Influence of age and sex on the longitudinal relaxation time, T1, of the lung in healthy never-smokers.

BACKGROUND: As several studies have provided evidence that lung disease affects the T1 of the human lung, our purpose was to investigate the effect of age on the T1-relaxation time in the lungs of healthy never-smokers, including group difference between sexes. MATERIALS AND METHODS: The Snapshot FLASH pulse sequence (inversion recovery with multiple gradient echo read-outs) was used to quantify lung T1 in 30 healthy never-smoking volunteers at 1.5 Tesla. Measurements were performed under breathhold of a tidal inspiration. Additionally, subjects underwent clinical MRI and pulmonary function tests. A linear regression model of T1 as a function of age and sex was tested. RESULTS: The slope of lung T1 at tidal end-inspiration as a function of age was statistically different between males and females (P < 0.001). In a linear regression model of T1 as a function of age and sex, females have slope of -4.1 ms/year (95% confidence interval [CI], [-5.2, -3.0]) at P < 0.001, and males -0.064 ms/year (95% CI, [-1.2, 1.1]) at P = 0.9, with a whole model R(2) = 0.83. CONCLUSION: The observed dependencies of lung T1 on age and sex are here attributed to a previously reported difference in blood T1 between sexes, and a previously reported decrease of pulmonary blood volume with increasing age. This may have implications for the interpretation of lung T1 measurements in both healthy individuals and patients.

Early mechanisms of neutrophil activation and transmigration in acute lung injury.

Introduction:Neutrophil transmigration is multifactorial and primarily driven by selectins and ß2-integrins (CD11b/CD18), whose expression are dependent on the underlying stimulus. Ventilator-induced lung injury (VILI) results in a predominantly CD18-independent mechanism of neutrophil recruitment, while direct endotoxin-induced lung injury results from a CD18-dependent mechanism. We previously observed that lack of NADPH oxidases DUOX1 and DUOX2 resulted in reduced neutrophil influx in a VILI model of lung injury but had no influence on neutrophil influx after LPS exposure. Based on these observations, we hypothesized that DUOX1/DUOX2 are an important component of CD18-independent mechanisms of neutrophil recruitment in the lung. Methods:We exposed Duoxa -/- (KO) mice and Duoxa +/+ (WT) mice to either an intratracheal exposure of lipopolysaccharide (LPS/endotoxin)-or high tidal volume ventilation and compared expression of neutrophil markers between groups. WT mice (129S6/SvEvTac) were obtained from Taconic Biosciences (One Discovery Drive Suite 304; Rensselaer, NY 1244) and were allowed to acclimatize for one week prior to study enrollment. KO mice were generated as previously described [Grasberger 2012] and bred in-house on a 129S6 background. We provided positive-pressure ventilation at a tidal volume of 10 ml/kg with 2 cmH20 positive end-expiratory pressure (PEEP). Mice were assigned to groups consisting of KO (n = 5) and WT (n = 5) in each group and divided into non-ventilated, positive-pressure ventilation, or LPS IT exposure groups. Positive-pressure ventilation was instituted for 4-h using a FlexiVent (Flexiware 8.1, Scireq, Montreal, QC, Canada). Lipopolysaccharide (Salmonella enterica serotype tryphimurium L6143, Millipore Sigma) was administered via an intratracheal (IT) route at a dose of 0.1 mg/kg. Mice were humanely euthanized at 4-h post-injection consistent with the UC Davis IAUCAC-approved protocol. Results:As previously observed, neutrophilic influx into the airways was significantly impaired in the Duoxa -/- (KO) mice after VILI, but not after LPS exposure. LPS-induced lung injury resulted in upregulation of CD11b+ neutrophils and shedding of CD62L and CD162 regardless of DUOX expression, whereas VILI resulted in upregulation of CD49+ neutrophils in the Duoxa +/+ (WT) mice but not the Duoxa -/- (KO) mice. Conclusion:Our data suggest DUOX is required for CD18-independent mechanisms of neutrophil recruitment in the lung induced by acute lung injury, but not for canonical CD18depedent mechanisms after LPS exposure.

Neutrophils exposed to A. phagocytophilum under shear stress fail to fully activate, polarize, and transmigrate across inflamed endothelium.

