Effect of epinephrine on neutrophil kinetics in rabbit lungs

The effect of epinephrine on neutrophil (PMN) kinetics in rabbit lungs was evaluated by measuring the retention of radiolabeled PMN's in the lung, the exchange rate between the marginated and circulating pools of PMN's, and the erythrocyte (RBC) transit time. Epinephrine treatment decreased RBC transit times and increased exchange rates in the regions with the shortest transit times but did not change the pulmonary recovery of radiolabeled PMN's. When regions of similar RBC transit time were compared, epinephrine did not affect PMN retention at short transit times but did produce greater retention at long transit times. These data suggest that the major effect of epinephrine was to increase the proportion of the lung having short RBC transit times and fast exchange rates between the marginated and circulating pools. However, this effect did not decrease the overall retention of PMN's most likely because it was balanced by recruitment of additional capillary segments, which increased PMN retention in regions with longer transit times.     

Neutrophil-associated lung injury after the infusion of activated plasma

Previous studies from our laboratory have shown that the infusion of zymosan-activated plasma (ZAP) caused large numbers of neutrophils (PMN) to accumulate in the lung. Although PMN are known to be activated by ZAP, it is unclear whether PMN delayed in the lung by ZAP infusion actually cause lung injury. The present study was designed to examine this question by measuring airway epithelial and endothelial injury. Airway epithelial injury was determined by depositing a known dose of fluorescein isothiocyanate-labeled dextran in the lung and measuring its appearance in the blood, and endothelial injury was measured by injecting colloidal carbon and measuring its accumulation in the microvasculature of the lung. The data show that ZAP infusion caused a mild epithelial and endothelial injury that did not increase either extravascular water or protein. This injury could be prevented either by depleting the animals of PMN or by pretreating them with indomethacin. In addition, the effect of ZAP infusion could be partially restored by transfusing donor PMN into the PMN-depleted animals. We conclude that ZAP infusion produces a mild lung injury that is dependent on PMN and the products of the cyclooxygenase pathway of arachidonic acid metabolism.     

Monocyte kinetics in rabbits

Previous studies of monocytes isolated from peripheral blood have suggested that the lung sequesters these cells and has an important role in monocyte kinetics. However, the lung also provides the first capillary bed encountered by these cells after intravenous injection. A major criticism of the previous reports is that the behavior of monocytes in the lung may be altered as a result of the isolation procedure. The present study addresses this question by comparing the distribution of isolated monocytes (87% pure) in various organs 10 min after they were injected into either the central venous or the arterial circulation. The data show that the extraction of monocytes on the first passage through the lung after intravenous injection was 86.5 +/- 1.5%. After the monocytes had circulated for 10 min, the lungs contained 35.5 +/- 2.5% of the cells after intravenous injection and 29.7 +/- 2.2% after intra-arterial injection (P greater than 0.05). The lung-to-blood recovery ratio after either intravenous or intra-arterial injection showed that the lung contained a marginating pool of monocytes that was five to seven times the size of the circulating pool. The retention of monocytes in a region of the lung was proportional to the regional erythrocyte transit time. The half-life of the radiolabeled monocytes in the circulation was approximately 25 h. We conclude that the lung contains a marginating pool of monocytes and speculate that they concentrate there in preparation for migration into the interstitium and air space of the lung.     

CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits

Neutrophil (PMN) migration in the systemic and pulmonary circulation of rabbits was compared by using different inflammatory stimuli to determine the role of the leukocyte adhesion complex, CD11/CD18, in each of these vascular beds. The adhesion complex was blocked by administering the anti-CD18 mAb 60.3. The data show that mAb 60.3 blocks PMN emigration into inflammatory foci in the abdominal wall produced by implanting sponges containing either hydrochloric acid, Streptococcus pneumoniae, Escherichia coli endotoxin, or PMA. mAb 60.3 also inhibited PMN emigration in response to peritoneal instillation of S. pneumoniae. The effect of mAb 60.3 on PMN emigration in the lungs varied depending upon the stimulus. PMN failed to migrate into the PMA-induced pneumonia; however, mAb 60.3 pretreatment only partially inhibited endotoxin-induced pneumonia and did not inhibit S. pneumoniae or hydrochloric acid-induced pneumonias. PMN lavaged from the alveolar spaces in the Streptococcal pneumonia had similar quantities of mAb 60.3 bound to their surfaces as the circulating PMN. We conclude that the CD11/CD18 complex mediates PMN adherence in the systemic circulation. However, PMN adherence in the pulmonary circulation may occur by either CD18-dependent or -independent mechanisms that are specific to the inciting stimulus.     

Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries

Dhadwal, Amit, Barry Wiggs, Claire M. Doerschuk, and RogerD. Kamm. Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries. J. Appl. Physiol. 83(5): 1711-1720, 1997.Atheoretical model is developed to simulate the flow of blood throughthe capillary network in a single alveolar septum. The objective is tostudy the influence of random variability in capillary dimension andcompliance on flow patterns and pressures within the network. Thecapillary bed is represented as an interconnected rectangular grid ofcapillary segments and junctions; blood flow is produced by applying apressure gradient across the network. Preferred flow channels are shownto be a natural consequence of random anatomic variability, the effectof which is accentuated at low transcapillary pressures. Thedistribution of pressure drops across single capillary segments widenswith increasing network variability and decreasing capillary transmuralpressure. Blockage of one capillary segment causes the pressure dropacross that segment to increase by 60%, but the increase falls to<10% at a distance of three segments. The factors that causenonuniform capillary blood flow through the capillary network arediscussed.

     

Neutrophil transit times through pulmonary capillaries: the effects of capillary geometry and fMLP-stimulation

The deformations of neutrophils as they pass through the pulmonary microcirculation affect their transit time, their tendency to contact and interact with the endothelial surface, and potentially their degree of activation. Here we model the cell as a viscoelastic Maxwell material bounded by constant surface tension and simulate indentation experiments to quantify the effects of (N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulation on its mechanical properties (elastic shear modulus and viscosity). We then simulate neutrophil transit through individual pulmonary capillary segments to determine the relative effects of capillary geometry and fMLP-stimulation on transit time. Indentation results indicate that neutrophil viscosity and shear modulus increase by factors of 3.4, for 10(-9) M fMLP, and 7.3, for 10(-6) M fMLP, over nonstimulated cell values, determined to be 30.8 Pa.s and 185 Pa, respectively. Capillary flow results indicate that capillary entrance radius of curvature has a significant effect on cell transit time, in addition to minimum capillary radius and neutrophil stimulation level. The relative effects of capillary geometry and fMLP on neutrophil transit time are presented as a simple dimensionless expression and their physiological significance is discussed.     

A statistical approach to computer processing of cryo-electron microscope images: virion classification and 3-D reconstruction

The scattering density of the virus is represented as a truncated weighted sum of orthonormal basis functions in spherical coordinates, where the angular dependence of each basis function has icosahedral symmetry. A statistical model of the image formation process is proposed and the maximum likelihood estimation method computed by an expectation-maximization algorithm is used to estimate the weights in the sum and thereby compute a 3-D reconstruction of the virus particle. If multiple types of virus particle are represented in the boxed images then multiple 3-D reconstructions are computed simultaneously without first requiring that the type of particle shown in each boxed image be determined. Examples of the procedure are described for viruses with known structure: (1). 3-D reconstruction of Flockhouse Virus from experimental images, (2). 3-D reconstruction of the capsid of Nudaurelia Omega Capensis Virus from synthetic images, and (3). 3-D reconstruction of both the capsid and the procapsid of Nudaurelia Omega Capensis Virus from a mixture of unclassified synthetic images.     

Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-1 and reactive oxygen species

This study evaluated the changes in the biomechanical properties of endothelial cells (ECs) induced by neutrophil adhesion and the roles of ICAM-1 and reactive oxygen species (ROS) in modulating these changes. Neutrophil adherence to 24-h TNF-alpha-activated pulmonary microvascular ECs induced an increase in the apparent stiffness of ECs within 2 min, measured with magnetic twisting cytometry. An anti-ICAM-1 Ab blocked the EC stiffening response without inhibiting neutrophil adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening response induced by neutrophils. The neutrophil-induced increase in the apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl radical scavenger), allopurinol (a xanthine oxidase inhibitor), or deferoxamine (an iron chelator), suggesting that ROS may be involved in mediating the EC stiffening response. The cellular sources of ROS were determined by measuring the oxidation of dichlorofluorescein. Neutrophil adherence to TNF-alpha-activated ECs induced ROS production only in ECs, and not in neutrophils. This ROS production in ECs was completely prevented by the anti-ICAM-1 Ab and partially inhibited by allopurinol. These results suggest that ICAM-1-mediated signaling events during neutrophil adherence may activate xanthine oxidase, which in turn mediates the ROS production in ECs that leads to stiffening. ROS generated in ECs on neutrophil adherence appear to mediate cytoskeletal remodeling, which may modulate subsequent inflammatory responses.     

Ab initio maximum likelihood reconstruction from cryo electron microscopy images of an infectious virion of the tailed bacteriophage P22 and maximum likelihood versions of Fourier Shell Correlation appropriate for measuring resolution of spherical or cylindrical objects

A maximum likelihood reconstruction method for an asymmetric reconstruction of the infectious P22 bacteriophage virion is described and demonstrated on a subset of the images used in [Lander, G.C., Tang, L., Casjens, S.R., Gilcrease, E.B., Prevelige, P., Poliakov, A., Potter, C.S., Carragher, B., Johnson, J.E., 2006. The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 312(5781), 1791–1795]. The method makes no assumptions at any stage regarding the structure of the phage tail or the relative rotational orientation of the phage tail and capsid but rather the structure and the rotation angle are determined as a part of the analysis. A statistical method for determining resolution consistent with maximum likelihood principles based on ideas for cylinders analogous to the ideas for spheres that are embedded in the Fourier Shell Correlation method is described and demonstrated on the P22 reconstruction. With a correlation threshold of .95, the resolution in the tail measured radially is greater than (33.3 Å) and measured axially is greater than (70.6 Å) both with probability p=0.02.     

Greater airway narrowing in immature than in mature rabbits during methacholine challenge

Shen, X., V. Bhargava, G. R. Wodicka, C. M. Doerschuk, S. J. Gunst, and R. S. Tepper. Greater airway narrowing in immature thanin mature rabbits during methacholine challenge. J. Appl. Physiol. 81(6): 2637-2643, 1996.It hasbeen demonstrated that methacholine (MCh) challenge produces a greaterincrease in lung resistance in immature than in mature rabbits (R. S. Tepper, X. Shen, E. Bakan, and S. J. Gunst.J. Appl. Physiol. 79: 1190-1198, 1995). To determine whether this maturational difference in the response to MCh was primarily related to changes in airway resistance (Raw) or changes in tissue resistance, we assessed airway narrowing in1-, 2-, and 6-mo-old rabbits during intravenous MCh challenge (0.01-5.0 mg/kg). Airway narrowing was determined frommeasurements of Raw in vivo and from morphometric measurements on lungsections obtained after rapidly freezing the lung after the MChchallenge. The fold increase in Raw was significantly greater for 1- and 2-mo-old animals than for 6-mo-old animals. Similarly, the degree of airway narrowing assessed morphometrically was significantly greaterfor 1- and 2-mo-old animals than for 6-mo-old animals. The foldincrease in Raw was highly correlated with the degree of airwaynarrowing assessed morphometrically(r² = 0.82, P < 0.001). We conclude that thematurational difference in the effect of MCh on lung resistance isprimarily caused by greater airway narrowing in the immature rabbits.