Samples were stored at -80ºC until analyzed. Serum creatine kinase was measured using an assay kit from Genzyme (Framingham, MA). Serum estradiol was measured by an ELISA from GenWay (San Diego, CA). Estradiol b-receptor presence on granulocytes was measured via flow cytometry using primary antibodies from Abcam (Cambridge, MA) and PeCy7 antibodies (secondary) from Santa Cruz (Santa Cruz, CA). RESULTS: No significant correlations between estrogen and CK response were found after an acute resistant exercise protocol. Moreover, no significant change in estradiol receptors were expressed on granulocytes after exercise. Creatine Kinase response, however, differed significantly between genders. Men had higher resting CK concentrations throughout all time points. Creatine Kinase response increased significantly after exercise in both men and women (p=0.008, F=9.798). Men had a significantly higher CK response at 24 hours post exercise than women. A significant condition/sex/time interaction was exhibited in CK response (p=0.02, F=4.547). Perceived general soreness presented a significant condition, sex interaction (p=0.01, F=9.532). DISCUSSION: Although no estradiol and CK response correlations were found in response to exercise, a significant difference in creatine kinase activity was present between men and women. This discrepancy of our results and findings in the literature may be due to the high variability between subjects in creatine kinase activity as well as estrogen concentrations. The lack of significance in change of estradiol receptor expression on granulocytes in response to exercise may be due to intracellular estradiol receptor staining and non-specific gating for granulocytes rather than additional staining for neutrophil markers. Because neutrophils are the initial cells present in the inflammatory response after strenuous exercise, staining for estrogen receptors on this cell type may allow for a better understanding of the effect of estrogen and its hypothesized protective effect against muscle damage. Furthermore, the mechanism of action may include estradiol receptor expression on the muscle fiber itself may play a role in the protective effects of estradiol rather than or in addition to expression on neutrophils. We have shown here that gender differences occur in CK activity as a marker of muscle damage in response to strenuous eccentric exercise, but may not be the result of estradiol concentration or estradiol receptor expression on granulocytes. Other variables should be examined in order to determine the mechanism involved in the difference in creatine kinase as a marker of muscle damage between men and women after heavy resistance exercise.

REGULATION OF CYTOSKELETAL ASSEMBLY: A STUDY OF THE INTERACTION OF FILAMIN AND F-ACTIN

Filamin is a high molecular weight (2 x 250,000) actin crosslinking protein found in a wide variety of cells and tissues. The most striking feature of filamin is its ability to crosslink F-actin filaments and cause ATP-independent gelation and contraction of F-actin solutions. The gelation of actin filaments by filamin involves binding to actin and crosslinking of the filaments by filamin self-association. In order to understand the role of filamin-actin interactions in the regulation of cytoskeletal assembly, two approaches were used. First, the structural relationship between self-association and actin-binding was examined using proteolytic fragments of filamin. Treatment of filamin with papain generated two major fragments, 90Kd and 180Kd. Upon incubation of the papain digest with F-actin and centrifugation at 100,000 x g, only the 180Kd fragment co-sedimented with F-actin. The binding of the 180Kd fragment, P180, was similar to native filamin in its sensitivity to ionic strength. Analytical gel filtration studies indicated that, unlike native filamin, P180 was monomeric and did not self-associate. Thermolysin treatment of P180 produced a 170Kd fragment, PT170, which no longer bound and co-sedimented with F-actin. These results suggested that filamin contained a discrete actin-binding domain. In order to locate the actin-binding domain, affinity purified antibodies to the papain and thermolysin sensitive regions of filamin were used in conjunction with filamin fragments generated by digestion with S. aureus V8 protease and elastase. The results indicated that the papain and thermolysin cleavage sites were close together, and, most likely, within 10Kd of one another. Taken together, these data suggest that filamin contains a discrete, internal actin-binding domain. The second approach was to use the non-crosslinking fragment P180 to develop a quantitative assay of filamin-actin binding. The binding of ('14)C-carboxyalkylated P180 was examined using the co-sedimentation assay. ('14)C-P180 binding to actin was equivalent to that of unlabelled P180 and exhibited comparable sensitivity of binding to changes in ionic strength. Within 5 min. of incubation the process had reached equilibrium. The specificity of binding was shown by the lack of binding of ('14)C-PT170. The binding of ('14)C-P180 was found to be a reversible and saturable process, with a K(,d) of 2 x 10('-7) M. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Clinical decisions related to CBC screening of asymptomatic full-term infants at risk for group B streptococcus disease