Anaplasma phagocytophilum is an obligate intracellular bacterium that has evolved mechanisms to hijack polymorphonuclear neutrophil (PMN) receptors and signaling pathways to bind, infect, and multiply within the host cell. E-selectin is upregulated during inflammation and is a requisite endothelial receptor that supports PMN capture, rolling, and activation of integrin-mediated arrest. Ligands expressed by PMN that mediate binding to endothelium via E-selectin include sialyl Lewis x (sLe(x))-expressing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1) and other glycolipids and glycoproteins. As A. phagocytophilum is capable of binding to sLe(x)-expressing ligands expressed on PMN, we hypothesized that acute bacterial adhesion to PMN would subsequently attenuate PMN recruitment during inflammation. We assessed the dynamics of PMN recruitment and migration under shear flow in the presence of a wild-type strain of A. phagocytophilum and compared it with a strain of bacteria that binds to PMN independent of PSGL-1. Acute bacterial engagement with PMN resulted in transient PMN arrest and minimal PMN polarization. Although the wild-type pathogen also signaled activation of beta2 integrins and elicited a mild intracellular calcium flux, downstream signals including PMN transmigration and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were inhibited. The mutant strain bound less well to PMN and failed to activate beta2 integrins and induce a calcium flux but did result in decreased PMN arrest and polarization that may have been partially mediated by a suppression of p38 MAPK activation. This model suggests that A. phagocytophilum binding to PMN under shear flow during recruitment to inflamed endothelium interferes with normal tethering via E-selectin and navigational signaling of transendothelial migration.

GlyCAM-1, a physiologic ligand for L-selectin, activates beta 2 integrins on naive peripheral lymphocytes.

Naive T cells are selectively recruited from the blood into peripheral lymph nodes during lymphocyte recirculation. L-selectin, a lectin-like receptor, mediates the initial attachment of lymphocytes to high endothelial venules (HEV) in lymph nodes. A subsequent step involving the activation of beta 2 integrins has been proposed to facilitate firm adhesion, but the activating signals are poorly understood. We report here that either antibody-mediated cross-linking of L-selectin on human lymphocytes or treatment of the cells with GlyCAM-1, an HEV-derived, secreted ligand for L-selectin, stimulates their binding to ICAM-1 through the beta 2 integrin pathway. Furthermore, GlyCAM-1 causes the rapid expression of a neoepitope on beta 2 integrins associated with a high-avidity state. Naive (CD45RA+), but not memory (CD45R0+) lymphocytes, respond to L-selectin cross-linking or GlyCAM-1 treatment. Thus, the complexing of L-selectin by specific ligands may provide key signals to naive lymphocytes, contributing to their selective recruitment into peripheral lymphoid organs.

Flow cytometric analysis and modeling of cell-cell adhesive interactions: the neutrophil as a model.

The immune function of granulocytes, monocytes, lymphocytes, and other specialized cells depends upon intercellular adhesion. In many cases the molecules mediating leukocyte cell adhesion belong to the Leu-CAM superfamily of adhesive molecules. To elucidate the events of homotypic aggregation in a quantitative fashion, we have examined the aggregation of neutrophils stimulated with formyl peptides, where aggregate formation is a transient reversible cell function. We have mathematically modeled the kinetics of aggregation using a linear model based on particle geometry and rates of aggregate formation and breakup. The time course was modeled as a three-phase process, each phase with distinct rate constants. Aggregate formation was measured on the flow cytometer; singlets and larger particles were distinguished using the intravital stain LDS-751. Aggregation proceeded rapidly after stimulation with formyl peptide (CHO-nle-leu-phe-nle-tyr-lys). The first phase lasted 30-60 s; this was modeled with the largest aggregation rate and smallest rate of disaggregation. Aggregate formation plateaued during the second phase which lasted up to 2.5 min. This phase was modeled with an aggregation rate nearly an order of magnitude less than that of the initial fast phase, whereas the disaggregation rate for this phase did not change significantly. A third phase where disaggregation predominated, lasted the remaining 2-3 min and was modeled with a four to fivefold increase of the disaggregation rate. The mechanism of cell-cell adhesion in the plateau phase was probed with the monoclonal antibody IB4 to the CD18 subunit of the adhesive receptor CR3. Based on these studies it appears that new aggregates do not form to a large degree after the first phase of aggregate formation is complete. However, new adhesive contact sites may form within the contact region of these adherent cells to keep the aggregates together.

T1 Relaxation Time in Lungs of Asymptomatic Smokers.

PURPOSE: Interest in using T1 as a potential MRI biomarker of chronic obstructive pulmonary disease (COPD) has recently increased. Since tobacco smoking is the major risk factor for development of COPD, the aim for this study was to examine whether tobacco smoking, pack-years (PY), influenced T1 of the lung parenchyma in asymptomatic current smokers. MATERIALS AND METHODS: Lung T1 measurements from 35 subjects, 23 never smokers and 12 current smokers were retrospectively analyzed from an institutional review board approved study. All 35 subjects underwent pulmonary function test (PFT) measurements and lung T1, with similar T1 measurement protocols. A backward linear model of T1 as a function of FEV1, FVC, weight, height, age and PY was tested. RESULTS: A significant correlation between lung T1 and PY was found with a negative slope of -3.2 ms/year (95% confidence interval [CI] [-5.8, -0.6], p = 0.02), when adjusted for age and height. Lung T1 shortens with ageing among all subjects, -4.0 ms/year (95%CI [-6.3, -1.7], p = 0.001), and among the never smokers, -3.7 ms/year (95%CI [-6.0, -1.3], p = 0.003). CONCLUSIONS: A correlation between lung T1 and PY when adjusted for both age and height was found, and T1 of the lung shortens with ageing. Accordingly, PY and age can be significant confounding factors when T1 is used as a biomarker in lung MRI studies that must be taken into account to detect underlying patterns of disease.

Induction of homotypic lymphocyte aggregation: evidence for a novel activation state of the beta1 integrin.

Intercellular adhesion of Jurkat lymphocytic cells was investigated by use of monoclonal antibodies 33B6 and 18D3, which bind to the beta1 integrin receptor. 33B6 induced homotypic aggregation of Jurkat cells, whereas 18D3 inhibited this aggregation. Jurkat cells could he induced to aggregate at low 33B6 concentrations corresponding to 5% beta1 integrin site occupancy, and the rate of aggregation was maximum at 30% occupancy. Simultaneous addition of mAb 18D3 and 33B6 demonstrated that the two antibodies mediate changes in the beta1 integrin activation state that are competitive in nature. Aggregation through beta1 integrin induced by 33B6 was reversed by subsequent addition of 18D3. To further examine the mechanism by which 33B6 and 18D3 affect cell adhesion function, we explored the binding of monoclonal antibody (mAb) 15/7. This mAb recognizes an activation epitope of the beta1 integrin and has been shown to sustain cell adhesion to vascular cell adhesion molecule 1 (VCAM-1) and fibronectin. Activation of Jurkat cells with Mn2+ caused a 2.5-fold increase in 15/7 binding but did not increase binding of 33B6. 33B6 partially blocked 15/7 binding to beta1 integrin on unstimulated and Mn2+-activated Jurkat cells. 18D3 did not affect mAb 15/7 binding. These results indicate that 33B6 and 18D3 modulated homotypic aggregation by inducing a novel activation state of the very late activation integrin distinct from the state recognized by 15/7, which supports cell binding to VCAM-1 and fibronectin.

Endocytosis of beta 2 integrins by stimulated human neutrophils analyzed by flow cytometry.

Flow cytometry and fluorescently labeled monoclonal antibodies were used to investigate endocytosis of human neutrophil beta 2 integrins following cellular activation. CD18 initially present on the cell surface cycled in two phases after exposure to formyl peptide or platelet-activating factor. The first phase lasted 3 min at 37 degrees C; after a lag, CD18 was specifically internalized at approximately 20%/min. Subsequently a second phase was detectable consisting of exponential reduction of internal fluorescence with a half-time of approximately 2 min, representing probe reexpression. At peak endocytosis approximately 40% of CD18 was internalized. All of the internalized CD18 was associated with alpha M (CR3); no endocytosis of alpha L (LFA-1) was observed. When neutrophils were stimulated with phorbol esters or calcium ionophore, CD18 was internalized much more slowly (t1/2 = 5 min) and probe was not reexpressed. Endocytosis of CD18 may participate in regulating neutrophil adhesiveness, removing activated receptors, or permitting receptor recycling.