Objective. Although complete blood count (CBC) changes occur with the development of clinical sepsis in newborns, the CBC has not been reported to be a sensitive predictor of sepsis in asymptomatic full-term newborn infants, nor has it been reported to be related to risk factors for sepsis or clinical decisions. The objective of this study was to evaluate the relationship between the WBC/I:T (immature:total neutrophil) ratio and maternal group B streptococcal (GBS) risk factors (rupture of membranes ≥18 hours, maternal temperature ≥100.4°F, maternal age ≤20 years, previous infant with invasive GBS disease, maternal GBS bacteriuria, and black ethnicity); and to evaluate the relationship between the WBC/I:T ratios and providers' clinical decisions (observe versus repeat the CBC or complete sepsis evaluation) in the asymptomatic full-term newborn at risk for early-onset GBS sepsis. ^ Methods. Medical records of infants admitted to the well baby nursery at a tertiary care teaching hospital in Houston, TX between 1/1/99 and 12/31/00 whose gestational ages were ≥35 weeks; who had mothers with GBS positive or unknown culture status and inadequate intrapartum antibiotic prophylaxis; and who had screening CBCs performed in the first 30 hours of life because of GBS risk were reviewed (n = 412). Demographic information, maternal GBS risk factors, CBC results, clinical decisions, and rationales for clinical decisions were collected. ^ Results. With the exception of black ethnicity (p = .0000, odds ratio = 0.213), no statistically significant differences in risk factors between infants with normal and abnormal WBC counts or normal and abnormal I:T ratios were found. Infants with abnormal WBCs had a significantly higher likelihood of having a CBC repeated (p = 0.002 for WBC). Providers documented the CBC result in the rationale for clinical decisions in 62% of the cases. ^ Conclusion. The CBC results were not related to maternal risk factors for GBS except for ethnicity. Black infants had significantly lower WBC levels than infants of other ethnicities, although this difference was clinically insignificant. Infants with abnormal WBCs had a significantly higher likelihood of undergoing repeat CBCs but not sepsis evaluations. Provider rationale was difficult to evaluate due to insufficient documentation. The screening CBC result did not impact the clinicians' decisions to initiate sepsis evaluations in this population. ^

Involvement of lipocalin 2 in leukemia and breast cancer

Human lipocalin 2 is described as the neutrophil gelatinase-associated lipocalin (NGAL). The lipocalin 2 gene encodes a small, secreted glycoprotein that possesses a variety of functions, of which the best characterized function is organic iron binding activity. Elevated NGAL expression has been observed in many human cancers including breast, colorectal, pancreatic and ovarian cancers. I focused on the characterization of NGAL function in chronic myelogenous leukemia (CML) and breast cancer. Using the leukemic xenograft mouse model, we demonstrated that over-expression of NGAL in K562 cells, a leukemic cell line, led to a higher apoptotic rate and an atrophy phenotype in the spleen of inoculated mice compared to K562 cells alone. These results indicate that NGAL plays a primary role in suppressing hematopoiesis by inducing apoptosis within normal hematopoietic cells. In the breast cancer project, we analyzed two microarray data sets of breast cancer cell lines ( n = 54) and primary breast cancer samples (n = 318), and demonstrated that high NGAL expression is significantly correlated with several tumor characteristics, including negative estrogen receptor (ER) status, positive HER2 status, high tumor grade, and lymph node metastasis. Ectopic NGAL expression in non-aggressive (ZR75.1 and MCF7) cells led to aggressive tumor phenotypes in vitro and in vivo. Conversely, knockdown of NGAL expression in various breast cancer cell lines by shRNA lentiviral infection significantly decreased migration, invasion, and metastasis activities of tumor cells both in vitro and in vivo . It has been previously reported that transgenic mice with a mutation in the region of trans-membrane domain (V664E) of HER2 develop mammary tumors that progress to lung metastasis. However, we observed that genetic deletion of the 24p3 gene, a mouse homolog of NGAL, in HER2 transgenic mice by breeding with 24p3-null mice resulted in a significant delay of mammary tumor formation and reduction of lung metastasis. Strikingly, we also found that treatment with affinity purified 24p3 antibodies in the 4T1 breast cancer mice strongly reduced lung metastasis. Our studies provide evidence that NGAL plays a critical role in breast cancer development and progression, and thus NGAL has potential as a new therapeutic target in breast cancer.^

The function of the murine complement *C3a and *C5a anaphylatoxin receptors in endotoxemia, bacteremia and septic shock