Beta2-integrins mediate stable adhesion in collisional interactions between neutrophils and ICAM-1-expressing cells.

The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and beta2-integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s(-1)) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s(-1) in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s(-1). The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed.

Preferential sites for stationary adhesion of neutrophils to cytokine-stimulated HUVEC under flow conditions.

Neutrophils form CD18-dependent adhesions to endothelial cells at sites of inflammation. This phenomenon was investigated under conditions of flow in vitro using isolated human neutrophils and monolayers of HUVEC. The efficiency of conversion of neutrophil rolling to stable adhesion in this model was >95%. Neither anti-CD11a nor anti-CD11b antibodies significantly altered the extent of this conversion, but a combination of both antibodies inhibited the arrest of rolling neutrophils by >95%. The efficiency of transendothelial migration of arrested neutrophils was >90%, and the site of transmigration was typically <6 microm from the site of stationary adhesion. Approximately 70% of transmigrating neutrophils migrated at tricellular corners between three adjacent endothelial cells. A model of neutrophils randomly distributed on endothelium predicted a significantly greater migration distance to these preferred sites of transmigration, but a model of neutrophils adhering to endothelial borders is consistent with observed distances. It appears that stable adhesions form very near tricellular corners.

P-selectin mediates neutrophil adhesion to endothelial cell borders.

During an acute inflammatory response, endothelial P-selectin (CD62P) can mediate the initial capture of neutrophils from the free flowing bloodstream. P-selectin is stored in secretory granules (Weibel-Palade bodies) and is rapidly expressed on the endothelial surface after stimulation with histamine or thrombin. Because neutrophil transmigration occurs preferentially at endothelial borders, we wished to determine whether P-selectin-dependent neutrophil capture (adhesion) occurs at endothelial cell borders. Under static or hydrodynamic flow (2 dyn/cm2) conditions, histamine (10(-4) M) or thrombin (0.2 U/mL) treatment induced preferential (> or = 75%) neutrophil adhesion to the cell borders of endothelial monolayers. Blocking antibody studies established that neutrophil adhesion was completely P-selectin dependent. P-selectin surface expression increased significantly after histamine treatment and P-selectin immunostaining was concentrated along endothelial borders. We conclude that preferential P-selectin expression along endothelial borders may be an important mechanism for targeting neutrophil migration at endothelial borders.

Preservation of spatial organization and antigenicity of leukocyte surface molecules by aldehyde fixation: flow cytometry and high-resolution FESEM studies of CD62L, CD11b, and Thy-1.

We used transmission and scanning electron microscopy in conjunction with immunogold labeling to study cell surface molecules for evidence of distribution-function relationships. Ascription of functional significance to surface distribution therefore requires preservation of cell morphology and maintenance of molecular expression and distribution through the multiple steps of cell preparation. These requirements prompted us to compare two methods for preparing leukocytes for analysis of surface molecule distribution: one method involved using low temperature to "stabilize" cell morphology and surface molecular organization through immunolabeling; the other involved fixation of the cells with dilute glutaraldehyde before their isolation and labeling. Binding of primary antibodies to several surface molecules, measured by flow cytometry, was comparable for cells prepared by the two methods. Cell morphology and molecular distributions, assessed by high-resolution field emission SEM, were likewise comparable. These results support the conclusion that cell morphologies and CAM distributions previously reported were not affected by exposure of the cells to low temperature through isolation and immunolabeling. Our additional observation that Thy-1 is expressed on both non-projecting and projecting membrane domains of mouse lymph node lymphocytes and rat thymocytes represents a third and new pattern of surface molecule distribution.

Enterococcus faecalis aggregation substance promotes opsonin-independent binding to human neutrophils via a complement receptor type 3-mediated mechanism.