The complement system functions as a major effector for both the innate and adaptive immune response. Activation of the complement cascade by either the classical, alternative, or lectin pathway promotes the proteolysis of C3 and C5 thereby generating C3a and C5a. Referred to as anaphylatoxins, the C3a and C5a peptides mediate biological effects upon binding to their respective receptors; C3a binds to the C3a receptor (C3aR) while C5a binds to the C5a receptor (C5aR, CD88). Both C3a and C5a are known for their broad proinflammatory effects. Elevated levels of both peptides have been isolated from patients with a variety of inflammatory diseases such as COPD, asthma, RA, SLE, and sepsis. Recent studies suggest that C5a is a critical component in the acquired neutrophil dysfunction, coagulopathy, and progressive multi-organ dysfunction characteristic of sepsis. The primary hypothesis of this dissertation was that preventing C3a-C3aR and C5a-C5aR mediated pro-inflammatory effects would improve survival in endotoxic, bacteremic and septic shock. To test this hypothesis, the murine C3aR and C5aR genes were disrupted. Following disruption of both the C3aR and C5aR genes, no abnormalities were identified other than the absence of their respective mRNA and protein. In models of both endotoxic and bacteremic shock, C3aR deficient mice suffered increased mortality when compared to their wild type littermates. C3aR deficient mice also had elevated circulating IL-1β levels. Using a model of sepsis, C3aR deficient mice had a higher circulating concentration of IL-6 and decreased peritoneal inflammatory infiltration. While these results were unexpected, they support an emerging role for C3a in immunomodulation. In contrast, following endotoxic or bacteremic shock, C5aR deficient mice experienced increased survival, less hemoconcentration and less thrombocytopenia. It was later determined that C5a mediated histamine release significantly contributes to host morbidity and mortality in bacteremic shock. These studies provide evidence that C5a functions primarily as a proinflammatory molecule in models of endotoxic and bacteremic shock. In the same models, C3a-C3aR interactions suppress the inflammatory response and protect the host. Collectively, these results present in vivo evidence that C3a and C5a have divergent biological functions. ^

Modeling iron-catecholates binding to NGAL protein

Neutrophil gelatinase associated lipocalin (NGAL) protein is attracting a great interest because of its antibacterial properties played upon modulating iron content in competition against iron acquisition processes developed by pathogenic bacteria that bind selective ferric iron chelators (siderophores). Besides its known high affinity to enterobactin, the most important siderophore, it has been recently shown that NGAL is able to bind Fe(III) coordinated by catechols. The selective binding of Fe(III)-catechol ligands to NGAL is here studied by using iron coordination structures with one, two, and three catecholate ligands. By means of a computational approach that consists of B3LYP/6-311G(d,p) quantum calculations for geometries, electron properties and electrostatic potentials of ligands, protein–ligand flexible docking calculations, analyses of protein–ligand interfaces, and Poisson–Boltzmann electrostatic potentials for proteins, we study the binding of iron catecholate ligands to NGAL as a central member of the lipocalin family of proteins. This approach provides a modeling basis for exploring in silico the selective binding of iron catecholates ligands giving a detailed picture of their interactions in terms of electrostatic effects and a network of hydrogen bonds in the protein binding pocket.

Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R−/− mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R−/− animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.

Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression

Leukocyte interactions with vascular endothelium during inflammation occur through discrete steps involving selectin-mediated leukocyte rolling and subsequent firm adhesion mediated by members of the integrin and Ig families of adhesion molecules. To identify functional synergy between selectin and Ig family members, mice deficient in both L-selectin and intercellular adhesion molecule 1 (ICAM-1) were generated. Leukocyte rolling velocities in cremaster muscle venules were increased significantly in ICAM-1-deficient mice during both trauma- and tumor necrosis factor α-induced inflammation, but rolling leukocyte flux was not reduced. Elimination of ICAM-1 expression in L-selectin-deficient mice resulted in a sharp reduction in the flux of rolling leukocytes during tumor necrosis factor α-induced inflammation. The observed differences in leukocyte rolling behavior demonstrated that ICAM-1 expression was required for optimal P- and L-selectin-mediated rolling. Increased leukocyte rolling velocities presumably translated into decreased tissue emigration because circulating neutrophil, monocyte, and lymphocyte numbers were increased markedly in L-selectin/ICAM-1-deficient mice. Furthermore, neutrophil emigration during acute peritonitis was reduced by 80% in the double-deficient mice compared with either L-selectin or ICAM-1-deficient mice. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling in vivo, which is essential for the generation of effective inflammatory responses.

Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P < 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n = 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.