Enterococcus faecalis aggregation substance (AS) mediates efficient adhesion between bacteria, thereby facilitating plasmid exchange as an integral part of a bacterial sex pheromone system. We examined the interaction of AS-bearing E. faecalis with human neutrophils (PMNs), an important component of the host defense system. AS promoted a markedly increased opsonin-independent bacterial binding to PMNs. Adhesion was dependent on the expression of the enterococcal Asc10 protein, which contains two Arg-Gly-Asp (RGD) sequences, and addition of exogenous RGD-containing peptides inhibited AS-mediated binding by 66%. AS-mediated adhesion was inhibited by 85% by anti-human complement receptor type 3 (CR3) monoclonal antibodies or by use of PMNs from a patient with leukocyte adhesion deficiency. However, AS-bearing E. faecalis cells were unable to bind to CHO-Mac-1 cells, expressing functionally active CR3, suggesting the potential need for additional PMN surface receptors for bacterial adhesion. Monoclonal antibodies against integrin-associated protein (CD47) and L-selectin, both of which may interact with CR3 and bind to ligands on E. faecalis, also inhibited AS-dependent binding. The non-opsonic binding of E. faecalis to PMNs may play an important role in this organism's pathogenesis.

Cutaneous insulin reaction resembling acanthosis nigricans.

We describe a patient who developed hyperkeratotic, verrucous plaques at sites of repeated insulin injections. One similar case has previously been reported. This phenomenon may be an unusual manifestation of the trophic actions of insulin.

E-selectin ligands as mechanosensitive receptors on neutrophils in health and disease.

Application of mechanical force to bonds between selectins and their ligands is a requirement for these adhesion receptors to optimally perform functions that include leukocyte tethering and activation of stable adhesion. Although all three selectins are reported to signal from the outside-in subsequent to ligand binding, E-selectin is unique in its capacity to bind multiple sialyl Lewis x presenting ligands and mediate slow rolling on the order of a micron per second. A diverse set of ligands are recognized by E-selectin in the mouse, including ESL-1, CD44 (HCELL), and PSGL-1 which are critical in transition from slow rolling to arrest and for efficient transendothelial migration. The molecular recognition process is different in humans as L-selectin is a major ligand, which along with glycolipids constitute more than half of the E-selectin receptors on human polymorphonuclear neutrophils (PMN). In addition, E-selectin is most efficient at raising the affinity and avidity of CD18 integrins that supports PMN deceleration and trafficking to sites of acute inflammation. The mechanism is only partially understood but known to involve a rise in cytosolic calcium and tyrosine phosphorylation that activates p38 MAP kinase and Syk kinase, both of which transduce signals from clustered E-selectin ligands. In this review we highlight the molecular recognition and mechanical requirements of this process to reveal how E-selectin confers selectivity and efficiency of signaling for extravasation at sites of inflammation and the mechanism of action of a new glycomimetic antagonist targeted to the lectin domain that has shown efficacy in blocking neutrophil activation and adhesion on inflamed endothelium.

HSP60 in heart failure: abnormal distribution and role in cardiac myocyte apoptosis.

Heat shock protein (HSP) 60 is a mitochondrial and cytosolic protein. Previously, we reported that HSP60 doubled in end-stage heart failure, even though levels of the protective HSP72 were unchanged. Furthermore, we observed that acute injury in adult cardiac myocytes resulted in movement of HSP60 to the plasma membrane. We hypothesized that the inflammatory state of heart failure would cause translocation of HSP60 to the plasma membrane and that this would provide a pathway for cardiac injury. Two models were used to test this hypothesis: 1) a rat model of heart failure and 2) human explanted failing hearts. We found that HSP60 localized to the plasma membrane and was also found in the plasma early in heart failure. Plasma membrane HSP60 localized to lipid rafts and was detectable on the cell surface with the use of both flow cytometry and confocal microscopy. Localization of HSP60 to the cell surface correlated with increased apoptosis. In heart failure, HSP60 is in the plasma membrane fraction, on the cell surface, and in the plasma. Membrane HSP60 correlated with increased apoptosis. Release of HSP60 may activate the innate immune system, promoting a proinflammatory state, including an increase in TNF-alpha. Thus abnormal trafficking of HSP60 to the cell surface may be an early trigger for myocyte loss and the progression of heart failure.